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Sample records for factor zapb stimulates

  1. Spatial resolution of two bacterial cell division proteins: ZapA recruits ZapB to the inner face of the Z-ring.

    PubMed

    Galli, Elisa; Gerdes, Kenn

    2010-06-01

    FtsZ, the essential regulator of bacterial cell division, is a dynamic cytoskeletal protein that forms helices that condense into the Z-ring prior to division. Two small coiled-coil proteins, ZapA and ZapB, are both recruited early to the Z-ring. We show here that ZapB is recruited to the Z-ring by ZapA. A direct interaction between ZapA and ZapB is supported by bacterial two-hybrid and in vitro interaction assays. Using high-resolution 3-D reconstruction microscopy, we find that, surprisingly, ZapB is located inside the Z-ring in virtually all cells investigated. We propose a molecular model in which ZapA increases lateral interactions between FtsZ proto-filaments and ZapB mediates further stabilization of this interaction by cross-linking ZapA molecules bound to adjacent FtsZ proto-filaments. Gene deletion and complementation assays show that ZapB can mitigate cell division and Z-ring assembly defects even in the absence of ZapA, raising the possibility that ZapB stimulates Z-ring assembly by two different mechanisms.

  2. Factors stimulating bone formation.

    PubMed

    Lind, M; Bünger, C

    2001-10-01

    The aim of this review is to describe major approaches for stimulating bone healing and to review other factors affecting bone healing. Spinal bone fusion after surgery is a demanding process requiring optimal conditions for clinical success. Bone formation and healing can be enhanced through various methods. Experimental studies have revealed an array of stimulative measures. These include biochemical stimulation by use of hormones and growth factors, physical stimulation through mechanical and electromagnetic measures, and bone grafting by use of bone tissue or bone substitutes. Newer biological techniques such as stem cell transplantation and gene therapy can also be used to stimulate bone healing. Apart from bone transplantation, clinical experience with the many stimulation modalities is limited. Possible areas for clinical use of these novel methods are discussed.

  3. Factors Associated with Speech-Sound Stimulability.

    ERIC Educational Resources Information Center

    Lof, Gregory L.

    1996-01-01

    This study examined stimulability in 30 children (ages 3 to 5) with articulation impairments. Factors found to relate to stimulability were articulation visibility, the child's age, the family's socioeconomic status, and the child's overall imitative ability. Perception, severity, otitis media history, language abilities, consistency of…

  4. In vivo organization of the FtsZ-ring by ZapA and ZapB revealed by quantitative super-resolution microscopy

    PubMed Central

    Buss, Jackson; Coltharp, Carla; Huang, Tao; Pohlmeyer, Chris; Wang, Shih-Chin; Hatem, Christine; Xiao, Jie

    2013-01-01

    Summary In most bacterial cells, cell division is dependent on the polymerization of the FtsZ protein to form a ring-like structure (Z-ring) at the midcell. Despite its essential role, the molecular architecture of the Z-ring remains elusive. In this work we examine the roles of two FtsZ-associated proteins, ZapA and ZapB, in the assembly dynamics and structure of the Z-ring in E. coli cells. In cells deleted of zapA or zapB, we observed abnormal septa and highly dynamic FtsZ structures. While details of these FtsZ structures are difficult to discern under conventional fluorescence microscopy, single-molecule based superresolution imaging method Photoactivated Localization Microscopy (PALM) reveals that these FtsZ structures arise from disordered arrangements of FtsZ clusters. Quantitative analysis finds these clusters are larger and comprise more molecules than a single FtsZ protofilament, and likely represent a distinct polymeric species that is inherent to the assembly pathway of the Z-ring. Furthermore, we find these clusters are not due to the loss of ZapB-MatP interaction in ΔzapA and ΔzapB cells. Our results suggest that the main function of ZapA and ZapB in vivo may not be to promote the association of individual protofilaments but to align FtsZ clusters that consist of multiple FtsZ protofilaments. PMID:23859153

  5. Stimulation of neutrophils by tumor necrosis factor

    SciTech Connect

    Klebanoff, S.J.; Vadas, M.A.; Harlan, J.M.; Sparks, L.H.; Gamble, J.R.; Agosti, J.M.; Waltersdorph, A.M.

    1986-06-01

    Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H/sub 2/O/sub 2/ production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

  6. The colony-stimulating factors and cancer.

    PubMed

    Metcalf, Donald

    2013-12-01

    The colony-stimulating factors (CSFs) are the master regulators of granulocyte and macrophage populations. There are four different aspects of the connection between the CSFs and cancer: (a) the CSFs can accelerate the regeneration of protective white cells damaged by chemotherapy; (b) the CSFs can mobilize stem cells to the peripheral blood in convenient numbers for transplantation; (c) the CSFs can enhance anticancer immune responses and (d) the CSFs are potentially involved in the genesis of the myeloid leukemias.

  7. The colony-stimulating factors and cancer.

    PubMed

    Metcalf, Donald

    2010-06-01

    The four colony-stimulating factors (CSFs) are glycoproteins that regulate the generation and some functions of infection-protective granulocytes and macrophages. Recombinant granulocyte-CSF (G-CSF) and granulocyte-macrophage-CSF (GM-CSF) have now been used to increase dangerously low white blood cell levels in many millions of cancer patients following chemotherapy. These CSFs also release haematopoietic stem cells to the peripheral blood, and these cells have now largely replaced bone marrow as more effective populations for transplantation to cancer patients who have treatment-induced bone marrow damage.

  8. The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

    PubMed Central

    Männik, Jaana; Castillo, Daniel E.; Yang, Da; Siopsis, George; Männik, Jaan

    2016-01-01

    Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregation in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. However, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication. PMID:26762981

  9. Granulocyte macrophage colony stimulating factor therapy for pulmonary alveolar proteinosis.

    PubMed

    Shende, Ruchira P; Sampat, Bhavin K; Prabhudesai, Pralhad; Kulkarni, Satish

    2013-03-01

    We report a case of 58 year old female diagnosed with Pulmonary Alveolar Proteinosis (PAP) with recurrence of PAP after 5 repeated whole lung lavage, responding to subcutaneous injections of Granulocyte Macrophage Colony Stimulating Factor therapy (GM-CSF). Thus indicating that GM-CSF therapy is a promising alternative in those requiring repeated whole lung lavage

  10. Macrophage colony-stimulating factor induces indirect angiogenesis in vivo.

    PubMed

    Phillips, G D; Aukerman, S L; Whitehead, R A; Knighton, D R

    1993-01-01

    The cytokine macrophage colony-stimulating factor was implanted in the rabbit cornea over a wide dose range (1 ng to 100 microg) to assay its angiogenic activity in vivo. Neovascularization occurred in a dose-dependent manner, and maximum angiogenesis occurred only with 100 microg. Histologic analysis revealed that the corneas were free of inflammation at the lower doses, but had slight inflammation at 50 and 100 microg. Nonspecific esterase staining of frozen sections and transmission electron microscopy revealed that the inflammatory cells were predominantly macrophages, with very few neutrophils present. This association of capillary formation with inflammation suggests an indirect mechanism of angiogenesis. The lack of neutrophils within the inflammatory cell infiltrate demonstrates that indirect angiogenesis can proceed without the local presence of neutrophils. This distinguishes macrophage colony-stimulating factor from other indirect-acting angiogenesis factors that have been identified to date.

  11. Rac regulates vascular endothelial growth factor stimulated motility.

    PubMed

    Soga, N; Connolly, J O; Chellaiah, M; Kawamura, J; Hruska, K A

    2001-01-01

    During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood. Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF. These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent

  12. Pituitary transcription factor Prop-1 stimulates porcine follicle-stimulating hormone beta subunit gene expression.

    PubMed

    Aikawa, Satoko; Kato, Takako; Susa, Takao; Tomizawa, Kyoko; Ogawa, Satoshi; Kato, Yukio

    2004-11-12

    Molecular cloning of the transcription factor that modulates the expression of porcine follicle-stimulating hormone beta subunit (FSHbeta) gene was performed by the yeast one-hybrid cloning system using the -852/-746 upstream region (Fd2) as a bait sequence. We eventually cloned a pituitary transcription factor, Prop-1, which has been identified as an upstream transcription factor of Pit-1 gene. Binding ability of Prop-1 to the bait sequence was confirmed using recombinant Prop-1, and the binding property was investigated by DNase I footprinting, revealing that Prop-1 certainly bound to the large AT-rich region throughout the Fd2. Co-transfection of Prop-1 expression vector together with a reporter gene fused with Fd2 in CHO cells demonstrated an attractive stimulation of reporter gene expression. Immunohistochemistry of adult porcine pituitary confirmed the colocalization of the Prop-1 and FSHbeta subunit. This study is the first to report that Prop-1 participates in the regulation of FSHbeta gene. The present finding will provide new insights into the development of pituitary cell lineage and combined pituitary hormone deficiency (CPHD), since why the defect of Prop-1 causes CPHD including gonadotropins (FSH and LH) has yet to be clarified.

  13. Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs

    PubMed Central

    Calatayud, S; Warner, T D; Mitchell, J A

    2002-01-01

    Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1β and TNF-α). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action. British Journal of Cancer (2002) 86, 1316–1321. DOI: 10.1038/sj/bjc/6600240 www.bjcancer.com © 2002 Cancer Research UK PMID:11953891

  14. Factors stimulating propagation of legionellae in cooling tower water

    SciTech Connect

    Yamamoto, Hiroyuki; Sugiura, Minoru; Kusunoki, Shinji; Ezaki, Takayuki; Ikedo, Masanari; Yabuuchi, Eiko )

    1992-04-01

    The authors survey of cooling tower water demonstrated that the highest density of legionellae, {ge}10{sup 4} CFU/100 ml, appeared in water containing protozoa, {ge}10{sup 2} MPN/100 ml, and heterotrophic bacteria, {ge}10{sup 6} CFU/100 ml, at water temperatures between 25 and 35C. Viable counts of legionellae were detected even in the winter samples, and propagation, up to 10{sup 5} CFU/100 ml, occurs in summer. The counts of legionellae correlated positively with increases in water temperature, pH, and protozoan counts, but not with heterotrophic bacterial counts. The water temperature of cooling towers may promote increases in the viable counts of legionellae, and certain microbes, e.g., protozoa or some heterotrophic bacteria, may be a factor stimulating the propagation of legionellae.

  15. Nerve growth factor: stimulation of polymorphonuclear leukocyte chemotaxis in vitro.

    PubMed Central

    Gee, A P; Boyle, M D; Munger, K L; Lawman, M J; Young, M

    1983-01-01

    Topical application of mouse nerve growth factor (NGF) to superficial skin wounds of mice has previously been shown to accelerate the rate of wound contraction. Results of the present study reveal that NGF in the presence of plasma is also chemotactic for human polymorphonuclear leukocytes in vitro, and the concentration of NGF required for this effect is similar to that which stimulates ganglionic neurite outgrowth. This property does not arise from liberation of the C5a fragment of complement, nor does it require the known enzymic activity of NGF. (NGF inactivated with diisopropyl fluorophosphate is equally active.) We conclude that NGF can display biological effects on cells of nonneural origin and function, and this feature might play a role in the early inflammatory response to injury. PMID:6580641

  16. Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

    SciTech Connect

    Connelly, P.A.; Sisk, R.B.; Johnson, R.M.; Garrison, J.C.

    1987-05-01

    Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent protein kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.

  17. The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors.

    PubMed Central

    Hill, C P; Osslund, T D; Eisenberg, D

    1993-01-01

    We have determined the three-dimensional structure of recombinant human granulocyte-colony-stimulating factor by x-ray crystallography. Phases were initially obtained at 3.0-A resolution by multiple isomorphous replacement and were refined by solvent flattening and by averaging of the electron density of the three molecules in the asymmetric unit. The current R factor is 21.5% for all data between 6.0- and 2.2-A resolution. The structure is predominantly helical, with 104 of the 175 residues forming a four-alpha-helix bundle. The only other secondary structure is also helical. In the loop between the first two long helices a four-residue 3(10)-helix is immediately followed by a 6-residue alpha-helix. Three residues in the short connection between the second and third bundle helices form almost one turn of left-handed helix. The up-up-down-down connectivity with two long crossover connections has been reported previously for five other proteins, which like granulocyte-colony-stimulating factor are all signaling ligands: growth hormone, granulocyte/macrophage-colony-stimulating factor, interferon beta, interleukin 2, and interleukin 4. Structural similarity among these growth factors occurs despite the absence of similarity in their amino acid sequences. Conservation of this tertiary structure suggests that these different growth factors might all bind to their respective sequence-related receptors in an equivalent manner. Images Fig. 2 PMID:7685117

  18. In vivo stimulation of granulopoiesis by recombinant human granulocyte colony-stimulating factor

    SciTech Connect

    Cohen, A.M.; Zsebo, K.M.; Inoue, H.; Hines, D.; Boone, T.C.; Chazin, V.R.; Tsai, L.; Ritch, T.; Souza, L.M.

    1987-04-01

    Osmotic pumps containing Escherichia coli-derived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased > 10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsa-stained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. After subcutaneous injection at /sup 35/S-labeled rhG-CSF doses of up to 10 ..mu..g x kg/sup -1/ x day/sup -1/ only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte and lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. injections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.

  19. Tissue factor: A potent stimulator of Von Willebrand factor synthesis by human umbilical vein endothelial cells

    PubMed Central

    Meiring, Muriel; Allers, W.; Le Roux, E.

    2016-01-01

    Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-α. Slightly lower levels of ADAMTS13 secretion were found with all treatments. This may explain the thrombogenicity of patients with inflammation where extremely high VWF levels and slightly lower ADAMTS13 levels are present. PMID:27766025

  20. Colony-Stimulating Factor-1 Signaling Suppresses Renal Crystal Formation

    PubMed Central

    Taguchi, Kazumi; Kitamura, Hiroshi; Yasui, Takahiro; Naiki, Taku; Hamamoto, Shuzo; Ando, Ryosuke; Mizuno, Kentaro; Kawai, Noriyasu; Tozawa, Keiichi; Asano, Kenichi; Tanaka, Masato; Miyoshi, Ichiro; Kohri, Kenjiro

    2014-01-01

    We recently reported evidence suggesting that migrating macrophages (Mϕs) eliminate renal crystals in hyperoxaluric mice. Mϕs can be inflammatory (M1) or anti-inflammatory (M2), and colony-stimulating factor-1 (CSF-1) mediates polarization to the M2Mϕ phenotype. M2Mϕs promote renal tissue repair and regeneration, but it is not clear whether these cells are involved in suppressing renal crystal formation. We investigated the role of M2Mϕs in renal crystal formation during hyperoxaluria using CSF-1–deficient mice, which lack M2Mϕs. Compared with wild-type mice, CSF-1–deficient mice had significantly higher amounts of renal calcium oxalate crystal deposition. Treatment with recombinant human CSF-1 increased the expression of M2-related genes and markedly decreased the number of renal crystals in both CSF-1–deficient and wild-type mice. Flow cytometry of sorted renal Mϕs showed that CSF-1 deficiency resulted in a smaller population of CD11b+F4/80+CD163+CD206hi cells, which represent M2-like Mϕs. Additionally, transfusion of M2Mϕs into CSF-1–deficient mice suppressed renal crystal deposition. In vitro phagocytosis assays with calcium oxalate monohydrate crystals showed a higher rate of crystal phagocytosis by M2-polarized Mϕs than M1-polarized Mϕs or renal tubular cells. Gene array profiling showed that CSF-1 deficiency resulted in disordered M2- and stone-related gene expressions. Collectively, our results provide compelling evidence for a suppressive role of CSF-1 signaling in renal crystal formation. PMID:24578130

  1. Granulocyte, granulocyte–macrophage, and macrophage colony-stimulating factors can stimulate the invasive capacity of human lung cancer cells

    PubMed Central

    Pei, X-H; Nakanishi, Y; Takayama, K; Bai, F; Hara, N

    1999-01-01

    We and other researchers have previously found that colony-stimulating factors (CSFs), which generally include granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), promote invasion by lung cancer cells. In the present study, we studied the effects of these CSFs on gelatinase production, urokinase plasminogen activator (uPA) production and their activity in human lung cancer cells. Gelatin zymographs of conditioned media derived from human lung adenocarcinoma cell lines revealed two major bands of gelatinase activity at 68 and 92 kDa, which were characterized as matrix metalloproteinase (MMP)-2 and MMP-9 respectively. Treatment with CSFs increased the 68- and 92-kDa activity and converted some of a 92-kDa proenzyme to an 82-kDa enzyme that was consistent with an active form of the MMP-9. Plasminogen activator zymographs of the conditioned media from the cancer cells showed that CSF treatment resulted in an increase in a 48–55 kDa plasminogen-dependent gelatinolytic activity that was characterized as human uPA. The conditioned medium from the cancer cells treated with CSFs stimulated the conversion of plasminogen to plasmin, providing a direct demonstration of the ability of enhanced uPA to increase plasmin-dependent proteolysis. The enhanced invasive behaviour of the cancer cells stimulated by CSFs was well correlated with the increase in MMPs and uPA activities. These data suggest that the enhanced production of extracellular matrix-degrading proteinases by the cancer cells in response to CSF treatment may represent a biochemical mechanism which promotes the invasive behaviour of the cancer cells. © 1999 Cancer Research Campaign PMID:10408691

  2. Colony-stimulating factor 1 potentiates lung cancer bone metastasis.

    PubMed

    Hung, Jaclyn Y; Horn, Diane; Woodruff, Kathleen; Prihoda, Thomas; LeSaux, Claude; Peters, Jay; Tio, Fermin; Abboud-Werner, Sherry L

    2014-04-01

    Colony-stimulating factor 1 (CSF1) is essential for osteoclastogenesis that mediates osteolysis in metastatic tumors. Patients with lung cancer have increased CSF1 in serum and high levels are associated with poor survival. Adenocarcinomas metastasize rapidly and many patients suffer from bone metastasis. Lung cancer stem-like cells sustain tumor growth and potentiate metastasis. The purpose of this study was to determine the role of CSF1 in lung cancer bone metastasis and whether inhibition of CSF1 ameliorates the disease. Human lung adenocarcinoma A549 cells were examined in vitro for CSF1/CSF1R. A549-luc cells were injected intracardiac in NOD/SCID mice and metastasis was assessed. To determine the effect of CSF1 knockdown (KD) in A549 cells on bone metastasis, cells were stably transfected with a retroviral vector containing short-hairpin CSF1 (KD) or empty vector (CT). Results showed that A549 cells express CSF1/CSF1R; CSF1 increased their proliferation and invasion, whereas soluble CSF1R inhibited invasion. Mice injected with A549-luc cells showed osteolytic bone lesions 3.5 weeks after injection and lesions increased over 5 weeks. Tumors recapitulated adenocarcinoma morphology and showed osteoclasts along the tumor/bone interface, trabecular, and cortical bone loss. Analyses of KD cells showed decreased CSF1 protein levels, reduced colony formation in soft agar assay, and decreased fraction of stem-like cells. In CSF1KD mice, the incidence of tumor metastasis was similar to controls, although fewer CSF1KD mice had metastasis in both hind limbs. KD tumors showed reduced CSF1 expression, Ki-67+ cells, and osteoclasts. Importantly, there was a low incidence of large tumors >0.1 mm(2) in CSF1KD mice compared with control mice (10% vs 62.5%). This study established a lung osteolytic bone metastasis model that resembles human disease and suggests that CSF1 is a key determinant of cancer stem cell survival and tumor growth. Results may lead to novel strategies to

  3. A microassay for colony-stimulating factor based on thymidine incorporation.

    PubMed Central

    Prystowsky, M. B.; Naujokas, M. F.; Ihle, J. N.; Goldwasser, E.; Fitch, F. W.

    1984-01-01

    A variety of growth factors and lectins were tested; only colony-stimulating factors CSF-1, Interleukin 3, and a T-lymphocyte GM CSF induced colony formation in semisolid medium and stimulated thymidine incorporation in liquid culture. All other growth factors and lectins were inactive in both assays. Factor-stimulated thymidine incorporation was detectable 24 hours after stimulation and reached maximal levels 4-6 days after stimulation. A convenient microassay for measuring CSF activity has been developed, enabling a large number of samples to be screened qualitatively in 2 days and permitting CSF activity to be measured quantitatively in 4-5 days. This microassay can supplement the clonal-cell assay method and be especially useful as an initial screening assay for CSF activity. PMID:6606982

  4. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

    PubMed Central

    Sehgal, I; Bailey, J; Hitzemann, K; Pittelkow, M R; Maihle, N J

    1994-01-01

    Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways. Images PMID:8049525

  5. Expression of interleukin-34 and colony stimulating factor-1 in the stimulated periodontal ligament cells with tumor necrosis factor-α.

    PubMed

    Kawabe, Mutsuki; Ohyama, Hideki; Kato-Kogoe, Nahoko; Yamada, Naoko; Yamanegi, Koji; Nishiura, Hiroshi; Hirano, Hirotugu; Kishimoto, Hiromitsu; Nakasho, Keiji

    2015-09-01

    Tumor necrosis factor-α (TNF-α) directly and indirectly plays a crucial role in osteoclastogenesis. However, the indirect effects of TNF-α on colony-stimulating factor-1 receptor (CSF-1R)-mediated osteoclastogenesis achieved via periodontal ligament (PDL) cells are not fully understood. We herein examined the potency of osteoclast differentiation and maturation induced by fivefold supernatants in the stimulated human PDL cells with a physiologically high concentration (10 ng/mL) of recombinant TNF-α to human peripheral blood monocytes/macrophages in the simultaneous presence of the receptor activator of nuclear factor kappa-B ligand. The number of tartrate-resistant acid phosphatase-positive cells with multiple nuclei, but not those with a single nucleus, was decreased by approximately 50% by neutralization with rabbit IgG against either interleukin-34 (IL-34) or CSF-1. Small and large amounts of IL34 and CSF1 transcripts were measured in the stimulated PDL cells using real-time polymerase chain reaction. The corresponding amounts of proteins to IL34 and CSF1 transcripts were observed in the stimulated PDL cells on immunohistochemical staining or Western blotting. Moreover, 0.13 ng/mL of IL-34 and 5.0 ng/mL of CSF-1 were measured in the supernatants of the stimulated PDL cells using an enzyme-linked immunosorbent assay. IL-34 derived from the stimulated PDL cells with TNF-α appeared to synergistically function with CSF-1 in the CSF-1R-mediated maturation of osteoclastogenesis.

  6. Identification of a unique B-cell-stimulating factor produced by a cloned dendritic cell.

    PubMed Central

    Clayberger, C; DeKruyff, R H; Fay, R; Cantor, H

    1985-01-01

    We describe a cloned dendritic cell, clone Den-1, which is a potent accessory cell for some B-cell responses. Clone Den-1 produces a unique lymphokine that induces polyclonal B-cell proliferation in the absence of other costimulators. This clone or factors produced by it also stimulate purified B cells to develop plaque-forming cell responses to type 2 antigens. The effect of this factor(s) on various B-cell populations and its relationship to previously described B-cell-stimulating factors is discussed. Images PMID:3871522

  7. Mechanically stimulated bone cells secrete paracrine factors that regulate osteoprogenitor recruitment, proliferation, and differentiation

    SciTech Connect

    Brady, Robert T.; O'Brien, Fergal J.; Hoey, David A.

    2015-03-27

    Bone formation requires the recruitment, proliferation and osteogenic differentiation of mesenchymal progenitors. A potent stimulus driving this process is mechanical loading, yet the signalling mechanisms underpinning this are incompletely understood. The objective of this study was to investigate the role of the mechanically-stimulated osteocyte and osteoblast secretome in coordinating progenitor contributions to bone formation. Initially osteocytes (MLO-Y4) and osteoblasts (MC3T3) were mechanically stimulated for 24hrs and secreted factors within the conditioned media were collected and used to evaluate mesenchymal stem cell (MSC) and osteoblast recruitment, proliferation and osteogenesis. Paracrine factors secreted by mechanically stimulated osteocytes significantly enhanced MSC migration, proliferation and osteogenesis and furthermore significantly increased osteoblast migration and proliferation when compared to factors secreted by statically cultured osteocytes. Secondly, paracrine factors secreted by mechanically stimulated osteoblasts significantly enhanced MSC migration but surprisingly, in contrast to the osteocyte secretome, inhibited MSC proliferation when compared to factors secreted by statically cultured osteoblasts. A similar trend was observed in osteoblasts. This study provides new information on mechanically driven signalling mechanisms in bone and highlights a contrasting secretome between cells at different stages in the bone lineage, furthering our understanding of loading-induced bone formation and indirect biophysical regulation of osteoprogenitors. - Highlights: • Physically stimulated osteocytes secrete factors that regulate osteoprogenitors. • These factors enhance recruitment, proliferation and osteogenic differentiation. • Physically stimulated osteoblasts secrete factors that also regulate progenitors. • These factors enhance recruitment but inhibit proliferation of osteoprogenitors. • This study highlights a contrasting

  8. STIMULATION OF DEFENSE FACTORS FOR OYSTERS DEPLOYED TO CONTAMINATED SITES IN PENSACOLA BAY, FLORIDA

    EPA Science Inventory

    A positive association between chemical contaminants and defense factors has been established for eastern oysters (Crassostrea virginica) from Florida, but it is unknown whether such factors can be stimulated through short-term exposure to contaminants in the field. Hatchery oyst...

  9. The Granulocyte-colony stimulating factor has a dual role in neuronal and vascular plasticity

    PubMed Central

    Wallner, Stephanie; Peters, Sebastian; Pitzer, Claudia; Resch, Herbert; Bogdahn, Ulrich; Schneider, Armin

    2015-01-01

    Granulocyte-colony stimulating factor (G-CSF) is a growth factor that has originally been identified several decades ago as a hematopoietic factor required mainly for the generation of neutrophilic granulocytes, and is in clinical use for that. More recently, it has been discovered that G-CSF also plays a role in the brain as a growth factor for neurons and neural stem cells, and as a factor involved in the plasticity of the vasculature. We review and discuss these dual properties in view of the neuroregenerative potential of this growth factor. PMID:26301221

  10. Platelet-derived growth factor stimulated mechanisms of glucosamine incorporation

    SciTech Connect

    Harrington, M.A.; Pledger, W.J. )

    1987-10-01

    Platelet-derived growth factor (PDGF) treatment of density-arrested BALB/c-3T3 cells results in increased ({sup 3}H)glucosamine (GlcN) incorporation into cellular material. The enhanced GlcN incorporation is not due to a preferential increase in proteoglycan synthesis as measured by ({sup 35}S)H{sub 2}SO{sub 4} incorporation. Approximately 50% of the GlcN incorporated in PDGF or platelet-poor plasma (PPP)-treated cultures enters N-linked glycoproteins. Addition of dolichol-phosphate (dolichol-P), a required intermediate in N-linked glycosylation, did not alter ({sup 3}H)GlcN incorporation in PDGF-treated cells but did increase incorporation in PPP-treated cultures to a level comparable to that observed for PDGF-treated cultures. PDGF-treated cultures contained twofold greater quantities of ({sup 3}H)GlcN dolichol intermediates and lipid-free glycoprotein. Over a 12-h time course 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) activity was similar in cultures treated with PDGF or PPP. Results of these studies reveal that enhanced protein glycosylation in response to PDGF treatment is not the result of a direct effect on HMG CoA reductase.

  11. Stimulation of human neutrophil leukocyte aerobic glucose metabolism by purified chemotactic factors.

    PubMed Central

    Goetzl, E J; Austen, K F

    1974-01-01

    The interaction of human neutrophils adherent to plastic petri dishes with the purified chemotactic factors C5a and kallikrein increased their rate of aerobic glycolysis 25-120% and the activity of their hexose monophosphate shunt (HMPS) 100-600%, reaching a plateau after 2 hr at 37 degrees C. The stimulation of either pathway required a chemotactically active stimulus since neither C5 nor prekallikrein or inactivated kallikrein could enhance metabolic activity. Marked suppression of the neutrophil chemotactic response by preincubation with a chemotactic factor to achieve deactivation, 5 x 10(-7) M diisopropyl fluorophosphate, or the neutrophil immobilizing factor (NIF) did not prevent the stimulation of HMPS activity or glycolysis by chemotactic factors. The metabolic inhibitors iodoacetate and 6-aminonicotinamide at concentrations which blocked enhancement of glycolysis or HMPS activity, respectively, partially suppressed the chemotactic response of neutrophils to the chemotactic factors. The capacity of a chemotactic factor to stimulate glucose metabolism of human neutrophils is associated with a maximal chemotactic response, but this stimulation is not alone sufficient for chemotaxis. Images PMID:11344574

  12. Granulocyte colony-stimulating factor (G-CSF) transiently suppresses mitogen-stimulated T-cell proliferative response

    PubMed Central

    Reyes, E; García-Castro, I; Esquivel, F; Hornedo, J; Cortes-Funes, H; Solovera, J; Alvarez-Mon, M

    1999-01-01

    Granulocyte colony-stimulation factor (G-CSF) is a cytokine that selectively promotes growth and maturation of neutrophils and may modulate the cytokine response to inflammatory stimuli. The purpose of this study was to examine the effect of G-CSF on ex vivo peripheral blood mononuclear cell (PBMC) functions. Ten patients with breast cancer were included in a clinical trial in which r-metHuG-CSF was administrered daily for 5 days to mobilize peripheral blood stem cells. Ten healthy women were also included as controls. Our data show that G-CSF treatment induces an increase in peripheral blood leucocyte, neutrophil, lymphocyte and monocyte counts. We have found a modulation in the percentages of CD19+, CD45+CD14+, CD4+CD45RA+ and CD4+CD45RO+ cells in PBMC fractions during G-CSF treatment. We have also found a significant reduction in the proliferative response of PBMC to mitogenic stimulation that reverted 14 days after the fifth and the last dose of G-CSF. Furthermore, it was not associated with significant changes in the pattern of cytokine production. The mechanism of this immunoregulatory effect is probably indirect since G-CSF receptor has not been found in T lymphocytes. This mechanism and its potential clinical applications remain to be elucidated. © 1999 Cancer Research Campaign PMID:10390001

  13. Epidermal growth factor (EGF) inhibits stimulated thyroid hormone secretion in the mouse

    SciTech Connect

    Ahren, B.

    1987-07-01

    It is known that epidermal growth factor (EGF) inhibits iodide uptake in the thyroid follicular cells and lowers plasma levels of thyroid hormones upon infusion into sheep and ewes. In this study, the effects of EGF on basal and stimulated thyroid hormone secretion were investigated in the mouse. Mice were pretreated with /sup 125/I and thyroxine; the subsequent release of /sup 125/I is an estimation of thyroid hormone secretion. It was found that basal radioiodine secretion was not altered by intravenous injection of EGF (5 micrograms/animal). However, the radioiodine secretion stimulated by both TSH (120 microU/animal) and vasoactive intestinal peptide (VIP; 5 micrograms/animal) were inhibited by EGF (5 micrograms/animal). At a lower dose level (0.5 microgram/animal), EGF had no influence on stimulated radioiodine secretion. In conclusion, EGF inhibits stimulated thyroid hormone secretion in the mouse.

  14. Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues.

    PubMed

    Revis-Gupta, S; Abdel-Ghany, M; Koland, J; Racker, E

    1991-07-15

    We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1. We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site. To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine. We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF. These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

  15. PGE2 is a UVR-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation

    PubMed Central

    Starner, Renny J.; McClelland, Lindy; Abdel-Malek, Zalfa; Fricke, Alex; Scott, Glynis

    2013-01-01

    Melanocyte proliferation, dendrite formation, and pigmentation are controlled by paracrine factors, particularly following exposure to ultraviolet radiation (UVR). Little is known about autocrine factors for melanocytes. Prostaglandins activate signaling pathways involved in growth, differentiation and apoptosis. Prostaglandin E2 (PGE2) is the most abundant prostaglandin released by keratinocytes following UVR, and stimulates the formation of dendrites in melanocytes. Synthesis of PGE2 is controlled by cPLA2, which releases arachidonic acid from membranes, and COX-2 and prostaglandin E2 synthases (PGES), which convert arachidonic acid to PGH2 and PGH2 to PGE2, respectively. In this report we show that multiple irradiations of human melanocytes with UVR stimulates tyrosinase activity, independent of expression of a functional melanocortin 1 receptor, suggesting the presence of a non-melanocortin autocrine factor. Irradiation of melanocytes activated cPLA2, the rate-limiting step in eicosanoid synthesis, and stimulated PGE2 secretion. PGE2 increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1–4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. These data suggest that relative levels or activity of these receptors controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes. PMID:20500768

  16. Childhood Conduct Problems and Other Early Risk Factors in Rural Adult Stimulant Users

    ERIC Educational Resources Information Center

    Kramer, Teresa L.; Han, Xiaotong; Leukefeld, Carl; Booth, Brenda M.; Edlund, Carrie

    2009-01-01

    Context: Understanding childhood risk factors associated with adult substance use and legal problems is important for treatment and prevention. Purpose: To examine the relationship of early substance use, conduct problems before age 15, and family history of substance abuse on adult outcomes in rural, stimulant users. Methods: Adult cocaine and…

  17. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR{sub 1}). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR{sub 1} and not PAR{sub 2}. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

  18. Administration of granulocyte colony-stimulating factor with radiotherapy promotes tumor growth by stimulating vascularization in tumor-bearing mice.

    PubMed

    Kim, Joong Sun; Son, Yeonghoon; Bae, Min Ji; Lee, Minyoung; Lee, Chang Geun; Jo, Wol Soon; Kim, Sung Dae; Yang, Kwangmo

    2015-07-01

    Although granulocyte-colony stimulating factor (G-CSF) is commonly used to support recovery from radiation-induced side-effects, the precise effects of G-CSF on colon cancer under radiotherapy remain poorly understood. In the present study, to investigate the effects of tumor growth following radiotherapy and G-CSF administration in a murine xenograft model of colon cancer, female BALB/c mice were injected with cells of a colon carcinoma cell line (CT26) with irradiation and G-CSF, alone or in combination. Mice received 2 Gy of focal radiation daily for 5 days and intraperitoneal injection of G-CSF (100 µg/kg/day) after irradiation for 7 days. Changes in the levels of myeloperoxidase (MPO), vascular endothelial growth factor (VEGF), matrix metalloproteinase type 9 (MMP-9) and CD31 were assessed in the mouse cancer induced by injection of colon cancer cells. We observed that G-CSF increased the number of circulating neutrophils, but facilitated tumor growth. However, G-CSF treatment did not affect radiation-induced cytotoxicity and cell viability in CT26 cells in vitro. Increased levels of myeloperoxidase, a neutrophil marker and those of vascular endothelial growth factor were observed in tumors with G-CSF supplementation. In addition, we found that increased levels of CD31 and matrix metalloproteinase-9 were correlated with the enhanced tumor growth after G-CSF treatment. Therefore, these data suggest that G-CSF may contribute to tumor growth and decrease the antitumor effect of radiotherapy, possibly by promoting vascularization in cancer lesions.

  19. Granulocyte colony-stimulating factor in the treatment of acute radiation syndrome: a concise review.

    PubMed

    Hofer, Michal; Pospíšil, Milan; Komůrková, Denisa; Hoferová, Zuzana

    2014-04-16

    This article concisely summarizes data on the action of one of the principal and best known growth factors, the granulocyte colony-stimulating factor (G-CSF), in a mammalian organism exposed to radiation doses inducing acute radiation syndrome. Highlighted are the topics of its real or anticipated use in radiation accident victims, the timing of its administration, the possibilities of combining G-CSF with other drugs, the ability of other agents to stimulate endogenous G-CSF production, as well as of the capability of this growth factor to ameliorate not only the bone marrow radiation syndrome but also the gastrointestinal radiation syndrome. G-CSF is one of the pivotal drugs in the treatment of radiation accident victims and its employment in this indication can be expected to remain or even grow in the future.

  20. Increased Expression of Hepatocyte Nuclear Factor 6 Stimulates Hepatocyte Proliferation during Mouse Liver Regeneration

    PubMed Central

    Tan, Yongjun; Yoshida, Yuichi; Hughes, Douglas E.; Costa, Robert H.

    2005-01-01

    Background & Aims The Hepatocyte Nuclear Factor 6 (HNF6 or ONECUT-1) protein is a cell-type specific transcription factor that regulates expression of hepatocyte-specific genes. Using hepatocytes for Chromatin Immunoprecipitation (ChIP) assays, the HNF6 protein was shown to associate with cell cycle regulatory promoters. Here, we examined whether increased levels of HNF6 stimulate hepatocyte proliferation during mouse liver regeneration. Methods Tail vein injection of adenovirus expressing the HNF6 cDNA (AdHNF6) was used to increase hepatic HNF6 levels during mouse liver regeneration induced by partial hepatectomy, and DNA replication was determined by Bromodeoxyuridine incorporation. Cotransfection and ChIP assays were used to determine transcriptional target promoters. Results Elevated expression of HNF6 during mouse liver regeneration causes a significant increase in the number of hepatocytes entering DNA replication (S-phase) and mouse hepatoma Hepa1-6 cells diminished for HNF6 levels by siRNA transfection exhibit a 50% reduction in S-phase following serum stimulation. This stimulation in hepatocyte S-phase progression was associated with increased expression of the hepatocyte mitogen Tumor Growth Factor α (TGFα) and the cell cycle regulators Cyclin D1 and Forkhead Box m1 (Foxm1) transcription factor. Cotransfection and ChIP assays show that TGFα, Cyclin D1, and HNF6 promoter regions are direct transcriptional targets of the HNF6 protein. Co-immunoprecipitation assays with regenerating mouse liver extracts reveal association between HNF6 and Foxm1 proteins and cotransfection assays show that HNF6 stimulates Foxm1 transcriptional activity. Conclusion These mouse liver regeneration studies show that increased HNF6 levels stimulate hepatocyte proliferation through transcriptional induction of cell cycle regulatory genes. PMID:16618419

  1. Imatinib mesylate inhibits platelet derived growth factor stimulated proliferation of rheumatoid synovial fibroblasts

    SciTech Connect

    Sandler, Charlotta; Joutsiniemi, Saima; Lindstedt, Ken A.; Juutilainen, Timo; Kovanen, Petri T.; Eklund, Kari K. . E-mail: kari.eklund@hus.fi

    2006-08-18

    Synovial fibroblast is the key cell type in the growth of the pathological synovial tissue in arthritis. Here, we show that platelet-derived growth factor (PDGF) is a potent mitogen for synovial fibroblasts isolated from patients with rheumatoid arthritis. Inhibition of PDGF-receptor signalling by imatinib mesylate (1 {mu}M) completely abrogated the PDGF-stimulated proliferation and inhibited approximately 70% of serum-stimulated proliferation of synovial fibroblasts. Similar extent of inhibition was observed when PDGF was neutralized with anti-PDGF antibodies, suggesting that imatinib mesylate does not inhibit pathways other than those mediated by PDGF-receptors. No signs of apoptosis were detected in synovial fibroblasts cultured in the presence of imatinib. These results suggest that imatinib mesylate specifically inhibits PDGF-stimulated proliferation of synovial fibroblasts, and that inhibition of PDGF-receptors could represent a feasible target for novel antirheumatic therapies.

  2. Tissue-engineered cartilage: the crossroads of biomaterials, cells and stimulating factors.

    PubMed

    Bhardwaj, Nandana; Devi, Dipali; Mandal, Biman B

    2015-02-01

    Damage to cartilage represents one of the most challenging tasks of musculoskeletal therapeutics due to its limited propensity for healing and regenerative capabilities. Lack of current treatments to restore cartilage tissue function has prompted research in this rapidly emerging field of tissue regeneration of functional cartilage tissue substitutes. The development of cartilaginous tissue largely depends on the combination of appropriate biomaterials, cell source, and stimulating factors. Over the years, various biomaterials have been utilized for cartilage repair, but outcomes are far from achieving native cartilage architecture and function. This highlights the need for exploration of suitable biomaterials and stimulating factors for cartilage regeneration. With these perspectives, we aim to present an overview of cartilage tissue engineering with recent progress, development, and major steps taken toward the generation of functional cartilage tissue. In this review, we have discussed the advances and problems in tissue engineering of cartilage with strong emphasis on the utilization of natural polymeric biomaterials, various cell sources, and stimulating factors such as biophysical stimuli, mechanical stimuli, dynamic culture, and growth factors used so far in cartilage regeneration. Finally, we have focused on clinical trials, recent innovations, and future prospects related to cartilage engineering.

  3. Granulocyte colony-stimulating factor-based stem cell mobilization in patients with sickle cell disease.

    PubMed

    Rosenbaum, Cara; Peace, David; Rich, Elizabeth; Van Besien, Koen

    2008-06-01

    Granulocyte colony-stimulating factor (G-CSF) has been reported to exacerbate vaso-occlusive crises in sickle cell disease. It has been recommended to avoid its use for stem cell mobilization in this population, yet autologous transplant is the standard of care and at times a life-saving treatment for patients with various hematologic malignancies such as relapsed aggressive lymphoma or multiple myeloma. We report 5 cases of patients with sickle cell disease and related hemoglobinopathies who underwent granulocyte-colony stimulating factor (G-CSF)-mobilization of peripheral blood stem cells (PBSC). Three of them developed manageable vaso-occlusive pain symptoms requiring parenteral narcotics alone. The 2 others had no complications. These cases demonstrate that stem cell mobilization using G-CSF, although complicated and not without risk, is feasible in patients with sickle cell syndromes.

  4. Stimulation of DNA and Collagen Synthesis by Autologous Growth Factor in Cultured Fetal Rat Calvaria

    NASA Astrophysics Data System (ADS)

    Canalis, Ernesto; Peck, William A.; Raisz, Lawrence G.

    1980-11-01

    Conditioned medium derived from organ or cell cultures prepared from 19- to 21-day fetal rat calvaria stimulated the incorporation of [3H]proline into collagen and of [3H]thymidine into DNA in organ cultures of the same tissue. Addition of cortisol enhanced the effect on collagen but not on DNA synthesis. These effects appeared to be due to a nondialyzable and heat-stable growth factor.

  5. Endothelium-Derived Hyperpolarizing Factor Mediates Bradykinin Stimulated Tissue Plasminogen Activator Release In Humans

    PubMed Central

    Rahman, Ayaz M.; Murrow, Jonathan R.; Ozkor, Muhiddin A.; Kavtaradze, Nino; Lin, Ji; De Staercke, Christine; Hooper, W. Craig; Manatunga, Amita; Hayek, Salim; Quyyumi, Arshed A.

    2014-01-01

    Aims Bradykinin stimulates tissue plasminogen activator (t-PA) release from human endothelium. Although bradykinin stimulates both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) release, the role of EDHF in t-PA release remains unexplored. This study sought to determine the mechanisms of bradykinin-stimulated t-PA release in the forearm vasculature of healthy human subjects. Methods In 33 healthy subjects (age 40.3±1.9 years) forearm blood flow (FBF) and t-PA release were measured at rest, and after intra-arterial infusions of bradykinin (400ng/min) and sodium nitroprusside (3.2 mg/min). Measurements were repeated after intra-arterial infusion of TEA (1 μmol/min), fluconazole (0.4 μmol.min-1.L-1), and NG-monomethyl-L-arginine (L-NMMA, 8 μmol/min) to block nitric oxide, and their combination in separate studies. Results Bradykinin significantly increased net t-PA release across the forearm (P<0.0001). Fluconazole attenuated both bradykinin-mediated vasodilation (-23.3±2.7% FBF, P<0.0001) and t-PA release (from 50.9±9.0 to 21.3±8.9 ng/min/100ml, P=0.02). TEA attenuated FBF (-14.7±3.2%, P=0.002) and abolished bradykinin-stimulated t-PA release (from 22.9+5.7 to - 0.8±3.6 ng/min/100ml, P=0.0002). L-NMMA attenuated FBF (P<0.0001), but did not inhibit bradykinin-induced t-PA release (P=NS). Conclusion Bradykinin-stimulated t-PA release is partly due to cytochrome P450-derived epoxides, and is inhibited by K+ca channel blockade. Thus, bradykinin stimulates both EDHF-dependent vasodilation and t-PA release. PMID:24925526

  6. Risk factors for stimulant use among homeless and unstably housed adult women

    PubMed Central

    Riley, Elise D.; Shumway, Martha; Knight, Kelly R.; Guzman, David; Cohen, Jennifer; Weiser, Sheri D.

    2015-01-01

    Background One of the most common causes of death among homeless and unstably housed women is acute intoxication where cocaine is present. While correlates of stimulant use have been determined in prior research, few studies have assessed risk factors of use specifically in this high-risk population. Methods We sampled biological women with a history of housing instability from community-based venues to participate in a cohort study. Baseline and 6-month follow-up data were used to determine the relative risk of stimulant use (crack cocaine, powder cocaine or methamphetamine) among individuals who did not use at baseline. Results Among 260 study participants, the median age was 47 years, 70% were women of color; 47% reported having unmet subsistence needs and 53% reported abstinence from stimulants at baseline. In analyses adjusting for baseline sociodemographics and drug treatment, the risk of using stimulants within 6 months was significantly higher among women who reported recent sexual violence (Adjusted Relative Risk [ARR] = 4.31; 95% CI:1.97–9.45), sleeping in a shelter or public place (ARR = 2.75; 95% CI:1.15–6.57), and using unprescribed opioid analgesics (ARR = 2.54; 95% CI:1.01–6.38). Conclusion We found that almost half of homeless and unstably housed women used stimulants at baseline and 14% of those who did not use began within 6 months. Addressing homelessness and sexual violence is critical to reduce stimulant use among impoverished women. PMID:26070454

  7. Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading

    NASA Technical Reports Server (NTRS)

    Decker, R. S.; Cook, M. G.; Behnke-Barclay, M.; Decker, M. L.

    1995-01-01

    Cultured adult rabbit cardiac myocytes treated with recombinant growth factors display enhanced rates of protein accumulation (ie, growth) in response to insulin and insulin-like growth factors (IGFs), but epidermal growth factor, acidic or basic fibroblast growth factor, and platelet-derived growth factor failed to increase contractile protein synthesis or growth of the heart cells. Insulin and IGF-1 increased growth rates by stimulating anabolic while simultaneously inhibiting catabolic pathways, whereas IGF-2 elevated growth modestly by apparently inhibiting lysosomal proteolysis. Neutralizing antibodies directed against either IGF-1 or IGF-2 or IGF binding protein 3 blocked protein accumulation. A monoclonal antibody directed against the IGF-1 receptor also inhibited changes in protein turnover provoked by recombinant human IGF-1 but not IGF-2. Of the other growth factors tested, only transforming growth factor-beta 1 increased the fractional rate of myosin heavy chain (MHC) synthesis, with beta-MHC synthesis being elevated and alpha-MHC synthesis being suppressed. However, the other growth factors were able to modestly stimulate the rate of DNA synthesis in this preparation. Bromodeoxyuridine labeling revealed that these growth factors increased DNA synthesis in myocytes and nonmyocytes alike, but the heart cells displayed neither karyokinesis or cytokinesis. In contrast, cocultures of cardiac myocytes and nonmyocytes and nonmyocyte-conditioned culture medium failed to enhance the rate of cardiac MHC synthesis or its accumulation, implying that quiescent heart cells do not respond to "conditioning" by cardiac nonmyocytes. These findings demonstrated that insulin and the IGFs promote passively loaded cultured adult rabbit heart cells to hypertrophy but suggest that other growth factors tested may be limited in this regard.

  8. Transcutaneous cervical vagal nerve stimulation modulates cardiac vagal tone and tumor necrosis factor-alpha.

    PubMed

    Brock, C; Brock, B; Aziz, Q; Møller, H J; Pfeiffer Jensen, M; Drewes, A M; Farmer, A D

    2016-12-12

    The vagus nerve is a central component of cholinergic anti-inflammatory pathways. We sought to evaluate the effect of bilateral transcutaneous cervical vagal nerve stimulation (t-VNS) on validated parameters of autonomic tone and cytokines in 20 healthy subjects. 24 hours after t-VNS, there was an increase in cardiac vagal tone and a reduction in tumor necrosis factor-α in comparison to baseline. No change was seen in blood pressure, cardiac sympathetic index or other cytokines. These preliminary data suggest that t-VNS exerts an autonomic and a subtle antitumor necrosis factor-α effect, which warrants further evaluation in larger controlled studies.

  9. [Lymphocyte transformation test following stimulation with a protein factor from neutrophilic granulocytes (PMNL) in psoriasis patients].

    PubMed

    Ruszczak, Z; Ciborska, L; Kaszuba, A

    1988-12-01

    The lymphocyte transformation test (LTT) was given to 20 healthy subjects and 43 patients with generalized psoriasis vulgaris: it was given right after stimulation with PHA (spontaneous) and after stimulation with allogenic and autogenic protein factor (NPF). NPF was isolated from secondary lysosome granules of peripheral blood neutrophils. The results were analyzed using computer statistic tests. No distinct differences were noticed between the spontaneous transformation test in psoriatic patients compared to the controls. After stimulation with PHA, the percentage of blast cells was significantly lower in patients with psoriasis. When allogenic and autogenic NPF was used for stimulation, the LTT values were significantly higher in the psoriasis group than in the control subjects. This fact points out the increase in sensitivity of lymphocytes to NPF in active psoriasis and the possibility of abnormal neutrophil-lymphocyte interactions in vivo. This phenomenon may be intensified when under the influence of bacterial or viral agents, or medicaments; the degranulation of secondary lysosome granules of neutrophils occurs, causing the release of NPF. These investigations support our opinion that psoriasis is a systemic disease and that NPF plays a considerable role in the psoriatic reaction.

  10. Stimulation of DNA synthesis in cultured primary human mesothelial cells by specific growth factors

    SciTech Connect

    Gabrielson, E.W.; Gerwin, B.I.; Harris, C.C.; Roberts, A.B.; Sporn, M.B.; Lechner, J.F.

    1988-08-01

    Monolayer cultures of human mesothelial cells made quiescent by serum deprivation are induced to undergo one round of DNA synthesis by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or transforming growth factor type beta 1 (TGF-beta 1). This one-time stimulation is independent of other serum components. The kinetics for induction of DNA synthesis observed for PDGF, EGF, and TGF-beta 1 are all similar to one another, with a peak of DNA synthesis occurring 24-36 h after the addition of the growth factors. Repetitive rounds of DNA synthesis and cell division do not ensue after addition of PDGF, EGF, or TGF-beta 1 alone or in combination; however, in media supplemented with chemically denatured serum, each of these factors is capable of sustaining continuous replication of mesothelial cells. Stimulation of growth by PDGF and TGF-beta 1 is unusual for an epithelial cell type, and indicates that mesothelial cells have growth regulatory properties similar to connective tissue cells.

  11. GROWTH REGULATING FACTOR5 stimulates Arabidopsis chloroplast division, photosynthesis, and leaf longevity.

    PubMed

    Vercruyssen, Liesbeth; Tognetti, Vanesa B; Gonzalez, Nathalie; Van Dingenen, Judith; De Milde, Liesbeth; Bielach, Agnieszka; De Rycke, Riet; Van Breusegem, Frank; Inzé, Dirk

    2015-03-01

    Arabidopsis (Arabidopsis thaliana) leaf development relies on subsequent phases of cell proliferation and cell expansion. During the proliferation phase, chloroplasts need to divide extensively, and during the transition from cell proliferation to expansion, they differentiate into photosynthetically active chloroplasts, providing the plant with energy. The transcription factor GROWTH REGULATING FACTOR5 (GRF5) promotes the duration of the cell proliferation period during leaf development. Here, it is shown that GRF5 also stimulates chloroplast division, resulting in a higher chloroplast number per cell with a concomitant increase in chlorophyll levels in 35S:GRF5 leaves, which can sustain higher rates of photosynthesis. Moreover, 35S:GRF5 plants show delayed leaf senescence and are more tolerant for growth on nitrogen-depleted medium. Cytokinins also stimulate leaf growth in part by extending the cell proliferation phase, simultaneously delaying the onset of the cell expansion phase. In addition, cytokinins are known to be involved in chloroplast development, nitrogen signaling, and senescence. Evidence is provided that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention after dark-induced senescence, which suggests that they also cooperate to stimulate chloroplast division and nitrogen assimilation. Taken together with the increased leaf size, ectopic expression of GRF5 has great potential to improve plant productivity.

  12. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors

    PubMed Central

    Bai, Huai; Forrester, John V.; Zhao, Min

    2015-01-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24 h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors. PMID:21524919

  13. Mechanism of kinase activation in the receptor for colony-stimulating factor 1.

    PubMed Central

    Lee, A W; Nienhuis, A W

    1990-01-01

    Receptor tyrosine kinases remain dormant until activated by ligand binding to the extracellular domain. Two mechanisms have been proposed for kinase activation: (i) ligand binding to the external domain of a receptor monomer may induce a conformational change that is transmitted across the cell membrane (intramolecular model) or (ii) the ligand may facilitate oligomerization, thereby allowing interactions between the juxtaposed kinase domains (intermolecular model). The receptor for colony-stimulating factor 1 was used to test these models. Large insertions at the junction between the external and transmembrane domains of the receptor, introduced by site-directed mutagenesis of the cDNA, were positioned to isolate the external domain and prevent transmembrane conformational propagation while allowing for receptor oligomerization. Such mutant receptors were expressed on the cell surface, bound ligand with high affinity, exhibited ligand-stimulated autophosphorylation, and signaled mitogenesis and cellular proliferation in the presence of ligand. A second experimental strategy directly tested the intermolecular model of ligand activation. A hybrid receptor composed of the external domain of human glycophorin A and the transmembrane and cytoplasmic domains of the colony-stimulating factor 1 receptor exhibited anti-glycophorin antibody-induced kinase activity that supported mitogenesis. Our data strongly support a mechanism of receptor activation based on ligand-induced receptor oligomerization. Images PMID:2169623

  14. Ex Vivo Assay of Electrical Stimulation to Rat Sciatic Nerves: Cell Behaviors and Growth Factor Expression.

    PubMed

    Du, Zhiyong; Bondarenko, Olexandr; Wang, Dingkun; Rouabhia, Mahmoud; Zhang, Ze

    2016-06-01

    Neurite outgrowth and axon regeneration are known to benefit from electrical stimulation. However, how neuritis and their surroundings react to electrical field is difficult to replicate by monolayer cell culture. In this work freshly harvested rat sciatic nerves were cultured and exposed to two types of electrical field, after which time the nerve tissues were immunohistologically stained and the expression of neurotrophic factors and cytokines were evaluated. ELISA assay was used to confirm the production of specific proteins. All cell populations survived the 48 h culture with little necrosis. Electrical stimulation was found to accelerate Wallerian degeneration and help Schwann cells to switch into migratory phenotype. Inductive electrical stimulation was shown to upregulate the secretion of multiple neurotrophic factors. Cellular distribution in nerve tissue was altered upon the application of an electrical field. This work thus presents an ex vivo model to study denervated axon in well controlled electrical field, bridging monolayer cell culture and animal experiment. It also demonstrated the critical role of electrical field distribution in regulating cellular activities.

  15. Multi-factorial modulation of IGD motogenic potential in MSF (migration stimulating factor).

    PubMed

    Ellis, Ian R; Jones, Sarah J; Staunton, David; Vakonakis, Ioannis; Norman, David G; Potts, Jennifer R; Milner, Caroline M; Meenan, Nicola A G; Raibaud, Sophie; Ohea, Go; Schor, Ana M; Schor, Seth L

    2010-09-10

    Migration Stimulating Factor (MSF) is a genetically truncated isoform of fibronectin (Fn). MSF is a potent stimulator of fibroblast migration, whereas full length Fn is devoid of motogenic activity. MSF and Fn contain four IGD motifs, located in the 3rd, 5th, 7th and 9th type I modules; these modules are referred to as (3)FnI, (5)FnI, (7)FnI and (9)FnI, respectively. We have previously reported that mutation of IGD motifs in modules (7)FnI and (9)FnI of MSF is sufficient to completely abolish the motogenic response of target adult skin fibroblasts. We now report that the IGD sequences in (3)FnI and (5)FnI are also capable of exhibiting motogenic activity when present within fragments of MSF. When present within (1-5)FnI, these sequences require the presence of serum or vitronectin for their motogenic activity to be manifest, whereas the IGD sequences in (7)FnI and (9)FnI are bioactive in the absence of serum factors. All MSF and IGD-containing peptides stimulated the phosphorylation of the integrin binding protein focal adhesion kinase (FAK) but did not necessarily affect migration. These results suggest that steric hindrance determines the motogenic activity of MSF and Fn, and that both molecules contain cryptic bioactive fragments.

  16. DC electric stimulation upregulates angiogenic factors in endothelial cells through activation of VEGF receptors.

    PubMed

    Bai, Huai; Forrester, John V; Zhao, Min

    2011-07-01

    Small direct current (DC) electric fields direct some important angiogenic responses of vascular endothelial cells. Those responses indicate promising use of electric fields to modulate angiogenesis. We sought to determine the regulation of electric fields on transcription and expression of a serial of import angiogenic factors by endothelial cells themselves. Using semi-quantitative PCR and ELISA we found that electric stimulation upregulates the levels of mRNAs and proteins of a number of angiogenic proteins, most importantly VEGF165, VEGF121 and IL-8 in human endothelial cells. The up-regulation of mRNA levels might be specific, as the mRNA encoding bFGF, TGF-beta and eNOS are not affected by DC electric stimulation at 24h time-point. Inhibition of VEGF receptor (VEGFR1 or VEGFR2) signaling significantly decreased VEGF production and completely abolished IL-8 production. DC electric stimulation selectively regulates production of some growth factors and cytokines important for angiogenesis through a feed-back loop mediated by VEGF receptors.

  17. Decrease in platelet activating factor stimulated phosphoinositide turnover during storage of human platelets in plasma

    SciTech Connect

    Carter, M.G.; Shukla, S.D. )

    1987-05-01

    Human platelet concentrate from the American Red Cross Blood Center was stored at 24{degree}C in a shaker and aliquots were taken out at time intervals aseptically. Platelet activating factor (PAF) stimulated turnover of phosphoinositide (PPI) was monitored by assaying {sup 32}P incorporation into phosphoinositides using platelet rich plasma (PRP). Platelets in PRP were incubated with 1 {times} 10{sup {minus}7} M PAF at 37{degree}C with gentle shaking and after 5 min their lipids were extracted and analysed by TLC for {sup 32}P-phosphoinositides. The percent stimulation of {sup 32}P incorporation by PAF (over control) into PPI was approximately 250, 100, 60, 25 and 20 on days 1, 2, 3, 5 and 6, respectively. This indicated a dramatic decrease in PAF responsive turnover of platelet PPI during storage. These findings have important implications in relation to PAF receptor activity and viability of platelets at different periods of storage.

  18. Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity.

    PubMed

    Lopes Bezerra, Leila M; Filler, Scott G

    2004-03-15

    Invasive aspergillosis causes significant mortality among patients with hematologic malignancies. This infection is characterized by vascular invasion and thrombosis. To study the pathogenesis of invasive aspergillosis, we investigated the interactions of Aspergillus fumigatus conidia and hyphae with endothelial cells in vitro. We found that both forms of the organism induced endothelial cell microfilament rearrangement and subsequent endocytosis. Conidia were endocytosed 2-fold more avidly than hyphae, and endocytosis was independent of fungal viability. Endocytosed conidia and hyphae caused progressive endothelial cell injury after 4 hours of infection. Live conidia induced more endothelial cell injury than did live hyphae. However, endothelial cell injury caused by conidia was dependent on fungal viability, whereas injury caused by hyphae was not, indicating that conidia and hyphae injure endothelial cells by different mechanisms. Neither live nor killed conidia increased tissue factor activity of endothelial cells. In contrast, both live and killed hyphae stimulated significant endothelial cell tissue factor activity, as well as the expression of tissue factor antigen on the endothelial cell surface. These results suggest that angioinvasion and thrombosis caused by A fumigatus hyphae in vivo may be due in part to endothelial cell invasion, induction of injury, and stimulation of tissue factor activity.

  19. PU/PTFE-stimulated monocyte-derived soluble factors induced inflammatory activation in endothelial cells.

    PubMed

    Xue, Yang; Liu, Xin; Sun, Jiao

    2010-03-01

    Polyurethane (PU) and polytetrafluoroethylene (PTFE) are two commonly used blood-contacting biomaterials. In the present study, we used a noncontact coculture model to evaluate the thrombosis-causing potential of monocyte-mediated PU and PTFE. We used human endothelial cells from umbilical cord (HUVECs) and human monocytes (THP1 cells). The THP1 cells were directly exposed to PU/PTFE, and the resultant cell-free supernatants were harvested for stimulating HUVECs. The treated HUVECs constituted the test group. HUVECs treated with supernatants of LPS-stimulated THP1 cells were used as the positive controls. To investigate the effects of the supernatant treatment on HUVECs, we measured the expression of the leukocyte-endothelial-cell adhesion molecules (CAMs) CD54 (ICAM-1), CD106 (VCAM-1), and CD62E (E-selectin) and evaluated the release of tissue factor (TF). The results demonstrated that both PU and PTFE induced the expressions of CD62E and TF. These activation effects were accompanied by activation of the NF-kappaB transcription factor. To further investigate the monocyte-derived soluble factors that might contribute to these effects, we evaluated the effects of the PU/PTFE stimulation on the expression of reactive oxygen species (ROS), TNF-alpha, IL-1beta, and IL-6 in monocyte monocultures. In comparison with the results for the negative control, both PU and PTFE significantly induced ROS release after 0.5h, while the expressions of TNF-alpha, IL-1beta, and IL-6 were variably increased after 24h. Our results suggest that the biomaterial induces monocytic activation and subsequently causes the release of soluble factors, which contribute to the inflammatory activation in HUVECs.

  20. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    PubMed

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

  1. POU domain transcription factors from different subclasses stimulate adenovirus DNA replication.

    PubMed Central

    Verrijzer, C P; Strating, M; Mul, Y M; van der Vliet, P C

    1992-01-01

    POU domain proteins constitute a family of eukaryotic transcription factors that exert critical functions during development. They contain a conserved 160 amino acids DNA binding domain, the POU domain. Genetic data have demonstrated that some POU domain proteins are essential for the proliferation of specific cell types, suggesting a possible role in DNA replication. In addition, the ubiquitous POU transcription factor Oct-1 or its isolated POU domain enhances adenovirus DNA replication. Here we compared the DNA binding specificities of POU domain proteins from different subclasses. They exhibit overlapping, yet distinct binding site preferences. Furthermore, purified Pit-1, Oct-1, Oct-2, Oct-6, Oct-4 and zebrafish POU[C] could all stimulate adenovirus DNA replication in a reconstituted in vitro system. Thus, activation appears to depend on a property common to most POU domain proteins. Adenovirus DNA replication is also stimulated by the transcription factor NFI/CTF. In contrast to NFI, the POU domain did not enhance binding of precursor terminal protein-DNA polymerase to the origin nor did it stabilize the preinitiation complex. These results suggest that the POU domain acts on a rate limiting step after formation of the preinitiation complex. Images PMID:1475198

  2. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

    SciTech Connect

    Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T. )

    1991-09-01

    Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.

  3. Highly metastatic 13762NF rat mammary adenocarcinoma cell clones stimulate bone marrow by secretion of granulocyte-macrophage colony-stimulating factor/interleukin-3 activity.

