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Sample records for factor zapb stimulates

  1. 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

  2. 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.

  3. Granulocyte colony stimulating factor in neutropenic patients with infective endocarditis

    PubMed Central

    Borgbjerg, B. M.; Hovgaard, D.; Laursen, J. B.; Aldershvile, J.

    1998-01-01

    A well known complication in the treatment of infectious endocarditis is development of neutropenia caused by treatment with antibiotics in high concentrations over long periods. Neutropenia often necessitates discontinuation of antibiotic treatment. Three patients with infectious endocarditis who developed neutropenia are reported. The patients were treated with granulocyte colony stimulating factor (G-CSF), a haematopoietic growth factor that stimulates neutrophils. G-CSF induced an immediate increase in white blood cell count, primarily neutrophils. G-CSF may be effective in ameliorating neutropenia in patients who receive antibiotics for treatment of infectious endocarditis.

 Keywords: granulocyte colony stimulating factor;  neutropenia;  endocarditis PMID:9505928

  4. Factors stimulating migration of holotrich protozoa into the rumen.

    PubMed

    Murphy, M R; Drone, P E; Woodford, S T

    1985-05-01

    The effects of feeding and various reticular infusions on ruminal holotrich concentrations were studied in an attempt to identify possible factors stimulating their migration into the rumen. It was concluded that glucose entering the reticulo-rumen shortly after feeding could stimulate migration of holotrich protozoa.

  5. 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

  6. 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

  7. 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

  8. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal

    PubMed Central

    Oatley, Jon M.; Oatley, Melissa J.; Avarbock, Mary R.; Tobias, John W.; Brinster, Ralph L.

    2009-01-01

    Summary Self-renewal and differentiation of spermatogonial stem cells (SSCs) provide the foundation for testis homeostasis, yet mechanisms that control their functions in mammals are poorly defined. We used microarray transcript profiling to identify specific genes whose expressions are augmented in the SSC-enriched Thy1+ germ cell fraction of mouse pup testes. Comparisons of gene expression in the Thy1+ germ cell fraction with the Thy1-depleted testis cell population identified 202 genes that are expressed 10-fold or higher in Thy1+ cells. This database provided a mining tool to investigate specific characteristics of SSCs and identify novel mechanisms that potentially influence their functions. These analyses revealed that colony stimulating factor 1 receptor (Csf1r) gene expression is enriched in Thy1+ germ cells. Addition of recombinant colony stimulating factor 1 (Csf1), the specific ligand for Csf1r, to culture media significantly enhanced the self-renewal of SSCs in heterogeneous Thy1+ spermatogonial cultures over a 63-day period without affecting total germ cell expansion. In vivo, expression of Csf1 in both pre-pubertal and adult testes was localized to clusters of Leydig cells and select peritubular myoid cells. Collectively, these results identify Csf1 as an extrinsic stimulator of SSC self-renewal and implicate Leydig and myoid cells as contributors of the testicular stem cell niche in mammals. PMID:19270176

  9. Macrophage Stimulating Protein Is a Novel Neurotrophic Factor

    PubMed Central

    Stella, Maria Cristina; Vercelli, Alessandro; Repici, Mariaelena; Follenzi, Antonia; Comoglio, Paolo M.

    2001-01-01

    Macrophage stimulating protein (MSP), also known as hepatocyte growth factor-like, is a soluble cytokine that belongs to the family of the plasminogen-related growth factors (PRGFs). PRGFs are α/β heterodimers that bind to transmembrane tyrosine kinase receptors. MSP was originally isolated as a chemotactic factor for peritoneal macrophages. Through binding to its receptor, encoded by the RON gene, it stimulates dissociation of epithelia and works as an inflammatory mediator by repressing the production of nitric oxide (NO). Here, we identify a novel role for MSP in the central nervous system. As a paradigm to analyze this function we chose the hypoglossal system of adult mice. We demonstrate in vivo that either administration of exogenous MSP or transplantation of MSP-producing cells at the proximal stump of the resected nerve is sufficient to prevent motoneuron atrophy upon axotomy. We also show that the MSP gene is expressed in the tongue, the target of the hypoglossal nerve, and that MSP induces biosynthesis of Ron receptor in the motoneuron somata. Finally, we show that MSP suppresses NO production in the injured hypoglossal nuclei. Together, these data suggest that MSP is a novel neurotrophic factor for cranial motoneurons and, by regulating the production of NO, may have a role in brain plasticity and regeneration. PMID:11359926

  10. Stimulants

    MedlinePlus

    Stimulants are drugs that increase your heart rate, breathing rate, and brain function. Some stimulants affect only a specific organ, such as the heart, lungs, brain, or nervous system. Epinephrine is a stimulant. It ...

  11. The neurohormone orexin stimulates hypoxia-inducible factor-1 activity.

    PubMed

    Sikder, Devanjan; Kodadek, Thomas

    2007-11-15

    Orexin A and Orexin B (also known as hypocretins) are neuropeptides that bind two related G-coupled protein receptors (OXR1 and OXR2) and thus induce wakefulness, food consumption, and locomotion. Conversely, deletion of the orexin gene in mice produces a condition similar to canine and human narcolepsy. Despite the central importance of the orexin system in regulating wakefulness and feeding behavior, little is known about the downstream signaling mechanisms that achieve these effects. In this study, genomics techniques are used to probe this question and reveal that orexin activates the hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor whose pathogenic role in stimulating angiogenesis in hypoxic tumors has been the focus of intense investigation. Orexin-stimulated HIF-1 activity is due to both increased HIF-1alpha gene transcription and a down-regulation of von Hippel-Lindau (VHL), the E3 ubiquitin ligase that mediates the turnover of HIF-1 via the ubiquitin-proteasome pathway. Orexin-mediated activation of HIF-1 results in increased glucose uptake and higher glycolytic activity, as expected from studies of hypoxic cells. However, orexin receptor-expressing cells somehow override the HIF-1-mediated preference for funneling pyruvate into anaerobic glycolysis and instead favor ATP production through the tricarboxylic acid cycle and oxidative phosphorylation. These findings implicate HIF-1 as an important transcription factor in the hormone-mediated regulation of hunger and wakefulness.

  12. Induction of transcription factors in somatosensory cortex after tactile stimulation.

    PubMed

    Mack, K J; Mack, P A

    1992-01-01

    Immediate early response genes have been shown to be inducible in the central nervous system after a variety of stimuli. Induction of these transcription factors in cerebral cortex by a physiological stimulus had not previously been demonstrated. In this study, tactile stimuli induced multiple transcription factors in the somatosensory cortex. Adult male rats were lightly anesthetized with urethane. Tactile stimuli was delivered by a paint brush gently stroking an animals whiskers on one side of its face for a 15 min period. Two h later, the animals were sacrificed. Cortex contralateral to the stimulation was compared with ipsilateral cortex using antibodies raised against immediate early response gene products NGFI-A, NGFI-B, and c-fos. The different transcription factors showed slightly different patterns of response to the tactile stimulus. However, the induction of immunohistochemical staining was most prominent in layer 4 with all antibodies under study. This increase in the number of cell bodies stained was less robust than that seen in the somatosensory cortex after a seizure, and showed more of a predominance in layer 4 cells. These data demonstrate that physiologic stimulation can induce immediate early response genes in cortical cells, and that multiple immediate early response genes react to a stimulus. PMID:1312199

  13. Granulocyte colony-stimulating factor and reproductive medicine: A review

    PubMed Central

    Cavalcante, Marcelo Borges; Costa, Fabrício DA Silva; Barini, Ricardo; Araujo Júnior, Edward

    2015-01-01

    Background: Recently, the use of granulocyte colony-stimulating factor (G-CSF) has been proposed to improve pregnancy outcomes in reproductive medicine. Objective: A systematic review of the current use of G-CSF in patients who have difficulty conceiving and maintaining pregnancy was performed. Materials and Methods: Two electronic databases (PubMed/ Medline and Scopus) were searched. Study selection, data extraction and quality assessment were performed in duplicate. The subject codes used were granulocyte colony-stimulating factor, G-CSF, recurrent miscarriage, IVF failure, and endometrium. Results: The search of electronic databases resulted in 215 citations (PubMed/ Medline: 139 and Scopus: 76), of which 38 were present in both databases. Of the remaining 177 publications, seven studies were included in the present review. Conclusion: Treatment with G-CSF is a novel proposal for immune therapy in patients with recurrent miscarriage and implantation failure following cycles of IVF. However, a larger number of well-designed studies are required for this treatment to be established. PMID:26131007

  14. Recombinant human granulocyte colony-stimulating factor reverts vascular dysfunction.

    PubMed

    Squadrito, F; Altavilla, D; Squadrito, G; Campo, G M; Ioculano, M; Serranò, M; Minutoli, L; Arlotta, M; Musolino, C; Saitta, A; Caputi, A P

    1997-01-01

    The aim of our study was to investigate the vascular effects of recombinant human granulocyte colony-stimulating factor (rh G-CSF) in a rat model of irreversible vascular failure. Male anesthetized rats were subjected to the clamping of the splanchnic arteries for 45 min. This surgical procedure resulted in an irreversible state of shock (splanchnic artery occlusion shock) characterized by high mortality rate (0% survival, 120 min following the release of clamps), a profound hypotension and vascular dysfunction consisting of a marked hyporeactivity to phenylephrine (PE 1 nM-10 microM) of aortic rings. Administration of recombinant human granulocyte colony-stimulating factor (20 micrograms/kg i.v. 5 min after the release of occlusion) increased survival rate (90% 4 h after the release of occlusion), blunted the profound hypotension and reverted the marked vascular dysfunction. Finally, rh G-CSF inhibited the activity of inducible nitric oxide synthase in peritoneal macrophages activated with endotoxin. Our data suggest that rh G-CSF may influence vascular function when low-flow states occur.

  15. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.

    PubMed Central

    Connolly, D T; Heuvelman, D M; Nelson, R; Olander, J V; Eppley, B L; Delfino, J J; Siegel, N R; Leimgruber, R M; Feder, J

    1989-01-01

    Vascular permeability factor (VPF) is an Mr 40-kD protein that has been purified from the conditioned medium of guinea pig line 10 tumor cells grown in vitro, and increases fluid permeability from blood vessels when injected intradermally. Addition of VPF to cultures of vascular endothelial cells in vitro unexpectedly stimulated cellular proliferation. VPF promoted the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The identity of the growth factor activity with VPF was established in four ways: (a) the molecular weight of the activity in preparative SDS-PAGE was the same as VPF (Mr approximately 40 kD); (b) multiple isoforms (pI greater than or equal to 8) for both VPF and the growth-promoting activity were observed; (c) a single, unique NH2-terminal amino acid sequence was obtained; (d) both growth factor and permeability-enhancing activities were immunoadsorbed using antipeptide IgG that recognized the amino terminus of VPF. Furthermore, 125I-VPF was shown to bind specifically and with high affinity to endothelial cells in vitro and could be chemically cross-linked to a high-molecular weight cell surface receptor, thus demonstrating a mechanism whereby VPF can interact directly with endothelial cells. Unlike other endothelial cell growth factors, VPF did not stimulate [3H]thymidine incorporation or promote growth of other cell types including mouse 3T3 fibroblasts or bovine smooth muscle cells. VPF, therefore, appears to be unique in its ability to specifically promote increased vascular permeability, endothelial cell growth, and angio-genesis. Images PMID:2478587

  16. 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

  17. 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.

  18. Colony-Stimulating Factors for Febrile Neutropenia during Cancer Therapy

    PubMed Central

    Bennett, Charles L.; Djulbegovic, Benjamin; Norris, LeAnn B.; Armitage, James O.

    2014-01-01

    A 55-year-old, previously healthy woman received a diagnosis of diffuse large-B-cell lymphoma after the evaluation of an enlarged left axillary lymph node obtained on biopsy. She had been asymptomatic except for the presence of enlarged axillary lymph nodes, which she had found while bathing. She was referred to an oncologist, who performed a staging evaluation. A complete blood count and test results for liver and renal function and serum lactate dehydrogenase were normal. Positron-emission tomography and computed tomography (PET–CT) identified enlarged lymph nodes with abnormal uptake in the left axilla, mediastinum, and retroperitoneum. Results on bone marrow biopsy were normal. The patient’s oncologist recommends treatment with six cycles of cyclophosphamide, doxorubicin, vincristine, and prednisone with rituximab (CHOP-R) at 21-day intervals. Is the administration of prophylactic granulocyte colony-stimulating factor (G-CSF) with the first cycle of chemotherapy indicated? PMID:23514290

  19. 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

  20. A case of MRSA controlled: predisposing factors and immune stimulation.

    PubMed

    Lamson, Davis W; Sadlon, Angela E

    2010-07-01

    Most treatments for methicillin-resistant Staphylococcus aureus (MRSA) focus on agents to eliminate the bacterium. Since MRSA infection is not universal, susceptibility factors are possible. Immune resistance could be lowered in such individuals; therefore, locating immune-inhibiting or immune-enhancing factors might decrease susceptibility. Such seemed to be the case in a 48-year-old female who presented with recurring MRSA despite multiple rounds of a variety of antibiotics. When the patient encountered an intensely stressful situation an outbreak of MRSA occurred. The patient had additional underlying health issues that suppressed her immune system and made her more susceptible to stress. Gluten allergy and hypothyroidism were discovered and alleviated but did not end the MRSA outbreaks. Implementation of a popular treatment from the 1930s, intravenous dilute hydrochloric acid (for immune stimulation), prevented most MRSA outbreaks when administered frequently. This case provides anecdotal support for the proposition that immune enhancement is a viable approach to forestall or clear recurring MRSA.

  1. Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines

    PubMed Central

    1986-01-01

    Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell- stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes. PMID:3086481

  2. 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

  3. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth

    PubMed Central

    1992-01-01

    Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub- nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate. PMID:1383237

  4. 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

  5. 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

  6. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

  7. American Society of Clinical Oncology guidelines for the use of hematopoietic colony-stimulating factors.

    PubMed

    Ozer, H

    1996-01-01

    The hematopoietic colony-stimulating factors have been introduced into clinical practice as additional supportive measures that can reduce the likelihood of neutropenic complications due to chemotherapy. Clinical benefit has been shown, but the high cost of colony-stimulating factors has led to concern about their appropriate use. The American Society of Clinical Oncology has established evidence-based, clinical practice guidelines for the use of colony-stimulating factors in patients who are not enrolled in clinical trials. An expert multidisciplinary panel reviewed the clinical data documenting the activity of colony-stimulating factors. For each common clinical situation, the panel formulated a guideline to encourage reasonable use of colony-stimulating factors to preserve effectiveness but discourage excess use when little marginal benefit is anticipated. Outcomes considered in evaluating colony stimulating factor benefit included duration of neutropenia, incidence of febrile neutropenia, incidence and duration of antibiotic use, frequency and duration of hospitalization, infectious mortality, chemotherapy dose intensity, chemotherapy efficacy, quality of life, colony-stimulating factor toxicity, and economic impact. To the extent that these data were available, the panel placed greatest value on survival benefit, reduction in rates of febrile neutropenia, decreased hospitalization, and reduced costs. Lesser value was placed on alterations in absolute neutrophil counts.

  8. Recombinant granulocyte colony-stimulating factor (G-CSF) in infectious diseases: still a debate.

    PubMed

    Wiedermann, F J; Mittermayr, M; Hoffmann, G; Schobersberger, W

    2001-02-15

    Granulocyte colony-stimulating factor (G-CSF), a central mediator of the endogenous response to infection and inflammation, is approved for use in the prevention of infection-related complications in patients with nonmyeloid malignancies during antineoplastic therapy associated with high risk of severe neutropenia. Administration of granulocyte colony-stimulating factor results in improvement of host defence paired with anti-inflammatory effects. There is evidence from animal and clinical studies that administration of granulocyte colony-stimulating factor may also be beneficial in non-neutropenic infections. This review focuses mainly on the results of different animal and clinical studies of granulocyte colony stimulating factor used in the treatment of severe infections and sepsis.

  9. Action of granulocyte-macrophage colony-stimulating factors: studies using a human leukemia cell line.

    PubMed Central

    Lusis, A J; Koeffler, H P

    1980-01-01

    Granulocyte-macrophage colony-stimulating factors (CSFs) have previously been shown to stimulate colony formation in soft agar culture by a human myelogenous leukemia cell line known as KG-1. We have used KG-1 cells as a model system to investigate the interaction of CSF with myeloid cells. We now report that exposure of KG-1 cells to human CSFs in liquid culture results in a rapid (within 3 hr) burst of RNA synthesis and, after a lag of about 10 hr, a stimulation of DNA and protein synthesis. RNA and protein synthesis were maximally stimulated about 2-fold and DNA synthesis was stimulated about 2.5-fold. The stimulation was specific; various growth factors, hormones, and mouse CSFs had no effect on KG-1 macromolecular synthesis. Treatment with CSF did not discernibly alter the morphological appearance of the KG-1 cells (primarily myeloblasts) nor did it qualitatively affect the pattern of newly synthesized proteins separable by one- and two-dimensional electrophoresis. Several myeloid leukemia cell lines that were not responsive to CSF in agar culture, including a dedifferentiated variant of KG-1, showed little or no stimulation of macromolecular synthesis upon exposure to CSF. We have used the CSF-dependent stimulation of macromolecular synthesis of KG-1 to develop a rapid, sensitive microassay for human CSFs. The assay, involving thymidine incorporation by the cells, should be useful for characterization and purification of human CSFs. Images PMID:6159645

  10. Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication.

    PubMed Central

    Guo, Z S; DePamphilis, M L

    1992-01-01

    The origins of DNA replication (ori) in simian virus 40 (SV40) and polyomavirus (Py) contain an auxiliary component (aux-2) composed of multiple transcription factor binding sites. To determine whether this component stimulated replication by binding specific transcription factors, aux-2 was replaced by synthetic oligonucleotides that bound a single transcription factor. Sp1 and T-antigen (T-ag) sites, which exist in the natural SV40 aux-2 sequence, provided approximately 75 and approximately 20%, respectively, of aux-2 activity when transfected into monkey cells. In cell extracts, only T-ag sites were active. AP1 binding sites could replace completely either SV40 or Py aux-2. Mutations that eliminated AP1 binding also eliminated AP1 stimulation of replication. Yeast GAL4 binding sites that strongly stimulated transcription in the presence of GAL4 proteins failed to stimulate SV40 DNA replication, although they did partially replace Py aux-2. Stimulation required the presence of proteins consisting of the GAL4 DNA binding domain fused to specific activation domains such as VP16 or c-Jun. These data demonstrate a clear role for transcription factors with specific activation domains in activating both SV40 and Py ori. However, no correlation was observed between the ability of specific proteins to stimulate promoter activity and their ability to stimulate origin activity. We propose that only transcription factors whose specific activation domains can interact with the T-ag initiation complex can stimulate SV40 and Py ori-core activity. Images PMID:1317005

  11. 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

  12. Luteinizing hormone releasing factor in rat hypophysial portal blood collected during electrical stimulation of the hypothalamus

    PubMed Central

    Harris, G. W.; Ruf, K. B.

    1970-01-01

    1. Ovulation was induced in Nembutal-blocked pro-oestrous rats by electrical stimulation of the hypothalamus. 2. The same type of electrical stimulation was applied during the collection of hypophysial portal blood. 3. Pooled hypophysial portal plasma from donors in pro-oestrus, oestrus and met-oestrus was assayed for ovarian ascorbic acid depleting (OAAD) activity. 4. Electrical stimulation of the hypothalamus increased the OAAD activity, believed to be due to luteinizing hormone releasing factor (LRF), in pro-oestrus and met-oestrus, but not in oestrus. 5. It is concluded that the hypothalamic nerve fibres responsible for releasing LRF into the hypophysial portal vessels are depleted of their store of this releasing factor, or are refractory to electrical stimulation, during oestrus. PMID:5499765

  13. 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...