    PubMed

    McGary, C T; Miele, M E; Welch, D R

    1995-12-01

    Circulating neutrophil (polymorphonuclear leukocyte levels rise 50-fold in 13762NF tumor-bearing rats in proportion to the tumor's metastatic potential. Purified tumor-elicited neutrophils enhance metastasis of syngeneic tumor cells when co-injected intravenously; however, circulating and phorbol ester-activated polymorphonuclear neutrophils do not. The purpose of this study was to elucidate the source of tumor-elicited neutrophils in metastatic tumor-bearing rats. We examined the bone marrow in rats bearing tumors of poorly, moderately, and highly metastatic cell clones. Marrow from rats with highly metastatic tumors had increased cellularity (100%), myeloid to erythroid ratio (10:1), and megakaryocytes compared with control rats (cellularity, approximately 80%; myeloid to erythroid ratio, 5:1), with marrows from rats with moderately metastatic tumors having intermediate values. This suggested production of a colony-stimulating factor by the metastatic cells. To confirm this, bone marrow colony formation from control and tumor-bearing rats was compared. Colony number increased in proportion to the metastatic potential of the tumor. Conditioned medium from metastatic cells supported growth of the granulocyte-macrophage colony-stimulating factor/interleukin-3-dependent 32Dcl3 cell line, but media from nonmetastatic or moderately metastatic cells did not. Antibodies to murine granulocyte-macrophage colony-stimulating factor neutralized 32Dcl3 growth in tumor cell conditioned medium. These results suggest production of a granulocyte-macrophage colony-stimulating factor or interleukin-3-like activity by highly metastatic 13762NF clones and implicate a possible role for colony-stimulating factors in regulating the metastatic potential of mammary adenocarcinoma cell clones.

  4. Corticotropin releasing factor stimulates cAMP formation in pituitary corticotropic tumor cells

    SciTech Connect

    Parenti, M.; Cantalamessa, L.; Catania, A.; Reschini, E.; Mueller, E.E.

    1984-01-23

    Addition of corticotropin-releasing factor (CRF) to membranes from two ACTH-secreting pituitary tumors strikingly increased in a dose-dependent fashion adenylate cyclase (AC) activity. Stimulation of AC activity by CRF in membranes from non-tumoral tissue adjacent to tumoral corticotrophs was considerably lower, and was lacking in membranes from a growth hormone secreting tumor. These data correlated well with in vivo pre-surgery and post-surgery ACTH responsiveness to CRF of the tumor bearing patients. Basal AC activity was higher in pituitary adenomas than in non-tumoral adjacent tissue.

  5. Neuroprotective Activities of Granulocyte-Macrophage Colony Stimulating Factor Following Controlled Cortical Impact

    PubMed Central

    Kelso, Matthew L.; Elliott, Bret R.; Haverland, Nicole A.; Mosley, R. Lee; Gendelman, Howard E.

    2014-01-01

    Neurodegeneration after traumatic brain injury (TBI) is facilitated by innate and adaptive immunity and can be harnessed to effect brain repair. In mice subjected to controlled cortical impact (CCI) we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI. PMID:25468272

  6. Brain-derived neurotrophic factor stimulates energy metabolism in developing cortical neurons.

    PubMed

    Burkhalter, Julia; Fiumelli, Hubert; Allaman, Igor; Chatton, Jean-Yves; Martin, Jean-Luc

    2003-09-10

    Brain-derived neurotrophic factor (BDNF) promotes the biochemical and morphological differentiation of selective populations of neurons during development. In this study we examined the energy requirements associated with the effects of BDNF on neuronal differentiation. Because glucose is the preferred energy substrate in the brain, the effect of BDNF on glucose utilization was investigated in developing cortical neurons via biochemical and imaging studies. Results revealed that BDNF increases glucose utilization and the expression of the neuronal glucose transporter GLUT3. Stimulation of glucose utilization by BDNF was shown to result from the activation of Na+/K+-ATPase via an increase in Na+ influx that is mediated, at least in part, by the stimulation of Na+-dependent amino acid transport. The increased Na+-dependent amino acid uptake by BDNF is followed by an enhancement of overall protein synthesis associated with the differentiation of cortical neurons. Together, these data demonstrate the ability of BDNF to stimulate glucose utilization in response to an enhanced energy demand resulting from increases in amino acid uptake and protein synthesis associated with the promotion of neuronal differentiation by BDNF.

  7. Platelet-derived growth factor (PDGF) stimulates glycogen synthase activity in 3T3 cells

    SciTech Connect

    Chan, C.P.; Bowen-Pope, D.F.; Ross, R.; Krebs, E.G.

    1986-05-01

    Hormonal regulation of glycogen synthase, an enzyme that can be phosphorylated on multiple sites, is often associated with changes in its phosphorylation state. Enzyme activation is conventionally monitored by determining the synthase activity ratio ((activity in the absence of glucose 6-P)/(activity in the presence of glucose 6-P)). Insulin causes an activation of glycogen synthase with a concomitant decrease in its phosphate content. In a previous report, the authors showed that epidermal growth factor (EGF) increases the glycogen synthase activity ratio in Swiss 3T3 cells. The time and dose-dependency of this response was similar to that of insulin. Their recent results indicate that PDGF also stimulates glycogen synthase activity. Enzyme activation was maximal after 30 min. of incubation with PDGF; the time course observed was very similar to that with insulin and EGF. At 1 ng/ml (0.03nM), PDGF caused a maximal stimulation of 4-fold in synthase activity ratio. Half-maximal stimulation was observed at 0.2 ng/ml (6 pM). The time course of changes in enzyme activity ratio closely followed that of /sup 125/I-PDGF binding. The authors data suggest that PDGF, as well as EFG and insulin, may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

  8. Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells.

    PubMed

    Badesch, D B; Lee, P D; Parks, W C; Stenmark, K R

    1989-04-14

    Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.

  9. Nerve growth factor and epidermal growth factor stimulate clusterin gene expression in PC12 cells.

    PubMed Central

    Gutacker, C; Klock, G; Diel, P; Koch-Brandt, C

    1999-01-01

    Clusterin (apolipoprotein J) is an extracellular glycoprotein that might exert functions in development, cell death and lipid transport. Clusterin gene expression is elevated at sites of tissue remodelling, such as differentiation and apoptosis; however, the signals responsible for this regulation have not been identified. We use here the clusterin gene as a model system to examine expression in PC12 cells under the control of differentiation and proliferation signals produced by nerve growth factor (NGF) and by epidermal growth factor (EGF) respectively. NGF induced clusterin mRNA, which preceded neurite outgrowth typical of neuronal differentiation. EGF also activated the clusterin mRNA, demonstrating that both proliferation and differentiation signals regulate the gene. To localize NGF- and EGF-responsive elements we isolated the clusterin promoter and tested it in PC12 cell transfections. A 2.5 kb promoter fragment and two 1.5 and 0.3 kb deletion mutants were inducible by NGF and EGF. The contribution to this response of a conserved activator protein 1 (AP-1) motif located in the 0.3 kb fragment was analysed by mutagenesis. The mutant promoter was not inducible by NGF or EGF, which identifies the AP-1 motif as an element responding to both factors. Binding studies with PC12 nuclear extracts showed that AP-1 binds to this sequence in the clusterin promoter. These findings suggest that NGF and EGF, which give differential gene regulation in PC12 cells, resulting in neuronal differentiation and proliferation respectively, use the common Ras/extracellular signal-regulated kinase/AP-1 signalling pathway to activate clusterin expression. PMID:10215617

  10. Evidence for multiple bone resorption-stimulating factors produced by normal human keratinocytes in culture.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Tashjian, A H

    1988-06-01

    Conditioned medium from cultured normal human foreskin keratinocytes enhanced the release of calcium from neonatal mouse calvaria in organ culture. Unfractionated keratinocyte-conditioned medium (KCM) stimulated bone resorption in a dose-dependent manner, but it did not increase the concentration of prostaglandin E2 (PGE2) in the bone culture medium until a maximal dose of KCM for resorption was used. Furthermore, inhibitors of PGE2 synthesis, indomethacin, ibuprofen, and piroxicam, did not inhibit KCM-induced calcium release. High concentrations of KCM increased cAMP production by calvaria in the presence of isobutylmethylxanthine, but the increase was small compared with that produced by a dose of bovine PTH that caused a similar level of bone resorption. The bone resorption-stimulating activity of KCM was not lost after incubation at 56 C for 60 min, but it was lost after heating at 100 C for 10 min. Fractionation of KCM by gel filtration chromatography revealed two distinct peaks of bone resorption-stimulating activity. One peak, KCMI, caused a significant increase in bone resorption at 2 micrograms protein/ml. KCMI did not increase medium PGE2, and inhibition of PGE2 synthesis in bone had no effect on KCMI-induced bone resorption. KCMI failed to increase cAMP production by human osteosarcoma SaOS-2 cells. Another peak, KCMII, caused a dose-dependent increase in bone resorption, and a significant increase in medium calcium was noted at a 20-fold lower concentration (0.1 microgram protein/ml) than with KCMI. In contrast to KCMI, the increase in bone resorption stimulated by KCMII was accompanied by a parallel increase in the production of PGE2, and inhibition of PGE2 synthesis completely inhibited the bone resorption-stimulating activity of KCMII. KCMII also caused an increase in cAMP production by SaOS-2 cells. We conclude that KCM contains at least two distinct bone resorption-stimulating factors, one of which acts via a PG-mediated mechanism and the other by

  11. Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis.

    PubMed

    Slungaard, A; Key, N S

    1994-10-14

    Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.

  12. Activation of Sterol Regulatory Element Binding Factors by Fenofibrate and Gemfibrozil Stimulates Myelination in Zebrafish

    PubMed Central

    Ashikawa, Yoshifumi; Nishimura, Yuhei; Okabe, Shiko; Sasagawa, Shota; Murakami, Soichiro; Yuge, Mizuki; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Oligodendrocytes are major myelin-producing cells and play essential roles in the function of a healthy nervous system. However, they are also one of the most vulnerable neural cell types in the central nervous system (CNS), and myelin abnormalities in the CNS are found in a wide variety of neurological disorders, including multiple sclerosis, adrenoleukodystrophy, and schizophrenia. There is an urgent need to identify small molecular weight compounds that can stimulate myelination. In this study, we performed comparative transcriptome analysis to identify pharmacodynamic effects common to miconazole and clobetasol, which have been shown to stimulate myelination by mouse oligodendrocyte progenitor cells (OPCs). Of the genes differentially expressed in both miconazole- and clobetasol-treated mouse OPCs compared with untreated cells, we identified differentially expressed genes (DEGs) common to both drug treatments. Gene ontology analysis revealed that these DEGs are significantly associated with the sterol biosynthetic pathway, and further bioinformatics analysis suggested that sterol regulatory element binding factors (SREBFs) might be key upstream regulators of the DEGs. In silico screening of a public database for chemicals associated with SREBF activation identified fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, as a drug that increases the expression of known SREBF targets, raising the possibility that fenofibrate may also stimulate myelination. To test this, we performed in vivo imaging of zebrafish expressing a fluorescent reporter protein under the control of the myelin basic protein (mbp) promoter. Treatment of zebrafish with fenofibrate significantly increased expression of the fluorescent reporter compared with untreated zebrafish. This increase was attenuated by co-treatment with fatostatin, a specific inhibitor of SREBFs, confirming that the fenofibrate effect was mediated via SREBFs. Furthermore, incubation of zebrafish

  13. Transforming growth factor Beta 1 stimulates profibrotic activities of luteal fibroblasts in cows.

    PubMed

    Maroni, Dulce; Davis, John S

    2012-11-01

    Luteolysis is characterized by angioregression, luteal cell apoptosis, and remodeling of the extracellular matrix characterized by deposition of collagen 1. Transforming growth factor beta 1 (TGFB1) is a potent mediator of wound healing and fibrotic processes through stimulation of the synthesis of extracellular matrix components. We hypothesized that TGFB1 stimulates profibrotic activities of luteal fibroblasts. We examined the actions of TGFB1 on luteal fibroblast proliferation, extracellular matrix production, floating gel contraction, and chemotaxis. Fibroblasts were isolated from the bovine corpus luteum. Western blot analysis showed that luteal fibroblasts expressed collagen 1 and prolyl 4-hydroxylase but did not express markers of endothelial or steroidogenic cells. Treatment of fibroblasts with TGFB1 stimulated the phosphorylation of SMAD2 and SMAD3. [(3)H]thymidine incorporation studies showed that TGFB1 caused concentration-dependent reductions in DNA synthesis in luteal fibroblasts and significantly (P < 0.05) reduced the proliferative effect of FGF2 and fetal calf serum. However, TGFB1 did not reduce the viability of luteal fibroblasts. Treatment of luteal fibroblasts with TGFB1 induced the expression of laminin, collagen 1, and matrix metalloproteinase 1 as determined by Western blot analysis and gelatin zymography of conditioned medium. TGFB1 increased the chemotaxis of luteal fibroblasts toward fibronectin in a transwell system. Furthermore, TGFB1 increased the fibroblast-mediated contraction of floating bovine collagen 1 gels. These results suggest that TGFB1 contributes to the structural regression of the corpus luteum by stimulating luteal fibroblasts to remodel and contract the extracellular matrix.

  14. Exploring bikeability in a metropolitan setting: stimulating and hindering factors in commuting route environments

    PubMed Central

    2012-01-01

    Background Route environments may influence people's active commuting positively and thereby contribute to public health. Assessments of route environments are, however, needed in order to better understand the possible relationship between active commuting and the route environment. The aim of this study was, therefore, to assess the potential associations between perceptions of whether the route environment on the whole hinders or stimulates bicycle commuting and perceptions of environmental factors. Methods The Active Commuting Route Environment Scale (ACRES) was used for the assessment of bicycle commuters' perceptions of their route environments in the inner urban parts of Greater Stockholm, Sweden. Bicycle commuters (n = 827) were recruited by advertisements in newspapers. Simultaneous multiple regression analyses were used to assess the relation between predictor variables (such as levels of exhaust fumes, noise, traffic speed, traffic congestion and greenery) and the outcome variable (hindering - stimulating route environments). Two models were run, (Model 1) without and (Model 2) with the item traffic: unsafe or safe included as a predictor. Results Overall, about 40% of the variance of hindering - stimulating route environments was explained by the environmental predictors in our models (Model 1, R2 = 0.415, and Model 2, R 2= 0.435). The regression equation for Model 1 was: y = 8.53 + 0.33 ugly or beautiful + 0.14 greenery + (-0.14) course of the route + (-0.13) exhaust fumes + (-0.09) congestion: all types of vehicles (p ≤ 0.019). The regression equation for Model 2 was y = 6.55 + 0.31 ugly or beautiful + 0.16 traffic: unsafe or safe + (-0.13) exhaust fumes + 0.12 greenery + (-0.12) course of the route (p ≤ 0.001). Conclusions The main results indicate that beautiful, green and safe route environments seem to be, independently of each other, stimulating factors for bicycle commuting in inner urban areas. On the other hand, exhaust fumes, traffic

  15. Stimulation of body weight increase and epiphyseal cartilage growth by insulin like growth factor

    NASA Technical Reports Server (NTRS)

    Ellis, S.

    1981-01-01

    The ability of insulin-like growth factor (IGF) to induce growth in hypophysectomized immature rats was tested by continuous infusion of the partially purified factor at daily doses of 6, 21, and 46 mU for an 8-day period. A dose-dependent growth of the proximal epiphyseal cartilage of the tibia and an associated stimulation of the primary spongiosa were produced by these amounts of IGF. The two highest doses of IGF also resulted in dose-dependent increases of body weight. Gel permeation of the sera at neutrality showed that the large-molecular-weight IGF binding protein was not induced by the infusion of IGF, whereas it ws generated in the sera of hypophysectomized rats that were infused with daily doses of 86 mU of human growth hormone.

  16. Treatment of leg ischemia with biodegradable gelatin hydrogel microspheres incorporating granulocyte colony-stimulating factor.

    PubMed

    Kawamura, Itta; Takemura, Genzou; Tsujimoto, Akiko; Watanabe, Takatomo; Kanamori, Hiromitsu; Esaki, Masayasu; Kobayashi, Hiroyuki; Takeyama, Toshiaki; Kawaguchi, Tomonori; Goto, Kazuko; Maruyama, Rumi; Fujiwara, Takako; Fujiwara, Hisayoshi; Tabata, Yasuhiko; Minatoguchi, Shinya

    2011-04-01

    Granulocyte colony-stimulating factor (G-CSF) is a potent angiogenic factor. We hypothesized that G-CSF-immersed gelatin hydrogel microspheres (G-CSF-GHMs) injected into the ischemic legs might continuously release a small amount of G-CSF to locally stimulate angiogenesis without unfavorable systemic effects. Just after ligation of the right femoral artery of BALB/c mice, recombinant human G-CSF (100-μg/kg)-immersed GHM was injected into the right hindlimb muscles; the controls included a saline-injected group, an intramuscularly injected G-CSF group, a subcutaneously injected G-CSG group, and an empty GHM-injected group. Eight weeks later, improvement of blood perfusion to the ischemic limb was significantly augmented in the G-CSF-GHM group compared with any of the control groups. Despite there being no increase in the serum concentration of G-CSF, in peripheral granulocytes, or in circulating endothelial progenitor cells, not only capillary but also arteriolar density was significantly increased in this group. Next, we started treatment with G-CSF-GHM 4 weeks after ligation to examine whether the treatment is effective if performed during the chronic stage of ischemia. The late treatment was also found to effectively improve blood flow in the ischemic leg. In conclusion, G-CSF-GHM administration is suggested to be a promising and readily usable approach to treating peripheral artery disease, applicable even during the chronic stage.

  17. Side-effects of subthalamic stimulation in Parkinson's disease: clinical evolution and predictive factors.

    PubMed

    Guehl, D; Cuny, E; Benazzouz, A; Rougier, A; Tison, F; Machado, S; Grabot, D; Gross, C; Bioulac, B; Burbaud, P

    2006-09-01

    Chronic bilateral high-frequency stimulation of the subthalamic nucleus (STN) is an alternative treatment for disabling forms of Parkinson's disease when on-off fluctuations and levodopa-induced dyskinesias compromise patients' quality of life. The aim of this study was to assess the evolution of side-effects during the first year of follow-up and search for clinical predictive factors accounting for their occurrence. We compared the frequency of side-effects at 3 and 12 months after surgery in a cohort of 44 patients. The off-medication scores of Unified Parkinson's Disease Rating Scale (UPDRS) II, III, axial symptoms, disease duration and age at surgery were retained for correlation analysis. Dysarthria/hypophonia, weight gain and postural instability were the most frequent chronic side-effects. Whereas dysarthria/hypophonia remained stable over time, weight gain and postural instability increased during the first year post-op. High axial and UPDRS II scores at surgery were predictive of dysarthria/hypophonia. Age and axial score at surgery were positively correlated with postural instability. Despite the occurrence of side-effects, the benefit/side-effects ratio of STN stimulation was largely positive during the first year of follow-up. Age, intensity of axial symptoms and UDPRS II off-medication score before surgery are predictive factors of dysarthria/hypophonia and postural instability after surgery.

  18. Acetylation impacts Fli-1-driven regulation of granulocyte colony stimulating factor.

    PubMed

    Lennard Richard, Mara L; Brandon, Danielle; Lou, Ning; Sato, Shuzo; Caldwell, Tomika; Nowling, Tamara K; Gilkeson, Gary; Zhang, Xian K

    2016-10-01

    Fli-1 has emerged as a critical regulator of inflammatory mediators, including MCP-1, CCL5, and IL-6. The cytokine, granulocyte colony stimulating factor (G-CSF) regulates neutrophil precursor maturation and survival, and activates mature neutrophils. Previously, a significant decrease in neutrophil infiltration into the kidneys of Fli-1(+/-) lupus-prone mice was observed. In this study, a significant decrease in G-CSF protein expression was detected in stimulated murine and human endothelial cells when expression of Fli-1 was inhibited. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and several regions within the proximal promoter are significantly enriched for Fli-1 binding. Transient transfection assays indicate that Fli-1 drives transcription from the G-CSF promoter and mutation of the Fli-1 DNA binding domain resulted in a 94% loss of transcriptional activation. Mutation of a known acetylation site, led to a significant increase in G-CSF promoter activation. The histone acetyltransferases p300/CBP and p300/CBP associated factor (PCAF) significantly decrease Fli-1 specific activation of the G-CSF promoter. Thus, acetylation appears to be an important mechanism behind Fli-1 driven activation of the G-CSF promoter. These results further support the theory that Fli-1 plays a major role in the regulation of several inflammatory mediators, ultimately affecting inflammatory disease pathogenesis.

  19. Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

    PubMed Central

    Schecter, A D; Giesen, P L; Taby, O; Rosenfield, C L; Rossikhina, M; Fyfe, B S; Kohtz, D S; Fallon, J T; Nemerson, Y; Taubman, M B

    1997-01-01

    Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury. PMID:9410905

  20. Promiscuous stimulation of ParF protein polymerization by heterogeneous centromere binding factors.

    PubMed

    Machón, Cristina; Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2007-11-16

    The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.

  1. Multiple Growth Factors, But Not VEGF, Stimulate Glycosaminoglycan Hyperelongation in Retinal Choroidal Endothelial Cells

    PubMed Central

    Al Gwairi, Othman; Osman, Narin; Getachew, Robel; Zheng, Wenhua; Liang, X-L.; Kamato, Danielle; Thach, Lyna; Little, Peter J.

    2016-01-01

    A major feature of early age-related macular degeneration (AMD) is the thickening of Bruch's membrane in the retina and an alteration in its composition with increased lipid deposition. In certain pathological conditions proteoglycans are responsible for lipid retention in tissues. Growth factors are known to increase the length of glycosaminoglycan chains and this can lead to a large increase in the interaction between proteoglycans and lipids. Using choroidal endothelial cells, we investigated the effects of a number of AMD relevant growth factors TGFβ, thrombin, PDGF, IGF and VEGF on proteoglycan synthesis. Cells were characterized as of endothelial origin using the specific cell markers endothelial nitric oxide synthesis and von Willebrand factor and imaged using confocal microscopy. Cells were treated with growth factors in the presence and absence of the appropriate inhibitors and were radiolabeled with [35S]-SO4. Proteoglycans were isolated by ion exchange chromatography and sized using SDS-PAGE. Radiosulfate incorporation was determined by the cetylpyridinium chloride (CPC) precipitation technique. To measure cellular glycosaminoglycan synthesizing capacity we added xyloside and assessed the xyloside-GAGs by SDS-PAGE. TGFβ, thrombin, PDGF & IGF dose-dependently stimulated radiosulfate incorporation and GAG elongation as well as xyloside-GAG synthesis, however VEGF treatment did not stimulate any changes in proteoglycan synthesis. VEGF did not increase pAKT but caused a large increase in pERK relative to the response to PDGF. Thus, AMD relevant agonists cause glycosaminoglycan hyperelongation of proteoglycans synthesised and secreted by retinal choroidal endothelial cells. The absence of a response to VEGF is intriguing and identifies proteoglycans as a novel potential target in AMD. Future studies will examine the relevance of these changes to enhanced lipid binding and the development of AMD. PMID:27570478

  2. Down-regulation of hypoxia-inducible factor-2 in PC12 cells by nerve growth factor stimulation.

    PubMed

    Naranjo-Suárez, Salvador; Castellanos, María Carmen; Alvarez-Tejado, Miguel; Vara, Alicia; Landázuri, Manuel O; del Peso, Luis

    2003-08-22

    Cellular responses to low oxygen tension are mediated, at least in part, by the activation of the hypoxia-inducible factors (HIFs). In the presence of oxygen, specific HIF residues become hydroxylated by the action of a recently described group of dioxygenases. These post-translational modifications target HIF for proteosomal degradation and prevent its transcriptional activity. Despite these detailed studies, little is known about the regulation of HIF by stimuli other than hypoxia. Here we report that, in rat pheochromocytoma PC12 cells, nerve growth factor (NGF) stimulation results in a decrease of both basal and hypoxia-induced levels of HIF-2 alpha protein. NGF treatment did not increase HIF-hydroxylase gene expression or activity, and the reduction of the HIF-2 alpha protein level upon stimulation was observed even in the presence of HIF-hydroxylase inhibitors such as deferoxamine or dimethyloxoglutarate. Thus, in contrast to the response to hypoxia, the effect of NGF on HIF-2 alpha protein levels is not mediated by the HIF hydroxilases. Quantitative real time (RT)-PCR showed that NGF stimulation results in a decrease of the HIF-2 alpha mRNA level similar to that found at the protein level. Interestingly, NGF effect was specific for HIF-2 alpha mRNA because it did not affect HIF-1 alpha mRNA levels. NGF treatment reduced HIF-2 alpha mRNA levels even in the presence of actinomycin D, suggesting an effect on mRNA stability. Finally, the effect of NGF on HIF2 alpha correlates with reduction of both basal and hypoxia-induced vascular endothelial growth factor mRNA levels. Reporter assays suggest that the reduced expression of hypoxia-inducible genes upon NGF treatment is related, at least in part, to the reduction of HIF-2 alpha protein. Hence, in PC12 cells the level of HIF-2 alpha protein and its effect on gene expression can be down-regulated by stimuli other than oxygen.

  3. Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors.

    PubMed

    Hah, Young-Sool; Jun, Jin-Su; Lee, Seong-Gyun; Park, Bong-Wook; Kim, Deok Ryong; Kim, Uk-Kyu; Kim, Jong-Ryoul; Byun, June-Ho

    2011-02-01

    Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF(121), VEGF(165), VEGF(189), and VEGF(206)), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF(165) elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF(165) resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.

  4. Perinatal factors associated with neonatal thyroid-stimulating hormone in normal newborns

    PubMed Central

    2016-01-01

    Purpose This study was to evaluate the effect of neonatal, maternal, and delivery factors on neonatal thyroid-stimulating hormone (TSH) of healthy newborns. Methods Medical records of 705 healthy infants born through normal vaginal delivery were reviewed. Neonatal TSH levels obtained by neonatal screening tests were analyzed in relation to perinatal factors and any associations with free thyroxine (FT4) and 17-α hydroxyprogesterone (17OHP) levels. Results An inverse relationship was found between TSH and sampling time after birth. Twin babies and neonates born by vacuum-assisted delivery had higher TSH levels than controls. First babies had higher TSH levels than subsequent babies. Birth weight, gestational age, maternal age and duration from the rupture of the membrane to birth were not related to neonatal TSH. There were no significant differences in TSH level according to sex, Apgar scores, labor induction, the presence of maternal disease and maternal medications. There was a positive association between TSH and 17OHP level but not between TSH and FT4 level. Multiple linear regression analyses showed that sampling time, mode of delivery, birth order, and 17OHP level were significant factors affecting neonatal TSH level. Conclusion Neonatal TSH levels of healthy normal newborns are related with multiple factors. Acute stress during delivery may influence the neonatal TSH level in early neonatal period. PMID:28164073

  5. The effect of diet on tumor necrosis factor stimulation of hepatic lipogenesis

    SciTech Connect

    Feingold, K.R.; Soued, M.; Serio, M.K.; Adi, S.; Moser, A.H.; Grunfeld, C. )

    1990-06-01

    In this study, we determined the effects of tumor necrosis factor (TNF) on serum lipid levels and hepatic lipid synthesis in animals whose diets and feeding conditions were varied to induce changes in baseline serum lipid levels and/or rates of hepatic lipid synthesis. In animals studied at both the nadir and peak of the diurnal cycle of hepatic lipid synthesis, TNF acutely increases serum triglyceride levels, stimulates hepatic fatty acid synthesis, and increases the quantity of newly synthesized fatty acids found in the serum. Similarly, in animals ingesting either high-sucrose or cholesterol-enriched diets, TNF induces the characteristic rapid increase in serum triglyceride levels, hepatic fatty acid synthesis, and quantity of labeled fatty acids in the serum. In animals fed a diet high in triglycerides, using either corn oil or lard, TNF stimulates hepatic fatty acid synthesis and increases the quantity of newly synthesized fatty acids in the serum, but serum triglyceride levels do not change. However, TNF inhibits gastric emptying, which results in a marked decrease in fat absorption in TNF-treated animals. It is likely that a decrease in the dietary contribution to serum triglyceride levels during high-triglyceride feeding counterbalances the increased hepatic contribution induced by TNF treatment. In animals fasted before TNF administration there was no acute change in either serum lipid levels, hepatic fatty acid synthesis, or the quantity of labeled fatty acids in the serum. Thus, TNF stimulates hepatic fatty acid synthesis and increases serum triglyceride levels under many diverse dietary conditions, suggesting that there is a strong linkage between the immune system and lipid metabolism that is independent of most dietary manipulations and may be of fundamental importance in the body's response to infection.

  6. Modulation of cultured porcine granulosa cell responsiveness to follicle stimulating hormone and epidermal growth factor

    SciTech Connect

    Buck, P.A.

    1986-01-01

    Ovarian follicular development is dependent upon the coordinated growth and differentiation of the granulosa cells which line the follicle. Follicle stimulating hormone (FSH) induces granulosa cell differentiation both in vivo and in vitro. Epidermal growth factor (EGF) stimulates granulosa cell proliferation in vitro. The interaction of these two effectors upon selected parameters of growth and differentiation was examined in monolayer cultures of porcine granulose cells. Analysis of the EGF receptor by /sup 125/I-EGF binding revealed that the receptor was of high affinity with an apparent dissociation constant of 4-6 x 10/sup -10/ M. The average number of receptors per cell varied with the state of differentiation both in vivo and in vitro; highly differentiated cells bound two-fold less /sup 125/I-EGF and this effect was at least partially induced by FSH in vitro. EGF receptor function was examined by assessing EGF effects on cell number and /sup 3/H-thymidine incorporation. EGF stimulated thymidine incorporation in both serum-free and serum-supplemented culture, but only in serum-supplemented conditions was cell number increased. EGF receptor function was inversely related to the state of differentiation and was attenuated by FSH. The FSH receptor was examined by /sup 125/I-FSH binding. EGF increased FSH receptor number, and lowered the affinity of the receptor. The function of these receptors was assessed by /sup 125/I-hCG binding and progesterone radioimmunoassay. If EGF was present continuously in the cultures. FSH receptor function was attenuated regardless of FSH receptor number. A preliminary effort to examine the mechanism of this interaction was performed by analyzing hormonally controlled protein synthesis with /sup 35/S-methionine labeling, SDS polyacrylamide gel electrophoresis and fluorography. FSH promoted the expression of a 27,000 dalton protein. This effect was attenuated by EGF.

  7. Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation

    PubMed Central

    Tong, Junfeng; Li, Laiji; Ballermann, Barbara; Wang, Zhixiang

    2016-01-01

    The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity. PMID:26816343

  8. Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

    2005-05-01

    Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

  9. Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells.

    PubMed

    Kadoya, Masatoshi; Yamamoto, Aihiro; Hamaguchi, Masahide; Obayashi, Hiroshi; Mizushima, Katsura; Ohta, Mitsuhiro; Seno, Takahiro; Oda, Ryo; Fujiwara, Hiroyoshi; Kohno, Masataka; Kawahito, Yutaka

    2014-06-06

    Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14(+) peripheral blood mononuclear cells (CD14(+) PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14(+) PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14(+) PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.

  10. Inhibitory effect of trichothecene mycotoxins on bovine platelets stimulated by platelet activating factor.

    PubMed Central

    Gentry, P A; Ross, M L; Bondy, G S

    1987-01-01

    Several species of fungi, which infect cereals and grains, can produce a class of compounds, known as trichothecene mycotoxins, which is characterized by a substituted epoxy-trichothecene ring structure. Cattle are susceptible to intoxication from feeds contaminated with T-2 toxin, one of the more potent trichothecene mycotoxins, while swine refuse to ingest feed contaminated with T-2 toxin. The bovine platelet has been used as a model cell system to evaluate the effects of T-2 toxin and its natural metabolites, HT-2 toxin and T-2 tetraol, on cell function in vitro. Due to the lipophilic nature of these mycotoxins, a biologically active phospholipid was used to stimulate the platelets in the presence and absence of the toxins. The mycotoxin T-2 toxin and its major metabolite HT-2 toxin inhibited platelet activating factor-stimulated bovine platelets, suspended in homologous plasma, in a concentration but not time dependent manner. Significant inhibition of platelet function (p less than 0.01) occurred with 135 ng T-2 toxin per 10(6) platelets and with 77 ng HT-2 toxin per 10(6) platelets. These mycotoxins exerted an additive inhibitory effect on the platelet aggregation response. In contrast, the minor metabolite T-2 tetraol had no inhibitory effect on platelet function and had no influence on the responses of T-2 toxin or HT-2 toxin when the mycotoxins were present together in the platelet suspensions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3453270

  11. Role of macrophage colony-stimulating factor (M-CSF) in human granulosa cells.

    PubMed

    Xu, Song; Zhang, Zhifen; Xia, Li-Xia; Huang, Jian

    2016-12-01

    Macrophage colony-stimulating factor (M-CSF) has been proved to have a positive role in the follicular development. We investigated its effect on human granulosa cells and found that M-CSF could stimulate the production of E2. The production of FSH receptors was enhanced by M-CSF in vitro in a dose-dependent manner with or without the addition of tamoxifen (p <0.05). Correspondingly, FSH was also able to coordinate the expression of M-CSF and its receptor (p <0.05). That maybe important to maintain the level of Nppc and the meiotic arrest of the oocyte. The protein p-JAK2 and p-STAT3 in JAK/STAT-signaling pathway elevated after the influence of M-CSF (p < 0.05). These results suggest that M-CSF has a role in regulating the response of granulosa cells to gonadotropins. Its function is associated with JAK/STAT-signaling pathway.

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

    PubMed

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

    2010-08-01

    Previously, dogs with canine parvovirus-induced neutropenia have not responded to treatment with recombinant human granulocyte-colony stimulating factor (rhG-CSF). However, recombinant canine G-CSF (rcG-CSF) has not been previously evaluated for treatment of parvovirus-induced neutropenia in dogs. We assessed the effectiveness of rcG-CSF in dogs with parvovirus-induced neutropenia with a prospective, open-label, nonrandomized clinical trial. Endpoints of our study were time to recovery of WBC and neutrophil counts, and duration of hospitalization. 28 dogs with parvovirus and neutropenia were treated with rcG-CSF and outcomes were compared to those of 34 dogs with parvovirus and neutropenia not treated with rcG-CSF. We found that mean WBC and neutrophil counts were significantly higher (P < 0.05) in the 28 dogs treated with rcG-CSF compared to disease-matched dogs not treated with rcG-CSF. In addition, the mean duration of hospitalization was reduced (P = 0.01) in rcG-CSF treated dogs compared to untreated dogs. However, survival times were decreased in dogs treated with rcG-CSF compared to untreated dogs. These results suggest that treatment with rcG-CSF was effective in stimulating neutrophil recovery and shortening the duration of hospitalization in dogs with parvovirus infection, but indicate the need for additional studies to evaluate overall safety of the treatment.

  13. Insulin-like growth factor I stimulates erythropoiesis in hypophysectomized rats

    SciTech Connect

    Kurtz, A.; Zapf, J.; Eckardt, K.U.; Clemons, G.; Froesch, E.R.; Bauer, C. )

    1988-10-01

    Stimulation of erythropoiesis during growth is necessary to ensure proportionality between erythrocyte mass and body mass. However, the way by which erythrocyte formation is adapted to body growth is still unknown. Growth arrest in hypophysectomized rats is accompanied by decreased erythropoiesis. The authors have, therefore, examined whether insulin-like growth factor I (IGF-I), the mediator of growth hormone effects on body growth, is able to restore erythropoiesis in these animals. Subcutaneous infusions of 120 {mu}g of recombinant human IGF-I per day in hypophysectomized rats led to increases in body weight, {sup 59}Fe incorporation into erythrocytes, and the number of reticulocytes that were similar to increases caused by infusions of 28 milliunits of human growth hormone per day. Body weight gain and {sup 59}Fe incorporation were linearly correlated. Like growth hormone, IGF-I also caused a significant rise in serum erythropoietin concentrations. However, the stimulatory effect on erythropoiesis occurred before serum erythropoietin levels had risen. These results demonstrate that IGF-I mediates the stimulatory effect of growth hormone on erythropoiesis in vivo and thus further support the somatomedin concept. They also show that IGF-I can stimulate erythropoiesis in an endocrine manner, and they suggest two possible routes of action: a direct one and an indirect one by means of enhanced erythropoietin production.

  14. The contribution of interindividual factors to variability of response in transcranial direct current stimulation studies

    PubMed Central

    Li, Lucia M.; Uehara, Kazumasa; Hanakawa, Takashi

    2015-01-01

    There has been an explosion of research using transcranial direct current stimulation (tDCS) for investigating and modulating human cognitive and motor function in healthy populations. It has also been used in many studies seeking to improve deficits in disease populations. With the slew of studies reporting “promising results” for everything from motor recovery after stroke to boosting memory function, one could be easily seduced by the idea of tDCS being the next panacea for all neurological ills. However, huge variability exists in the reported effects of tDCS, with great variability in the effect sizes and even contradictory results reported. In this review, we consider the interindividual factors that may contribute to this variability. In particular, we discuss the importance of baseline neuronal state and features, anatomy, age and the inherent variability in the injured brain. We additionally consider how interindividual variability affects the results of motor-evoked potential (MEP) testing with transcranial magnetic stimulation (TMS), which, in turn, can lead to apparent variability in response to tDCS in motor studies. PMID:26029052

  15. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    PubMed

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  16. Requirement of Src kinase Lyn for induction of DNA synthesis by granulocyte colony-stimulating factor.

    PubMed

    Corey, S J; Dombrosky-Ferlan, P M; Zuo, S; Krohn, E; Donnenberg, A D; Zorich, P; Romero, G; Takata, M; Kurosaki, T

    1998-02-06

    Treatment of cells with granulocyte colony-stimulating factor (G-CSF) leads to tyrosine phosphorylation of cellular proteins. G-CSF stimulates both the activation of protein tyrosine kinases Lyn, Jak1, and Jak2 and the association of these enzymes with the G-CSF receptor. Wild-type, lyn-deficient, and syk-deficient chicken B lymphocyte cell lines were transfected with the human G-CSF receptor, and stable transfectants were studied. G-CSF-dependent tyrosyl phosphorylation of Jak1 and Jak2 occurred in all three cell lines. Wild-type and syk-deficient transfectants responded to G-CSF in a dose-responsive fashion with increased thymidine incorporation, but none of the clones of lyn-deficient transfectants did. Ectopic expression of Lyn, but not that of c-Src, in the lyn-deficient cells restored their mitogenic responsiveness to G-CSF. Ectopic expression in wild-type cells of the kinase-inactive form of Lyn, but not of the kinase-inactive form of Jak2, inhibited thymidine incorporation in response to G-CSF. These studies show that the absence of Lyn results in the loss of mitogenic signaling in the G-CSF signaling pathway and that activation of Jak1 or Jak2 is not sufficient to cause mitogenesis.

  17. Pancreatic adenocarcinoma upregulated factor serves as adjuvant by activating dendritic cells through stimulation of TLR4

    PubMed Central

    Yang, Benjamin; Lee, Je-Jung; Lee, Hyun-Ju; Lee, Jaemin; Jung, In Duk; Han, Hee Dong; Lee, Seung-Hyun; Koh, Sang Seok; Wu, T.-C.; Park, Yeong-Min

    2015-01-01

    Dendritic cell (DC) based cancer vaccines represent a promising immunotherapeutic strategy against cancer. To enhance the modest immunogenicity of DC vaccines, various adjuvants are often incorporated. Particularly, most of the common adjuvants are derived from bacteria. In the current study, we evaluate the use of a human pancreatic cancer derived protein, pancreatic adenocarcinoma upregulated factor (PAUF), as a novel DC vaccine adjuvant. We show that PAUF can induce activation and maturation of DCs and activate NFkB by stimulating the Toll-like receptor signaling pathway. Furthermore, vaccination with PAUF treated DCs pulsed with E7 or OVA peptides leads to generation of E7 or OVA-specific CD8+ T cells and memory T cells, which correlate with long term tumor protection and antitumor effects against TC-1 and EG.7 tumors in mice. Finally, we demonstrated that PAUF mediated DC activation and immune stimulation are dependent on TLR4. Our data provides evidence supporting PAUF as a promising adjuvant for DC based therapies, which can be applied in conjunction with other cancer therapies. Most importantly, our results serve as a reference for future investigation of human based adjuvants. PMID:26336989

  18. Expression and purification of canine granulocyte colony-stimulating factor (cG-CSF).

    PubMed

    Yamamoto, Akira; Iwata, Akira; Saito, Toshiki; Watanabe, Fumiko; Ueda, Susumu

    2009-08-15

    Canine granulocyte colony-stimulating factor (cG-CSF) with modification of cysteine at position 17 to serine was expressed in Brevibacillus choshinensis HPD31. cG-CSF secreted into the culture medium was purified by ammonium sulfate precipitation and consecutive column chromatography, using butyl sepharose and DEAE sepharose. Biological activity of the recombinant cG-CSF was 8.0 x 10(6) U/mg protein, as determined by its stimulatory effect on NFS-60 cell proliferation. Purified cG-CSF was subcutaneously administered once a day for two successive days to dogs (1, 5, 25, or 125 microg). Neutrophil count increased the following day in all dogs except those administered the lowest dose (1 microg). No severe side effects were observed in dogs after administration of cG-CSF.

  19. Autopsy of anaplastic carcinoma of the pancreas producing granulocyte colony-stimulating factor.

    PubMed

    Hayashi, Haruna; Eguchi, Noriaki; Sumimoto, Kyoku; Matsumoto, Kenta; Azakami, Takahiro; Sumida, Tomonori; Tamura, Tadamasa; Sumii, Masaharu; Uraoka, Naohiro; Shimamoto, Fumio

    2016-08-01

    A 50-year-old man presented to a nearby hospital with high fever and anorexia. An abdominal tumor was detected, and he was referred to our hospital. A pancreatic tumor was detected by computed tomography and abdominal ultrasonography. He had high fever, leukocytosis, and high serum granulocyte colony-stimulating factor (G-CSF). We performed a tumor biopsy and histological examination revealed anaplastic carcinoma of the pancreas. Based on the diagnosis, we initiated chemotherapy using gemcitabine plus S-1. However, the tumor rapidly progressed and he deteriorated and died 123 days after admission. As immunohistochemical study showed positive staining for G-CSF in the tumor cell, we diagnosed the tumor producing G-CSF during autopsy. Anaplastic carcinoma of the pancreas producing G-CSF is very rare, with 10 cases, including ours, reported in the literature.

  20. Circulating macrophage colony-stimulating factor is not reduced in malignant osteopetrosis.

    PubMed

    Orchard, P J; Dahl, N; Aukerman, S L; Blazar, B R; Key, L L

    1992-01-01

    Malignant osteopetrosis is a disorder characterized by a deficiency in osteoclast number or function. In one animal model of osteopetrosis, the op/op mouse, macrophage colony-stimulating factor (M-CSF) is absent, and the administration of M-CSF corrects the defects. We evaluated the serum of 13 patients with malignant osteopetrosis by an M-CSF radioimmunoassay to determine if a quantitative M-CSF deficiency existed in these patients. All patients had M-CSF present in levels equal to or higher than control serum. In addition, serum from 6 osteopetrotic patients was tested in a bioassay to determine if the M-CSF present is biologically active, and in all cases there was demonstrable activity in these samples. We provide evidence that deficiency of circulating M-CSF is unlikely to be a major contributor to the etiologic basis for the majority of children with malignant osteopetrosis.

  1. Using Student-Centred Learning Environments to Stimulate Deep Approaches to Learning: Factors Encouraging or Discouraging Their Effectiveness

    ERIC Educational Resources Information Center

    Baeten, Marlies; Kyndt, Eva; Struyven, Katrien; Dochy, Filip

    2010-01-01

    This review outlines encouraging and discouraging factors in stimulating the adoption of deep approaches to learning in student-centred learning environments. Both encouraging and discouraging factors can be situated in the context of the learning environment, in students' perceptions of that context and in characteristics of the students…

  2. Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

    SciTech Connect

    Nakashima, S.; Suganuma, A.; Sato, M.; Tohmatsu, T.; Nozawa, Y. )

    1989-08-15

    Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When (3H) AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of (3H)AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of (3H)AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate).

  3. Stimulation of prostaglandin E/sub 2/ production by phorbol esters and epidermal growth factor in porcine thyroid cells

    SciTech Connect

    Kasai, K.; Hiraiwa, M.; Emoto, T.; Akimoto, K.; Takaoka, T.; Shimoda, S.I.

    1987-07-13

    Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E/sub 2/ production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E/sub 2/ production by the cells in dose related fashion. PMA stimulated prostaglandin E/sub 2/ production over fifty-fold with the dose of 10/sup -7/ M compared with control. EGF (10/sup -7/ M) also stimulated it about ten-fold. The ED/sub 50/ values of PMA and EGF were respectively around 1 x 10/sup -9/ M and 5 x 10/sup -10/ M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E/sub 2/ production from 1 to 24-h incubation. The release of radioactivity from (/sup 3/H)-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E/sub 2/ production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells. 36 references, 2 figures, 1 table.

  4. Bioengineered sequential growth factor delivery stimulates brain tissue regeneration after stroke.

    PubMed

    Wang, Yuanfei; Cooke, Michael J; Sachewsky, Nadia; Morshead, Cindi M; Shoichet, Molly S

    2013-11-28

    Stroke is a leading cause of disability with no effective regenerative treatment. One promising strategy for achieving tissue repair involves the stimulation of endogenous neural stem/progenitor cells through sequential delivery of epidermal growth factor (EGF) followed by erythropoietin (EPO). Yet currently available delivery strategies such as intracerebroventricular (ICV) infusion cause significant tissue damage. We designed a novel delivery system that circumvents the blood brain barrier and directly releases growth factors to the brain. Sequential release of the two growth factors is a key in eliciting tissue repair. To control release, we encapsulate pegylated EGF (EGF-PEG) in poly(lactic-co-glycolic acid) (PLGA) nanoparticles and EPO in biphasic microparticles comprised of a PLGA core and a poly(sebacic acid) coating. EGF-PEG and EPO polymeric particles are dispersed in a hyaluronan methylcellulose (HAMC) hydrogel which spatially confines the particles and attenuates the inflammatory response of brain tissue. Our composite-mediated, sequential delivery of EGF-PEG and EPO leads to tissue repair in a mouse stroke model and minimizes damage compared to ICV infusion.

  5. Free bone graft reconstruction of irradiated facial tissue: Experimental effects of basic fibroblast growth factor stimulation

    SciTech Connect

    Eppley, B.L.; Connolly, D.T.; Winkelmann, T.; Sadove, A.M.; Heuvelman, D.; Feder, J. )

    1991-07-01

    A study was undertaken to evaluate the potential utility of basic fibroblast growth factor in the induction of angiogenesis and osseous healing in bone previously exposed to high doses of irradiation. Thirty New Zealand rabbits were evaluated by introducing basic fibroblast growth factor into irradiated mandibular resection sites either prior to or simultaneous with reconstruction by corticocancellous autografts harvested from the ilium. The fate of the free bone grafts was then evaluated at 90 days postoperatively by microangiographic, histologic, and fluorochrome bone-labeling techniques. Sequestration, necrosis, and failure to heal to recipient osseous margins was observed both clinically and histologically in all nontreated irradiated graft sites as well as those receiving simultaneous angiogenic stimulation at the time of graft placement. No fluorescent activity was seen in these graft groups. In the recipient sites pretreated with basic fibroblast growth factor prior to placement of the graft, healing and reestablishment of mandibular contour occurred in nearly 50 percent of the animals. Active bone formation was evident at cortical margins adjacent to the recipient sites but was absent in the more central cancellous regions of the grafts.

  6. Human Granulocyte Macrophage Colony-Stimulating Factor Enhances Antibiotic Susceptibility of Pseudomonas aeruginosa Persister Cells

    PubMed Central

    Choudhary, Geetika S.; Yao, Xiangyu; Wang, Jing; Peng, Bo; Bader, Rebecca A.; Ren, Dacheng

    2015-01-01

    Bacterial persister cells are highly tolerant to antibiotics and cause chronic infections. However, little is known about the interaction between host immune systems with this subpopulation of metabolically inactive cells, and direct effects of host immune factors (in the absence of immune cells) on persister cells have not been studied. Here we report that human granulocyte macrophage-colony stimulating factor (GM-CSF) can sensitize the persister cells of Pseudomonas aeruginosa PAO1 and PDO300 to multiple antibiotics including ciprofloxacin, tobramycin, tetracycline, and gentamicin. GM-CSF also sensitized the biofilm cells of P. aeruginosa PAO1 and PDO300 to tobramycin in the presence of biofilm matrix degrading enzymes. The DNA microarray and qPCR results indicated that GM-CSF induced the genes for flagellar motility and pyocin production in the persister cells, but not the normal cells of P. aeruginosa PAO1. Consistently, the supernatants from GM-CSF treated P. aeruginosa PAO1 persister cell suspensions were found cidal to the pyocin sensitive strain P. aeruginosa PAK. Collectively, these findings suggest that host immune factors and bacterial persisters may directly interact, leading to enhanced susceptibility of persister cells to antibiotics. PMID:26616387

  7. Adjunctive granulocyte colony-stimulating factor for treatment of septic shock due to melioidosis.

    PubMed

    Cheng, Allen C; Stephens, Dianne P; Anstey, Nicholas M; Currie, Bart J

    2004-01-01

    Melioidosis, caused by the intracellular pathogen Burkholderia pseudomallei, is endemic in northern Australia and Southeast Asia. Risk factors for this infection have also been associated with functional neutrophil defects. Because of this, granulocyte colony-stimulating factor (G-CSF) was adopted for use in patients with septic shock due to melioidosis in December 1998. We compared the mortality rates from before and after the introduction of G-CSF therapy at the Royal Darwin Hospital (Darwin, Australia) during the period of 1989-2002. The mortality rate decreased from 95% to 10% after the introduction of G-CSF. Risk factors, the duration of illness before presentation, and the severity of illness were similar in both groups. A smaller decrease in mortality among patients in the intensive care unit who did not have melioidosis was observed, suggesting that other changes in management did not account for the magnitude of the benefit seen. We conclude that G-CSF may have contributed to the reduction in the mortality rate among patients with septic shock due to melioidosis.

  8. Factors to predict positive results of gonadotropin releasing hormone stimulation test in girls with suspected precocious puberty.

    PubMed

    Nam, Hyo-Kyoung; Rhie, Young Jun; Son, Chang Sung; Park, Sang Hee; Lee, Kee-Hyoung

    2012-02-01

    Sometimes, the clinical findings and the results of the gonadotropin-releasing hormone (GnRH) stimulation test are inconsistent in girls with early breast development and bone age advancement. We aimed to investigate the factors predicting positive results of the GnRH stimulation test in girls with suspected central precocious puberty (CPP). We reviewed the records of 574 girls who developed breast budding before the age of 8 yr and underwent the GnRH stimulation test under the age of 9 yr. Positive results of the GnRH stimulated peak luteinizing hormone (LH) level were defined as 5 IU/L and over. Girls with the initial positive results (n = 375) showed accelerated growth, advanced bone age and higher serum basal LH, follicle-stimulating hormone, and estradiol levels, compared to those with the initial negative results (n = 199). Girls with the follow-up positive results (n = 64) showed accelerated growth and advanced bone age, compared to those with the follow-up negative results. In the binary logistic regression, the growth velocity ratio was the most significant predictive factor of positive results. We suggest that the rapid growth velocity is the most useful predictive factor for positive results in the GnRH stimulation test in girls with suspected precocious puberty.

  9. Cloning and expression of porcine Colony Stimulating Factor-1 (CSF-1) and Colony Stimulating Factor-1 Receptor (CSF-1R) and analysis of the species specificity of stimulation by CSF-1 and Interleukin 34

    PubMed Central

    Gow, Deborah J.; Garceau, Valerie; Kapetanovic, Ronan; Sester, David P.; Fici, Greg J.; Shelly, John A.; Wilson, Thomas L.; Hume, David A.

    2012-01-01

    Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists. To enable such studies, we cloned and expressed active pig CSF-1. To provide a bioassay, pig CSF-1R was expressed in the factor-dependent Ba/F3 cell line. On this transfected cell line, recombinant porcine CSF-1 and human CSF-1 had identical activity. Mouse CSF-1 does not interact with the human CSF-1 receptor but was active on pig. By contrast, porcine CSF-1 was active on mouse, human, cat and dog cells. IL-34 was previously shown to be species-specific, with mouse and human proteins demonstrating limited cross-species activity. The pig CSF-1R was equally responsive to both mouse and human IL-34. Based upon the published crystal structures of CSF-1/CSF-1R and IL34/CSF-1R complexes, we discuss the molecular basis for the species specificity. PMID:22974529

  10. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells.

    PubMed Central

    Chen, J K; Hoshi, H; McKeehan, W L

    1987-01-01

    Myo-intimal proteoglycan metabolism is thought to be important in blood vessel homeostasis, blood clotting, atherogenesis, and atherosclerosis. Human platelet-derived transforming growth factor type beta (TGF-beta) specifically stimulated synthesis of at least two types of chondroitin sulfate proteoglycans in nonproliferating human adult arterial smooth muscle cells in culture. Stimulation of smooth muscle cell proteoglycan synthesis by smooth muscle cell growth promoters (epidermal growth factor, platelet-derived growth factor, and heparin-binding growth factors) was less than 20% of that elicited by TGF-beta. TGF-beta neither significantly stimulated proliferation of quiescent smooth muscle cells nor inhibited proliferating cells. The extent of TGF-beta stimulation of smooth muscle cell proteoglycan synthesis was similar in both nonproliferating and growth-stimulated cells. TGF-beta, which is a reversible inhibitor of endothelial cell proliferation, had no comparable effect on endothelial cell proteoglycan synthesis. These results are consistent with the hypothesis that TGF-beta is a cell-type-specific regulator of proteoglycan synthesis in human blood vessels and may contribute to the myo-intimal accumulation of proteoglycan in atherosclerotic lesions. Images PMID:3474655

  11. Synergy of interleukin 1 and granulocyte colony-stimulating factor: in vivo stimulation of stem-cell recovery and hematopoietic regeneration following 5-fluorouracil treatment of mice

    SciTech Connect

    Moore, M.A.S.; Warren, D.J.

    1987-10-01

    The human bladder carcinoma cell line 5637 produces hematopoietic growth factors (granulocyte and granulocyte/macrophage colony-stimulating factors (G-CSF and GM-CSF)) and hemopoietin 1, which synergizes with CSFs to stimulate colony formation by primitive hematopoietic stem cells in 5-fluorouracil-treated mouse bone marrow. Molecular and functional properties of hemopoietin 1 identified it as identical to interleukin 1..cap alpha.. (IL-1..cap alpha..). When bone marrow cells from 5-fluorouracil-treated mice were cultured in suspension for 7 days with recombinant human IL-1..cap alpha.. and/or G-CSF, it was found that the two factors synergized to enhance recovery of myelopoietic cells and colony-forming cells of both high and low proliferative potential. G-CSF alone did not sustain these populations, but the combination had greater-than-additive stimulating capacity. In vivo, 5-fluorouracil (150 mg/kg) produced profound myelosuppression and delayed neutrophil regeneration for up to 2 weeks in C3H/HeJ mice. Daily administration of recombinant human G-CSF or human IL-1..cap alpha.. accelerated recovery of stem cells, progenitor cells, and blood neutrophils by up to 4 days in 5-fluorouracil-treated C3H/HeJ and B6D2F/sub 1/ mice. The combination of IL-1..cap alpha.. and G-CSF acted synergistically, reducing neutropenia and accelerating recovery of normal neutrophil numbers by up to 7 days. These results indicate the possible therapeutic potential of combination therapy with IL-1 and hematopoietic growth factors such as G-CSF in the treatment of chemotherapy- or radiation-induced myelosuppression.

  12. Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit.

    PubMed

    Jubinsky, P T; Laurie, A S; Nathan, D G; Yetz-Aldepe, J; Sieff, C A

    1994-12-15

    To determine the expression and function of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha chain (GMR alpha) during hematopoiesis and on leukemic cells, monoclonal antibodies were raised by immunizing mice with cells expressing high levels of human GMR alpha. A pool of five antibodies isolated from three different mice was used to characterize GMR alpha. This antibody pool (anti-GMR alpha) immunoprecipitated a protein with the expected molecular weight of GMR alpha from COS cells transiently transfected with the GMR alpha gene. In factor-dependent cells, GMR alpha existed as a phosphoprotein. However, its phosphorylation was not stimulated by the presence of GM-CSF. Anti-GMR alpha inhibited the GM-CSF-dependent growth of cell lines and normal bone marrow cells and inhibited the binding of iodinated GM-CSF to its receptor. Cell surface expression of GMR alpha was examined using anti-GMR alpha and flow cytometry. GMR alpha was readily detectable on both blood monocytes and neutrophils. In adherence-depleted normal bone marrow, two separate populations expressed GMR alpha. The most positive cells were predominantly macrophages, whereas the cells that expressed less GMR alpha were largely myelocytes and metamyelocytes. A small population of lin-CD34+ or CD34+CD38- cells also expressed GMR alpha, but they were not capable of significant growth in colony-forming assays. In contrast, the majority of lin-CD34+ and CD34+CD38- cells were GMR alpha-, yet they produced large numbers of myeloid and erythroid colonies in the same assay. Malignant cells from patients with leukemia were also tested for GMR alpha expression. All of the myeloid leukemias and only rare lymphoid leukemias surveyed tested positive for GMR alpha. These results show that anti-GMR alpha is useful for the functional characterization of the GMR alpha and for the detection of myeloid leukemia and that GMR alpha is expressed on certain lineages throughout hematopoietic

  13. Arecoline-stimulated connective tissue growth factor production in human buccal mucosal fibroblasts: Modulation by curcumin.

    PubMed

    Deng, Yi-Ting; Chen, Hsin-Ming; Cheng, Shih-Jung; Chiang, Chun-Pin; Kuo, Mark Yen-Ping

    2009-09-01

    Connective tissue growth factor (CTGF) is associated with the onset and progression of fibrosis in many human tissues. Areca nut (AN) chewing is the most important etiological factor in the pathogenesis of oral submucous fibrosis (OSF). We immunohistochemically examined the expression of CTGF protein in 20 cases of OSF and found positive CTGF staining in fibroblasts and endothelial cells in all cases. Western blot analysis showed that arecoline, a main alkaloid found in AN, stimulated CTGF synthesis in a dose- and time-dependent manner in buccal mucosal fibroblasts. Constitutive overexpression of CTGF during AN chewing may enhance the fibrotic activity in OSF and play a role in the pathogenesis of OSF. Pretreatment with NF-kappaB inhibitor Bay 11-7082, JNK inhibitor SP600125, p38 MAPK inhibitor SB203580 and antioxidant N-acetyl-l-cysteine, but not ERK inhibitor PD98059, significantly reduced arecoline-induced CTGF synthesis. Furthermore, curcumin completely inhibited arecoline-induced CTGF synthesis and the inhibition is dose-dependent. These results indicated that arecoline-induced CTGF synthesis was mediated by ROS, NF-kappaB, JNK, P38 MAPK pathways and curcumin could be a useful agent in controlling OSF.

  14. Factor Analysis of Low-Frequency Repetitive Transcranial Magnetic Stimulation to the Temporoparietal Junction for Tinnitus

    PubMed Central

    Li, Bei; Wang, Meiye; Li, Ming; Yin, Shankai

    2016-01-01

    Objectives. We investigated factors that contribute to suppression of tinnitus after repetitive transcranial magnetic stimulation (rTMS). Methods. A total of 289 patients with tinnitus underwent active 1 Hz rTMS in the left temporoparietal region. A visual analog scale (VAS) was used to assess tinnitus loudness. All participants were interviewed regarding age, gender, tinnitus duration, laterality and pitch, audiometric parameters, sleep, and so forth. The resting motor thresholds (RMTs) were measured in all patients and 30 age- and gender-matched volunteers. Results. With respect to different factors that contribute to tinnitus suppression, we found improvement in the following domains: shorter duration, normal hearing (OR: 3.25, 95%CI: 2.01–5.27, p = 0.001), and without sleep disturbance (OR: 2.51, 95%CI: 1.56–4.1, p = 0.005) adjusted for age and gender. The patients with tinnitus lasting less than 1 year were more likely to show suppression of tinnitus (OR: 2.77, 95%CI: 1.48–5.19, p = 0.002) compared to those with tinnitus lasting more than 5 years. Tinnitus patients had significantly lower RMTs compared with healthy volunteers. Conclusion. Active low-frequency rTMS results in a significant reduction in the loudness of tinnitus. Significant tinnitus suppression was shown in subjects with shorter tinnitus duration, with normal hearing, and without sleep disturbance. PMID:27847647

  15. Characterisation of a Novel Fc Conjugate of Macrophage Colony-stimulating Factor

    PubMed Central

    Gow, Deborah J; Sauter, Kristin A; Pridans, Clare; Moffat, Lindsey; Sehgal, Anuj; Stutchfield, Ben M; Raza, Sobia; Beard, Philippa M; Tsai, Yi Ting; Bainbridge, Graeme; Boner, Pamela L; Fici, Greg; Garcia-Tapia, David; Martin, Roger A; Oliphant, Theodore; Shelly, John A; Tiwari, Raksha; Wilson, Thomas L; Smith, Lee B; Mabbott, Neil A; Hume, David A

    2014-01-01

    We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule. PMID:24962162

  16. Colony-Stimulating Factor-1 Promotes Kidney Growth and Repair via Alteration of Macrophage Responses

    PubMed Central

    Alikhan, Maliha A.; Jones, Christina V.; Williams, Timothy M.; Beckhouse, Anthony G.; Fletcher, Anne L.; Kett, Michelle M.; Sakkal, Samy; Samuel, Chrishan S.; Ramsay, Robert G.; Deane, James A.; Wells, Christine A.; Little, Melissa H.; Hume, David A.; Ricardo, Sharon D.

    2011-01-01

    Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury. PMID:21762674

  17. Monocyte activation following systemic administration of granulocyte-macrophage colony-stimulating factor.

    PubMed

    Chachoua, A; Oratz, R; Hoogmoed, R; Caron, D; Peace, D; Liebes, L; Blum, R H; Vilcek, J

    1994-04-01

    Twenty-four patients with solid malignancies were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) on a Phase 1b trial. The objective of the study was to evaluate the effects of GM-CSF on peripheral blood monocyte activation. GM-CSF was administered by subcutaneous injection daily for 14 days. Immune parameters measured were monocyte cytotoxicity against the human colon carcinoma (HT29) cell line, serum tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and in vitro TNF-alpha and IL-1 beta induction. All patients were evaluable for toxicity. Fifteen patients were evaluable for immunologic response. Treatment with GM-CSF led to a statistically significant enhancement in direct monocyte cytotoxicity against HT29 cells. There was no increase in serum TNF-alpha or IL-1 beta and no consistent in vitro induction of TNF-alpha or IL-1 beta from monocytes posttreatment. Treatment was well tolerated overall. We conclude that treatment with GM-CSF can lead to enhanced monocyte cytotoxicity. Further studies are in progress to evaluate the effect of GM-CSF on other parameters of monocyte functions.

  18. Characterisation of a novel Fc conjugate of macrophage colony-stimulating factor.

    PubMed

    Gow, Deborah J; Sauter, Kristin A; Pridans, Clare; Moffat, Lindsey; Sehgal, Anuj; Stutchfield, Ben M; Raza, Sobia; Beard, Philippa M; Tsai, Yi Ting; Bainbridge, Graeme; Boner, Pamela L; Fici, Greg; Garcia-Tapia, David; Martin, Roger A; Oliphant, Theodore; Shelly, John A; Tiwari, Raksha; Wilson, Thomas L; Smith, Lee B; Mabbott, Neil A; Hume, David A

    2014-09-01

    We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule.

  19. Colony-stimulating factor-1 promotes kidney growth and repair via alteration of macrophage responses.

    PubMed

    Alikhan, Maliha A; Jones, Christina V; Williams, Timothy M; Beckhouse, Anthony G; Fletcher, Anne L; Kett, Michelle M; Sakkal, Samy; Samuel, Chrishan S; Ramsay, Robert G; Deane, James A; Wells, Christine A; Little, Melissa H; Hume, David A; Ricardo, Sharon D

    2011-09-01

    Colony-stimulating factor (CSF)-1 controls the survival, proliferation, and differentiation of macrophages, which are recognized as scavengers and agents of the innate and the acquired immune systems. Because of their plasticity, macrophages are endowed with many other essential roles during development and tissue homeostasis. We present evidence that CSF-1 plays an important trophic role in postnatal organ growth and kidney repair. Notably, the injection of CSF-1 postnatally enhanced kidney weight and volume and was associated with increased numbers of tissue macrophages. Moreover, CSF-1 promotes postnatal renal repair in mice after ischemia-reperfusion injury by recruiting and influencing macrophages toward a reparative state. CSF-1 treatment rapidly accelerated renal repair with tubular epithelial cell replacement, attenuation of interstitial fibrosis, and functional recovery. Analysis of macrophages from CSF-1-treated kidneys showed increased expression of insulin-like growth factor-1 and anti-inflammatory genes that are known CSF-1 targets. Taken together, these data suggest that CSF-1 is important in kidney growth and the promotion of endogenous repair and resolution of inflammatory injury.

  20. Simulation of epiretinal prostheses - Evaluation of geometrical factors affecting stimulation thresholds

    PubMed Central

    2011-01-01

    Background An accurate understanding of the electrical interaction between retinal prostheses and retinal tissue is important to design effective devices. Previous studies have used modelling approaches to simulate electric fields generated by epiretinal prostheses in saline and to simulate retinal ganglion cell (RGC) activation using passive or/and active biophysical models of the retina. These models have limited scope for studying an implanted human retinal prosthesis as they often do not account for real geometry and composition of the prosthesis-retina interface. This interface consists of real dimensions and location of stimulation and ground electrodes that are separated by the retinal tissue and surrounded by physiological fluids. Methods In this study, we combined the prosthesis-retina interface elements into a framework to evaluate the geometrical factors affecting stimulation thresholds for epiretinal prostheses used in clinical human trials, as described by Balthasar et al. in their Investigative Ophthalmology and Visual Science (IOVS) paper published in 2008 using the Argus I epiretinal implants. Finite element method (FEM) based computations were used to estimate threshold currents based on a threshold criterion employing a passive electric model of the retina. Results Threshold currents and impedances were estimated for different electrode-retina distances. The profiles and the values for thresholds and impedances obtained from our simulation framework are within the range of measured values in the only elaborate published clinical trial until now using Argus I epiretinal implants. An estimation of resolution for the electrodes used in these trials was provided. Our results reiterate the importance of close proximity between electrodes and retina for safe and efficient retinal stimulation. Conclusions The validation of our simulation framework being relevant for epiretinal prosthesis research is derived from the good agreement of the computed trends

  1. Macrophage colony-stimulating factor induces prolactin expression in rat pituitary gland.

    PubMed

    Hoshino, Satoya; Kurotani, Reiko; Miyano, Yuki; Sakahara, Satoshi; Koike, Kanako; Maruyama, Minoru; Ishikawa, Fumio; Sakatai, Ichiro; Abe, Hiroyuki; Sakai, Takafumi

    2014-06-01

    We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.

  2. Neuropeptide W stimulates adrenocorticotrophic hormone release via corticotrophin-releasing factor but not via arginine vasopressin.

    PubMed

    Yogo, Kosuke; Oki, Yutaka; Iino, Kazumi; Yamashita, Miho; Shibata, Shoko; Hayashi, Chiga; Sasaki, Shigekazu; Suenaga, Toshiko; Nakahara, Daiichiro; Nakamura, Hirotoshi

    2012-01-01

    Neuropeptide W (NPW) was isolated as an endogenous ligand for NPBWR1, an orphan G protein-coupled receptor localized in the rat brain, including the paraventricular nucleus. It has been reported that central administration of NPW stimulates corticosterone secretion in rats. We hypothesized that NPW activates the hypothalamic-pituitary-adrenal (HPA) axis via corticotrophin-releasing factor (CRF) and/or arginine vasopressin (AVP). NPW at 1 pM to 10 nM did not affect basal or ACTH-induced corticosterone release from dispersed rat adrenocortical cells, or basal and CRF-induced ACTH release from dispersed rat anterior pituitary cells. In conscious and unrestrained male rats, intravenous administration of 2.5 and 25 nmol NPW did not affect plasma ACTH levels. However, intracerebroventricular (icv) administration of 2.5 and 5.0 nmol NPW increased plasma ACTH levels in a dose-dependent manner at 15 min after stimulation (5.0 vs. 2.5 nmol NPW vs. vehicle: 1802 ± 349 vs. 1170 ± 204 vs. 151 ± 28 pg/mL, respectively, mean ± SEM). Pretreatment with astressin, a CRF receptor antagonist, inhibited the increase in plasma ACTH levels induced by icv administration of 2.5 nmol NPW at 15 min (453 ± 176 vs. 1532 ± 343 pg/mL, p<0.05) and at 30 min (564 ± 147 vs. 1214 ± 139 pg/mL, p<0.05) versus pretreatment with vehicle alone. However, pretreatment with [1-(β-mercapto-β, β-cyclopentamethylenepropionic acid), 2-(Ο-methyl)tyrosine]-arg-vasopressin, a V1a/V1b receptor antagonist, did not affect icv NPW-induced ACTH release at any time (p>0.05). In conclusion, we suggest that central NPW activates the HPA axis by activating hypothalamic CRF but not AVP.

  3. Granulocyte colony-stimulating factor in repeated IVF failure, a randomized trial.

    PubMed

    Aleyasin, Ashraf; Abediasl, Zhila; Nazari, Atefeh; Sheikh, Mahdi

    2016-06-01

    Recent studies have revealed key roles for granulocyte colony-stimulating factor (GCSF) in embryo implantation process and maintenance of pregnancy, and some studies showed promising results by using local intrauterine infusion of GCSF in patients undergoing in vitro fertilization (IVF). This multicenter, randomized, controlled trial included 112 infertile women with repeated IVF failure to evaluate the efficacy of systemic single-dose subcutaneous GCSF administration on IVF success in these women. In this study, the Long Protocol of ovarian stimulation was used for all participants. Sealed, numbered envelopes assigned 56 patients to receive subcutaneous 300 µg GCSF before implantation and 56 in the control group. The implantation (number of gestational sacs on the total number of transferred embryos), chemical pregnancy (positive serum β-HCG), and clinical pregnancy (gestational sac and fetal heart) rates were compared between the two groups. This trial is registered at www.irct.ir (IRCT201503119568N11). The successful implantation (18% vs 7.2%, P=0.007), chemical pregnancy (44.6% vs 19.6%, P=0.005), and clinical pregnancy (37.5% vs 14.3%, P=0.005) rates were significantly higher in the intervention group than in the control group. After adjustment for participants' age, endometrial thickness, good-quality oocyte counts, number of transferred embryos, and anti-Mullerian hormone levels, GCSF treatment remained significantly associated with successful implantation (OR=2.63, 95% CI=1.09-6.96), having chemical pregnancy (OR= 2.74, 95% CI=1.11-7.38) and clinical pregnancy (OR=2.94, 95% CI=1.23-8.33). In conclusion, administration of single-dose systemic subcutaneous GCSF before implantation significantly increases the IVF success, implantation, and pregnancy rates in infertile women with repeated IVF failure.

  4. Immunostimulation using granulocyte- and granulocyte-macrophage colony stimulating factor in patients with severe sepsis and septic shock.

    PubMed

    Schefold, Joerg C

    2011-01-01

    Sepsis is associated with failure of multiple organs, including failure of the immune system. The resulting 'sepsis-associated immunosuppression' resembles a state of immunological anergy that is characterized by repeated 'infectious hits', prolonged multiple-organ failure, and death. As a consequence, adjunctive treatment approaches using measures of immunostimulation with colony-stimulating factors (CSFs) were tested in animal experiments and clinical trials. Herein, data from randomized clinical trials will be discussed in the context of a recently published meta-analysis investigating the effects of granulocyte- and granulocyte-macrophage colony-stimulating factor therapy in patients with severe sepsis and septic shock.

  5. Prognostic factors in the prediction of chronic wound healing by electrical stimulation.

    PubMed

    Cukjati, D; Robnik-Sikonja, M; Rebersek, S; Kononenko, I; Miklavcic, D

    2001-09-01

    The aim of the study is to determine the effects of wound, patient and treatment attributes on the wound healing rate and to propose a system for wound healing rate prediction. Predicting the wound healing rate from the initial wound, patient and treatment data collected in a database of 300 chronic wounds is not possible. After considering weekly follow-ups, it was determined that the best prognostic factors are weekly follow-ups of the wound healing process, which alone were found to predict accurately the wound healing rate after a minimum follow-up period of four weeks (at least five measurements of wound area). After combining the follow-ups with wound, patient and treatment attributes, the minimum follow-up period was reduced to two weeks (at least three measurements of wound area). After a follow-up period of two weeks, it was possible to predict the wound healing rate of an independent test set of chronic wounds with a relative squared error of 0.347, and after three weeks, with a relative squared error of 0.181 (using regression trees with linear equations in its leaves). Regression trees with a relative squared error close to 0 produce better prediction than with an error closer to 1. Results show that the type of treatment is just one of many prognostic factors. Arranged in order of decreasing prediction capability, prognostic factors are: wound size, patient's age, elapsed time from wound appearance to the beginning of the treatment, width-to-length ratio, location and type of treatment. The data collected support former findings that the biphasic- and direct-current stimulation contributes to faster healing of chronic wounds. The model of wound healing dynamics aids the prediction of chronic wound healing rate, and hence helps with the formulation of appropriate treatment decisions.

  6. Differential processing of colony-stimulating factor 1 precursors encoded by two human cDNAs.

    PubMed Central

    Rettenmier, C W; Roussel, M F

    1988-01-01

    The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo. Images PMID:3264877

  7. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    PubMed

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  8. The combination of stem cell factor and granulocyte-colony stimulating factor for chronic stroke treatment in aged animals

    PubMed Central

    2012-01-01

    Background Stroke occurs more frequently in the elderly population and presents the number one leading cause of persistent disability worldwide. Lack of effective treatment to enhance brain repair and improve functional restoration in chronic stroke, the recovery phase of stroke, is a challenging medical problem to be solved in stroke research. Our early study has revealed the therapeutic effects of stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF) on chronic stroke in young animals. However, whether this treatment is effective and safe to the aged population remains to be determined. Methods Cortical brain ischemia was produced in aged C57BL mice or aged spontaneously hypertensive rats. SCF+G-CSF or equal volume of vehicle solution was subcutaneously injected for 7 days beginning at 3–4 months after induction of cortical brain ischemia. Using the approaches of biochemistry assays, flow cytometry, pathology, and evaluation of functional outcome, several doses of SCF+G-CSF have been examined for their safety and efficiency on chronic stroke in aged animals. Results All tested doses did not show acute or chronic toxicity in the aged animals. Additionally, SCF+G-CSF treatment in chronic stroke of aged animals mobilized bone marrow stem cells and improved functional outcome in a dose-dependent manner. Conclusions SCF+G-CSF treatment is a safe and effective approach to chronic stroke in the aged condition. This study provides important information needed for developing a new therapeutic strategy to improve the health of older adults with chronic stroke. PMID:23254113

  9. Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL.

    PubMed

    Liu, Xiao-Yun; Gonzalez-Toledo, Maria E; Fagan, Austin; Duan, Wei-Ming; Liu, Yanying; Zhang, Siyuan; Li, Bin; Piao, Chun-Shu; Nelson, Lila; Zhao, Li-Ru

    2015-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5 days and repeated 4 times with 1-4 month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

  10. Adiponectin stimulates Wnt inhibitory factor-1 expression through epigenetic regulations involving the transcription factor specificity protein 1.

    PubMed

    Liu, Jing; Lam, Janice B B; Chow, Kim H M; Xu, Aimin; Lam, Karen S L; Moon, Randall T; Wang, Yu

    2008-11-01

    Adiponectin (ADN) is an adipokine possessing growth inhibitory activities against various types of cancer cells. Our previous results demonstrated that ADN could impede Wnt/beta-catenin-signaling pathways in MDA-MB-231 human breast carcinoma cells [Wang,Y. et al. (2006) Adiponectin modulates the glycogen synthase kinase-3 beta/beta-catenin signaling pathway and attenuates mammary tumorigenesis of MDA-MB-231 cells in nude mice. Cancer Res., 66, 11462-11470]. Here, we extended our studies to elucidate the effects of ADN on regulating the expressions of Wnt inhibitory factor-1 (WIF1), a Wnt antagonist frequently silenced in human breast tumors. Our results showed that ADN time dependently stimulated WIF1 gene and protein expressions in MDA-MB-231 cells. Overexpression of WIF1 exerted similar inhibitory effects to those of ADN on cell proliferations, nuclear beta-catenin activities, cyclin D1 expressions and serum-induced phosphorylations of Akt and glycogen synthase kinase-3 beta. Blockage of WIF1 activities significantly attenuated the suppressive effects of ADN on MDA-MB-231 cell growth. Furthermore, our in vivo studies showed that both supplementation of recombinant ADN and adenovirus-mediated overexpression of this adipokine substantially enhanced WIF1 expressions in MDA-MB-231 tumors implanted in nude mice. More interestingly, we found that ADN could alleviate methylation of CpG islands located within the proximal promoter region of WIF1, possibly involving the specificity protein 1 (Sp1) transcription factor and its downstream target DNA methyltransferase 1 (DNMT1). Upon ADN treatment, the protein levels of both Sp1 and DNMT1 were significantly decreased. Using silencing RNA approaches, we confirmed that downregulation of Sp1 resulted in an increased expression of WIF1 and decreased methylation of WIF1 promoter. Taken together, these data suggest that ADN might elicit its antitumor activities at least partially through promoting WIF1 expressions.

  11. Cloning and expression of feline colony stimulating factor receptor (CSF-1R) and analysis of the species specificity of stimulation by colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34)

    PubMed Central

    Gow, Deborah J.; Garceau, Valerie; Pridans, Clare; Gow, Adam G.; Simpson, Kerry E.; Gunn-Moore, Danielle; Hume, David A.

    2013-01-01

    Colony stimulating factor (CSF-1) and its receptor, CSF-1R, have been previously well studied in humans and rodents to dissect the role they play in development of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, IL-34 has been described in several species. In this study, we have cloned and expressed the feline CSF-1R and examined the responsiveness to CSF-1 and IL-34 from a range of species. The results indicate that pig and human CSF-1 and human IL-34 are equally effective in cats, where both mouse CSF-1 and IL-34 are significantly less active. Recombinant human CSF-1 can be used to generate populations of feline bone marrow and monocyte derived macrophages that can be used to further dissect macrophage-specific gene expression in this species, and to compare it to data derived from mouse, human and pig. These results set the scene for therapeutic use of CSF-1 and IL-34 in cats. PMID:23260168

  12. Structure of the SH3 domain of human osteoclast-stimulating factor at atomic resolution

    SciTech Connect

    Chen, Liqing Wang, Yujun; Wells, David; Toh, Diana; Harold, Hunt; Zhou, Jing; DiGiammarino, Enrico; Meehan, Edward J.

    2006-09-01

    The crystal structure of the SH3 domain of human osteoclast-stimulating factor has been determined and refined to the ultrahigh resolution of 1.07 Å. The structure at atomic resolution provides an accurate framework for structure-based design of its inhibitors. Osteoclast-stimulating factor (OSF) is an intracellular signaling protein, produced by osteoclasts themselves, that enhances osteoclast formation and bone resorption. It is thought to act via an Src-related signaling pathway and contains SH3 and ankyrin-repeat domains which are involved in protein–protein interactions. As part of a structure-based anti-bone-loss drug-design program, the atomic resolution X-ray structure of the recombinant human OSF SH3 domain (hOSF-SH3) has been determined. The domain, residues 12–72, yielded crystals that diffracted to the ultrahigh resolution of 1.07 Å. The overall structure shows a characteristic SH3 fold consisting of two perpendicular β-sheets that form a β-barrel. Structure-based sequence alignment reveals that the putative proline-rich peptide-binding site of hOSF-SH3 consists of (i) residues that are highly conserved in the SH3-domain family, including residues Tyr21, Phe23, Trp49, Pro62, Asn64 and Tyr65, and (ii) residues that are less conserved and/or even specific to hOSF, including Thr22, Arg26, Thr27, Glu30, Asp46, Thr47, Asn48 and Leu60, which might be key to designing specific inhibitors for hOSF to fight osteoporosis and related bone-loss diseases. There are a total of 13 well defined water molecules forming hydrogen bonds with the above residues in and around the peptide-binding pocket. Some of those water molecules might be important for drug-design approaches. The hOSF-SH3 structure at atomic resolution provides an accurate framework for structure-based design of its inhibitors.

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

    PubMed Central

    Vanz, Ana LS; Renard, Gaby; Palma, Mario S; Chies, Jocelei M; Dalmora, Sérgio L; Basso, Luiz A; Santos, Diógenes S

    2008-01-01

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

  14. A pre-formed Pyrogenic Factor Released by Lipopolysaccharide Stimulated Macrophages

    PubMed Central

    Zampronio, A. R.; Melo, M. C. C.; Silva, C. A. A.; Pelá, I. R.; Hopkins, S. J.

    1994-01-01

    The aim of this study was to investigate the pyrogenic activity of factor(s) released by rat peritoneal macrophages following a brief stimulation with LPS. The effect of this factor on the number of circulating leukocytes and serum Fe, Cu and Zn levels, was also evaluated. The possibility that the content of interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF) in the supernatant could explain the observations was investigated. Supernatant produced over a period of 1 h by peritoneal macrophages, following a 30 min incubation with LPS at 37°C, was ultrafiltered through a 10 000 MW cut-off Amicon membrane, sterilized, and concentrated 2.5, 5, 10 and 20 times. The intravenous (i.v.) injection of this supernatant induced a concentration-dependent fever in rats with a maximal response at 2 h. The pyrogenic activity was produced by macrophages elicited with thioglycollate and by resident cells. The supernatants also induced neutrophilia and reduction in Fe and Zn 6 h after the injection. Absence of activity in boiled supernatants, or supernatants from macrophages incubated at 4°C with LPS, indicates that LPS was not responsible for the activity. In vitro treatment with indomethacin (Indo), dexamethasone (Dex), or cycloheximide (Chx) did not modify the release of pyrogenic activity into the supernatant or its effects on the reduction in serum metal levels. Although Chx abolished the production of mediator(s) inducing neutrophilia, and Dex reduced the induction of IL-1β, TNF and IL-6, injection of the highest concentration of these cytokines detected in the supernatants did not induce fever. In vivo treatment with Dex, but not Indo, abolished the fever induced by the supernatant. These results suggest that macrophages contain pre-formed pyrogenic mediator(s), not related to IL-1β, IL-6 or TNF, that acts indirectly and independently of prostaglandtn. It also seems likely that the pyrogenic activity is related to the factor responsible for the reduction of serum Fe

  15. Biphasic Stimulation of Translational Activity Correlates with Induction of Translation Elongation Factor 1 Subunit [alpha] upon Wounding in Potato Tubers.

    PubMed Central

    Morelli, J. K.; Shewmaker, C. K.; Vayda, M. E.

    1994-01-01

    Potato (Solanum tuberosum) tubers exhibit an increase in translational activity in response to mechanical wounding. The response is biphasic, with an initial stimulation apparent within the first 2 h after wounding and a second increase occurring 12 to 24 h after wounding. Increased activity is apparent by measurement of protein synthesis both in vivo and in vitro using a cell-free extract. Accumulation of the translational elongation factor 1 subunit [alpha] (EF-1[alpha]) parallels translational activity. Changes in the steady-state level of EF-1[alpha] mRNA, and expression of a chimeric EF-1[alpha] promoter/[beta]-glucuronidase construct in transgenic potato tubers, indicate that the gene encoding EF-1[alpha] is transcribed during both periods of translational stimulation. These results indicate that stimulation of translational activity is coordinated with increased expression and accumulation of translation factors. PMID:12232374

  16. Immunocytochemical localization of latent transforming growth factor-beta1 activation by stimulated macrophages

    NASA Technical Reports Server (NTRS)

    Chong, H.; Vodovotz, Y.; Cox, G. W.; Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1999-01-01

    Transforming growth factor-beta1 (TGF-beta) is secreted in a latent form consisting of mature TGF-beta noncovalently associated with its amino-terminal propeptide, which is called latency associated peptide (LAP). Biological activity depends upon the release of TGF-beta from the latent complex following extracellular activation, which appears to be the key regulatory mechanism controlling TGF-beta action. We have identified two events associated with latent TGF-beta (LTGF-beta) activation in vivo: increased immunoreactivity of certain antibodies that specifically detect TGF-beta concomitant with decreased immunoreactivity of antibodies to LAP. Macrophages stimulated in vitro with interferon-gamma and lipopolysaccharide reportedly activate LTGF-beta via cell membrane-bound protease activity. We show through dual immunostaining of paraformaldehyde-fixed macrophages that such physiological TGF-beta activation is accompanied by a loss of LAP immunoreactivity with concomitant revelation of TGF-beta epitopes. The induction of TGF-beta immunoreactivity colocalized with immunoreactive betaglycan/RIII in activated macrophages, suggesting that LTGF-beta activation occurs on the cell surface. Confocal microscopy of metabolically active macrophages incubated with antibodies to TGF-beta and betaglycan/RIII prior to fixation supported the localization of activation to the cell surface. The ability to specifically detect and localize LTGF-beta activation provides an important tool for studies of its regulation.

  17. Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor by Bacillus subtilis SCK6

    PubMed Central

    Bashir, Shaista; Sadaf, Saima; Ahmad, Sajjad; Akhtar, Muhammad Waheed

    2015-01-01

    This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer. PMID:26881203

  18. Electrical stimulation drives chondrogenesis of mesenchymal stem cells in the absence of exogenous growth factors

    PubMed Central

    Kwon, Hyuck Joon; Lee, Gyu Seok; Chun, Honggu

    2016-01-01

    Electrical stimulation (ES) is known to guide the development and regeneration of many tissues. However, although preclinical and clinical studies have demonstrated superior effects of ES on cartilage repair, the effects of ES on chondrogenesis remain elusive. Since mesenchyme stem cells (MSCs) have high therapeutic potential for cartilage regeneration, we investigated the actions of ES during chondrogenesis of MSCs. Herein, we demonstrate for the first time that ES enhances expression levels of chondrogenic markers, such as type II collagen, aggrecan, and Sox9, and decreases type I collagen levels, thereby inducing differentiation of MSCs into hyaline chondrogenic cells without the addition of exogenous growth factors. ES also induced MSC condensation and subsequent chondrogenesis by driving Ca2+/ATP oscillations, which are known to be essential for prechondrogenic condensation. In subsequent experiments, the effects of ES on ATP oscillations and chondrogenesis were dependent on extracellular ATP signaling via P2X4 receptors, and ES induced significant increases in TGF-β1 and BMP2 expression. However, the inhibition of TGF-β signaling blocked ES-driven condensation, whereas the inhibition of BMP signaling did not, indicating that TGF-β signaling but not BMP signaling mediates ES-driven condensation. These findings may contribute to the development of electrotherapeutic strategies for cartilage repair using MSCs. PMID:28004813

  19. 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.

  20. 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.

  1. NUTRITIONAL FACTORS STIMULATING THE FORMATION OF LYSINE DECARBOXYLASE IN ESCHERICHIA COLI

    PubMed Central

    Maretzki, Andrew; Mallette, M. F.

    1962-01-01

    Maretzki, Andrew (Pennsylvania State University, University Park) and M. F. Mallette. Nutritional factors stimulating the formation of lysine decarboxylase in Escherichia coli. J. Bacteriol. 83:720–726. 1962 — Inclusion of complex nitrogen sources in the induction medium was shown to be necessary for the synthesis of appreciable amounts of l-lysine decarboxylase by Escherichia coli B. Hy-case, a commercial acid hydrolyzate of casein, was especially effective in enzyme production, which was assayed manometrically after lysis of the bacteria from without by bacteriophage. Partial fractionation of the Hy-case, identification of the free amino acids, and addition of these amino acids to test media revealed stimulatory effects by methionine, threonine, proline, leucine, and tyrosine. A full complement of amino acids did not match the enzyme levels reached in the presence of Hy-case. Certain peptide fractions obtained from this mixture supplemented the effects of the amino acids in such a way as to suggest direct incorporation of peptide rather than transport or protective roles. Added purines, pyrimidines, iron, and water-soluble vitamins were without effect. Neither carbohydrates nor phosphorylated materials could be detected in the stimulatory fractions. PMID:14469751

  2. Granulocyte colony-stimulating factor increases the platelet volume in peripheral stem cell apheresis donors.

    PubMed

    Ihara, Akihiro; Matsui, Keiko; Minami, Ryouta; Uchida, Shuzou; Ueda, Shuji; Nishiura, Tetsuo

    2008-01-01

    We investigated the short-term influence of granulocyte colony-stimulating factor (G-CSF) administration on platelet counts and platelet indices in 12 donors (8 males and 4 females; median age 34 years, range 16-49) for peripheral stem cell transplantation using an automated blood cell analyzer. On day 3 (D3) compared with D0, 11 donors with normal laboratory and physical findings showed increases in platelet indices (chi(2) = 12.0, p = 0.0025). Furthermore, mean platelet volume (MPV) was significantly increased (p = 0.04). Also, platelet count decreased, and platelet distribution width and platelet-large cell ratio were increased, but these were not significant. On the contrary, 1 donor with abnormal laboratory findings who had large platelets (MPV 11.4 fl) before G-CSF administration showed decreases in platelet indices (MPV 10.3 fl) on D3, although platelet count (18.2 x 10(4)/microl) decreased after G-CSF administration. G-CSF administration induces an inflammatory process with endothelial cell activation. This is probably the reason why platelet volume increases after G-CSF use. This is the first report showing that G-CSF administration immediately induces increases in large platelets in peripheral stem cell transplant donors before harvest.

  3. 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.

  4. Protective, restorative, and therapeutic properties of recombinant colony-stimulating factors

    SciTech Connect

    Talmadge, J.E.; Tribble, H.; Pennington, R.; Bowersox, O.; Schneider, M.A.; Castelli, P.; Black, P.L.; Abe, F. )

    1989-06-01

    Pretreatment of mice with recombinant murine (rM) colony-stimulating factor-granulocyte-macrophage (CSF-gm) or recombinant human (rH) CSF-g provides partial protection from the lethal effects of ionizing radiation or the alkylating agent cyclophosphamide (CTX). In addition, these agents can significantly prolong survival if administered following lethal doses of irradiation or CTX. To induce protective activity, cytokines were injected 20 hours before lethal irradiation or CTX administration. To accelerate recovery from lethal irradiation, the cytokines must be administered shortly following irradiation, and the induction of maximal levels of activity is dependent on chronic administration. In contrast, because of their longer half-lives, accelerated recovery from alkylating agents requires a delay of at least 24 to 48 hours to allow complete clearance of CTX before administration of a CSF. Studies quantitating peripheral blood leukocytes and bone marrow cellularity as well as colony-forming units per culture (CFU-C) frequency and CFU-C per femur revealed a significant correlation between these parameters and the ability to survive lethal irradiation.

  5. Endotoxin down-modulates granulocyte colony-stimulating factor receptor (CD114) on human neutrophils.

    PubMed

    Hollenstein, U; Homoncik, M; Stohlawetz, P J; Marsik, C; Sieder, A; Eichler, H G; Jilma, B

    2000-07-01

    During infection, the development of nonresponsiveness to granulocyte colony-stimulating factor (G-CSF) may be influenced by the down-modulation of G-CSF receptor (G-CSFR) by cytokines. This down-modulation was studied during experimental human endotoxemia. Healthy volunteers received either 2 ng/kg endotoxin (lipopolysaccharide [LPS], n=20) or placebo (n=10) in a randomized, controlled trial. Endotoxin infusion increased the mean fluorescence intensity of the neutrophil activation marker CD11b >300% after 1 h (P<.001 vs. placebo). LPS infusion down-modulated G-CSFR expression in as early as 60 min (-17%; P=.001 vs. placebo). Down-modulation was almost maximal at 90 min and persisted for 6 h (-50% from baseline; P<.0001 vs. placebo). Plasma levels of G-CSF started to increase only after G-CSFR down-modulation had occurred and peaked 37-fold above baseline at 4 h (P<.0001 vs. placebo). In conclusion, LPS down-modulates G-CSFR expression in humans, which may render neutrophils less responsive to the effects of G-CSF and, thereby, compromise host defense mechanisms.

  6. The use of granulocyte-colony-stimulating factor in volunteer unrelated hemopoietic stem cell donors.

    PubMed

    Pamphilon, Derwood; Nacheva, Elisabeth; Navarrete, Cristina; Madrigal, Alejandro; Goldman, John

    2008-07-01

    Granulocyte-colony-stimulating factor (G-CSF) is used for the mobilization of hemopoietic stem cells in healthy donors. It has a number of common side effects such as bone pain, which resolve rapidly after administration is discontinued. Recent publications have raised concern that it might act as a trigger for the development of hematologic malignancy in susceptible individuals, possibly by causing genomic instability, but to date there is no evidence that healthy volunteer donors who receive G-CSF are at any increased risk. Ongoing studies aim to confirm whether or not G-CSF can cause chromosomal abnormalities in healthy donors. In the UK, the British Bone Marrow Registry and Anthony Nolan Trust give G-CSF to donors who have agreed to donate peripheral blood stem cells. It is recommended by the UK Registries at present that all stem cell donors are given updated information explaining the current uncertainties with regard to the use of G-CSF before they give informed consent to its administration. This information is based on a statement agreed by the World Marrow Donor Association for use by individual donor registries. Further, it is our current practice that all donors who have received G-CSF, as well as marrow donors who do not, should be under regular review for at least 10 years to allow the occurrence of any long-term adverse events to be documented.

  7. Proepithelin stimulates growth plate chondrogenesis via nuclear factor-kappaB-p65-dependent mechanisms.

    PubMed

    Wu, Shufang; Zang, Weijin; Li, Xu; Sun, Hongzhi

    2011-07-08

    Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.

  8. Interleukin 1 stimulates platelet-activating factor production in cultured human endothelial cells.

    PubMed Central

    Bussolino, F; Breviario, F; Tetta, C; Aglietta, M; Mantovani, A; Dejana, E

    1986-01-01

    Monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of platelet-activating factor (PAF) in cultured human vascular endothelial cells (HEC). The product was identified as PAF by its behavior in chromatographic systems, its recovery of biological activity, and its physico-chemical properties and susceptibility to lipases. The response of HEC to IL-1 was concentration-dependent, took more than 2 h to become apparent, and decreased after 18 h of incubation. Most of the PAF produced was cell-associated and only a small amount (about 25% of the total) was released in the culture medium. To study the mechanism of IL-1-induced HEC-PAF production we tested the activity of 1-O-alkyl-sn-glycero-3-phosphocholine:acetyl/coenzyme A acetyltransferase in HEC. Acetyltransferase activity measured in IL-1-stimulated HEC lysates showed a three to five times greater maximum velocity, but the same Michaelis constant, as untreated cells. The regulation of PAF generation in HEC by IL-1 may be an important aspect of the two-way interaction between immunocompetent cells and vascular tissue. PMID:2872233

  9. Neuroprotection of Granulocyte Colony-Stimulating Factor for Early Stage Parkinson's Disease.

    PubMed

    Tsai, Sheng-Tzung; Chu, Sung-Chao; Liu, Shu-Hsin; Pang, Cheng-Yoong; Hou, Ting-Wen; Lin, Shinn-Zong; Chen, Shin-Yuan

    2017-03-13

    Parkinson's disease (PD) is a slowly progressive neurodegenerative disease. Both medical and surgical choices provide symptomatic treatment. Granulocyte colony-stimulating factor (G-CSF), a conventional treatment for hematological diseases, has demonstrated its effectiveness in acute and chronic neurological diseases through its anti-inflammatory and antiapoptosis mechanisms. Based on previous in vitro and in vivo studies, we administered a lower dose (3.3 μg/kg) G-CSF injection for 5 days and six courses for 1 year in early-stage PD patients as a phase I trial. The four PD patient's mean unified PD rating scale motor scores in medication off status remained stable from 23 before the first G-CSF injection to 22 during the 2-year follow-up. 3,4-Dihydroxy-6-18F-fluoro-l-phenylalanine (18F-DOPA) positron emission tomography (PET) studies also revealed an annual 3.5% decrease in radiotracer uptake over the caudate nucleus and 7% in the putamen, both slower than those of previous reports of PD. Adverse effects included transient muscular-skeletal pain, nausea, vomiting, and elevated liver enzymes. Based on this preliminary report, G-CSF seems to alleviate disease deterioration for early stage PD patients. The effectiveness of G-CSF was possibly due to its amelioration of progressive dopaminergic neuron degeneration.

  10. Multipronged attenuation of macrophage-colony stimulating factor signaling by Epstein-Barr virus BARF1

    SciTech Connect

    Shim, Ann Hye-Ryong; Chang, Rhoda Ahn; Chen, Xiaoyan; Longnecker, Richard; He, Xiaolin

    2014-10-02

    The ubiquitous EBV causes infectious mononucleosis and is associated with several types of cancers. The EBV genome encodes an early gene product, BARF1, which contributes to pathogenesis, potentially through growth-altering and immune-modulating activities, but the mechanisms for such activities are poorly understood. We have determined the crystal structure of BARF1 in complex with human macrophage-colony stimulating factor (M-CSF), a hematopoietic cytokine with pleiotropic functions in development and immune response. BARF1 and M-CSF form a high-affinity, stable, ring-like complex in both solution and the crystal, with a BARF1 hexameric ring surrounded by three M-CSF dimers in triangular array. The binding of BARF1 to M-CSF dramatically reduces but does not completely abolish M-CSF binding and signaling through its cognate receptor FMS. A three-pronged down-regulation mechanism is proposed to explain the biological effect of BARF1 on M-CSF:FMS signaling. These prongs entail control of the circulating and effective local M-CSF concentration, perturbation of the receptor-binding surface of M-CSF, and imposition of an unfavorable global orientation of the M-CSF dimer. Each prong may reduce M-CSF:FMS signaling to a limited extent but in combination may alter M-CSF:FMS signaling dramatically. The downregulating mechanism of BARF1 underlines a viral modulation strategy, and provides a basis for understanding EBV pathogenesis.

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

    PubMed

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

    2015-07-21

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

  12. Biosimilar granulocyte-colony-stimulating factor for healthy donor stem cell mobilization: need we be afraid?

    PubMed

    Bonig, Halvard; Becker, Petra S; Schwebig, Arnd; Turner, Matthew

    2015-02-01

    Biosimilars are approved biologics with comparable quality, safety, and efficacy to a reference product. Unlike generics, which are chemically manufactured copies of small-molecule drugs with relatively simple chemical structures, the biosimilar designation is applied to drugs that are produced by living organisms, implying much more difficult to control manufacturing and purification procedures. To account for these complexities, the European Medicines Agency (EMA), the US Food and Drug Administration, the Australian Therapeutic Goods Administration, and other regulatory authorities have devised and implemented specific, markedly more demanding pathways for the evaluation and approval of biosimilars. To date, several biosimilars have been approved, including versions of somatropin, erythropoietin, and granulocyte-colony-stimulating factor (G-CSF), and several biosimilar monoclonal antibodies are currently in development. The reference G-CSF product (Neupogen, Amgen) has been used for many years for prevention and treatment of neutropenia and also for mobilization of peripheral blood stem cells (PBSCs). However, concerns have been raised about the safety and efficacy of biosimilar G-CSF during PBSC mobilization procedures, especially in healthy donors. This article reviews the available evidence on the use of biosimilar G-CSF in this setting. Aggregate clinical evidence supports the assessment by the EMA of biosimilar and originator G-CSF as highly biologically similar, with respect to desired and undesired effects.

  13. 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.

  14. Granulocyte-Macrophage Colony-Stimulating Factor Is Neuroprotective in Experimental Traumatic Brain Injury

    PubMed Central

    Tan, Xin L.; Wright, David K.; Liu, Shijie J.; Semple, Bridgette D.; Johnston, Leigh; Jones, Nigel C.; Cook, Andrew D.; Hamilton, John A.; O'Brien, Terence J.

    2014-01-01

    Abstract Traumatic brain injury (TBI) is an international health concern with a complex pathogenesis resulting in major long-term neurological, neurocognitive, and neuropsychiatric outcomes. Although neuroinflammation has been identified as an important pathophysiological process resulting from TBI, the function of specific inflammatory mediators in the aftermath of TBI remains poorly understood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an inflammatory cytokine that has been reported to have neuroprotective effects in various animal models of neurodegenerative disease that share pathological similarities with TBI. The importance of GM-CSF in TBI has yet to be studied, however. We examined the role of GM-CSF in TBI by comparing the effects of a lateral fluid percussion (LFP) injury or sham injury in GM-CSF gene deficient (GM-CSF-/-) versus wild-type (WT) mice. After a 3-month recovery interval, mice were assessed using neuroimaging and behavioral outcomes. All mice given a LFP injury displayed significant brain atrophy and behavioral impairments compared with those given sham-injuries; however, this was significantly worse in the GM-CSF-/- mice compared with the WT mice. GM-CSF-/- mice given LFP injury also had reduced astrogliosis compared with their WT counterparts. These novel findings indicate that the inflammatory mediator, GM-CSF, may have significant protective properties in the chronic sequelae of experimental TBI and suggest that further research investigating GM-CSF and its potential benefits in the injured brain is warranted. PMID:24392832

  15. Pedagogical Factors Stimulating the Self-Development of Students' Multi-Dimensional Thinking in Terms of Subject-Oriented Teaching

    ERIC Educational Resources Information Center

    Andreev, Valentin I.

    2014-01-01

    The main aim of this research is to disclose the essence of students' multi-dimensional thinking, also to reveal the rating of factors which stimulate the raising of effectiveness of self-development of students' multi-dimensional thinking in terms of subject-oriented teaching. Subject-oriented learning is characterized as a type of learning where…

  16. An interleukin-1 receptor antagonist blocks lipopolysaccharide-induced colony-stimulating factor production and early endotoxin tolerance.

    PubMed Central

    Henricson, B E; Neta, R; Vogel, S N

    1991-01-01

    In this report, administration of a recombinant interleukin-1 receptor antagonist protein to mice was found to inhibit induction of colony-stimulating factor as well as induction of early endotoxin tolerance by lipopolysaccharide. These findings provide direct evidence that interleukin-1 is an intermediate in these two lipopolysaccharide-induced phenomena. PMID:1825485

  17. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration.

    PubMed

    Dise, Rebecca S; Frey, Mark R; Whitehead, Robert H; Polk, D Brent

    2008-01-01

    Regulated intestinal epithelial cell migration plays a key role in wound healing and maintenance of a healthy gastrointestinal tract. Epidermal growth factor (EGF) stimulates cell migration and wound closure in intestinal epithelial cells through incompletely understood mechanisms. In this study we investigated the role of the small GTPase Rac in EGF-induced cell migration using an in vitro wound-healing assay. In mouse colonic epithelial (MCE) cell lines, EGF-stimulated wound closure was accompanied by a doubling of the number of cells containing lamellipodial extensions at the wound margin, increased Rac membrane translocation in cells at the wound margin, and rapid Rac activation. Either Rac1 small interfering (si)RNA or a Rac1 inhibitor completely blocked EGF-stimulated wound closure. Whereas EGF failed to activate Rac in colon cells from EGF receptor (EGFR) knockout mice, stable expression of wild-type EGFR restored EGF-stimulated Rac activation and migration. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or Src family kinases reduced EGF-stimulated Rac activation. Cotreatment of cells with both inhibitors completely blocked EGF-stimulated Rac activation and localization to the leading edge of cells and lamellipodial extension. Our results present a novel mechanism by which the PI3K and Src signaling cascades cooperate to activate Rac and promote intestinal epithelial cell migration downstream of EGFR.

  18. Metal: ATP characteristics of insulin- and epidermal growth factor-stimulated phosphorylation in detergent extracts of rat liver plasma membranes.

    PubMed

    Uhing, R J; Exton, J H

    1986-09-01

    The metal: ATP characteristics of insulin- and epidermal growth factor-(EGF)-stimulated protein kinase activities were examined in Nonidet P40 extracts of rat liver plasma membranes. The two kinase activities were capable of utilizing either manganese or magnesium, although differences were observed. Insulin-stimulated 32P incorporation into an Mr 95 000 protein exhibited a higher affinity for ATP in the presence of manganese compared to magnesium. At 200 microM ATP, insulin stimulated 32P incorporation into the Mr 95 000 protein 3- to 5-fold after 5 min in the presence of either metal. At 1 mM ATP, insulin-stimulated 32P incorporation was significantly greater in the presence of magnesium. In contrast, EGF-stimulated 32P incorporation into an Mr 170 000 protein exhibited similar ATP dependencies in the presence of magnesium or manganese. Basal phosphorylation of the Mr 170 000 protein was 2- to 3-fold higher in the presence of manganese, however. Since the higher basal phosphorylation persisted after chromatography on wheat germ lectin-Sepharose, it may represent an inherent activity of the receptor kinase. In the presence of magnesium: ATP, low concentrations of manganese enhanced both insulin- and EGF-stimulated phosphorylation of angiotensin II suggesting involvement of a second metal binding site which regulates the kinase activity. The results presented show major differences in the metal: ATP properties of the two major hormonally regulated protein kinase activities observed in detergent-extracted liver membranes.

  19. 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.

  20. Effect of maternal and neonatal factors on cord blood thyroid stimulating hormone

    PubMed Central

    Lakshminarayana, Sheetal G.; Sadanandan, Nidhish P.; Mehaboob, A. K.; Gopaliah, Lakshminarayana R.

    2016-01-01

    Background: Congenital hypothyroidism (CH) is most common preventable cause of mental retardation in children. Cord blood Thyroid Stimulating Hormone (CBTSH) level is an accepted screening tool for CH. Objectives: To study CBTSH profile in neonates born at tertiary care referral center and to analyze the influence of maternal and neonatal factors on their levels. Design: Cross retrospective sectional study. Methods: Study population included 979 neonates (males = 506 to females = 473). The CBTSH levels were estimated using electrochemiluminescence immunoassay on Cobas analyzer. Kit based cut-offs of TSH level were used for analysis. All neonates with abnormal CBSTH levels, were started on levothyroxine supplementation 10 μg/Kg/day and TSH levels were reassessed as per departmental protocol. Results: The mean CBTSH was 7.82 μIU/mL (Range 0.112 to 81.4, SD = 5.48). The mean CBTSH level was significantly higher in first order neonates, neonates delivered by assisted vaginal delivery and normal delivery, delivered at term or preterm, neonates with APGAR score <5 and those needing advanced resuscitation after birth. The CBTSH level >16.10 and <1.0 μIU/mL was found in 4.39 % and 1.02 % neonates respectively. The prevalence rate of CBTSH level >16.1 μIU/mL was significantly higher in neonates delivered by assisted vaginal delivery and normal delivery, term and preterm neonates, APAGR score of <5, presence of fetal distress, need for resuscitation beyond initial steps and in those with birth weight of <1.5 Kg. Three neonates were confirmed to have CH after retesting of TSH level. Conclusions: The CBTSH estimation is an easy, non-invasive method for screening for CH. The cutoff level of CB TSH (μIU/mL) >16.10 and <1.0 led to a recall of 5.41% of neonates which is practicable given the scenario in our Country. The mode of delivery and perinatal stress factors have a significant impact on CBTSH levels and any rise to be seen in the light of these factors. The prevalence

  1. Involvement of Connective Tissue Growth Factor (CTGF) in Insulin-like Growth Factor-I (IGF1) Stimulation of Proliferation of a Bovine Mammary Epithelial Cell Line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin-like growth factor I (IGF1) plays an important role in mammary gland development and lactation in part by stimulating proliferation of the milk-producing epithelial cells. In this study, we used the bovine mammary epithelial cell line MAC-T cells as a model to understand the mechanism by whi...

  2. Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells.

    PubMed

    Vianney, John-Mary; Miller, Damon A; Spitsbergen, John M

    2014-11-07

    Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor required for survival of neurons in the central and peripheral nervous system. Specifically, GDNF has been characterized as a survival factor for spinal motor neurons. GDNF is synthesized and secreted by neuronal target tissues, including skeletal muscle in the peripheral nervous system; however, the mechanisms by which GDNF is synthesized and released by skeletal muscle are not fully understood. Previous results suggested that cholinergic neurons regulate secretion of GDNF by skeletal muscle. In the current study, GDNF production by skeletal muscle myotubes following treatment with acetylcholine was examined. Acetylcholine receptors on myotubes were identified with labeled alpha-bungarotoxin and were blocked using unlabeled alpha-bungarotoxin. The question of whether electrical stimulation has a similar effect to that of acetylcholine was also investigated. Cells were stimulated with voltage pulses; at 1 and 5 Hz frequencies for times ranging from 30 min to 48 h. GDNF content in myotubes and GDNF in conditioned culture medium were quantified by enzyme-linked immunosorbant assay. Results suggest that acetylcholine and short-term electrical stimulation reduce GDNF secretion, while treatment with carbachol or long-term electrical stimulation enhances GDNF production by skeletal muscle.

  3. Macrophage colony-stimulating factor gene expression in vascular cells and in experimental and human atherosclerosis.

    PubMed Central

    Clinton, S. K.; Underwood, R.; Hayes, L.; Sherman, M. L.; Kufe, D. W.; Libby, P.

    1992-01-01

    The infiltration of monocytes into the vascular wall and their transformation into lipid-laden foam cells characterizes early atherogenesis. Macrophages are also present in more advanced human atherosclerotic plaques and can produce many mediators that may contribute to lesion formation and progression. Macrophage colony-stimulating factor (MCSF) enhances the proliferation and differentiation of monocyte progenitors and is required for the survival and activation of mature monocytes and macrophages. The authors therefore examined the expression of the MCSF gene in cultured human vascular endothelial (EC) and smooth muscle cells (SMC) as well as in atheromatous lesions from rabbits and humans. Growth arrested EC and SMC contain a low level of MCSF mRNA. Bacterial lipopolysaccharide (LPS), recombinant human interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) induced MCSF mRNA accumulation in a concentration-dependent manner in both EC and SMC. These stimuli induced large increases in MCSF mRNA with peak induction between 4-8 hours after treatment. LPS, IL-1 alpha, and TNF alpha stimulated EC and SMC also showed increased fluorescent antibody staining for MCSF protein and released immunoreactive MCSF in a time-dependent manner. In contrast, phorbol 12-myristate 13-acetate (PMA) was a less potent inducer of MCSF gene expression and iron-oxidized low-density lipoproteins (ox-LDL) did not increase consistently MCSF mRNA or the synthesis and secretion of immunoreactive protein. Northern analysis of mRNA isolated from the atheromatous aorta of rabbits fed a 1% cholesterol diet for 10 weeks showed elevated MCSF mRNA compared with controls. Immunostaining of atheromatous arterial lesions of rabbits demonstrated MCSF protein in association with intimal SMC as well as macrophages. Furthermore, polymerase chain reaction (PCR) analysis of MCSF mRNA in human atheromata showed higher levels than found in nonatherosclerotic arteries and veins. Since the

  4. Immobilized transition metals stimulate contact activation and drive factor XII-mediated coagulation

    PubMed Central

    Mutch, N.J.; Waters, E.K.; Morrissey, J. H.

    2012-01-01

    Summary Background Upon contact with an appropriate surface, factor XII (FXII) undergoes autoactivation or cleavage by kallikrein. Zn2+ is known to facilitate binding of FXII and the cofactor, high molecular weight kininogen (HK), to anionic surfaces. Objectives To investigate whether transition metals immobilized on liposome surfaces can initiate coagulation via the contact pathway. Methods & Results Liposomes containing a metal ion-chelating lipid (DOGS-NTA) were prepared by membrane extrusion (20% DOGS-NTA, 40% phosphatidylcholine, 10% phosphatidylserine, and 30% phosphatidylethanolamine). Ni2+ immobilized on such liposomes accelerated clotting in normal, but not FXI- or FXII-deficient plasma. Results were comparable to a commercial aPTT reagent. Charging such liposomes with other transition metals revealed differences in their procoagulant capacity, with Ni2+> Cu2+> Co2+ and Zn2+. Plasma could be depleted of FXI, FXII and HK by adsorption with Ni2+-containing beads, resulting in delayed clot times. Consistent with this, FXI, FXII and HK bound to immobilized Ni2+ or Cu2+ with high affinity as determined by surface plasmon resonance. In the presence of Ni2+-bearing liposomes, Km and kcat values derived for autoactivation of FXII and prekallikrein, as well as for activation of FXII by kallikrein or prekallikrein by FXIIa, were similar to literature values in the presence of dextran sulfate. Conclusions Immobilized Ni2+ and Cu2+ bind FXII, FXI and HK with high affinity and stimulate activation of the contact pathway, driving FXII-mediated coagulation. Activation of the contact system by immobilized transition metals may have implications during pathogenic infection or in individuals exposed to high levels of pollution. PMID:22905925

  5. Clonidine stimulates atrial natriuretic factor (ANF) release in water-deprived rats.

    PubMed

    Baranowska, B; Tremblay, J; Gutkowska, J

    1988-01-01

    To determine the effect of clonidine, an alpha 2-adrenergic agonist, on atrial natriuretic factor (ANF) release during water deprivation, plasma immunoreactive ANF (IR-ANF) arginine vasopressin, diuresis and natriuresis were measured in rats which had been deprived of water for 24 and 48 hr after intravenous (IV) administration of 50 micrograms clonidine. In normally-hydrated rats clonidine produced a marked elevation of plasma IR-ANF from 40.5 +/- 4.6 pg/ml to 1064 +/- 22 pg/ml (mean +/- SEM) and sodium excretion from 73.3 +/- 6.8 microEq to 723.4 +/- 62.3 microEq. Clonidine evoked an increase in plasma IR-ANF from 16.6 +/- 5.9 pg/ml to 229.5 +/- 60 pg/ml (mean +/- SEM) after 24 hr water deprivation and from 13.6 +/- 7.4 pg/ml to 104.8 +/- 21 pg/ml (mean +/- SEM) after 48 hr water deprivation. Clonidine did not induce any significant changes in vasopressin levels. During 24 hr and 48 hr water deprivation vasopressin rose from 3.1 +/- 0.3 pg/ml to 7.3 +/- 1.3 pg/ml and 8.4 +/- 0.6 pg/ml (mean +/- SEM), respectively. In normally-hydrated rats clonidine produced a marked diuresis and natriuresis. These effects and urinary cGMP excretion were significantly inhibited by anti-ANF antibodies. Clonidine caused a significant increase in urine output in 24 hr water-deprived rats but the response was markedly lower than that seen in normally-hydrated rats. In conclusion, clonidine stimulates ANF release both in normally-hydrated and water-deprived rats. The diuretic effect of clonidine appears to be related to ANF release but not to inhibition of vasopressin.

  6. Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium

    PubMed Central

    1990-01-01

    Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF- alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states. PMID:1972179

  7. Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium.

    PubMed

    Slungaard, A; Vercellotti, G M; Walker, G; Nelson, R D; Jacob, H S

    1990-06-01

    Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF-alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states.

  8. Skin impedance is not a factor in transcutaneous electrical nerve stimulation effectiveness

    PubMed Central

    Vance, Carol GT; Rakel, Barbara A; Dailey, Dana L; Sluka, Kathleen A

    2015-01-01

    Objective Transcutaneous electrical nerve stimulation (TENS) is a nonpharmacological intervention used to manage pain using skin surface electrodes. Optimal electrode placement is unclear. We hypothesized that better analgesia would occur if electrodes were placed over sites with lower skin impedance. Optimal site selection (OSS) and sham site selection (SSS) electrode sites on the forearm were identified using a standard clinical technique. Methods Experiment 1 measured skin impedance in the forearm at OSS and SSS. Experiment 2 was a crossover design double-blind randomized controlled trial comparing OSS-TENS, SSS-TENS, and placebo TENS (P-TENS) to confirm differences in skin impedance between OSS and SSS, and measure change in pressure pain threshold (PPT) following a 30-minute TENS treatment. Healthy volunteers were recruited (ten for Experiment 1 [five male, five female] and 24 for Experiment 2 [12 male, 12 female]). TENS was applied for 30 minutes at 100 Hz frequency, 100 µs pulse duration, and “strong but nonpainful” amplitude. Results Experiment 1 results demonstrate significantly higher impedance at SSS (17.69±1.24 Ω) compared to OSS (13.53±0.57 Ω) (P=0.007). For Experiment 2, electrode site impedance was significantly higher over SSS, with both the impedance meter (P=0.001) and the TENS unit (P=0.012) compared to OSS. PPT change was significantly greater for both OSS-TENS (P=0.024) and SSS-TENS (P=0.025) when compared to P-TENS. PPT did not differ between the two active TENS treatments (P=0.81). Conclusion Skin impedance is lower at sites characterized as optimal using the described technique of electrode site selection. When TENS is applied at adequate intensities, skin impedance is not a factor in attainment of hypoalgesia of the forearm in healthy subjects. Further investigation should include testing in patients presenting with painful conditions. PMID:26316808

  9. Factors predicting incremental administration of antihypertensive boluses during deep brain stimulator placement for Parkinson's disease.

    PubMed

    Rajan, Shobana; Deogaonkar, Milind; Kaw, Roop; Nada, Eman Ms; Hernandez, Adrian V; Ebrahim, Zeyd; Avitsian, Rafi

    2014-10-01

    Hypertension is common in deep brain stimulator (DBS) placement predisposing to intracranial hemorrhage. This retrospective review evaluates factors predicting incremental antihypertensive use intraoperatively. Medical records of Parkinson's disease (PD) patients undergoing DBS procedure between 2008-2011 were reviewed after Institutional Review Board approval. Anesthesia medication, preoperative levodopa dose, age, preoperative use of antihypertensive medications, diabetes mellitus, anxiety, motor part of the Unified Parkinson's Disease Rating Scale score and PD duration were collected. Univariate and multivariate analysis was done between each patient characteristic and the number of antihypertensive boluses. From the 136 patients included 60 were hypertensive, of whom 32 were on angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), told to hold on the morning of surgery. Antihypertensive medications were given to 130 patients intraoperatively. Age (relative risk [RR] 1.01; 95% confidence interval [CI] 1.00-1.02; p=0.005), high Joint National Committee (JNC) class (p<0.0001), diabetes mellitus (RR 1.4; 95%CI 1.2-17; p<0.0001) and duration of PD >10 years (RR 1.2; 95%CI 1.1-1.3; p=0.001) were independent predictors for antihypertensive use. No difference was noted in the mean dose of levodopa (p=0.1) and levodopa equivalent dose (p=0.4) between the low (I/II) and high severity (III/IV) JNC groups. Addition of dexmedetomidine to propofol did not influence antihypertensive boluses required (p=0.38). Intraoperative hypertension during DBS surgery is associated with higher age group, hypertensive, diabetic patients and longer duration of PD. Withholding ACEI or ARB is an independent predictor of hypertension requiring more aggressive therapy. Levodopa withdrawal and choice of anesthetic agent is not associated with higher intraoperative antihypertensive medications.

  10. Effect of thrombopoietin and granulocyte colony-stimulating factor on platelets and polymorphonuclear leukocytes.

    PubMed

    Schattner, M; Pozner, R G; Gorostizaga, A B; Lazzari, M A

    2000-07-15

    Thrombopoietin (TPO) and granulocyte colony-stimulating factor (G-CSF) may be administered together in aplastic patients. We evaluated the effect of both cytokines alone or combined on platelets and polymorphonuclear leukocytes (PMN) functional responses. TPO, G-CSF, or the combination of both cytokines, induced neither platelet nor PMN activation. TPO but not G-CSF synergized with threshold ADP concentrations to induce maximal aggregation and ATP release. The synergistic effect of TPO with ADP was not modified by the presence of G-CSF. Flow cytometry studies have shown that thrombin-induced loss of GPIb from platelet surface was significantly increased by pretreatment of platelets with TPO, G-CSF, or both cytokines. P-selectin expression induced by thrombin was augmented by TPO, but not by G-CSF. Coincubation of the cells with TPO and G-CSF did not modify the values obtained with TPO alone. Expression of CD11b on PMN surface was augmented by G-CSF or fMLP. G-CSF-treated PMN increased the effect of fMLP on CD11b expression. TPO did not modify either basal levels of CD11b or the increased expression induced by G-CSF or fMLP. Incubation of PMN with both cytokines showed no differences compared to G-CSF alone. Platelet-PMN aggregates induced by thrombin in whole blood were augmented by TPO. G-CSF alone neither synergized with thrombin nor changed the results observed with TPO. These data show that in vitro functional responses of platelets, or PMN induced by TPO or G-CSF alone, were neither further increased nor inhibited by treatment of the cells with both cytokines.

  11. Elevated levels of macrophage colony-stimulating factor in human fracture healing.

    PubMed

    Sarahrudi, Kambiz; Mousavi, Mehdi; Thomas, Anita; Eipeldauer, Stefan; Vécsei, Vilmos; Pietschmann, Peter; Aharinejad, Seyedhossein

    2010-05-01

    Macrophage colony-stimulating factor (M-CSF) plays a unique role in bone remodeling. However, to our knowledge, no data on the role of M-CSF in fracture healing in humans have been published so far. This study addressed this issue. One hundred and thirteen patients with long-bone fractures were included in the study and divided into two groups, according to their course of fracture healing. The first group contained 103 patients with normal fracture healing. Ten patients with impaired fracture healing formed the second group of the study. Volunteers donated blood samples as control. Serum samples were collected over a period of 6 months, following a standardized time schedule. In addition, M-CSF levels were measured in fracture hematoma and serum of 11 patients with bone fractures. M-CSF concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Fracture hematoma contained significantly higher M-CSF concentrations compared to M-CSF concentrations in patient's serum. M-CSF levels in fracture hematoma and in patient's serum were both significantly higher than M-CSF concentrations measured in serum of healthy controls. Highly elevated M-CSF serum concentrations were found in patients with physiological fracture healing over the entire observation period. Significant differences in the M-CSF serum concentration between patients with normal fracture healing and patients with impaired fracture healing were not observed. This study indicates, for the first time, to our knowledge, a possible local and systemic involvement of M-CSF in humans during fracture healing.

  12. Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor alpha.

    PubMed

    McFaul, S J; Bowman, P D; Villa, V M; Gutierrez-Ibanez, M J; Johnson, M; Smith, D

    1994-11-01

    Incubation of human mononuclear leukocytes (MNL) with human stroma-free hemolysate (SFH), purified adult hemoglobin Ao (HbAo), and oxidized HbAo (METHb) caused MNL to release compounds into the supernate that mediated neutrophil (polymorphonuclear leukocytes, PMN) chemotaxis and PMN adherence to human umbilical vein endothelial cells (HUVEC). Chemotaxis and PMN adherence to HUVEC were reduced significantly when supernates were preincubated with neutralizing antibodies to interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha), respectively, suggesting that IL-8 and TNF-alpha played significant roles in mediating these activities. Greatest chemotactic activity was observed in supernates of MNL treated with HbAo; while greatest PMN/endothelial cell (EC) adherence activity was observed in supernates of MNL treated with METHb. Furthermore, PMN/EC adherence activity was a function of METHb content in each hemoglobin solution. PMN chemotaxis, PMN adherence to HUVEC, and cytokine release increased as a function of increasing incubation time. Chemotactic activity was detected in HbAo-treated and METHb-treated MNL supernates after incubation for 6 hours and was maximal by 10 hours. IL-8 was detected in both HbAo and METHb-MNL supernates by 4 hours. PMN/EC adherence activity was detected in HbAo-MNL supernates at 10 hours and in METHb-MNL supernates at 4 hours. TNF-alpha was detected in METHb and HbAo-MNL supernates at 4 and 12 hours, respectively. These results suggest that hemoglobin solutions stimulate MNL to release IL-8 and TNF-alpha in quantities sufficient to induce PMN chemotaxis and PMN adherence to HUVEC. This is a US government work. There are no restrictions on its use.

  13. Granulocyte colony-stimulating factor receptor expression on human transitional cell carcinoma of the bladder.

    PubMed Central

    Tachibana, M.; Miyakawa, A.; Uchida, A.; Murai, M.; Eguchi, K.; Nakamura, K.; Kubo, A.; Hata, J. I.

    1997-01-01

    Receptors for granulocyte colony-stimulating factor (G-CSFRs) have been confirmed on the cell surfaces of several non-haematopoietic cell types, including bladder cancer cells. This observation has naturally led to the hypothesis that the expression of G-CSFR on these cells may enhance their growth by G-CSF. In this study, the expression of G-CSFR was determined in both established human bladder cancer cell lines and primary bladder cancers. We studied five different human bladder cancer cell lines (KU-1, KU-7, T-24, NBT-2 and KK) and 26 newly diagnosed bladder tumours. G-CSFR mRNA expressions on cultured cell lines were determined using the reverse transcriptase polymerase chain reaction (RT-PCR) method. Furthermore, the G-CSFR binding experiments on the cultured cell lines were conducted using the Na(125)I-labelled G-CSF ligand-binding assay method. Moreover, the G-CSFR mRNA expressions on primary bladder tumour specimens were assessed using the in situ RT-PCR method. Three out of the five cultured cell lines (KU-1, NBT-2 and KK) exhibited G-CSFR mRNA signals when the RT-PCR method was used. The G-CSFR binding experiments showed an equilibrium dissociation constant (K[d]) of 490 pM for KU-1, 340 pM for NBT-2 and 103 pM for KK cells. With in situ RT-PCR, the tumour cells of 6 out of 26 primary bladder tumour specimens (23.1%) presented positive G-CSFR mRNA signals. Thus, in this study, G-CSFR expression was frequently observed on bladder cancer cells. Therefore, the clinical use of G-CSF for patients with bladder cancer should be selected with great care. Images Figure 1 Figure 3 Figure 4 PMID:9166942

  14. Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei*

    PubMed Central

    Greene, Amy Styer; Hajduk, Stephen L.

    2016-01-01

    Human innate immunity against the veterinary pathogen Trypanosoma brucei brucei is conferred by trypanosome lytic factors (TLFs), against which human-infective T. brucei gambiense and T. brucei rhodesiense have evolved resistance. TLF-1 is a subclass of high density lipoprotein particles defined by two primate-specific apolipoproteins: the ion channel-forming toxin ApoL1 (apolipoprotein L1) and the hemoglobin (Hb) scavenger Hpr (haptoglobin-related protein). The role of oxidative stress in the TLF-1 lytic mechanism has been controversial. Here we show that oxidative processes are involved in TLF-1 killing of T. brucei brucei. The lipophilic antioxidant N,N′-diphenyl-p-phenylenediamine protected TLF-1-treated T. brucei brucei from lysis. Conversely, lysis of TLF-1-treated T. brucei brucei was increased by the addition of peroxides or thiol-conjugating agents. Previously, the Hpr-Hb complex was postulated to be a source of free radicals during TLF-1 lysis. However, we found that the iron-containing heme of the Hpr-Hb complex was not involved in TLF-1 lysis. Furthermore, neither high concentrations of transferrin nor knock-out of cytosolic lipid peroxidases prevented TLF-1 lysis. Instead, purified ApoL1 was sufficient to induce lysis, and ApoL1 lysis was inhibited by the antioxidant DPPD. Swelling of TLF-1-treated T. brucei brucei was reminiscent of swelling under hypotonic stress. Moreover, TLF-1-treated T. brucei brucei became rapidly susceptible to hypotonic lysis. T. brucei brucei cells exposed to peroxides or thiol-binding agents were also sensitized to hypotonic lysis in the absence of TLF-1. We postulate that ApoL1 initiates osmotic stress at the plasma membrane, which sensitizes T. brucei brucei to oxidation-stimulated osmotic lysis. PMID:26645690

  15. Use of Granulocyte Colony–Stimulating Factor During Pregnancy in Women With Chronic Neutropenia

    PubMed Central

    Boxer, Laurence A.; Bolyard, Audrey Anna; Kelley, Merideth L.; Marrero, Tracy M.; Phan, Lan; Bond, Jordan M.; Newburger, Peter E.; Dale, David C.