  14. 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

  15. Insulin-like growth factor-1 stimulation of lymphopoiesis.

    PubMed Central

    Clark, R; Strasser, J; McCabe, S; Robbins, K; Jardieu, P

    1993-01-01

    We show that treatment of adult mice with recombinant human insulin-like growth factor 1 (rhIGF-1) induces striking modifications in lymphocyte number and function. 9-mo-old male mice received rhIGF-1 (4 mg/kg per d) or its excipient by subcutaneous infusion from osmotic minipumps for 7 or 14 d. Mice were weighed daily and bled at sacrifice; the spleen and thymus were harvested and single cell suspensions were made for analysis of cell phenotype and cell number. The responses of splenocytes to mitogens (concanavalin A, lipopolysaccharide, and pokeweed mitogen), alloantigens and dinitrophenyl ovalbumin were measured. After either 7 or 14 d of treatment, rhIGF-1 had an overall whole-body anabolic effect, resulting in increased body and organ weights with prominent increases in the weight of the spleen and thymus. Furthermore, the rhIGF-1 treated mice were normoglycemic but had reduced blood urea nitrogens, again reflecting the anabolic activity of rhIGF-1. The increased spleen and thymus weights were associated with a large increase in the number of lymphocytes in both organs. In addition to an increase in T cells, specifically CD4+ T cells, a dramatic increase in splenic B cells was also observed. This increase was accompanied by an enhanced responsiveness to dinitrophenyl ovalbumin resulting in increased immunoglobulin production. However, despite the increases in cellularity, there was a decrease in the in vitro responses of spleen cells to mitogens after 7 d of rhIGF-1 treatment. In contrast, treatment with rhIGF-1 for 14 d increased both the cell number and mitogenic responses of splenocytes suggesting that some time is required for the cells populating the peripheral organs to gain mitogenic responsiveness. It is clear from these data that rhIGF-1, at doses that have whole-body anabolic activity, can expand cell number in lymphoid tissue in a normal adult mouse. These dual effects of rhIGF-1, of increasing lymphocyte number and activity, indicate that, in a

  16. RAS is required for epidermal growth factor-stimulated arachidonic acid release in rat-1 fibroblasts.

    PubMed

    Warner, L C; Hack, N; Egan, S E; Goldberg, H J; Weinberg, R A; Skorecki, K L

    1993-12-01

    Previous studies have provided suggestive evidence for an interaction between ras activation and signalling pathways involved in agonist-stimulated arachidonic acid release in a variety of cell systems. In order to clarify this interaction, we have measured epidermal growth factor (EGF)-stimulated arachidonic acid release in rat-1 fibroblasts transfected with the N-17 dominant negative mutation of ras. Cells transfected with the N-17 ras mutant, display a markedly attenuated arachidonic acid-release response to EGF, compared to sham-transfected and non-transfected cells. In contrast, the response to phorbol myristate acetate (PMA) was not attenuated in the N-17-mutant expressing cells. No differences were detected between sham-transfected and N-17 mutant expressing cells in levels of immunodetectable EGF receptor, cytosolic phospholipase A2 or mitogen-activated protein (MAP) kinase. Attenuation of EGF-stimulated arachidonic acid release in the N-17 mutant expressing cells, was accompanied by a marked diminution in EGF-stimulated tyrosine phosphorylation of MAP kinase. We conclude that the signalling pathway involved in epidermal growth factor-stimulated arachidonic acid release is similar to the signalling pathway for mitogenic responses to epidermal growth factor and requires ras activation, likely followed by a downstream cascade of kinases eventuating in MAP kinase activation.

  17. 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…

  18. Wnt3a upregulates transforming growth factor-β-stimulated VEGF synthesis in osteoblasts.

    PubMed

    Natsume, Hideo; Tokuda, Haruhiko; Matsushima-Nishiwaki, Rie; Kato, Kenji; Yamakawa, Kengo; Otsuka, Takanobu; Kozawa, Osamu

    2011-07-01

    It is recognized that Wnt3a affects bone metabolism via the canonical Wnt/β-catenin signalling pathway. We have previously shown that transforming growth factor-β (TGF-β) stimulates the synthesis of vascular endothelial growth factor (VEGF) via p44/p42 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TGF-β-stimulated VEGF synthesis in these cells. Wnt3a, which alone had little effect on the VEGF levels, significantly enhanced the TGF-β-stimulated VEGF release. Lithium chloride and SB216763, inhibitors of glycogen synthase kinase 3β, markedly amplified the TGF-β-stimulated VEGF release. Wnt3a failed to affect the TGF-β-induced phosphorylation of Smad2, p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. Wnt3a and lithium chloride strengthened the VEGF mRNA expression induced by TGF-β. These results strongly suggest that Wnt3a upregulates VEGF synthesis stimulated by TGF-β via activation of the canonical pathway in osteoblasts.

  19. 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.

  20. Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

    PubMed

    Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

    2016-09-15

    We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. PMID:27609291

  1. Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

    PubMed

    Tessner, T G; Rock, C O; Kalmar, G B; Cornell, R B; Jackowski, S

    1991-09-01

    Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.

  2. 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.

  3. Wnt3a regulates tumor necrosis factor-α-stimulated interleukin-6 release in osteoblasts.

    PubMed

    Natsume, Hideo; Tokuda, Haruhiko; Adachi, Seiji; Matsushima-Nishiwaki, Rie; Kato, Kenji; Minamitani, Chiho; Otsuka, Takanobu; Kozawa, Osamu

    2011-01-01

    It is recognized that Wnt pathways regulate bone metabolism. We have previously shown that tumor necrosis factor-α (TNF-α) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase)/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TNF-α-stimulated IL-6 synthesis in these cells. Wnt3a, which alone did not affect the IL-6 levels, significantly suppressed the TNF-α-stimulated IL-6 release. Lithium Chloride (LiCl), which is an inhibitor of GSK3β, markedly reduced the TNF-α-stimulated IL-6 release, similar to the results with Wnt3a. The suppression by Wnt3a or LiCl was also observed in the intracellular protein levels of IL-6 elicited by TNF-α. Wnt3a failed to affect the TNF-α-induced phosphorylation of p44/p42 MAP kinase, Akt, IκB or NFκB. Either Wnt3a or LiCl failed to reduce, rather increased the IL-6 mRNA expression stimulated by TNF-α. Lactacystin, a proteasome inhibitor, and bafilomycin A1, a lysosomal protease inhibitor, significantly restored the suppressive effect of Wnt3a on TNF-α-stimulated IL-6 release. Taken together, our results strongly suggest that Wnt3a regulates IL-6 release stimulated by TNF-α at post-transcriptional level in osteoblasts.

  4. Insulin-like growth factor-I stimulates IL-10 production in human T cells.

    PubMed

    Kooijman, Ron; Coppens, Astrid

    2004-10-01

    There is vast body of evidence that the insulin-like growth factor (IGF)-I exerts immunomodulatory effects in vitro and in vivo. In vitro studies indicate that stimulatory effects of IGF-I may be exerted through augmentation of inflammatory cytokine production. To further explore the immunomodulatory effects of IGF-I through regulation of cytokine production, we tested the in vitro effects of IGF-I on the secretion of inflammatory T helper cell type 1 (Th1) and Th2 cytokines by human peripheral blood mononuclear cells (PBMC). To this end, PBMC were stimulated with the T cell mitogen phytohemagglutinin (PHA), and cytokines in the culture media were assessed after 18, 42, 66, and 80 h of culture. We found that IGF-I stimulated the secretion of the Th2 cytokine interleukin (IL)-10 by 40-70% in PHA-stimulated PBMC. In addition, we observed a small stimulatory effect (15%) on the secretion of another Th2 cytokine IL-4. The secretion of IL-2, IL-5, IL-6, interferon-gamma, and the inflammatory cytokines IL-1beta, IL-8, and tumor necrosis factor alpha was not or was hardly affected. IL-10 secretion was also stimulated in purified T cells, and we established that IGF-I also stimulated IL-10 mRNA expression by 100-150%. The monocyte-activating bacterial cell-wall product lipopolysaccharide induced IL-10 production in PBMC, but this was not affected by IGF-I. As IL-10 predominantly exerts anti-inflammatory actions and suppresses Th1-dependent immune responses, our results indicate that IGF-I may exert inhibitory actions on inflammatory and Th1-mediated cellular immune responses through stimulation of IL-10 production in T cells.

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

    PubMed Central

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

    1991-01-01

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

  6. The macrophage-colony stimulating factor gene is a growth factor-inducible immediate early gene in fibroblasts.

    PubMed

    Ryseck, R P; Macdonald-Bravo, H; Bravo, R

    1991-02-01

    Polypeptide growth factors rapidly induce the expression of a group of genes during the onset of cell proliferation. We report that one of these genes, which is induced by several mitogens in NIH 3T3 cells, is identical to the gene for macrophage-colony stimulating factor (M-CSF). In contrast to other immediate early genes, the expression of the M-CSF gene lasted for several hours. Run-on assays demonstrated that the increased level of M-CSF mRNA following stimulation was mainly due to transcriptional activation. Our results support the notion that the products of the immediate early genes are not all mediators of fibroblasts growth but that some play an important role in other physiological responses such as wound repair. PMID:1712227

  7. Osteoporosis in severe congenital neutropenia treated with granulocyte colony-stimulating factor.

    PubMed

    Bishop, N J; Williams, D M; Compston, J C; Stirling, D M; Prentice, A

    1995-04-01

    Recombinant human granulocyte colony-stimulating factor (G-CSF) has substantially improved life expectancy for children with severe congenital neutropenia (SCN). Severe osteoporosis, reported in this population, may relate to the disease process, or be a therapeutic side-effect. This report details bone loss, quantitated absorptiometrically and histomorphometrically, in a child with SCN and vertebral collapse, and the positive response to anabolic steroid and bisphosphonate therapy.

  8. [Successful treatment of agranulocytosis caused by carbimazole using recombinant granulocyte-macrophage colony stimulating factor].

    PubMed

    Kreze, A; Kuviková, A; Laca, L; Kompis, S; Dobáková, M; Babusík, P; Matecek, L; Pekárová, E

    1995-07-01

    The authors describe a patient with hyperthyroidism who developed after three weeks' treatment with carbimazole agranulocytosis and the condition was complicated by septic shock. The authors used for treatment in addition to antibiotics and corticoids, for the first time in their practice, human recombinant granulocyte macrophage colonies stimulating factor (rHuGM-CSF) which produced a very favourable effect. In the meantime the patient was prepared for goitrectomy, operated and discharged in a satisfactory condition to have ambulatory treatment.

  9. 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.

  10. 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

  11. Differential and synergistic effects of mechanical stimulation and growth factor presentation on vascular wall function

    PubMed Central

    Liang, Mao-Shih; Koobatian, Maxwell T.; Lei, Pedro; Swartz, Daniel D.; Andreadis, Stelios T.

    2013-01-01

    We investigated the hypothesis that immobilizing TGF-β1 within fibrin hydrogels may act in synergy with cyclic mechanical stimulation to enhance the properties of vascular grafts. To this end, we engineered a fusion TGF-β1 protein that can covalently anchor to fibrin during polymerization upon the action of factor XIII. We also developed a 24-well based bioreactor in which vascular constructs can be mechanically stimulated by distending the silastic mandrel in the middle of each well. TGF-β1 was either conjugated to fibrin or supplied in the culture medium and the fibrin based constructs were cultured statically for a week followed by cyclic distention for another week. The tissues were examined for myogenic differentiation, vascular reactivity, mechanical properties and ECM content. Our results showed that some aspects of vascular function were differentially affected by growth factor presentation vs. pulsatile force application, while others were synergistically enhanced by both. Overall, this two-prong biomimetic approach improved ECM secretion, vascular reactivity and mechanical properties of vascular constructs. These findings may be applied in other tissue engineering applications such as cartilage, tendon or cardiac regeneration where growth factors TGF-β1 and mechano-stimulation play critical roles. PMID:23810080

  12. A bioassay system for two types of colony stimulating factor in human serum.

    PubMed

    Francis, G E

    1980-07-01

    The biological assay of factors with granulocyte-macrophage colony stimulating activity in human serum poses special problems. Assays based on colony counts suggest, often erroneously, that serum lacks colony stimulating factors (CSF) but interacts with other materials to potentiate or produce factors. Although the scoring of total clone numbers reveals the presence of CSF in serum, this is still no sufficient for the interpretation of the effects of mixing materials containing CSF, because increments in clone numbers are not directly proportional to increments in CSF. This is particularly important in serum assays because two types of activity are present, one which stimulates progenitor cells directly, and another which results from the interation of serum and bone marrow adherent CSF-producing) cells or peripheral blood leucocytes, indicating the presence of both direct acting and "adherent cell dependent" CSF. A biological assay is described which uses analysis of dose-response curves of clone formation in agar culture, and allows simultaneous assay of both types of activity. The criteria for the selection of suitable target progenitor cell populations are discussed.

  13. Acidic fibroblast growth factor and keratinocyte growth factor stimulate fetal rat pulmonary epithelial growth.

    PubMed

    Deterding, R R; Jacoby, C R; Shannon, J M

    1996-10-01

    We have shown that pulmonary epithelial growth and differentiation can occur if pulmonary mesenchyme is replaced with a mixture of growth factors [total growth medium (TGM)] that consists of adult rat bronchoalveolar lavage fluid, insulin, epidermal growth factor (EGF), cholera toxin (CT), acidic fibroblast growth factor (aFGF), and fetal bovine serum. In the present study, we have defined the importance of specific components of TGM. Day 14 fetal rat distal lung epithelium, devoid of mesenchyme, was enrobed in growth factor-depleted Matrigel and cultured for 5 days in various soluble factors. We found that deleting aFGF or CT from TGM significantly reduced DNA synthesis. Epithelial proliferation was not significantly different when keratinocyte growth factor (KGF) replaced aFGF in TGM. KGF, however, required the presence of a basal medium containing CT, insulin, and serum for optimal proliferation. We then added specific growth factors to the basal medium and showed that aFGF and KGF were more potent mitogens than EGF, transforming growth factor-alpha, and hepatocyte growth factor. Additionally, basal medium + KGF also allowed progression to a distal alveolar phenotype. We conclude that aFGF and KGF may be important mediators in epithelial-mesenchymal interactions. PMID:8897895

  14. 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.

  15. GROWTH REGULATING FACTOR5 Stimulates Arabidopsis Chloroplast Division, Photosynthesis, and Leaf Longevity1[OPEN

    PubMed Central

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

    2015-01-01

    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. PMID:25604530

  16. Attractant and stimulant factors for oviposition of Culex pipiens fatigans in natural breeding-sites*

    PubMed Central

    Ikeshoji, Toshiaki

    1966-01-01

    The breeding of mosquito larvae in the field is determined by the ovipositing behaviour of the gravid females. Investigation of the chemical factors that induce oviposition is therefore important for understanding mosquito ecology. These substances may also prove to be useful in assessing and controlling mosquito populations. The author has demonstrated two chemical factors, an ovipositing attractant and an ovipositing stimulant, in surface-water. The ovipositing attractant, extracted from surface-water by distillation and extraction with diethyl ether, was found to be quite effective when used to recapture known numbers of gravid mosquitos released in a large calf-shed. The presence of the stimulant factor was established by forcing gravid females to touch the testing water with tarsi and proboscis. After such contact, they began oviposition three times more rapidly on surface-water than on tap-water. The importance of these factors was demonstrated in the larval populations of Culex pipiens fatigans in pit-latrines in Rangoon, Burma. PMID:5298039

  17. Growth factor(s) produced during infection with an adenovirus variant stimulates proliferation of nonestablished epithelial cells.

    PubMed

    Quinlan, M P; Sullivan, N; Grodzicker, T

    1987-05-01

    Infection of primary baby rat kidney cells with an adenovirus variant that encodes only the 12S gene of the E1A region, adenovirus type 5 (Ad5) 12S, results in the production of a growth factor that stimulates primary epithelial cells to proliferate. Increased epithelial cell DNA synthesis and proliferation is detectable between 24 and 36 hr after the addition of conditioned medium from Ad5 12S infected cells and not from cells infected with an E1A deletion mutant virus, Ad5 dl312. This mitogenic factor(s) is effective in the absence of serum and can override the inhibitory effect of serum on primary epithelial cells. Furthermore, there is a requirement for the continued presence of the growth factor(s) in the Ad5 12S conditioned medium to maintain epithelial cell proliferation, and the conditioned medium can maintain these cells in a proliferative state for at least 6 wk. The stimulatory activity in Ad5 12S conditioned medium is associated with large molecular weight complexes, from which it can be released by 4 M NaCl. Several characteristics of the growth factor(s) indicate that it is a unique mitogen for epithelial cells. PMID:2953026

  18. Tyrosine dephosphorylation of nuclear proteins mimics transforming growth factor {beta}1 stimulation of {alpha}2(I) collagen gene expression

    SciTech Connect

    Greenwel, P.; Hu, Wei; Ramirez, F.; Kohanski, R.A.

    1995-12-01

    This report describes how the transforming growth factor {beta}1 (TGF-{beta}1) stimulates the transcription of the gene coding for collagen I (COL1A2). The report goes on to correlate tyrosine dephosphorylation, increased binding of a transcriptional complex and TGF-{beta}1 stimulation of gene expression. 33 refs., 8 figs., 1 tab.

  19. Priming effect of platelet activating factor on leukotriene C4 from stimulated eosinophils of asthmatic patients.

    PubMed Central

    Shindo, K.; Koide, K.; Hirai, Y.; Sumitomo, M.; Fukumura, M.

    1996-01-01

    BACKGROUND: Eosinophils from asthmatic patients are known to release greater amounts of leukotrienes than normal eosinophils when stimulated by the calcium ionophore A23187. The effect of platelet activating factor (PAF) in priming eosinophils was investigated. METHODS: Eosinophils were obtained from 18 asthmatic patients and 18 healthy donors. Cells separated by the Percoll gradients were incubated with PAF (C-18) for 30 minutes and then stimulated with the calcium ionophore A23187 (2.5 microM) for 15 minutes. The amount of leukotriene C4 (LTC4) in supernatants was measured using a combination of high pressure liquid chromatography and radioimmunoassay. RESULTS: The mean (SD) amount of LTC4 released by eosinophils from asthmatic patients upon stimulation with the calcium ionophore A23187 alone was 27.9 (9.9) ng/10(6) cells (n = 6). The amount of LTC4 released following stimulation with the calcium ionophore A23187 after pretreatment with PAF (1, 5, and 10 microM) was 57.2 (8.9), 75.1 (14.3), and 52.6 (10.7) ng/10(6) cells (n = 6), respectively. Trace amounts of LTC4 (0.9 (0.02) ng/10(6) cells, n = 6) were detected in the supernatant of the cells after stimulation by PAF alone (5 microM). The amount of LTC4 released upon stimulation by calcium ionophore A23187 alone in eosinophils from healthy donors was 10.3 (3.7) ng/10(6) cells (n = 4). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with PAF at concentrations of 1, 5, and 10 microM were 11.9 (3.5), 17.8 (5.6), and 12.7 (5.1) ng/10(6) cells (n = 4), respectively. Trace amounts of LTC4 (0.6 (0.02) ng/10(6) cells, n = 4) were detected in the supernatant of the cells upon stimulation with PAF alone (5 microM). The amounts of LTC4 released upon stimulation with calcium ionophore A23187 after pretreatment with lyso-PAF at concentrations of 1, 5, and 10 microM (n = 4 or 6) were 30.8 (5.2), 22.9 (5.1), and 27.3 (4.3) ng/10(6) cells (n = 6) from the eosinophils of asthmatic

  20. Corticotropin-releasing factor administered centrally, but not peripherally, stimulates hippocampal acetylcholine release.

    PubMed

    Day, J C; Koehl, M; Le Moal, M; Maccari, S

    1998-08-01

    In addition to corticotropin-releasing factor's well-known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin-releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin-releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin-releasing factor (0.5-5.0 microg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose-dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin-releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin-releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin-releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress.

  1. Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans.

    PubMed Central

    Xie, Z. P.; Staehelin, C.; Vierheilig, H.; Wiemken, A.; Jabbouri, S.; Broughton, W. J.; Vogeli-Lange, R.; Boller, T.

    1995-01-01

    Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots. PMID:12228558

  2. Corticotropin-releasing factor administered centrally, but not peripherally, stimulates hippocampal acetylcholine release.