    2014-01-01

    Objective To report outcomes associated with the administration of granulocyte colony–stimulating factor (G-CSF) to women with chronic neutropenia during pregnancy. Methods We conducted an observational study of women of child-bearing potential with congenital, cyclic, idiopathic, or autoimmune neutropenia enrolled in the Severe Chronic Neutropenia International Registry to determine outcomes of pregnancies, without and with chronic G-CSF therapy, 1999–2014. Treatment decisions were made by the patients’ personal physicians. A research nurse conducted telephone interviews of all enrolled U.S. women of child-bearing potential using a standard questionnaire. Comparisons utilized Fisher’s exact test analysis and Student’s t-test. Results One-hundred seven women reported 224 pregnancies, 124 without G-CSF therapy and 100 on chronic G-CSF therapy (median dose: 1.0 mcg/kg/day, range 0.02–8.6 mcg/kg/day). There were no significant differences in adverse events between the groups considering all pregnancies or individual mothers, e.g., spontaneous terminations (all pregnancies: no G-CSF 27/124, G-CSF 13/100; P=0.11, Fisher’s exact test,), preterm labors (all pregnancies, no G-CSF 9/124, G-CSF 2/100, P=0.12,). A study with at least 300 per group would be needed to detect a difference in these events with 80% statistical power (alpha=0.05). Four newborns of mothers with idiopathic or autoimmune neutropenia not on G-CSF (4/101) had life-threatening infections, whereas there were no similar events (0/90) in the treated group, but this difference was also not statistically significant. (p=0.124). Adverse events in the neonates were similar for the two groups. Conclusions This observational study showed no significant adverse effects of administration of G-CSF to women with severe chronic neutropenia during pregnancy. PMID:25560125

  16. Vascular endothelial growth factor directly stimulates tumour cell proliferation in non-small cell lung cancer.

    PubMed

    Devery, Aoife M; Wadekar, Rekha; Bokobza, Sivan M; Weber, Anika M; Jiang, Yanyan; Ryan, Anderson J

    2015-09-01

    Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is found predominantly on endothelial cells. The purpose of this study was to determine the role of VEGFR2 expression in NSCLC cells. NSCLC cells and tissue sections were stained for VEGFR2 expression by immunohistochemistry (IHC). Immunoblotting and ELISA were used to determine the activation and inhibition of VEGFR2 and its downstream signalling pathways. Five-day proliferation assays were carried out in the presence or absence of VEGF. IHC analysis of NSCLC demonstrated tumour cell VEGFR2 expression in 20% of samples. Immunoblot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation was apparent in NSCLC cells, and was associated with increased tumor cell proliferation. Cediranib treatment or siRNA against VEGFR2 inhibited VEGF-dependent increases in cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine. Our data suggest that a subset of NSCLC tumour cells express functional VEGFR2 which can act to promote VEGF-dependent tumour cell growth. In this tumour subset, therapies targeting VEGFR2 signalling, such as cediranib, have the potential to inhibit both tumour cell proliferation and angiogenesis.

  17. Purification of a Factor from Human Placenta That Stimulates Capillary Endothelial Cell Protease Production, DNA Synthesis, and Migration

    NASA Astrophysics Data System (ADS)

    Moscatelli, David; Presta, Marco; Rifkin, Daniel B.

    1986-04-01

    A protein that stimulates the production of plasminogen activator and latent collagenase in cultured bovine capillary endothelial cells has been purified 106-fold from term human placenta by using a combination of heparin affinity chromatography, ion-exchange chromatography, and gel chromatography. The purified molecule has a molecular weight of 18,700 as determined by NaDodSO4/PAGE under both reducing and nonreducing conditions. The purified molecule stimulates the production of plasminogen activator and latent collagenase in a dose-dependent manner between 0.1 and 10 ng of protein/ml. The purified protein also stimulates DNA synthesis and chemotaxis in capillary endothelial cells in the same concentration range. Thus, this molecule has all of the properties predicted for an angiogenic factor.

  18. Biosynthesis of platelet-activating factor by cultured rat Kupffer cells stimulated with calcium ionophore A23187.

    PubMed Central

    Chao, W; Siafaka-Kapadai, A; Olson, M S; Hanahan, D J

    1989-01-01

    Cultured rat Kupffer cells synthesize and release platelet-activating factor (PAF) when stimulated with calcium ionophore A23187. The production of PAF is concentration- and time-dependent and, based upon [3H]serotonin release assays, approx. 1.0 pmol of PAF is formed per 8 x 10(6) cells during 10 min of ionophore stimulation. It is suggested that Kupffer cells are important cellular components which produce and release PAF in order to facilitate communication between hepatic sinusoidal and parenchymal cells. Further, it is suggested that such mediator production in response to reticulo-endothelial cell stimulation causes the hepatic glycogenolytic response previously in the isolated perfused rat liver. PMID:2494988

  19. Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

    PubMed

    Kelly, J M; Moir, A J G; Carlson, K; Yang, Y; MacNeil, S; Haycock, J W

    2006-02-01

    alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

  20. Granulocyte-Colony-Stimulating Factor Stimulation of Bone Marrow Mesenchymal Stromal Cells Promotes CD34+ Cell Migration Via a Matrix Metalloproteinase-2-Dependent Mechanism

    PubMed Central

    Ponte, Adriana López; Ribeiro-Fleury, Tatiana; Chabot, Valérie; Gouilleux, Fabrice; Langonné, Alain; Hérault, Olivier; Charbord, Pierre

    2012-01-01

    Human hematopoietic stem/progenitor cells (HSPCs) can be mobilized into the circulation using granulocyte-colony stimulating factor (G-CSF), for graft collection in view of hematopoietic transplantation. This process has been related to bone marrow (BM) release of serine proteases and of the matrix metalloproteinase-9 (MMP-9). Yet, the role of these mediators in HSC egress from their niches remains questionable, because they are produced by nonstromal cells (mainly neutrophils and monocytes/macrophages) that are not a part of the niche. We show here that the G-CSF receptor (G-CSFR) is expressed by human BM mesenchymal stromal/stem cells (MSCs), and that G-CSF prestimulation of MSCs enhances the in vitro trans-stromal migration of CD34+ cells. Zymography analysis indicates that pro-MMP-2 (but not pro-MMP-9) is expressed in MSCs, and that G-CSF treatment increases its expression and induces its activation at the cell membrane. We further demonstrate that G-CSF-stimulated migration depends on G-CSFR expression and is mediated by a mechanism that involves MMPs. These results suggest a molecular model whereby G-CSF infusion may drive, by the direct action on MSCs, HSPC egress from BM niches via synthesis and activation of MMPs. In this model, MMP-2 instead of MMP-9 is implicated, which constitutes a major difference with mouse mobilization models. PMID:22651889

  1. Stimulation of tumor necrosis factor alpha production in human monocytes by inhibitors of protein phosphatase 1 and 2A

    PubMed Central

    1992-01-01

    The protein phosphatase 1 and 2A inhibitor, okadaic acid, has been shown to stimulate many cellular functions by increasing the phosphorylation state of phosphoproteins. In human monocytes, okadaic acid by itself stimulates tumor necrosis factor alpha (TNF-alpha) mRNA accumulation and TNF-alpha synthesis. Calyculin A, a more potent inhibitor of phosphatase 1, has similar effects. TNF-alpha mRNA accumulation in okadaic acid-treated monocytes is due to increased TNF- alpha mRNA stability and transcription rate. The increase in TNF-alpha mRNA stability is more remarkable in okadaic acid-treated monocytes than the mRNA stability of other cytokines, such as interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6. Gel retardation studies show the stimulation of AP-1, AP-2, and NF-kappa B binding activities in okadaic acid-stimulated monocytes. This increase may correlate with the increase in TNF-alpha mRNA transcription rate. In addition to the stimulation of TNF-alpha secretion by monocytes, okadaic acid appears to modulate TNF-alpha precursor processing, as indicated by a marked increase in the cell-associated 26-kD precursor. These results suggest that active basal phosphorylation/dephosphorylation occurs in monocytes, and that protein phosphatase 1 or 2A is important in regulating TNF-alpha gene transcription, translation, and posttranslational modification. PMID:1324971

  2. Growth differentiation factor-9 stimulates progesterone synthesis in granulosa cells via a prostaglandin E2/EP2 receptor pathway.

    PubMed

    Elvin, J A; Yan, C; Matzuk, M M

    2000-08-29

    Growth differentiation factor-9 (GDF-9), an oocyte-secreted member of the transforming growth factor beta superfamily, progesterone receptor, cyclooxygenase 2 (Cox2; Ptgs2), and the EP2 prostaglandin E(2) (PGE(2)) receptor (EP2; Ptgerep2) are required for fertility in female but not male mice. To define the interrelationship of these factors, we used a preovulatory granulosa cell culture system in which we added recombinant GDF-9, prostaglandins, prostaglandin receptor agonists, or cyclooxygenase inhibitors. GDF-9 stimulated Cox2 mRNA within 2 h, and PGE(2) within 6 h; however, progesterone was not increased until 12 h after addition of GDF-9. This suggested that Cox2 is a direct downstream target of GDF-9 but that progesterone synthesis required an intermediate. To determine whether prostaglandin synthesis was required for progesterone production, we analyzed the effects of PGE(2) and cyclooxygenase inhibitors on this process. PGE(2) can stimulate progesterone synthesis by itself, although less effectively than GDF-9 (3-fold vs. 6-fold increase over 24 h, respectively). Furthermore, indomethacin or NS-398, inhibitors of Cox2, block basal and GDF-9-stimulated progesterone synthesis. However, addition of PGE(2) to cultures containing both GDF-9 and NS-398 overrides the NS-398 block in progesterone synthesis. To further define the PGE(2)-dependent pathway, we show that butaprost, a specific EP2 agonist, stimulates progesterone synthesis and overrides the NS-398 block. In addition, GDF-9 stimulates EP2 mRNA synthesis by a prostaglandin- and progesterone-independent pathway. Thus, GDF-9 induces an EP2 signal transduction pathway which appears to be required for progesterone synthesis in cumulus granulosa cells. These studies further demonstrate the importance of oocyte-somatic cell interactions in female reproduction.

  3. Neuronal expression of nuclear transcription factor MafG in the rat medulla oblongata after baroreceptor stimulation.

    PubMed

    Kumaki, Iku; Yang, Dawei; Koibuchi, Noriyuki; Takayama, Kiyoshige

    2006-03-06

    The medulla oblongata is the site of central baroreceptive neurons in mammals. These neurons express specific basic-leucine zipper transcription factors (bZIP) after baroreceptor stimulation. Previously we showed that activation of baroreceptors induced expression of nuclear transcription factors c-Fos and FosB in central baroreceptive neurons. Here we studied the effects of baroreceptor stimulation on induction of MafG, a member of small Maf protein family that functions as dimeric partners for various bZIP transcription factors by forming transcription-regulating complexes, in the rat medulla oblongata. To determine whether gene expression of MafG is induced by stimulation of arterial baroreceptors, we examined the expression of its mRNA by semi-quantitative reverse transcription-PCR method and its gene product by immunohistochemistry. We found that the number of MafG transcripts increased significantly in the medulla oblongata after baroreceptor stimulation. MafG-immunoreactive neurons were distributed in the nucleus tractus solitarii, the dorsal motor nucleus of the vagus nerve, the ambiguous nucleus and the ventrolateral medulla. The numbers of MafG-immunoreactive neurons in these nuclei were significantly greater in test rats than in saline-injected control rats. We also found approximately 20% of MafG-immunoreactive neurons coexpress FosB after baroreceptor stimulation. Our results suggest that MafG cooperates with FosB to play critical roles as an immediate early gene in the signal transduction of cardiovascular regulation mediated by baroreceptive signals in the medulla oblongata.

  4. Effects of low- and high-frequency repetitive magnetic stimulation on neuronal cell proliferation and growth factor expression: A preliminary report.

    PubMed

    Lee, Ji Yong; Park, Hyung Joong; Kim, Ji Hyun; Cho, Byung Pil; Cho, Sung-Rae; Kim, Sung Hoon

    2015-09-14

    Repetitive magnetic stimulation is a neuropsychiatric and neurorehabilitation tool that can be used to investigate the neurobiology of sensory and motor functions. Few studies have examined the effects of repetitive magnetic stimulation on the modulation of neurotrophic/growth factors and neuronal cells in vitro. Therefore, the current study examined the differential effects of repetitive magnetic stimulation on neuronal cell proliferation as well as various growth factor expression. Immortalized mouse neuroblastoma cells were used as the cell model in this study. Dishes of cultured cells were randomly divided into control, sham, low-frequency (0.5Hz, 1Tesla) and high-frequency (10Hz, 1Tesla) groups (n=4 dishes/group) and were stimulated for 3 days. Expression of neurotrophic/growth factors, Akt and Erk was investigated by Western blotting analysis 3 days after repetitive magnetic stimulation. Neuroblastoma cell proliferation was determined with a cell counting assay. There were differences in cell proliferation based on stimulus frequency. Low-frequency stimulation did not alter proliferation relative to the control, while high-frequency stimulation elevated proliferation relative to the control group. The expression levels of brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) and platelet-derived growth factor (PDGF) were elevated in the high-frequency magnetic stimulation group. Akt and Erk expression was also significantly elevated in the high-frequency stimulation group, while low-frequency stimulation decreased the expression of Akt and Erk compared to the control. In conclusion, we determined that different frequency magnetic stimulation had an influence on neuronal cell proliferation via regulation of Akt and ERK signaling pathways and the expression of growth factors such as BDNF, GDNF, NT-3 and PDGF. These findings represent a promising opportunity to gain insight into how different

  5. In vivo production of macrophage migration inhibition and stimulation factors during the inductive phase of the alloimmune response

    SciTech Connect

    Suslov, A.P.; Yazova, A.K.; Berkova, N.P.

    1986-12-01

    This paper offers a study of the production of macrophage migration inhibition factor (MIF), and also of the alternative macrophage migration stimulation factor (MSF), in vivo. Mice were injected with mouse spleen cells, irradiated with a dose of 1500 rads. The animals were divided into three groups, two of which were injected for a second time with irradiated mouse spleen cells. Samples of all fractions obtained by electrophoresis of sera of unimmunized mice had no significant effect of macrophage migration, while unfractionated sera of immunized mice obtained after a second injection of alloantigen as a rule stimulated macrophage migration. The results are evidence that T cells may function in vivo during the period before development of the antigen-specific proliferative response of T cells. The technique used to approach the problem, described in this study, can be used for preparative isolation of purified MIF and MSF without contamination by embryonic calf serum proteins which are usually present in culture in vitro.

  6. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    SciTech Connect

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  7. Integrin α6β4 Promotes Autocrine Epidermal Growth Factor Receptor (EGFR) Signaling to Stimulate Migration and Invasion toward Hepatocyte Growth Factor (HGF).

    PubMed

    Carpenter, Brittany L; Chen, Min; Knifley, Teresa; Davis, Kelley A; Harrison, Susan M W; Stewart, Rachel L; O'Connor, Kathleen L

    2015-11-06

    Integrin α6β4 is up-regulated in pancreatic adenocarcinomas where it contributes to carcinoma cell invasion by altering the transcriptome. In this study, we found that integrin α6β4 up-regulates several genes in the epidermal growth factor receptor (EGFR) pathway, including amphiregulin (AREG), epiregulin (EREG), and ectodomain cleavage protease MMP1, which is mediated by promoter demethylation and NFAT5. The correlation of these genes with integrin α6β4 was confirmed in The Cancer Genome Atlas Pancreatic Cancer Database. Based on previous observations that integrin α6β4 cooperates with c-Met in pancreatic cancers, we examined the impact of EGFR signaling on hepatocyte growth factor (HGF)-stimulated migration and invasion. We found that AREG and EREG were required for autocrine EGFR signaling, as knocking down either ligand inhibited HGF-mediated migration and invasion. We further determined that HGF induced secretion of AREG, which is dependent on integrin-growth factor signaling pathways, including MAPK, PI3K, and PKC. Moreover, matrix metalloproteinase activity and integrin α6β4 signaling were required for AREG secretion. Blocking EGFR signaling with EGFR-specific antibodies or an EGFR tyrosine kinase inhibitor hindered HGF-stimulated pancreatic carcinoma cell chemotaxis and invasive growth in three-dimensional culture. Finally, we found that EGFR was phosphorylated in response to HGF stimulation that is dependent on EGFR kinase activity; however, c-Met phosphorylation in response to HGF was unaffected by EGFR signaling. Taken together, these data illustrate that integrin α6β4 stimulates invasion by promoting autocrine EGFR signaling through transcriptional up-regulation of key EGFR family members and by facilitating HGF-stimulated EGFR ligand secretion. These signaling events, in turn, promote pancreatic carcinoma migration and invasion.

  8. Granulocyte Colony-stimulating Factor Producing Anaplastic Carcinoma of the Pancreas: Case Report and Review of the Literature.

    PubMed

    Vinzens, Sarah; Zindel, Joel; Zweifel, Martin; Rau, Tilman; Gloor, Beat; Wochner, Annette

    2017-01-01

    We report on the case of a 67-year-old man with granulocyte colony-stimulating factor (G-CSF) producing anaplastic carcinoma of the pancreas. Preoperative routine tests revealed an elevated white blood cell (WBC) count of 25.2 G/l, consisting almost exclusively of neutrophilic granulocytes (23.31 G/l) with a predominance of segmented neutrophils (78% of all neutrophilic granulocytes), and elevated levels of C-reactive protein at 87 mg/l. Upon surgery, local tumour infiltration was more extensive than expected from preoperative imaging. However, no peritoneal dissemination was found and curative resection was attempted. Only seven days after the operation, signs of relapse were seen upon computed tomograpy. Histology revealed an undifferentiated anaplastic carcinoma, on the basis of a poorly differentiated ductal adenocarcinoma. Immunohistochemistry demonstrated G-CSF and G-CSF-Receptor expression in some CD68-positive syncytial macrophages. Granulocyte colony-stimulating factor (G-CSF) in serum was elevated at 5.6 pg/ml, which further raised to 43 pg/ml one week after FOLFIRINOX chemotherapy (oxaliplatin, irinotecan, 5-fluorouracil), while WBC decreased from 103.3 G/l to 59.3 G/l. Granulocyte macrophage-colony stimulating factor (GM-CSF) in serum was normal (<0.5 pg/ml). The patient died on postoperative day 34.

  9. Glucose and Insulin Stimulate Lipogenesis in Porcine Adipocytes: Dissimilar and Identical Regulation Pathway for Key Transcription Factors

    PubMed Central

    Hua, Zhang Guo; Xiong, Lu Jian; Yan, Chen; Wei, Dai Hong; YingPai, ZhaXi; Qing, Zhao Yong; Lin, Qiao Zi; Fei, Feng Ruo; Ling, Wang Ya; Ren, Ma Zhong

    2016-01-01

    Lipogenesis is under the concerted action of ChREBP, SREBP-1c and other transcription factors in response to glucose and insulin. The isolated porcine preadipocytes were differentiated into mature adipocytes to investigate the roles and interrelation of these transcription factors in the context of glucose- and insulin-induced lipogenesis in pigs. In ChREBP-silenced adipocytes, glucose-induced lipogenesis decreased by ~70%, however insulin-induced lipogenesis was unaffected. Moreover, insulin had no effect on ChREBP expression of unperturbed adipocytes irrespective of glucose concentration, suggesting ChREBP mediate glucose-induced lipogenesis. Insulin stimulated SREBP-1c expression and when SREBP-1c activation was blocked, and the insulin-induced lipogenesis decreased by ~55%, suggesting SREBP-1c is a key transcription factor mediating insulin-induced lipogenesis. LXRα activation promoted lipogenesis and lipogenic genes expression. In ChREBP-silenced or SREBP-1c activation blocked adipocytes, LXRα activation facilitated lipogenesis and SREBP-1c expression, but had no effect on ChREBP expression. Therefore, LXRα might mediate lipogenesis via SREBP-1c rather than ChREBP. When ChREBP expression was silenced and SREBP-1c activation blocked simultaneously, glucose and insulin were still able to stimulated lipogenesis and lipogenic genes expression, and LXRα activation enhanced these effects, suggesting LXRα mediated directly glucose- and insulin-induced lipogenesis. In summary, glucose and insulin stimulated lipogenesis through both dissimilar and identical regulation pathway in porcine adipocytes. PMID:27871177

  10. RhoC Mediates Epidermal Growth Factor-Stimulated Migration and Invasion in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Tumur, Zohra; Katebzadeh, Shahbaz; Guerra, Carlos; Bhushan, Lokesh; Alkam, Tursun; Henson, Bradley S.

    2015-01-01

    Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where it has been shown to promote tumor cell invasion upon phosphorylation. One mechanism by which EGFR promotes tumor progression is by activating signal cascades that lead to loss of E-cadherin, a transmembrane glycoprotein of the cell-cell adherence junctions; however mediators of these signaling cascades are not fully understood. One such mediator, RhoC, is activated upon a number of external stimuli, such as epidermal growth factor (EGF), but its role as a mediator of EGF-stimulated migration and invasion has not been elucidated in HNSCC. In the present study, we investigate the role of RhoC as a mediator of EGF-stimulated migration and invasion in HNSCC. We show that upon EGF stimulation, EGFR and RhoC were strongly activated in HNSCC. This resulted in activation of the phosphatidylinositol 3-Kinase Akt pathway (PI3K-Akt), phosphorylation of GSK-3β at the Ser9 residue, and subsequent down regulation of E-cadherin cell surface expression resulting in increased tumor cell invasion. Knockdown of RhoC restored E-cadherin expression and inhibited EGF-stimulated migration and invasion. This is the first report in HNSCC demonstrating the role RhoC plays in mediating EGF-stimulated migration and invasion by down-regulating the PI3K-Akt pathway and E-cadherin expression. RhoC may serve as a treatment target for HNSCC. PMID:25622907

  11. Transforming growth factor-beta 1 stimulates synthesis of proteoglycan aggregates in calf articular cartilage organ cultures

    SciTech Connect

    Morales, T.I. )

    1991-04-01

    Previous work showed that transforming growth factor-beta 1 (TGF-beta 1), added alone to bovine cartilage organ cultures, stimulated (35S)sulfate incorporation into macromolecular material but did not investigate the fidelity of the stimulated system to maintain synthesis of cartilage-type proteoglycans. This paper provides evidence that chondrocytes synthesize the appropriate proteoglycan matrix under TGF-beta 1 stimulation: (1) there is a coordinated increase in hyaluronic acid and proteoglycan monomer synthesis, (2) link-stable proteoglycan aggregates are assembled, (3) the hybrid chondroitin sulfate/keratan sulfate monomeric species is synthesized, and (4) there is an increase in protein core synthesis. Some variation in glycosylation patterns was observed when proteoglycans synthesized under TGF-beta 1 stimulation were compared to those synthesized under basal conditions. Thus comparing TGF-beta 1 to basal samples respectively, the monomers were larger (Kav on Sepharose CL-2B = 0.29 vs 0.41), the chondroitin sulfate chains were longer by approximately 3.5 kDa, the percentage of total glycosaminoglycan in keratan sulfate increased slightly from approximately 4% (basal) to approximately 6%, and the unsulfated disaccharide decreased from 28% (basal) to 12%. All of these variations are in the direction of a more anionic proteoglycan. Since the ability of proteoglycans to confer resiliency to the cartilage matrix is directly related to their anionic nature, these changes would presumably have a beneficial effect on tissue function.

  12. Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling.

    PubMed

    Frey, Mark R; Golovin, Anastasia; Polk, D Brent

    2004-10-22

    Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.

  13. Insulin-like growth factor I stimulates lipid oxidation, reduces protein oxidation, and enhances insulin sensitivity in humans.

    PubMed Central

    Hussain, M A; Schmitz, O; Mengel, A; Keller, A; Christiansen, J S; Zapf, J; Froesch, E R

    1993-01-01

    To elucidate the effects of insulin-like growth factor I (IGF-I) on fuel oxidation and insulin sensitivity, eight healthy subjects were treated with saline and recombinant human (IGF-I (10 micrograms/kg.h) during 5 d in a crossover, randomized fashion, while receiving an isocaloric diet (30 kcal/kg.d) throughout the study period. On the third and fourth treatment days, respectively, an L-arginine stimulation test and an intravenous glucose tolerance test were performed. A euglycemic, hyperinsulinemic clamp combined with indirect calorimetry and a glucose tracer infusion were performed on the fifth treatment day. IGF-I treatment led to reduced fasting and stimulated (glucose and/or L-arginine) insulin and growth hormone secretion. Basal and stimulated glucagon secretion remained unchanged. Intravenous glucose tolerance was unaltered despite reduced insulin secretion. Resting energy expenditure and lipid oxidation were both elevated, while protein oxidation was reduced, and glucose turnover rates were unaltered on the fifth treatment day with IGF-I as compared to the control period. Enhanced lipolysis was reflected by elevated circulating free fatty acids. Moreover, insulin-stimulated oxidative and nonoxidative glucose disposal (i.e., insulin sensitivity) were enhanced during IGF-I treatment. Thus, IGF-I treatment leads to marked changes in lipid and protein oxidation, whereas, at the dose used, carbohydrate metabolism remains unaltered in the face of reduced insulin levels and enhanced insulin sensitivity. Images PMID:8227340

  14. Chicken tumor necrosis-like factor. I. In vitro production by macrophages stimulated with Eimeria tenella or bacterial lipopolysaccharide.

    PubMed

    Zhang, S; Lillehoj, H S; Ruff, M D

    1995-08-01

    HD11, a transformed avian macrophage cell line, and chicken peripheral blood leukocyte-derived macrophages (PBL-M phi) were stimulated with bacterial endotoxin lipopolysaccharide (LPS) or Eimeria tenella sporozoites and merozoites. The specific cytotoxicities of the culture supernatants against different target cell lines were measured, and the kinetics of tumor necrosis-like factor (TNF) production by HD11 and PBL-M phi were also measured. The results showed that HD11 and PBL-M phi secreted a TNF-like factor when stimulated with Eimeria parasites or LPS. A time- and dose-dependent TNF-like factors production by PBL-M phi was observed poststimulation with Eimeria parasites. Chicken TNF-like factor preferentially kills CHCC OU-2 cells, a fibroblast cell line of chicken origin, when compared to LM cells, a murine cell line used for mammalian TNF. This study indicates that chicken M phi produce a significant level of TNF-like factor following coccidial infection.

  15. Immunomodulation of Bu-Zhong-Yi-Qi-Tang on in vitro granulocyte colony-stimulating-factor and tumor necrosis factor-alpha production by peripheral blood mononuclear cells.

    PubMed

    Kao, S T; Yang, S L; Hsieh, C C; Yang, M D; Wang, T F; Lin, J G

    2000-11-01

    Bu-Zhong-Yi-Qi-Tang (BZYQT) is a Chinese medicine, and has been used for the treatment of hepatocellular carcinoma (HCC) patients. At present, we still do not fully understand the effects of BZYQT on the cellular physiology. Present in vitro study demonstrated that BZYQT is capable of increasing granulocyte colony-stimulating-factor (G-CSF) and tumor necrosis factor-alpha (TNF-alpha) production by peripheral blood mononuclear cells (PBMC) in healthy volunteers and patients with HCC. The productions of G-CSF and TNF-alpha by PBMC of volunteers were significantly stimulated by more than 125 microg/ml of BZYQT. G-CSF levels stimulated by PBMC of healthy volunteers were higher than in PBMC of the HCC patients when more than 625 microg/ml of BZYQT was administrated. The reason may be due to the impaired immunologic reactivity of mononuclear cells in HCC patients. However, the production levels of TNF-alpha in HCC patients can be stimulated to levels as high as those in healthy volunteers. When adding high concentration (3.125 mg/ml) of BZYQT to the cultured PBMC, the increments of G-CSF and TNF-alpha production decreased although there were no obvious changes in the number of metabolic active PBMC changed. TNF-alpha andG-CSF are known to play important roles in the biological defensive mechanism. These findings show that BZYQT is a unique formula for the stimulation of PBMC to produce G-CSF and TNF-alpha. Administration of BZYQT may be beneficial for patients with HCC to modulate these cytokines.

  16. Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism

    PubMed Central

    McElroy, Steven J.; Frey, Mark R.; Yan, Fang; Edelblum, Karen L.; Goettel, Jeremy A.; John, Sutha; Polk, D. Brent

    2008-01-01

    Tumor necrosis factor (TNF) and epidermal growth factor (EGF) are key regulators in the intricate balance maintaining intestinal homeostasis. Previous work from our laboratory shows that TNF attenuates ligand-driven EGF receptor (EGFR) phosphorylation in intestinal epithelial cells. To identify the mechanisms underlying this effect, we examined EGFR phosphorylation in cells lacking individual TNF receptors. TNF attenuated EGF-stimulated EGFR phosphorylation in wild-type and TNFR2−/−, but not TNFR1−/−, mouse colon epithelial (MCE) cells. Reexpression of wild-type TNFR1 in TNFR1−/− MCE cells rescued TNF-induced EGFR inhibition, but expression of TNFR1 deletion mutant constructs lacking the death domain (DD) of TNFR1 did not, implicating this domain in EGFR downregulation. Blockade of p38 MAPK, but not MEK, activation of ERK rescued EGF-stimulated phosphorylation in the presence of TNF, consistent with the ability of TNFR1 to stimulate p38 phosphorylation. TNF promoted p38-dependent EGFR internalization in MCE cells, suggesting that desensitization is achieved by reducing receptor accessible to ligand. Taken together, these data indicate that TNF activates TNFR1 by DD- and p38-dependent mechanisms to promote EGFR internalization, with potential impact on EGF-induced proliferation and migration key processes that promote healing in inflammatory intestinal diseases. PMID:18467504

  17. Insulin-like growth factor-I-stimulated Akt phosphorylation and oligodendrocyte progenitor cell survival require cholesterol-enriched membranes.

    PubMed

    Romanelli, Robert J; Mahajan, Kedar R; Fulmer, Clifton G; Wood, Teresa L

    2009-11-15

    Previously we showed that insulin-like growth factor-I (IGF-I) promotes sustained phosphorylation of Akt in oligodendrocyte progenitor cells (OPCs) and that Akt phosphorylation is required for survival of these cells. The direct mechanisms, however, by which IGF-I promotes Akt phosphorylation are currently undefined. Recently, cholesterol-enriched membranes (CEMs) have been implicated in regulation of growth factor-mediated activation of the PI3K/Akt pathway and survival of mature oligodendrocytes; however, less is know about their role in OPC survival. In the present study, we investigate the role of CEMs in IGF-I-mediated Akt phosphorylation and OPC survival. We report that acute disruption of membrane cholesterol with methyl-beta-cyclodextrin results in altered OPC morphology and inhibition of IGF-I-mediated Akt phosphorylation. We also report that long-term inhibition of cholesterol biosynthesis with 25-hydroxycholesterol blocks IGF-I stimulated Akt phosphorylation and cell survival. Moreover, we show that the PI3K regulatory subunit, p85, Akt, and the IGF-IR are sequestered within cholesterol-enriched fractions in steady-state stimulation of the IGF-IR and that phosphorylated Akt and IGF-IR are present in cholesterol-enriched fractions with IGF-I stimulation. Together, the results of these studies support a role for CEMs or "lipid rafts" in IGF-I-mediated Akt phosphorylation and provide a better understanding of the mechanisms by which IGF-I promotes OPC survival.

  18. [LED LIGHTING AS A FACTOR FOR THE STIMULATION OF THE HORMONE SYSTEM].

    PubMed

    Deynego, V N; Kaptsov, V A

    2015-01-01

    There are considered questions of non-visual effects of blue LED light sources on hormonal systems (cortisol, glucose, insulin) providing the high human performance. In modern conditions hygiene strategy for child and adolescent health strategy was shown to be replaced by a strategy of light stimulation of the hormonal profile. There was performed a systematic analysis of the axis "light stimulus-hypothalamus-pituitary-adrenals-cortisol-glucose-insulin". The elevation of the content of cortisol leads to the increase of the glucose level in the blood and the stimulation of the production of insulin, which can, like excessive consumption of food, give rise to irreversible decline in the number of insulin receptors on the cell surface, and thus--to a steady reduction in the ability of cells to utilize glucose, i.e. to type 2 diabetes or its aggravation.

  19. Pharmacological analysis for mechanisms of GPI-80 release from tumour necrosis factor-alpha-stimulated human neutrophils.

    PubMed

    Nitto, Takeaki; Araki, Yoshihiko; Takeda, Yuji; Sendo, Fujiro

    2002-10-01

    1 GPI-80, a glycosylphosphatidylinositol (GPI)-anchored protein initially identified on human neutrophils, plays a role(s) in the regulation of beta2 integrin function. Previous studies have shown that GPI-80 is sublocated in secretory vesicles. It is also found in soluble form in the synovial fluid of rheumatoid arthritis patients, and in the culture supernatant of formyl-methionyl-leucyl-phenylalanine-stimulated neutrophils. To understand the behaviour of GPI-80 under conditions of stimulation, we investigated the effects of tumour necrosis factor (TNF)-alpha on its expression and release. We also probed the mechanism of its release with various pharmacologic tools. 2 TNF-alpha induced the release of GPI-80 from human neutrophils in a concentration- and time-dependent manner (in the range of 1-100 u ml(-1) and 30-120 min, respectively), but did not affect surface GPI-80 levels. 3 Cytochalasin B, genistein, and SB203580 but not PD98059 inhibited TNF-alpha-stimulated GPI-80 release and neutrophil adherence at the same concentration. In addition, TNF-alpha-induced GPI-80 release was inhibited by blocking monoclonal antibodies specific to components of Mac-1 (CD11b and CD18). 4 Antioxidants (pyrrolidine dithiocarbamate and N-acetyl-L-cysteine) inhibited GPI-80 release by TNF-alpha stimulation, but superoxide dismutase did not. Antioxidants but not superoxide dismutase reduced an intracellular oxidation state. 5 These findings indicate that TNF-alpha-stimulated GPI-80 release from human neutrophils depends upon adherence via beta2 integrins. They also suggest that cytochalasin B, genistein, and SB203580 inhibit GPI-80 release by suppressing signals for cell adherence, rather than by a direct effect on its secretion. Finally, we suggest that GPI-80 release involves an intracellular change in a redox state.

  20. Factors involved in the relaxation of female pig urethra evoked by electrical field stimulation.

    PubMed Central

    Werkström, V.; Persson, K.; Ny, L.; Bridgewater, M.; Brading, A. F.; Andersson, K. E.

    1995-01-01

    1. Non-adrenergic, non-cholinergic (NANC) relaxations induced by electrical field stimulation (EFS) were studied in pig isolated urethra. The mechanism for relaxation was characterized by measurement of cyclic nucleotides and by study of involvement of different subsets of voltage-operated calcium channels (VOCCs). 2. EFS evoked frequency-dependent and tetrodotoxin-sensitive relaxations in the presence of propranolol (1 microM), phentolamine (1 microM) and scopolamine (1 microM). At low frequencies (< 12 Hz), relaxations were rapid, whereas at high (> 12 Hz) frequencies distinct biphasic relaxations were evoked. The latter consisted of a rapidly developing first phase followed by a more long-lasting second phase. 3. Treatment with the NO-synthesis inhibitor NG-nitro-L-arginine (L-NOARG; 0.3 mM) inhibited relaxations at low frequencies of stimulation. At high frequencies (> 12 Hz) only the first relaxation phase was affected. 4. Measurement of cyclic nucleotides in preparations subjected to continuous nerve-stimulation, revealed an increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels from 1.3 +/- 0.3 to 3.0 +/- 0.4 pmol mg-1 protein (P < 0.01). In the presence of L-NOARG, there was a significant decrease in cyclic GMP content to control. However, there was no increase in cyclic GMP content in response to EFS. Levels of cyclic AMP remained unchanged following EFS. 5. Treatment with the N-type VOCC-inhibitor, omega-conotoxin GVIA (0.1 microM) reduced NO-dependent relaxations, the effect being most pronounced at low frequencies (1-4 Hz) of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8564225

  1. Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin

    SciTech Connect

    Parries, G.; Hoebel, R.; Racker, E.

    1987-05-01

    Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradyknin to activate phospholipase C-mediated phosphoinositide hydrolysis. In (/sup 3/H)inositol-labeled rat-1 fibroblasts, PDGF resulted in a 2-fold increase in the level of (/sup 3/H)inositol trisphosphate (InsP/sub 3/) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of (/sup 3/H)inositol monophosphate (InsP/sub 1/) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either (/sup 3/H)InsP/sub 3/ or (/sup 3/H)InsP/sub 1/. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21/sup ras/ and the loss of PDGF-stimulated (/sup 3/H)InsP/sub 1/ accumulation. The authors propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.

  2. Transforming growth factor-alpha in vivo stimulates epithelial cell proliferation in digestive tissues of suckling rats.

    PubMed Central

    Hormi, K; Lehy, T

    1996-01-01

    BACKGROUND: The role that exogenous transforming growth factor-alpha (TGF-alpha) may exert on cell proliferation in vivo is poorly understood. AIM: To investigate the effect of rat TGF-alpha on epithelial cell proliferation in all suckling rat digestive tissues and to compare it with that of rat epidermal growth factor (EGF). ANIMAL AND METHODS: TGF-alpha and EGF were given three times daily either subcutaneously (10 or 20 micrograms/kg) or intraperitoneally (100 micrograms/kg) to rats from the ninth postnatal day. Cell proliferation was assessed through 5-bromo- 2-deoxyuridine incorporation and estimation of labelling indices. RESULTS: For both growth factors, the highest dose given for only two days significantly increased stomach and intestinal weights compared with controls (p < 0.05 to p < 0.001). The proliferative responded depended on the dose given, colonic mucosa being the most sensitive whereas oxyntic mucosa remained unresponsive. TGF-alpha was as potent as EGF in stimulating epithelial cell proliferation in antral, duodenal, and colonic mucosae. However, EGF was more active on oesophageal and jejunal cell proliferation whereas TGF-alpha was more active on pancreatic exocrine cell proliferation and the differences between the two growth factor treated groups were significant. CONCLUSIONS: These results prove for the first time the stimulating effect in vivo of exogenous rat TGF-alpha on epithelial cell proliferation in rat digestive tissues during the developmental period and support a functional role for TGF-alpha at that time. PMID:8944561

  3. Stimulation of human monocytes with macrophage colony-stimulating factor induces a Grb2-mediated association of the focal adhesion kinase pp125FAK and dynamin.

    PubMed Central

    Kharbanda, S; Saleem, A; Yuan, Z; Emoto, Y; Prasad, K V; Kufe, D

    1995-01-01

    Macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. In the present studies using human monocytes, we show that M-CSF induces interaction of the Grb2 adaptor protein with the focal adhesion kinase pp125FAK. The results demonstrate that tyrosine-phosphorylated pp125FAK directly interacts with the SH2 domain of Grb2. The findings indicate that a pYENV site at Tyr-925 in pp125FAK is responsible for this interaction. We also demonstrate that the Grb2-FAK complex associates with the GTPase dynamin. Dynamin interacts with the SH3 domains of Grb2 and exhibits M-CSF-dependent tyrosine phosphorylation in association with pp125FAK. These findings suggest that M-CSF-induced signaling involves independent Grb2-mediated pathways, one leading to Ras activation and another involving pp125FAK and a GTPase implicated in receptor internalization. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7597091

  4. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation.

    PubMed

    Frelinger, A L; Torres, A S; Caiafa, A; Morton, C A; Berny-Lang, M A; Gerrits, A J; Carmichael, S L; Neculaes, V B; Michelson, A D

    2016-01-01

    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications.

  5. Comparative proteome analysis of Tumor necrosis factor α-stimulated human Vascular Smooth Muscle Cells in response to melittin

    PubMed Central

    2013-01-01

    Background Bee venom has been used to relieve pain and to treat inflammatory diseases, including rheumatoid arthritis, in humans. To better understand the mechanisms of the anti-inflammatory and anti-atherosclerosis effect of bee venom, gel electrophoresis and mass spectrometry were used to identify proteins whose expression was altered in human Vascular Smooth Muscle Cells (hVSMCs) stimulated by tumor necrosis factor alpha after 12 h in the presence of melittin. Results To obtain valuable insights into the anti-inflammatory and anti-atherosclerosis mechanisms of melittin, two-dimensional (2-D) gel electrophoresis and MALDI-TOF/TOF were used. The proteome study, we showed 33 significant proteins that were differentially expressed in the cells treated with tumor necrosis factor alpha and melittin. Thirteen proteins were significantly increased in the cells treated with tumor necrosis factor alpha, and those proteins were reduced in the cells treated with melittin. Five of the proteins that showed increased expression in the cells treated with tumor necrosis factor alpha are involved in cell migration, including calreticulin, an essential factor of development that plays a role in transcription regulation. The proteins involved in cell migration were reduced in the melittin treated cells. The observed changes in the expression of GRP75, prohibitin, and a select group of other proteins were validated with reverse transcribed-PCR. It was confirmed that the observed change in the protein levels reflected a change in the genes level. In addition, the phosphorylation of EGFR and ERK was validated by analyzing the protein pathway. Conclusion Taken together, these data established that the expression of some proteins was significantly changed by melittin treatment in tumor necrosis factor alpha stimulated the cells and provided insights into the mechanism of the melittin function for its potential use as an anti-inflammatory agent. PMID:23651618

  6. Glucose Stimulation of Transforming Growth Factor-β Bioactivity in Mesangial Cells Is Mediated by Thrombospondin-1

    PubMed Central

    Poczatek, Maria H.; Hugo, Christian; Darley-Usmar, Victor; Murphy-Ullrich, Joanne E.

    2000-01-01

    Glucose is a key factor in the development of diabetic complications, including diabetic nephropathy. The development of diabetic glomerulosclerosis is dependent on the fibrogenic growth factor, transforming growth factor-β (TGF-β). Previously we showed that thrombospondin-1 (TSP-1) activates latent TGF-β both in vitro and in vivo. Activation occurs as the result of specific interactions of latent TGF-β with TSP-1, which potentially alter the conformation of latent TGF-β. As glucose also up-regulates TSP-1 expression, we hypothesized that the increased TGF-β bioactivity observed in rat and human mesangial cells cultured with high glucose concentrations is the result of latent TGF-β activation by autocrine TSP-1. Glucose-induced bioactivity of TGF-β in mesangial cell cultures was reduced to basal levels by peptides from two different sequences that antagonize activation of latent TGF-β by TSP, but not by the plasmin inhibitor, aprotinin. Furthermore, glucose-dependent stimulation of matrix protein synthesis was inhibited by these antagonist peptides. These studies demonstrate that glucose stimulation of TGF-β activity and the resultant matrix protein synthesis are dependent on the action of autocrine TSP-1 to convert latent TGF-β to its biologically active form. These data suggest that antagonists of TSP-dependent TGF-β activation may be the basis of novel therapeutic approaches for ameliorating diabetic renal fibrosis. PMID:11021838

  7. The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells.

    PubMed

    Yang, Chao-Huei; Tsao, Chiung-Fang; Ko, Wang-Sheng; Chiou, Ya-Ling

    2016-01-09

    In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%-99% after 48 h (p < 0.05) and induced G₁/G₀ cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials.

  8. The Oligo Fucoidan Inhibits Platelet-Derived Growth Factor-Stimulated Proliferation of Airway Smooth Muscle Cells

    PubMed Central

    Yang, Chao-Huei; Tsao, Chiung-Fang; Ko, Wang-Sheng; Chiou, Ya-Ling

    2016-01-01

    In the pathogenesis of asthma, the proliferation of airway smooth muscle cells (ASMCs) is a key factor in airway remodeling and causes airway narrowing. In addition, ASMCs are also the effector cells of airway inflammation. Fucoidan extracted from marine brown algae polysaccharides has antiviral, antioxidant, antimicrobial, anticlotting, and anticancer properties; however, its effectiveness for asthma has not been elucidated thus far. Platelet-derived growth factor (PDGF)-treated primary ASMCs were cultured with or without oligo-fucoidan (100, 500, or 1000 µg/mL) to evaluate its effects on cell proliferation, cell cycle, apoptosis, and Akt, ERK1/2 signaling pathway. We found that PDGF (40 ng/mL) increased the proliferation of ASMCs by 2.5-fold after 48 h (p < 0.05). Oligo-fucoidan reduced the proliferation of PDGF-stimulated ASMCs by 75%–99% after 48 h (p < 0.05) and induced G1/G0 cell cycle arrest, but did not induce apoptosis. Further, oligo-fucoidan supplementation reduced PDGF-stimulated extracellular signal-regulated kinase (ERK1/2), Akt, and nuclear factor (NF)-κB phosphorylation. Taken together, oligo-fucoidan supplementation might reduce proliferation of PDGF-treated ASMCs through the suppression of ERK1/2 and Akt phosphorylation and NF-κB activation. The results provide basis for future animal experiments and human trials. PMID:26761017

  9. Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures.

    PubMed

    Kwon, Tae-Ho; Kim, Young-Sook; Lee, Jae-Hwa; Yang, Moon-Sik

    2003-09-01

    A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 microg l(-1) after 10 d' cultivation.

  10. Macrophage colony-stimulating factor mRNA and protein in atherosclerotic lesions of rabbits and humans.

    PubMed Central

    Rosenfeld, M. E.; Ylä-Herttuala, S.; Lipton, B. A.; Ord, V. A.; Witztum, J. L.; Steinberg, D.

    1992-01-01

    In this study, the authors demonstrate the expression of mRNA and the presence of protein for macrophage colony-stimulating factor (MCSF) in atherosclerotic lesions from humans and rabbits. In situ hybridization of serial sections of human fatty streaks demonstrated expression of MCSF mRNA by cells dispersed throughout the lesions. Immunocytochemical staining with a panel of MCSF-specific antibodies showed extensive cell-associated staining of all of the cell types in the lesions. Immunocytochemical studies of atherosclerotic lesions from Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits demonstrated a similar cell-associated pattern of staining. There was no MCSF-specific staining of aortas from normal rabbits or of cultured aortic smooth muscle cells from either humans or rabbits. Macrophage-derived foam cells (MFC) were isolated from the aortas of ballooned, cholesterol-fed rabbits. A Northern blot demonstrated that RNA isolated from the MFC hybridized with a human cDNA probe for MCSF. RNA from alveolar macrophages isolated simultaneously from the same rabbits did not hybridize with the MCSF probe. Conditioned media from an 18- to 24-hour incubation of the MFC contained colony-stimulating activity as demonstrated in a mouse bone marrow culture assay. Most of this colony-stimulating activity was neutralized by preincubating the conditioned media with an MCSF-specific antibody. Images Figure 2 Figure 1 Figure 1 Figure 3 PMID:1739123

  11. Excess glucose induces hypoxia-inducible factor-1α in pancreatic cancer cells and stimulates glucose metabolism and cell migration

    PubMed Central

    Liu, Zhiwen; Jia, Xiaohui; Duan, Yijie; Xiao, Huijie; Sundqvist, Karl-Gösta; Permert, Johan; Wang, Feng

    2013-01-01

    Pancreatic cancer patients frequently show hyperglycemia, but it is uncertain whether hyperglycemia stimulates pancreatic cancer cells. We have investigated whether excess glucose induces hypoxia-inducible factor-1α (HIF-1α) and stimulates glucose metabolism and cell migration in pancreatic cancer cells. We studied wild-type (wt) MiaPaCa2 pancreatic cancer cells and a MiaPaCa2 subline (namely si-MiaPaCa2) that had HIF-1α-specific small interfering RNA. Wt-MiaPaCa2 cells are known to be HIF-1α-positive in hypoxia and HIF-1α-negative in normoxia, whereas si-MiaPaCa2 cells are devoid of HIF-1α in both normoxia and hypoxia. We incubated these cells with different amounts of glucose and determined HIF-1α mRNA and protein by real-time polymerase chain reaction and western blotting. We determined glucose consumption, lactate production and intracellular hexokinase-II and ATP to assess glucose metabolisms and determined pyruvate dehydrogenase kinase-1, reactive oxygen species and fumarate to assess mitochondrial activities. Further, we studied cell migration using a Boyden chamber. Excess glucose (16.7−22.2mM) increased HIF-1α in hypoxic wt-MiaPaCa2 cells. HIF-1α expression increased ATP contents and inhibited mitochondrial activities. Extracellular glucose and hypoxia stimulated glucose metabolisms independent of HIF-1α. Excess glucose stimulated the migration of wt- and si-MiaPaCa2 cells in both normoxia and hypoxia. Thus, glucose stimulated cell migration independent of HIF-1α. Nevertheless, hypoxic wt-MiaPaCa2 cells showed greater migrating ability than their si-MiaPaCa2 counterparts. We conclude that (1) excess glucose increases HIF-1α and ATP in hypoxic wt-MiaPaCa2 cells, (2) extracellular glucose and hypoxia regulate glucose metabolisms independent of HIF-1α and (3) glucose stimulates cell migration by mechanisms that are both dependent on HIF-1α and independent of it. PMID:23377827

  12. Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

    PubMed Central

    Hartmann, G.; Krug, A.; Waller-Fontaine, K.; Endres, S.

    1996-01-01

    BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for

  13. Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

    PubMed Central

    Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

    2014-01-01

    It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

  14. Platelet-activating factor stimulates metabolism of phosphoinositides via phospholipase A2 in primary cultured rat hepatocytes

    SciTech Connect

    Okayasu, T.; Hasegawa, K.; Ishibashi, T.

    1987-07-01

    Addition of platelet-activating factor (PAF) to cells doubly labeled with (/sup 14/C)glycerol plus (/sup 3/H)arachidonic acid resulted in a transient decrease of (/sup 14/C)glycerol-labeled phosphatidylinositol (PI) and a transient increase of (/sup 14/C)glycerol-labeled lysophosphatidylinositol (LPI). (/sup 3/H)Arachidonate-labeled PI, on the other hand, decreased in a time-dependent manner. The radioactivity in phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, and phosphatidylserine did not change significantly. The /sup 3/H//sup 14/C ratio decreased in PI in a time-dependent manner, suggesting the involvement of a phospholipase A2 activity. Although PAF also induced a gradual increase of diacylglycerol (DG), the increase of (/sup 14/C)glycerol-labeled DG paralleled the loss of triacyl (/sup 14/C)glycerol and the /sup 3/H//sup 14/C ratio of DG was 16 times smaller than that of PI. Thus, DG seemed not to be derived from PI. In myo- (/sup 3/H)inositol-prelabeled cells, PAF induced a transient decrease of (/sup 3/H)phosphatidylinositol-4,5-bis-phosphate (TPI) and (/sup 3/H)phosphatidylinositol-4-phosphate (DPI) at 1 min. PAF stimulation of cultured hepatocytes prelabeled with /sup 32/Pi induced a transient decrease of (/sup 32/P)polyphosphoinositides at 20 sec to 1 min. (/sup 32/P)LPI appeared within 10 sec after stimulation and paralleled the loss of (/sup 32/P)PI. (/sup 3/H)Inositol triphosphate, (/sup 3/H)inositol diphosphate, and (/sup 3/H)inositol phosphate, which increased in a time-dependent manner upon stimulation with adrenaline, did not accumulate with the stimulation due to PAF. These observations indicate that PAF causes degradation of inositol phospholipids via phospholipase A2 and induces a subsequent resynthesis of these phospholipids.

  15. Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells.

    PubMed

    Beazely, Michael A; Alan, Jamie K; Watts, Val J

    2005-01-01

    Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin- and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.

  16. Effect of Granulocyte Colony-Stimulating Factor Immobilized by the Electron-Beam Synthesis Nanotechnology on Reparative Regeneration of Spermatogenous Tissue.

    PubMed

    Borovskaya, T G; Dygai, A M; Shchemerova, Yu A; Kamalova, S I; Mashanova, V A; Vychuzhanina, A V; Poluektova, M E; Madonov, P G; Kinsht, D N; Goldberg, V E

    2016-09-01

    Effectiveness of the granulocyte colony-stimulating factor immobilized by using electronbeam synthesis nanotechnology was investigated on the model of experimental testicular failure caused by the toxic effect on stem spermatogonia. Administration of the drug to experimental paclitaxel-treated animals increased the number of sources of the proliferative pool of spermatogenesis and its productivity. The effectiveness of immobilized granulocyte colony-stimulating factor was based on its ability to stimulate reparative regeneration of the spermatogenic tissue, which manifested in a decrease in spermatogenic layer maturity and increase in the number of microenvironment cells. Effectiveness of the immobilized form of the drug was superior to that of non-immobilized form.

  17. Mechanochemical switching between growth and differentiation during fibroblast growth factor-stimulated angiogenesis in vitro: role of extracellular matrix

    PubMed Central

    1989-01-01

    The angiogenic factor, basic fibroblast growth factor (FGF), either stimulates endothelial cell growth or promotes capillary differentiation depending upon the microenvironment in which it acts. Analysis of various in vitro models of spontaneous angiogenesis, in combination with time-lapse cinematography, demonstrated that capillary tube formation was greatly facilitated by promoting multicellular retraction and cell elevation above the surface of the rigid culture dish or by culturing endothelial cells on malleable extracellular matrix (ECM) substrata. These observations suggested to us that mechanical (i.e., tension-dependent) interactions between endothelial cells and ECM may serve to regulate capillary development. To test this hypothesis, FGF-stimulated endothelial cells were grown in chemically defined medium on bacteriological (nonadhesive) dishes that were precoated with different densities of fibronectin. Extensive cell spreading and growth were promoted by fibronectin coating densities that were highly adhesive (greater than 500 ng/cm2), whereas cell rounding, detachment, and loss of viability were observed on dishes coated with low fibronectin concentrations (less than 100 ng/cm2). Intermediate fibronectin coating densities (100-500 ng/cm2) promoted cell extension, but they could not completely resist cell tractional forces. Partial retraction of multicellular aggregates resulted in cell shortening, cessation of growth, and formation of branching tubular networks within 24-48 h. Multicellular retraction and subsequent tube formation also could be elicited on highly adhesive dishes by overcoming the mechanical resistance of the substratum using higher cell plating numbers. Dishes coated with varying concentrations of type IV collagen or gelatin produced similar results. These results suggest that ECM components may act locally to regulate the growth and pattern- regulating actions of soluble FGF based upon their ability to resist cell-generated mechanical

  18. Selective Endothelin-B Receptor Stimulation Increases Vascular Endothelial Growth Factor in the Rat Brain during Postnatal Development.

    PubMed

    Leonard, M G; Prazad, P; Puppala, B; Gulati, A

    2015-11-01

    Endothelin, vascular endothelial growth factor and nerve growth factor play important roles in development of the central nervous system. ET(B) receptors have been shown to promote neurovascular remodeling in the adult ischemic brain through an increase in VEGF and NGF. It is possible that ET(B) receptors may be involved in postnatal development of the brain through VEGF and NGF. In the present study, the brains of male rat pups on postnatal days 1, 7, 14 and 28 were analyzed for expression of ET(B) receptors, VEGF and NGF. In order to determine the effect of ET(B) receptor stimulation, a separate group of pups were administered saline or ET(B) receptor agonist, IRL-1620, on day 21, and their brains were analyzed on day 28. The intensity of ET(B) receptor and VEGF staining in the vasculature as well as the number of blood vessels of normal pups increased with age and was significantly higher on postnatal day 14 compared to day 1 and day 7. In contrast, both ET(B) and NGF staining intensity in the cortex and subventricular zones decreased (P<0.01) at postnatal day 14 compared to earlier time points. Stimulation of ET(B) receptors resulted in a significant increase in VEGF and ET(B) intensity both in the vasculature and the brain (P<0.05), however, IRL-1620 did not produce any change in NGF expression. Results indicate that ET(B) receptors appear to play a role in the development of the CNS and selective stimulation of ET(B) receptors enhances VEGF but not NGF in the postnatal rat brain.

  19. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    SciTech Connect

    Story, M.T. )

    1989-05-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

  20. Hematological and hepatic effects of vascular epidermal growth factor (VEGF) used to stimulate hair growth in an animal model

    PubMed Central

    2013-01-01

    Background Alopecia areata is the hair loss usually reversible, in sharply defined areas. The treatment of alopecia using growth factors shows interesting activity in promoting hair growth. In this concept, VEGF (vascular endothelial growth factor) is a marker of angiogenesis, stimulating hair growth by facilitating the supply of nutrients to the hair follicle, increasing follicular diameter. The aim of this study was the evaluation of a topical gel enriched with VEGF liposomes on the hair growth stimulation and its toxicological aspects. Methods Mesocricetus auratus were randomly divided into three groups. Control group was treated with Aristoflex® gel, 1% group with the same gel but added 1% VEGF and 3% group with 3% VEGF. Biochemical, hematological and histological analyses were done. Results At the end of the experiment (15th day of VEGF treatment) efficacy was determined macroscopically by hair density dermatoscopy analysis, and microscopically by hair diameter analysis. They both demonstrated that hair of the VEGF group increased faster and thicker than control. On the other hand, biochemical and hematological results had shown that VEGF was not 100% inert. Conclusions VEGF increased hair follicle area, but more studies are necessary to confirm its toxicity. PMID:24168457

  1. Efficacy of Intrauterine infusion of granulocyte colony stimulating factor on patients with history of implantation failure: A randomized control trial

    PubMed Central

    Eftekhar, Maryam; Miraj, Sepideh; Farid Mojtahedi, Maryam; Neghab, Nosrat

    2016-01-01

    Background: Although pregnancy rate in in vitro fertilization-embryo transfer (IVF-ET) cycles has been increased over the preceding years, but the majority of IVF-ET cycles still fail. Granulocyte colony stimulating factor (GCSF) is a glycoprotein that stimulates cytokine growth factor and induces immune system which may improve pregnancy rate in women with history of implantation failure. Objective: The aim of this study was to evaluate GCSF ability to improve pregnancy rate in women with history of implantation failure Materials and Methods: 0.5 ml (300 µg/ml) GCSF was infused intrauterine in intervention group. Pregnancy outcomes were assessed based on clinical pregnancy. Results: The mean age of participants was 31.95±4.71 years old. There were no significant differences between demographic characteristics in two groups (p>0.05). The pregnancy outcome in GCSF group was improved significantly (p=0.043). Conclusion: GCSF can improve pregnancy outcome in patients with history of implantation failure. PMID:27981253

  2. Sitagliptin attenuates inflammatory responses in lipopolysaccharide-stimulated cardiomyocytes via nuclear factor-κB pathway inhibition.

    PubMed

    Lin, Chien-Hung; Lin, Chung-Ching

    2016-06-01

    Glucagon-like peptide-1 (GLP-1) and GLP-1 receptors (GLP-1Rs) are responsible for glucose homeostasis, and have been shown to reduce inflammation in preclinical studies. The aim of the present study was to determine whether sitagliptin, an inhibitor of the enzyme dipeptidyl peptidase-4 (DPP-4), as a GLP-1 receptor agonist, exerts an anti-inflammatory effect on cardiomyoblasts during lipopolysaccharide (LPS) stimulation. Exposure to LPS increased the expression levels of tumor necrosis factor (TNF)-α, interleukin-6 (IL)-6 and IL-1β in H9c2 cells, and also resulted in elevations in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) nuclear translocation. Treatment with the DPP-4 inhibitor sitagliptin dose-dependently downregulated the mRNA levels of IL-6, COX-2 and iNOS in LPS-stimulated H9c2 cells. In addition, sitagliptin inhibited the increased protein expression of IL-6, TNF-α and IL-1β. NF-κB mRNA expression was reduced and its translocation to the nucleus was suppressed by treatment with sitagliptin. The present results demonstrated that sitagliptin exerts a beneficial effect on cardiomyoblasts exposed to LPS by inhibiting expression of inflammatory mediators and suppressing NF-κB activation. These findings indicate that the DPP-4 inhibitor sitagliptin may serve a function in cardiac remodeling attributed to sepsis-induced inflammation.

  3. The efficiency of granulocyte colony-stimulating factor in hemorrhagic mucositis and febrile neutropenia resulted from methotrexate toxicity.

    PubMed

    Ozkol, Hatice Uce; Toptas, Tayfur; Calka, Omer; Akdeniz, Necmettin

    2015-01-01

    Methotrexate (MTX) remains one of the most frequently used anti-metabolite agents in dermatology. MTX is an analog of folate that competitively and irreversibly inhibits dihydrofolate reductase. Oral mucositis is a common side effect of chemotherapy drugs and is characterized by erythema, pain, poor oral intake, pseudomembranous destruction, open ulceration and hemorrhage of the oral mucosa. In this paper, we report a 32-year-old female with a case of mucositis due to MTX intoxication that resulted from an overdose for rheumatoid arthritis. The patient had abdominal pain, vomiting, and nausea. During follow-up, the patient's white blood cell count was found to be 0.9 × 10(9)/L (4-10 × 10(9)/L). The patient developed fever exceeding 40 °C. The patient was consulted to the hematology service. They suggested using granulocyte colony-stimulating factor for febrile neutropenia. On the fifth day of treatment, the white blood cell count reached 5.3 × 10(9)/L and the patient's fever and mucositis started to resolve. Here, we presented a case of hemorrhagic mucositis and febrile neutropenia resulted from high-dose MTX that responded very well to granulocyte colony-stimulating factor treatment and we reviewed the literature.

  4. Growth factor- and cytokine-stimulated endothelial progenitor cells in post-ischemic cerebral neovascularization

    PubMed Central

    Peplow, Philip V.

    2014-01-01

    Endothelial progenitor cells are resident in the bone marrow blood sinusoids and circulate in the peripheral circulation. They mobilize from the bone marrow after vascular injury and home to the site of injury where they differentiate into endothelial cells. Activation and mobilization of endothelial progenitor cells from the bone marrow is induced via the production and release of endothelial progenitor cell-activating factors and includes specific growth factors and cytokines in response to peripheral tissue hypoxia such as after acute ischemic stroke or trauma. Endothelial progenitor cells migrate and home to specific sites following ischemic stroke via growth factor/cytokine gradients. Some growth factors are less stable under acidic conditions of tissue ischemia, and synthetic analogues that are stable at low pH may provide a more effective therapeutic approach for inducing endothelial progenitor cell mobilization and promoting cerebral neovascularization following ischemic stroke. PMID:25317152

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

    PubMed

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

    2016-04-01

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

  6. Vitamin D Receptor Deficiency and Low Vitamin D Diet Stimulate Aortic Calcification and Osteogenic Key Factor Expression in Mice

    PubMed Central

    Schmidt, Nadine; Brandsch, Corinna; Kühne, Hagen; Thiele, Alexandra; Hirche, Frank; Stangl, Gabriele I.

    2012-01-01

    Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR−/−) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR−/−) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D3 over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas. PMID:22536373

  7. Energy absorption buildup factors, exposure buildup factors and Kerma for optically stimulated luminescence materials and their tissue equivalence for radiation dosimetry

    NASA Astrophysics Data System (ADS)

    Singh, Vishwanath P.; Badiger, N. M.

    2014-11-01

    Optically stimulated luminescence (OSL) materials are sensitive dosimetric materials used for precise and accurate dose measurement for low-energy ionizing radiation. Low dose measurement capability with improved sensitivity makes these dosimeters very useful for diagnostic imaging, personnel monitoring and environmental radiation dosimetry. Gamma ray energy absorption buildup factors and exposure build factors were computed for OSL materials using the five-parameter Geometric Progression (G-P) fitting method in the energy range 0.015-15 MeV for penetration depths up to 40 mean free path. The computed energy absorption buildup factor and exposure buildup factor values were studied as a function of penetration depth and incident photon energy. Effective atomic numbers and Kerma relative to air of the selected OSL materials and tissue equivalence were computed and compared with that of water, PMMA and ICRU standard tissues. The buildup factors and kerma relative to air were found dependent upon effective atomic numbers. Buildup factors determined in the present work should be useful in radiation dosimetry, medical diagnostics and therapy, space dosimetry, accident dosimetry and personnel monitoring.