    PubMed

    Day, J C; Koehl, M; Le Moal, M; Maccari, S

    1998-08-01

    In addition to corticotropin-releasing factor's well-known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin-releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin-releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin-releasing factor (0.5-5.0 microg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose-dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin-releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin-releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin-releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress. PMID:9681452

  3. Effect of in vivo infusion of granulocyte colony-stimulating factor on immune function.

    PubMed

    Valente, John F; Alexander, J Wesley; Li, Bing-Guo; Noel, J Gregory; Custer, David A; Ogle, James D; Ogle, Cora K

    2002-01-01

    As the applications of hematopoietic growth factors increase, their complex impact on host defense and immune responses continues to unfold. The effect of the administration of granulocyte colony-stimulating factor (G-CSF) on bacterial defense, proliferation of lymphocytes, and cytokine production by lymphocytes and peripheral blood mononuclear cells (PBMC) was studied. The effect of G-CSF administration on the phenotype of the cells in the major hematopoietic organs was studied as well. ACI rats were given 10 mg/kg/day G-CSF or vehicle daily for 4 days. Isolated bone marrow neutrophils and enterocytes from treated animals showed a greater bactericidal activity than controls. Proliferation of mitogen-stimulated lymphocytes and PBMC was reduced in G-CSF-treated animals. The production of proinflammatory cytokines, tumor necrosis factor (TNF), and interleukin 6 (IL-6) by lymphocytes and PBMC was reduced by G-CSF pretreatment. G-CSF administration caused an increase in IL-4 (Th2 cytokine) release and a decrease in interferon-gamma (IFNgamma, Th1 cytokine) release by mitogen-stimulated lymphocytes. Cytometric analysis of cells in the progenitor cell region indicated a large increase in immature cells in the bone marrow of G-CSF-treated animals compared with sham along with an increase in B cells and a decrease in polymorphonuclear leukocytes (PMNs). In addition, cytometric analysis showed a large increase in PMNs in blood and splenocytes of the treated animals compared with sham. This study confirms and extends previous observations that G-CSF administration has a number of effects that might simultaneously enhance host defense while reducing the risk of developing uncontrolled systemic inflammation. This may also be efficacious in prolonging graft survival and reducing graft vs. host disease.

  4. Regulation of cytosolic phospholipase A2 phosphorylation and eicosanoid production by colony-stimulating factor 1.

    PubMed

    Xu, X X; Rock, C O; Qiu, Z H; Leslie, C C; Jackowski, S

    1994-12-16

    A colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell line (BAC1.2F5) and peritoneal macrophages were used to investigate the relationship between growth factor-dependent phosphorylation/activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) and arachidonic acid metabolism. The addition of CSF-1 to quiescent BAC1.2F5 cells was followed by the rapid phosphorylation, electrophoretic gel retardation, and stable increase in the specific activity of cPLA2 that correlated with the activation of ERK kinases. cPLA2 phosphorylation depended on the presence of growth factor and persisted throughout the cell cycle. CSF-1 inhibited prostaglandin E2 production and did not enhance arachidonic acid release or increase the levels of lysophosphatidylcholine or glycerophosphocholine. Treatment of BAC1.2F5 cells with the calcium ionophore A23187 plus CSF-1 did not stimulate eicosanoid release. Instead, CSF-1 enhanced the rate of exogenous arachidonic acid incorporation into phosphatidylcholine and its subsequent transfer to phosphatidylethanolamine suggesting that higher rates of arachidonic acid acylation may contribute to the suppression of prostaglandin production. In peritoneal macrophages, ERK kinase activity was stimulated and cPLA2 was phosphorylated and activated in response to CSF-1. However, CSF-1 did not trigger eicosanoid release but did augment arachidonic acid mobilization and prostaglandin E2 production elicited by zymosan and A23187. Thus, cPLA2 phosphorylation/activation and calcium mobilization are not the only determinants for eicosanoid release, and additional components in differentiated tissue macrophages are also required.

  5. Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors

    PubMed Central

    de Souza, Devandir Antonio; Borges, Antonio Carlos; Santana, Ana Carolina; Oliver, Constance; Jamur, Maria Célia

    2015-01-01

    Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization. PMID:26633538

  6. Trefoil Factor 1 Stimulates Both Pancreatic Cancer and Stellate Cells and Increases Metastasis

    PubMed Central

    Arumugam, Thiruvengadam; Brandt, Will; Ramachandran, Vijaya; Moore, Tood T.; Wang, Huamin; May, Felicity E.; Westley, Bruce R.; Hwang, Rosa F.; Logsdon, Craig D.

    2015-01-01

    Objectives Trefoil factor 1 (TFF1) is a stable secretory protein expressed widely in the gastrointestinal mucosa that is also expressed in pancreatic ductal adenocarcinoma (PDAC). In the current study, we documented the extent and timing of TFF1 expression and investigated the effects of TFF1 on PDAC cells and stellate cells, the primary cells of the PDAC stroma. Methods Trefoil factor 1 expression in pancreatic cancer tissues and cell lines was analyzed using microarray, quantitative reverse transcriptase–polymerase chain reaction, and immunohistochemistry. The effects of recombinant TFF1 on cell growth, migration, and invasion of pancreatic cancer cell lines and immortalized human pancreatic stellate cells (HPSCs) were analyzed using MTS and Matrigel-coated invasion chambers. In vivo studies were also conducted in which Mpanc-96 cells stably expressing TFF1 were implanted orthotopically into nude mice. Results Trefoil factor 1 was highly increased in preneoplastic lesions. Recombinant TFF1 stimulated motility of both cancer and HPSCs. In contrast, only HPSC cell growth was increased by TFF1. In vivo studies showed that overexpression of TFF1 in PDAC cells did not affect primary tumor growth but greatly increased metastasis. Conclusions The present data demonstrate that TFF1 influences both PDAC cells and stellate cells and stimulates metastasis. PMID:21747314

  7. Inositol lipid metabolism in vasopressin stimulated hepatocytes from rats infused with tumor necrosis factor

    SciTech Connect

    Spitzer, J.A.; Rodriguez de Turco, E.B. )

    1989-05-30

    We studied the effect of i.v. infusion of human recombinant tumor necrosis factor alpha (rHuTNF alpha, Cetus, 15 micrograms/100 g bw over 3 h) on vasopressin (VP)-stimulated {sup 32}P-inositol lipid turnover and the release of {sup 3}H-inositol phosphates in isolated rat hepatocytes. The early VP-induced decrease (within 30 s) in {sup 32}P-phosphatidylinositol 4-phosphate and {sup 32}P-phosphatidylinositol 4,5-bisphosphate labeling was significantly reduced (-40%) and at the same time the uptake of {sup 32}P into phosphatidic acid was 50% lower than in saline-infused (matched control) rats. Within 5 min of VP-stimulation, lower {sup 32}P phosphatidylinositol (-40%) and higher {sup 32}P-phosphatidic acid (+30%) labeling were observed in rHuTNF alpha-infused rats. Infusion of rHuTNF alpha also affected the VP-induced release of {sup 3}H-inositol phosphates. The accumulation of {sup 3}H-inositol-labeled water soluble products was decreased by 25% and 17% at 30 s and 10 min, respectively. These data show that rHuTNF alpha mimics early perturbations induced by Escherichia coli endotoxin infusion in VP-stimulated inositol lipid metabolism in rat hepatocytes.

  8. 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

  9. 1,25-Dihydroxyvitamin D3 stimulates rat osteoblastic cells to release a soluble factor that increases osteoclastic bone resorption.

    PubMed Central

    McSheehy, P M; Chambers, T J

    1987-01-01

    Although 1,25-dihydroxyvitamin D3 stimulates osteoclastic bone resorption in vivo and in organ culture, the mechanism by which it effects this stimulation is unknown. We have recently found that the agent does not stimulate resorption by osteoclasts mechanically disaggregated from bone and incubated on slices of cortical bone. This suggests that the osteoclasts were removed by disaggregation from the influence of some cell type, present in intact bone, that mediates hormone responsiveness. We therefore tested the ability of osteoblastic cells derived from neonatal rat calvariae and of cloned, hormone-responsive osteosarcoma cells (UMR106) to restore hormone responsiveness to unresponsive populations of osteoclasts. We found that osteoblastic cells from both sources induced a two- to fourfold stimulation of osteoclastic bone resorption in the presence of 1,25-dihydroxyvitamin D3. Stimulation was observed at concentrations of 10(-10) M and above. Actinomycin D and cycloheximide did not affect bone resorption by osteoclasts incubated alone, but abolished the capacity of osteoblastic cells to stimulate osteoclastic resorption in the presence of 1,25-dihydroxyvitamin D3. When calvarial cells or osteoblastlike UMR cells were incubated with the hormone, they produced a factor in cell-free supernatants that stimulated bone resorption by disaggregated osteoclasts. These experiments suggest that 1,25-dihydroxyvitamin D3 stimulates bone resorption through a primary action on osteoblastic cells, that are induced by the hormone to produce a factor that stimulates osteoclastic bone resorption. PMID:3611354

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

    PubMed

    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

  11. 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

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

    PubMed

    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. Factors Related to Outcomes of Subthalamic Deep Brain Stimulation in Parkinson's Disease

    PubMed Central

    Kim, Hae Yu; Chang, Won Seok; Kang, Dong Wan; Sohn, Young Ho; Lee, Myung Sik

    2013-01-01

    Objective Subthalamic nucleus (STN) deep brain stimulation (DBS) is an effective treatment of choice for patients with advanced idiopathic Parkinson's disease (PD) who have motor complication with medication. The objectives of this study are to analyze long-term follow-up data of STN DBS cases and to identify the factors related to outcomes. Methods Fifty-two PD patients who underwent STN DBS were followed-up for more than 3 years. The Unified Parkinsons Disease Rating Scale (UPDRS) and other clinical profiles were assessed preoperatively and during follow-up. A linear regression model was used to analyze whether factors predict the results of STN DBS. We divided the study individuals into subgroups according to several factors and compared subgroups. Results Preoperative activity of daily living (ADL) and the magnitude of preoperative levodopa response were shown to predict the improvement in UPDRS part II without medication, and preoperative ADL and levodopa equivalent dose (LED) were shown to predict the improvement in UPDRS part II with medication. In UPDRS part III with medication, the magnitude of preoperative levodopa response was a predicting factor. Conclusion The intensity of preoperative levodopa response was a strong factor for motor outcome. And preoperative ADL and LED were strong factors for ADL improvement. More vigorous studies should be conducted to elucidate how levodopa-induced motor complications are ameliorated after STN DBS. PMID:24175026

  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. Fibroblast growth factor 2-stimulated proliferation is lower in muscle precursor cells from old rats.

    PubMed

    Jump, Seth S; Childs, Tom E; Zwetsloot, Kevin A; Booth, Frank W; Lees, Simon J

    2009-06-01

    In aged skeletal muscle, impairments in regrowth and regeneration may be explained by a decreased responsiveness of muscle precursor cells (MPCs) to environmental cues such as growth factors. We hypothesized that impaired responsiveness to fibroblast growth factor 2 (FGF2) in MPCs from old animals would be explained by impaired FGF2 signalling. We determined that 5-bromo-2'-deoxyuridine (BrdU) incorporation and cell number increase less in MPCs from 32- compared with 3-month-old rats. In the presence of FGF2, we demonstrated that there were age-associated differential expression patterns for FGF receptor 1 and 2 mRNAs. Measurement of downstream signalling revealed that that mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2)-extracellular signal-regulated kinase 1/2, protein kinase C and p38 were FGF2-driven pathways in MPCs. Uniquely, protein kinase C signalling was shown to play the largest role in FGF2-stimulated proliferation in MPCs. c-Jun N-terminal kinase (JNK) signalling was ruled out as an FGF2-stimulated proliferation pathway in MPCs. Inhibition of JNK had no effect on FGF2 signalling to BrdU incorporation, and FGF2 treatment was associated with increased phosphorylation of p38, which inhibits, rather than stimulates, BrdU incorporation in MPCs. Surprisingly, the commonly used vehicle, dimethyl sulphoxide, rescued proliferation in MPCs from old animals. These findings provide insight for the development of effective treatment strategies that target the age-related impairments of MPC proliferation in old skeletal muscle. PMID:19270036

  16. 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.

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

    PubMed

    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

  18. 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

  19. Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.

    PubMed Central

    Yui, M. A.; Brissette, W. H.; Brennan, D. C.; Wuthrich, R. P.; Rubin-Kelley, V. E.

    1991-01-01

    Abnormal macrophages in MRL-lpr mice are implicated in the pathogenesis of autoimmune disease. These mice die of lupus nephritis by 5 to 6 months of age. This study reports that MRL-lpr mice have an increased level of circulating macrophage colony-stimulating factor (M-CSF) detectable as early as 1 week of age. Macrophage colony-stimulating factor decreased between 2 and 4 months and then steadily increased beginning at 4 months of age. In contrast, M-CSF was not detected in sera from congenic MRL-++ mice, normal C3H/FeJ mice, two other mouse strains with the lpr gene (B6-lpr and C3H-lpr), or another lupus model, the NZB/W mouse. These observations indicate that the lpr gene alone is not responsible for inducing this growth factor, and elevated M-CSF is not required for all forms of murine lupus. The entire source of serum M-CSF is not clear. The unique T cells regulated by the lpr gene are not responsible for the increased serum M-CSF levels, as no M-CSFs could be detected in supernatants from cultured lymph nodes from MRL-lpr mice, and the steady-state levels of M-CSF mRNA in lymph nodes and spleens in MRL-lpr, C3H-lpr mice and in their respective congenic strains were similar. The steady-state M-CSF mRNA transcripts in liver, lung, and bone marrow in MRL-lpr, MRL-++, and C3H/FeJ mice were also similar. Macrophage colony-stimulating factor transcripts were clearly elevated in the kidneys of MRL-lpr mice, suggesting a renal source of circulating M-CSF. The increase of M-CSF might be responsible for the increased numbers and enhanced functions of macrophages, which in turn cause tissue destruction in MRL-lpr mice. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:1867317

  20. Mechanisms of amphibian macrophage development: characterization of the Xenopus laevis colony-stimulating factor-1 receptor.

    PubMed

    Grayfer, Leon; Edholm, Eva-Stina; Robert, Jacques

    2014-01-01

    Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1R tissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidney CSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liver CSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrow CSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrow CSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpole macrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.

  1. 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.

  2. 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

  3. Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

    PubMed Central

    Griner, Samantha E.; Joshi, Jayashree P.; Nahta, Rita

    2015-01-01

    Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable overexpression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression. PMID:23085437

  4. RUNX1 haploinsufficiency results in granulocyte colony-stimulating factor hypersensitivity.

    PubMed

    Chin, D W L; Sakurai, M; Nah, G S S; Du, L; Jacob, B; Yokomizo, T; Matsumura, T; Suda, T; Huang, G; Fu, X-Y; Ito, Y; Nakajima, H; Osato, M

    2016-01-01

    RUNX1/AML1 is among the most commonly mutated genes in human leukemia. Haploinsufficiency of RUNX1 causes familial platelet disorder with predisposition to myeloid malignancies (FPD/MM). However, the molecular mechanism of FPD/MM remains unknown. Here we show that murine Runx1(+/-) hematopoietic cells are hypersensitive to granulocyte colony-stimulating factor (G-CSF), leading to enhanced expansion and mobilization of stem/progenitor cells and myeloid differentiation block. Upon G-CSF stimulation, Runx1(+/-) cells exhibited a more pronounced phosphorylation of STAT3 as compared with Runx1(+/+) cells, which may be due to reduced expression of Pias3, a key negative regulator of STAT3 signaling, and reduced physical sequestration of STAT3 by RUNX1. Most importantly, blood cells from a FPD patient with RUNX1 mutation exhibited similar G-CSF hypersensitivity. Taken together, Runx1 haploinsufficiency appears to predispose FPD patients to MM by expanding the pool of stem/progenitor cells and blocking myeloid differentiation in response to G-CSF. PMID:26745853

  5. 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

  6. 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.

  7. 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.

  8. Processing of newly synthesized cachectin/tumor necrosis factor in endotoxin-stimulate macrophages

    SciTech Connect

    Jue, Dae-Myung; Sherry, B.; Luedke, C.; Manogue, K.R.; Cerami, A. )

    1990-09-11

    The biosynthesis and processing of cachetin/tumor necrosis factor (TNF) were examined in the murine macrophage-like cell line RAW 264.7. Lipipolysaccharide-stimulated cells secreted both glycosylated and nonglycosylated 17-kilodalton (kDa) mature cachectin/TNF into the culture medium. Secreted cachectin/TNF was derived from membrane-associated precursors that were precipitated by polyclonal antisera raised against either the mature protein or synthetic peptide fragments of the 79 amino acid cachectin/TNF prohormone sequence. About half of the precursors were N-glycosylated, apparently cotranslationally. The cachectin/TNF precursors were then proteolytically cleaved to release soluble mature cytokine into the medium, while the membrane-bound 14-kDa prosequence remained cell associated. During the period of LPS stimulation, the amount of macrophage cell surface cachectin/TNF remained at a low level, suggesting that both nonglycosylated and glycosylated precursors of cachectin/TNF are efficiently cleaved by these cells. These findings suggest the presence of a unique mechanism for the secretion of cachectin/TNF.

  9. 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

  10. Possible mechanisms and function of nuclear trafficking of the colony-stimulating factor-1 receptor.

    PubMed

    Rovida, Elisabetta; Dello Sbarba, Persio

    2014-10-01

    Receptor tyrosine kinases (RTK) have long being studied with respect to the "canonical" signaling. This includes ligand-induced activation of a receptor tyrosine kinase at the cell surface that leads to receptor dimerization, followed by its phosphorylation in the intracellular domain and activation. The activated receptor then recruits cytoplasmic signaling molecules including other kinases. Activation of the downstream signaling cascade frequently leads to changes in gene expression following nuclear translocation of downstream targets. However, RTK themselves may localize within the nucleus, as either full-length molecules or cleaved fragments, with or without their ligands. Significant differences in this mechanism have been reported depending on the individual RTK, cellular context or disease. Accumulating evidences indicate that the colony-stimulating factor-1 receptor (CSF-1R) may localize within the nucleus. To date, however, little is known about the mechanism of CSF-1R nuclear shuttling, as well as the functional role of nuclear CSF-1R.

  11. Neutrophil kinetics of recombinant human granulocyte colony-stimulating factor-induced neutropenia in rats

    SciTech Connect

    Okada, Yuji; Kawagishi, Mayumi; Kusaka, Masaru )

    1990-01-01

    Single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately induced a decrease in the number of circulating neutrophils in rats. This neutropenia occurred 10 minutes after the injection but disappeared 40 minutes after injection. This transient neutropenia was dose-dependently induced by rhG-CSF and also induced by repeated injections. We studied the kinetics of circulating neutrophils in transient neutropenia. rhG-CSF markedly decreased the number of {sup 3}H-diisopropylfluorophosphate ({sup 3}H-DFP) labeled neutrophils in the circulation 10 minutes after injection but the labeled neutrophils recovered to near the control level 40 minutes after the injection. These results indicate that the neutrophil margination accounts for the neutrophenia and the marginated neutrophils return to the circulation.

  12. Some human factors issues in enhanced vision system: an experimental approach through stimulation techniques

    NASA Astrophysics Data System (ADS)

    Leger, Alain; Fleury, Lionel; Aymeric, Bruno

    1996-05-01

    Among the numerous human factors issues related to Enhanced Vision Systems the decision making process appears quite critical for certification purpose. An experimental setup based on a simplified aircraft environment including a HUD was developed in the framework of the FANSTIC II program. A stimulation technique based on recordings of IR sensors obtained during weather penetration test flight were used to study visual cues involved in decision process during approaches on IR imagery. A study of visual scanning strategies was also conducted in order to follow dynamically the process. Results show a good consistency in the pattern of visual cues used by different pilots in making their decision. Decision delays were found to be in the region of 5 - 6 seconds with little difference between FLIR and visible images. The introduction of symbology superimposed on the imagery sensibly modify visual scanning patterns. In this case, scanning is deeply influenced by pilot's previous experience.

  13. 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. PMID:27498938

  14. Establishment and characterization of a human thyroid carcinoma cell line (HOTHC) producing colony stimulating factor.