  8. Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells

    SciTech Connect

    Kakudo, Natsuko . E-mail: kakudon@takii.kmu.ac.jp; Shimotsuma, Ayuko; Kusumoto, Kenji

    2007-07-27

    Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), a key transcription factor in adipogenesis, was upregulated. The results of FGF-2 supplementation suggest the potential applications of FGF-2 and ASCs in adipose tissue regeneration.

  9. Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T.

    PubMed

    Sarilmiser, Hande Kazak; Ates, Ozlem; Ozdemir, Gonca; Arga, Kazim Yalcin; Oner, Ebru Toksoy

    2015-04-01

    Levan is a bioactive fructan polymer that is mainly associated with high-value applications where exceptionally high purity requirements call for well-defined cultivation conditions. In this study, microbial levan production by the halophilic extremophile Halomonas smyrnensis AAD6(T) was investigated systematically. For this, different feeding strategies in fed-batch cultures were employed and fermentation profiles of both shaking and bioreactor cultures were analyzed. Initial carbon and nitrogen source concentrations, production pH, NaCl and nitrogen pulses, nitrogen and phosphorous limitations, trace elements and thiamine contents of the basal production medium were found to affect the levan yields at different extends. Boric acid was found to be the most effective stimulator of levan production by increasing the sucrose utilization three-fold and levan production up to five-fold. This significant improvement implied the important role of quorum sensing phenomenon and its regulatory impact on levan production mechanism. Levan produced by bioreactor cultures under conditions optimized within this study was found to retain its chemical structure. Moreover, its biocompatibility was assessed for a broad concentration range. Hence H. smyrnensis AAD6(T) has been firmly established as an industrially important resource microorganism for high-quality levan production.

  10. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing.

    PubMed

    Jettanacheawchankit, Suwimon; Sasithanasate, Siriruk; Sangvanich, Polkit; Banlunara, Wijit; Thunyakitpisal, Pasutha

    2009-04-01

    Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [(3)H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol; and Carbopol containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 - 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

  11. Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells

    SciTech Connect

    Uzumaki, Hiroya; Okabe, Tetsuro; Sasaki, Norio; Hagiwara, Koichi; Takaku, Fumimaro; Tobita, Masahito; Yasukawa, Kaoru ); Ito, Seiga ); Umezawa, Yoshimi )

    1989-12-01

    Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, the authors synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the {sup 125}I-labeled mutein of human G-CSF (KW-2228). The specific binding of {sup 125}I-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time- and temperature-dependent, with maximal binding at 4{degree}C after a 24-hr incubation. When the authors examined the ability of hematopoietic growth factors to inhibit {sup 125}I-labeled KW-2228 binding, they found that KW-2228 and intact human G-CSF ihibited {sup 125}I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colony-stimulating factor did not. Scatchard analysis revealed a single receptor type. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species that could be specifically cross-linked to {sup 125}I-labeled KW-2228. Human trophoblastic cells, T3M-3, also possessed a single receptor for G-CSF. They have identified the receptor for human G-CSF on human placental membranes and trophoblastic cells.

  12. Stimulation of chick embryo cartilage sulfate and thymidine uptake: comparison of human serum, purified somatomedins, and other growth factors.

    PubMed

    Jennings, J; Buchanan, F; Freeman, D; Garland, J T

    1980-11-01

    We have compared the stimulation of sulfate and thymidine uptake into 10-day-old embryonic chick cartilage by normal human serum, partially purified somatomedins (Sm) A and B, homogeneous insulin-like growth factors (IGFs) I and II, and several other substances. With the exception of epidermal growth factor, all growth factors ((GFs) were assayed in the absence of other protein. Pelvic rudiments were preincubated in buffer for 6 h and then incubated for 24 h with the GF or serum, with labels added for the final 6 h. Human serum enhanced cartilage uptake of both thymidine and sulfate. There was a dose-dependent stimulation of thymidine uptake by Sm A or B (0.05--2 microgram/ml) and IGF I or II (0.5--20 ng/ml). Unlike serum, neither Sms nor IGFs increased SO4 uptake under these conditions. Bovine GH (10--500 ng/ml), albumin (100-1000 ng/ml), fibroblast GF (1--100 ng/ml), and epidermal GF (1--100 ng/ml) were inactive for both thymidine and sulfate. When a shorter incubation was used (7 h), Sm A enhanced SO4 uptake, and discrimination was increased by preincubation of the rudiments in buffer for 24 h. With this procedure, IGF I (0.5 ng/ml) was nearly equipotent to 5% serum. On a weight basis, IGF I was more active than either Sm A or IGF II. The data suggest that assay conditions are crucial for demonstration of Sm activity. Appropriate conditions may be different for isolated GF than for a complex medium such as serum. The results further suggest that with certain protocols, the responsiveness of chick embryo cartilage is qualitatively similar to that of hypophysectomized rat cartilage.

  13. The La protein counteracts cisplatin-induced cell death by stimulating protein synthesis of anti-apoptotic factor Bcl2

    PubMed Central

    Heise, Tilman; Kota, Venkatesh; Brock, Alexander; Morris, Amanda B.; Rodriguez, Reycel M.; Zierk, Avery W.; Howe, Philip H.; Sommer, Gunhild

    2016-01-01

    Up-regulation of anti-apoptotic factors is a critical mechanism of cancer cell resistance and often counteracts the success of chemotherapeutic treatment. Herein, we identified the cancer-associated RNA-binding protein La as novel factor contributing to cisplatin resistance. Our data demonstrate that depletion of the RNA-binding protein La in head and neck squamous cell carcinoma cells (HNSCC) increases the sensitivity toward cisplatin-induced cell death paralleled by reduced expression of the anti-apoptotic factor Bcl2. Furthermore, it is shown that transient expression of Bcl2 in La-depleted cells protects against cisplatin-induced cell death. By dissecting the underlying mechanism we report herein, that the La protein is required for Bcl2 protein synthesis in cisplatin-treated cells. The RNA chaperone La binds in close proximity to the authentic translation start site and unwinds a secondary structure embedding the authentic AUG. Altogether, our data support a novel model, whereby cancer-associated La protein contributes to cisplatin resistance by stimulating the translation of anti-apoptotic factor Bcl2 in HNSCC cells. PMID:27105491

  14. Clinical Factors Underlying the Inter-individual Variability of the Resting Motor Threshold in Navigated Transcranial Magnetic Stimulation Motor Mapping.

    PubMed

    Sollmann, Nico; Tanigawa, Noriko; Bulubas, Lucia; Sabih, Jamil; Zimmer, Claus; Ringel, Florian; Meyer, Bernhard; Krieg, Sandro M

    2017-01-01

    Correctly determining individual's resting motor threshold (rMT) is crucial for accurate and reliable mapping by navigated transcranial magnetic stimulation (nTMS), which is especially true for preoperative motor mapping in brain tumor patients. However, systematic data analysis on clinical factors underlying inter-individual rMT variability in neurosurgical motor mapping is sparse. The present study examined 14 preselected clinical factors that may underlie inter-individual rMT variability by performing multiple regression analysis (backward, followed by forward model comparisons) on the nTMS motor mapping data of 100 brain tumor patients. Data were collected from preoperative motor mapping of abductor pollicis brevis (APB), abductor digiti minimi (ADM), and flexor carpi radialis (FCR) muscle representations among these patients. While edema and age at exam in the ADM model only jointly reduced the unexplained variance significantly, the other factors kept in the ADM model (gender, antiepileptic drug intake, and motor deficit) and each of the factors kept in the APB and FCR models independently significantly reduced the unexplained variance. Hence, several clinical parameters contribute to inter-individual rMT variability and should be taken into account during initial and follow-up motor mappings. Thus, the present study adds basic evidence on inter-individual rMT variability, whereby some of the parameters are specific to brain tumor patients.

  15. The La protein counteracts cisplatin-induced cell death by stimulating protein synthesis of anti-apoptotic factor Bcl2.

    PubMed

    Heise, Tilman; Kota, Venkatesh; Brock, Alexander; Morris, Amanda B; Rodriguez, Reycel M; Zierk, Avery W; Howe, Philip H; Sommer, Gunhild

    2016-05-17

    Up-regulation of anti-apoptotic factors is a critical mechanism of cancer cell resistance and often counteracts the success of chemotherapeutic treatment. Herein, we identified the cancer-associated RNA-binding protein La as novel factor contributing to cisplatin resistance. Our data demonstrate that depletion of the RNA-binding protein La in head and neck squamous cell carcinoma cells (HNSCC) increases the sensitivity toward cisplatin-induced cell death paralleled by reduced expression of the anti-apoptotic factor Bcl2. Furthermore, it is shown that transient expression of Bcl2 in La-depleted cells protects against cisplatin-induced cell death. By dissecting the underlying mechanism we report herein, that the La protein is required for Bcl2 protein synthesis in cisplatin-treated cells. The RNA chaperone La binds in close proximity to the authentic translation start site and unwinds a secondary structure embedding the authentic AUG. Altogether, our data support a novel model, whereby cancer-associated La protein contributes to cisplatin resistance by stimulating the translation of anti-apoptotic factor Bcl2 in HNSCC cells.

  16. Induction of monocyte migration by recombinant macrophage colony-stimulating factor.

    PubMed

    Wang, J M; Griffin, J D; Rambaldi, A; Chen, Z G; Mantovani, A

    1988-07-15

    Human recombinant macrophage-CSF (M-CSF) induced migration across polycarbonate or nitrocellulose filters of human peripheral blood monocytes. Checkerboard analysis of M-CSF-induced migration, performed by seeding different cytokine concentrations above and below the filter, revealed that the locomotory response involved chemotaxis, though some gradient-independent augmentation of migration occurred. Polymixin B did not affect M-CSF chemotaxis and M-CSF was active on monocytes from the LPS-unresponsive mouse strain C3H/HeJ. These findings rule out a contribution of minute endotoxin contamination, below the sensitivity of the Limulus assay, in M-CSF chemotaxis. Rabbit anti-M-CSF antibodies inhibited the chemotactic activity of recombinant M-CSF, thus further indicating that the M-CSF molecule was indeed responsible for chemotaxis. M-CSF preparations encoded by 224 or 522 amino acid cDNA clones were equally effective in inducing monocyte migration. Recombinant M-CSF did not elicit a migratory response in large granular lymphocytes and in endothelial cells under conditions in which appropriate reference attractants were active. A modest stimulation of migration of polymorphonuclear leukocytes, inhibitable by antibodies, was observed at high cytokine concentrations (10 to 100 times higher than those required for monocyte locomotion). The maximal polymorphonuclear leukocytes response evoked by M-CSF was small compared to that evoked by reference chemoattractants or to that evoked by the same cytokine in monocytes. Hence, M-CSF is a potent chemoattractant for mononuclear phagocytes and exerts its action preferentially on cells of the monocyte-macrophage lineage. M-CSF, produced locally by activated macrophages, may play a role in the selective recruitment from the blood compartment of mononuclear phagocytes to amplify resistance against certain noxious agents.

  17. Breast cancer cells induce osteoclast formation by stimulating host IL-11 production and downregulating granulocyte/macrophage colony-stimulating factor.

    PubMed

    Morgan, Hayley; Tumber, Anthony; Hill, Peter A

    2004-05-01

    Breast cancer cells frequently metastasize to the skeleton, where they induce OCL formation and activity, resulting in extensive bone destruction. However, the mechanisms by which breast cancer cells mediate increased osteolysis remain unclear. To elucidate this point, we investigated how 3 human breast cancer cell lines, MDA-MB-231, MDA-MB-435 and MCF-7, induce OCL formation using a murine osteoblast-spleen cell coculture system and compared their effects with a human colorectal cancer cell line, HCT-15; a human lung cancer cell line, HT-1080; and a normal human breast cell line, HME. The breast cancer cell lines supported OCL formation only when osteoblasts were present in spleen cell cocultures, whilst the non-breast cancer cell lines and the normal breast cell line, HME, had no effect. Fractionation of BCCM by ultrafiltration established that osteoclastogenic activity was associated with factors having m.w. >3 kDa. Breast cancer cell lines produced primarily PTHrP, with lesser amounts of IL-6, IL-11 and TNF-alpha. The effect of BCCM on OCL formation in osteoblast-spleen cell cocultures was partially prevented by a neutralising antibody to human PTHrP and completely prevented by a neutralising antibody to either murine IL-11 or the murine IL-11 receptor; neutralising antibodies to human IL-6, IL-11 or TNF-alpha were without effect. BCCM or human PTHrP induced an increase in murine osteoblast IL-11 mRNA and protein production, effects that were prevented in the presence of a neutralising antibody to human PTHrP. The osteoclastogenic activity of IL-11 was mediated by enhancing osteoblast production of PGE(2) effects, which were abrogated by an inhibitor of cyclooxygenase. PGE(2) apparently enhanced OCL formation by downregulating GM-CSF production by spleen cells since recombinant murine GM-CSF inhibited OCL formation and a neutralising antibody to murine GM-CSF blocked these inhibitory effects. We conclude that breast cancer cells induce OCL formation by

  18. The novel growth factor, progranulin, stimulates mouse cholangiocyte proliferation via sirtuin-1-mediated inactivation of FOXO1.

    PubMed

    Frampton, Gabriel; Ueno, Yoshiyuki; Quinn, Matthew; McMillin, Matthew; Pae, Hae Yong; Galindo, Cheryl; Leyva-Illades, Dinorah; DeMorrow, Sharon

    2012-12-01

    Progranulin (PGRN), a secreted growth factor, regulates the proliferation of various epithelial cells. Its mechanism of action is largely unknown. Sirtuin 1 (Sirt1) is a protein deacetylase that is known to regulate the transcriptional activity of the forkhead receptor FOXO1, thereby modulating the balance between proapoptotic and cell cycle-arresting genes. We have shown that PGRN is overexpressed in cholangiocarcinoma and stimulates proliferation. However, its effects on hyperplastic cholangiocyte proliferation are unknown. In the present study, the expression of PGRN and its downstream targets was determined after bile duct ligation (BDL) in mice and in a mouse cholangiocyte cell line after stimulation with PGRN. The effects of PGRN on cholangiocyte proliferation were assessed in sham-operated (sham) and BDL mice treated with PGRN or by specifically knocking down endogenous PGRN expression using Vivo-Morpholinos or short hairpin RNA. PGRN expression and secretion were upregulated in proliferating cholangiocytes isolated after BDL. Treatment of mice with PGRN increased biliary mass and cholangiocyte proliferation in vivo and in vitro and enhanced cholangiocyte proliferation observed after BDL. PGRN treatment decreased Sirt1 expression and increased the acetylation of FOXO1, resulting in the cytoplasmic accumulation of FOXO1 in cholangiocytes. Overexpression of Sirt1 in vitro prevented the proliferative effects of PGRN. Conversely, knocking down PGRN expression in vitro or in vivo inhibited cholangiocyte proliferation. In conclusion, these data suggest that the upregulation of PGRN may be a key feature stimulating cholangiocyte proliferation. Modulating PGRN levels may be a viable technique for regulating the balance between ductal proliferation and ductopenia observed in a variety of cholangiopathies.

  19. Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters.

    PubMed Central

    Du, H; Roy, A L; Roeder, R G

    1993-01-01

    Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad-ML) promoter by binding to an E-box motif located at position -60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix-loop-helix protein. In this report, we demonstrate a 25-to 30-fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine-rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV-1 promoter, as well as those in the Ad-ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co-transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43-dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV-1 and Ad-ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear. Images PMID:8440240

  20. Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

    PubMed Central

    Baier-Bitterlich, G; Uberall, F; Bauer, B; Fresser, F; Wachter, H; Grunicke, H; Utermann, G; Altman, A; Baier, G

    1996-01-01

    T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation

  1. Trophic factors and cell therapy to stimulate brain repair after ischaemic stroke

    PubMed Central

    Gutiérrez-Fernández, María; Fuentes, Blanca; Rodríguez-Frutos, Berta; Ramos-Cejudo, Jaime; Vallejo-Cremades, María Teresa; Díez-Tejedor, Exuperio

    2012-01-01

    Brain repair involves a compendium of natural mechanisms that are activated following stroke. From a therapeutic viewpoint, reparative therapies that encourage cerebral plasticity are needed. In the last years, it has been demonstrated that modulatory treatments for brain repair such as trophic factor- and stem cell-based therapies can promote neurogenesis, gliogenesis, oligodendrogenesis, synaptogenesis and angiogenesis, all of which having a beneficial impact on infarct volume, cell death and, finally, and most importantly, on the functional recovery. However, even when promising results have been obtained in a wide range of experimental animal models and conditions these preliminary results have not yet demonstrated their clinical efficacy. Here, we focus on brain repair modulatory treatments for ischaemic stroke, that use trophic factors, drugs with trophic effects and stem cell therapy. Important and still unanswered questions for translational research ranging from experimental animal models to recent and ongoing clinical trials are reviewed here. PMID:22452968

  2. Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

    PubMed Central

    Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

    2013-01-01

    Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

  3. [Signaling of granulocyte macrophage colony stimulating factor and its clinical application: host-defense and organ protection].

    PubMed

    Uchida, Kanji

    2013-03-01

    Granulocyte macrophage colony stimulating factor (GM-CSF) is a cytokine with multipotent properties. It has not only an activity to generate both granulocyte and macrophage lineages in the bone marrow, but also is capable of inducing terminal maturation of alveolar macrophages that is central for pulmonary host defense and pulmonary surfactant homeostasis. GM-CSF can stimulate mature myeloid cells (i.e. neutrophils and monocytes) with a known mechanism called "priming" to efficiently eliminate invading pathogens. Several clinical trials to evaluate therapeutic efficacy of GM-CSF in patients with diseases related to functional impairment of mature myeloid cells were reported. Inhalation of GM-CSF improved clinical severity of pulmonary alveolar proteionosis. Administration of GM-CSF for patients with immune compromised situation such as sepsis showed marginal benefits so far. Several animal experiments indicated neuroprotective effect of GM-CSE In the clinical setting, establishing reliable biomarkers to distinguish patients who will have benefit by administering GM-CSF may maximize its clinical efficacy.

  4. Garlic (Allium sativum) stimulates lipopolysaccharide-induced tumor necrosis factor-alpha production from J774A.1 murine macrophages.

    PubMed

    Sung, Jessica; Harfouche, Youssef; De La Cruz, Melissa; Zamora, Martha P; Liu, Yan; Rego, James A; Buckley, Nancy E

    2015-02-01

    Garlic (Allium sativum) is known to have many beneficial attributes such as antimicrobial, antiatherosclerotic, antitumorigenetic, and immunomodulatory properties. In the present study, we investigated the effects of an aqueous garlic extract on macrophage cytokine production by challenging the macrophage J774A.1 cell line with the garlic extract in the absence or presence of lipopolysaccharide (LPS) under different conditions. The effect of allicin, the major component of crushed garlic, was also investigated. Using enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction, it was found that garlic and synthetic allicin greatly stimulated tumor necrosis factor-alpha (TNF-α) production in macrophages treated with LPS. The TNF-α secretion levels peaked earlier and were sustained for a longer time in cells treated with garlic and LPS compared with cells treated with LPS alone. Garlic acted in a time-dependent manner. We suggest that garlic, at least partially via its allicin component, acts downstream from LPS to stimulate macrophage TNF-α secretion.

  5. Effects of a granulocyte colony stimulating factor, Neulasta, in mini pigs exposed to total body proton irradiation

    PubMed Central

    Sanzari, Jenine K.; Krigsfeld, Gabriel S.; Shuman, Anne L.; Diener, Antonia K.; Lin, Liyong; Mai, Wilfried; Kennedy, Ann R.

    2015-01-01

    Astronauts could be exposed to solar particle event (SPE) radiation, which is comprised mostly of proton radiation. Proton radiation is also a treatment option for certain cancers. Both astronauts and clinical patients exposed to ionizing radiation are at risk for white blood cell (WBC) loss, which are the body’s main defense against infection. In this report, the effect of Neulasta treatment, a granulocyte colony stimulating factor, after proton radiation exposure is discussed. Mini pigs exposed to total body proton irradiation at a dose of 2 Gy received 4 treatments of either Neulasta or saline injections. Peripheral blood cell counts and thromboelastography parameters were recorded up to 30 days post-irradiation. Neulasta significantly improved white blood cell (WBC), specifically neutrophil, loss in irradiated animals by approximately 60% three days after the first injection, compared to the saline treated irradiated animals. Blood cell counts quickly decreased after the last Neulasta injection, suggesting a transient effect on WBC stimulation. Statistically significant changes in hemostasis parameters were observed after proton radiation exposure in both the saline and Neulasta treated irradiated groups, as well internal organ complications such as pulmonary changes. In conclusion, Neulasta treatment temporarily alleviates proton radiation-induced WBC loss, but has no effect on altered hemostatic responses. PMID:25909052

  6. Production of mouse granulocyte-macrophage colony-stimulating factor by gateway technology and transgenic rice cell culture.

    PubMed

    Liu, Yu-Kuo; Huang, Li-Fen; Ho, Shin-Lon; Liao, Chun-Yu; Liu, Hsin-Yi; Lai, Ying-Hui; Yu, Su-May; Lu, Chung-An

    2012-05-01

    To establish a production platform for recombinant proteins in rice suspension cells, we first constructed a Gateway-compatible binary T-DNA destination vector. It provided a reliable and effective method for the rapid directional cloning of target genes into plant cells through Agrobacterium-mediated transformation. We used the approach to produce mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) in a rice suspension cell system. The promoter for the αAmy3 amylase gene, which is induced strongly by sugar depletion, drove the expression of mGM-CSF. The resulting recombinant protein was fused with the αAmy3 signal peptide and was secreted into the culture medium. The production of rice-derived mGM-CSF (rmGM-CSF) was scaled up successfully in a 2-L bioreactor, in which the highest yield of rmGM-CSF was 24.6 mg/L. Due to post-translational glycosylation, the molecular weight of rmGM-CSF was larger than that of recombinant mGM-CSF produced in Escherichia coli. The rmGM-CSF was bioactive and could stimulate the proliferation of a murine myeloblastic leukemia cell line, NSF-60.

  7. Oral phosphorus supplementation secondarily increases circulating fibroblast growth factor 23 levels at least partially via stimulation of parathyroid hormone secretion.

    PubMed

    Takasugi, Satoshi; Akutsu, Miho; Nagata, Masashi

    2014-01-01

    Oral phosphorus supplementation stimulates fibroblast growth factor 23 (FGF23) secretion; however, the underlying mechanism remains unclear. The aim of this study was to investigate the involvement of parathyroid hormone (PTH) in increased plasma FGF23 levels after oral phosphorus supplementation in rats. Rats received single dose of phosphate with concomitant subcutaneous injection of saline or human PTH (1-34) after treatment with cinacalcet or its vehicle. Cinacalcet is a drug that acts as an allosteric activator of the calcium-sensing receptor and reduces PTH secretion. Plasma phosphorus and PTH levels significantly increased 1 h after oral phosphorus administration and returned to basal levels within 3 h, while plasma FGF23 levels did not change up to 2 h post-treatment, but rather significantly increased at 3 h after administration and maintained higher levels for at least 6 h compared with the 0 time point. Plasma PTH and FGF23 levels were significantly lower in the cinacalcet-treated rats than in the vehicle-treated rats. Plasma phosphorus levels were significantly higher in the cinacalcet-treated rats than in the vehicle-treated rats at 2, 3, 4, and 6 h after oral phosphorus administration. Furthermore, rats treated with cinacalcet+human PTH (1-34) showed transiently but significantly higher plasma FGF23 levels at 3 h after oral phosphorus administration compared with cinacalcet-treated rats. These results suggest that oral phosphorus supplementation secondarily increases circulating FGF23 levels at least partially by stimulation of PTH secretion.

  8. The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis

    PubMed Central

    Arenz, Stefan; Abdelshahid, Maha; Sohmen, Daniel; Payoe, Roshani; Starosta, Agata L.; Berninghausen, Otto; Hauryliuk, Vasili; Beckmann, Roland; Wilson, Daniel N.

    2016-01-01

    Under stress conditions, such as nutrient starvation, deacylated tRNAs bound within the ribosomal A-site are recognized by the stringent factor RelA, which converts ATP and GTP/GDP to (p)ppGpp. The signaling molecules (p)ppGpp globally rewire the cellular transcriptional program and general metabolism, leading to stress adaptation. Despite the additional importance of the stringent response for regulation of bacterial virulence, antibiotic resistance and persistence, structural insight into how the ribosome and deacylated-tRNA stimulate RelA-mediated (p)ppGpp has been lacking. Here, we present a cryo-EM structure of RelA in complex with the Escherichia coli 70S ribosome with an average resolution of 3.7 Å and local resolution of 4 to >10 Å for RelA. The structure reveals that RelA adopts a unique ‘open’ conformation, where the C-terminal domain (CTD) is intertwined around an A/T-like tRNA within the intersubunit cavity of the ribosome and the N-terminal domain (NTD) extends into the solvent. We propose that the open conformation of RelA on the ribosome relieves the autoinhibitory effect of the CTD on the NTD, thus leading to stimulation of (p)ppGpp synthesis by RelA. PMID:27226493

  9. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  10. Effects of a granulocyte colony stimulating factor, Neulasta, in mini pigs exposed to total body proton irradiation

    NASA Astrophysics Data System (ADS)

    Sanzari, Jenine K.; Krigsfeld, Gabriel S.; Shuman, Anne L.; Diener, Antonia K.; Lin, Liyong; Mai, Wilfried; Kennedy, Ann R.

    2015-04-01

    Astronauts could be exposed to solar particle event (SPE) radiation, which is comprised mostly of proton radiation. Proton radiation is also a treatment option for certain cancers. Both astronauts and clinical patients exposed to ionizing radiation are at risk for loss of white blood cells (WBCs), which are the body's main defense against infection. In this report, the effect of Neulasta treatment, a granulocyte colony stimulating factor, after proton radiation exposure is discussed. Mini pigs exposed to total body proton irradiation at a dose of 2 Gy received 4 treatments of either Neulasta or saline injections. Peripheral blood cell counts and thromboelastography parameters were recorded up to 30 days post-irradiation. Neulasta significantly improved WBC loss, specifically neutrophils, in irradiated animals by approximately 60% three days after the first injection, compared to the saline treated, irradiated animals. Blood cell counts quickly decreased after the last Neulasta injection, suggesting a transient effect on WBC stimulation. Statistically significant changes in hemostasis parameters were observed after proton radiation exposure in both the saline and Neulasta treated irradiated groups, as well as internal organ complications such as pulmonary changes. In conclusion, Neulasta treatment temporarily alleviates proton radiation-induced WBC loss, but has no effect on altered hemostatic responses.

  11. 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

  12. Weekly CODE chemotherapy with recombinant human granulocyte colony-stimulating factor for relapsed or refractory small cell lung cancer.

    PubMed

    Sato, K; Tsuchiya, S; Minato, K; Sunaga, N; Ishihara, S I; Makimoto, T; Naruse, I; Hoshino, H; Watanabe, S; Saitoh, R; Mori, M

    2000-01-01

    We used cisplatin, vincristine, doxorubicin, and etoposide (CODE) plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) weekly for salvage chemotherapy in relapsed or refractory small cell lung cancer (SCLC). We reviewed the medical charts of patients between January 1993 and December 1996 at the National Nishi-Gunma Hospital. Twenty patients were treated with salvage chemotherapy. The overall response rate was 55.0%. The median survival time of extensive disease patients from the start of CODE therapy was 23 weeks and the 1-year survival rate was 21.0%. Toxicities were severe, especially in myelosuppression. CODE could be selected as a salvage therapy for chemotherapy- relapsed SCLC cases.

  13. Survival enhancement and hemopoietic regeneration following radiation exposure: therapeutic approach using glucan and granulocyte colony-stimulating factor

    SciTech Connect

    Patchen, M.L.; MacVittie, T.J.; Solberg, B.D.; Souza, L.M.

    1990-01-01

    C3H/HeN female mice were exposed to whole-body cobalt-60 radiation and administered soluble glucan (5 mg i.v. at 1 h following exposure), recombinant human granulocyte colony-stimulating factor or both agents. Treatments were evaluated for their ability to enhance hemopoietic regeneration, and to increase survival after radiation-induced myelosuppression. Both glucan and G-CSF enhanced hemopoietic regeneration alone; however, greater effects were observed in mice receiving both agents. For example, on day 17 following a sublethal 6.5-Gy radiation exposure, mice treated with saline, G-CSF, glucan, or both agents, respectively, exhibited 36%, 65%, 50%, and 78% of normal bone marrow cellularity, and 84%, 175%, 152%, and 212% of normal splenic cellularity.

  14. Antigen-and ionophore-stimulated synthesis of platelet-activating factor by the cloned mast cell line, MC9

    SciTech Connect

    Musch, M.W.; Billah, M.M.; Siegel, M.I.

    1987-05-14

    MC9 mast cells stimulated by a soluble (calcium ionophore A23187) or by an Fc epsilon-receptor agonist (IgE plus hapten) produce platelet activating factor (PAF). MC9 cells incorporate either exogenous (/sup 3/H)acetic acid or (/sup 3/H)lyso-PAF into PAF. PAF was identified by mobility on thin layer chromatography, platelet aggregatory activity inhibitable by known PAF antagonists, and by enzymatic modification. Quantified by aggregation of rabbit platelets, MC9 cells produce 6 pmoles PAF/10(6) cells. MC9 cells express acetyltransferase activity of 0.19 nmole/5 min-mg protein. Analysis of MC9 phospholipids by HPLC showed that MC9 cells contain large amounts of phosphatidylcholine (82 nmoles/10(7) cells) but contain little ether-linked phosphatidylcholine (4 nmoles/10(7) cells).

  15. Fludarabine, cyclophosphamide and rituximab plus granulocyte macrophage colony-stimulating factor as frontline treatment for patients with chronic lymphocytic leukemia.

    PubMed

    Strati, Paolo; Ferrajoli, Alessandra; Lerner, Susan; O'Brien, Susan; Wierda, William; Keating, Michael J; Faderl, Stefan

    2014-04-01

    Fludarabine, cyclophosphamide and rituximab (FCR), the standard of care for the frontline treatment of patients with chronic lymphocytic leukemia (CLL), is associated with a high rate of neutropenia and infectious complications. Granulocyte macrophage colony-stimulating factor (GM-CSF) reduces myelosuppression and can potentiate rituximab activity. We conducted a clinical trial combining GM-CSF with FCR for frontline treatment of 60 patients with CLL. Eighty-six percent completed all six courses and 18% discontinued GM-CSF for toxicity: grade 3-4 neutropenia was observed in 30% of cycles, and severe infections in 16% of cases. The overall response rate was 100%. Both median event-free survival (EFS) and overall survival (OS) have not been reached. Longer EFS was associated with favorable cytogenetics. GM-CSF led to a lower frequency of infectious complications than in the historical FCR group, albeit similar EFS and OS.

  16. Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft.

    PubMed

    Tayebi, H; Lapierre, V; Saas, P; Lienard, A; Sutton, L; Milpied, N; Attal, M; Cahn, J Y; Kuentz, M; Blaise, D; Hervé, P; Tiberghien, P; Robinet, E

    2001-09-01

    In a randomized study that compared human leucocyte antigen-identical allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G-CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G-CSF-induced increase in B-cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B-cell activation could contribute to the accelerated kinetics of immuno-haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft-versus-host disease observed after PBSC transplantation.

  17. Successful mobilization of peripheral blood stem cells in children with cancer using plerixafor (Mozobil) and granulocyte-colony stimulating factor.

    PubMed

    Avramova, Boryana E; Yordanova, Maya N; Konstantinov, Dobrin N; Bobev, Dragan G

    2011-01-01

    This paper describes the successful mobilization of peripheral blood stem cells for autologous transplantation in three children with malignant diseases by using plerixafor (Mozobil; Genzyme Corporation, Cambridge, MA) and granulocyte-colony stimulating factor (G-CSF) after failed previous mobilizations. A median sixfold increase in the number of circulating CD34+ cells after plerixafor treatment as compared with the baseline level was observed. An optimal CD34+ cell count for transplantation with one or two leukapheresis sessions was achieved. Mobilization using plerixafor was found to be safe with no adverse events. Therefore, the combination of G-CSF and plerixafor in children results in effective increases in peripheral CD34+ cell counts and reduces the risk of mobilization failure.

  18. Successful mobilization of peripheral blood stem cells in children with cancer using plerixafor (Mozobil™) and granulocyte-colony stimulating factor

    PubMed Central

    Avramova, Boryana E; Yordanova, Maya N; Konstantinov, Dobrin N; Bobev, Dragan G

    2011-01-01

    This paper describes the successful mobilization of peripheral blood stem cells for autologous transplantation in three children with malignant diseases by using plerixafor (Mozobil™; Genzyme Corporation, Cambridge, MA) and granulocyte-colony stimulating factor (G-CSF) after failed previous mobilizations. A median sixfold increase in the number of circulating CD34+ cells after plerixafor treatment as compared with the baseline level was observed. An optimal CD34+ cell count for transplantation with one or two leukapheresis sessions was achieved. Mobilization using plerixafor was found to be safe with no adverse events. Therefore, the combination of G-CSF and plerixafor in children results in effective increases in peripheral CD34+ cell counts and reduces the risk of mobilization failure. PMID:21966213

  19. Effects of methylmercury on primary cultured rat hepatocytes: Cell injury and inhibition of growth factor stimulated DNA synthesis

    SciTech Connect

    Tanno, Keiichi; Fukazawa, Toshiyuki; Tajima, Shizuko; Fujiki, Motoo )

    1992-08-01

    Many more studies deal with the toxicity of methylmercury on nervous tissue than on its toxicity to the liver. Methylmercury accumulates in the liver in higher concentrations than brain and the liver has the primary function of detoxifying methylmercury. According to recent studies, hepatocyte mitochondrial membranes are destroyed by methylmercury and DNA synthesis is inhibited by methylmercury during hepatocyte regeneration. Methylmercury alters the membrane ion permeability of isolate skate hepatocytes, and inhibits the metal-sensitive alcohol dehydrogenase and glutathione reductase of primary cultured rat hepatocytes. However, little is known about the effect of methylmercury on hepatocyte proliferation in primary cultured rat hepatocytes. We therefore used the primary cultured rat hepatocytes to investigate the effects of methylmercury on cell injury and growth factor stimulate DNA synthesis. The primary effect of methylmercury is to inhibit hepatocyte proliferation rather than to cause direct cell injury. 16 refs., 4 figs.

  20. GABAergic stimulation regulates the phenotype of hippocampal interneurons through the regulation of brain-derived neurotrophic factor.

    PubMed

    Marty, S; Berninger, B; Carroll, P; Thoenen, H

    1996-03-01

    Gamma-Aminobutyric acid (GABA) switches from enhancing to repressing brain-derived neurotrophic factor (BDNF) mRNA synthesis during the maturation of hippocampal neurons in vitro. Interneurons do not produce BDNF themselves, but BDNF enhances their differentiation. Therefore, the question arose whether hippocampal interneurons regulate their phenotype by regulating BDNF expression and release from adjacent cells. The GABA(A) receptor agonist muscimol and BDNF increased the size and neuropeptide Y (NPY) immunoreactivity of hippocampal interneurons. However, GABAergic stimulation failed to increase NPY immunoreactivity in cultures from BDNF knockout embryos. At later developmental stages, when GABA represses BDNF synthesis, treatment with muscimol induced a decrease in cell size and NPY immunoreactivity of interneurons. Interneurons might thus control their phenotype through the regulation of BDNF synthesis in, and release from, their target neurons.

  1. Construction of recombinant Escherichia coli strains for secretory expression of artificial genes for human granulocyte-macrophage colony stimulating factor

    SciTech Connect

    Petrovskaya, L.E.; Ruzin, A.V.; Shingarova, L.N.; Korobko, V.G.

    1995-11-01

    A number of recombinant plasmids for expression of artificial genes encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. A hybrid gene was obtained that contains a sequence encoding the leader peptide and a tandem of two IgG-binding domains of protein A from Staphylococcus aureus coupled, through an enteropepdidase linker, to a synthetic gmcsf gene. The construction enables Escherichia coli to carry out biosynthesis of the hybrid protein and its subsequent transport into the periplasmic space of bacteria. Another hybrid gene, combining sequences for the signal peptide of the E. coli outer membrane protein OmpA and GM-CSF, was obtained using polymerase chain reaction. The localization of the mature protein produced by the hybrid gene was found to depend on the strength of the promoter used. 39 refs., 6 figs.

  2. RhoA GTPase oxidation stimulates cell proliferation via nuclear factor-κB activation.

    PubMed

    Kim, Jae-Gyu; Kwon, Hyung-Joo; Wu, Guang; Park, Yohan; Lee, Jae-Yong; Kim, Jaebong; Kim, Sung-Chan; Choe, Myoen; Kang, Seung Goo; Seo, Goo-Young; Kim, Pyeung-Hyeun; Park, Jae-Bong

    2017-02-01

    Reactive oxygen species (ROS) produced by many kinds of stimuli are essential for cellular signaling including cell proliferation. The dysregulation of ROS, therefore, is related to a variety of diseases including cancer. However, it was not clearly elucidated how ROS regulate cell proliferation and tumorigenesis. In this study, we investigated a mechanism by which the oxidation of RhoA GTPase regulates nuclear factor-κB (NF-κB) and cell proliferation. Hydrogen peroxide activated NF-κB and RhoA GTPase, but did not activate RhoA C16/20A mutant, an oxidation-resistant form. Remarkably, the oxidation of RhoA reduced its affinity towards RhoGDI, leading to the dissociation of RhoA-RhoGDI complex. Si-Vav2, a guanine nucleotide exchange factor (GEF), inhibited RhoA activation upon hydrogen peroxide. The oxidized RhoA (oxRhoA)-GTP was readily bound to IκB kinase γ (IKKγ), whereas oxidized RhoGDI did not bind to IKKγ. The oxRhoA-GTP bound to IKKγ activated IKKβ, leading to IκB phosphorylation and degradation, consequently NF-κB activation. Hydrogen peroxide induced cell proliferation, but RhoA C16/20A mutant suppressed cell proliferation and tumorigenesis. Conclusively, RhoA oxidation at Cys16/20 is critically involved in cell proliferation and tumorigenesis through NF-κB activation in response to ROS.

  3. The Probiotic Mixture VSL#3 Accelerates Gastric Ulcer Healing by Stimulating Vascular Endothelial Growth Factor

    PubMed Central

    Dharmani, Poonam; De Simone, Claudio; Chadee, Kris

    2013-01-01

    Studies assessing the effect and mechanism of probiotics on diseases of the upper gastrointestinal tract (GI) including gastric ulcers are limited despite extensive work and promising results of this therapeutic option for other GI diseases. In this study, we investigated the mechanisms by which the probiotic mixture VSL#3 (a mixture of eight probiotic bacteria including Lactobacilli, Bifidobacteria and Streptococcus species) heals acetic acid induced gastric ulcer in rats. VSL#3 was administered orally at low (6×109 bacteria) or high (1.2×1010 bacteria) dosages from day 3 after ulcer induction for 14 consecutive days. VSL#3 treatments significantly enhanced gastric ulcer healing in a dose-dependent manner. To assess the mechanism(s) whereby VSL#3 exerted its protective effects, we quantified the gene expression of several pro-inflammatory cytokines, protein and expression of stomach mucin-Muc5ac, regulatory cytokine-IL-10, COX-2 and various growth factors. Of all the components examined, only expression and protein production of VEGF was increased 332-fold on day 7 in the ulcerated tissues of animals treated with VSL#3. Predictably, animals treated with VEGF neutralizing antibody significantly delayed gastric ulcer healing in VSL#3 treated animals. This is the first report to demonstrate high efficacy of the probiotic mixture VSL#3 in enhancing gastric ulcer healing. Probiotic efficacy was effective at higher concentrations of VSL#3 by specifically increasing the expression and production of angiogenesis promoting growth factors, primarily VEGF. PMID:23484048

  4. Vascular endothelial growth factor stimulates bone repair by promoting angiogenesis and bone turnover.

    PubMed

    Street, John; Bao, Min; deGuzman, Leo; Bunting, Stuart; Peale, Franklin V; Ferrara, Napoleone; Steinmetz, Hope; Hoeffel, John; Cleland, Jeffrey L; Daugherty, Ann; van Bruggen, Nicholas; Redmond, H Paul; Carano, Richard A D; Filvaroff, Ellen H

    2002-07-23

    Several growth factors are expressed in distinct temporal and spatial patterns during fracture repair. Of these, vascular endothelial growth factor, VEGF, is of particular interest because of its ability to induce neovascularization (angiogenesis). To determine whether VEGF is required for bone repair, we inhibited VEGF activity during secondary bone healing via a cartilage intermediate (endochondral ossification) and during direct bone repair (intramembranous ossification) in a novel mouse model. Treatment of mice with a soluble, neutralizing VEGF receptor decreased angiogenesis, bone formation, and callus mineralization in femoral fractures. Inhibition of VEGF also dramatically inhibited healing of a tibial cortical bone defect, consistent with our discovery of a direct autocrine role for VEGF in osteoblast differentiation. In separate experiments, exogenous VEGF enhanced blood vessel formation, ossification, and new bone (callus) maturation in mouse femur fractures, and promoted bony bridging of a rabbit radius segmental gap defect. Our results at specific time points during the course of healing underscore the role of VEGF in endochondral vs. intramembranous ossification, as well as skeletal development vs. bone repair. The responses to exogenous VEGF observed in two distinct model systems and species indicate that a slow-release formulation of VEGF, applied locally at the site of bone damage, may prove to be an effective therapy to promote human bone repair.

  5. Purified human platelet-derived growth factor receptor has ligand-stimulated tyrosine kinase activity.

    PubMed Central

    Bishayee, S; Ross, A H; Womer, R; Scher, C D

    1986-01-01

    The platelet-derived growth factor receptor (PDGF-R), a 180-kDa single-chain polypeptide, was purified from membranes of the human osteogenic sarcoma cell line MG-63. Purification was achieved by treatment of membranes with PDGF and ATP, followed by solubilization with nonionic detergent and successive chromatography on solid-phase anti-phosphotyrosine monoclonal antibody and DEAE-cellulose. The PDGF-R, which was estimated to be 50-80% pure by NaDodSO4/polyacrylamide gel electrophoresis of 32P-labeled preparations, was free of contaminating epidermal growth factor receptor and had no detectable phosphatase activity. It specifically bound 125I-labeled PDGF, a reaction quantified by binding of the ligand-PDGF-R complex to the anti-phosphotyrosine antibody. The purified receptor displayed PDGF-stimulatable tyrosine kinase activity, assayed by autophosphorylation of PDGF-R at tyrosine residues and by phosphorylation of angiotensin II. The Km for ATP in the autophosphorylation reaction was 7.5 microM. Addition of PDGF did not change the Km but increased the Vmax 1.7-fold. Images PMID:3018745

  6. Biologic Activity of Autologous, Granulocyte-Macrophage Colony Stimulating Factor Secreting Alveolar Soft Parts Sarcoma and Clear Cell Sarcoma Vaccines

    PubMed Central

    Goldberg, John; Fisher, David E.; Demetri, George D.; Neuberg, Donna; Allsop, Stephen A.; Fonseca, Catia; Nakazaki, Yukoh; Nemer, David; Raut, Chandrajit P.; George, Suzanne; Morgan, Jeffrey A.; Wagner, Andrew J.; Freeman, Gordon J.; Ritz, Jerome; Lezcano, Cecilia; Mihm, Martin; Canning, Christine; Hodi, F. Stephen; Dranoff, Glenn

    2015-01-01

    Purpose Alveolar soft parts sarcoma (ASPS) and clear cell sarcoma (CCS) are rare mesenchymal malignancies driven by chromosomal translocations that activate members of the microphthalmia transcription factor (MITF) family. However, in contrast to malignant melanoma, little is known about their immunogenicity. To learn more about the host response to ASPS and CCS, we conducted a phase I clinical trial of vaccination with irradiated, autologous sarcoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor (GM-CSF). Experimental Design Metastatic tumors from ASPS and CCS patients were resected, processed to single cell suspensions, transduced with a replication defective adenoviral vector encoding GM-CSF, and irradiated. Immunizations were administered subcutaneously and intradermally weekly times three and then every other week. Results Vaccines were successfully manufactured for 11 of the 12 enrolled patients. Eleven subjects received from 3 to 13 immunizations. Toxicities were restricted to grade 1–2 skin reactions at inoculation sites. Vaccination elicited local dendritic cell infiltrates and stimulated T cell mediated delayed type-hypersensitivity reactions to irradiated, autologous tumor cells. Antibody responses to tissue-type plasminogen activator (tTPA) and angiopoietins-1/2 were detected. Tumor biopsies showed programmed death-1 (PD-1) positive CD8+ T cells in association with PD ligand-1 (PD-L1) expressing sarcoma cells. No tumor regressions were observed. Conclusions Vaccination with irradiated, GM-CSF secreting autologous sarcoma cell vaccines is feasible, safe, and biologically active. Concurrent targeting of angiogenic cytokines and antagonism of the PD-1 negative regulatory pathway might intensify immune-mediated tumor destruction. PMID:25805798

  7. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

    PubMed

    Lorber, Barbara; Berry, Martin; Logan, Ann

    2005-04-01

    In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.

  8. Chronic recurrent multifocal osteomyelitis with Crohn's disease exacerbation and vasculitis after granulocyte colony-stimulating factor therapy.

    PubMed

    Manners, P; Robbins, P

    2000-10-01

    Chronic recurrent multifocal osteomyelitis (CRMO), of unknown etiology, is characterized by recurring non-suppurative lesions of bone in multiple sites, and has been considered to be self-limiting. Reported therapies include prolonged antibiotics, corticosteroids and anti-inflammatory medications. This case is presented to illustrate the following: 1) CRMO may be severe, on-going, and unresponsive to treatment; 2) it may be associated with Crohns' disease; 3) the use of granulocyte colony-stimulating factor (G-CSF) may be associated with severe gastrointestinal vasculitis. A male was treated from ages 11-20 years for CRMO (manifesting as multiple bone lesions), with therapies of variable efficacy (anti-inflammatories, antibiotics, corticosteroids, gammaglobulin and methotrexate). With increasing disruption to his life, a 10-day course of granulocyte colony-stimulating factor (G-CSF) was given with benefit seen on magnetic resonance imaging (MRI). With exacerbation of symptoms one month later, G-CSF was re-commenced but ceased after 3 weeks because of abdominal pain, rectal blood loss, and progression of bone lesions with subsequent removal of portions of ileum, colon and appendix, which showed vasculitis. Months later, a colonoscopy revealed perianastomotic ulcers and continuing gastroenterological ulceration not unlike Crohn's disease. With azathioprine, gut and bone symptoms improved. We conclude that 1) CRMO may adversely affect life for years; 2) proven treatments are unavailable; 3) gastroenterological vasculitis/ Crohn's may be associated with CRMO; 4) MRI is useful for monitoring CRMO; 5) In this patient, G-CSF seemed beneficial initially, but later, vasculitis (possibly Crohn's) manifested, leading to bowel resection; 6) Crohn's disease may have been present for years, masked by corticosteroid, and unmasked by reduction of steroids and use of G-CSF.

  9. Effect of epidermal growth factor on follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles.

    PubMed

    Lin, Jin-xing; Jia, Yu-dong; Zhang, Cai-qiao

    2011-11-01

    The development of ovarian follicular cells is controlled by multiple circulating and local hormones and factors, including follicle-stimulating hormone (FSH) and epidermal growth factor (EGF). In this study, the stage-specific effect of EGF on FSH-induced proliferation of granulosa cells was evaluated in the ovarian follicles of egg-laying chickens. Results showed that EGF and its receptor (EGFR) mRNAs displayed a high expression in granulosa cells from the prehierarchical follicles, including the large white follicle (LWF) and small yellow follicle (SYF), and thereafter the expression decreased markedly to the stage of the largest preovulatory follicle. SYF represents a turning point of EGF/EGFR mRNA expression during follicle selection. Subsequently the granulosa cells from SYF were cultured to reveal the mediation of EGF in FSH action. Cell proliferation was remarkably increased by treatment with either EGF or FSH (0.1-100 ng/ml). This result was confirmed by elevated proliferating cell nuclear antigen (PCNA) expression and decreased cell apoptosis. Furthermore, EGF-induced cell proliferation was accompanied by increased mRNA expressions of EGFR, FSH receptor, and the cell cycle-regulating genes (cyclins D1 and E1, cyclin-dependent kinases 2 and 6) as well as decreased expression of luteinizing hormone receptor mRNA. However, the EGF or FSH-elicited effect was reversed by simultaneous treatment with an EGFR inhibitor AG1478. In conclusion, EGF and EGFR expressions manifested stage-specific changes during follicular development and EGF mediated FSH-induced cell proliferation and retarded cell differentiation in the prehierarchical follicles. These expressions thus stimulated follicular growth before selection in the egg-laying chicken.

  10. SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons.

    PubMed

    Morii, Hiroshi; Shiraishi-Yamaguchi, Yoko; Mori, Nozomu

    2006-09-01

    Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.

  11. Role of granulocyte-macrophage colony stimulating factor (GM-CSF) in the pathogenesis of adult pulmonary histiocytosis X.

    PubMed Central

    Tazi, A; Bonay, M; Bergeron, A; Grandsaigne, M; Hance, A J; Soler, P

    1996-01-01

    BACKGROUND: Pulmonary histiocytosis X is a disorder characterised by the presence of destructive granulomas preferentially involving distal bronchioles, that contain numerous activated Langerhans' cells. Recent studies have shown that granulocyte-macrophage colony stimulating factor (GM-CSF), which is produced by normal bronchiolar epithelium, may play an important part in the distribution and differentiation of Langerhans' cells. The aim of this study was to evaluate the role of this factor in the pathogenesis of pulmonary histiocytosis X. METHODS: Four patients with pulmonary histiocytosis X were examined by immunohistochemical techniques for GM-CSF and CD1a surface molecules. RESULTS: In early lesions the epithelium of bronchioles affected by the disease was strongly positive for GM-CSF and infiltrated by numerous CD1a+ Langerhans' cells organised into granulomas. In contrast, the expression of GM-CSF was substantially lower in bronchioles not affected by the disease, and these bronchioles contained few Langerhans' cells. When destruction by histiocytosis X lesions was more advanced, only remnants of bronchiolar epithelium could occasionally be identified; these remained strongly reactive for GM-CSF. Langerhans' cells within granulomas also moderately expressed this cytokine. CONCLUSIONS: These results support the hypothesis that GM-CSF could be one of the factors responsible for the local accumulation of lymphostimulatory Langerhans' cells in early lesions of pulmonary histiocytosis X. Images PMID:8693443

  12. Inhibition of colony-stimulating factor (CSF) production by postburn serum: negative feedback inhibition mediated by lactoferrin.

    PubMed

    Peterson, V M; Ambruso, D R; Emmett, M; Bartle, E J

    1988-11-01

    Fatal infections in severely burned patients are often preceded by a decline in the production of colony-stimulating factor (CSF) and the proliferation of granulocyte-macrophage stem cells (CFU-GM), and overwhelming sepsis is often associated with leukopenia. The underlying mechanisms accounting for these granulopoietic defects are poorly understood, but the fact that postburn serum has been shown to inhibit CSF production suggests that a humoral factor or factors may play a role. Previous work has demonstrated that plasma levels of lactoferrin (LF), a known inhibitor of CSF production, are elevated following burn injury. To determine if LF is responsible for serum-mediated inhibition of CSF production, serial plasma levels of LF were measured in 18 burn patients using an enzyme-linked immunoabsorbent assay (ELISA). LF was elevated within 24 hours of injury and was associated with an absolute granulocytosis which rapidly declined, reaching a nadir at postburn days 3 through 5. Postburn serum, especially when collected during the first 24 hours following burn injury, inhibited in vitro CSF production by normal human peripheral blood mononuclear cells. Pre-incubation of postburn serum with an LF antibody restored normal CSF production. These data suggest that LF may play an important role in the regulation of postburn granulopoiesis.

  13. Macrophage colony-stimulating factor expressed in non-cancer tissues provides predictive powers for recurrence in hepatocellular carcinoma

    PubMed Central

    Kono, Hiroshi; Fujii, Hideki; Furuya, Shinji; Hara, Michio; Hirayama, Kazuyoshi; Akazawa, Yoshihiro; Nakata, Yuuki; Tsuchiya, Masato; Hosomura, Naohiro; Sun, Chao

    2016-01-01

    AIM To investigate the role of macrophage colony-stimulating factor (M-CSF) in patients with hepatocellular carcinoma (HCC) after surgery. METHODS Expression of M-CSF, distribution of M2 macrophages (MΦs), and angiogenesis were assessed in the liver, including tumors and peritumoral liver tissues. The prognostic power of these factors was assessed. Mouse isolated hepatic MΦs or monocytes were cultured with media containing M-CSF. The concentration of vascular endothelial growth factor (VEGF) in media was assessed. Furthermore, the role of the M-CSF-matured hepatic MΦs on proliferation of the vascular endothelial cell (VEC) was investigated. RESULTS A strong correlation between the expressions of M-CSF and CD163 was observed in the peritumoral area. Also, groups with high density of M-CSF, CD163 or CD31 showed a significantly shorter time to recurrence (TTR) than low density groups. Multivariate analysis revealed the expression of M-CSF or hepatic M2MΦs in the peritumoral area as the most crucial factor responsible for shorter TTR. Moreover, the expression of M-CSF and hepatic M2MΦs in the peritumoral area had better predictable power of overall survival. Values of VEGF in culture media were significantly greater in the hepatic MΦs compared with the monocytes. Proliferation of the VEC was greatest in the cells co-cultured with hepatic MΦs when M-CSF was present in media. CONCLUSION M-CSF increases hepatocarcinogenesis, most likely by enhancing an angiogenic factor derived from hepatic MΦ and could be a useful target for therapy against HCC. PMID:27818593

  14. Kick-starting the cell cycle: From growth-factor stimulation to initiation of DNA replication

    NASA Astrophysics Data System (ADS)

    Aguda, Baltazar D.

    2001-03-01

    The essential genes, proteins and associated regulatory networks involved in the entry into the mammalian cell cycle are identified, from activation of growth-factor receptors to intracellular signal transduction pathways that impinge on the cell cycle machinery and ultimately on the initiation of DNA replication. Signaling pathways mediated by the oncoproteins Ras and Myc induce the activation of cyclin-dependent kinases CDK4 and CDK2, and the assembly and firing of pre-replication complexes require a collaboration among E2F, CDK2, and Cdc7 kinase. A proposed core mechanism of the restriction point, the major checkpoint prior to commitment to DNA synthesis, involves cyclin E/CDK2, the phosphatase Cdc25A, and the CDK inhibitor p27Kip1.

  15. Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration

    SciTech Connect

    Maheshwari, Gargi; Wiley, H Steven ); Lauffenburger, Douglas A.

    2001-12-24

    Autocrine receptor/ligand signaling loops were first identified in tumor cells, where it was found that transformation of cells resulted in overexpression of certain growth factors leading to unregulated proliferation of the tumor cells (Sporn and Todaro, 1980). However, in the ensuing decades autocrine signaling has been found to operate in numerous physiological situations (Sporn and Roberts, 1992), including wound healing (Tokumaru et al., 2000), angiogenesis (Seghezzi et al., 1998), and tissue organization during development (Wasserman and Freeman, 1998) and reproductive cycles (Xie et al., 1997). Although it is becoming evident that autocrine loops play crucial roles in regulation of cell function within tissue contexts, it is unclear whether their effects on cell responses are different from the effects of the same ligand presented in exogenous or paracrine manner.

  16. Characterization of mechanical behavior of an epithelial monolayer in response to epidermal growth factor stimulation

    SciTech Connect

    Yang, Ruiguo; Chen, Jennifer Y.; Xi, Ning; Lai, King Wai Chiu; Qu, Chengeng; Fung, Carmen Kar Man; Penn, Lynn S.; Xi, Jun

    2012-03-10

    Cell signaling often causes changes in cellular mechanical properties. Knowledge of such changes can ultimately lead to insight into the complex network of cell signaling. In the current study, we employed a combination of atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D) to characterize the mechanical behavior of A431 cells in response to epidermal growth factor receptor (EGFR) signaling. From AFM, which probes the upper portion of an individual cell in a monolayer of cells, we observed increases in energy dissipation, Young's modulus, and hysteresivity. Increases in hysteresivity imply a shift toward a more fluid-like mechanical ordering state in the bodies of the cells. From QCM-D, which probes the basal area of the monolayer of cells collectively, we observed decreases in energy dissipation factor. This result suggests a shift toward a more solid-like state in the basal areas of the cells. The comparative analysis of these results indicates a regionally specific mechanical behavior of the cell in response to EGFR signaling and suggests a correlation between the time-dependent mechanical responses and the dynamic process of EGFR signaling. This study also demonstrates that a combination of AFM and QCM-D is able to provide a more complete and refined mechanical profile of the cells during cell signaling. -- Highlights: Black-Right-Pointing-Pointer The EGF-induced cellular mechanical response is regionally specific. Black-Right-Pointing-Pointer The EGF-induced cellular mechanical response is time and dose dependent. Black-Right-Pointing-Pointer A combination of AFM and QCM-D provides a more complete mechanical profile of cells.

  17. Stimulation of the multiplication of Micrococcus luteus by an autocrine growth factor.

    PubMed

    Mukamolova, G V; Kormer, S S; Kell, D B; Kaprelyants, A S

    1999-07-01

    Viable cells of Micrococcus luteus secrete a proteineous growth factor (Rpf) which promotes the resuscitation of dormant, nongrowing cells to yield normal, colony-forming bacteria. When washed M. luteus cells were used as an inoculum, there was a pronounced influence of Rpf on the true lag phase and cell growth on lactate minimal medium. In the absence of Rpf, there was no increase in colony-forming units for up to 10 days. When the inoculum contained less than 10(5) cells ml-1, macroscopically observable M. luteus growth was not obtained in succinate minimal medium unless Rpf was added. Incubation of M. luteus in the stationary phase for 100 h resulted in a failure of the cells to grow in lactate minimal medium from inocula of small size although the viability of these cells was close to 100% as estimated using agar plates made from lactate minimal medium or rich medium. The underestimation of viable cells by the most-probable-number (MPN) method in comparison with colony-forming units was equivalent to the requirement that at least 10(5) cells grown on succinate medium, 10(3) cells from old stationary phase, or approximately 10-500 washed cells are required per millilitre of inoculum for growth to lead to visible turbidity. The addition of Rpf in the MPN dilutions led to an increase of the viable cell numbers estimated to approximately the same levels as those determined by colony-forming units. Thus, a basic principle of microbiology - "one cell-one culture" - may not be applicable in some circumstances in which the metabolic activity of "starter" cells is not sufficient to produce enough autocrine growth factor to support cell multiplication.

  18. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro.

    PubMed Central

    Papapetropoulos, A.; Desai, K. M.; Rudic, R. D.; Mayer, B.; Zhang, R.; Ruiz-Torres, M. P.; García-Cardeña, G.; Madri, J. A.; Sessa, W. C.

    1997-01-01

    Angiogenesis is a complex process involving endothelial cell (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-beta 1. Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-beta 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs. Images Figure 2 Figure 4 Figure 5 PMID:9137106

  19. The production of lymphocyte mitogenic factor and migration–inhibition factor by antigen-stimulated lymphocytes of subjects with grass pollen allergy

    PubMed Central

    Maini, R. N.; Dumonde, D. C.; Faux, J. A.; Hargreave, F. E.; Pepys, J.

    1971-01-01

    Peripheral blood lymphocytes of twenty-three subjects with grass pollen allergy were cultured with grass pollen antigen for 3 days. After harvesting, the culture supernatants were added to fresh autologous lymphocytes which were maintained in culture for 6 days. Cellular uptake of [3H]thymidine was measured during the sixth day of culture, and revealed that the lymphocyte culture supernatants stimulated greater thymidine uptake than expected from the lymphocyte transformation response to corresponding amounts of antigen. The supernatant factor which mediated this effect was termed `lymphocyte mitogenic factor' by analogy with a similar response of lymphocytes in clinical and experimental delayed hypersensitivity. Lymphocyte culture supernatants were also tested for migration–inhibition factor by their ability to inhibit the migration of guinea-pig macrophages. The majority of `allergic' supernatants contained a lymphocyte mitogenic factor active at 1/3 dilution (14/22) and 1/12 dilution (19/21) in contrast to supernatants derived from non-allergic subjects (2/16 and 1/17 respectively). The production of lymphocyte mitogenic factor corresponded to the occurrence of antigen-induced lymphocyte transformation (allergic: 18/22; non-allergic: 1/14); but only a minority of allergic supernatants contained a migration–inhibition factor (6/20). Clinical analysis revealed that migration–inhibition factor was particularly associated with the milder forms of allergy and with a past history of desensitization by depot injection of emulsified pollen antigen. In contrast, lymphocyte transformation and the production of mitogenic factor were uniformly distributed among the various categories of allergic subjects, all of whom had immediate (reaginic) hypersensitivity, but only three of whom had delayed hypersensitivity. The demonstration of lymphocyte mitogenic factor in a clinical state dominated by immediate hypersensitivity supported the view that antigen

  20. Stimulation of Leishmania tropica protein kinase CK2 activities by platelet-activating factor (PAF).

    PubMed

    Dutra, Patricia M L; Vieira, Danielle P; Meyer-Fernandes, Jose R; Silva-Neto, Mario A C; Lopes, Angela H

    2009-09-01

    Leishmania tropica is one of the causative agents of cutaneous leishmaniasis. Platelet-activating factor (PAF) is a phospholipid mediator in diverse biological and pathophysiological processes. Here we show that PAF promoted a three-fold increase on ecto-protein kinase and a three-fold increase on the secreted kinase activity of L. tropica live promastigotes. When casein was added to the reaction medium, along with PAF, there was a four-fold increase on the ecto-kinase activity. When live L. tropica promastigotes were pre-incubated for 30 min in the presence of PAF-plus casein, a six-fold increase on the secreted kinase activity was observed. Also, a protein released from L. tropica promastigotes reacted with polyclonal antibodies for the mammalian CK2 alpha catalytic subunit. Furthermore, in vitro mouse macrophage infection by L. tropica was doubled when promastigotes were pre-treated for 2 h with PAF. Similar results were obtained when the interaction was performed in the presence of purified CK2 or casein. TBB and DRB, CK2 inhibitors, reversed PAF enhancement of macrophage infection by L. tropica. WEB 2086, a competitive PAF antagonist, reversed all PAF effects here described. This study shows for the first time that PAF promotes the activation of two isoforms of CK2, secreted and membrane-bound, correlating these activities to infection of mouse macrophages.

  1. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

    PubMed

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

  2. Transgenic expression of the proneural transcription factor Ascl1 in Müller glia stimulates retinal regeneration in young mice

    PubMed Central

    Ueki, Yumi; Wilken, Matthew S.; Cox, Kristen E.; Chipman, Laura; Jorstad, Nikolas; Sternhagen, Kristen; Simic, Milesa; Ullom, Kristy; Nakafuku, Masato; Reh, Thomas A.

    2015-01-01

    Müller glial cells are the source of retinal regeneration in fish and birds; although this process is efficient in fish, it is less so in birds and very limited in mammals. It has been proposed that factors necessary for providing neurogenic competence to Müller glia in fish and birds after retinal injury are not expressed in mammals. One such factor, the proneural transcription factor Ascl1, is necessary for retinal regeneration in fish but is not expressed after retinal damage in mice. We previously reported that forced expression of Ascl1 in vitro reprograms Müller glia to a neurogenic state. We now test whether forced expression of Ascl1 in mouse Müller glia in vivo stimulates their capacity for retinal regeneration. We find that transgenic expression of Ascl1 in adult Müller glia in undamaged retina does not overtly affect their phenotype; however, when the retina is damaged, the Ascl1-expressing glia initiate a response that resembles the early stages of retinal regeneration in zebrafish. The reaction to injury is even more pronounced in Müller glia in young mice, where the Ascl1-expressing Müller glia give rise to amacrine and bipolar cells and photoreceptors. DNaseI-seq analysis of the retina and Müller glia shows progressive reduction in accessibility of progenitor gene cis-regulatory regions consistent with the reduction in their reprogramming. These results show that at least one of the differences between mammal and fish Müller glia that bears on their difference in regenerative potential is the proneural transcription factor Ascl1. PMID:26483457

  3. Contribution of granulocyte colony-stimulating factor to the acute mobilization of endothelial precursor cells by vascular disrupting agents.

    PubMed

    Shaked, Yuval; Tang, Terence; Woloszynek, Jill; Daenen, Laura G; Man, Shan; Xu, Ping; Cai, Shi-Rong; Arbeit, Jeffrey M; Voest, Emile E; Chaplin, David J; Smythe, Jon; Harris, Adrian; Nathan, Paul; Judson, Ian; Rustin, Gordon; Bertolini, Francesco; Link, Daniel C; Kerbel, Robert S

    2009-10-01

    Vascular disrupting agents (VDA) cause acute shutdown of abnormal established tumor vasculature, followed by massive intratumoral hypoxia and necrosis. However, a viable rim of tumor tissue invariably remains from which tumor regrowth rapidly resumes. We have recently shown that an acute systemic mobilization and homing of bone marrow-derived circulating endothelial precursor (CEP) cells could promote tumor regrowth following treatment with either a VDA or certain chemotherapy drugs. The molecular mediators of this systemic reactive host process are unknown. Here, we show that following treatment of mice with OXi-4503, a second-generation potent prodrug derivative of combretastatin-A4 phosphate, rapid increases in circulating plasma vascular endothelial growth factor, stromal derived factor-1 (SDF-1), and granulocyte colony-stimulating factor (G-CSF) levels are detected. With the aim of determining whether G-CSF is involved in VDA-induced CEP mobilization, mutant G-CSF-R(-/-) mice were treated with OXi-4503. We found that as opposed to wild-type controls, G-CSF-R(-/-) mice failed to mobilize CEPs or show induction of SDF-1 plasma levels. Furthermore, Lewis lung carcinomas grown in such mice treated with OXi-4503 showed greater levels of necrosis compared with tumors treated in wild-type mice. Evidence for rapid elevations in circulating plasma G-CSF, vascular endothelial growth factor, and SDF-1 were also observed in patients with VDA (combretastatin-A4 phosphate)-treated cancer. These results highlight the possible effect of drug-induced G-CSF on tumor regrowth following certain cytotoxic drug therapies, in this case using a VDA, and hence G-CSF as a possible therapeutic target.

  4. Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.

    PubMed

    Wiktor-Jedrzejczak, W; Bartocci, A; Ferrante, A W; Ahmed-Ansari, A; Sell, K W; Pollard, J W; Stanley, E R

    1990-06-01

    Osteopetrotic (op/op) mutant mice suffer from congenital osteopetrosis due to a severe deficiency of osteoclasts. Furthermore, the total number of mononuclear phagocytes is extremely low in affected mice. Serum, 11 tissues, and different cell and organ conditioned media from op/op mice were shown to be devoid of biologically active colony-stimulating factor 1 (CSF-1), whereas all of these preparations from littermate control +/+ and +/op mice contained the growth factor. The deficiency was specific for CSF-1 in that serum or conditioned media from op/op mice possessed elevated levels of at least three other macrophage growth factors. Partial correction of the op/op defect was observed following intraperitoneal implantation of diffusion chambers containing L929 cells, which in culture produce CSF-1 as their sole macrophage growth factor. No rearrangement of the CSF-1 gene in op/op mice was detected by Southern analysis. However, in contrast to control lung fibroblasts, which contained 4.6- and 2.3-kilobase CSF-1 mRNAs, only the 4.6-kilobase species was detected in op/op cells. An alteration in the CSF-1 gene is strongly implicated as the primary defect in op/op mice because they do not contain detectable CSF-1, their defect is correctable by administration of CSF-1, the op locus and the CSF-1 gene map within the same region of mouse chromosome 3, their CSF-1 mRNA biosynthesis is altered, and the op/op phenotype is consistent with the phenotype expected in a CSF-1 deficient mouse.

  5. Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1

    PubMed Central

    Zwicker, Stephanie; Bureik, Daniela; Bosma, Madeleen; Martinez, Gisele Lago; Almer, Sven

    2016-01-01

    Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1, RPTP-ζ), an additional IL-34 receptor, was recently identified. Here, we aimed to assess PTPRZ1 expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of PTPRZ1 was higher in non-IBD colon compared to ileum. PTPRZ1 expression was not altered with inflammation in IBD, however, correlated to IL34, CSF1, and CSF1R. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor α (TNF-α), IL-1β, interferon γ (IFN-γ), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that PTPRZ1 was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions. PMID:27898738

  6. Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1.

    PubMed

    Zwicker, Stephanie; Bureik, Daniela; Bosma, Madeleen; Martinez, Gisele Lago; Almer, Sven; Boström, Elisabeth A

    2016-01-01

    Differential intestinal expression of the macrophage growth factors colony stimulating factor-1 (CSF-1), interleukin (IL)-34, and their shared CSF-1 receptor (CSF-1R) in inflammatory bowel disease (IBD) has been shown. Diverse expression between CSF-1 and IL-34, suggest that IL-34 may signal via an alternate receptor. Receptor-type protein-tyrosine phosphatase ζ (PTPRZ1, RPTP-ζ), an additional IL-34 receptor, was recently identified. Here, we aimed to assess PTPRZ1 expression in IBD and non-IBD intestinal biopsies. Further, we aimed to investigate cellular PTPRZ1 and CSF-1R expression, and cytokine- and chemokine responses by IL-34 and CSF-1. The expression of PTPRZ1 was higher in non-IBD colon compared to ileum. PTPRZ1 expression was not altered with inflammation in IBD, however, correlated to IL34, CSF1, and CSF1R. The expression patterns of PTPRZ1 and CSF-1R differed in peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and intestinal epithelial cell line. PBMCs and monocytes of the same donors responded differently to IL-34 and CSF-1 with altered expression of tumor-necrosis factor α (TNF-α), IL-1β, interferon γ (IFN-γ), IL-13, IL-8, and monocyte chemotactic protein-1 (MCP-1) levels. This study shows that PTPRZ1 was expressed in bowel tissue. Furthermore, CSF-1R protein was detected in an intestinal epithelial cell line and donor dependently in primary PBMCs, monocytes, and macrophages, and first hints also suggest an expression in these cells for PTPRZ1, which may mediate IL-34 and CSF-1 actions.

  7. Neural Growth Factor Stimulates Proliferation of Spinal Cord Derived-Neural Precursor/Stem Cells

    PubMed Central

    Han, Youngmin

    2016-01-01

    Objective Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs. Methods NPSCs (2×104) were suspended in 100 µL of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained. Results The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group. Conclusion SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in

  8. Metabolic remodeling of the tumor microenvironment: migration stimulating factor (MSF) reprograms myofibroblasts toward lactate production, fueling anabolic tumor growth.

    PubMed

    Carito, Valentina; Bonuccelli, Gloria; Martinez-Outschoorn, Ubaldo E; Whitaker-Menezes, Diana; Caroleo, Maria Cristina; Cione, Erika; Howell, Anthony; Pestell, Richard G; Lisanti, Michael P; Sotgia, Federica

    2012-09-15

    Migration stimulating factor (MSF) is a genetically truncated N-terminal isoform of fibronectin that is highly expressed during mammalian development in fetal fibroblasts, and during tumor formation in human cancer-associated myofibroblasts. However, its potential functional role in regulating tumor metabolism remains unexplored. Here, we generated an immortalized fibroblast cell line that recombinantly overexpresses MSF and studied their properties relative to vector-alone control fibroblasts. Our results indicate that overexpression of MSF is sufficient to confer myofibroblastic differentiation, likely via increased TGF-b signaling. In addition, MSF activates the inflammation-associated transcription factor NFκB, resulting in the onset of autophagy/mitophagy, thereby driving glycolytic metabolism (L-lactate production) in the tumor microenvironment. Consistent with the idea that glycolytic fibroblasts fuel tumor growth (via L-lactate, a high-energy mitochondrial fuel), MSF fibroblasts significantly increased tumor growth, by up to 4-fold. Mechanistic dissection of the MSF signaling pathway indicated that Cdc42 lies downstream of MSF and fibroblast activation. In accordance with this notion, Cdc42 overexpression in immortalized fibroblasts was sufficient to drive myofibroblast differentiation, to provoke a shift towards glycolytic metabolism and to promote tumor growth by up to 2-fold. In conclusion, the MSF/Cdc42/NFκB signaling cascade may be a critical druggable target in preventing "Warburg-like" cancer metabolism in tumor-associated fibroblasts. Thus, MSF functions in the metabolic remodeling of the tumor microenvironment by metabolically reprogramming cancer-associated fibroblasts toward glycolytic metabolism.

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

    PubMed Central

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

    2013-01-01

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

  10. Intranasal Delivery of Granulocyte Colony-Stimulating Factor Enhances Its Neuroprotective Effects Against Ischemic Brain Injury in Rats.

    PubMed

    Sun, Bao-liang; He, Mei-qing; Han, Xiang-yu; Sun, Jing-yi; Yang, Ming-feng; Yuan, Hui; Fan, Cun-dong; Zhang, Shuai; Mao, Lei-lei; Li, Da-wei; Zhang, Zong-yong; Zheng, Cheng-bi; Yang, Xiao-yi; Li, Yang V; Stetler, R Anne; Chen, Jun; Zhang, Feng

    2016-01-01

    Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor with strong neuroprotective properties. However, it has limited capacity to cross the blood-brain barrier and thus potentially limiting its protective capacity. Recent studies demonstrated that intranasal drug administration is a promising way in delivering neuroprotective agents to the central nervous system. The current study therefore aimed at determining whether intranasal administration of G-CSF increases its delivery to the brain and its neuroprotective effect against ischemic brain injury. Transient focal cerebral ischemia in rat was induced with middle cerebral artery occlusion. Our resulted showed that intranasal administration is 8-12 times more effective than subcutaneous injection in delivering G-CSF to cerebrospinal fluid and brain parenchyma. Intranasal delivery enhanced the protective effects of G-CSF against ischemic injury in rats, indicated by decreased infarct volume and increased recovery of neurological function. The neuroprotective mechanisms of G-CSF involved enhanced upregulation of HO-1 and reduced calcium overload following ischemia. Intranasal G-CSF application also promoted angiogenesis and neurogenesis following brain ischemia. Taken together, G-CSF is a legitimate neuroprotective agent and intranasal administration of G-CSF is more effective in delivery and neuroprotection and could be a practical approach in clinic.

  11. Sublethal doses of dinophysistoxin-1 and okadaic acid stimulate secretion of inflammatory factors on innate immune cells: Negative health consequences.

    PubMed

    Del Campo, Miguel; Zhong, Ta-Ying; Tampe, Ricardo; García, Lorena; Lagos, Néstor

    2017-02-01

    One of the proposed mechanisms to explain why Diarrhetic Shellfish Poison (DSP) toxins are tumor promoters is founded on the capacity of these toxins to increase TNF-α secretion. Although macrophages are the principal cells in the activation of the inflammatory response, the immune profile that Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) trigger in these cells has not been fully explored. We have therefore investigated the effect of various concentrations of both toxins on the activity of several inflammatory factors. Our results demonstrate that OA and DTX-1, at sublethal doses, stimulate secretion of inflammatory factors. Nevertheless DTX-1 was more potent than OA in increasing TNF-α and IL-6 as well as their dependent chemokines KC, MCP-1, LIX, MIP-1 α, MIP-1 β and MIP-2. On the other hand, secretion of IFN-γ and the anti-inflammatory cytokines, IL-4 and IL-10, was unaffected. In addition, DTX-1 also raises matrix metalloproteinase-9 (MMP-9) activity. In this study, for the first time the effect of OA and DTX-1 over the secretion of pro-inflammatory and carcinogenic signals in macrophages are compared, showing that DTX-1 is ten times more potent that OA. The inflammatory profile produced by DTX-1 is shown for the first time. The safe limit regulation should be changed to DSP toxins zero tolerance in the shellfish to be consumed by humans.

  12. BET bromodomain proteins mediate downstream signaling events following growth factor stimulation in human lung fibroblasts and are involved in bleomycin-induced pulmonary fibrosis.

    PubMed

    Tang, Xiaoyan; Peng, Ruoqi; Ren, Yonglin; Apparsundaram, Subramanium; Deguzman, Jeremy; Bauer, Carla M; Hoffman, Ann F; Hamilton, Shannon; Liang, Zhenmin; Zeng, Hang; Fuentes, Maria E; Demartino, Julie A; Kitson, Christopher; Stevenson, Christopher S; Budd, David C

    2013-01-01

    Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

  13. Efficient Process Development of Recombinant Human Granulocyte Colony-Stimulating Factor (rh-GCSF) Production in Escherichia coli

    PubMed Central

    Babaeipour, Valiollah; Khanchezar, Sirwan; Mofid, Mohammad Reza; Pesaran Hagi Abbas, Mahdi

    2015-01-01

    Background: The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. Methods: An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h-1 at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. Results: Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l-1 and 32.1 g.l-1. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g-1 DCW and 1.83 g.l-1.h-1, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen® and PD-Grastim showed that all proteins have a similar secondary structure. Conclusion: Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity. PMID:25864815

  14. Brain-derived neurotrophic factor--a major player in stimulation-induced homeostatic metaplasticity of human motor cortex?

    PubMed

    Mastroeni, Claudia; Bergmann, Til Ole; Rizzo, Vincenzo; Ritter, Christoph; Klein, Christine; Pohlmann, Ines; Brueggemann, Norbert; Quartarone, Angelo; Siebner, Hartwig Roman

    2013-01-01

    Repetitive transcranial magnetic stimulation (rTMS) of the human motor hand area (M1HAND) can induce lasting changes in corticospinal excitability as indexed by a change in amplitude of the motor-evoked potential. The plasticity-inducing effects of rTMS in M1HAND show substantial inter-individual variability which has been partially attributed to the val(66)met polymorphism in the brain-derived neurotrophic factor (BDNF) gene. Here we used theta burst stimulation (TBS) to examine whether the BDNF val(66)met genotype can be used to predict the expression of TBS-induced homeostatic metaplasticity in human M1HAND. TBS is a patterned rTMS protocol with intermittent TBS (iTBS) usually inducing a lasting increase and continuous TBS (cTBS) a lasting decrease in corticospinal excitability. In three separate sessions, healthy val(66)met (n = 12) and val(66)val (n = 17) carriers received neuronavigated cTBS followed by cTBS (n = 27), cTBS followed by iTBS (n = 29), and iTBS followed by iTBS (n = 28). Participants and examiner were blinded to the genotype at the time of examination. As expected, the first TBS intervention induced a decrease (cTBS) and increase (iTBS) in corticospinal excitability, respectively, at the same time priming the after effects caused by the second TBS intervention in a homeostatic fashion. Critically, val(66)met carriers and val(66)val carriers showed very similar response patterns to cTBS and iTBS regardless of the order of TBS interventions. Since none of the observed TBS effects was modulated by the BDNF val(66)met polymorphism, our results do not support the notion that the BDNF val(66)met genotype is a major player with regard to TBS-induced plasticity and metaplasticity in the human M1HAND.

  15. Tumour necrosis factor-α and its receptors in the beneficial effects of vagal stimulation after myocardial infarction in rats.

    PubMed

    Kong, Shan-Shan; Liu, Jin-Jun; Hwang, Tyzh-Chang; Yu, Xiao-Jiang; Lu, Yi; Zang, Wei-Jin

    2011-05-01

    1. Acute myocardial infarction (AMI) often activates the sympathetic system and inhibits the vagal system. Long-term vagal nerve stimulation (VNS) exerts several beneficial effects on the ischaemic heart, including an anti-inflammatory effect. The aim of the present study was to investigate whether short-term VNS during AMI could inhibit tumour necrosis factor (TNF)-α expression and the effect of TNF receptor (TNFR), key components in inflammatory responses to AMI, in a rodent model. 2. Adult male Sprague-Dawley rats were divided into four groups, namely a control (C), VNS (S), AMI (M) and an AMI group subjected to prior VNS (MS). In the S and MS groups, the right vagus nerve was stimulated electrically for 4 h; in the M and MS groups, AMI was induced by occlusion of the left anterior descending coronary artery. Haemodynamic data were monitored continuously using a multichannel physiological recorder. Lactate dehydrogenase (LDH) leakage, creatine kinase (CK) leakage and infarct size were determined. The expression of TNF-α and its receptors were analysed by reverse transcription-polymerase chain reaction, western blotting and ELISA. 3. Compared with the control group, rats in the M group had low blood pressure, high left ventricular (LV) end-diastolic pressure, a depressed maximum dP/dt of LV pressure, higher LDH and CK leakage, a larger infarct size, increased TNF-α levels and an increased TNFR1/TNFR2 ratio. However, these presumably harmful effects of AMI were all significantly ameliorated by VNS during AMI (MS group). 4. In conclusion, VNS can rectify ischaemia-induced cardiac dysfunction partly via inhibition of a TNF-α-mediated signalling pathway.

  16. Effect of recombinant human macrophage colony-stimulating factor 1 on immunopathology of experimental brucellosis in mice.

    PubMed Central

    Doyle, A G; Halliday, W J; Barnett, C J; Dunn, T L; Hume, D A

    1992-01-01

    Brucella abortus injected into CBA mice replicated primarily in the spleen and liver, reaching a peak bacterial count in both organs about 7 days postinfection. The organism was eliminated from the liver but declined to a chronic phase in the spleen. The infection caused hepatosplenomegaly. An influx of macrophages into the two organs was monitored by quantitative Northern (RNA blot) analysis of the macrophage-specific marker lysozyme mRNA. Lysozyme mRNA was detectable in spleen and increased three- to fourfold during infection. In liver, lysozyme mRNA was initially undetectable, but at about the peak of infection it reached a level comparable to that in the spleen. Macrophage colony-stimulating factor 1 (CSF-1) has been reported to be elevated in the circulation of animals infected with B. abortus and is known to stimulate monocytopoiesis. To investigate the role of CSF-1 in pathogenesis, we studied the effect of further increasing the CSF-1 concentration by administration of recombinant human CSF-1. Since the infection is characterized by several distinct phases, recombinant human CSF-1 was administered at defined times relative to these phases. Pronounced effects were observed only when CSF-1 administration was begun during the developing acute phase. The consequences were decreased bacterial numbers in the spleen but an increase in the liver, reduced antibody generation, and increased hepatosplenomegaly. A feature of many chronic intracellular infections is immunosuppression. B. abortus caused a substantial diminution of responsiveness of spleen cells to T-cell mitogens, particularly concanavalin A. This action was mimicked by CSF-1 treatment of the animals prior to spleen cell isolation. The results suggest that CSF-1 plays a role in macrophage recruitment in brucellosis and that recruited macrophages contribute to the immunopathology and immunosuppression. PMID:1548070

  17. Macrophage inflammatory protein-1 delta: a novel osteoclast stimulating factor secreted by renal cell carcinoma bone metastasis.

    PubMed

    Kominsky, Scott L; Abdelmagid, Samir M; Doucet, Michele; Brady, Kelly; Weber, Kristy L

    2008-03-01

    Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1 delta (MIP-1 delta), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1 delta expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1 delta, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1 delta stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1 delta treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1 delta significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1 delta expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.

  18. CREB, AP‐1, ternary complex factors and MAP kinases connect transient receptor potential melastatin‐3 (TRPM3) channel stimulation with increased c‐Fos expression

    PubMed Central

    Rubil, Sandra; Rössler, Oliver G.

    2016-01-01

    Background and Purpose The rise in intracellular Ca2+ stimulates the expression of the transcription factor c‐Fos. Depending on the mode of entry of Ca2+ into the cytosol, distinct signal transducers and transcription factors are required. Here, we have analysed the signalling pathway connecting a Ca2+ influx via activation of transient receptor potential melastatin‐3 (TRPM3) channels with enhanced c‐Fos expression. Experimental Approach Transcription of c‐Fos promoter/reporter genes that were integrated into the chromatin via lentiviral gene transfer was analysed in HEK293 cells overexpressing TRPM3. The transcriptional activation potential of c‐Fos was measured using a GAL4‐c‐Fos fusion protein. Key Results The signalling pathway connecting TRPM3 stimulation with enhanced c‐Fos expression requires the activation of MAP kinases. On the transcriptional level, three Ca2+‐responsive elements, the cAMP‐response element and the binding sites for the serum response factor (SRF) and AP‐1, are essential for the TRPM3‐mediated stimulation of the c‐Fos promoter. Ternary complex factors are additionally involved in connecting TRPM3 stimulation with the up‐regulation of c‐Fos expression. Stimulation of TRPM3 channels also increases the transcriptional activation potential of c‐Fos. Conclusions and Implications Signalling molecules involved in connecting TRPM3 with the c‐Fos gene are MAP kinases and the transcription factors CREB, SRF, AP‐1 and ternary complex factors. As c‐Fos constitutes, together with other basic region leucine zipper transcription factors, the AP‐1 transcription factor complex, the results of this study explain TRPM3‐induced activation of AP‐1 and connects TRPM3 with the biological functions regulated by AP‐1. © 2015 The British Pharmacological Society PMID:26493679

  19. Macrophage colony stimulating factor regulation by nuclear factor kappa B: a relevant pathway in human immunodeficiency virus type 1 infected macrophages.

    PubMed

    Kogan, Michael; Haine, Valerie; Ke, Yuxong; Wigdahl, Brian; Fischer-Smith, Tracy; Rappaport, Jay

    2012-03-01

    Macrophage colony stimulating factor (M-CSF) is a cytokine that promotes monocyte differentiation and survival. When overexpressed, M-CSF contributes to pathology in a wide variety of diseases, including osteoporosis, obesity, certain human cancers, and in human immunodeficiency virus type 1 (HIV-1) infection, particularly with respect to monocyte/macrophage infection and the development of HIV-1 associated central nervous system disorders. In this study, our aim was to expand the current knowledge of M-CSF regulation, focusing on nuclear factor kappa B (NF-κB), a transcription factor playing a prominent role during inflammation and HIV-1 infection. Our results suggest that tumor necrosis factor alpha (TNF-α) promotes M-CSF secretion in primary macrophages and activates the -1310/+48 bp M-CSF promoter in Mono-Mac 1 cells. Inhibitors of the NF-κB pathway diminish this response. We identified four putative NF-κB and four CCAAT-enhancer-binding protein beta binding sites within the M-CSF promoter. Our findings, using promoter constructs mutated at individual NF-κB sites within the M-CSF promoter region, suggest that these sites are redundant with respect to NF-κB regulation. TNF-α treatment promoted NF-κB p65 binding to the M-CSF promoter in phorbol 12-myristate 13-acetate (PMA) treated U937 cells chronically infected with HIV-1 (U1 cells), but not in PMA treated uninfected U937 cells, suggesting that the presence of HIV-1 increases the NF-κB response. In conclusion, our findings demonstrate that NF-κB induces M-CSF expression on a promoter level via multiple functional NF-κB binding sites and that this pathway is likely relevant in HIV-1 infection of macrophages.

  20. Role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in gastric ulcer healing in mice.

    PubMed

    Kawahara, Y; Nakase, Y; Isomoto, Y; Matsuda, N; Amagase, K; Kato, S; Takeuchi, K

    2011-08-01

    We examined the role of macrophage colony-stimulating factor (M-CSF)-dependent macrophages in the healing of gastric ulcers in mice. Male M-CSF-deficient (op/op) and M-CSF-expressing heterozygote (+/?) mice were used. Gastric ulcers were induced by thermal cauterization under ether anesthesia, and healing was observed for 14 days after ulceration. The numbers of macrophages and microvessels in the gastric mucosa were determined immunohistochemically with anti-CD68 and anti-CD31 antibodies, respectively. Expression of tumor necrosis factor (TNF)-α, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF) mRNA was determined via real-time reverse transcription-polymerase chain reaction (RT-PCR), and the mucosal content of prostaglandin (PG) E(2) was determined via enzyme immunoassay on day 10 after ulceration. The healing of gastric ulcers was significantly delayed in op/op mice compared with +/? mice. Further, significantly fewer macrophages were observed in the normal gastric mucosa of op/op mice than in +/? mice. Ulcer induction caused a marked accumulation of macrophages around the ulcer base in +/? mice, but this response was attenuated in op/op mice. The mucosal PGE(2) content as well as the expression of COX-2, VEGF, and TNF-α mRNA were all upregulated in the ulcerated area of +/? mice but significantly suppressed in op/op mice. The degree of vascularization in the ulcerated area was significantly lower in op/op mice than in +/? mice. Taken together, these results suggest that M-CSF-dependent macrophages play an important role in the healing of gastric ulcers, and that this action may be associated with angiogenesis promoted by upregulation of COX-2/PGE(2) production.

  1. Sulodexide down-regulates the release of cytokines, chemokines, and leukocyte colony stimulating factors from human macrophages: role of glycosaminoglycans in inflammatory pathways of chronic venous disease.

    PubMed

    Mannello, Ferdinando; Ligi, Daniela; Canale, Matteo; Raffetto, Joseph D

    2014-01-01

    Chronic venous disease (CVeD) is a debilitating condition that affects millions of individuals worldwide. The condition can result in varicose veins, or advance to severe skin changes and venous ulceration. The fundamental basis for CVeD is inflammation within the venous circulation and that it is subjected to increased hydrostatic pressure resulting in increased ambulatory venous pressure. The inflammation involves leukocytes, in particular macrophages and monocytes, inflammatory modulators and chemokines, cytokine expression, growth factors, metalloproteinase (MMP) activity, and many regulatory pathways that perpetuate inflammation. Sulodexide (SDX) is a glycosaminoglycan with pro-fibrinolytic and anti-thrombotic properties. We have previously demonstrated that SDX inhibits the secretion of pro-zymogen MMP-9 from human leukocytes without displacing high molecular complexes of MMP-9. The anti-inflammatory properties of SDX on activated leukocytes have not been well established. We hypothesized that SDX will reduce the secretion of inflammatory mediators from lipopolysaccharide (LPS)-stimulated macrophages. Therefore, we evaluated the effects of SDX on LPS-stimulated macrophage secretion of various inflammatory and anti-inflammatory cytokines, chemokines, and colony stimulating factors. We used microplatebased multiplex immunoassays. LPS-stimulated macrophages in vitro caused a substantial increase of interleukins, tumor necrosis factor, interferon, chemokines and colony stimulating factors. The addition of SDX caused both a dose-dependent and dose-independent decrease in nearly all of the inflammatory cytokines, chemokines and colony stimulating factors. These findings suggest that SDX has a significant effect on the release of inflammatory mediators from macrophages, and may be useful in the treatment of early and advanced CVeD.

  2. Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6.

    PubMed Central

    Rose, T M; Bruce, A G

    1991-01-01

    Oncostatin M (OSM), a glycoprotein of Mr approximately 28,000 produced by activated monocyte and T-lymphocyte cell lines, was previously identified by its ability to inhibit the growth of cells from melanoma and other solid tumors. We have detected significant similarities in the primary amino acid sequences and predicted secondary structures of OSM, leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). Analysis of the genes encoding these proteins revealed a shared exon organization, suggesting evolutionary descent from a common ancestral gene. Using a panel of DNAs from somatic cell hybrids, we have shown that OSM, like LIF, is located on human chromosome 22. We have also demonstrated that OSM has the ability to inhibit the proliferation of murine M1 myeloid leukemic cells and can induce their differentiation into macrophage-like cells, a function shared by LIF, G-CSF, and IL-6. We propose that OSM, LIF, G-CSF, and IL-6 are structurally related members of a cytokine family that have in common the ability to modulate differentiation of a variety of cell types. Images PMID:1717982

  3. Diagnostic Power of Vascular Endothelial Growth Factor and Macrophage Colony-Stimulating Factor in Breast Cancer Patients Based on ROC Analysis

    PubMed Central

    Głażewska, Edyta Katarzyna; Będkowska, Grażyna Ewa; Chorąży, Przemysław; Szmitkowski, Maciej; Ławicki, Sławomir

    2016-01-01

    Breast cancer (BC) is the most common malignancy in women. Vascular endothelial growth factor (VEGF) has been described as an important regulator of angiogenesis which plays a vital role in the progression of tumor. Macrophage colony-stimulating factor (M-CSF) is a cytokine whose functions include regulation of hematopoietic lineages cells growth, proliferation, and differentiation. We investigated the diagnostic significance of these parameters in comparison to CA15-3 in BC patients and in relation to the control group (benign breast tumor and healthy women). Plasma levels of the tested parameters were determined by ELISA and CA15-3 was determined by CMIA. VEGF was shown to be comparable to CA15-3 values of sensitivity in BC group and, what is more important, higher values in early stages of BC. VEGF was also the only parameter which has statistically significant AUC in all stages of cancer. M-CSF has been shown to be comparable to CA15-3 and VEGF, specificity, and AUC values only in stages III and IV of BC. These results indicate the usefulness and high diagnostic power of VEGF in the detection of BC. Also, it occurred to be the best candidate for cancer diagnostics in stages I and II of BC and in the differentiation between BC and benign cases. PMID:27445439

  4. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  5. Spatial factors and muscle spindle input influence the generation of neuromuscular responses to stimulation of the human foot

    NASA Astrophysics Data System (ADS)

    Layne, Charles S.; Forth, Katharine E.; Abercromby, Andrew F. J.

    2005-05-01

    Removal of the mechanical pressure gradient on the soles leads to physiological adaptations that ultimately result in neuromotor degradation during spaceflight. We propose that mechanical stimulation of the soles serves to partially restore the afference associated with bipedal loading and assists in attenuating the negative neuromotor consequences of spaceflight. A dynamic foot stimulus device was used to stimulate the soles in a variety of conditions with different stimulation locations, stimulation patterns and muscle spindle input. Surface electromyography revealed the lateral side of the sole elicited the greatest neuromuscular response in ankle musculature, followed by the medial side, then the heel. These responses were modified by preceding stimulation. Neuromuscular responses were also influenced by the level of muscle spindle input. These results provide important information that can be used to guide the development of a "passive" countermeasure that relies on sole stimulation and can supplement existing exercise protocols during spaceflight.

  6. Nerve growth factor and brain-derived neurotrophic factor but not granulocyte colony-stimulating factor, nimodipine and dizocilpine, require ATP for neuroprotective activity after oxygen-glucose deprivation of primary neurons.

    PubMed

    Ferenz, Katja B; Gast, Ronald E; Rose, Karsten; Finger, Indra E; Hasche, Anja; Krieglstein, Josef

    2012-04-11

    In previous work, we have demonstrated by radiolabeling, mass spectrometry and site-directed mutagenesis that nerve growth factor (NGF) as well as brain-derived neurotrophic factor (BDNF) and fibroblast growth factor 2 (FGF2) are capable of ATP-binding and that this binding appears to be essential for their neuroprotective activity. In this study, we attempted to shed some light on the question whether ATP is a general prerequisite for neuroprotection. Therefore, we used the non-ATP-binding granulocyte colony-stimulating factor (GCSF), the calcium antagonist nimodipine and the NMDA antagonist dizocilpine to find out whether they need ATP for neuroprotection comparable to NGF and BDNF. However, ATP was not necessary for the neuroprotective effects of GCSF, nimodipine and dizocilpine on primary cultures of rat cortical neurons damaged by oxygen-glucose deprivation whereas neuroprotection was demonstrable for NGF and BDNF only when ATP was present in the culture medium at a concentration higher than ca. 0.4nmol/l. In circular dichroism studies ATP caused changes of the secondary structure of NGF but not of GCSF. Taken together, we suggest that ATP is not a general prerequisite for neuroprotectivity but some growth factors like NGF and BDNF can stimulate their receptors only if they have bound ATP.

  7. Vaccination with Irradiated Autologous Melanoma Cells Engineered to Secrete Human Granulocyte--Macrophage Colony-Stimulating Factor Generates Potent Antitumor Immunity in Patients with Metastatic Melanoma

    NASA Astrophysics Data System (ADS)

    Soiffer, Robert; Lynch, Thomas; Mihm, Martin; Jung, Ken; Rhuda, Catherine; Schmollinger, Jan C.; Hodi, F. Stephen; Liebster, Laura; Lam, Prudence; Mentzer, Steven; Singer, Samuel; Tanabe, Kenneth K.; Benedict Cosimi, A.; Duda, Rosemary; Sober, Arthur; Bhan, Atul; Daley, John; Neuberg, Donna; Parry, Gordon; Rokovich, Joseph; Richards, Laurie; Drayer, Jan; Berns, Anton; Clift, Shirley; Cohen, Lawrence K.; Mulligan, Richard C.; Dranoff, Glenn

    1998-10-01

    We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte--macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte--macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.

  8. In vitro stimulation of vascular endothelial growth factor by borate-based glass fibers under dynamic flow conditions.

    PubMed

    Chen, Sisi; Yang, Qingbo; Brow, Richard K; Liu, Kun; Brow, Katherine A; Ma, Yinfa; Shi, Honglan

    2017-04-01

    Bioactive borate glass has been recognized to have both hard and soft tissue repair and regeneration capabilities through stimulating both osteogenesis and angiogenesis. However, the underlying biochemical and cellular mechanisms remain unclear. In this study, dynamic flow culturing modules were designed to simulate the micro-environment near the vascular depletion and hyperplasia area in wound-healing regions, thus to better investigate the mechanisms underlying the biocompatibility and functionality of borate-based glass materials. Glass fibers were dosed either upstream or in contact with the pre-seeded cells in the dynamic flow module. Two types of borate glasses, doped with (1605) or without (13-93B3) CuO and ZnO, were studied along with the silicate-based glass, 45S5. Substantial fiber dissolution in cell culture medium was observed, leading to the release of ions (boron, sodium and potassium) and the deposition of a calcium phosphate phase. Different levels of vascular endothelial growth factor secretion were observed from cells exposed to these three glass fibers, and the copper/zinc containing borate 1605 fibers exhibited the most positive influence. These results indicate that dynamic studies of in vitro bioactivity provide useful information to understand the in vivo response to bioactive borate glasses.

  9. Macrophage Colony Stimulating Factor Derived from CD4+ T Cells Contributes to Control of a Blood-Borne Infection

    PubMed Central

    de Melo, Gabrielly L.; Anidi, Chioma; Hamburger, Rebecca; Pham, Jennifer

    2016-01-01

    Dynamic regulation of leukocyte population size and activation state is crucial for an effective immune response. In malaria, Plasmodium parasites elicit robust host expansion of macrophages and monocytes, but the underlying mechanisms remain unclear. Here we show that myeloid expansion during P. chabaudi infection is dependent upon both CD4+ T cells and the cytokine Macrophage Colony Stimulating Factor (MCSF). Single-cell RNA-Seq analysis on antigen-experienced T cells revealed robust expression of Csf1, the gene encoding MCSF, in a sub-population of CD4+ T cells with distinct transcriptional and surface phenotypes. Selective deletion of Csf1 in CD4+ cells during P. chabaudi infection diminished proliferation and activation of certain myeloid subsets, most notably lymph node-resident CD169+ macrophages, and resulted in increased parasite burden and impaired recovery of infected mice. Depletion of CD169+ macrophages during infection also led to increased parasitemia and significant host mortality, confirming a previously unappreciated role for these cells in control of P. chabaudi. This work establishes the CD4+ T cell as a physiologically relevant source of MCSF in vivo; probes the complexity of the CD4+ T cell response during type 1 infection; and delineates a novel mechanism by which T helper cells regulate myeloid cells to limit growth of a blood-borne intracellular pathogen. PMID:27923070

  10. Effect of granulocyte colony stimulating factor (G-CSF) on IVF outcomes in infertile women: An RCT

    PubMed Central

    Eftekhar, Maryam; Hosseinisadat, Robabe; Baradaran, Ramesh; Naghshineh, Elham

    2016-01-01

    Background: Despite major advances in assisted reproductive techniques, the implantation rates remain relatively low. Some studies have demonstrated that intrauterine infusion of granulocyte colony stimulating factor (G-CSF) improves implantation in infertile women. Objective: To assess the G-CSF effects on IVF outcomes in women with normal endometrial thickness. Materials and methods: In this randomized controlled clinical trial, 100 infertile women with normal endometrial thickness who were candidate for IVF were evaluated in two groups. Exclusion criteria were positive history of repeated implantation failure (RIF), endocrine disorders, severe endometriosis, congenital or acquired uterine anomaly and contraindication for G-CSF (renal disease, sickle cell disease, or malignancy). In G-CSF group (n=50), 300 µg trans cervical intrauterine of G-CSF was administered at the oocyte retrieval day. Controls (n=50) were treated with standard protocol. Chemical, clinical and ongoing pregnancy rates, implantation rate, and miscarriage rate were compared between groups. Results: Number of total and mature oocytes (MII), two pronuclei (2PN), total embryos, transferred embryos, quality of transferred embryos, and fertilization rate did not differ significantly between two groups. So there were no significant differences between groups in chemical, clinical and ongoing pregnancy rate, implantation rate, and miscarriage rate Conclusion: our result showed in normal IVF patients with normal endometrial thickness, the intrauterine infusion of G-CSF did not improve pregnancy outcomes. PMID:27326420

  11. Structure of macrophage colony stimulating factor bound to FMS: Diverse signaling assemblies of class III receptor tyrosine kinases

    SciTech Connect

    Chen, Xiaoyan; Liu, Heli; Focia, Pamela J.; Shim, Ann Hye-Ryong; He, Xiaolin

    2009-06-12

    Macrophage colony stimulating factor (M-CSF), through binding to its receptor FMS, a class III receptor tyrosine kinase (RTK), regulates the development and function of mononuclear phagocytes, and plays important roles in innate immunity, cancer and inflammation. We report a 2.4 {angstrom} crystal structure of M-CSF bound to the first 3 domains (D1-D3) of FMS. The ligand binding mode of FMS is surprisingly different from KIT, another class III RTK, in which the major ligand-binding domain of FMS, D2, uses the CD and EF loops, but not the {beta}-sheet on the opposite side of the Ig domain as in KIT, to bind ligand. Calorimetric data indicate that M-CSF cannot dimerize FMS without receptor-receptor interactions mediated by FMS domains D4 and D5. Consistently, the structure contains only 1 FMS-D1-D3 molecule bound to a M-CSF dimer, due to a weak, hydrophilic M-CSF:FMS interface, and probably a conformational change of the M-CSF dimer in which binding to the second site is rendered unfavorable by FMS binding at the first site. The partial, intermediate complex suggests that FMS may be activated in two steps, with the initial engagement step distinct from the subsequent dimerization/activation step. Hence, the formation of signaling class III RTK complexes can be diverse, engaging various modes of ligand recognition and various mechanistic steps for dimerizing and activating receptors.

  12. Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats

    PubMed Central

    Okubo, Masamichi; Yamanaka, Hiroki; Kobayashi, Kimiko; Dai, Yi; Kanda, Hirosato; Yagi, Hideshi; Noguchi, Koichi

    2016-01-01

    Peripheral nerve injury induces proliferation of microglia in the spinal cord, which can contribute to neuropathic pain conditions. However, candidate molecules for proliferation of spinal microglia after injury in rats remain unclear. We focused on the colony-stimulating factors (CSFs) and interleukin-34 (IL-34) that are involved in the proliferation of the mononuclear phagocyte lineage. We examined the expression of mRNAs for macrophage-CSF (M-CSF), granulocyte macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF) and IL-34 in the dorsal root ganglion (DRG) and spinal cord after spared nerve injury (SNI) in rats. RT-PCR and in situ hybridization revealed that M-CSF and IL-34, but not GM- or G-CSF, mRNAs were constitutively expressed in the DRG, and M-CSF robustly increased in injured-DRG neurons. M-CSF receptor mRNA was expressed in naive rats and increased in spinal microglia following SNI. Intrathecal injection of M-CSF receptor inhibitor partially but significantly reversed the proliferation of spinal microglia and in early phase of neuropathic pain induced by SNI. Furthermore, intrathecal injection of recombinant M-CSF induced microglial proliferation and mechanical allodynia. Here, we demonstrate that M-CSF is a candidate molecule derived from primary afferents that induces proliferation of microglia in the spinal cord and leads to induction of neuropathic pain after peripheral nerve injury in rats. PMID:27071004

  13. Alpha-Melanocyte-Stimulating Hormone Attenuates Behavioral Effects of Corticotropin-Releasing Factor in Isolated Guinea Pig Pups

    PubMed Central

    Miller, Emily; Deak, Terrence; Hennessy, Michael B.

    2016-01-01

    During a 3-hr period of social isolation in a novel environment, guinea pig pups exhibit an initial active phase of behavioral responsiveness, characterized primarily by vocalizing, which is then followed by a stage of passive responsiveness in which pups display a distinctive crouch, eye-closing, and extensive piloerection. Prior treatment of pups with alpha-melanocyte-stimulating hormone (α-MSH) reduces each of the passive behaviors. The onset of passive responding during separation can be accelerated with peripheral injection of corticotropin-releasing factor (CRF). To examine whether CRF produces its effects through a mechanism similar to that of prolonged separation, we examined the effect of administering α-MSH to pups injected with CRF. As expected, CRF markedly enhanced passive responding during a 60-min period of separation. α-MSH delivered by either intracerebroventricular infusion or intraperitoneal injection significantly reduced each of the passive behavioral responses without significantly affecting active behavior. These findings, together with previous results indicating that it is the anti-inflammatory property of α-MSH that is responsible for its behavioral effects during prolonged separation, suggest that peripheral CRF speeds the induction of passive responding through a mechanism involving enhanced proinflammatory activity. PMID:19492314

  14. Peripheral blood morphologic changes after high-dose antineoplastic chemotherapy and recombinant human granulocyte colony-stimulating factor administration.

    PubMed

    Kerrigan, D P; Castillo, A; Foucar, K; Townsend, K; Neidhart, J

    1989-09-01

    The peripheral blood morphologic findings in 17 patients with cancer who had received high-dose cytotoxic chemotherapy followed by recombinant human-granulocyte colony-stimulating factor (rh-GCSF) were reviewed and compared with a control group of patients who received only high-dose chemotherapy. Both groups showed dysmyelopoiesis (abnormal granulation and nuclear lobulation) in the granulocytic series during the period of bone marrow recovery that followed the cytotoxic chemotherapy. Most of these morphologic abnormalities were more prominent in the rh-GCSF-treated group. Monocytic cells in both groups showed prominent vacuolation and immature nuclei. The percentages and absolute numbers of large granular lymphocytes were increased in the rh-GCSF group compared with the control group. No quantitative or qualitative abnormalities of eosinophilic or basophilic granulocytes were detected in either group. Both groups showed nonspecific red blood cell abnormalities, and large platelets were present in half of the control group smears. This report provides the first detailed peripheral blood morphologic description in patients treated with rh-GCSF and high-dose chemotherapy.

  15. Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter.

    PubMed

    Susa, Takao; Nakayama, Michie; Kitahara, Kousuke; Kimoto, Fuyuko; Kato, Takako; Kato, Yukio

    2007-06-08

    Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.

  16. Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor.

    PubMed

    Sadallah, S; Lach, E; Schwarz, S; Gratwohl, A; Spertini, O; Schifferli, J A

    1999-01-01

    Complement receptor type 1 is expressed by erythrocytes and most leukocytes. A soluble form is shed from the leukocytes and found in plasma (sCR1). sCR1 is a powerful inhibitor of complement. We report an increased sCR1 in the plasma of leukemia patients, up to levels producing measurable complement inhibition. Half of the 180 patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) had sCR1 levels above the normal range. The highest levels were observed in T-ALL (17 patients). The complement function of a T-ALL serum was improved by blocking sCR1 with a specific mAb (3D9). Measurements in 16 peripheral stein cell donors before and after granulocyte colony-stimulating factor (G-CSF) administration showed an increase in sCR1 (before, 43.8+/-15.4; at day 5, 118.3+/-44.7 ng/mL; P < 0.0001). This increase paralleled the increase in total leukocyte counts and was concomitant with de novo leukocyte mRNA CR1 expression in all three individuals tested. Whether pharmacological intervention may be used to up-regulate sCR1 so as to inhibit complement in vivo should be further investigated.

  17. Terbinafine: effects on platelet-derived growth factor-stimulated smooth muscle cells in vitro and myointimal proliferation in vivo

    SciTech Connect

    McCarthy, L.; Van Halen, R.G.; St. Denny, I.H.; Glinka, K.G.; Handley, D.A.; Stuetz, A.; Nemecek, G.M.

    1987-05-01

    Terbinafine (T; (E)-N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine), an antimycotic agent with antimitogenic activity in fibroblasts, was examined for its effects on platelet-derived growth factor (PDGF)-stimulated aortic smooth muscle cell DNA synthesis in vitro and myointimal proliferation in vivo. Exposure of smooth muscle cells to 1-25 ..mu..M T resulted in a concentration-dependent inhibition of PDGF-induced mitogenesis as determined by (/sup 3/H)thymidine incorporation or cell number. The IC/sub 50/ for T was approximately 5 ..mu..M. The inhibitory effect of terbinafine persisted in the presence of 0.4-8.0 ..mu..g/ml cholesterol or 130 ..mu..g/ml mevalonate. Administration of T to rats for 2 d before and 14 d after balloon catheter carotid injury resulted in a 40% decrease in lesion area. These observations indicate that T is both a potent in vitro antagonist of the smooth muscle cell mitogenic response to PDGF and an effective, well-tolerated, orally active inhibitor of myointimal proliferation in vivo.

  18. Antifungal activity of recombinant human macrophage colony-stimulating factor in models of acute and chronic candidiasis in the rat.

    PubMed

    Vitt, C R; Fidler, J M; Ando, D; Zimmerman, R J; Aukerman, S L

    1994-02-01

    Models of acute and chronic candidiasis were developed in Fischer 344 rats to evaluate the therapeutic efficacy of recombinant human macrophage colony-stimulating factor (rhM-CSF) alone and in combination with the antifungal agent fluconazole. In the acute model, rats were challenged by intravenous injection with 2 x 10(6) Candida albicans, approximately 4 times the LD50. Daily subcutaneous (sc) bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 0.3 mg/kg of fluconazole improved the median survival time from 5 days (32% survival) with fluconazole alone to > 30 days (88% survival) in the rhM-CSF- and fluconazole-treated rats. In the chronic model, daily sc bolus injections of rhM-CSF for 10 days plus a single sc bolus dose of 1.0 mg/kg of fluconazole decreased the median titer of C. albicans cultured from the kidneys by 10-fold at 15 and 30 days after infection. These studies showed that rhM-CSF treatment improved the therapeutic outcome in both the acute and chronic rat model of candidiasis when used with fluconazole, a standard fungistatic agent.

  19. 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

  20. Granulocyte-macrophage colony stimulating factor: Evaluation of biopharmaceutical formulations by stability-indicating RP-LC method and bioassay.

    PubMed

    Leal, Diogo Paim; Souto, Ricardo Bizogne; Schutkoski, Renato; Bergamo, Ana Cláudia; Dalmora, Sérgio Luiz

    2011-07-01

    The granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic cells and activates granulocytes and macrophages. A reversed-phase liquid chromatography (RP-LC) method was validated for the assessing of the stability of non-glycosylated recombinant rhGM-CSF (Molgramostim) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C(4) column (250 mm × 4.6 mm i.d.), maintained at 45 °C. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA in acetonitrile, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 29.2 min, and was linear over the concentration range of 2-300 μg/mL (r(2) = 0.9992). Specificity was established in degradation studies. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p < 0.05). The method was applied to the assessment of rhGM-CSF and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay. It is concluded that the employment of RP-LC in conjunction with current methods allows a great improvement in monitoring stability, quality control and thereby assures the therapeutic efficacy.

  1. Association between Disability and Psychological Factors and Dose of Neuromuscular Electrical Stimulation in Subjects with Rheumatoid Arthritis

    PubMed Central

    Piva, Sara R.; Lasinski, Stephanie; Almeida, Gustavo JM; Fitzgerald, G. Kelley; Delitto, Anthony

    2013-01-01

    Background The therapeutic effect of neuromuscular electrical stimulation (NMES) on muscle strengthening and hypertrophy depends on its dose. Patients must tolerate high doses of NMES to maximize gains in muscle function. It is unknown why some patients are able to achieve high NMES dose while others are not. Disability and psychological attributes may play a role in a patient’s tolerance of NMES dose. Purpose To explore if disability and psychological attributes associate with the ability to achieve high doses of NMES in patients with rheumatoid arthritis (RA). Methods Cross-sectional study. Forty subjects with RA participated in 2 sessions of NMES intervention to the quadriceps muscles. The highest NMES dose achieved by each subject was recorded. Dose was defined as the torque produced by the NMES as a percentage of the torque produced during a maximum voluntary isometric contraction. Subjects were then grouped in high or low NMES dose. Variables investigated in this study included disability, pain coping strategies, pain acceptance, sense of mastery or control, anxiety, and depression. Correlations were sought between these factors and NMES dose. Main Results In unadjusted models, disability, coping self-statements, catastrophizing, and anxiety were predictors of NMES dose. In adjusted models only disability (OR = 0.17 [95% CI: 0.04, 0.77]) and catastrophizing (OR = 0.85 [95% CI: 0.72, 0.99]) predicted NMES dose. Conclusion Patients with RA with lower disability and lower catastrophising achieve higher doses of NMES. Identifying factors associated with achieving high NMES dose may guide strategies to improve effectiveness of this intervention. PMID:24967156

  2. Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

    PubMed Central

    Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W.; Hou, Rong; Shen, Wei

    2015-01-01

    It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro. PMID:26375397

  3. Epidermal growth factor inhibits radioiodine uptake but stimulates deoxyribonucleic acid synthesis in newborn rat thyroids grown in nude mice

    SciTech Connect

    Ozawa, S.; Spaulding, S.W. )

    1990-08-01

    We have studied the effect of altering the level of circulating epidermal growth factor (EGF) on the function and growth of newborn rat thyroids transplanted into nude mice. Preliminary studies confirmed that sialoadenectomy reduced circulating EGF levels in nude mice (from 0.17 +/- 0.02 to 0.09 +/- 0.02 ng/ml), and that ip injection of 5 micrograms EGF raised EGF levels (the peak level of 91.7 +/- 3.3 ng/ml was achieved at 30 min, with a subsequent half-life of about 1 h). The radioiodine uptake by newborn rat thyroid transplants in the sialoadenectomized and sham-operated animals correlated inversely with the circulating EGF levels determined when the mice were killed (r = -0.99). Low-dose TSH treatment (0.1 microU/day) generally stimulated the radioiodine uptake, but high-dose TSH groups (100 microU/day) were not significantly different from the control group. The 5-day nuclear (3H)thymidine labeling index was 6.8 +/- 0.5% IN newborn rat thyroid transplants grown in sialoadenectomized animals, 13.1 +/- 0.3% in sham-operated animals, and 16.8 +/- 0.5% in nude mice receiving 5 micrograms EGF ip daily. In general, both low-dose and high-dose TSH promoted DNA synthesis under low EGF conditions but were ineffective in the presence of higher levels of EGF. Adult rat thyroid transplants showed no significant responses. Although sialoadenectomy may alter other factors besides EGF, it appears that changes in the levels of circulating EGF within the physiological range affect the function and growth of newborn rat thyroid transplants. Circulating EGF may play a role in thyroid maturation and may also be involved in the regulation of thyroid function throughout life.

  4. Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca).

    PubMed

    Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W; Hou, Rong; Shen, Wei

    2015-01-01

    It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.

  5. Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-defective adenovirus induces a sustained increase in circulating peripheral blood neutrophils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of immunomodulators is a promising area for biotherapeutic, prophylactic, and metaphylactic use to prevent and combat infectious disease, particularly during periods of peak disease incidence. Cytokines, including granulocyte colony-stimulating factor (G-CSF), are one class of compounds that...

  6. Predicting erythroid response to recombinant erythropoietin plus granulocyte colony-stimulating factor therapy following a single subcutaneous bolus in patients with myelodysplasia.

    PubMed

    Bowen, David; Hyslop, Ann; Keenan, Norene; Groves, Michael; Culligan, Dominic; Johnson, Peter; Shaw, Ann; Geddes, Fiona; Evans, Patricia; Porter, John; Cavill, Ivor

    2006-05-01

    We randomized 21 patients with low-risk myelodysplastic syndromes (MDS) to receive a single subcutaneous bolus of recombinant erythropoietin (epoietin) +/- granulocyte-colony stimulating factor (G-CSF), or placebo and monitored erythropoietic response over 7 days. In this small study, the reticulocyte response at day 7 was highly predictive of subsequent response to a therapeutic trial of epoietin + G-CSF.

  7. Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

    PubMed Central

    Xu, W D; Firestein, G S; Taetle, R; Kaushansky, K; Zvaifler, N J

    1989-01-01

    A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity. Images PMID:2646320

  8. Epidermal growth factor stimulates mouse placental lactogen I but inhibits mouse placental lactogen II secretion in vitro.

    PubMed Central

    Yamaguchi, M; Ogren, L; Endo, H; Thordarson, G; Kensinger, R; Talamantes, F

    1992-01-01

    This study was undertaken to determine whether epidermal growth factor (EGF) regulates the secretion of mouse placental lactogen (mPL)-I and mPL-II. Primary cell cultures were prepared from placentas from days 7, 9, and 11 of pregnancy and cultured for up to 5 days. Addition of EGF (20 ng/ml) to the medium resulted in significant stimulation of mPL-I secretion by the second day of culture in cells from days 7 and 9 of pregnancy and significant inhibition of mPL-II secretion by the third or fourth day of culture in cells from days 7, 9, and 11. Dose-response studies carried out with cells from day 7 of pregnancy demonstrated that the minimum concentration of EGF that stimulated mPL-I secretion and inhibited mPL-II secretion was 1.0 ng/ml. EGF did not affect the DNA content of the cells or cell viability, assessed by trypan blue exclusion, nor did it have a general effect on protein synthesis. There are three types of PL-containing giant cells in mouse placental cell cultures: cells that contain either mPL-I or mPL-II and cells that contain both hormones. Immunocytochemical analysis and the reverse hemolytic plaque assay indicated that EGF treatment was accompanied by a significant increase in the number of cells that produce mPL-I, but among the PL cells that contained mPL-I, there was no change in the fraction of cells that contained only mPL-I or the fraction that contained both mPL-I and mPL-II. In contrast, EGF treatment did affect the distribution of mPL-II among PL cells. In control cultures, about 75% of the cells that contained mPL-II also contained mPL-I, but in EGF-treated cultures, all of the cells that contained mPL-II also contained mPL-I. These data suggest that EGF regulates mPL-I and mPL-II secretion at least partly by regulating PL cell differentiation. PMID:1454826

  9. Insulin response sequence-dependent and -independent mechanisms mediate effects of insulin on glucocorticoid-stimulated insulin-like growth factor binding protein-1 promoter activity.

    PubMed

    Gan, Lixia; Pan, Haiyun; Unterman, Terry G

    2005-10-01

    IGF binding protein-1 (IGFBP-1) gene expression is stimulated by glucocorticoids and suppressed by insulin in the liver. Insulin response sequences (IRSs) mediate effects of insulin on basal promoter function, whereas glucocorticoids stimulate promoter activity through a contiguous glucocorticoid response element. Here we examined the role of IRS-dependent and -independent mechanisms in mediating insulin and glucocorticoids effects on IGFBP-1 promoter activity. Dexamethasone (Dex) stimulates IGFBP-1 promoter activity in HepG2 cells, and mutation of IRSs reduces this effect, indicating that IRS-associated factors enhance glucocorticoid effects on promoter function. Conversely, insulin inhibits basal promoter activity by 40% and Dex-stimulated promoter activity by 65%, indicating that glucocorticoids enhance the ability of insulin to suppress promoter activity. Mutation of IRSs completely disrupts the insulin effect on basal promoter activity and reduces but does not abolish inhibition of Dex-stimulated promoter activity, indicating that insulin suppresses glucocorticoid-stimulated promoter activity through both IRS-dependent and -independent mechanisms. IRS-independent effects of insulin are context dependent because insulin does not suppress glucocorticoid-stimulated activity of a promoter containing multiple glucocorticoid response elements. Cotransfection studies indicate that suppression of peroxisomal proliferator-activated receptor-gamma coactivator-1alpha, an insulin-regulated coactivator of the glucocorticoid receptor, is not required for this effect of insulin. Studies with pharmacological inhibitors indicate that both phosphatidylinositol-3' kinase and mitogen-activated kinase kinase pathways contribute to IRS-independent effects. These studies indicate that glucocorticoids and IRS-associated factors function together to mediate effects of insulin and glucocorticoids on promoter activity and that glucocorticoid treatment creates a complex environment in

  10. Platelet-derived growth factor stimulation of (/sup 3/H)-glucosamine incorporation in density-arrested BALB/c-3T3 cells

    SciTech Connect

    Harrington, M.A.; Wharton, W.; Pledger, W.J.

    1987-01-01

    G/sub 0//G/sub 1/ traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G/sub 0//G/sub 1/. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. The authors have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10 fold more (/sup 3/H)-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca/sub 3/ (PO/sub 4/)/sub 2/ and was not due to cell-cycle traverse. Stimulation of (/sup 3/H)-GlcN incorporation by PDGF was time dependent, and increased incorporation of (/sup 3/H)-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.

  11. Molecular cloning of LIM homeodomain transcription factor Lhx2 as a transcription factor of porcine follicle-stimulating hormone beta subunit (FSHβ) gene.

    PubMed

    Kato, Takako; Ishikawa, Akio; Yoshida, Saishu; Sano, Yoshiya; Kitahara, Kousuke; Nakayama, Michie; Susa, Takao; Kato, Yukio

    2012-01-01

    We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone β subunit gene (Fshβ) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LβT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LβT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshβ, it is possible that LHX2 controls the synthesis of FSH at the transcription level.

  12. Increased susceptibility to liver injury after hemorrhagic shock in rats chronically fed ethanol: role of nuclear factor-kappa B, interleukin-6, and granulocyte colony-stimulating factor.

    PubMed

    Ono, Masafumi; Yu, Bi; Hardison, Edith G; Mastrangelo, Mary-Ann A; Tweardy, David J

    2004-06-01

    Chronic ethanol use preceding severe trauma and hemorrhagic shock (HS) is associated with an increased incidence of multiorgan failure (MOF) and death; however, the molecular basis for this increased susceptibility is unknown. We previously demonstrated that production of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF), mediated by nuclear factor-kappa B (NF-kappa B), each make essential contributions to organ injury and inflammation in a rodent model of controlled HS, and we proposed in this study to examine the hypothesis that the increased susceptibility to MOF after shock/trauma in the setting of chronic ethanol use is due to an exaggerated activation of NF-kappa B and production of these proinflammatory cytokines. We observed increased HS-induced liver injury 4 h after resuscitation in rats fed the ethanol-containing Lieber-DeCarli liquid diet for 8 weeks compared with rats fed the control liquid diet (3-fold increase in serum alanine aminotransferase [ALT], P = 0.008, and 2-fold increase in focal liver necrosis, P = 0.005). The increased liver injury in the ethanol-fed HS rats was accompanied by a 70% increase in liver NF-kappa B activation (P < 0.05), a 3- to 5-fold increase in hepatocyte and Kupffer cell production of IL-6 and G-CSF (P < 0.05 for each), and a 2-fold increase in neutrophil infiltration (P < 0.005) compared with the control diet-fed HS rats. Thus, increased susceptibility to HS-induced liver injury in the setting of chronic ethanol use may be mediated, at least in part, by increased NF-kappa B activation resulting in increased local production of IL-6 and G-CSF and increased infiltration of neutrophils, which can damage liver cells directly and contribute to impaired sinusoidal blood flow.

  13. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  14. Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1.

    PubMed

    Giacco, Ferdinando; Perruolo, Giuseppe; D'Agostino, Elio; Fratellanza, Giorgio; Perna, Enzo; Misso, Saverio; Saldalamacchia, Gennaro; Oriente, Francesco; Fiory, Francesca; Miele, Claudia; Formisano, Salvatore; Beguinot, Francesco; Formisano, Pietro

    2006-11-01

    Platelet components have found successful clinical utilization to initiate or to accelerate tissue-repair mechanisms. However, the molecular pathways by which platelet factors contribute to tissue regeneration have not been fully elucidated. We have studied the effect of thrombin-activated platelets (TAPs) on cell growth in vivo and in cultured cell systems. Application of TAPs to ulcerative skin lesions of diabetic patients induced local activation of ERK1/2 and Akt/PKB. Moreover, when applied to cultured human skin fibroblasts, TAPs promoted cell growth and DNA synthesis and activated platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 receptor tyrosine kinases. PDGF was released by TAPs and rapidly achieved a plateau. At variance, the release of IGF-1 was mainly provided by the TAPs-stimulated fibroblasts and progressively increased up to 48 h. The PDGF-R blocker Ag1296 reduced the activation of Akt/PKB and, at a lesser extent, of ERK1/2. Conversely, inhibition of IGF-1 signaling by Ag1024 and expression of a dominant-negative IGF-1R mutant selectively reduced the stimulation of ERK1/2 by TAPs and fibroblast-released factors, with minor changes of Akt/PKB activity. Thus, platelet factors promote fibroblast growth by acutely activating Akt/PKB and ERK1/2. Sustained activation of ERK1/2, however, requires autocrine production of IGF-1 by TAPs-stimulated fibroblasts.

  15. Recombinant human follicle-stimulating hormone and transforming growth factor-alpha enhance in vitro maturation of porcine oocytes.

    PubMed

    Mito, Tomomi; Yoshioka, Koji; Noguchi, Michiko; Yamashita, Shoko; Hoshi, Hiroyoshi

    2013-07-01

    The biological functions of recombinant human follicle-stimulating hormone (FSH) and transforming growth factor-α (TGF-α) on in vitro maturation of porcine oocytes were investigated. Cumulus-oocyte complexes were matured in defined porcine oocyte medium containing 0-0.1 IU/ml FSH in the presence or absence of 10 ng/ml TGF-α. The percentage of oocytes reaching metaphase II was significantly higher with the addition of 0.01-0.1 IU/ml FSH compared with no addition, and was further enhanced in the presence of TGF-α. The rates of sperm penetration and blastocyst formation were significantly higher with the addition of 0.05-0.1 IU/ml FSH compared with no addition after in vitro fertilization and embryo culture. There was no beneficial effect of FSH and TGF-α on nuclear maturation of denuded oocytes. The specific EGF receptor inhibitor, AG1478, completely inhibited TGF-α-induced meiotic resumption, but did not completely prevent the stimulatory effect of FSH. Addition of both FSH and TGF-α significantly enhanced cumulus expansion compared with no addition. When cumulus expansion-related genes (HAS2, HAPLN1, and VCAN) mRNA expression in COCs was measured during in vitro maturaiton, addition of both of FSH and TGF-α upregulated the expression of HAS2 mRNA after 20 hr culture and HAPLN1 mRNA after 44 hr culture compared with no addition. Expression of VCAN mRNA was significantly higher in the presence of FSH compared with addition of TGF-α alone. These results suggest that FSH and TGF-α synergistically enhance porcine oocyte maturation via cumulus cells, and act through different signaling pathways.

  16. Granulocyte-Colony Stimulating Factor (G-CSF) for stroke: an individual patient data meta-analysis

    PubMed Central

    England, Timothy J.; Sprigg, Nikola; Alasheev, Andrey M.; Belkin, Andrey A.; Kumar, Amit; Prasad, Kameshwar; Bath, Philip M.