    PubMed

    Ishiwata, Isamu; Ono, Isao; Kiguchi, Kazushige; Ishiwata, Chieko; Soma, Masayuki; Ishikawa, Hiroshi

    2005-09-01

    A cell line designated HOTHC was established from an anaplastic carcinoma (giant cell type) of the thyroid gland of 80-year-old woman. The HOTHC line grew rapidly in multilayer without contact inhibition, and more than 120 serial passages were made within 27 months. The cells were spindle or polygonal in shape and revealed neoplastic and pleomorphic features. These cells were characterized as containing coloid droplets and poorly developed rough-endoplasmic reticulum in the cytoplasm. Doubling time was about 24 hours and plating efficiency was about 70%. The karyotype exhibits hyperploidy and marker chromosomes, and the modal chromosome number ranged between 77-90. The HOTHC cells were transplanted into the subcutis of BALB/c nude mice and produced anaplatic carcinomas (giant cell type) resembling the original tumor. The HOTHC cells produced colony stimulating factor (CSF) and caused granulocytosis in the mice. PMID:17022149

  15. 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…

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

    NASA Astrophysics Data System (ADS)

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

    1993-04-01

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

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

    PubMed Central

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

    1993-01-01

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

  18. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

  19. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    PubMed

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1. PMID:27025961

  20. 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.

  1. Epithelial differentiation of metanephric mesenchymal cells after stimulation with hepatocyte growth factor or embryonic spinal cord.

    PubMed Central

    Karp, S L; Ortiz-Arduan, A; Li, S; Neilson, E G

    1994-01-01

    Mammalian kidney emerges from metanephric mesenchyme following the insertion of a migrating ureteric bud. The pattern morphology of mesenchymal specialization during tubular segmentation is remarkably complex, and the relative contribution of pattern gradients from the microenvironment versus the instructive role of individual cells is not known. We have started to examine the differentiation of metanephric mesenchyme using cultures of metanephric ridge (MMR) cells from day 13.5 mouse embryos to investigate the conversion of mesenchyme toward kidney epithelium in vitro. One of our mesenchymal clones, MMR1, expresses little Pax2, uvomorulin, or cytokeratin but does express neural cell adhesion molecule, bc12, and desmin; these are properties consistent with an early stem cell. Coculture of MMR1 cells with embryonic spinal cord leads to the induction of a more differentiated cell phenotype characterized by decreased expression of neural cell adhesion molecule, the appearance of uvomorulin, and the emergence of cytokeratin, all consistent with an evolution toward epithelium. We were also able to detect the hepatocyte growth factor receptor c-met on MMR1 cells by indirect immunofluorescence. When MMR1 cells were stimulated with hepatocyte growth factor, neural cell adhesion molecule expression decreased and uvomorulin appeared. This effect of hepatocyte growth factor, as a single cytokine, may be important in the early assemblage of kidney, since we were able to detect mRNA transcripts encoding c-met from mouse embryo metanephric kidneys. Images PMID:8202482

  2. Osteoblast-derived paracrine factors regulate angiogenesis in response to mechanical stimulation.

    PubMed

    Liu, Chao; Cui, Xin; Ackermann, Thomas M; Flamini, Vittoria; Chen, Weiqiang; Castillo, Alesha B

    2016-07-11

    Angiogenesis is a process by which new blood vessels emerge from existing vessels through endothelial cell sprouting, migration, proliferation, and tubule formation. Angiogenesis during skeletal growth, homeostasis and repair is a complex and incompletely understood process. As the skeleton adapts to mechanical loading, we hypothesized that mechanical stimulation regulates "osteo-angio" crosstalk in the context of angiogenesis. We showed that conditioned media (CM) from osteoblasts exposed to fluid shear stress enhanced endothelial cell proliferation and migration, but not tubule formation, relative to CM from static cultures. Endothelial cell sprouting was studied using a dual-channel collagen gel-based microfluidic device that mimics vessel geometry. Static CM enhanced endothelial cell sprouting frequency, whereas loaded CM significantly enhanced both frequency and length. Both sprouting frequency and length were significantly enhanced in response to factors released from osteoblasts exposed to fluid shear stress in an adjacent channel. Osteoblasts released angiogenic factors, of which osteopontin, PDGF-AA, IGBP-2, MCP-1, and Pentraxin-3 were upregulated in response to mechanical loading. These data suggest that in vivo mechanical forces regulate angiogenesis in bone by modulating "osteo-angio" crosstalk through release of paracrine factors, which we term "osteokines". PMID:27332785

  3. 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. PMID:23933523

  4. Colony-stimulating factors for the management of neutropenia in cancer patients.

    PubMed

    Dale, David C

    2002-01-01

    Neutropenia and its subsequent infectious complications represent the most common dose-limiting toxicity of cancer chemotherapy. Febrile neutropenia (FN) occurs with common chemotherapy regimens in 25 to 40% of treatment-naive patients, and its severity depends on the dose intensity of the chemotherapy regimen, the patient's prior history of either radiation therapy or use of cytotoxic treatment, and comorbidities. The occurrence of FN often causes subsequent chemotherapy delays or dose reductions. It may also lengthen hospital stay, increase monitoring, diagnostic and treatment costs, and reduce patient quality of life. A decade after their introduction, colony-stimulating factors (CSFs) such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are now an integral part of the prevention of potentially life-threatening FN; however, only G-CSF has US Food and Drug Administration approval for use in chemotherapy-induced neutropenia. These adjunctive agents accelerate formation of neutrophils from committed progenitors, thereby reducing the duration and severity of neutropenia. Important uses of CSFs in oncology are prevention of FN after chemotherapy, treatment of febrile neutropenic episodes and support following bone marrow transplantation, and collection of CSF-mobilised peripheral blood progenitor cells. G-CSF is used more frequently than GM-CSF for all of these indications because of fewer associated adverse effects. Clinical trials to date have not demonstrated a significant effect on overall survival or disease-free survival, which is most likely to be due to small sample size and lack of power to prove effect. However, they have demonstrated clinical utility in allowing the delivery of planned chemotherapy dose on schedule, an important clinical goal especially in curative tumour settings. The high cost of these agents limits their widespread use. Current American Society of Clinical Oncology

  5. Epidermal growth factor and gonadotropin-releasing hormone stimulate proliferation of enriched population of gonadotropes.

    PubMed

    Childs, G V; Unabia, G

    2001-02-01

    Recent studies of epidermal growth factor (EGF) receptors on gonadotropes show that they appear early in the estrous cycle on immature gonadotropes, most of which could be identified by LH messenger RNA only. As diestrous gonadotropes translate the messenger RNAs, the percentages of LH and FSH cells with EGF receptors increase to reach a peak during proestrus. To learn more about the function of EGF in gonadotrope regulation, parallel studies of its mitogenic potential were conducted. To test this in a cell growth assay, we initially developed a protocol for enrichment of gonadotropes by counterflow centrifugation (elutriation). Analysis of immunolabeled cells in the enriched fraction showed that the population contained 90-95% cells with LH and/or FSH antigens. Less than 4% have TSH or PRL antigens, and less than 7% have ACTH antigens. About 15% of the enriched population expressed GH antigens in male rats and nearly 30% of the population express GH in females. This agrees with the known hormone storage overlap between these cells, especially in proestrous female rats. The MTT cell growth/cell death assay was then used to test the mitogenic potential of EGF, GnRH, and activin. This assay showed a linear relationship between plated cell numbers and optical density of the media after the MTT reaction was run. The enriched gonadotropes were plated in 96-microwell trays and grown for 3-4 days in the presence of defined media alone (no serum), or defined media containing 0.5-10 ng/ml EGF, 0.5-1 nM GnRH, 60 ng/ml activin or two of these factors. In all of the 12 experiments, each of the factors stimulated a 3- to 10-fold increase in optical density values, depending on the dose of the stimulating factor. The effects of any two factors were not additive. Because the MTT assays do not discriminate between mitogenic effects and enhanced cell survival, a second group of tests was run with mixed cultures of pituitary cells from diestrous female rats. These cells were

  6. 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.

  7. The role of stem cell factor and granulocyte-colony stimulating factor in brain repair during chronic stroke.

    PubMed

    Piao, Chun-Shu; Gonzalez-Toledo, Maria E; Xue, Yue-Qiang; Duan, Wei-Ming; Terao, Satoshi; Granger, D Neil; Kelley, Roger E; Zhao, Li-Ru

    2009-04-01

    Chronic stroke is a highly important but under-investigated scientific problem in neurologic research. We have reported earlier that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) treatment during chronic stroke improves functional outcomes. Here we have determined the contribution of bone marrow-derived cells in angiogenesis and neurogenesis, which are enhanced by SCF+G-CSF treatment during chronic stroke. Using bone marrow tracking, flow cytometry, 2-photon live brain imaging, and immunohistochemistry, we observed that the levels of circulating bone marrow stem cells (BMSCs) (CD34+/c-kit+) were significantly increased by SCF+G-CSF treatment. In addition, live brain imaging revealed that numerous bone marrow-derived cells migrate into the brain parenchyma in the treated mice. We also found that bone marrow-derived cells, bone marrow-derived endothelial cells, vascular density, and bone marrow-derived neurons were significantly augmented by SCF+G-CSF. It is interesting that, in addition to the increase in bone marrow-derived endothelial cells, the number of bone marrow-derived pericytes was reduced after SCF+G-CSF treatment during chronic stroke. These data suggest that SCF+G-CSF treatment can enhance repair of brain damage during chronic stroke by mobilizing BMSCs, and promoting the contribution of bone marrow-derived cells to angiogenesis and neurogenesis. PMID:19209180

  8. 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

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

    2012-12-01

    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.

  9. Mesenchymal cell chondrogenesis is stimulated by basement membrane matrix and inhibited by age-associated factors.

    PubMed

    Bradham, D M; Passaniti, A; Horton, W E

    1995-07-01

    During development of the embryonic limb, differentiation of mesenchymal progenitor cells into chondrocytes is regulated by cell shape, extracellular matrix, and growth and differentiation factors. In this study, reconstituted basement membrane (Matrigel) prepared from mouse Englebreth-Holm-Swarm tumor tissue was found to stimulate mesenchymal cell chondrogenesis in vitro and the production of cartilage at ectopic sites in athymic mice. The rate of chondrogenesis of mesenchymal cells from chick limb bud was increased four-fold by the addition of 400 micrograms/ml Matrigel to the media of micromass cultures, and this activity was not blocked by neutralizing antibodies to transforming growth factor-beta (TGF-beta) or fibroblast growth factor (FGF). Mesenchymal cells cultured on Matrigel, but not laminin or collagen type I or IV, formed spheres of condensed cells which stained with Alcian blue. Chick limb-bud mesenchymal cells suspended in Matrigel prepared from tumors grown in C57 mice aged 3, 12, or 26 months formed disks of hyaline cartilage within 2 weeks with wet weights of 59.1 mg, 35.7 mg, and 21.4 mg, indicating that the Matrigel from the old animals was less biologically active. In agreement with the in vivo data, Alcian blue staining of proteoglycan was over two-fold higher in micromass cultures supplemented with the Matrigel from young animals than in cultures treated with the Matrigel from old mice. A high-salt wash preparation of Matrigel from tumors grown in old mice increased the rate of chondrogenesis and cartilage production, suggesting that an inhibitor of chondrogenesis is produced by the old host. Thus, Matrigel contains chondrogenic activity distinct from TGF-beta or FGF. The aged host may produce factors that are inhibitory to mesenchymal cell differentiation and adversely affect cartilage formation and repair.

  10. Human macrophage colony-stimulating factor induces macrophage colonies after L-phenylalanine methylester treatment of human marrow.

    PubMed

    Rosenfeld, C S; Evans, C; Shadduck, R K

    1990-11-01

    Macrophage-colony stimulating factor (M-CSF) has well-known effects on murine bone marrow, but its colony stimulating activity for human bone marrow is controversial. After treatment of human bone marrow with L-phenylalanine methylester (PME), macrophage-colonies (CFU-M) were induced by M-CSF in a dose-dependent fashion. The optimal concentration of recombinant human-macrophage colony stimulating factor (rhM-CSF) was 1,000 U/mL. Purified human urine M-CSF had colony stimulating activity similar to rhM-CSF. Further studies were performed to determine the factors responsible for the enhanced CFU-M formation from PME treated marrow. Compared with nylon wool and carbonyl iron monocyte depletion methods, PME eliminated significantly more monocytes and myeloid cells. This observation suggested that these cells may release hematopoietic inhibitory factors for CFU-M. Low concentrations (1%) but not normal (10%) concentrations of blood monocytes were inhibitory (mean inhibition, 48%) to CFU-M. High concentrations of monocytes (50%) augmented CFU-M colonies. HL-60 conditioned media was used to simulate secretory products of early myeloid cells. HL-60 conditioned media (1%) inhibited CFU-M formation but not granulocyte macrophage or granulocyte colonies. We conclude that M-CSF has colony stimulating activity for human marrow that can be recognized after removal of inhibitory cells by PME treatment. PMID:2224128

  11. 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

  12. 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.

  13. Gene Expression in Mouse Thyrotrope Adenoma: Transcription Elongation Factor Stimulates Proliferation.

    PubMed

    Gergics, Peter; Christian, Helen C; Choo, Monica S; Ajmal, Adnan; Camper, Sally A

    2016-09-01

    Thyrotrope hyperplasia and hypertrophy are common responses to primary hypothyroidism. To understand the genetic regulation of these processes, we studied gene expression changes in the pituitaries of Cga(-/-) mice, which are deficient in the common α-subunit of TSH, LH, and FSH. These mice have thyrotrope hypertrophy and hyperplasia and develop thyrotrope adenoma. We report that cell proliferation is increased, but the expression of most stem cell markers is unchanged. The α-subunit is required for secretion of the glycoprotein hormone β-subunits, and mutants exhibit elevated expression of many genes involved in the unfolded protein response, consistent with dilation and stress of the endoplasmic reticulum. Mutants have elevated expression of transcription factors that are important in thyrotrope function, such as Gata2 and Islet 1, and those that stimulate proliferation, including Nupr1, E2f1, and Etv5. We characterized the expression and function of a novel, overexpressed gene, transcription elongation factor A (SII)-like 5 (Tceal5). Stable expression of Tceal5 in a pituitary progenitor cell line is sufficient to increase cell proliferation. Thus, Tceal5 may act as a proto-oncogene. This study provides a rich resource for comparing pituitary transcriptomes and an analysis of gene expression networks. PMID:27580811

  14. 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.

  15. An Analysis of Scalp Thickness and Other Novel Risk Factors for Deep Brain Stimulator Infections

    PubMed Central

    Nehrbass, Elena; McInerney, James

    2016-01-01

    Introduction: Deep brain stimulator (DBS) infections are a persistent problem for patients undergoing this procedure. They may require further surgery, treatment with antibiotics, or even removal of the device. To date, no consensus exists on the best practices to avoid DBS infections or what factors predispose patients to an eventual infection. The goal of this study was to examine several patient factors for association with DBS infection. Methods: A single-center, single-surgeon quality improvement database was queried. All patients who experienced an infection were identified. The primary variable analyzed was scalp thickness. Other pre-specified, secondary variables included routine intraoperative cultures, operative time, diagnosis, and age. Results: None of the independent variables examined were significantly associated with DBS infections. Only two of the 46 infections qualified as surgical site infections as defined by the Centers for Disease Control. Conclusion: DBS infections are independent of all of the predictor variables analyzed. Surgical site infections, according to traditional definitions, are not the optimal definition for evaluating DBS infections/erosions. New studies must examine new variables that are not routinely gathered in this population. Also, because of the rare event rates and difficulty in randomizing patients to exposures, a large, multicenter registry may be the optimal study design to solve this clinical problem. PMID:27774360

  16. Identification of osteoblast stimulating factor 5 as a negative regulator in the B-lymphopoietic niche.

    PubMed

    Fujita, Natsuko; Ichii, Michiko; Maeda, Tetsuo; Saitoh, Norimitsu; Yokota, Takafumi; Yamawaki, Kengo; Kakitani, Makoto; Tomizuka, Kazuma; Oritani, Kenji; Kanakura, Yuzuru

    2015-11-01

    Recent studies have revealed the crucial role of the niche which supports B-lymphocyte differentiation from hematopoietic stem cells. In this study, we aimed to identify a novel regulator of B lymphopoiesis secreted in the specific niche using the signal sequence trap method. Among the identified proteins from MS5 stromal cells, expression of pleiotrophin, placental proliferin 2, and osteoblast stimulating factor 5 (OSF-5) was dominantly high in several stromal cell lines. We found that OSF-5 suppressed early B lymphopoiesis in transgenic mice producing the target protein. The number of pre-B and immature B cells was reduced by more than half compared with control in the transgenic mice. In vitro studies showed that a secreted variant of OSF-5 inhibited the proliferation and colony formation of pre-B cells, whereas cell-intrinsic form had no influence on B lymphopoiesis. The main components of the B-lymphopoietic niche, osteoblasts in mice and mesenchymal cells in humans, are primary producers of OSF-5. These results define a novel mechanism of B lymphopoiesis in bone marrow. In the specific niche, B-lymphocyte differentiation is fine-tuned by negative regulators as well as supportive factors. PMID:26213229

  17. 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.

  18. In vivo stimulation of a chimeric promoter by binding sites for nuclear factor I.

    PubMed Central

    Knox, J J; Rebstein, P J; Manoukian, A; Gronostajski, R M

    1991-01-01

    Nuclear factor I (NFI) is composed of a family of site-specific DNA-binding proteins which recognize a DNA-binding site with the consensus sequence TGGC/A(N)5GCCAA. Binding sites for NFI have previously been shown to stimulate mRNA synthesis in vitro when present upstream of the TATA box of the adenovirus major late promoter (AdMLP). We have examined the effect of NFI-binding sites on transcription in vivo in transiently transfected HeLa and COS cells. An NFI-binding site isolated from the human genome activated expression from the minimal AdMLP in vivo in both the absence and presence of the simian virus 40 enhancer. A point mutation that decreased NFI binding affinity for the site in vitro reduced expression to near the basal level of the AdMLP. Several NFI-binding sites which differed in their spacer and flanking sequences were tested for their ability to activate expression in vivo. The ability of these sites to activate expression correlated with the strength of NFI binding in vitro. An NFI-binding site stimulated expression equally well when placed from 33 to 65 bp upstream of the TATA box. However, expression dropped to basal levels when the site was located from 71 to 77 bp upstream of the TATA box. These studies indicate that an NFI-binding site in this chimeric promoter activates expression in vivo only if located within a critical distance of the TATA box. PMID:1903836

  19. Collagen degradation and platelet-derived growth factor stimulate the migration of vascular smooth muscle cells.

    PubMed

    Stringa, E; Knäuper, V; Murphy, G; Gavrilovic, J

    2000-06-01

    Cell migration is a key event in many biological processes and depends on signals from both extracellular matrix and soluble motogenic factors. During atherosclerotic plaque development, vascular smooth muscle cells migrate from the tunica media to the intima through a basement membrane and interstitial collagenous matrix and proliferate to form a neointima. Matrix metalloproteinases have previously been implicated in neointimal formation and in this study smooth muscle cell adhesion and migration on degraded collagen have been evaluated. Vascular smooth muscle cells adhered to native intact collagen type I and to its first degradation by-product, 3/4 fragment (generated by collagenase-3 cleavage), unwound at 35 degrees C to mimic physiological conditions. PDGF-BB pre-treatment induced a fourfold stimulation of smooth muscle cell motility on the collagen 3/4 fragment whereas no increase in smooth muscle cell motility on collagen type I was observed. Cell migration on collagen type I was mediated by alpha2 integrin, whereas PDGF-BB-stimulated migration on the 3/4 collagen fragment was dependent on alphavbeta3 integrin. alphavbeta3 integrin was organised in clusters concentrated at the leading and trailing edges of the cells and was only expressed when cells were exposed to the 3/4 collagen fragment. Tyrphostin A9, an inhibitor of PDGF receptor-beta tyrosine kinase activity, resulted in complete abolition of migration of PDGF-BB treated cells on collagen type I and 3/4 fragment. These results strongly support the hypothesis that the cellular migratory response to soluble motogens can be regulated by proteolytic modification of the extracellular matrix. PMID:10806116

  20. Role of macrophages in serum colony-stimulating factor induction by Lactobacillus casei in mice.

    PubMed Central

    Nanno, M; Shimizu, T; Mike, A; Ohwaki, M; Mutai, M

    1988-01-01

    Heat-killed Lactobacillus casei YIT9018 (LC9018), when injected intravenously into mice at a dose of 4 to 40 mg/kg, induced the production of serum colony-stimulating factor (CSF). Since this induction was observed in both C3H/HeJ and C3H/HeN mice, LC9018 was considered to act differently from lipopolysaccharide. The amount of serum CSF induced by LC9018 in nude mice and whole-body-X-ray-irradiated mice was similar to that in control mice, but the induction of serum CSF was suppressed by the previous administration of carrageenan, indicating that macrophages, but not T cells, were responsible for serum CSF induction by LC9018. To determine whether macrophages themselves produce CSF or help other cells produce CSF in response to LC9018, we prepared adherent cells from the peritoneal cavity of normal mice and examined CSF activity in their conditioned media. Peritoneal adherent cells did not produce CSF without LC9018, but when cultivated with 1 mg of LC9018 per ml, they produced CSF at the same time that serum CSF was induced after the intravenous administration of LC9018. Additionally, in vitro-induced CSF formed macrophage, granulocyte, and mixed colonies, as serum CSF did. CSF production by peritoneal adherent cells was completely inhibited by cycloheximide (50 micrograms/ml), and neither the elimination of T cells from the peritoneal adherent cells by treating them with anti-Thy-1.2 antibody plus complement nor the addition of T cells affected CSF production. These results suggest that heat-killed LC9018 induces serum CSF in mice via direct stimulation of macrophages to produce CSF de novo. PMID:3123388

  1. 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. PMID:26980809

  2. Role of macrophage colony-stimulating factor in the development of neointimal thickening following arterial injury.

    PubMed

    Mishra, Vivek; Sinha, Satyesh K; Rajavashisth, Tripathi B

    2016-01-01

    Evidence suggests that macrophage colony-stimulating factor (M-CSF) participates critically in atherosclerosis; little is known about the role of M-CSF in the development of neointimal hyperplasia following mechanical vascular injury. We examined the expression of M-CSF and its receptor, c-fms, in rodent and rabbit models of arterial injury. Injured rat carotid arteries expressed 3- to 10-fold higher levels of M-CSF and c-fms mRNA and protein following balloon injury as compared to uninjured arteries. In the rabbit, M-CSF protein expression was greatest in neointimal smooth muscle cells (SMCs) postinjury, with some expression in medial SMCs. M-CSF-positive SMCs exhibited markers of proliferation. At 30days postinjury, neointimal SMCs in the adjacent healed area near the border between injured and uninjured zone lost both proliferative activity and overexpression of M-CSF. The presence of induced M-CSF and c-fms expression correlated with the initiation of SMCs proliferation. M-CSF stimulated incorporation of [(3)H] thymidine in human aortic smooth muscle cells in a concentration-dependent manner. Serum-free conditioned medium from aortic SMCs also promoted DNA synthesis, and this effect was blocked by M-CSF specific antibody. To test further the role of M-CSF in vivo, we induced arterial injury by placing a periadventitial collar around the carotid arteries in compound mutant mice lacking apolipoprotein apoE (apoE(-/-)) and M-CSF. Loss of M-CSF abolished the neointimal hyperplastic response to arterial injury in apoE(-/-) mice. Local delivery of M-CSF to the injured artery restored neointimal proliferation, suggesting a critical role of M-CSF for the development of neointimal thickening following arterial injury. PMID:27135205

  3. [Relationship between PTEN mutations and protein kinase B phosphorylation caused by insulin or recombinant human epidermal growth factor stimulation].

    PubMed

    Zhong, Hailan; Hu, Xianfu; Lin, Jianhua

    2016-08-01

    Objective To study the effect of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) mutations on protein kinase B (Akt) phosphorylation of CNE-1 nasopharyngeal carcinoma cell line. Methods CNE-1 cells were cultured in RPMI1640 medium containing 100 mL/L fetal calf serum, and then transfected with wild-type PTEN (wtPTEN), mutant PTEN C124S and mutant PTEN G129E plasmid separately. After overnight serum starvation, the cells were stimulated with 0.15 IU/mL insulin or 0.3 μg/mL recombinant human epidermal growth factor (rhEGF). At last, Akt phosphorylation was evaluated by Western blotting. Results Insulin or rhEGF stimulation led to Akt activation in CNE-1 cells. The wtPTEN inhibited insulin- or rhEGF-stimulated phosphorylation of Akt. PTEN C124S mutant activated insulin-stimulated phosphorylation of Akt, but not rhEGF-stimulated phosphorylation of Akt. PTEN G129E mutant inhibited insulin-stimulated phosphorylation of Akt. Conclusion The wtPTEN inhibited insulin- or rhEGF-stimulated phosphorylation of Akt, while PTEN C124S and G129E mutants failed to activate the phosphorylation of Akt consistently. This suggested PTEN mutations might not be correlated with activated Akt.

  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. Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib.

    PubMed

    Dewar, Andrea L; Cambareri, Antony C; Zannettino, Andrew C W; Miller, Bernadette L; Doherty, Kathleen V; Hughes, Timothy P; Lyons, A Bruce

    2005-04-15

    Imatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl-expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)-binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF-induced proliferation of a cytokine-dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.

  6. Melatonin decreases muscular oxidative stress and inflammation induced by strenuous exercise and stimulates growth factor synthesis.

    PubMed

    Borges, Leandro da Silva; Dermargos, Alexandre; da Silva Junior, Edenilson Pinto; Weimann, Eleine; Lambertucci, Rafael Herling; Hatanaka, Elaine

    2015-03-01

    Strenuous exercise is detrimental to athletes because of the overproduction of reactive oxygen species. Melatonin, a classic antioxidant, has been shown to exhibit beneficial effects regarding intense exercise and tissue repair. In this study, we evaluated the onset and resolution of inflammation in melatonin-treated and nontreated rats subjected to a strenuous exercise session. We also analyzed the formation of thiobarbituric acid reactive substances (TBARS) and the activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Control and treated rats were subjected to exhaustive exercise after a period of 10 days of melatonin treatment (20 mg/dL). Plasma and muscle levels of tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), cytokine-induced neutrophil chemoattractant-2-alpha/beta (CINC-2α/β), l-selectin, macrophage inflammatory protein-3-alpha (MIP-3α), and vascular endothelial growth factor (VEGF) were measured prior to, immediately after, and 2 hr after exercise. Our data revealed decreases in the muscle concentrations of IL-1β (35%), TNF-α (13%), IL-6 (48%), and TBARS (40%) in the melatonin-treated group compared with the control group. We also observed decreases in the plasma concentrations of IL-1β (17%) in the melatonin-treated group. VEGF-α concentrations and SOD activity increased by 179% and 22%, respectively, in the melatonin-treated group compared with the control group. We concluded that muscle inflammation and oxidative stress resulting from exhaustive exercise were less severe in the muscles of melatonin-treated animals than in the muscles of control animals. Thus, melatonin treatment may reverse exercise-induced skeletal muscle inflammation and stimulate growth factor synthesis.

  7. CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

    PubMed Central

    Yang, Z; Carter, C D; Miller, M S; Bochsler, P N

    1995-01-01

    The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml). PMID:7528735

  8. 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.

  9. Regulation of urokinase-type plasminogen activator gene transcription by macrophage colony-stimulating factor.

    PubMed Central

    Stacey, K J; Fowles, L F; Colman, M S; Ostrowski, M C; Hume, D A

    1995-01-01

    The mouse urokinase-type plasminogen activator (uPA) gene was used as a model macrophage colony-stimulating factor 1 (CSF-1)-inducible gene to investigate CSF-1 signalling pathways. Nuclear run-on analysis showed that induction of uPA mRNA by CSF-1 and phorbol myristate acetate (PMA) was at the transcriptional level in bone marrow-derived macrophages. CSF-1 and PMA synergized strongly in the induction of uPA mRNA, showing that at least some components of CSF-1 action are mediated independently of protein kinase C. Promoter targets of CSF-1 signalling were investigated with NIH 3T3 cells expressing the human CSF-1 receptor (c-fms). uPA mRNA was induced in these cells by treatment with CSF-1, and a PEA3/AP-1 element at -2.4 kb in the uPA promoter was involved in this response. Ets transcription factors can act through PEA3 sequences, and the involvement of Ets factors in the induction of uPA was confirmed by use of a dominant negative Ets-2 factor. Expression of the DNA binding domain of Ets-2 fused to the lacZ gene product prevented CSF-1-mediated induction of uPA mRNA in NIH 3T3 cells expressing the CSF-1 receptor. Examination of ets-2 mRNA expression in macrophages showed that it was also induced synergistically by CSF-1 and PMA. In the macrophage cell line RAW264, the uPA PEA3/AP-1 element mediated a response to both PMA and cotransfected Ets-2. uPA promoter constructs were induced 60- to 130-fold by Ets-2 expression, and the recombinant Ets-2 DNA binding domain was able to bind to the uPA PEA3/AP-1 element. This work is consistent with a proposed pathway for CSF-1 signalling involving sequential activation of fms, ras, and Ets factors. PMID:7760840

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

    PubMed

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

    1999-05-01

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

  11. 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.

  12. 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.

  13. Regulatory elements responsible for inducible expression of the granulocyte colony-stimulating factor gene in macrophages.

    PubMed Central

    Nishizawa, M; Nagata, S

    1990-01-01

    Granulocyte colony-stimulating factor (G-CSF) plays an essential role in granulopoiesis during bacterial infection. Macrophages produce G-CSF in response to bacterial endotoxins such as lipopolysaccharide (LPS). To elucidate the mechanism of the induction of G-CSF gene in macrophages or macrophage-monocytes, we have examined regulatory cis elements in the promoter of mouse G-CSF gene. Analyses of linker-scanning and internal deletion mutants of the G-CSF promoter by the chloramphenicol acetyltransferase assay have indicated that at least three regulatory elements are indispensable for the LPS-induced expression of the G-CSF gene in macrophages. When one of the three elements was reiterated and placed upstream of the TATA box of the G-CSF promoter, it mediated inducibility as a tissue-specific and orientation-independent enhancer. Although this element contains a conserved NF-kappa B-like binding site, the gel retardation assay and DNA footprint analysis with nuclear extracts from macrophage cell lines demonstrated that nuclear proteins bind to the DNA sequence downstream of the NF-kappa B-like element, but not to the conserved element itself. The DNA sequence of the binding site was found to have some similarities to the LPS-responsive element which was recently identified in the promoter of the mouse class II major histocompatibility gene. Images PMID:1691438

  14. Granulocyte colony-stimulating factor-producing hepatocellular carcinoma with abrupt changes

    PubMed Central

    Nagata, Hiroaki; Komatsu, Shuhei; Takaki, Wataru; Okayama, Tokunari; Sawabe, Yasunori; Ishii, Michiaki; Kishimoto, Mitsuo; Otsuji, Eigo; Konosu, Hiroshi

    2016-01-01

    Granulocyte colony-stimulating factor (G-CSF)-producing tumor is one of the rare types of cancer clinically characterized by an elevated fever and white blood cell (WBC) increment. Although G-CSF producing tumors have been reported in several types of cancer including those of the lungs, cervix and bladder, G-CSF producing hepatocellular carcinoma is extremely rare. Here, we report the case of a rapidly growing and poorly differentiated hepatocellular carcinoma producing G-CSF. The patient showed symptoms of continuous high fever, stomach pain and cough, and high serum WBC counts, C-reactive protein (CRP) and G-CSF levels were found in laboratory tests. After a radical hepatectomy, the patient completely recovered from the above symptoms and inflammatory state. The serum levels of G-CSF were reduced to normal levels after radical surgery. An immunohistochemical analysis revealed the overexpression of G-CSF in the cytoplasm of certain hepatocellular carcinoma (HCC) cell. The patient’s serum WBC, CRP and G-CSF levels remained within normal levels in the six months after surgery without recurrence. This is the 9th case report of G-CSF producing hepatocellular carcinoma in English literature. We review the clinical characteristics of the G-CSF producing HCC and discuss a possible treatment strategy. PMID:27777880

  15. 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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2015-01-01

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

  18. Neonatal thyroid-stimulating hormone level is influenced by neonatal, maternal, and pregnancy factors.

    PubMed

    Trumpff, Caroline; Vandevijvere, Stefanie; Moreno-Reyes, Rodrigo; Vanderpas, Jean; Tafforeau, Jean; Van Oyen, Herman; De Schepper, Jean

    2015-11-01

    The percentage of newborns with a neonatal whole blood thyroid-stimulating hormone (TSH) greater than 5 mIU/L has been used as an indicator of iodine deficiency at the population level. However, TSH levels in newborns may be influenced by many factors other than iodine status. The objective of this study was to identify neonatal, maternal, and pregnancy-related determinants of neonatal TSH levels in a retrospective cohort study. The study sample included 313 Belgian mothers and their 4- to 5-year-old children. The children had a neonatal TSH concentration between 0 and 15 mIU/L at neonatal screening, and blood samples were collected 3 to 5 days after birth. Children with suspected congenital hypothyroidism (neonatal TSH level >15 mIU/L), prematurely born (i.e., <37 weeks), or with a low birth weight (i.e., <2500 g) were excluded. Information about maternal and birth-related determinants was collected from the neonatal screening center via a self-administered questionnaire filled in by the mother together with the child's health booklet. Higher TSH levels were found in spring and winter compared to summer and autumn (P = .011). Higher TSH levels were associated with lifetime smoking behavior (up to child birth) in the mother (P = .005), lower weight gain during pregnancy (P = .014), and longer pregnancies (P = .003). This study showed that several neonatal, maternal, and pregnancy-related determinants are influencing neonatal TSH level. PMID:26428622

  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. 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.

  2. Exogenous nerve growth factor stimulates choline acetyltransferase activity in aging Fischer 344 male rats.

    PubMed

    Williams, L R

    1991-01-01

    The effect of age and exogenous nerve growth factor (NGF) infusion on choline acetyltransferase (ChAT) specific activity is examined in microdissections of cerebral and hippocampal cortices, and the cholinergic nuclei of the medial septum and diagonal band of Broca (MS/DB), the nucleus basalis magnocellularis (NBM), and striatum of Fischer 344 male rats. Significant, 20% losses in ChAT activity are found in the MS/DB and striatum of 24-month-old rats (n = 21) compared to 4-month-old animals, but there is no apparent loss of enzyme activity in the NBM. Loss of ChAT activity in the MS/DB is only observed in animals older than 19 months of age, while a striatal deficit is found in animals older than 7 months. Treatment for 2 weeks with NGF at 1.2 micrograms/day results in significant 70% increases of ChAT activity in the MS/DB and striatum of 24-month-old rats compared to untreated and vehicle-treated 4-month-old rats, but does not stimulate activity in the NBM. Sensitivity of ChAT activity in the MS/DB and striatum to exogenous NGF increases with age. These experiments indicate that in the MS/DB, NBM, and striatum of Fischer 344 male rat there is an age-associated, differential regulation of ChAT enzyme activity and sensitivity to exogenous NGF.

  3. 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.

  4. Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

    PubMed Central

    Rosa, Renata G.; Joazeiro, Paulo P.; Bianco, Juares; Kunz, Manuela; Weber, Joanna F.; Waldman, Stephen D.

    2014-01-01

    The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation. PMID:25126941

  5. Colony-stimulating Factor-1 Receptor Utilizes Multiple Signaling Pathways to Induce Cyclin D2 Expression

    PubMed Central

    Dey, Arunangsu; She, Hongyun; Kim, Leopold; Boruch, Allan; Guris, Deborah L.; Carlberg, Kristen; Sebti, Saïd M.; Woodley, David T.; Imamoto, Akira; Li, Wei

    2000-01-01

    Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src−/− bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes. PMID:11071910

  6. 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…

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

    PubMed Central

    1994-01-01

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

  8. Synergistic action of the benzene metabolite hydroquinone on myelopoietic stimulating activity of granulocyte/macrophage colony-stimulating factor in vitro.

    PubMed Central

    Irons, R D; Stillman, W S; Colagiovanni, D B; Henry, V A

    1992-01-01

    The effects of in vitro pretreatment with benzene metabolites on colony-forming response of murine bone marrow cells stimulated with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) were examined. Pretreatment with hydroquinone (HQ) at concentrations ranging from picomolar to micromolar for 30 min resulted in a 1.5- to 4.6-fold enhancement in colonies formed in response to rGM-CSF that was due to an increase in granulocyte/macrophage colonies. The synergism equaled or exceeded that reported for the effects of interleukin 1, interleukin 3, or interleukin 6 with GM-CSF. Optimal enhancement was obtained with 1 microM HQ and was largely independent of the concentration of rGM-CSF. Pretreatment with other authentic benzene metabolites, phenol and catechol, and the putative metabolite trans, trans-muconaldehyde did not enhance growth factor response. Coadministration of phenol and HQ did not enhance the maximal rGM-CSF response obtained with HQ alone but shifted the optimal concentration to 100 pM. Synergism between HQ and rGM-CSF was observed with nonadherent bone marrow cells and lineage-depleted bone marrow cells, suggesting an intrinsic effect on recruitment of myeloid progenitor cells not normally responsive to rGM-CSF. Alterations in differentiation in a myeloid progenitor cell population may be of relevance in the pathogenesis of acute myelogenous leukemia secondary to drug or chemical exposure. PMID:1570288

  9. 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.

  10. Variables affecting production of monocyte chemotactic factor 1 from human leukocytes stimulated with Cryptococcus neoformans.