    2016-01-01

    Granulocyte colony stimulating factor (G-CSF) may enhance recovery from stroke through neuroprotective mechanisms if administered early, or neurorepair if given later. Several small trials suggest administration is safe but effects on efficacy are unclear. We searched for randomised controlled trials (RCT) assessing G-CSF in patients with hyperacute, acute, subacute or chronic stroke, and asked Investigators to share individual patient data on baseline characteristics, stroke severity and type, end-of-trial modified Rankin Scale (mRS), Barthel Index, haematological parameters, serious adverse events and death. Multiple variable analyses were adjusted for age, sex, baseline severity and time-to-treatment. Individual patient data were obtained for 6 of 10 RCTs comprising 196 stroke patients (116 G-CSF, 80 placebo), mean age 67.1 (SD 12.9), 92% ischaemic, median NIHSS 10 (IQR 5–15), randomised 11 days (interquartile range IQR 4–238) post ictus; data from three commercial trials were not shared. G-CSF did not improve mRS (ordinal regression), odds ratio OR 1.12 (95% confidence interval 0.64 to 1.96, p = 0.62). There were more patients with a serious adverse event in the G-CSF group (29.6% versus 7.5%, p = 0.07) with no significant difference in all-cause mortality (G-CSF 11.2%, placebo 7.6%, p = 0.4). Overall, G-CSF did not improve stroke outcome in this individual patient data meta-analysis. PMID:27845349

  17. MicroRNA-181b stimulates inflammation via the nuclear factor-κB signaling pathway in vitro.

    PubMed

    Wang, Yazhen; Mao, Genxiang; Lv, Yuandong; Huang, Qingdong; Wang, Guofu

    2015-10-01

    Acute lung injury (ALI) is characterized by severe lung edema and an increase in the inflammatory reaction. Considerable evidence has indicated that microRNAs (miRNAs or miRs) are involved in various human diseases; however, the expression profile and function of miRNAs in ALI have been rarely reported. The present study used miRNA microarray and reverse transcription-quantitative polymerase chain reaction to demonstrate that miR-181b is the one of the most significantly upregulated miRNA after lipopolysaccharide (LPS) stimulation in human bronchial epithelial cells, BEAS-2B. To elaborate the role of miR-181b in ALI, an assay was performed to investigate the overexpression of miR-181b in BEAS-2B cells, and the expression of inflammatory factors was then analyzed. The overexpression of miR-181b resulted in the induction of an increment in interleukin (IL)-6 levels. p65 was identified to be a primary component of NF-κB, since it was upregulated in the miR-181b overexpression in the BEAS-2B cells, while pyrrolidine dithiocarbamate, a specific inhibitor of NF-κB, was found to be able to abrogate the upregulation of the expression of p65. In conclusion, the findings of the present study suggested that miR-181b may be involved in the process of LPS-induced inflammation in BEAS-2B cells by activating the NF-κB signaling pathway, which implies that it may serve as a potential therapeutic target for ALI.

  18. Herbal medicine "sho-saiko-to" induces in vitro granulocyte colony-stimulating factor production on peripheral blood mononuclear cells.

    PubMed

    Yamashiki, M; Asakawa, M; Kayaba, Y; Kosaka, Y; Nishimura, A

    1992-01-01

    The herbal medicine "Sho-saiko-to (Xiao-Chai-Hu-Tang)" has been used in China for about 3000 years for the treatment of pyretic diseases. This medicine is now available as one of the prescribing drugs approved by the Ministry of Health and Welfare of Japan, and has also been widely used for patients with chronic viral liver disease as one of biological response modifiers in the field of Japan's Western Medicine. However, its mode of action has not been fully described. In the present in vitro study, we added "Sho-saiko-to" (TJ-9, Tsumura, Tokyo) to the culture of peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers, and observed a dose-dependent increase in the production of granulocyte colony-stimulating factor (G-CSF). The same experiment was conducted using other herbal medicines "Dai-saiko-to" (TJ-8) and "Saiko-keishi-to" (TJ-10) which showed similar effects, or "Sho-seiryu-to" (TJ-19) which consists of very different compounds and shows different efficacy. The increases of G-CSF production were similar when "Sho-saiko-to" (TJ-9) or one of the 2 reference drugs (TJ-8 and 10) was added, whereas the increase when the control drug "Sho-seiryu-to" (TJ-19) was added, was quite small. This result shows that G-CSF induction is not a common effect of herbal medicines, but a specific effect of TJ-8, 9, and 10. Among these 3 drugs the increase produced by "Sho-saiko-to" was the largest. Based on this result, we conclude that administration of "Sho-saiko-to" may be useful not only for the treatment of chronic liver disease, but also for malignant diseases and acute infectious diseases where G-CSF is efficacious.

  19. Adrenaline administration promotes the efficiency of granulocyte colony stimulating factor-mediated hematopoietic stem and progenitor cell mobilization in mice.

    PubMed

    Chen, Chong; Cao, Jiang; Song, Xuguang; Zeng, Lingyu; Li, Zhenyu; Li, Yong; Xu, Kailin

    2013-01-01

    A high dose of granulocyte colony stimulating factor (G-CSF) is widely used to mobilize hematopoietic stem and progenitor cells (HSPC), but G-CSF is relatively inefficient and may cause adverse effects. Recently, adrenaline has been found to play important roles in HSPC mobilization. In this study, we explored whether adrenaline combined with G-CSF could induce HSPC mobilization in a mouse model. Mice were treated with adrenaline and either a high or low dose of G-CSF alone or in combination. Peripheral blood HSPC counts were evaluated by flow cytometry. Levels of bone marrow SDF-1 were measured by ELISA, the transcription of CXCR4 and SDF-1 was measured by real-time RT-PCR, and CXCR4 protein was detected by Western blot. Our results showed that adrenaline alone fails to mobilize HSPCs into the peripheral blood; however, when G-CSF and adrenaline are combined, the WBC counts and percentages of HSPCs are significantly higher compared to those in mice that received G-CSF alone. The combined use of adrenaline and G-CSF not only accelerated HSPC mobilization, but also enabled the efficient mobilization of HSPCs into the peripheral blood at lower doses of G-CSF. Adrenaline/G-CSF treatment also extensively downregulated levels of SDF-1 and CXCR4 in mouse bone marrow. These results demonstrated that adrenaline combined with G-CSF can induce HSPC mobilization by down-regulating the CXCR4/SDF-1 axis, indicating that the use of adrenaline may enable the use of reduced dosages or durations of G-CSF treatment, minimizing G-CSF-associated complications.

  20. Biweekly docetaxel and vinorelbine with granulocyte colony-stimulating factor support for patients with anthracycline-resistant metastatic breast cancer.

    PubMed

    Gómez-Bernal, Amalia; Cruz, Juan Jesús; Olaverri, Amaya; Arizcun, Alberto; Martín, Teresa; Rodríguez, Cesar A; Martín, Germán; Fonseca, Emilio; Sánchez, Pedro

    2005-01-01

    This phase II trial evaluated the efficacy and toxicity of vinorelbine 25 mg/m2 plus docetaxel 60 mg2/m administered on day 1, every 2 weeks with granulocyte colony-stimulating factor support (G-CSF, 5 microg/kg/day, days 3-7) as primary prophylaxis in patients with histologically confirmed metastatic breast cancer (MBC) and previously treated with anthracyclines in the adjuvant or in the first-line setting. A total of 48 patients received 352 cycles (median 8, range 2-10). All patients were included in the efficacy and safety evaluation on an intent-to-treat analysis. Eight patients (17%) showed a complete response and 14 patients (29%) showed a partial response. Overall response rate was 46% [95% confidence interval (CI) 33-60]. The median duration of response was 10.0 months. With a median follow-up of 18.0 months, the median time to progression was 11.9 months and the median overall survival was 27.1 months. The most frequently reported grade 3/4 hematological toxicity was neutropenia (19% of patients, 4% of cycles). Febrile neutropenia was reported in six patients (13%) and 7 cycles (2%), but no toxic deaths were reported. The most common grade 3/4 non-hematological toxicity was asthenia (17% of patients, 6% of cycles) and nail toxicity (15% of patients, 3% of cycles). In conclusion, biweekly docetaxel plus vinorelbine with G-CSF support is active and well tolerated as chemotherapy for patients with MBC resistant to anthracyclines. G-CSF support is recommended for lowering the incidence and severity of neutropenia and febrile neutropenia.

  1. Survival enhancement and hemopoietic regeneration following radiation exposure: Therapeutic approach using glucan and granulocyte colony-stimulating factor

    SciTech Connect

    Patchen, M.L.; MacVittie, T.J.; Solberg, B.D.; Souza, L.M. )

    1990-10-01

    C3H/HeN female mice were exposed to whole-body cobalt-60 radiation and administered soluble glucan (5 mg i.v. at 1 h following exposure), recombinant human granulocyte colony-stimulating factor (G-CSF; 2.5 micrograms/day s.c., days 3-12 following exposure), or both agents. Treatments were evaluated for their ability to enhance hemopoietic regeneration, and to increase survival after radiation-induced myelosuppression. Both glucan and G-CSF enhanced hemopoietic regeneration alone; however, greater effects were observed in mice receiving both agents. For example, on day 17 following a sublethal 6.5-Gy radiation exposure, mice treated with saline, G-CSF, glucan, or both agents, respectively, exhibited 36%, 65%, 50%, and 78% of normal bone marrow cellularity, and 84%, 175%, 152%, and 212% of normal splenic cellularity. At this same time, granulocyte-macrophage colony-forming cell (GM-CFC) values in saline, G-CSF, glucan, or combination-treated mice, respectively, were 9%, 46%, 26%, and 57% of normal bone marrow values, and 57%, 937%, 364%, and 1477% of normal splenic values. Endogenous spleen colony formation was also increased in all treatment groups, with combination-treated mice exhibiting the greatest effects. Likewise, although both glucan and G-CSF alone enhanced survival following an 8-Gy radiation exposure, greatest survival was observed in mice treated with both agents. These studies suggest that glucan, a macrophage activator, can synergize with G-CSF to further accelerate hemopoietic regeneration and increase survival following radiation-induced myelosuppression.

  2. 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

  3. Colony-stimulating factor-1 plays a major role in the development of reproductive function in male mice.

    PubMed

    Cohen, P E; Hardy, M P; Pollard, J W

    1997-10-01

    Colony-stimulating factor-1 (CSF-1) is the principal regulator of cells of the mononuclear phagocytic lineage that includes monocytes, tissue macrophages, microglia, and osteoclasts. Macrophages are found throughout the reproductive tract of both males and females and have been proposed to act as regulators of fertility at several levels. Mice homozygous for the osteopetrosis mutation (csfm[op]) lack CSF-1 and, consequently, have depleted macrophage numbers. Further analysis has revealed that male csfm(op)/csfm(op) mice have reduced mating ability, low sperm numbers, and 90% lower serum testosterone levels. The present studies show that this low serum testosterone is due to reduced testicular Leydig cell steroidogenesis associated with severe ultrastructural abnormalities characterized by disrupted intracellular membrane structures. In addition, the Leydig cells from csfm(op)/ csfm(op) males have diminished amounts of the steroidogenic enzyme proteins P450 side chain cleavage, 3beta-hydroxysteroid dehydrogenase, and P450 17alpha-hydroxylase-lyase, with associated reductions in the activity of all these steroidogenic enzymes, as well as in 17beta-hydroxysteroid dehydrogenase. The CSF-1-deficient males also have reduced serum LH and disruption of the normal testosterone negative feedback response of the hypothalamus, as demonstrated by the failure to increase LH secretion in castrated males and their lack of response to exogenous testosterone. However, these males are responsive to GnRH and LH treatment. These studies have identified a novel role for CSF-1 in the development and/or regulation of the male hypothalamic-pituitary-gonadal axis.

  4. Granulocyte colony-stimulating factor-producing pancreatic anaplastic carcinoma in ascitic fluid at initial diagnosis: A case report.

    PubMed

    Kubota, Nao; Naito, Yoshiki; Kawahara, Akihiko; Taira, Tomoki; Yamaguchi, Tomohiko; Yoshida, Tomoko; Abe, Hideyuki; Takase, Yorihiko; Fukumitsu, Chihiro; Murata, Kazuya; Ishida, Yusuke; Okabe, Yoshinobu; Kimura, Yoshizo; Tanigawa, Masahiko; Mihara, Yutaro; Nakayama, Masamichi; Yamaguchi, Rin; Akiba, Jun; Yano, Hirohisa

    2017-02-10

    Granulocyte colony-stimulating factor (G-CSF)-producing pancreatic tumors are extremely rare. These tumors have an aggressive clinical course and no established treatment. Here, we report an autopsy case of G-CSF-production in pancreatic anaplastic carcinoma (PAC). A 72-year-old woman presented with a large pancreatic head mass and multiple liver metastases. Laboratory data showed leukocytosis (leukocyte count 113.3 × 10(3) /µL) and high serum G-CSF levels (441 pg/mL; normal range: <39.0 pg/mL). The ascitic fluid was submitted to our pathology laboratory at initial diagnosis. Cytopathology showed that smears from the ascitic fluid were highly cellular and contained numerous malignant cells, mainly in loose groupings. Occasional pseudoglandular formations and giant cells were also present. The malignant cells were round, and no spindle-shaped cells were visible. The nuclei were round to ovoid with coarsely granular chromatin and large prominent nucleoli. Upon immunocytochemistry, tumor cells were positive for G-CSF and vimentin; there was no E-cadherin expression. Histopathological examination of the tumor showed a mixed composition of adenocarcinomatous and sarcomatous regions. Upon immunohistochemistry, both components were positive for G-CSF. Few CD34-positive myeloblasts were observed in the bone marrow. Thus, we diagnosed this as a case of G-CSF production in PAC with leukocytosis. To the best of our knowledge, this is the first report on G-CSF expression immunocytochemically confirmed in PAC. Diagn. Cytopathol. © 2017 Wiley Periodicals, Inc.

  5. Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products.

    PubMed

    Gao, Kai; Rao, Chunming; Tao, Lei; Han, Chunmei; Shi, Xinchang; Wang, Lan; Fan, Wenhong; Yu, Lei; Wang, Junzhi

    2012-03-01

    This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.

  6. Mechanical loading by fluid shear stress of myotube glycocalyx stimulates growth factor expression and nitric oxide production.

    PubMed

    Juffer, Petra; Bakker, Astrid D; Klein-Nulend, Jenneke; Jaspers, Richard T

    2014-07-01

    Skeletal muscle fibers have the ability to increase their size in response to a mechanical overload. Finite element modeling data suggest that mechanically loaded muscles in vivo may experience not only tensile strain but also shear stress. However, whether shear stress affects biological pathways involved in muscle fiber size adaptation in response to mechanical loading is unknown. Therefore, our aim was twofold: (1) to determine whether shear stress affects growth factor expression and nitric oxide (NO) production by myotubes, and (2) to explore the mechanism by which shear stress may affect myotubes in vitro. C2C12 myotubes were subjected to a laminar pulsating fluid flow (PFF; mean shear stress 0.4, 0.7 or 1.4 Pa, 1 Hz) or subjected to uni-axial cyclic strain (CS; 15 % strain, 1 Hz) for 1 h. NO production during 1-h PFF or CS treatment was quantified using Griess reagent. The glycocalyx was degraded using hyaluronidase, and stretch-activated ion channels (SACs) were blocked using GdCl3. Gene expression was analyzed immediately after 1-h PFF (1.4 Pa, 1 Hz) and at 6 h post-PFF treatment. PFF increased IGF-I Ea, MGF, VEGF, IL-6, and COX-2 mRNA, but decreased myostatin mRNA expression. Shear stress enhanced NO production in a dose-dependent manner, while CS induced no quantifiable increase in NO production. Glycocalyx degradation and blocking of SACs ablated the shear stress-stimulated NO production. In conclusion, shear stress activates signaling pathways involved in muscle fiber size adaptation in myotubes, likely via membrane-bound mechanoreceptors. These results suggest that shear stress exerted on myofiber extracellular matrix plays an important role in mechanotransduction in muscle.

  7. Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin

    PubMed Central

    Pass, Harvey I.; Lavilla, Carmencita; Canino, Claudia; Goparaju, Chandra; Preiss, Jordan; Noreen, Samrah; Blandino, Giovanni; Cioce, Mario

    2016-01-01

    In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo, we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo. These anticancer effects correlated with a reduced number of CSF-1Rpos cells, in tumors excised from the treated mice. Depletion of the CD45pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components. PMID:27486763

  8. Efficacy of LL-37 and granulocyte colony-stimulating factor in a neutropenic murine sepsis due to Pseudomonas aeruginosa.

    PubMed

    Cirioni, Oscar; Ghiselli, Roberto; Tomasinsig, Linda; Orlando, Fiorenza; Silvestri, Carmela; Skerlavaj, Barbara; Riva, Alessandra; Rocchi, Marco; Saba, Vittorio; Zanetti, Margherita; Scalise, Giorgio; Giacometti, Andrea

    2008-10-01

    A promising therapeutic strategy for the management of severe Pseudomonas infection in neutropenic patients may result from the coadministration of colony-stimulating factors (CSFs) that help maintain immune competence and antimicrobial peptides, a novel generation of adjunctive therapeutic agents with antimicrobial and anti-inflammatory properties. A promising peptide with these properties is LL-37, the only member of the cathelicidin family of antimicrobial peptides found in humans. BALB/c male mice were rendered neutropenic by intraperitoneal administration of cyclophosphamide on days -4 and -2 preinfection. Septic shock was induced at time 0 by intraperitoneal injection of 2x10 colony-forming units of P. aeruginosa American Type Culture Collection (ATCC) 27853. All animals were randomized to receive intravenously isotonic sodium chloride solution, 1 mg/kg of LL-37, 20 mg/kg of imipenem, 0.1 mg/kg of granulocyte CSF (G-CSF), 1 mg/kg of LL-37+0.1 mg/kg of G-CSF, or 20 mg/kg of imipenem+0.1 mg/kg of G-CSF. Lethality and bacterial growth in blood, peritoneum, spleen, liver, and kidney were evaluated. All regimens were significantly superior to controls at reducing the mouse lethality rate and bacterial burden in organs. Particularly, the combination between LL-37 and G-CSF was the most effective in protecting neutropenic mice from the onset of sepsis and in vitro significantly reduced the apoptosis of neutrophils. Combination therapy between LL-37 and G-CSF is a promising therapeutic strategy for the management of severe Pseudomonas infection complicated by neutropenia.

  9. Granulocyte-colony stimulating factor improves Parkinson's disease associated with co-morbid depression: An experimental exploratory study

    PubMed Central

    Prakash, Ajay; Chopra, Kanwaljit; Medhi, Bikash

    2013-01-01

    Introduction: The present study was designed to evaluate the effect of granulocyte-colony stimulating factor (G-CSF) in the treatment of Parkinson's disease (PD), the second most common neurodegenerative disease characterized by muscle and movement disorder, often associated with depression. PD is very difficult to treat. Hence, the present study was aimed to evaluate the effect of G-CSF in PD associated with depression. Materials and Methods: Adult Wistar male rats weighing about 180-250 g were selected and divided into five groups in parallel designed method namely; control group (n = 5); sham operated group (n = 5); Vehicle group (n = 5); G-CSF group (70 μg/kg, s.c.) (n = 5) and L-DOPA group (n = 5). The rats were treated with 6-hydroxydopamine (6-OHDA) on day 0 and then treatment was continued for 14 day of L-DOPA/carbidopa, whereas G-CSF (70 μg/kg, s.c.) was given from day 1 to 6. Thereafter, adhesive removal and forced swim tests were conducted to evaluate the behavioral outcome of G-CSF treatment. The finding was correlated and analyzed with Nissl staining findings for the final conclusion. Results: The behavioral parameters were assessed and found to be ameliorate the symptoms of Parkinson's and reduced the depression like behavior in PD. The histological findings were supported the behavioral findings and showed pathological improvement. Conclusion: As a preliminary work, the present study first time suggested that G-CSF have a potential role in PD and associated depression. PMID:24347771

  10. Use of granulocyte-macrophage colony-stimulating factor in two children treated with cord blood transplantation.

    PubMed

    Vowels, M R; Tiedemann, K; Lam-Po-Tang, R; Tucker, D P

    1994-01-01

    Cord blood contains stem cells in amounts similar to or slightly less than those present in a bone marrow collection to be used for bone marrow transplantation (BMT). Too few cord blood transplants (CBT) have yet been performed to define the ability to achieve engraftment and the rate of engraftment. Two cord blood transplants have been performed using granulocyte-macrophage colony stimulating factor (GM-CSF) to hasten engraftment. Two children, aged 5 and 6 years received a CBT using HLA-identical stem cells collected at the birth of a sibling. One child had X-linked lymphoproliferative disease (XLP), and the other, acute lymphoblastic leukemia in second complete remission. One had an ABO and one an Rh blood group mismatch. Conditioning therapy consisted of cyclophosphamide, melphalan, and antithymocyte globulin or busulphan and cyclophosphamide. Graft-versus-host disease prophylaxis was methotrexate and cyclosporine or cyclosporine. Both children were given GM-CSF at 5 micrograms/kg/day from day 1 until the absolute neutrophil count (ANC) reached 1.0 x 10(9)/L for 3 consecutive days. If this level was not reached by day 14, the dose of GM-CSF was doubled. Both children engrafted rapidly, with ANCs reaching 0.5 x 10(9)/L in 12 and 16 days. Engraftment was confirmed by blood group in both and sex chromosome typing in one. Both children developed mild GVHD localized to skin, which resolved with steroid therapy. The child with XLP was cured and has survived for 34 months; the second child has survived 27 months with normal marrow function but has had a relapse of leukemia.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor

    PubMed Central

    Pizzey, A. R.; Linch, D. C.

    1993-01-01

    The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 ± 10% (mean ± S.E.M., n = 13) and 182 ± 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b β2-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation. PMID:18475524

  12. Growth hormone stimulates protein synthesis in bovine skeletal muscle cells without altering insulin-like growth factor-I mRNA expression.

    PubMed

    Ge, X; Yu, J; Jiang, H

    2012-04-01

    Growth hormone is a major stimulator of skeletal muscle growth in animals, including cattle. In this study, we determined whether GH stimulates skeletal muscle growth in cattle by direct stimulation of proliferation or fusion of myoblasts, by direct stimulation of protein synthesis, or by direct inhibition of protein degradation in myotubes. We also determined whether these direct effects of GH are mediated by IGF-I produced by myoblasts or myotubes. Satellite cells were isolated from cattle skeletal muscle and were allowed to proliferate as myoblasts or induced to fuse into myotubes in culture. Growth hormone at 10 and 100 ng/mL increased protein synthesis in myotubes (P < 0.05), but had no effect on protein degradation in myotubes or proliferation of myoblasts (P > 0.05). Insulin-like growth factor-I at 50 and 500 ng/mL stimulated protein synthesis (P < 0.01), and this effect of IGF-I was much greater than that of GH (P < 0.05). Besides stimulating protein synthesis, IGF-I at 50 and 500 ng/mL also inhibited protein degradation in myotubes (P < 0.01), and IGF-I at 500 ng/mL stimulated proliferation of myoblasts (P < 0.05). Neither GH nor IGF-I had effects on fusion of myoblasts into myotubes (P > 0.1). These data indicate that GH and IGF-I have largely different direct effects on bovine muscle cells. Growth hormone at 10 and 100 ng/mL had no effect on IGF-I mRNA expression in either myoblasts or myotubes (P > 0.1). This lack of effect was not because the cultured myoblasts or myotubes were not responsive to GH; GH receptor mRNA was detectable in them and the expression of the cytokine-inducible SH2-containing protein (CISH) gene, a well-established GH target gene, was increased by GH in bovine myoblasts (P < 0.05). Overall, the data suggest that GH stimulates skeletal muscle growth in cattle in part through stimulation of protein synthesis in the muscle and that this stimulation is not mediated through increased IGF-I mRNA expression in the muscle.

  13. Glia cell stimulating factor (GSF): a new lymphokine. Part 1. Cellular sources and partial purification of murine GSF, role of cytoskeleton and protein synthesis in its production.

    PubMed

    Fontana, A; Dubs, R; Merchant, R; Balsiger, S; Grob, P J

    1982-01-01

    The effect of activated-lymphocyte supernatant on glia cells was investigated. When treated in vitro with Concanavalin A (ConA), murine spleen cells released a soluble product, termed glia cell stimulating factor (GSF), which stimulated RNA and DNA synthesis in cultured murine glia cells. Furthermore, GSF appeared to promote the maturation of undifferentiated glia cells to astrocytes having a high content of glial fibrillary acidic protein. GSF secretion occurred after a lag period of 16 hours and proceeded at a constant rate for more than 48 hours. This GSF produced by ConA-stimulated murine lymphocytes has an apparent molecular weight between 60,000 and 80,000. Antigenic stimulation of primed lymph node cells with BGG resulted in a similar GSF production. Cellular sources of mitogen-induced GSF were investigated by using isolated lymphoid populations. GSF release by ConA-activated pure T-lymphocytes reconstituted with peritoneal macrophages was equivalent to that of unseparated spleen cells, whereas GSF production by T-lymphocytes alone was low. Macrophages alone did not elaborate detectable levels of GSF. GSF was also secreted by enriched -B-lymphocytes populations stimulated by Protein A. Formation of GSF was suppressed when cytochalasin B or cyclo-heximide was added to the cultures, while colchicine failed to have any effect. DNA synthesis is not required for GSF production as determined by resistence to treatment with mitomycin C. The data indicate that the GSF production and secretion mechanism is much like that described for other lymphokines.

  14. Regulation by intracellular Ca sup 2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

    SciTech Connect

    Miyata, Yoshihiko Tokyo Metropolitan Inst. of Medical Science ); Nishida, Eisuke; Sakai, Hikoichi ); Koyasu, Shigeo; Yahara, Ichiro )

    1989-04-01

    Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes and stimulate the fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells. An increase in intracellular Ca{sup 2+} concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca{sup 2+} or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca{sup 2+} and cAMP. {sup 125}I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca{sup 2+}- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.

  15. PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium †

    PubMed Central

    London, Fredda S.; Marcinkiewicz, Mariola; Walsh, Peter N.

    2008-01-01

    We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (Kd 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (Kd 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients. PMID:16752917

  16. p-ERK1/2 is a predictive factor of response to erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes

    PubMed Central

    Frisan, Emilie; Pawlikowska, Patrycja; Pierre-Eugène, Cecile; Viallon, Vivian; Gibault, Laure; Park, Sophie; Mayeux, Patrick; Dreyfus, François; Porteu, Françoise; Fontenay, Michaëla

    2010-01-01

    Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45−/CD71+/GPA−cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents. PMID:20823131

  17. Insulin-induced hypoglycemia stimulates corticotropin-releasing factor and arginine vasopressin secretion into hypophysial portal blood of conscious, unrestrained rams.

    PubMed Central

    Caraty, A; Grino, M; Locatelli, A; Guillaume, V; Boudouresque, F; Conte-Devolx, B; Oliver, C

    1990-01-01

    Insulin-induced hypoglycemia (IIH) is a strong stimulator of pituitary ACTH secretion. The mechanisms by which IIH activates the corticotrophs are still controversial. Indeed, in rats the variations of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) secretion in hypophysial portal blood (HPB) during IIH have been diversely appreciated. This may be due to the stressful conditions required for portal blood collection in rats. We studied the effects of IIH on the secretion of CRF and AVP in HPB and on the release of ACTH and cortisol in peripheral plasma in conscious, unrestrained, castrated rams. After the injection of a low (0.2 IU/kg) or high dose (2 IU/kg) of insulin, ACTH and cortisol levels in peripheral plasma increased in a dose-related manner. After injection of the low dose of insulin, CRF and AVP secretion in HPB were equally stimulated. After injection of the high dose of insulin, CRF secretion was further stimulated, while AVP release was dramatically increased. These results suggest that when the hypoglycemia is moderate, CRF is the main factor triggering ACTH release, and that the increased AVP secretion potentiates the stimulatory effect of CRF. When hypoglycemia is deeper, AVP secretion becomes predominant and may by itself stimulate ACTH release. Images PMID:2161426

  18. MbIDGF, a novel member of the imaginal disc growth factor family in Mamestra brassicae, stimulates cell proliferation in two lepidopteran cell lines without insulin.

    PubMed

    Zhang, Jun; Iwai, Sachio; Tsugehara, Taketo; Takeda, Makio

    2006-07-01

    Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway.

  19. The effects of vitamin D binding protein-macrophage activating factor and colony-stimulating factor-1 on hematopoietic cells in normal and osteopetrotic rats.

    PubMed

    Benis, K A; Schneider, G B

    1996-10-15

    Osteopetrosis is a heterogeneous group of bone disorders characterized by the failure of osteoclasts to resorb bone and by several immunological defects including macrophage dysfunction. Two compounds, colony-stimulating factor-1 (CSF-1) and vitamin D-binding protein-macrophage activating factor (DBP-MAF) were used in the present study to evaluate their effects on the peritoneal population of cells and on cells within the bone marrow microenvironment in normal and incisors absent (ia) osteopetrotic rats. Previous studies in this laboratory have demonstrated that administration of DBP-MAF to newborn ia animals results in a substantial increase in bone marrow cavity size due to upregulated osteoclast function. To study the effects of these compounds on the macrophage/osteoclast precursors, DBP-MAF, CSF-1, and the combination of these compounds were given to newborn ia and normal littermate animals. Both the normal and mutant phenotypes responded similarly when treated with these compounds. Rats exhibited a profound shift toward the macrophage lineage from the neutrophil lineage when compared with vehicle-treated control animals after treatment with these compounds. In the in vivo peritoneal lavage study, animals received injections of CSF-1, DBP-MAF or DBP-MAF/CSF-1 over a 4-week period. The various types of cells in the peritoneal cavity were then enumerated. The in vitro study consisted of cells isolated from the bone marrow microenvironment and cultured on feeder layers of CSF-1, DBP-MAF, or DBP-MAF/CSF-1 for colony enumeration. The increase in macrophage numbers at the expense of neutrophil numbers could be seen in both the in vivo and in vitro experiments. The macrophage/osteoclast and neutrophil lineages have a common precursor, the granulocyte/macrophage colony-forming cell (GM-CFC). With the addition of CSF-1, the GM-CFC precursor may be induced into the macrophage/osteoclast lineage rather than the granulocyte lineage. This increased pool of cells in the

  20. Targeting the colony stimulating factor 1 receptor alleviates two forms of Charcot-Marie-Tooth disease in mice.

    PubMed

    Klein, Dennis; Patzkó, Ágnes; Schreiber, David; van Hauwermeiren, Anemoon; Baier, Michaela; Groh, Janos; West, Brian L; Martini, Rudolf

    2015-11-01

    See Scherer (doi:10.1093/awv279) for a scientific commentary on this article.Charcot-Marie-Tooth type 1 neuropathies are inherited disorders of the peripheral nervous system caused by mutations in Schwann cell-related genes. Typically, no causative cure is presently available. Previous preclinical data of our group highlight the low grade, secondary inflammation common to distinct Charcot-Marie-Tooth type 1 neuropathies as a disease amplifier. In the current study, we have tested one of several available clinical agents targeting macrophages through its inhibition of the colony stimulating factor 1 receptor (CSF1R). We here show that in two distinct mouse models of Charcot-Marie-Tooth type 1 neuropathies, the systemic short- and long-term inhibition of CSF1R by oral administration leads to a robust decline in nerve macrophage numbers by ∼70% and substantial reduction of the typical histopathological and functional alterations. Interestingly, in a model for the dominant X-linked form of Charcot-Marie-Tooth type 1 neuropathy, the second most common form of the inherited neuropathies, macrophage ablation favours maintenance of axonal integrity and axonal resprouting, leading to preserved muscle innervation, increased muscle action potential amplitudes and muscle strengths in the range of wild-type mice. In another model mimicking a mild, demyelination-related Charcot-Marie-Tooth type 1 neuropathy caused by reduced P0 (MPZ) gene dosage, macrophage blockade causes an improved preservation of myelin, increased muscle action potential amplitudes, improved nerve conduction velocities and ameliorated muscle strength. These observations suggest that disease-amplifying macrophages can produce multiple adverse effects in the affected nerves which likely funnel down to common clinical features. Surprisingly, treatment of mouse models mimicking Charcot-Marie-Tooth type 1A neuropathy also caused macrophage blockade, but did not result in neuropathic or clinical improvements

  1. New Insight into Atherosclerosis in Hemodialysis Patients: Overexpression of Scavenger Receptor and Macrophage Colony-Stimulating Factor Genes

    PubMed Central

    Nishida, Miki; Ando, Minoru; Iwamoto, Yusuke; Tsuchiya, Ken; Nitta, Kosaku

    2016-01-01

    Background Scavenger receptors (SRs) play a pivotal role in atherogenesis. The mechanism of atherosclerosis, which is specific to hemodialysis (HD) patients, was studied on the basis of SR gene expressions. Methods The gene expressions of SR type A (SR-A) and CD36 were studied in peripheral monocytes by real-time reverse transcription polymerase chain reaction. Data were compared between HD (n = 30) and age-matched control subjects (n = 10). Serum levels of macrophage colony-stimulating factor (M-CSF) were measured with enzyme-linked immunosorbent assay to test its role in SR expression. The statistical differences and associations between two continuous variables were assessed using the Mann-Whitney U test and Pearson's correlation coefficient, respectively. Results The relative quantities of SR mRNAs were significantly greater in HD patients than in controls [median (interquartile range): SR-A, 1.67 (0.96-2.76) vs. 0.90 (0.60-1.04), p = 0.0060; CD36, 1.09 (0.88-1.74) vs. 0.74 (0.64-0.99), p = 0.0255]. The serum concentration of M-CSF was significantly higher in HD patients than in controls [1, 121 (999-1,342) vs. 176 (155-202) pg/ml, p < 0.0001]. In addition, the relative quantity of M-CSF mRNA was significantly greater in HD patients than in controls [0.79 (0.42-1.53) vs. 0.42 (0.28-0.66), p = 0.0392]. The serum M-CSF levels were positively correlated with both the relative quantity of SR-A mRNA (r2 = 0.1681, p = 0.0086) and that of CD36 mRNA (r2 = 0.1202, p = 0.0284) in all subjects (n = 40). Conclusion HD patients are predisposed to atherosclerosis as a consequence of their enhanced monocyte SR expressions. SRs and M-CSF are potential therapeutic targets for atherosclerosis in this high-risk population. PMID:27721822

  2. Antimetastatic effect of recombinant human macrophage-colony-stimulating factor against lung and liver metastatic B16 melanoma.

    PubMed

    Sakurai, T; Yamada, M; Simamura, S; Motoyoshi, K

    1997-03-01

    We studied the effect of recombinant human macrophage-colony-stimulating factor (rhM-CSF) on the formation of lung and liver metastases following the i.v. injection of the B16 melanoma subline (B16 LiLu) into mice. When rhM-CSF was administered before the B16 inoculation, the number of tumor metastases decreased in the lung and liver. However, the administration of rhM-CSF after B16 inoculation did not produce an antimetastatic effect in the lung, but did in the liver, B16 cells labeled with 5-[125I]-iodo-2'-deoxyuridine (125I-dUrd) were injected and the arrest of tumor cell emboli was examined in the capillary beds of the lung and liver of mice treated with either vehicle or rhM-CSF. In both groups, there were the same numbers of B16 cells in both the lung and the liver 3 minutes after the B16 injection, and almost all tumor cells died within 24 h. However, the number of cells surviving in the lung was decreased in mice injected with rhM-CSF (37%). There was no difference in the number of cells in the livers of mice treated either with vehicle or rhM-CSF in the first 24 h after tumor cell injection. The administration of rhM-CSF increased NK 1.1+ cells in the mouse spleen and facilitated NK activity in vivo. At the same time, the administration of an anti-NK 1.1 antibody blocked the antimetastatic effect of rhM-CSF in the lung but not in the liver. The antibody was effective only when it was injected before the B16 inoculation. These results suggest that the antimetastatic effect of rhM-CSF in the lung was mediated by NK 1.1+ cells within 24 h of B16 injection. In contrast, the antimetastatic effect of rhM-CSF in the liver was mediated not only by NK 1.1+ cells but also by other antimetastatic systems such as macrophages.

  3. The role of individual cysteine residues in the processing, structure, and function of human macrophage colony-stimulating factor.

    PubMed

    Deng, P; Wang, Y L; Pattengale, P K; Rettenmier, C W

    1996-11-12

    The shortest form of human macrophage colony-stimulating factor (M-CSF alpha, CSF-1(256) is expressed on the cell surface as a homodimeric type I transmembrane glycoprotein. The seven cysteine residues in CSF-1(256) form three intrachain disulfide bonds (Cys7-Cys90, Cys48-Cys139, and Cys 102-Cys146), and one interchain disulfide bond (Cys31-Cys31). To examine the role of the seven cysteine residues in CSF-1(256), we replaced each half-cystine by a serine using site-directed mutagenesis, and stably expressed the mutated genes in mouse NIH 3T3 cells. We showed that each of the seven cysteines of CSF-1(256) is essential for its biological activity. Our data further show that substitution of Cys48 or Cys139 totally blocked dimer formation and cell surface expression of CSF-1(256), and that substitution of Cys102 and Cys146 severely impaired CSF-1 dimer formation and cell surface expression. In contrast, substitution of Cys7 or Cys90 affected CSF-1 dimer formation to a lesser degree but did not significantly affect cell surface expression of CSF-1. Furthermore, disruption of the interchain disulfide bond led to efficient cell surface expression of monomeric CSF-1. All of the cell surface expressed mutant CSF-1 proteins, either dimeric or monomeric, still underwent efficient ectodomain cleavage. The electrophoretic mobilities of the cleaved dimeric ectodomains of these mutant CSF-1 proteins on SDS-PAGE exhibited distinctly different patterns as compared with the wild type. Substitution of either Cys7 or Cys90 produced the same shift, while substitution of either Cys102 or Cys146 resulted in a shift distinct from that caused by substitution of Cys7 or Cys90. These data suggest that replacement of either of a pair of intrachain half-cystine residues results in similar conformational changes, and may provide a novel method for mapping intrachain disulfide bonds in dimeric proteins.

  4. Sustained in vivo activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) using HEPES buffer.

    PubMed

    Kasraian, K; Kuzniar, A; Earley, D; Kamicker, B J; Wilson, G; Manion, T; Hong, J; Reiber, C; Canning, P

    2001-08-01

    The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6-8) and at temperatures of approximately 40 degrees C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance. Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the "control" formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24-30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the T(M) (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the T(M) was 48 degrees C (onset approximately 40 degrees C), while bG-CSF formulated in 1 M HEPES buffer has a T(M) of 59 degrees C (onset approximately 50 degrees C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological

  5. Fibroblast growth factor stimulates angiotensin converting enzyme expression in vascular smooth muscle cells. Possible mediator of the response to vascular injury.

    PubMed Central

    Fishel, R S; Thourani, V; Eisenberg, S J; Shai, S Y; Corson, M A; Nabel, E G; Bernstein, K E; Berk, B C

    1995-01-01

    Angiotensin converting enzyme (ACE) activity contributes to the vascular response to injury because ACE inhibition limits neointima formation in rat carotid arteries after balloon injury. To investigate the mechanisms by which ACE may contribute to vascular smooth muscle cell (VSMC) proliferation, we studied expression of ACE in vivo after injury and in vitro after growth factor stimulation. ACE activity 14 d after injury was increased 3.6-fold in the injured vessel. ACE expression, measured by immunohistochemistry, became apparent at 7 d in the neointima and at 14 d was primarily in the most luminal neointimal cells. To characterize hormones that induce ACE in vivo, cultured VSMC were exposed to steroids and growth factors. Among steroids, only glucocorticoids stimulated ACE expression with an 8.0 +/- 2.1-fold increase in activity and a 6.5-fold increase in mRNA (30 nM dexamethasone for 72 h). Among growth factors tested, only fibroblast growth factor (FGF) stimulated ACE expression (4.2 +/- 0.7-fold increase in activity and 1.6-fold increase in mRNA in response to 10 ng/ml FGF for 24 h). Dexamethasone and FGF were synergistic at the indicated concentrations inducing 50.6 +/- 12.4-fold and 32.5-fold increases in activity and mRNA expression, respectively. In addition, when porcine iliac arteries were transfected with recombinant FGF-1 (in the absence of injury), ACE expression increased in neointimal VSMC, to the same extent as injured, nontransfected arteries. The data suggest a temporal sequence for the response to injury in which FGF induces ACE, ACE generates angiotensin II, and angiotensin II stimulates VSMC growth in concert with FGF. Images PMID:7814638

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

    PubMed

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

    2016-03-01

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

  7. Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene

    SciTech Connect

    Benjamin, C.W.; Tarpley, W.G.; Gorman, R.R.

    1987-01-01

    Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. The authors now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A/sub 2/ and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E/sub 2/ (PGE/sub 2/) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 ..mu..M arachidonate resulted in identical levels of PGE/sub 2/ release. The lack of PDGF-stimulated PGE/sub 2/ release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much /sup 125/I-labeled PDGF as control cells and are stimulated to incorporate (/sup 3/H)thymidine and to proliferate after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A/sub 2/ activities are inhibited in the EJ-ras-transfected cells.

  8. Two squamous cell carcinomas not associated with humoral hypercalcemia produce a potent bone resorption-stimulating factor which is interleukin-1 alpha.

    PubMed

    Fried, R M; Voelkel, E F; Rice, R H; Levine, L; Gaffney, E V; Tashjian, A H

    1989-08-01

    Conditioned medium (CM) from two squamous cell carcinoma cell lines, SCC-9 and SCC-13, stimulated bone resorption in neonatal mouse calvariae in organ culture. Enhanced bone resorption induced by CM was associated with an increased production of prostaglandin-E2 (PGE2) by the calvariae. Complete inhibition of stimulated PGE2 synthesis by indomethacin only partially inhibited bone resorption-stimulating activity (BRSA) in the CM. Neither SCC-9 nor SCC-13 CM stimulated cAMP production in rat osteosarcoma cells (ROS 17/2.8). The BRSA in CM was completely inhibited by an antibody to interleukin-1 alpha (IL-1 alpha). Fractionation of SCC-9 CM by gel filtration and HPLC ion exchange chromatography revealed a single peak of BRSA and PGE2 synthesis-stimulating activity at 17-20K (termed SCMII). In mouse calvariae, SCMII increased medium Ca2+ and PGE2 in a dose-dependent manner at concentrations from 20 ng protein/ml to a maximum of 500 ng protein/ml. Preincubation of SCMII with antibody to IL-1 alpha completely inhibited SCMII-induced bone resorption. SCMII also enhanced thymocyte proliferation with activity that was equivalent to 353 U/ml IL-1. Antibodies to IL-1 beta and tumor necrosis factor had no effect on SCMII-induced bone resorption. Using specific enzyme-linked immunosorbent assays for IL-1 alpha and IL-1 beta, IL-1 alpha was measured in high concentrations in both crude and partially purified fractions of SCC-9 and SCC-13 CM. In contrast, IL-1 beta was either undetectable or present in amounts below those that stimulate bone resorption. In addition, SCMII did not enhance cAMP production in bone cells. We conclude that the BRSA produced by the two squamous cell carcinoma cell lines SCC-9 and SCC-13 is IL-1 alpha.

  9. Anti-granulocyte-macrophage colony-stimulating factor autoantibodies are a risk factor for central nervous system infection by Cryptococcus gattii in otherwise immunocompetent patients.

    PubMed

    Saijo, Tomomi; Chen, Jianghan; Chen, Sharon C-A; Rosen, Lindsey B; Yi, Jin; Sorrell, Tania C; Bennett, John E; Holland, Steven M; Browne, Sarah K; Kwon-Chung, Kyung J

    2014-03-18

    Cryptococcosis is caused by either Cryptococcus neoformans or C. gattii. While cryptococcal meningoencephalitis is caused mostly by C. neoformans in immunocompromised patients, the risk factors remain unclear for patients with no known immune defect. Recently, anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies were detected in the plasma of seven "immunocompetent" cryptococcosis patients, and the cryptococcal strains from these patients were reported as C. neoformans (three strains), C. gattii (one strain), and Cryptococcus (three strains not identified to the species level). We identified all three strains that had not been identified to the species level as C. gattii. Notably, the three strains that were reported as C. neoformans but were unavailable for species confirmation originated from Sothern California and Thailand where C. gattii is endemic. Most clinical laboratories designate C. neoformans without distinguishing between the two species; hence, these three strains could have been C. gattii. Since C. gattii infects more immunocompetent patients than C. neoformans, we pursued the possibility that this antibody may be more prevalent in patients infected with C. gattii than in those infected with C. neoformans. We screened the plasma of 20 healthy controls and 30 "immunocompetent" patients with cryptococcal meningoencephalitis from China and Australia (multiple ethnicities). Anti-GM-CSF autoantibodies were detected only in the plasma of seven patients infected by C. gattii and one healthy volunteer and in none infected by C. neoformans. While plasma from these C. gattii patients completely prevented GM-CSF-induced p-STAT5 in normal human peripheral blood mononuclear cells (PBMCs), plasma from one healthy volunteer positive for anti-GM-CSF autoantibodies caused only partial blockage. Our results suggest that anti-GM-CSF autoantibodies may predispose otherwise immunocompetent individuals to meningoencephalitis caused by C. gattii but

  10. Involvement of JAK1, JAK2, and JAK3 in Stimulation of Functional Activity of Mesenchymal Progenitor Cells by Fibroblast Growth Factor.

    PubMed

    Zyuz'kov, G N; Zhdanov, V V; Udut, E V; Miroshnichenko, L A; Simanina, E V; Polyakova, T Yu; Stavrova, L A; Udut, V V; Minakova, M Yu; Dygai, A M

    2016-12-01

    We studied the involvement of individual JAK kinases in the realization of the growth potential of mesenchymal precursors under the effect of fibroblast growth factor. The important role of JAK2 and JAK3 in determining the initial level of mitotic activity of progenitor cells and participation of JAK1 in this process under conditions of cytokine stimulation of progenitor cells were demonstrated. Specific inhibitors of these kinases reduced the yield of fibroblast CFU and the rate of their division. Moreover, blockade of JAK1, JAK2, and JAK3 under the effect of fibroblast growth factor was accompanied by an increase in the intensity of progenitor cell differentiation.

  11. The Priming Effects of Tumor Necrosis Factor and Interleukin-1 on Canine Neutrophils Stimulated with Interleukin-8

    DTIC Science & Technology

    1991-08-01

    production .............................. 65 1 CHAPTER I. INTRODUCTION Cytokines Cytokines are a family of heterogeneous glycoproteins that stimulate many...digest invading bacteria can destroy surrounding healthy tissues. Unexpectedly high levels of Interleukin-8 have keen demonstrated in lesions of psoriasis ...cell responsible for localizing, digesting, and killing invading microorganisms. It is also the prevalent leukocyte in the peripheral circulation. The

  12. Cartilage–Specific Over-Expression of CCN Family Member 2/Connective Tissue Growth Factor (CCN2/CTGF) Stimulates Insulin-Like Growth Factor Expression and Bone Growth

    PubMed Central

    Tomita, Nao; Hattori, Takako; Itoh, Shinsuke; Aoyama, Eriko; Yao, Mayumi; Yamashiro, Takashi; Takigawa, Masaharu

    2013-01-01

    Previously we showed that CCN family member 2/connective tissue growth factor (CCN2) promotes the proliferation, differentiation, and maturation of growth cartilage cells in vitro. To elucidate the specific role and molecular mechanism of CCN2 in cartilage development in vivo, in the present study we generated transgenic mice overexpressing CCN2 and analyzed them with respect to cartilage and bone development. Transgenic mice were generated expressing a ccn2/lacZ fusion gene in cartilage under the control of the 6 kb-Col2a1-enhancer/promoter. Changes in cartilage and bone development were analyzed histologically and immunohistologically and also by micro CT. Primary chondrocytes as well as limb bud mesenchymal cells were cultured and analyzed for changes in expression of cartilage–related genes, and non-transgenic chondrocytes were treated in culture with recombinant CCN2. Newborn transgenic mice showed extended length of their long bones, increased content of proteoglycans and collagen II accumulation. Micro-CT analysis of transgenic bones indicated increases in bone thickness and mineral density. Chondrocyte proliferation was enhanced in the transgenic cartilage. In in vitro short-term cultures of transgenic chondrocytes, the expression of col2a1, aggrecan and ccn2 genes was substantially enhanced; and in long-term cultures the expression levels of these genes were further enhanced. Also, in vitro chondrogenesis was strongly enhanced. IGF-I and IGF-II mRNA levels were elevated in transgenic chondrocytes, and treatment of non-transgenic chondrocytes with recombinant CCN2 stimulated the expression of these mRNA. The addition of CCN2 to non-transgenic chondrocytes induced the phosphorylation of IGFR, and ccn2-overexpressing chondrocytes showed enhanced phosphorylation of IGFR. Our data indicates that the observed effects of CCN2 may be mediated in part by CCN2-induced overexpression of IGF-I and IGF-II. These findings indicate that CCN2-overexpression in

  13. Epidermal growth factor (EGF)-stimulated inositol phosphate formation in hepatocytes is abolished by pertussis toxin and phorbol esters

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1987-05-01

    The EGF-stimulated rise in intracellular Ca/sup 2 +/ (Ca/sup 2 +/)/sub i/ and Ca/sup 2 +/-dependent protein phosphorylation events in isolated hepatocytes are blocked by pertussis toxin and phorbol ester pretreatment. The present study characterized the EGF-stimulated formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P/sub 3/) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P/sub 3/) in hepatocytes using HPLC methodology to separate the InsP/sub 3/ isomers. Both 66 nM EGF and 10 nM angiotensin II (ANG II) caused a rapid increase in the Ins(1,4,5)P/sub 3/ isomer although EGF-stimulated formation was smaller. At a concentration of ANG II (0.1 nM) which gave an equivalent rise in (Ca/sup 2 +/)/sub i/ as 66 nM EGF, the kinetics and magnitude of Ins(1,4,5)P/sub 3/ formation were similar. EGF or ANG II-stimulated formation of the Ins(1,3,4)P/sub 3/ isomer was more gradual and increased beyond the level of Ins(1,4,5)P/sub 3/ after 60 sec. The initial EGF and ANG II-stimulated increase in both InsP/sub 3/ isomers was not affected by removing external Ca/sup 2 +/ with a 10-fold excess of EGTA. Pretreatment of rats with pertussis toxin for 72 hrs blocked the ability of EGF to increase Ins(1,4,5)P/sub 3/ but did not affect the increase due to ANG II. Three main pretreatment of cells with 1 ..mu..g/ml phorbol 12-myristate-13-acetate (PMA) also inhibited the EGF-stimulated Ins(1,4,5)P/sub 3/ formation. PMA slightly attenuated Ins(1,4,5)P/sub 3/ formation stimulated by 0.1 nM ANG II but not enough to affect the Ca/sup 2 +/ signal. These data suggest that the signal transduction system used by EGF receptors to increase Ins (1,4,5)P/sub 3/ in hepatocytes is somehow different from that used by ANG II receptors.

  14. Production of colony-stimulating factors (CSFs) during infection: separate determinations of macrophage-, granulocyte-, granulocyte-macrophage-, and multi-CSFs.

    PubMed Central

    Cheers, C; Haigh, A M; Kelso, A; Metcalf, D; Stanley, E R; Young, A M

    1988-01-01

    After infection of mice with Listeria monocytogenes, elevated levels of colony-stimulating factors (CSFs) in the serum were quantitated by six different assays: ability to stimulate colony formation, the proliferation of 2 suspension of bone marrow cells (both measuring total colony-stimulating activity), a radioimmunoassay for macrophage-CSF (CSF-1), the WEHI-3B differentiation assay for granulocyte-CSF, and proliferation of 32D-c1-3 and FDC-P1 cell lines (specific for multi-CSF and either multi- or granulocyte-macrophage-CSFs, respectively). The great bulk of serum colony-stimulating activity represented macrophage- and granulocyte-CSFs, with small but measurable amounts of granulocyte-macrophage-CSF. The degree of elevation of serum CSF depended on the infecting dose used and the numbers of bacteria growing in the spleens and livers of the two mouse strains compared, i.e., L. monocytogenes-resistant C57BL/10 and susceptible BALB/cJ. The increase in serum CSFs occurred before the peak in bone marrow granulocyte-macrophage progenitors and before the reduction in bacterial numbers which follows the onset of specific cell-mediated immunity. PMID:3257205

  15. Macrophage colony-stimulating factor-induced macrophage differentiation promotes regrowth in atrophied skeletal muscles and C2C12 myotubes.

    PubMed

    Dumont, Nicolas A; Frenette, Jérôme

    2013-02-01

    Skeletal muscle injury and regeneration are closely associated with an inflammatory reaction that is usually characterized by sequential recruitment of neutrophils and monocytes or macrophages. Selective macrophage depletion models have shown that macrophages are essential for complete regeneration of muscle fibers after freeze injuries, toxin injuries, ischemia-reperfusion, and hindlimb unloading and reloading. Although there is growing evidence that macrophages possess major myogenic capacities, it is not known whether the positive effects of macrophages can be optimized to stimulate muscle regrowth. We used in vivo and in vitro mouse models of atrophy to investigate the effects of stimulating macrophages with macrophage colony-stimulating factor (M-CSF) on muscle regrowth. When atrophied soleus muscles were injected intramuscularly with M-CSF, we observed a 1.6-fold increase in macrophage density and a faster recovery in muscle force (20%), combined with an increase in muscle fiber diameter (10%), after 7 days of reloading, compared with PBS-injected soleus muscles. Furthermore, coculture of atrophied myotubes with or without bone marrow-derived macrophages (BMDM) and/or M-CSF revealed that the combination of BMDMs and M-CSF was required to promote myotube growth (15%). More specifically, M-CSF promoted the anti-inflammatory macrophage phenotype, which in turn decreased protein degradation and MuRF-1 expression by 25% in growing myotubes. These results indicate that specific macrophage subsets can be stimulated to promote muscle cell regrowth after atrophy.

  16. Alterations in T cell-derived colony-stimulating factors associated with GVH-induced immune deficiency

    SciTech Connect

    Hakim, F.T.; Pluznik, D.H.; Shearer, G.M. )

    1990-04-01

    Injection of parental C57BL/10 spleen cells into unirradiated immune-competent (B10 x B10.BR)F1 hosts has been demonstrated to produce a graft-vs.-host-induced immune deficiency in T cell-mediated functions, including mitogen or alloantigen stimulated proliferation or cytotoxic T cell generation. The production of T cell-derived lymphokines affecting hematopoiesis was also altered during GVH. During the first two weeks of GVH, IL-3 and particularly GM-CSF were produced spontaneously; in subsequent weeks, the spontaneous production dropped to normal or subnormal levels. CSF content in concanavalin A-stimulated splenic supernatants was reduced at weeks 1-2, and declined to less than 5% of normal levels by 3-4 weeks of GVH. This decline in CSF content was correlated with a decrease in immune function as assessed by concanavalin A-stimulated IL-2 production and by generation of cytotoxic T lymphocytes. Concurrent with the recovery of immune function during GVH weeks 8-15, mitogen-stimulated production of CSF returned to normal levels. In addition to the decrease in CSF production identified in acute suppressive GVH, CSF content in concanavalin A-stimulated splenic supernatants was also decreased in chronic stimulatory GVH, generated in the strain combination (B6 x B6bm1)F1----(B6bm1 x B6bm12)F1. This decrease in CSF production correlated with a decrease in self-restricted T helper cell function. Finally, a decrease in both immune function and CSF production capacity was observed in the acute GVH following allogeneic (minor histocompatibility loci) bone marrow transplantation into irradiated hosts.

  17. Pegylated granulocyte colony-stimulating factor conveys long-term neuroprotection and improves functional outcome in a model of Parkinson's disease.

    PubMed

    Frank, Tobias; Klinker, Florian; Falkenburger, Björn H; Laage, Rico; Lühder, Fred; Göricke, Bettina; Schneider, Armin; Neurath, Hartmud; Desel, Herbert; Liebetanz, David; Bähr, Mathias; Weishaupt, Jochen H

    2012-06-01

    Recent proof-of-principle data showed that the haematopoietic growth factor granulocyte colony-stimulating factor (filgrastim) mediates neuroprotection in rodent models of Parkinson's disease. In preparation for future clinical trials, we performed a preclinical characterization of a pegylated derivative of granulocyte colony-stimulating factor (pegfilgrastim) in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease. We determined serum and cerebrospinal fluid drug levels after subcutaneous injection. A single injection of pegfilgrastim was shown to achieve stable levels of granulocyte colony-stimulating factor in both serum and cerebrospinal fluid with substantially higher levels compared to repetitive filgrastim injections. Leucocyte blood counts were only transiently increased after repeated injections. We demonstrated substantial dose-dependent long-term neuroprotection by pegfilgrastim in both young and aged mice, using bodyweight-adjusted doses that are applicable in clinical settings. Importantly, we found evidence for the functionally relevant preservation of nigrostriatal projections by pegfilgrastim in our model of Parkinson's disease, which resulted in improved motor performance. The more stable levels of pegylated neuroprotective proteins in serum and cerebrospinal fluid may represent a general advantage in the treatment of chronic neurodegenerative diseases and the resulting longer injection intervals are likely to improve patient compliance. In summary, we found that pegylation of a neuroprotective growth factor improved its pharmacokinetic profile over its non-modified counterpart in an in vivo model of Parkinson's disease. As the clinical safety profile of pegfilgrastim is already established, these data suggest that evaluation of pegfilgrastim in further Parkinson's disease models and ultimately clinical feasibility studies are warranted.

  18. 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.

  19. Glial cell line-derived neurotrophic factor family ligands enhance capsaicin-stimulated release of calcitonin gene-related peptide from sensory neurons.

    PubMed

    Schmutzler, B S; Roy, S; Hingtgen, C M

    2009-06-16

    The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are a group of peptides that have been implicated as important factors in inflammation, since they are released in increased amounts during inflammation and induce thermal hyperalgesia upon injection. Mouse isolated sensory neurons in culture and freshly dissociated spinal cord slices were used to examine the enhancement in stimulated-release of the neuropeptide, calcitonin gene-related peptide (CGRP), as a measure of sensitization. Exposure of isolated sensory neurons in culture to GDNF, neurturin, and artemin enhanced the capsaicin-stimulated release of immunoreactive calcitonin gene-related peptide (iCGRP) two- to threefold, but did not increase potassium-stimulated release of iCGRP. A similar profile of sensitization was observed in freshly dissociated spinal cord slices. Persephin, another member of the GFL family thought to be important in development, was unable to induce an enhancement in the release of iCGRP. These results demonstrate that specific GFLs are important mediators affecting sensory neuronal sensitivity, likely through modulation of the capsaicin receptor. The sensitization of sensory neurons during inflammation, and the pain and neurogenic inflammation resulting from this sensitization, may be due in part to the effects of these selected GFLs.

  20. Aberrant Low Expression of A20 in Tumor Necrosis Factor-α-stimulated SLE Monocytes Mediates Sustained NF-κB Inflammatory Response.

    PubMed

    Shi, Xiaowei; Qian, Tian; Li, Min; Chen, Fangru; Chen, Yan; Hao, Fei

    2015-01-01

    The aberrantly activated monocytes and nuclear factor-kappaB (NF-κB) pathway contribute to the pathogenesis of systemic lupus erythematosus (SLE), and the aberrantly activated NF-κB is associated with defects in the anti-inflammatory A20 in SLE. However, whether SLE monocytes express A20 and whether the A20 expression under sustained proinflammatory stimulation is altered to contribute to the uncontrolled NF-κB inflammatory response are unclear. In this study, we found that the freshly isolated monocytes from SLE patients and healthy controls did not differ in expression levels of IL-1β, IκBα and A20. After TNF-α stimulation for 48 h, the monocytes from both groups expressed higher levels of IL-1β and IκBα than the monocytes without TNF-α treatment. Although the increased levels of NF-κB were observed in the nucleus of both the SLE and control monocytes after 24 h of TNF-α stimulation, the enhancement in SLE monocytes was significantly more robust than in the control monocytes. In addition, while the p-IκBα level in healthy monocytes was increased, the p-IκBα level in SLE monocytes was slightly decreased after TNF-α stimulation. Interestingly, after TNF-α treatment, the A20 expression in SLE monocytes was not markedly altered compared with the untreated SLE monocytes; moreover, the SLE monocytes expressed significantly lower A20 than healthy monocytes with TNF-α treatment at each time point. Results in this study demonstrate that TNF-α activates a significant NF-κB inflammatory response in SLE monocytes, which is at least partially mediated by the aberrantly low expression of A20 upon TNF-α stimulation, contributing to the prolonged inflammatory response in SLE.

  1. Generation of reactive oxygen species (ROS) is a key factor for stimulation of macrophage proliferation by ceramide 1-phosphate

    SciTech Connect

    Arana, Lide; Gangoiti, Patricia; Ouro, Alberto; Rivera, Io-Guane; Ordonez, Marta; Trueba, Miguel; Lankalapalli, Ravi S.; Bittman, Robert; Gomez-Munoz, Antonio

    2012-02-15

    We previously demonstrated that ceramide 1-phosphate (C1P) is mitogenic for fibroblasts and macrophages. However, the mechanisms involved in this action were only partially described. Here, we demonstrate that C1P stimulates reactive oxygen species (ROS) formation in primary bone marrow-derived macrophages, and that ROS are required for the mitogenic effect of C1P. ROS production was dependent upon prior activation of NADPH oxidase by C1P, which was determined by measuring phosphorylation of the p40phox subunit and translocation of p47phox from the cytosol to the plasma membrane. In addition, C1P activated cytosolic calcium-dependent phospholipase A{sub 2} and protein kinase C-{alpha}, and NADPH oxidase activation was blocked by selective inhibitors of these enzymes. These inhibitors, and inhibitors of ROS production, blocked the mitogenic effect of C1P. By using BHNB-C1P (a photolabile caged-C1P analog), we demonstrate that all of these C1P actions are caused by intracellular C1P. It can be concluded that the enzyme responsible for C1P-stimulated ROS generation in bone marrow-derived macrophages is NADPH oxidase, and that this enzyme is downstream of PKC-{alpha} and cPLA{sub 2}-{alpha} in this pathway. -- Highlights: Black-Right-Pointing-Pointer Ceramide 1-phosphate (C1P) stimulates reactive oxygen species (ROS) formation. Black-Right-Pointing-Pointer The enzyme responsible for ROS generation by C1P in macrophages is NADPH oxidase. Black-Right-Pointing-Pointer NADPH oxidase lies downstream of cPLA{sub 2}-{alpha} and PKC-{alpha} in this pathway. Black-Right-Pointing-Pointer ROS generation is essential for the stimulation of macrophage proliferation by C1P.

  2. Interferon regulatory factor-two restricts expression of interferon-stimulated genes to the endometrial stroma and glandular epithelium of the ovine uterus.

    PubMed

    Choi, Y; Johnson, G A; Burghardt, R C; Berghman, L R; Joyce, M M; Taylor, K M; Stewart, M D; Bazer, F W; Spencer, T E

    2001-10-01

    Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

  3. Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2.