    PubMed Central

    Levitz, S M; North, E A; Jiang, Y; Nong, S H; Kornfeld, H; Harrison, T S

    1997-01-01

    The chemokine monocyte chemoattractant protein 1 (MCP-1) is produced predominantly by mononuclear phagocytes and stimulates recruitment into infected tissues of blood monocytes and T cells. These cell types are thought to be critical to host defenses against infections due to Cryptococcus neoformans, a major cause of disease in persons with AIDS and other disorders of cell-mediated immunity. Accordingly, in the present study, we examined the conditions under which human monocytes and bronchoalveolar macrophages (BAM) are stimulated by C. neoformans to produce MCP-1. C. neoformans was a potent inducer of MCP-1 release from monocytes, with levels of chemokine secreted similar to that seen following stimulation with lipopolysaccharide (LPS). BAM, in contrast, were stimulated by LPS, but not by C. neoformans, to secrete MCP-1. A peak in MCP-1 mRNA was seen 8 h following cryptococcal stimulation of monocytes. Nine strains of C. neoformans stimulated monocytes to release MCP-1, and there was only modest variation between strains. However, when an individual strain was used, the capacity of C. neoformans to stimulate monocyte MCP-1 release did vary, depending upon the conditions used to grow the fungal stimuli. Finally, C. neoformans stimulated comparable quantities of MCP-1 release in monocytes from donors with and without human immunodeficiency virus infection. These data establish C. neoformans as a potent stimulator of MCP-1 in monocytes, but not in BAM. The failure of C. neoformans to stimulate MCP-1 in BAM, if occurring in vivo, could result in a diminished cell-mediated inflammatory response following inhalation of airborne fungi. PMID:9038295

  11. Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor

    PubMed Central

    Tan, Yaohong; Shao, Hongwei; Eton, Darwin; Yang, Zhe; Alonso-Diaz, Luis; Zhang, Hongkun; Schulick, Andrew; Livingstone, Alan S.; Yu, Hong

    2008-01-01

    Objective Granulocyte colony-stimulating factor (G-CSF) mobilizes bone marrow mononuclear cells into the peripheral circulation. Stromal cell-derived factor-1 (SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue. We hypothesize that SDF-1 will boost the pro-angiogenic effect of G-CSF. Methods and results NIH 3T3 cells retrovirally transduced with SDF-1α gene (NIH 3T3/SDF-1) were used to deliver SDF-1 in vitro and in vivo. Endothelial progenitor cells (EPCs) co-cultured with NIH 3T3/SDF-1 cells using cell culture inserts migrated faster and were less apoptotic compared to those not exposed to SDF-1. NIH 3T3/SDF-1 (106 cells) were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. G-CSF (25 μg/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system. The perfusion ratio of ischemic/non-ischemic limb increased to 0.57±0.03 and 0.50±0.06 with the treatment of either SDF-1 or G-CSF only, respectively, 3 weeks after surgery, which was significantly higher than the saline-injected control group (0.41±0.01, P<0.05). Combined treatment with both SDF-1 and G-CSF resulted in an even better perfusion ratio of 0.69±0.08 (P<0.05 versus the single treatment groups). Mice were sacrificed 21 days after surgery. Immunostaining and Western blot assay of the tissue lysates showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1. CD34+ cells were detected with immunostaining, capillary density was assessed with alkaline phosphatase staining, and the apoptosis of muscle cells was viewed using an in situ cell death detection kit. More CD34+ cells, increased capillary density, and less apoptotic muscle cells were found in both G-CSF and SDF-1 treated group (P<0.05 versus other groups). Conclusion Combination of G-CSF-mediated progenitor cell

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

    PubMed Central

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

    2013-01-01

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

  13. Granulocyte-colony stimulating factor therapy to induce neovascularization in ischemic heart disease.

    PubMed

    Ripa, Rasmus Sejersten

    2012-03-01

    Cell based therapy for ischemic heart disease has the potential to reduce post infarct heart failure and chronic ischemia. Treatment with granulocyte-colony stimulating factor (G-CSF) mobilizes cells from the bone marrow to the peripheral blood. Some of these cells are putative stem or progenitor cells. G-CSF is injected subcutaneously. This therapy is intuitively attractive compared to other cell based techniques since repeated catheterizations and ex vivo cell purification and expansion are avoided. Previous preclinical and early clinical trials have indicated that treatment with G-CSF leads to improved myocardial perfusion and function in acute or chronic ischemic heart disease. The hypothesis of this thesis is that patient with ischemic heart disease will benefit from G-CSF therapy. We examined this hypothesis in two clinical trials with G-CSF treatment to patients with either acute myocardial infarction or severe chronic ischemic heart disease. In addition, we assed a number of factors that could potentially affect the effect of cell based therapy. Finally, we intended to develop a method for in vivo cell tracking in the heart. Our research showed that subcutaneous G-CSF along with gene therapy do not improve myocardial function in patients with chronic ischemia despite a large increase in circulation bone marrow-derived cells. Also, neither angina pectoris nor exercise capacity was improved compared to placebo treatment. We could not identify differences in angiogenic factors or bone marrow-derived cells in the blood that could explain the neutral effect of G-CSF. Next, we examined G-CSF as adjunctive therapy following ST segment elevation myocardial infarction. We did not find any effect of G-CSF neither on the primary endpoint--regional myocardial function--nor on left ventricular ejection fraction (secondary endpoint) compared to placebo treatment. In subsequent analyses, we found significant differences in the types of cells mobilized from the bone marrow

  14. Modulation of the endocannabinoid system: vulnerability factor and new treatment target for stimulant addiction.

    PubMed

    Olière, Stéphanie; Joliette-Riopel, Antoine; Potvin, Stéphane; Jutras-Aswad, Didier

    2013-01-01

    Cannabis is one of the most widely used illicit substance among users of stimulants such as cocaine and amphetamines. Interestingly, increasing recent evidence points toward the involvement of the endocannabinoid system (ECBS) in the neurobiological processes related to stimulant addiction. This article presents an up-to-date review with deep insights into the pivotal role of the ECBS in the neurobiology of stimulant addiction and the effects of its modulation on addictive behaviors. This article aims to: (1) review the role of cannabis use and ECBS modulation in the neurobiological substrates of psychostimulant addiction and (2) evaluate the potential of cannabinoid-based pharmacological strategies to treat stimulant addiction. A growing number of studies support a critical role of the ECBS and its modulation by synthetic or natural cannabinoids in various neurobiological and behavioral aspects of stimulants addiction. Thus, cannabinoids modulate brain reward systems closely involved in stimulants addiction, and provide further evidence that the cannabinoid system could be explored as a potential drug discovery target for treating addiction across different classes of stimulants. PMID:24069004

  15. 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...

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

    PubMed Central

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

    1990-01-01

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

  17. 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

    2014-11-01

    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.

  18. In-vivo blockage of neutrophil migration by LPS is mimicked by a factor released from LPS-stimulated macrophages.

    PubMed Central

    Cunha, F. Q.; Souza, G. E.; Souza, C. A.; Cerqueira, B. C.; Ferreira, S. H.

    1989-01-01

    The present study was performed to determine the effect of an intravenous injection of the macrophage-derived neutrophil chemotactic factor (MNCF) (Cunha & Ferreira 1986) on neutrophil migration to rat peritoneal cavities, which were challenged with chemotactic stimuli. Macrophage monolayers stimulated by LPS release a factor (MW greater than 10,000 D) which, when injected intravenously, blocked neutrophil migration in carrageenin-induced peritonitis. This inhibition was dependent on dose and lasted more than 2 h. It was not due to neutropaenia, hypotension or LPS contamination. Neutrophil migration induced by LPS, MNCF, the Gram-negative bacterium Pseudomonas aeruginosa was also blocked by intravenous administration of the factor. Intravenous injection of recombinant interleukin 1 beta or tumour necrosis factor-alpha, present in the samples of the factor, failed to reproduce the described inhibitory effect on neutrophil migration. The release of this factor by LPS-stimulated macrophage monolayers was inhibited by dexamethasone but not by indomethacin. It is suggested that the failure of neutrophils to migrate during septicaemia may be the result of a continuous release of chemotactic factors in the circulation, particularly of the macrophage-derived neutrophil chemotactic factor(s). PMID:2647118

  19. Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation.

    PubMed

    Langlois, W J; Sasaoka, T; Saltiel, A R; Olefsky, J M

    1995-10-27

    Insulin and epidermal growth factor receptors transmit signals for cell proliferation and gene regulation through formation of active GTP-bound p21ras mediated by the guanine nucleotide exchange factor Sos. Sos is constitutively bound to the adaptor protein Grb2 and growth factor stimulation induces association of the Grb2/Sos complex with Shc and movement of Sos to the plasma membrane location of p21ras. Insulin or epidermal growth factor stimulation induces a rapid increase in p21ras levels, but after several minutes levels decline toward basal despite ongoing hormone stimulation. Here we show that deactivation of p21ras correlates closely with phosphorylation of Sos and dissociation of Sos from Grb2, and that inhibition of mitogen-activated protein (MAP) kinase kinase (also known as extracellular signal-related kinase (ERK) kinase, or MEK) blocks both events, resulting in prolonged p21ras activation. These data suggest that a negative feedback loop exists whereby activation of the Raf/MEK/MAP kinase cascade by p21ras causes Sos phosphorylation and, therefore, Sos/Grb2 dissociation, limiting the duration of p21ras activation by growth factors. A serine/threonine kinase downstream of MEK (probably MAP kinase) mediates this desensitization feedback pathway.

  20. Effect of recombinant human granulocyte colony-stimulating factor on granulocytopenia in mice induced by irradiation

    SciTech Connect

    Fushiki, M.; Ono, K.; Sasai, K.; Shibamoto, Y.; Tsutsui, K.; Nishidai, T.; Takahashi, M.; Abe, M. )

    1990-02-01

    We report the effect of human granulocyte colony-stimulating factor (hG-CSF) on the recovery from granulocytopenia induced by irradiation. Female 9-week old C3H/He mice were used. The irradiation schedule was as follows: Group 1 and 2 received whole-body irradiation of 1 Gy and 5 Gy, respectively, on day 0; Group 3 and 4 received whole-body irradiation of 0.5 and 1.0 Gy, respectively, for 5 consecutive days; Group 5 received upper hemibody irradiation of 3 Gy for 5 consecutive days. Daily subcutaneous injections of G-CSF (3 x 10(5) Unit/mouse) or 0.3 ml of saline to each group were started from the day after the first irradiation and continued for 18 days. Mice were sampled randomly from each group, and the total number of leukocytes, erythrocytes of peripheral blood, nucleated cells in femur, and spleen weight were counted and measured, respectively, on day 0, 3, 5, 7, 9, 12, and 18. The leukocyte counts decreased with an increase in radiation doses. In Group 1 and 2 mice, G-CSF enhanced the leukocyte count more than saline. In Group 3 mice, the recovery of leukocytopenia was facilitated by G-CSF, but in Group 4 mice, G-CSF had no effect on the leukocyte count decrease or on leukocytopenia recovery. In Group 5 mice, G-CSF greatly affected leukocytopenia recovery. Increase in spleen weight paralleled the peripheral leukocyte count. Daily administration of recombinant hG-CSF accelerated the granulocytopenia recovery which was induced by irradiation, and it may be a useful therapeutic agent for treating myelosuppressive cases.

  1. Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response.

    PubMed

    Mitrasinovic, Olivera M; Robinson, Christopher C; Tenen, Daniel G; Lee, Yuen Ling; Poon, Clara; Murphy, Greer M

    2004-08-01

    The receptor for macrophage colony-stimulating factor (M-CSFR; c-fms) is expressed at increased levels by microglia in Alzheimer's disease (AD) and in mouse models for AD. Increased expression of M-CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M-CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac-1 integrin alpha subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c-fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c-fms construct resulted in expression of the c-fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M-CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)-1alpha macrophage inflammatory protein (MIP-1alpha), and trends toward increased production of IL-6 and M-CSF. These findings demonstrate that microglial overexpression of M-CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M-CSFR could contribute to the inflammatory response observed in AD brain.

  2. The effects of granulocyte colony-stimulating factor in preclinical models of infection and acute inflammation.

    PubMed

    Marshall, John C

    2005-12-01

    The cytokine granulocyte colony-stimulating factor (G-CSF) is a potent endogenous trigger for the release of neutrophils from bone marrow stores and for their activation for enhanced antimicrobial activity. G-CSF has been widely evaluated in preclinical models of acute illness, with generally promising though divergent results. A recombinant G-CSF molecule has recently undergone clinical trials to assess its efficacy as an adjuvant therapy in community-acquired and nosocomial pneumonia, however, these studies failed to provide convincing evidence of benefit. We undertook a systematic review of the published literature reporting the effects of modulation of G-CSF in preclinical in vivo models to determine whether evidence of differential efficacy might explain the disappointing results of human studies and point to disease states that might be more likely to benefit from G-CSF therapy. G-CSF has been evaluated in 86 such studies involving a variety of different models. The strongest evidence of benefit was seen in studies involving intraperitoneal challenge with live organisms; benefit was evident whether the agent was given before or after challenge. G-CSF demonstrates anti-inflammatory activity in models of systemic challenge with viable organisms or endotoxin, but only when the agent is given before challenge; evidence of benefit after challenge was minimal. Preclinical models of intrapulmonary challenge only show efficacy when the cytokine is administered before the infectious challenge, and suggested harm in gram-negative pneumonia resulting from challenge with Escherichia coli or Klebsiella. There is little evidence for therapeutic efficacy in noninfectious models of acute illness. We conclude that the most promising populations for evaluation of G-CSF are neutropenic patients with invasive infection and patients with intra-abdominal infection, particularly those with the syndrome of tertiary, or recurrent, peritonitis. Significant variability in the design

  3. Clinical safety of tbo-filgrastim, a short-acting human granulocyte colony-stimulating factor.

    PubMed

    Pettengell, Ruth; Bias, Peter; Mueller, Udo; Lang, Nicole

    2016-06-01

    The recombinant human granulocyte colony-stimulating factor (G-CSF) known as filgrastim (Tevagrastim(®), Ratiograstim(®), Biograstim(®)) in Europe (approved in 2008) and tbo-filgrastim (Granix(®)) in the USA (approved in 2012; Teva Pharmaceutical Industries Ltd., Petach Tikva, Israel) is indicated to reduce the duration of severe neutropenia in patients with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs associated with a clinically significant incidence of febrile neutropenia. This article presents pooled clinical data for tbo-filgrastim compared with Neupogen(®) (Amgen, Thousand Oaks, CA, USA) as well as tbo-filgrastim post-marketing safety data. The safety and efficacy of tbo-filgrastim were evaluated in three phase III studies in 677 patients receiving myelosuppressive chemotherapy and study drug (348 patients with breast cancer, 237 with lung cancer, 92 with non-Hodgkin lymphoma). In each study, the efficacy of tbo-filgrastim was similar to that of Neupogen. Overall, 633 (93.5 %) patients receiving the study drug experienced 6093 treatment-emergent adverse events (AEs), most of which were related to chemotherapy. Adverse events related to the study drug (tbo-filgrastim or Neupogen) were experienced by 185 (27.3 %) patients; 19 (2.8 %) had severe drug-related AEs, 5 (0.7 %) had drug-related serious AEs, and 6 (0.9 %) discontinued the study due to drug-related AEs. Overall, the most common drug-related AEs were bone pain (7.1 %), myalgia (4.0 %), and asthenia (4.4 %). The post-marketing safety profile of tbo-filgrastim was consistent with that observed during the clinical studies. The availability of tbo-filgrastim, a G-CSF with safety and efficacy comparable to those of Neupogen, provides physicians with an alternative treatment option for supportive care of patients with non-myeloid malignancies receiving myelosuppressive chemotherapy. PMID:26780505

  4. L1CAM stimulates glioma cell motility and proliferation through the fibroblast growth factor receptor.

    PubMed

    Mohanan, Vishnu; Temburni, Murali K; Kappes, John C; Galileo, Deni S

    2013-04-01

    The L1CAM cell adhesion/recognition molecule (L1, CD171) and fibroblast growth factor receptor (FGFR) both are expressed by human high-grade glioma cells, but their potential actions in controlling cell behavior have not been linked. L1 actions in cancer cells have been attributed mainly to integrin receptors, and we demonstrated previously that L1-stimulated glioma cell migration correlates with integrin expression, increased focal adhesion kinase activation and focal complex turnover. Our analyses of datasets revealed FGFR is overexpressed in glioma regardless of grade, while ADAM10 metalloprotease expression increases with glioma grade. Here, we used dominant-negative and short hairpin RNA approaches to inhibit the activation of FGFR1 and expression of L1, respectively. An L1 peptide that inhibits L1-FGFR interaction and PD173074, a chemical inhibitor of FGFR1 activity, also were used to elucidate the involvement of L1-FGFR interactions on glioma cell behavior. Time-lapse cell motility studies and flow cytometry cell cycle analyses showed that L1 operates to increase glioma cell motility and proliferation through FGFR activation. Shutdown of both L1 expression and FGFR activity in glioma cells resulted in a complete termination of cell migration in vitro. These studies show for the first time that soluble L1 ectodomain (L1LE) acts on glioma cells through FGFRs, and that FGFRs are used by glioma cells for increasing motility as well as proliferation in response to activation by L1LE ligand. Thus, effective treatment of high-grade glioma may require simultaneous targeting of L1, FGFRs, and integrin receptors, which would reduce glioma cell motility as well as proliferation.

  5. NF-κB family of transcription factors: biochemical players of CD28 co-stimulation.

    PubMed

    Tuosto, Loretta

    2011-03-30

    The signalling pathways that lead from antigen-receptor and/or co-receptor triggering to the activation of transcription factors of the NF-κB family have a crucial role in the regulation of immune responses. The understanding of the molecular mechanisms that control NF-κB activation in lymphocytes has, therefore, important implications for the therapy of immune diseases. CD28 is one of the most important co-stimulatory receptors necessary for full T lymphocyte activation. CD28-mediated signals lower T cell receptor (TCR) activation threshold, thus leading to the enhancement of several T cell functions, including cytokine production, cell cycle progression, survival and regulation of both cytotoxic and humoral T cell responses. However, most of these pathways are under the tight control of TCR and may be bypassed by repeated antigen stimulation. On the contrary, the activation of the NF-κB pathway and NF-κB-regulated genes is a unique feature of CD28. The ultimate nature of CD28 signalling relies on its cytoplasmic tail and on its ability to recruit several signalling mediators involved in coupling CD28 to distinct NF-κB cascades. This review will focus on the current knowledge of the molecular mechanisms whereby CD28 co-operates with the TCR in activating NF-κB. We also describe recent finding on the existence of autonomous signals emanating from CD28, which through a non-conventional NF-κB cascade may account for the critical role of CD28 in regulating cytokine/chemokine production and T cell survival.

  6. Characterization of the receptor binding determinants of granulocyte colony stimulating factor.

    PubMed Central

    Young, D. C.; Zhan, H.; Cheng, Q. L.; Hou, J.; Matthews, D. J.

    1997-01-01

    We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF. The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild-type G-CSF and the E19A mutant were expressed in E. coli. The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF. Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex. Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor. PMID:9194183

  7. Use of granulocyte colony-stimulating factor: a survey among Italian medical oncologists.

    PubMed

    Danova, Marco; Rosti, Giovanni; De Placido, Sabino; Bencardino, Katia; Venturini, Marco

    2005-12-01

    In October 2003, the Italian Association of Medical Oncology (AIOM) published its own guidelines on the use of granulocyte colony-stimulating factor (G-CSF). The present survey was conducted during the same period with the aim of collecting data on the current use of G-CSF to provide a starting point for future evaluations of the implementation of AIOM guidelines. From October 2003 to January 2004, 1591 AIOM members were asked to complete a questionnaire based on specific clinical scenarios, regarding the use of G-CSF for primary and secondary prophylaxis and treatment of neutropenia. The rate of response was 22%. For primary prophylaxis, the majority of physicians avoid using G-CSF, with no difference in cases of adjuvant, curative or palliative chemotherapy (CT). In fact, 67.2% to 74.9% would 'rarely or never' use G-CSF in the proposed clinical scenarios. In chemosensitive tumors, rather than reducing CT doses, 55.7% would use G-CSF as a secondary prophylaxis after afebrile neutropenia (AN), and 68.8% after febrile neutropenia (FN). In elderly patients experiencing FN, 35.7% would reduce the adjuvant CT doses and 23.1% would change the regimen. Most oncologists would use G-CSF to treat neutropenia, and the median duration of G-CSF treatment is less than 1 week and would depend on neutrophil count. Our survey shows that Italian oncologists are particularly oriented towards the use of G-CSF in clinical practice to maintain the CT dose intensity, and are sensitive to the prevention and treatment of not only FN, but also AN. Finally, Italian medical oncologists appear to be very cautious in introducing G-CSF when treating elderly patients. PMID:16273232

  8. 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

  9. 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.

  10. Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

    PubMed

    Jungck, David; Knobloch, Jürgen; Körber, Sandra; Lin, Yingfeng; Konradi, Jürgen; Yanik, Sarah; Stoelben, Erich; Koch, Andrea

    2015-06-01

    Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential

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

    PubMed

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

    2015-04-01

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

  12. Nerve growth factor stimulates the hydrolysis of glycosylphosphatidylinositol in PC-12 cells: A mechanism of protein kinase C regulation

    SciTech Connect

    Chan, B.L.; Saltiel, A.R. ); Chao, M.V. )

    1989-03-01

    Treatment of PC-12 pheochromocytoma cells with nerve growth factor (NGF) results in the differentiation of these cells into a sympathetic neuron-like phenotype. Although the initial intracellular signals elicited by NGF remain unknown, some of the cellular effects of NGF are similar to those of other growth factors, such as insulin. The authors have investigated the involvement of a newly identified inositol-containing glycolipid in signal transduction for the actions of NGF. NGF stimulates the rapid generation of a species of diacylglycerol that is labeled with ({sup 3}H)myristate but not with ({sup 3}H)arachidonate. NGF stimulates ({sup 3}H)myristate- or ({sup 32}P)phosphate-labeled phosphatidic acid production over the same time course. Although NGF alone has no effect on the turnover of inositol phospholipids, it does stimulate the hydrolysis of glycosylphosphatidylinositol. The NGF-dependent cleavage of this lipid is accompanied by an increase in the accumulation of its polar head group, an inositol phosphate glycan, which is generated within 30-60 sec of NGF treatment. In an unresponsive PC-12 mutant cell line, neither the diacylglycerol nor inositol phosphate glycan response is detected. A possible role for the NGF-stimulated diacylglycerol is suggested by the inhibition of NGF-dependent c-fos induction by staurosporin, a potent inhibitor of protein kinase C. These results suggest that, like insulin, some of the cellular effects of NGF may be mediated by the phospholipase C-catalyzed hydrolysis of glycosylphosphatidylinositol.

  13. Association between colony-stimulating factor 1 receptor gene polymorphisms and asthma risk.

    PubMed

    Shin, Eun Kyong; Lee, Shin-Hwa; Cho, Sung-Hwan; Jung, Seok; Yoon, Sang Hyuk; Park, Sung Woo; Park, Jong Sook; Uh, Soo Taek; Kim, Yang Ki; Kim, Yong Hoon; Choi, Jae-Sung; Park, Byung-Lae; Shin, Hyoung Doo; Park, Choon-Sik

    2010-09-01

    Colony-stimulating factor 1 receptor (CSF1R) is expressed in monocytes/macrophages and dendritic cells. These cells play important roles in the innate immune response, which is regarded as an important aspect of asthma development. Genetic alterations in the CSF1R gene may contribute to the development of asthma. We investigated whether CSF1R gene polymorphisms were associated with the risk of asthma. Through direct DNA sequencing of the CSF1R gene, we identified 28 single nucleotide polymorphisms (SNPs) and genotyped them in 303 normal controls and 498 asthmatic patients. Expression of CSF1R protein and mRNA were measured on CD14-positive monocytes and neutrophils in peripheral blood of asthmatic patients using flow cytometry and real-time PCR. Among the 28 polymorphisms, two intronic polymorphism (+20511C>T and +22693T>C) were associated with the risk of asthma by logistic regression analysis. The frequencies of the minor allele at CSF1R +20511C>T and +22693T>C were higher in asthmatic subjects than in normal controls (4.6 vs. 7.7%, p = 0.001 in co-dominant and dominant models; 16.4 vs. 25.8%, p = 0.0006 in a recessive model). CSF1R mRNA levels in neutrophils of the asthmatic patients having the +22693CC allele were higher than in those having the +22693TT allele (p = 0.026). Asthmatic patients with the +22693CC allele also showed significantly higher CSF1R expression on CD14-positive monocytes and neutrophils than did those with the +22693TT allele (p = 0.045 and p = 0.044). The +20511C>T SNP had no association with CSF1R mRNA or protein expression. In conclusion, the minor allele at CSF1R +22693T>C may have a susceptibility effect in the development of asthma, via increased CSF1R protein and mRNA expression in inflammatory cells.

  14. 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

  15. Protective effects of granulocyte colony-stimulating factor on endotoxin shock in mice with retrovirus-induced immunodeficiency syndrome.

    PubMed

    Toki, S; Hiromatsu, K; Aoki, Y; Makino, M; Yoshikai, Y

    1997-10-01

    Mice with retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS) were hypersensitive to lipopolysaccharide (LPS)-induced lethal shock accompanied by marked elevations of systematic interleukin 1beta (IL-beta) and interferon gamma (IFN-gamma) after LPS challenge. Pretreatment with 10 microg of recombinant human granulocyte colony-stimulating factor (rhG-CSF) protected MAIDS mice from hypersensitivity to LPS-induced lethal shock and this protection was concomitant with suppression of IFN-gamma production.

  16. Insulin and insulin-like growth factor I (IGF-I) stimulate GLUT4 glucose transporter translocation in Xenopus oocytes.

    PubMed Central

    Mora, S; Kaliman, P; Chillarón, J; Testar, X; Palacín, M; Zorzano, A

    1995-01-01

    1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation. Images Figure 2 Figure 3 PMID:7575481

  17. 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

  18. 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.

  19. Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.

    PubMed

    Fueger, Patrick T; Schisler, Jonathan C; Lu, Danhong; Babu, Daniella A; Mirmira, Raghavendra G; Newgard, Christopher B; Hohmeier, Hans E

    2008-05-01

    Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.

  20. Actinobacillus actinomycetemcomitans serotype b-specific polysaccharide antigen stimulates production of chemotactic factors and inflammatory cytokines by human monocytes.

    PubMed Central

    Yamaguchi, N; Yamashita, Y; Ikeda, D; Koga, T

    1996-01-01

    Serotype b-specific polysaccharide antigen (SPA) was extracted from whole cells of Actinobacillus actinomycetemcomitans Y4 by autoclaving and purified by chromatography on DEAE-Sephadex A-25 and Sephacryl S-300. SPA induced the release of monocyte and leukocyte chemotactic factors by human monocytes. Polymyxin B had almost no effect on the release of monocyte chemotactic factor, but a monoclonal antibody against SPA markedly inhibited it. Human monocytes stimulated with SPA exhibited the increased mRNA expression of monocyte chemoattractant protein 1 (MCP-1) and a neutrophil chemotactic factor, interleukin-8 (IL-8). On the other hand, SPA induced the release of IL-1, IL-6, and tumor necrosis factor (TNF) and enhanced the expression of IL-1alpha, IL-1beta, IL-6, and TNF alpha (TNF-alpha) mRNAs. Human monocytes expressed MCP-1 and IL-8 mRNAs when stimulated by human recombinant IL-1alpha, I1-1beta, IL-6, and TNF-alpha, suggesting that these inflammatory cytokines induced by SPA might participate in the production of chemotactic factors in human monocytes. PMID:8698480

  1. Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha.

    PubMed Central

    Takashiba, S; Takigawa, M; Takahashi, K; Myokai, F; Nishimura, F; Chihara, T; Kurihara, H; Nomura, Y; Murayama, Y

    1992-01-01

    Inflammatory mediators produced by cells in the gingiva have been implicated in the initiation and progression of periodontal disease, a common infectious disease. In this study, we examined the biological activity of neutrophil chemotactic factors and the kinetics of expression of interleukin-8 (IL-8) mRNA derived from normal gingival fibroblasts in response to inflammatory mediators in an in vitro model. Gingival fibroblasts stimulated by either recombinant human interleukin-1 beta or recombinant human tumor necrosis factor alpha produced neutrophil chemotactic factors after 4 h, whereas expression of cell-derived IL-8 mRNA was detected within 1 h after stimulation. Furthermore, in a neutralization assay, rabbit anti-recombinant human IL-8 antiserum inhibited neutrophil chemotactic activity to basal levels. These results provide evidence that gingival fibroblasts synthesize potent chemotactic factors such as IL-8 in the presence of the inflammatory mediators interleukin-1 beta and tumor necrosis factor alpha. The activity of these factors may contribute to neutrophil-mediated processes in the pathogenesis of periodontal disease. Images PMID:1452358

  2. [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.

  3. Stimulating effect of space flight factors on Artemia cysts: comparison with irradiation by gamma rays

    SciTech Connect

    Gaubin, Y.; Pianezzi, B.; Gasset, G.; Plannel, H.; Kovalev, E.E.

    1986-06-01

    The Artemia cyst, a gastrula in dormant state, is a very suitable material to investigate the individual effects of HZE cosmic particles. Monolayers of Artemia cysts, sandwiched with nuclear emulsions, flew aboard the Soviet biosatellite Cosmos 1129. The space flight stimulated the developmental capacity expressed by higher percentages of emergence, hatching, and alive nauplii at day 4-5. A greater mean life span was reported in Artemias developed from Artemia cysts hit by the cosmic heavy ions. On Earth, Artemia cysts were exposed to 1, 10, 100, 200 and 400 Gy of gamma (gamma) rays. A stimulating effect on developmental capacity was observed for 10 Gy; the mean life span was significantly increased for this dose. These results are discussed in comparison with previous investigations performed on Earth and in space.

  4. Enhanced expression of DNA topoisomerase II by recombinant human granulocyte colony-stimulating factor in human leukemia cells.

    PubMed

    Towatari, M; Ito, Y; Morishita, Y; Tanimoto, M; Kawashima, K; Morishima, Y; Andoh, T; Saito, H

    1990-11-15

    The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on DNA topoisomerase II (topo II) expression was studied in two human acute myelogenous leukemia cell lines, NKM-1 and NOMO-1, which express G-CSF receptor and proliferate in response to exogenous G-CSF. Northern blot analysis revealed that the level of topo II mRNA in 16-h stimulated cells in serum-free medium with G-CSF (10 ng/ml) was approximately 2-fold higher than that in cells without G-CSF. Enhanced topo II mRNA expression was detectable within 3 h after the addition of G-CSF. Topo II activity in crude nuclear extracts from 16-h G-CSF-stimulated cells was also found to be approximately 2-fold greater than that from unstimulated cells. According to in vitro cytotoxic assay, the sensitivity of G-CSF-stimulated cells to intercalating (daunorubicin) and nonintercalating (etoposide) topo II-targeting drugs increased significantly, whereas no enhancement of sensitivity was observed with an alkylating agent (4-hydroperoxycyclophosphamide). The augmented drug sensitivity observed was not due to the increased level of drug transport, as suggested by the similar extent of [3H]etoposide uptake between G-CSF-stimulated and unstimulated cells. By measuring the topo II mRNA and the cytotoxicity of the above mentioned drugs, we obtained essentially the same results in G-CSF-responsive leukemia cells isolated from three acute myeloblastic leukemia patients, as observed in the cultured cell lines. These findings strongly suggest that the sensitivity to "topo II-targeting drugs" could be augmented by exogenous G-CSF through elevated topo II activity in G-CSF-responsive leukemia cells. PMID:1699657

  5. 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

  6. Differential effects of culture conditions on the migration pattern of stromal cell-derived factor-stimulated hematopoietic stem cells.

    PubMed

    Weidt, Corinna; Niggemann, Bernd; Hatzmann, Wolfgang; Zänker, Kurt S; Dittmar, Thomas

    2004-01-01

    Evidence is mounting that hematopoietic stem cells (HSCs) play a critical role in bone marrow regeneration and tissue renewal, for which migration is an obvious prerequisite. Computer-aided analysis and a three-dimensional collagen matrix assay enabled us to analyze single-cell migratory characteristics of stromal cell-derived factor-1 alpha (SDF-1 alpha)-stimulated cord blood-derived HSCs. We defined and resolved specific migratory parameters in spontaneous and SDF-1 alpha-induced migration of these cells. The addition of interleukin 6 to the culture medium led to differential SDF-1 alpha-stimulated migratory response, which comprised a recruitment of nonmoving cells and an increase in speed and frequency of pauses but a decrease in pause duration. We were thus able to decipher the exact parameters that result in an increase in the migration of HSCs and demonstrate that extensive analysis of single-cell behavior is elementary in the study of stem cell migration.

  7. Identification of key factors in Accelerated Low Water Corrosion through experimental simulation of tidal conditions: influence of stimulated indigenous microbiota.

    PubMed

    Marty, Florence; Gueuné, Hervé; Malard, Emilie; Sánchez-Amaya, José M; Sjögren, Lena; Abbas, Ben; Quillet, Laurent; van Loosdrecht, Mark C M; Muyzer, Gerard

    2014-01-01

    Biotic and abiotic factors favoring Accelerated Low Water Corrosion (ALWC) on harbor steel structures remain unclear warranting their study under controlled experimental tidal conditions. Initial stimulation of marine microbial consortia by a pulse of organic matter resulted in localized corrosion and the highest corrosion rates (up to 12-times higher than non-stimulated conditions) in the low water zone, persisting after nine months exposure to natural seawater. Correlations between corrosion severity and the abundance and composition of metabolically active sulfate-reducing bacteria (SRB) indicated the importance and persistence of specific bacterial populations in accelerated corrosion. One phylotype related to the electrogenic SRB Desulfopila corrodens appeared as the major causative agent of the accelerated corrosion. The similarity of bacterial populations related to sulfur and iron cycles, mineral and tuberculation with those identified in ALWC support the relevance of experimental simulation of tidal conditions in the management of steel corrosion exposed to harbor environments.

  8. Granulocyte/macrophage colony-stimulating factor is an intrinsic keratinocyte-derived growth factor for human melanocytes in UVA-induced melanosis.

    PubMed

    Imokawa, G; Yada, Y; Kimura, M; Morisaki, N

    1996-01-15

    Recently we demonstrated that endothelins secreted from human keratinocytes act as intrinsic mitogens and melanogens for human melanocytes in UVB-induced melanosis. We show here that UVA-induced melanosis is associated with other keratinocyte-derived growth factors, secretion of which is specifically stimulated after exposure of human keratinocytes to UVA. Medium conditioned by UVA-exposed human keratinocytes elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of endothelin-1 and interleukin (IL)-1 alpha in the conditioned medium by ELISA, both of which are major keratinocyte-derived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human keratinocytes to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were significantly increased in the conditioned medium of human keratinocytes after exposure to UVA at a dose of 1.0 J/cm2. The gel chromatographic profile of UVA-exposed keratinocyte-conditioned medium demonstrated that there were two factors (P-1 and P-2) with molecular masses of approx. 20 and 1 kDa respectively that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by [14C]thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to basic fibroblast growth factor or stem cell factor. Binding and proliferation assays with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF(Kd 2.11 nM; binding sites, 2.5-3.5 x 10(4) per cell), and recombinant GM

  9. 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

  10. Differences in the kinetics of activation of protein kinases and extracellular signal-related protein kinase 1 in colony-stimulating factor 1-stimulated and lipopolysaccharide-stimulated macrophages.

    PubMed Central

    Jaworowski, A; Christy, E; Yusoff, P; Byrne, R; Hamilton, J A

    1996-01-01

    To determine the relevance of mitogen-activated protein kinase activity to macrophage proliferation, we measured the stimulation of myelin basic protein (MBP) kinase and extracellular signal-related protein kinase (ERK) activity in a macrophage cell line (BAC1.2F5), bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM). By using an 'ingel' MBP kinase assay the activities of renaturable MBP kinases were detected, including several with molecular masses similar to those of ERK-1 and ERK-2. These represented a minor fraction of total activity and were not activated to an appreciable extent by colony-stimulating factor 1 (CSF-1). By using a sensitive and specific immune-complex kinase assay, activation of ERK-1 by CSF-1 and lipopolysaccharide (LPS) was demonstrated. Two kinetically distinct pathways of ERK-1 activation by CSF-1 were resolved, with peak activations occurring at 5 and 15 min. The kinetics and degree of activation were similar in BMM, BAC1.2F5 cells and RPM. LPS activated ERK-1 with a single peak at 10-15 min, corresponding to the later peak of activation by CSF-1. Thus there was no strict correlation between ERK activation and macrophage proliferation. PMID:9003393

  11. Tumor Necrosis Factor α Stimulates Osteoclast Differentiation by a Mechanism Independent of the Odf/Rankl–Rank Interaction

    PubMed Central

    Kobayashi, Kanichiro; Takahashi, Naoyuki; Jimi, Eijiro; Udagawa, Nobuyuki; Takami, Masamichi; Kotake, Shigeru; Nakagawa, Nobuaki; Kinosaki, Masahiko; Yamaguchi, Kyoji; Shima, Nobuyuki; Yasuda, Hisataka; Morinaga, Tomonori; Higashio, Kanji; Martin, T. John; Suda, Tatsuo

    2000-01-01

    Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF–dependent bone marrow macrophages (M-BMMφ) appeared within 3 d. Tartrate-resistant acid phosphatase–positive osteoclasts were also formed when M-BMMφ were further cultured for 3 d with mouse tumor necrosis factor α (TNF-α) in the presence of M-CSF. Osteoclast formation induced by TNF-α was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti–RANK (ODF/RANKL receptor) antibody. Experiments using M-BMMφ prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-α. Osteoclasts induced by TNF-α formed resorption pits on dentine slices only in the presence of IL-1α. These results demonstrate that TNF-α stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL–RANK system. TNF-α together with IL-1α may play an important role in bone resorption of inflammatory bone diseases. PMID:10637272

  12. Early recognition in the Rhizobium meliloti-alfalfa symbiosis: root exudate factor stimulates root adsorption of homologous rhizobia.

    PubMed Central

    Wall, L G; Favelukes, G

    1991-01-01

    Adsorption of Rhizobium meliloti to alfalfa roots before their infection and nodule formation shows the specificity of the symbiotic association (G. Caetano-Anollés and G. Favelukes, Appl. Environ. Microbiol. 52:377-382, 1986). The time course of specific adsorption of R. meliloti (10(3) to 10(4) cells per ml) to roots shows an initial lag period of 3 h, suggesting that either or both symbionts must become conditioned for the adsorption process. Preincubation of R. meliloti L5-30 for 3 h with dialyzed alfalfa root exudate (RE) markedly increased early adsorption of rhizobia to alfalfa roots. The activity in RE was linked to a nondialyzable, thermolabile, trypsin-sensitive factor(s), very different from the root-exuded flavonoid compounds also involved in early Rhizobium-legume interactions. The lack of activity in the RE from plants grown in 5 mM NO3- suggested its negative regulation by the nitrogen nutritional status of the plant. Preincubation of R. meliloti with heterologous clover RE did not stimulate adsorption of rhizobial cells to roots. A short pretreatment of RE with homologous (but not heterologous) strains eliminated the stimulatory activity from solution. The stimulation of adsorption of R. meliloti to alfalfa roots was strongly dependent on the growth phase of the rhizobia, being greater at the late exponential stage. Nevertheless, the capacity of R. meliloti L5-30 to eliminate from solution the stimulatory activity in RE appeared to be constitutive in the rhizobia. The low concentration of rhizobial cells used in these experiments was critical to detect the stimulation of adsorption. The early interaction of spontaneously released alfalfa root macromolecular factor(s) and free-living R. meliloti, which shows the specificity and regulatory properties characteristic of infection and nodulation, would be an initial recognition event in the rhizosphere which triggers the process of symbiotic association. PMID:2045369

  13. Dendritic Cell-Lymphocyte Cross Talk Downregulates Host Restriction Factor SAMHD1 and Stimulates HIV-1 Replication in Dendritic Cells

    PubMed Central

    Biedma, Marina Elizabeth; Lederle, Alexandre; Peressin, Maryse; Lambotin, Mélanie; Proust, Alizé; Decoville, Thomas; Schmidt, Sylvie; Laumond, Géraldine

    2014-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication in dendritic cells (DCs) is restricted by SAMHD1. This factor is counteracted by the viral protein Vpx; Vpx is found in HIV-2 and simian immunodeficiency virus (SIV) from sooty mangabeys (SIVsm) or from macaques (SIVmac) but is absent from HIV-1. We previously observed that HIV-1 replication in immature DCs is stimulated by cocultivation with primary T and B lymphocytes, suggesting that HIV-1 restriction in DCs may be overcome under coculture conditions. Here, we aimed to decipher the mechanism of SAMHD1-mediated restriction in DC-lymphocyte coculture. We found that coculture with lymphocytes downregulated SAMHD1 expression and was associated with increased HIV-1 replication in DCs. Moreover, in infected DC-T lymphocyte cocultures, DCs acquired maturation status and secreted type 1 interferon (alpha interferon [IFN-α]). The blockade of DC-lymphocyte cross talk by anti-ICAM-1 antibody markedly inhibited the stimulation of HIV-1 replication and prevented the downregulation of SAMHD1 expression in cocultured DCs. These results demonstrate that, in contrast to purified DCs, cross talk with lymphocytes downregulates SAMHD1 expression in DCs, triggering HIV-1 replication and an antiviral immune response. Therefore, HIV-1 replication and immune sensing by DCs should be investigated in more physiologically relevant models of DC/lymphocyte coculture. IMPORTANCE SAMHD1 restricts HIV-1 replication in dendritic cells (DCs). Here, we demonstrate that, in a coculture model of DCs and lymphocytes mimicking early mucosal HIV-1 infection, stimulation of HIV-1 replication in DCs is associated with downregulation of SAMHD1 expression and activation of innate immune sensing by DCs. We propose that DC-lymphocyte cross talk occurring in vivo modulates host restriction factor SAMHD1, promoting HIV-1 replication in cellular reservoirs and stimulating immune sensing. PMID:24574390

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

    PubMed

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

    2003-07-15

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

  15. 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

  16. Amifostine plus granulocyte colony-stimulating factor therapy enhances recovery from supralethal radiation exposures: preclinical experience in animals models.