    PubMed

    Fujino, Hiromichi; West, Kimberly A; Regan, John W

    2002-01-25

    Recently we have shown that the FP(B) prostanoid receptor, a G-protein-coupled receptor that couples to Galpha(q), activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-mediated transcriptional activation (Fujino, H., and Regan, J. W. (2001) J. Biol. Chem. 276, 12489-12492). We now report that the EP(2) and EP(4) prostanoid receptors, which couple to Galpha(s), also activate Tcf/Lef signaling. By using a Tcf/Lef-responsive luciferase reporter gene, transcriptional activity was stimulated approximately 10-fold over basal by 1 h of treatment with prostaglandin E(2) (PGE(2)) in HEK cells that were stably transfected with the human EP(2) and EP(4) receptors. This stimulation of reporter gene activity was accompanied by a PGE(2)-dependent increase in the phosphorylation of both glycogen synthase kinase-3 (GSK-3) and Akt kinase. H-89, an inhibitor of protein kinase A (PKA), completely blocked the agonist-dependent phosphorylation of GSK-3 in both EP(2)- and EP(4)-expressing cells. However, H-89 pretreatment only blocked PGE(2)-stimulated Lef/Tcf reporter gene activity by 20% in EP(4)-expressing cells compared with 65% inhibition in EP(2)-expressing cells. On the other hand wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had the opposite effect and inhibited PGE(2)-stimulated reporter gene activity to a much greater extent in EP(4)-expressing cells as compared with EP(2)-expressing cells. These findings indicate that the activation of Tcf/Lef signaling by EP(2) receptors occurs primarily through a PKA-dependent pathway, whereas EP(4) receptors activate Tcf/Lef signaling mainly through a phosphatidylinositol 3-kinase-dependent pathway. This is the first indication of a fundamental difference in the signaling potential of EP(2) and EP(4) prostanoid receptors.

  4. Influence of a ras oncogene on platelet-derived growth factor (PDGF)-stimulated phosphoinositide hydrolysis in murine fibroblasts

    SciTech Connect

    Parries, G.; Racker, E.

    1986-05-01

    The authors have examined the effects of transfection of rat-1 fibroblasts with the ras oncogene on the metabolism of phosphatidylinositol (PI). Incubation of (/sup 3/H)inositol-labeled rat-1 cells with PDGF resulted in a 2- to 3-fold increase in (/sup 3/H)IP3 levels within 90 s. In the presence of 25 mM Li+, (/sup 3/H)IP1 levels were increased 8-fold after 30 min. In contrast, incubation of ras-transfected fibroblasts (EJ-2 line) with PDGF had little or no effect on the level of either (/sup 3/H)IP3 or (/sup 3/H)IP1. Similar stimulations by PDGF were observed in NIH 3T3 cells, but not in Kirsten virus-transformed or Harvey ras-transfected cell lines. On the other hand, NIH 3T3 cells transfected with v-src responded to PDGF by stimulation of PI turnover similar to the parent cell line. In NIH 3T3 cells transfected with an expression vector containing the v-Ha-ras gene under transcriptional control of the glucocorticoid-inducible mouse mammary tumor virus promoter, the PDGF stimulation of (/sup 3/H)inositol incorporation into PI was reduced from 10-fold in the absence of dexamethasone to 1.8-fold when the cells were pretreated for 26 h with 2 ..mu..M dexamethasone. In the parental 3T3 cells PDGF stimulation was reduced by about 40% in the presence of dexamethasone. In the absence of PDGF the rate of PI turnover (i.e., the kinetics of (/sup 3/H)IP1 accumulation in the presence of Li+) in EJ-2 cells was similar to that in rat-1 cells. Thus, in the presence of PDGF, the rate of PI turnover in rat-1 cells was several fold higher than in the transfected cells. These results suggest that the ras gene product (p21) may exert an inhibitory effect on PDGF-stimulated phosphoinositide metabolism.

  5. The Gottingen Minipig Is a Model of the Hematopoietic Acute Radiation Syndrome: G-Colony Stimulating Factor Stimulates Hematopoiesis and Enhances Survival From Lethal Total-Body γ-Irradiation

    SciTech Connect

    Moroni, Maria; Ngudiankama, Barbara F.; Christensen, Christine; Olsen, Cara H.; Owens, Rossitsa; Lombardini, Eric D.; Holt, Rebecca K.; Whitnall, Mark H.

    2013-08-01

    Purpose: We are characterizing the Gottingen minipig as an additional large animal model for advanced drug testing for the acute radiation syndrome (ARS) to enhance the discovery and development of novel radiation countermeasures. Among the advantages provided by this model, the similarities to human hematologic parameters and dynamics of cell loss/recovery after irradiation provide a convenient means to compare the efficacy of drugs known to affect bone marrow cellularity and hematopoiesis. Methods and Materials: Male Gottingen minipigs, 4 to 5 months old and weighing 9 to 11 kg, were used for this study. We tested the standard off-label treatment for ARS, rhG-CSF (Neupogen, 10 μg/kg/day for 17 days), at the estimated LD70/30 total-body γ-irradiation (TBI) radiation dose for the hematopoietic syndrome, starting 24 hours after irradiation. Results: The results indicated that granulocyte colony stimulating factor (G-CSF) enhanced survival, stimulated recovery from neutropenia, and induced mobilization of hematopoietic progenitor cells. In addition, the administration of G-CSF resulted in maturation of monocytes/macrophages. Conclusions: These results support continuing efforts toward validation of the minipig as a large animal model for advanced testing of radiation countermeasures and characterization of the pathophysiology of ARS, and they suggest that the efficacy of G-CSF in improving survival after total body irradiation may involve mechanisms other than increasing the numbers of circulating granulocytes.

  6. NF-kappaB inhibitor dehydroxymethylepoxyquinomicin suppresses osteoclastogenesis and expression of NFATc1 in mouse arthritis without affecting expression of RANKL, osteoprotegerin or macrophage colony-stimulating factor.

    PubMed

    Kubota, Tetsuo; Hoshino, Machiko; Aoki, Kazuhiro; Ohya, Keiichi; Komano, Yukiko; Nanki, Toshihiro; Miyasaka, Nobuyuki; Umezawa, Kazuo

    2007-01-01

    Inhibition of NF-kappaB is known to be effective in reducing both inflammation and bone destruction in animal models of arthritis. Our previous study demonstrated that a small cell-permeable NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), suppresses expression of proinflammatory cytokines and ameliorates mouse arthritis. It remained unclear, however, whether DHMEQ directly affects osteoclast precursor cells to suppress their differentiation to mature osteoclasts in vivo. The effect of DHMEQ on human osteoclastogenesis also remained elusive. In the present study, we therefore examined the effect of DHMEQ on osteoclastogenesis using a mouse collagen-induced arthritis model, and using culture systems of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis, and of osteoclast precursor cells from peripheral blood of healthy volunteers. DHMEQ significantly suppressed formation of osteoclasts in arthritic joints, and also suppressed expression of NFATc1 along the inner surfaces of bone lacunae and the eroded bone surface, while serum levels of soluble receptor activator of NF-kappaB ligand (RANKL), osteoprotegerin and macrophage colony-stimulating factor were not affected by the treatment. DHMEQ also did not suppress spontaneous expression of RANKL nor of macrophage colony-stimulating factor in culture of fibroblast-like synovial cells obtained from patients with rheumatoid arthritis. These results suggest that DHMEQ suppresses osteoclastogenesis in vivo, through downregulation of NFATc1 expression, without significantly affecting expression of upstream molecules of the RANKL/receptor activator of NF-kappaB/osteoprotegerin cascade, at least in our experimental condition. Furthermore, in the presence of RANKL and macrophage colony-stimulating factor, differentiation and activation of human osteoclasts were also suppressed by DHMEQ, suggesting the possibility of future application of NF-kappaB inhibitors to rheumatoid arthritis

  7. Regulation of the nuclear export of the transcription factor NFATc1 by protein kinases after slow fibre type electrical stimulation of adult mouse skeletal muscle fibres.

    PubMed

    Shen, Tiansheng; Cseresnyés, Zoltán; Liu, Yewei; Randall, William R; Schneider, Martin F

    2007-03-01

    The transcription factor nuclear factor of activated T cells (NFAT)c1 has been shown to be involved in turning on slow skeletal muscle fibre gene expression. Previous studies from our laboratory have characterized the stimulation pattern-dependent nuclear import and resting shuttling of NFATc1-green fluorescent protein (GFP) in flexor digitorum brevis (FDB) muscle fibres from adult mouse. In this study, we use viral expression of the transcription factor NFATc1-GFP fusion protein to investigate the mechanisms underlying the nuclear export of the NFATc1-GFP that accumulated in the nuclei of cultured dissociated adult mouse FDB muscle fibres during slow-twitch fibre type electrical stimulation. In these studies, we found that inhibition of either glycogen synthase kinase 3beta (GSK3beta) or casein kinase 1 or 2 (CK1/2) markedly slowed the decay of nuclear NFATc1-GFP after cessation of muscle fibre electrical stimulation, whereas inhibition of casein kinase 1delta, p38 mitogen-activated protein kinase, c-Jun N-terminal kinase and protein kinase A had little effect. Simultaneous inhibition of GSK3beta and CK1/2 completely blocked the nuclear export of NFATc1-GFP after muscle activity. We also developed a simplified model of NFATc1 phosphorylation/dephosphorylation and nuclear fluxes, and used this model to simulate the observed time courses of nuclear NFATc1-GFP with and without NFATc1 kinase inhibition. Our results suggest that GSK3beta and CK1/2 are the major protein kinases that contribute to the removal of NFATc1 that accumulates in muscle fibre nuclei during muscle activity, and that GSK3beta and CK1/2 are responsible for phosphorylating NFATc1 in muscle nuclei in a complementary or synergistic fashion.

  8. Administration of colony stimulating factor-1 corrects some macrophage, dental, and skeletal defects in an osteopetrotic mutation (toothless, tl) in the rat.

    PubMed

    Marks, S C; Wojtowicz, A; Szperl, M; Urbanowska, E; MacKay, C A; Wiktor-Jedrzejczak, W; Stanley, E R; Aukerman, S L

    1992-01-01

    The toothless (tl/tl) mutation in the rat results in a paucity of osteoclasts and osteopetrosis that cannot be corrected by bone marrow transplantation. In the present study we demonstrate that tl/tl rats also have profound deficiencies of femoral, peritoneal, and pleural cavity macrophages. Furthermore, the macrophage colony stimulating activity of post-endotoxin sera from tl/tl rats is substantially reduced, suggesting that, as in the case of the op mutation in mice, the basis of the tl mutation is a deficiency of the macrophage growth factor, colony stimulating factor-1 (CSF-1). Consistent with this suggestion, treatment of tl/tl rats from birth for up to six weeks with CSF-1 reduced the osteopetrosis, increased body weight, and permitted tooth eruption. In addition, CSF-1 treatment induced large numbers of osteoclasts in tl/tl bones and macrophages in the peritoneal cavity and bone marrow. Persistence of metaphyseal sclerosis, however, indicated that the disease was not totally corrected by this treatment. These studies indicate that the basis of the tl mutation is most likely another CSF-1 deficiency, and further emphasize the role of this growth factor in osteoclast differentiation.

  9. Development of multiple necrotizing enteritis induced by a tumor necrosis factor-like cytokine from lipopolysaccharide-stimulated peritoneal macrophages in rats.

    PubMed Central

    Torimoto, K.; Sato, N.; Okubo, M.; Yagihashi, A.; Wada, Y.; Hara, I.; Hayasaka, H.; Kikuchi, K.

    1990-01-01

    We report the development of an animal model of multiple necrotizing enteritis (MNE) in rats. When rats were injected directly with a culture supernatant of lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages into the abdominal aorta, the overt pathologic lesions of MNE developed within 30 minutes after injection. The rats showed an elevated level of blood fibrinogen degradation product content even 30 minutes after injection. Furthermore the rats that were pretreated intravenously with heparin sulfate did not develop MNE, indicating the acute disturbances of blood microcirculation in the intestine. Multiple necrotizing enteritis was developed also by the injection with recombinant tumor necrosis factor (rTNF) but rarely was observed with even a high dose of recombinant interleukin-1 (rIL-1) or platelet-activating factor (PAF). The supernatant was cytotoxic in vitro to TNF-susceptible LM and many other cells but was less cytotoxic to the TNF-resistant LR line. Partial purification of the supernatant suggested that the supernatant contained a cytokine that has biochemical features of TNF. Furthermore polyclonal anti-TNF antibody could inhibit not only the cytotoxicity in vitro but also MNE development in vivo by this factor. These data strongly indicate that MNE possibly could be caused by a TNF-like cytokine produced by macrophages that are stimulated by the endotoxin. Images Figure 1 PMID:2240161

  10. Adherent cells in granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived dendritic cell culture system are qualified dendritic cells.

    PubMed

    Li, Gong-Bo; Lu, Guang-Xiu

    2010-01-01

    A widely-used method for generating dendritic cell (DC) is to culture bone marrow cells in granulocyte-macrophage colony-stimulating factor (GM-CSF)-containing medium for 6-10 days. Usually, non-adherent cells are used as qualified dendritic cells while the adherent ones are discarded as "non-dendritic cells" or macrophages. In this study, we show that the adherent cells are nearly identical to the non-adherent cells in both dendritic cell surface markers expression and main dendritic cell-related functions, hence to prove that these "junk cells" are actually qualified dendritic cells.

  11. Bacillus cereus brain abscesses occurring in a severely neutropenic patient: successful treatment with antimicrobial agents, granulocyte colony-stimulating factor and surgical drainage.

    PubMed

    Sakai, C; Iuchi, T; Ishii, A; Kumagai, K; Takagi, T

    2001-07-01

    Multiple brain and liver abscesses developed immediately after Bacillus cereus bacteremia in a neutropenic patient with acute lymphoblastic leukemia. After even 8 weeks of antimicrobial chemotherapy together with administration of granulocyte colony-stimulating factor, every infectious process disappeared but the patient's headache has still persisted. Because the wall of one brain abscess became thin and was in danger of rupturing into the ventricle, surgical drainage was performed, resulting in disappearance of headache and resolution of brain abscess. The present case indicates that a combined medical and surgical approach is mandatory to treat patients with brain abscesses.

  12. Prostaglandin E2 inhibits platelet-derived growth factor-stimulated cell proliferation through a prostaglandin E receptor EP2 subtype in rat hepatic stellate cells.

    PubMed

    Koide, Shigeki; Kobayashi, Yoshimasa; Oki, Yutaka; Nakamura, Hirotoshi

    2004-09-01

    Prostaglandin (PG) E2 inhibits hepatic stellate cell (HSC) mitogenesis. PGE-specific receptors are divided into four subtypes that are coupled either to Ca2+ mobilization (EP1 and EP3) or to the stimulation of adenyl cyclase (EP2 and EP4). The aims of the current study were to identify PGE receptor subtypes in cultured rat HSC and to examine which PGE receptor subtype(s) mediates the inhibitory effect of PGE2 on platelet-derived growth factor (PDGF)-stimulated proliferation. Reverse transcription-polymerase chain reaction analysis was performed to detect PGE receptor subtype mRNA expression. Cell proliferation was determined by measuring [3H]thymidine incorporation, and intracellular cyclic AMP was measured by radioimmunoassay. Cultured rat HSC expressed mRNAs for all four subtypes of PGE receptor. PGE2- and EP2-selective agonist produced dose-dependent inhibitory effects on PDGF-stimulated proliferation. Neither EP1-, EP3-, nor EP4-selective agonists showed any inhibitory effect. An adenylate cyclase inhibitor strongly blunted the inhibition of DNA synthesis elicited by PGE2 and the EP2 agonist. The EP2 agonist generated higher and more prolonged increases in intracellular cyclic AMP than the EP4 agonist. Activation of the PGE EP2 receptor has an antiproliferative effect in HSC that may be mediated by cyclic AMP-related signal transduction pathways.

  13. The high mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms.

    PubMed

    Du, W; Maniatis, T

    1994-11-22

    The high mobility group protein HMG I(Y) stimulates the binding of a specific isoform of the activating transcription factor 2 (ATF-2(195)) to the interferon beta (IFN-beta) gene promoter. HMG I(Y) specifically interacts with the basic-leucine zipper region of ATF-2(195), and HMG I(Y) binds to two sites immediately flanking the ATF-2 binding site of the IFN-beta promoter. Here, we show that HMG I(Y) can stimulate the binding of ATF-2(195), at least in part, by promoting ATF-2 dimerization. In addition, we report the characterization of a naturally occurring isoform of ATF-2 (ATF-2(192)) that binds specifically to the IFN-beta promoter but is unable to interact with HMG I(Y). Remarkably, HMG I(Y) inhibits the binding of ATF-2(192) to the IFN-beta promoter. Thus, the ability of HMG I(Y) to specifically interact with ATF-2 correlates with its ability to stimulate ATF-2 binding to the IFN-beta promoter. Comparisons of the amino acid sequences of the basic-leucine zipper domains of ATF-2(195) and ATF-2(192) suggest that HMG I(Y) interacts with a short stretch of basic amino acids near the amino terminus of the basic-leucine zipper domain of ATF-2(195).

  14. Assaying the granulocyte-macrophage colony-stimulating factor (GM-CSF) as a mitogen of immature cells in fetal blood cultures.

    PubMed

    Costa, D; Borrell, A; Jou, J M; Besón, I; Soler, A; Carrió, A; Margarit, E; Caballín, R; Ballesta, F; Fortuny, A

    1999-01-01

    Based on the presence of immature cells in fetal blood, and in an attempt to shorten the cytogenetic reporting time, three simultaneous one-day culture regimes were established in 23 fetal blood samples: (a) the standard phytohemagglutinin (PHA)-stimulated lymphocytes culture, (b) a culture using the granulocyte-macrophage colony-stimulating factor (GM-CSF) as an alternative mitogen, and (c) an unstimulated culture. Diagnostic success rates achieved by these three methods were as follows: 43 per cent (95 per cent CI: 23-64) (GM-CSF), 30 per cent (95 per cent CI: 12-49) (PHA) and 9 per cent (unstimulated). These three regimes were also assayed in three-day cultures giving 100 per cent diagnostic success rate for the PHA and GM-CSF, and 62 per cent (95 per cent CI: 41-83) for the unstimulated. A moderate correlation was found between the initial concentration of cultured erythroblasts and the metaphase count in one-day GM-CSF-stimulated (r=0.43, p=0.01) and unstimulated (r=0.35, p=0.05) cultures, suggesting that erythroblasts may be in part responsible for the mitotic index observed in these two regime cultures. In conclusion, our experience suggests that immature cells in fetal blood may be successfully cultured for diagnostic purposes.

  15. Predicting stimulant medication response in ADHD: evidence from an integrated profile of neuropsychological, psychophysiological and clinical factors.

    PubMed

    Hermens, Daniel F; Cooper, Nicholas J; Kohn, Michael; Clarke, Simon; Gordon, Evian

    2005-03-01

    There have been significant advances in understanding the neurobiology of Attention-Deficit/Hyperactivity Disorder (ADHD) and it is timely to examine the ability of biological and psychological markers to predict medication response in this disorder. We evaluated prediction of medication response in adolescent ADHD using neuropsychological testing and psychophysiological measures of central and autonomic function. Fifty ADHD adolescents participated in pre- and post-stimulant medication testing. Separately ranked performance in auditory oddball and visual Working Memory (WM) tasks determined 20 "responders" and 20 "non-responders" with 10 "neutrals" excluded from the discriminant function analyses (DFA). For both oddball and WM performance rankings, the two groups did not differ in age, sex, or handedness. However, responders did have higher levels of symptomatology than non-responders at baseline. Pre-stimulant medication psychophysiology variables were used as predictors in each DFA. Oddball performance correctly classified 85.0% of responders and 95.0% of non-responders. Better response was associated with increased resting beta power (left posteriorly), delayed oddball target N1 (frontally), decreased oddball target P2 (left posteriorly) and decreased WM distractor P3 (right frontally). Working memory performance classified 80.0% of responders and 90.0% of non-responders, with a broadly similar profile of psychophysiological predictors. These finding indicate the value of integrating neuropsychological and psychophysiological data in predicting medication response in ADHD.

  16. Stimulation of neuronal KATP channels by cGMP-dependent protein kinase: involvement of ROS and 5-hydroxydecanoate-sensitive factors in signal transduction

    PubMed Central

    Chai, Yongping

    2010-01-01

    The ATP-sensitive potassium (KATP) channel couples intracellular metabolic state to membrane excitability. Recently, we demonstrated that neuronal KATP channels are functionally enhanced by activation of a nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) signaling cascade. In this study, we further investigated the intracellular mechanism underlying PKG stimulation of neuronal KATP channels. By performing single-channel recordings in transfected HEK293 and neuroblastoma SH-SY5Y cells, we found that the increase of Kir6.2/SUR1 (i.e., the neuronal-type KATP) channel currents by PKG activation in cell-attached patches was diminished by 5-hydroxydecanoate (5-HD), an inhibitor of the putative mitochondrial KATP channel; N-(2-mercaptopropionyl)glycine, a reactive oxygen species (ROS) scavenger, and catalase, a hydrogen peroxide (H2O2)-decomposing enzyme. These reagents also ablated NO-induced KATP channel stimulation and prevented the shifts in the single-channel open- and closed-time distributions resulting from PKG activation and NO induction. Bath application of H2O2 reproduced PKG stimulation of Kir6.2/SUR1 but did not activate tetrameric Kir6.2LRKR368/369/370/371AAAA channels. Moreover, neither the PKG activator nor exogenous H2O2 was able to enhance the function of KATP channels in the presence of Ca2+ chelators and calmodulin antagonists, whereas the stimulatory effect of H2O2 was unaffected by 5-HD. Altogether, in this report we provide novel evidence that activation of PKG stimulates neuronal KATP channels by modulating intrinsic channel gating via a 5-HD-sensitive factor(s)/ROS/Ca2+/calmodulin signaling pathway that requires the presence of the SUR1 subunit. This signaling pathway may contribute to neuroprotection against ischemic injury and regulation of neuronal excitability and neurotransmitter release by modulating the function of neuronal KATP channels. PMID:20053925

  17. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades.

    PubMed

    Sancho, Veronica; Berna, Marc J; Thill, Michelle; Jensen, R T

    2011-12-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal (GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin (CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA was present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCK(A)-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125(FAK) and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ's direct association with AKT, RafA, RafC and Lyn. These results show for the first time the PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth).

  18. Venlafaxine treatment stimulates expression of brain-derived neurotrophic factor protein in frontal cortex and inhibits long-term potentiation in hippocampus.

    PubMed

    Cooke, J D; Grover, L M; Spangler, P R

    2009-09-15

    Antidepressant action may involve stimulation of brain-derived neurotrophic factor (BDNF). BDNF also regulates long-term potentiation (LTP). We hypothesized that the 5-HT and norepinephrine reuptake inhibitor, venlafaxine, would stimulate BDNF expression and alter LTP more effectively than the selective 5-HT reuptake inhibitor, citalopram. To test this, we administered venlafaxine or citalopram to rats for 1 or 3 weeks; control rats received vehicle only. We measured BDNF protein in hippocampal and frontal cortex homogenates, and serum. We assessed LTP in area cornu ammonis region 1 (CA1) of in vitro hippocampal brain slices. We also examined input/output function to determine if basal synaptic transmission in area CA1 was altered. Compared to vehicle control, frontal cortex BDNF protein was significantly greater after three, but not one, weeks of venlafaxine treatment. In contrast, citalopram (1 or 3 weeks) did not stimulate BDNF. The stimulatory effect of venlafaxine treatment on BDNF was superimposed on a general time-dependent decrease in expression which was seen in both vehicle control and citalopram-treated animals. LTP was significantly impaired in slices from venlafaxine-treated rats after both 1 and 3 weeks of treatment, but LTP appeared normal in slices from citalopram-treated and vehicle control rats. The LTP impairment caused by venlafaxine treatment was independent of changes in BDNF: LTP was impaired after only 1 week of treatment, prior to any effect on BDNF, and LTP magnitude was not correlated with BDNF protein concentration. Input/output function was significantly but equally reduced after 3 weeks of citalopram, venlafaxine, or control treatment. Decreased BDNF protein in citalopram and vehicle control animals, and decreased input/output function may be consequences of individual housing of animals, which we used to ensure proper dosing. Venlafaxine stimulation of BDNF and inhibition of LTP may be related to the reported effectiveness of

  19. PKCθ activation in pancreatic acinar cells by gastrointestinal hormones/neurotransmitters and growth factors is needed for stimulation of numerous important cellular signaling cascades

    PubMed Central

    Sancho, Veronica; Berna, Marc J.; Thill, Michelle; Jensen, R. T.

    2011-01-01

    The novel PKCθ isoform is highly expressed in T-cells, brain and skeletal muscle and originally thought to have a restricted distribution. It has been extensively studied in T-cells and shown to be important for apoptosis, T-cell activation and proliferation. Recent studies showed its presence in other tissues and importance in insulin signaling, lung surfactant secretion, intestinal barrier permeability, platelet and mast-cell functions. However, little information is available for PKCθ activation by gastrointestinal(GI) hormones/neurotransmitters and growth factors. In the present study we used rat pancreatic acinar cells to explore their ability to activate PKCθ and the possible interactions with important cellular mediators of their actions. Particular attention was paid to cholecystokinin(CCK), a physiological regulator of pancreatic function and important in pathological processes affecting acinar function, like pancreatitis. PKCθ-protein/mRNA were present in the pancreatic acini, and T538-PKCθ phosphorylation/activation was stimulated only by hormones/neurotransmitters activating phospholipase C. PKCθ was activated in time- and dose-related manner by CCK, mediated 30% by high-affinity CCKA-receptor activation. CCK stimulated PKCθ translocation from cytosol to membrane. PKCθ inhibition (by pseudostrate-inhibitor or dominant negative) inhibited CCK- and TPA-stimulation of PKD, Src, RafC, PYK2, p125FAK and IKKα/β, but not basal/stimulated enzyme secretion. Also CCK- and TPA-induced PKCθ activation produced an increment in PKCθ’s direct association with AKT, RafA, RafC and Lyn. These results show for the first time PKCθ presence in pancreatic acinar cells, its activation by some GI hormones/neurotransmitters and involvement in important cell signaling pathways mediating physiological responses (enzyme secretion, proliferation, apoptosis, cytokine expression, and pathological responses like pancreatitis and cancer growth). PMID:21810446

  20. Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

    PubMed

    Zhang, Z; Harkiss, G D; Hopkins, J; Woodall, C J

    2002-08-01

    Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

  1. Stimulation causing the double effect as in dyspraxia, a universal mechanism of disease, and auto-electrocution, the fatal factor.

    PubMed

    Baggot, M G

    1993-05-01

    Almost every action in the body involves the nervous system which uses electricity for its speed and versatility. Operating and controlling muscular activity is a major function of the nervous system. Muscles are paired so every normal contraction requires a reciprocal relaxation of its doppelganger (Newton said 'Equal and opposite'). Minor failures of co-operation, i.e. neuromuscular dysfunction make common survivable diseases. Multiple and major stimuli can create chaotic conflicts within these couplings and lead to fatalities. Excessive, abnormal, even ordinary stimulation will cause the (irritable?) partner(s) which should relax also to contract. Thus function is impaired or impossible. In the limbs faulty neuromuscular co-ordination is obvious as stiffness, Parkinson's Disease, Erb's Palsy etc. Less evident, it is even more important in the cardiovascular, gastrointestinal, genitourinary and respiratory systems.

  2. Transcription factor Sp1 is necessary for basal calmodulin gene transcription and for its selective stimulation by insulin.

    PubMed

    Solomon, S S; Palazzolo, M R; Takahashi, T; Raghow, R

    1997-11-01

    Insulin positively regulates transcription of rat calmodulin (CaM) I gene and activates the low Km cyclic AMP (cAMP) phosphodiesterase (PDE). To elucidate the mechanism of transcriptional regulation, rat hepatoma (H-411E) cells were transfected with DNA constructs containing the putative CaM promoters coupled to a luciferase reporter and challenged with insulin. Activation of the full length 1835 bp rat CaM I promoter containing all three Sp1 sites or truncated promoters with combinations of one to three of the Sp1 sites was studied in Sp1 deficient Drosophilia SL2 cells and in SL2 cells co-transfected with an Sp1 expression vector and re-challenged with insulin. Our results demonstrate that Sp1 is obligatory for basal activation of the CaM promoter. The maximal insulin stimulation of CaM promoter is elicited only if it contains at least two Sp1 sites.

  3. Activation of Epidermal Growth Factor Receptor Mediates Mucin Production Stimulated by p40, a Lactobacillus rhamnosus GG-derived Protein*

    PubMed Central

    Wang, Lihong; Cao, Hailong; Liu, Liping; Wang, Bangmao; Walker, W. Allan; Acra, Sari A.; Yan, Fang

    2014-01-01

    The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfrwa5 mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury. PMID:24895124

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

    PubMed Central

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

    2015-01-01

    Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. PMID:26229149

  5. Attenuated Listeria monocytogenes Vectors Overcome Suppressive Plasma Factors During HIV Infection to Stimulate Myeloid Dendritic Cells to Promote Adaptive Immunity and Reactivation of Latent Virus

    PubMed Central

    Miller, Elizabeth A.; Spadaccia, Meredith R.; Norton, Thomas; Demmler, Morgan; Gopal, Ramya; O'Brien, Meagan; Landau, Nathaniel; Dubensky, Thomas W.; Lauer, Peter; Brockstedt, Dirk G.

    2015-01-01

    Abstract HIV-1 infection is characterized by myeloid dendritic cell (DC) dysfunction, which blunts the responsiveness to vaccine adjuvants. We previously showed that nonviral factors in HIV-seropositive plasma are partially responsible for mediating this immune suppression. In this study we investigated recombinant Listeria monocytogenes (Lm) vectors, which naturally infect and potently activate DCs from seronegative donors, as a means to overcome DC dysfunction associated with HIV infection. Monocyte-derived DCs were cocultured with plasma from HIV-infected donors (HIV-moDCs) to induce a dysregulated state and infected with an attenuated, nonreplicative vaccine strain of Lm expressing full length clade B consensus gag (KBMA Lm-gag). Lm infection stimulated cytokine secretion [interleukin (IL)-12p70, tumor necrosis factor (TNF)-α, and IL-6] and Th-1 skewing of allogeneic naive CD4 T cells by HIV-moDCs, in contrast to the suppressive effects observed by HIV plasma on moDCs on toll-like receptor ligand stimulation. Upon coculture of “killed” but metabolically active (KBMA) Lm-gag-infected moDCs from HIV-infected donors with autologous cells, expansion of polyfunctional, gag-specific CD8+ T cells was observed. Reactivation of latent proviruses by moDCs following Lm infection was also observed in models of HIV latency in a TNF-α-dependent manner. These findings reveal the unique ability of Lm vectors to contend with dysregulation of HIV-moDCs, while simultaneously possessing the capacity to activate latent virus. Concurrent stimulation of innate and adaptive immunity and disruption of latency may be an approach to reduce the pool of latently infected cells during HIV infection. Further study of Lm vectors as part of therapeutic vaccination and eradication strategies may advance this evolving field. PMID:25376024

  6. Single-chain bifunctional vascular endothelial growth factor (VEGF)-follicle-stimulating hormone (FSH)-C-terminal peptide (CTP) is superior to the combination therapy of recombinant VEGF plus FSH-CTP in stimulating angiogenesis during ovarian folliculogenesis.

    PubMed

    Trousdale, Rhonda K; Pollak, Susan V; Klein, Jeffrey; Lobel, Leslie; Funahashi, Yasuhiro; Feirt, Nikki; Lustbader, Joyce W

    2007-03-01

    Infertility technologies often employ exogenous gonadotropin therapy to increase antral follicle production. In an effort to enhance ovarian response, several long-acting FSH therapies have been developed including an FSH-C-terminal peptide (CTP), where the FSH subunits are linked by the CTP moiety from human chorionic gonadotropin, which is responsible for the increased half-life of human chorionic gonadotropin. We found that administration of FSH-CTP for ovarian hyperstimulation in rats blunted ovarian follicle vascular development. In women, reduced ovarian vasculature has been associated with lower pregnancy rates. We were interested in determining whether vascular endothelial growth factor (VEGF) therapy could enhance ovarian angiogenesis in FSH-CTP-treated rats. Coadministration of systemic FSH-CTP plus recombinant VEGF was compared with treatment with a novel, single-chain bifunctional VEGF-FSH-CTP (VFC) analog. For VFC, the FSH portion targets the protein to the ovary and stimulates follicle growth, whereas VEGF enhances local vascular development. Both in vitro and in vivo studies confirm the dual FSH and VEGF action of the VFC protein. Evaluation of ovarian follicle development demonstrates that administration of combination therapy using VEGF and FSH-CTP failed to increase follicle vasculature above levels seen with FSH-CTP monotherapy. However, treatment with VFC significantly increased follicle vascular development while concurrently increasing the number of large antral follicles produced. In conclusion, we report the production and characterization of a long-acting, bifunctional VEGF-FSH-CTP protein that is superior to combination therapy for enhancing VEGF activity in the ovary and stimulating follicular angiogenesis in rats.

  7. Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

    PubMed

    Zhao, Limin; Sullivan, Michelle N; Chase, Marlee; Gonzales, Albert L; Earley, Scott

    2014-06-01

    Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

  8. Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression.

    PubMed Central

    Donohue, P J; Feng, S L; Alberts, G F; Guo, Y; Peifley, K A; Hsu, D K; Winkles, J A

    1996-01-01

    Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair. PMID:8870641

  9. Krüppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans

    PubMed Central

    Czakai, Kristin; Leonhardt, Ines; Dix, Andreas; Bonin, Michael; Linde, Joerg; Einsele, Hermann; Kurzai, Oliver; Loeffler, Jürgen

    2016-01-01

    Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation. PMID:27346433

  10. Basic fibroblast growth factor promotes stem Leydig cell development and inhibits LH-stimulated androgen production by regulating microRNA expression.

    PubMed

    Liu, Hui; Yang, Yan; Zhang, Lei; Liang, Rui; Ge, Ren-Shan; Zhang, Yufei; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2014-10-01

    Leydig cells are the primary source of testosterone in the testes, and their steroidogenic function is strictly controlled by the hypothalamus-pituitary-gonad axis. Emerging evidence has indicated that fibroblast growth factors play a role in regulating stem Leydig cell development and steroidogenesis, but little is known about the regulatory mechanism. Using a seminiferous tubule culture system, we demonstrated that basic fibroblast growth factor (bFGF) can promote stem Leydig cell proliferation and commitment toward differentiation in testosterone-producing Leydig cells. However, these promoting effects decreased with an increase in the bFGF dose. Previous studies have reported that bFGF inhibits luteinizing hormone (LH)-stimulated androgen production by downregulating the mRNA expression of steroidogenic genes in immature Leydig cells. However, the expression levels of 677 microRNAs did not change significantly during the LH-mediated process of testosterone synthesis. Five microRNAs (miR-29a, -29c, -142-3p, -451 and -335) were identified, and their expression in immature Leydig cells was regulated simultaneously by bFGF and LH. These results suggested that the inhibition of LH-stimulated androgen production may be modulated by a change in bFGF-mediated microRNA expression, which further impacts the signaling pathway of testosterone biosynthesis and steroidogenic gene expression.

  11. Proximal tubule-derived Colony Stimulating Factor-1 mediates polarization of renal macrophages and dendritic cells, and recovery in acute kidney injury

    PubMed Central

    Wang, Yinqiu; Chang, Jian; Yao, Bing; Niu, Aolei; Kelly, Emily; Breeggemann, Matthew C.; Abboud Werner, Sherry L.; Harris, Raymond C.; Zhang, Ming-Zhi

    2015-01-01

    Infiltrating cells play an important role in both the development of and recovery from acute kidney injury (AKI). Macrophages and renal dendritic cells are of particular interest because they can exhibit distinctly different functional phenotypes, broadly characterized as proinflammatory (M1) or tissue reparative (M2). Resident renal macrophages and dendritic cells participate in recovery from AKI in response to either ischemia/reperfusion or a model of selective proximal tubule injury induced by diphtheria toxin-induced apoptosis in transgenic mice expressing the human diphtheria toxin receptor on proximal tubule cells. Colony Stimulating Factor-1 (CSF-1) is an important factor mediating the recovery from AKI, and CSF-1 can stimulate macrophage and dendritic cell proliferation and polarization during the recovery phase of AKI. The kidney, and specifically the proximal tubule, is a major source of intrarenal CSF-1 production in response to AKI. We induced selective deletion of proximal tubule CSF-1 to determine its role in expansion and proliferation of renal macrophages and dendritic cells and in recovery from AKI. In both models of AKI, there was decreased M2 polarization, delayed functional and structural recovery and increased tubulointerstitial fibrosis. Thus, intrarenal CSF-1 is an important mediator of macrophage/dendritic cell polarization and recovery from AKI. PMID:26422503

  12. Identification of a LPS-induced TNF-α factor (LITAF) from mollusk Solen grandis and its expression pattern towards PAMPs stimulation.

    PubMed

    Yang, Dinglong; Wei, Xiumei; Yang, Jianmin; Yang, Jialong; Xu, Jie; Fang, Jinghui; Wang, Sheng; Liu, Xiangquan

    2013-10-01

    Lipopolysaccharide-induced TNF-α factor (LITAF) is one of the most important transcription factors mediating TNF-α transcription. In the present study, a LITAF gene (designated as SgLITAF) was identified from razor clams Solen grandis. The full-length cDNA of SgLITAF was of 1476 bp, encoding a polypeptide of 130 amino acids showed high similarity to other known LITAFs. SgLITAF encoded a LITAF domain and the Zn(2+)-binding motifs in the domain were well conserved. The mRNA transcripts of SgLITAF were detected in all tested tissues of healthy razor clams, including mantle, gill, gonad, hemocytes, muscle and hepatopancreas, and with the highest expression level in hepatopancreas. The expression level of SgLITAF in hemocytes was significantly up-regulated (P < 0.01) after razor clams were stimulated by LPS or β-1, 3-glucan, but no obvious fluctuation of SgLITAF mRNA expression was observed after PGN stimulation. All the results indicated that there might be a LITAF-regulated TNF-α signaling pathway existing in S. grandis, which involved in the immune response not only against gram-negative bacteria but also towards fungi.

  13. Macrophage production during murine listeriosis: colony-stimulating factor 1 (CSF-1) and CSF-1-binding cells in genetically resistant and susceptible mice.

    PubMed Central

    Cheers, C; Stanley, E R

    1988-01-01

    The concentration of the macrophage-specific colony-stimulating factor (CSF-1) and the numbers of bone marrow and spleen cells with specific receptors for that factor have been investigated in a number of mouse strains under normal conditions and after infection with the facultative intracellular bacterium Listeria monocytogenes. The CSF-1 concentration in serum and tissue was markedly elevated in infected mice, the degree of stimulation reflecting the dose of L. monocytogenes. The CSF-1 titer did not correlate with genetic resistance or susceptibility of the mice to L. monocytogenes. In contrast to the effect of lipopolysaccharide, Listeria infection was able to increase the level of CSF-1 in the lipopolysaccharide nonresponder strain C3H/HeJ. In line with earlier findings on colony-forming cells, cells bearing receptors for CSF-1 in uninfected susceptible BALB/cJ mice were only half those in resistant C57BL/6J mice. After infection the majority of these cells disappeared from the bone marrow and spleen cells of both resistant and susceptible mice. The number of CSF-1 receptor-bearing cells in the normal bone marrow may determine the degree of resistance to L. monocytogenes. PMID:3262588

  14. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

    PubMed Central

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  15. A Bilayer Construct Controls Adipose-Derived Stem Cell Differentiation into Endothelial Cells and Pericytes without Growth Factor Stimulation

    DTIC Science & Technology

    2011-01-01

    cells and pericytes. Gene expression analysis revealed strong upregulation of endothelial markers, CD31, and von Willebrand factor, up to day 11 in...phenotype, but a subset of the ASC expressed pericyte markers. The NG2 gene was upregulated over 11 days with corresponding evidence for the cell...surface marker. Platelet- derived growth factor receptor beta gene expression decreased as the multipotent ASC differentiated up to day 7. Increased

  16. 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.

  17. Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum. Mediation by adenosine 3':5'-monophosphate.

    PubMed

    Drezner, M K; Eisenbarth, G S; Neelon, F A; Lebovitz, H E

    1975-02-13

    The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of alpha-amino [1-14C] isobutyrate or [1-14C] cycloleucine than control cartilages incubated in medium alone. Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate amino-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r=0.977). Theophylline and prostaglandin E1, WHICH RAISE CARTILAGE CYCLIC AMP also increase amino-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyryl cyclic AMP and n6, 02'-dibutyryl cyclic AMP increase cartilage amino-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.

  18. Activation of the human. beta. sub 2 -interferon/hepatocyte-stimulating factor/interleukin 6 promoter by cytokines, viruses, and second messenger agonists

    SciTech Connect

    Ray, A.; Tatter, S.B.; May, L.T.; Sehgal, P.B. )

    1988-09-01

    The hallmark of {beta}{sub 2}-interferon (IFN-{beta}{sub 2})/hepatocyte-stimulating factor/interleukin 6 gene expression is its inducibility in different types of human cells (fibroblasts, monocytes, epithelial cells, and endothelial cells) by different stimuli, which include cytokines such as tumor necrosis factor, interleukin 1 (IL-1) and platelet-derived growth factor, different viruses, and bacterial products such as endotoxin. The activation by cytokines, viruses, and second messenger agonists of the IFN-{beta}{sub 2} promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was studied after transfection into HeLa cells. A chimeric gene containing IFN-{beta}{sub 2} DNA from -1180 to +13 linked to the CAT gene was inducible {approx}10-fold by phorbol 12-myristate 13-acetate (PMA), followed, in decreasing order, by pseudorabies and Sendai viruses; serum; the cytokines tumor necrosis factor, IL-1, and epidermal growth factor; the cAMP agonists BrcAMP and forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine; poly(I){center dot}poly(C); 1,2-diacylglycerol and the calcium ionophore A23187. The region between -225 and -113 in IFN-{beta}{sub 2}, which contains DNA motifs similar to the regulatory elements in the human c-fos gene, appears to contain the major cis-acting regulatory elements responsible for the activation of the IFN-{beta}{sub 2} promoter by several different cytokines, viruses, and second messenger agonists.

  19. Differential expression of HIV-1 interfering factors in monocyte-derived macrophages stimulated with polarizing cytokines or interferons

    NASA Astrophysics Data System (ADS)

    Jiménez, Viviana Cobos; Booiman, Thijs; de Taeye, Steven W.; van Dort, Karel A.; Rits, Maarten A. N.; Hamann, Jörg; Kootstra, Neeltje A.

    2012-10-01

    HIV-1 replication in macrophages can be regulated by cytokines and infection is restricted in macrophages activated by type I interferons and polarizing cytokines. Here, we observed that the expression levels of the cellular factors Trim5α, CypA, APOBEC3G, SAMHD-1, Trim22, tetherin and TREX-1, and the anti-HIV miRNAs miR-28, miR-150, miR-223 and miR-382 was upregulated by IFN-α and IFN-β in macrophages, which may account for the inhibiting effect on viral replication and the antiviral state of these cells. Expression of these factors was also increased by IFN-γ +/- TNF-α, albeit to a lesser extent; yet, HIV-1 replication in these cells was not restricted at the level of proviral synthesis, indicating that these cellular factors only partially contribute to the observed restriction. IL-4, IL-10 or IL-32 polarization did not affect the expression of cellular factors and miRNAs, suggesting only a limited role for these cellular factors in restricting HIV-1 replication in macrophages.

  20. Sunlight Triggers Cutaneous Lupus through a Colony Stimulating Factor-1 (CSF-1) Dependent Mechanism in MRL-Faslpr mice

    PubMed Central

    Menke, Julia; Hsu, Mei-Yu; Byrne, Katelyn T.; Lucas, Julie A.; Rabacal, Whitney A.; Croker, Byron P.; Zong, Xiao-Hua; Stanley, E. Richard; Kelley, Vicki R.

    2008-01-01

    Sunlight (UVB) triggers cutaneous (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø) -mediated mechanism in MRL-Faslpr mice. By constructing mutant MRL-Faslpr strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex-vivo gene transfer to deliver CSF-1 intra-dermally, we determined that CSF-1 induces CLE in lupus-susceptible, MRL-Faslpr mice, but not in lupus-resistant, BALB/c mice. Notably, UVB incites an increase in Mø, apoptosis in the skin and CLE in MRL-Faslpr, but not in CSF-1-deficient MRL-Faslpr mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Mø that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Faslpr, but not lupus-resistant BALB/c mice. Taken together, we envision CSF-1 as the “match” and lupus-susceptibility as the “tinder” leading to CLE. PMID:18981160

  1. Neuroprotective and Angiogenic Effects of Bone Marrow Transplantation Combined With Granulocyte Colony-Stimulating Factor in a Mouse Model of Amyotrophic Lateral Sclerosis

    PubMed Central

    Ohta, Yasuyuki; Nagai, Makiko; Miyazaki, Kazunori; Tanaka, Nobuhito; Kawai, Hiromi; Mimoto, Takafumi; Morimoto, Nobutoshi; Kurata, Tomoko; Ikeda, Yoshio; Matsuura, Tohru; Abe, Koji

    2011-01-01

    Bone marrow (BM) cells from amyotrophic lateral sclerosis (ALS) patients show significantly reduced expression of several neurotrophic factors. Monotherapy with either wild-type (WT) BM transplantation (BMT) or granulocyte colony-stimulating factor (GCSF) has only a small clinical therapeutic effect in an ALS mouse model, due to the phenomenon of neuroprotection. In this study, we investigated the clinical benefits of combination therapy using BMT with WT BM cells, plus GCSF after disease onset in ALS mice [transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation]. Combined treatment with BMT and GCSF delayed disease progression and prolonged the survival of G93A mice, whereas BMT or GCSF treatment alone did not. Histological study of the ventral horns of lumbar cords from G93A mice treated with BMT and GCSF showed a reduction in motor neuron loss coupled with induced neuronal precursor cell proliferation, increased expression of neurotrophic factors (glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, vascular endothelial growth factor and angiogenin), and neovascularization compared with controls (vehicle only). Compared with G93A microglial cells, most BM-derived WT cells differentiated into microglial cells and strongly expressed neurotrophic factors, combined BMT and GCSF treatment led to the replacement of G93A microglial cells with BM-derived WT cells. These results indicate combined treatment with BMT and GCSF has potential neuroprotective and angiogenic effects in ALS mice, induced by the replacement of G93A microglial cells with BM-derived WT cells. Furthermore, this is the first report showing the effects of combined BMT and GCSF treatment on blood vessels in ALS. PMID:26998403

  2. Tumor Necrosis Factor-alpha Stimulates the Overproduction of Intestinal Apolipoprotein B48-containing Very Low Density Lipoproproteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tumor necrosis factor-alpha(a)(TNFa), a proinflammatory cytokine, is involved in obesity-associated pathologies including type 2 diabetes and atherosclerosis. TNFa enhanced postprandial apoB48-VLDL1 overproduction by about 89% compared with the control after 90 min olive oil loading; TNFa did not si...

  3. Expression of murine and human granulocyte-macrophage colony-stimulating factors in S. cerevisiae: mutagenesis of the potential glycosylation sites.

    PubMed Central

    Miyajima, A; Otsu, K; Schreurs, J; Bond, M W; Abrams, J S; Arai, K

    1986-01-01

    Murine (m) and human (h) granulocyte--macrophage colony-stimulating factors (GM-CSF) have been expressed in large quantities in Saccharomyces cerevisiae using a secretion vector containing the promoter and leader sequences of the mating pheromone alpha-factor. Functionally active mGM-CSF was identified by a proliferation assay with a factor-dependent cell line and by a granulocyte--macrophage colony formation assay using bone marrow cells. The activity of hGM-CSF was confirmed by stimulation of granulocyte--macrophage colony formation using human cord blood cells. Murine GM-CSF with various apparent mol. wts (13, 18, 24, 34 and 40 kd, as well as a smear of higher mol. wts) was detected in yeast culture medium by protein blotting using a rat monoclonal antibody specific for the mGM-CSF N-terminal region peptide. Protein blotting using a rat monoclonal antibody specific for the hGM-CSF N-terminal region demonstrated that a 15.6-kd and higher mol. wt heterogeneous species were secreted. Mutations introduced at each of the two potential N-linked glycosylation sites in mGM-CSF showed that the 13-kd protein is not glycosylated and the major 18-kd protein is mainly glycosylated at the more C-terminal site, whereas the heterogeneous higher mol. wt species were not affected by the mutations. The N-terminal amino acid of the 13-kd protein was shown to be Ser which was four amino acids in the C-terminal direction from the fusion point. Images Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:3525148

  4. Oestrogen requires the insulin-like growth factor-I receptor for stimulation of prolactin synthesis via mitogen-activated protein kinase.

    PubMed

    Arroba, A I; Frago, L M; Argente, J; Chowen, J A

    2005-02-01

    Sex steroids and growth factors interact at the intracellular level in a variety of tissues to control numerous physiological functions. Oestrogen is known to stimulate prolactin synthesis and secretion, but the effect of insulin-like growth factor (IGF)-I is less clear. We used GH3 cells, a somatolactotroph cell line, to study the interaction of 17beta-oestradiol (E(2)) and IGF-I on prolactin protein levels and the intracellular mechanisms involved. Cell cultures were treated with E(2) (10 nM) and/or IGF-I (10 ng/ml) for 8 h. The real-time reverse transcriptase-polymerase chain reaction, Western blot and enzyme-immunoassay were used to determine changes in prolactin mRNA and protein levels. At this time-point, there were no significant changes in cell number, prolactin mRNA expression, or the amount of secreted prolactin. However, E(2) increased intracellular prolactin concentrations. IGF-I alone had no effect, but blocked the stimulatory effect of E(2). MAPK (ERK1/2) activation, as determined by Western blot analysis, increased with both E(2) and IGF-I, but not with the combination of these factors. The MAPK inhibitor PD98059 blocked the ability of E(2) to increase intracellular prolactin concentrations. Similarly, the IGF-I receptor antagonist, JB1, blocked the effect of E(2) on prolactin synthesis and MAPK activation, as did the oestrogen receptor antagonist ICI182 780. These results suggest that, to stimulate prolactin synthesis, E(2) activates the MAPK cascade and that this requires the presence of both oestrogen and IGF-I receptors.

  5. Endogenous factors enhance HIV infection of tissue naive CD4 T cells by stimulating high molecular mass APOBEC3G complex formation.

    PubMed

    Kreisberg, Jason F; Yonemoto, Wes; Greene, Warner C

    2006-04-17

    Human immunodeficiency virus (HIV) can infect resting CD4 T cells residing in lymphoid tissues but not those circulating in peripheral blood. The molecular mechanisms producing this difference remain unknown. We explored the potential role of the tissue microenvironment and its influence on the action of the antiviral factor APOBEC3G (A3G) in regulating permissivity to HIV infection. We found that endogenous IL-2 and -15 play a key role in rendering resident naive CD4 T cells susceptible to HIV infection. Infection of memory CD4 T cells also requires endogenous soluble factors, but not IL-2 or -15. A3G is found in a high molecular mass complex in HIV infection-permissive, tissue-resident naive CD4 T cells but resides in a low molecular mass form in nonpermissive, blood-derived naive CD4 T cells. Upon treatment with endogenous soluble factors, these cells become permissive for HIV infection, as low molecular mass A3G is induced to assemble into high molecular mass complexes. These findings suggest that in lymphoid tissues, endogenous soluble factors, likely including IL-2 and -15 and others, stimulate the formation of high molecular mass A3G complexes in tissue-resident naive CD4 T cells, thereby relieving the potent postentry restriction block for HIV infection conferred by low molecular mass A3G.

  6. Thyroid Stimulating Hormone Receptor (TSHR) Intron 1 Variants Are Major Risk Factors for Graves' Disease in Three European Caucasian Cohorts

    PubMed Central

    Jurecka-Lubieniecka, Beata; Franaszczyk, Maria; Kula, Dorota; Krajewski, Paweł; Karamat, Muhammad A.; Simmonds, Matthew J.; Franklyn, Jayne A.; Gough, Stephen C. L.; Jarząb, Barbara; Bednarczuk, Tomasz

    2010-01-01

    Background The thyroid stimulating hormone receptor (TSHR) gene is an established susceptibility locus for Graves' disease (GD), with recent studies refining association to two single nucleotide polymorphisms (SNPs), rs179247 and rs12101255, within TSHR intron 1. Methodology and Principal Findings We aimed to validate association of rs179247 and rs12101255 in Polish and UK Caucasian GD case-control subjects, determine the mode of inheritance and to see if association correlates with specific GD clinical manifestations. We investigated three case-control populations; 558 GD patients and 520 controls from Warsaw, Poland, 196 GD patients and 198 controls from Gliwice, Poland and 2504 GD patients from the UK National collection and 2784 controls from the 1958 British Birth cohort. Both rs179247 (P = 1.2×10−2–6.2×10−15, OR = 1.38–1.45) and rs12101255 (P = 1.0×10−4–3.68×10−21, OR = 1.47–1.87) exhibited strong association with GD in all three cohorts. Logistic regression suggested association of rs179247 is secondary to rs12101255 in all cohorts. Inheritance modeling suggested a co-dominant mode of inheritance in all cohorts. Genotype-phenotype correlations provided no clear evidence of association with any specific clinical characteristics. Conclusions We have validated association of TSHR intron 1 SNPs with GD in three independent European cohorts and have demonstrated that the aetiological variant within the TSHR is likely to be in strong linkage disequilibrium with rs12101255. Fine mapping is now required to determine the exact location of the aetiological DNA variants within the TSHR. PMID:21124799

  7. A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity.

    PubMed

    Pham, X H; Reddy, M K; Ehtesham, N Z; Matta, B; Tuteja, N

    2000-10-01

    DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.

  8. Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene.

    PubMed

    Vetter, M L; Martin-Zanca, D; Parada, L F; Bishop, J M; Kaplan, D R

    1991-07-01

    Nerve growth factor (NGF) promotes the survival and differentiation of specific populations of neurons. The molecular mechanisms by which cells respond to NGF are poorly understood, but two clues have emerged recently. First, NGF rapidly stimulates tyrosine phosphorylation of several unidentified proteins in the NGF-responsive pheochromocytoma cell line PC12 [Maher, P. (1988) Proc. Natl. Acad. Sci. USA 85, 6788-6791]. Second, the protein-tyrosine kinase encoded by the protooncogene trk (p140trk), a member of the receptor class of tyrosine kinases, becomes activated and phosphorylated on tyrosine after NGF treatment of PC12 cells [Kaplan, D. R., Martin-Zanca, D. & Parada, L. F. (1991) Nature (London) 350, 158-160]. We now report that NGF rapidly induces tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), and we present evidence that the responsible tyrosine kinase is either p140trk or a closely associated protein. Treatment of responsive cells with NGF elicited phosphorylation of PLC-gamma 1 on tyrosine and serine. PLC-gamma 1 immunoprecipitated from NGF-stimulated cells was phosphorylated in vitro by coprecipitating protein kinase activity, and the phosphorylations occurred principally on tyrosine. The responsible kinase could be depleted from cellular lysates by antibodies specific for p140trk. This procedure also depleted a 140-kDa protein that normally coprecipitated with PLC-gamma 1 and became phosphorylated on tyrosine in vivo in response to NGF. Analysis of tryptic peptides from PLC-gamma 1 indicated that the residues phosphorylated in vitro by p140trk-associated kinase activity were largely congruent with those phosphorylated in vivo after NGF treatment. Our findings identify PLC-gamma 1 as a likely substrate for the trk-encoded tyrosine kinase, and they provide a link between NGF-dependent activation of p140trk and the stimulation of intracellular second messenger pathways.

  9. Effects of brain-derived neurotrophic factor (BDNF) and electrical stimulation on survival and function of cochlear spiral ganglion neurons in deafened, developing cats.

    PubMed

    Leake, Patricia A; Stakhovskaya, Olga; Hetherington, Alexander; Rebscher, Stephen J; Bonham, Ben

    2013-04-01

    Both neurotrophic support and neural activity are required for normal postnatal development and survival of cochlear spiral ganglion (SG) neurons. Previous studies in neonatally deafened cats demonstrated that electrical stimulation (ES) from a cochlear implant can promote improved SG survival but does not completely prevent progressive neural degeneration. Neurotrophic agents combined with an implant may further improve neural survival. Short-term studies in rodents have shown that brain-derived neurotrophic factor (BDNF) promotes SG survival after deafness and may be additive to trophic effects of stimulation. Our recent study in neonatally deafened cats provided the first evidence of BDNF neurotrophic effects in the developing auditory system over a prolonged duration Leake et al. (J Comp Neurol 519:1526-1545, 2011). Ten weeks of intracochlear BDNF infusion starting at 4 weeks of age elicited significant improvement in SG survival and larger soma size compared to contralateral. In the present study, the same deafening and BDNF infusion procedures were combined with several months of ES from an implant. After combined BDNF + ES, a highly significant increase in SG numerical density (>50 % improvement re: contralateral) was observed, which was significantly greater than the neurotrophic effect seen with ES-only over comparable durations. Combined BDNF + ES also resulted in a higher density of myelinated radial nerve fibers within the osseous spiral lamina. However, substantial ectopic and disorganized sprouting of these fibers into the scala tympani also occurred, which may be deleterious to implant function. EABR thresholds improved (re: initial thresholds at time of implantation) on the chronically stimulated channels of the implant. Terminal electrophysiological studies recording in the inferior colliculus (IC) revealed that the basic cochleotopic organization was intact in the midbrain in all studied groups. In deafened controls or after ES-only, lower IC

  10. Roles of tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor, platelet-activating factor, and arachidonic acid metabolites in interleukin-1-induced resistance to infection in neutropenic mice.

    PubMed Central

    Vogels, M T; Hermsen, C C; Huys, H L; Eling, W M; van der Meer, J W

    1994-01-01

    Treatment with a single low dose (80 to 800 ng) of interleukin-1 (IL-1) 24 h before a lethal bacterial challenge in granulocytopenic and in normal mice enhances nonspecific resistance. The mechanism behind this protection has only partially been elucidated. Since IL-1 induces production of tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-activating factor (PAF), and arachidonic acid metabolites, we investigated the potential role of these substances in IL-1-induced protection. Low doses of murine TNF-alpha but not of human TNF-alpha enhanced survival, suggesting an effect via the type II TNF receptor rather than the type I TNF receptor, which has little species specificity. In line with this TNF-alpha-induced protection from infection, pretreatment with a low dose of a rat anti-murine TNF-alpha monoclonal antibody tended to inhibit IL-1-induced protection, suggesting a role of TNF-alpha as a mediator of IL-1-induced enhanced resistance to infection. Pretreatment with higher doses of anti-TNF-alpha, however, showed a dose-related protective effect per se, which could be further enhanced by a suboptimal dose of IL-1. A combination of optimal doses of anti-TNF-alpha and IL-1 produced an increase in survival similar to that produced by separate pretreatments. This lack of further enhancement of survival by combined optimal pretreatments suggests a similar mechanism of protection, most likely attenuation of deleterious effects of overproduced proinflammatory cytokines like TNF-alpha during lethal infection. Pretreatment with different doses of GM-CSF before a lethal Pseudomonas aeruginosa challenge in neutropenic mice did not enhance survival. Different doses of WEB 2170, a selective PAF receptor antagonist, of MK-886, a selective inhibitor of leukotriene biosynthesis, or of several cyclooxygenase inhibitors did not reduce the protective effect of IL-1 pretreatment. We conclude that IL-1-induced nonspecific

  11. Mobilization and collection of CD34+ cells for autologous transplantation of peripheral blood hematopoietic progenitor cells in children: analysis of two different granulocyte-colony stimulating factor doses

    PubMed Central

    Eid, Kátia Aparecida de Brito; Miranda, Eliana Cristina Martins; Aguiar, Simone dos Santos

    2015-01-01

    Introduction The use of peripheral hematopoietic progenitor cells (HPCs) is the cell choice in autologous transplantation. The classic dose of granulocyte-colony stimulating factor (G-CSF) for mobilization is a single daily dose of 10 μg/kg of patient body weight. There is a theory that higher doses of granulocyte-colony stimulating factor applied twice daily could increase the number of CD34+ cells collected in fewer leukapheresis procedures. Objective The aim of this study was to compare a fractionated dose of 15 μg G-CSF/kg of body weight and the conventional dose of granulocyte-colony stimulating factor in respect to the number of leukapheresis procedures required to achieve a minimum collection of 3 × 106 CD34+ cells/kg body weight. Methods Patients were divided into two groups: Group 10 – patients who received a single daily dose of 10 μg G-CSF/kg body weight and Group 15 – patients who received a fractioned dose of 15 μg G-CSF/kg body weight daily. The leukapheresis procedure was carried out in an automated cell separator. The autologous transplantation was carried out when a minimum number of 3 × 106 CD34+ cells/kg body weight was achieved. Results Group 10 comprised 39 patients and Group 15 comprised 26 patients. A total of 146 apheresis procedures were performed: 110 (75.3%) for Group 10 and 36 (24.7%) for Group 15. For Group 10, a median of three (range: 1–7) leukapheresis procedures and a mean of 8.89 × 106 CD34+ cells/kg body weight (±9.59) were collected whereas for Group 15 the corresponding values were one (range: 1–3) and 5.29 × 106 cells/kg body weight (±4.95). A statistically significant difference was found in relation to the number of apheresis procedures (p-value <0.0001). Conclusions To collect a minimum target of 3 × 106 CD34+ cells/kg body weight, the administration of a fractionated dose of 15 μg G-CSF/kg body weight significantly decreased the number of leukapheresis procedures performed. PMID:26041417

  12. Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions.

    PubMed

    Kidaj, Dominika; Wielbo, Jerzy; Skorupska, Anna

    2012-03-20

    Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.

  13. Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host.

    PubMed

    Pila, Emmanuel A; Gordy, Michelle A; Phillips, Valerie K; Kabore, Alethe L; Rudko, Sydney P; Hanington, Patrick C

    2016-05-10

    Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

  14. Abnormal Responses of Myeloid Progenitor Cells to GM-CSF (Granulocyte-Macrophage-Colony-Stimulating Factor) in Human Cyclic Neutropenia

    DTIC Science & Technology

    1989-04-01

    Continut on reverse if necessar and jentify by bi).nrbe,; FIELD GROUP SUBG ROUP Ne utropen ia, granu Iocyte-macrop ae ofnyS imu £ating factor (GM...be _3 eosinophilic were also detected occasionally (< 5% of total col- , onies) among CFU-GM grown with > 1.0 nmol/liter rhGM- CSF. Whereas the clonal

  15. Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host

    PubMed Central

    Pila, Emmanuel A.; Gordy, Michelle A.; Phillips, Valerie K.; Kabore, Alethe L.; Rudko, Sydney P.; Hanington, Patrick C.

    2016-01-01

    Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni. Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection. PMID:27114544

  16. 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.

  17. Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes.

    PubMed Central

    Alvaro-Gracia, J M; Zvaifler, N J; Firestein, G S

    1990-01-01

    The effects of a broad array of cytokines, individually and in combination, were determined on separate functions (proliferation, collagenase production, and granulocyte macrophage colony-stimulating factor [GM-CSF] production) and phenotype (expression of class II MHC antigens) of cultured fibroblast-like RA synoviocytes. The following recombinant cytokines were used: IL-1 beta, IL-2, IL-3, IL-4, IFN-gamma, tumor necrosis factor (TNF)-alpha, GM-CSF, and macrophage colony-stimulating factor (M-CSF). Only IFN-gamma induced HLA-DR (but not HLA-DQ) expression. TNF-alpha inhibited IFN-gamma-mediated HLA-DR expression (46.7 +/- 4.1% inhibition) and HLA-DR mRNA accumulation. This inhibitory effect was also observed in osteoarthritis synoviocytes. Only TNF-alpha and IL-1 increased synoviocyte proliferation (stimulation index 3.60 +/- 1.03 and 2.31 +/- 0.46, respectively). IFN-gamma (but none of the other cytokines) inhibited TNF-alpha-induced proliferation (70 +/- 14% inhibition) without affecting the activity of IL-1. Only IL-1 beta and TNF-alpha induced collagenase production (from less than 0.10 U/ml to 1.10 +/- 0.15 and 0.72 +/- 0.24, respectively). IFN-gamma decreased TNF-alpha-mediated collagenase production (69