    PubMed

    Patchen, M L

    1995-01-01

    A murine model was used to explore whether the cytoprotective agent amifostine (WR-2721) can be used to protect a critical fraction of haemopoietic stem cells against radiation, and whether granulocyte colony-stimulating factor (G-CSF) can then be used to stimulate the protected cells to proliferate and reconstitute the haematopoietic system. Groups of C3H/HeN mice treated with 200 mg/kg amifostine i.p. 30 min before 60Co irradiation and/or 125 micrograms/kg G-CSF subcutaneously from days 1-16 post irradiation were compared. The dose reduction factor (DRF) of the combination of amifostine and G-CSF from LD50/30 values was greater than the sum of the DRFs for amifostine and G-CSF individually. Acceleration of recovery bone marrow and splenic multipotent stem cells (CFU-s) and granulocyte-macrophage progenitor cells (GM-CFC), as well as of peripheral blood red and white cells and platelets, was greatest in mice treated with amifostine plus G-CSF. These studies suggest that amifostine and recombinant haematopoietic growth factors can be used in combination to reduce myelosuppression and lethality associated with radiation or radiomimetic drugs

  17. 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.

  18. Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration

    PubMed Central

    Kurtagic, Elma; Rich, Celeste B.; Buczek-Thomas, Jo Ann; Nugent, Matthew A.

    2015-01-01

    Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment. PMID:26672607

  19. Dehydrodiisoeugenol, an isoeugenol dimer, inhibits lipopolysaccharide-stimulated nuclear factor kappa B activation and cyclooxygenase-2 expression in macrophages.

    PubMed

    Murakami, Yukio; Shoji, Masao; Hirata, Atsushi; Tanaka, Shoji; Yokoe, Ichiro; Fujisawa, Seiichiro

    2005-02-15

    o-Methoxyphenols such as eugenol and isoeugenol exhibit anti-oxidant and anti-inflammatory activities, but at higher concentrations act as oxidants and potent allergens. We recently demonstrated the eugenol dimer bis-eugenol to be an efficient inhibitor of lipopolysaccharide (LPS)-induced inflammatory cytokine expression in macrophages without cytotoxicity. This result suggested that dimer compound of o-methoxyphenols may possess anti-inflammatory activity. Thus, we further synthesized dehydrodiisoeugenol and alpha-diisoeugenol from isoeugenols, and investigated whether these dimers could inhibit LPS-stimulated nuclear factor kappa B (NF-kappaB) activation and cyclooxygenase (COX)-2 gene expression, both of which are closely involved in inflammation and mutagenesis. The expression of the COX-2 gene was strongly inhibited by dehydrodiisoeugenol in RAW264.7 murine macrophages stimulated with LPS. In contrast, isoeugenol and alpha-diisoeugenol did not inhibit it. Dehydrodiisoeugenol also significantly inhibited LPS-stimulated phosphorylation-dependent proteolysis of inhibitor kappaB-alpha and transcriptional activity of NF-kappaB in the cells. These findings suggest that dehydrodiisoeugenol acts as a potent anti-inflammatory agent.

  20. Granulocyte-Colony Stimulating Factor (G-CSF) Improves Motor Recovery in the Rat Impactor Model for Spinal Cord Injury

    PubMed Central

    Dittgen, Tanjew; Pitzer, Claudia; Plaas, Christian; Kirsch, Friederike; Vogt, Gerhard; Laage, Rico; Schneider, Armin

    2012-01-01

    Granulocyte-colony stimulating factor (G-CSF) improves outcome after experimental SCI by counteracting apoptosis, and enhancing connectivity in the injured spinal cord. Previously we have employed the mouse hemisection SCI model and studied motor function after subcutaneous or transgenic delivery of the protein. To further broaden confidence in animal efficacy data we sought to determine efficacy in a different model and a different species. Here we investigated the effects of G-CSF in Wistar rats using the New York University Impactor. In this model, corroborating our previous data, rats treated subcutaneously with G-CSF over 2 weeks show significant improvement of motor function. PMID:22253813

  1. Transcription factors involved in glucose-stimulated insulin secretion of pancreatic beta cells

    SciTech Connect

    Shao, Shiying; Fang, Zhong; Yu, Xuefeng; Zhang, Muxun

    2009-07-10

    GSIS, the most important function of pancreatic beta cell, is essential for maintaining the glucose homeostasis. Transcription factors are known to control different biological processes such as differentiation, proliferation and apoptosis. In pancreas, some transcription factors are involved in regulating the function of beta cells. In this review, the role of these transcription factors including Pdx-1, FoxO1, SREBP-1c, and MafA in GSIS is highlighted. The related molecular mechanisms are analyzed as well. Furthermore, the association between the role of transcription factors in GSIS and the development of T2DM is discussed.

  2. Enhancement of in vitro and in vivo anti-tumor activity of anti-GD2 monoclonal antibody 220-51 against human neuroblastoma by granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

    PubMed

    Fukuda, M; Horibe, K; Furukawa, K

    1998-10-01

    We have evaluated the anti-tumor effect of anti-GD2 mouse monoclonal antibody (mAb) 220-51 against human neuroblastoma cell line TGW in vitro and in vivo. The mAb 220-51 was able to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) using human effector cells. In the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte ADCC was significantly augmented in vitro. When mAb 220-51 was administered to tumor-bearing nude mice, tumor growth was significantly inhibited as compared with untreated controls. Administration of recombinant murine GM-CSF in combination with mAb 220-51 significantly enhanced the anti-tumor effect of mAb in vivo. Recombinant human granulocyte colony-stimulating factor (G-CSF) combined with mAb 220-51 was also able to enhance it, although granulocyte ADCC was not affected by the presence of recombinant human G-CSF in vitro. Moreover, GM-CSF and G-CSF work additively to enhance the anti-tumor effect of mAb 220-51 in vivo. The GM-CSF and G-CSF may have a clinical potency in immunotherapy with anti-GD2 mAb for the treatment of neuroblastoma.

  3. 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

  4. Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

    PubMed

    Bunse, C E; Tischer, S; Lahrberg, J; Oelke, M; Figueiredo, C; Blasczyk, R; Eiz-Vesper, B

    2016-07-01

    Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation. PMID:26990855

  5. Down-regulation by resveratrol of basic fibroblast growth factor-stimulated osteoprotegerin synthesis through suppression of Akt in osteoblasts.

    PubMed

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

    2014-10-06

    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.

  6. Mechanisms of leukocyte accumulation and activation in chorioamnionitis: interleukin 1 beta and tumor necrosis factor alpha enhance colony stimulating factor 2 expression in term decidua.

    PubMed

    Arcuri, Felice; Toti, Paolo; Buchwalder, Lynn; Casciaro, Alessandra; Cintorino, Marcella; Schatz, Frederick; Rybalov, Basya; Lockwood, Charles J

    2009-05-01

    Chorioamnionitis is a major cause of prematurity as well as perinatal morbidity and mortality. The present study observed a marked increase in immunohistochemical staining for Colony Stimulating Factor 2 (CSF2; also known as granulocyte macrophage-colony stimulating factor), a potent neutrophil and macrophage chemoattractant and activator, in the decidua of patients with CAM compared with controls (n = 8; P = .001). To examine the regulation of this CSF2, cultured decidual cells primed with estradiol (E2) or E2 plus medroxyprogesterone acetate, were exposed to tumor necrosis factor-alpha or interleukin-1beta and secreted CSF2 measured by ELISA. Levels of CSF2 in E2 plus MPA-treated cultures increased 18- and 245-fold following treatment with TNF or IL1B (n = 7, P < .05). Quantitative RT-PCR demonstrated parallel changes in mRNA levels. This study reveals that CSF2 is strongly expressed in decidua from patients with CAM and indicates TNF or IL1B as important regulators of CAM-related decidual leukocyte infiltration and activation.

  7. Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.

    PubMed Central

    Kuwabara, K; Ogawa, S; Matsumoto, M; Koga, S; Clauss, M; Pinsky, D J; Lyn, P; Leavy, J; Witte, L; Joseph-Silverstein, J

    1995-01-01

    Wound repair and tumor vascularization depend upon blood vessel growth into hypoxic tissue. Although hypoxia slows endothelial cell (EC) proliferation and suppresses EC basic fibroblast growth factor (bFGF) expression, we report that macrophages (MPs) exposed to PO2 approximately 12-14 torr (1 torr = 133.3 Pa) synthesize and release in a time-dependent manner platelet-derived growth factor (PDGF) and acidic/basic FGFs (a/bFGFs), which stimulate the growth of hypoxic ECs. Chromatography of hypoxic MP-conditioned medium on immobilized heparin with an ascending NaCl gradient resolved three peaks of mitogenic activity: activity of the first peak was neutralized by antibody to PDGF; activity of the second peak was neutralized by antibody to aFGF; and activity of the third peak was neutralized by antibody to bFGF. Metabolically labeled lysates and supernatants from MPs exposed to hypoxia showed increased synthesis and release of immunoprecipitable PDGF and a/bFGF in the absence of changes in cell viability. Possible involvement of a heme-containing oxygen sensor in MP elaboration of growth factors was suggested by the induction of bFGF and PDGF by normoxic MPs exposed to nickel or cobalt, although metabolic inhibitors such as sodium azide were without effect. These results suggest a paracrine model in which hypoxia stimulates MP release of PDGF and a/bFGF, inducing EC proliferation and potentially promoting angiogenesis in hypoxic environments. Images Fig. 1 Fig. 3 Fig. 4 PMID:7538678

  8. Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

    PubMed Central

    Bijnsdorp, I V; Capriotti, F; Kruyt, F A E; Losekoot, N; Fukushima, M; Griffioen, A W; Thijssen, V L; Peters, G J

    2011-01-01

    Background: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. Methods: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1=CT-CM, RT112/TP=RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT–PCR, ELISA and blocking antibodies. Results: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 μ TPI and 100 μ L-dR, blocked migration and reduced the invasion by 50–70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-α, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. Conclusion: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy. PMID:21386840

  9. Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial.

    PubMed

    Spiel, Alexander O; Bartko, Johann; Schwameis, Michael; Firbas, Christa; Siller-Matula, Jolanta; Schuetz, Matthias; Weigl, Manuela; Jilma, Bernd

    2011-04-01

    Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events. PMID:21301783

  10. Lipoteichoic acid and interleukin 1 stimulate synergistically production of hepatocyte growth factor (scatter factor) in human gingival fibroblasts in culture.

    PubMed Central

    Sugiyama, A; Arakaki, R; Ohnishi, T; Arakaki, N; Daikuhara, Y; Takada, H

    1996-01-01

    Lipoteichoic acids (LTA) from various gram-positive bacteria, including oral streptococci such as Streptococcus sanguis, enhanced the production of hepatocyte growth factor (HGF) (scatter factor) by human gingival fibroblasts in culture, whereas lipopolysaccharides (LPS) from various gram-negative bacteria did not. In contrast, LPS induced interleukin 1 activity in human gingival epithelial cells in culture, while LTA had little effect. LTA and recombinant human interleukin 1 alpha enhanced synergistically the production of HGF/SF in human gingival fibroblast cultures. Recombinant human HGF, in turn, enhanced the proliferation of human gingival epithelial cells in culture. PMID:8606111

  11. Analysis of the mechanisms involved in the stimulation of neutrophil apoptosis by tumour necrosis factor

    PubMed Central

    Salamone, Gabriela; Trevani, Analía; Martínez, Diego; Vermeulen, Mónica; Gamberale, Romina; Fernández-Calotti, Paula; Raiden, Silvina; Giordano, Mirta; Geffner, Jorge

    2004-01-01

    We have previously reported that human neutrophils pretreated with tumour necrosis factor-α (TNF-α) and then exposed to a variety of agents such as immune complexes, zymosan, phorbol 12-myristate 13-acetate (PMA), C5a, fMLP, or granulocyte–macrophage colony-stimulating factor (GM-CSF), undergo a dramatic stimulation of apoptosis, suggesting that TNF-α is able to prime an apoptotic death programme which can be rapidly triggered by different stimuli. We report here that this response involves the participation of Mac-1 (CD11b/CD18), is dependent on caspases 3, 8 and 9, and is associated with both a loss of mitochondrial transmembrane potential and a down-regulation in expression of the anti-apoptotic protein, Mcl-1. Interestingly, we also found that the anti-apoptotic cytokine interleukin-1 (IL-1) improves the ability of TNF-α to promote apoptosis, supporting the notion than TNF-α, acting together with IL-1, may favour the depletion of neutrophils from the inflammatory areas during the course of acute inflammation. PMID:15500622

  12. 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.

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

    PubMed Central

    LIN, CHIEN-HUNG; LIN, CHUNG-CHING

    2016-01-01

    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. PMID:27284355

  14. Bone pain from granulocyte colony stimulating factor: does clinical trial sponsorship by a pharmaceutical company influence its reporting?

    PubMed

    Aldairy, Y; Nguyen, P L; Jatoi, A

    2011-01-01

    It is alleged that pharmaceutical companies sometimes unfairly present clinical trial results. To our knowledge, studies have not explored whether such alleged unfair reporting also occurs in the testing of palliative care agents in cancer patients, a particularly vulnerable group. Therefore, a systematic search was conducted to retrieve all published, prospective clinical trials that used granulocyte colony stimulating factor starting in 2003. Because granulocyte colony stimulating factor can cause severe bone pain - a concerning but historically under-reported symptom in cancer patients - this symptom was assessed to determine whether differences in reporting occurred based on pharmaceutical company-sponsorship. A total of 239 published clinical trials met the present study's eligibility criteria and were retrievable. Within this entire group of studies, 65 (27%) were pharmaceutical company-sponsored, and only 31 (13%) reported on bone pain. However, pharmaceutical company-sponsored trials reported on bone pain at a higher rate compared with other studies: 23% versus 9% (P= 0.005), and this conclusion did not change after adjusting for dose, use of the slow release formulation and year of publication. The reporting of adverse events from cancer symptom control and palliative care interventions should be improved - especially in trials not sponsored by pharmaceutical companies.

  15. Role of vascular endothelial growth factor in the stimulation of cellular invasion and signaling of breast cancer cells.

    PubMed

    Price, D J; Miralem, T; Jiang, S; Steinberg, R; Avraham, H

    2001-03-01

    The expression of vascular endothelial growth factor (VEGF) by breast tumors has been previously correlated with a poor prognosis in the pathogenesis of breast cancer. Furthermore, VEGF secretion is a prerequisite for tumor development. Although most of the effects of VEGF have been shown to be attributable to the stimulation of endothelial cells, we present evidence here that breast tumor cells are capable of responding to VEGF. We show that VEGF stimulation of T-47D breast cancer cells leads to changes in cellular signaling and invasion. VEGF increases the cellular invasion of T-47D breast cancer cells on Matrigel/ fibronectin-coated transwell membranes by a factor of two. Northern analysis for the expression of the known VEGF receptors shows the presence of moderate levels of Flt-1 and low levels of Flk-1/KDR mRNAs in a variety of breast cancer cell lines. T-47D breast cancer cells bind 125I-labeled VEGF with a Kd of 13 x 10(-9) M. VEGF induces the activation of the extracellular regulated kinases 1,2 as well as activation of phosphatidylinositol 3'-kinase, Akt, and Forkhead receptor L1. These findings in T-47D breast cancer cells strongly suggest an autocrine role for VEGF contributing to the tumorigenic phenotype.

  16. Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth.

    PubMed

    Koch, Sylvia; Garcia Gonzalez, Omar; Assfalg, Robin; Schelling, Adrian; Schäfer, Patrick; Scharffetter-Kochanek, Karin; Iben, Sebastian

    2014-01-01

    Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients.

  17. Cockayne syndrome protein A is a transcription factor of RNA polymerase I and stimulates ribosomal biogenesis and growth

    PubMed Central

    Koch, Sylvia; Garcia Gonzalez, Omar; Assfalg, Robin; Schelling, Adrian; Schäfer, Patrick; Scharffetter-Kochanek, Karin; Iben, Sebastian

    2014-01-01

    Mutations in the Cockayne syndrome A (CSA) protein account for 20% of Cockayne syndrome (CS) cases, a childhood disorder of premature aging and early death. Hitherto, CSA has exclusively been described as DNA repair factor of the transcription-coupled branch of nucleotide excision repair. Here we show a novel function of CSA as transcription factor of RNA polymerase I in the nucleolus. Knockdown of CSA reduces pre-rRNA synthesis by RNA polymerase I. CSA associates with RNA polymerase I and the active fraction of the rDNA and stimulates re-initiation of rDNA transcription by recruiting the Cockayne syndrome proteins TFIIH and CSB. Moreover, compared with CSA deficient parental CS cells, CSA transfected CS cells reveal significantly more rRNA with induced growth and enhanced global translation. A previously unknown global dysregulation of ribosomal biogenesis most likely contributes to the reduced growth and premature aging of CS patients. PMID:24781187

  18. 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.

  19. A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells.

    PubMed

    Hack, N; Clayman, P; Skorecki, K

    1990-08-01

    We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A2(PLA2) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [3H]- or [14C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 microM GDP beta S and 108% augmentation with 100 microM GTP gamma S). GTP gamma S alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTP gamma S. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA2 by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.

  20. Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance.

    PubMed

    Lupien, Mathieu; Meyer, Clifford A; Bailey, Shannon T; Eeckhoute, Jérôme; Cook, Jennifer; Westerling, Thomas; Zhang, Xiaoyang; Carroll, Jason S; Rhodes, Daniel R; Liu, X Shirley; Brown, Myles

    2010-10-01

    Estrogen receptor α (ERα) expression in breast cancer is predictive of response to endocrine therapy; however, resistance is common in ERα-positive tumors that overexpress the growth factor receptor ERBB2. Even in the absence of estrogen, ERα can be activated by growth factors, including the epidermal growth factor (EGF). EGF induces a transcriptional program distinct from estrogen; however, the mechanism of the stimulus-specific response is unknown. Here we show that the EGF-induced ERα genomic targets, its cistromes, are distinct from those induced by estrogen in a process dependent on the transcription factor AP-1. The EGF-induced ERα cistrome specifically regulates genes found overexpressed in ERBB2-positive human breast cancers. This provides a potential molecular explanation for the endocrine therapy resistance seen in ERα-positive breast cancers that overexpress ERBB2. These results suggest a central role for ERα in hormone-refractory breast tumors dependent on growth factor pathway activation and favors the development of therapeutic strategies completely antagonizing ERα, as opposed to blocking its estrogen responsiveness alone.

  1. 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. PMID:25454698

  2. 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.

  3. Stimulated production of steroids in Inonotus obliquus by host factors from birch.

    PubMed

    Wang, Lian-Xia; Lu, Zhen-Ming; Geng, Yan; Zhang, Xiao-Mei; Xu, Guo-Hua; Shi, Jin-Song; Xu, Zheng-Hong

    2014-12-01

    Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

  4. 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.

  5. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    SciTech Connect

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H. )

    1988-09-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using ({sup 32}P)phosphorylase {alpha} as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at {approx}5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.

  6. Thyrotropin inhibits while insulin, epidermal growth factor and tetradecanoyl phorbol acetate stimulate insulin-like growth factor binding protein secretion from sheep thyroid cells.

    PubMed

    Eggo, M C; Bachrach, L K; Brown, A L; Burrow, G N

    1991-01-01

    Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function. PMID:1722684

  7. Inducible tumor necrosis factor (TNF) receptor-associated factor-1 expression couples the canonical to the non-canonical NF-κB pathway in TNF stimulation.

    PubMed

    Choudhary, Sanjeev; Kalita, Mridul; Fang, Ling; Patel, Kershaw V; Tian, Bing; Zhao, Yingxin; Edeh, Chukwudi B; Brasier, Allan R

    2013-05-17

    The NF-κB transcription factor mediates the inflammatory response through distinct (canonical and non-canonical) signaling pathways. The mechanisms controlling utilization of either of these pathways are largely unknown. Here we observe that TNF stimulation induces delayed NF-κB2/p100 processing and investigate the coupling mechanism. TNF stimulation induces TNF-associated factor-1 (TRAF-1) that directly binds NF-κB-inducing kinase (NIK) and stabilizes it from degradation by disrupting its interaction with TRAF2·cIAP2 ubiquitin ligase complex. We show that TRAF1 depletion prevents TNF-induced NIK stabilization and reduces p52 production. To further examine the interactions of TRAF1 and NIK with NF-κB2/p100 processing, we mathematically modeled TRAF1·NIK as a coupling signaling complex and validated computational inference by siRNA knockdown to show non-canonical pathway activation is dependent not only on TRAF1 induction but also NIK stabilization by forming TRAF1·NIK complex. Thus, these integrated computational-experimental studies of TNF-induced TRAF1 expression identified TRAF1·NIK as a central complex linking canonical and non-canonical pathways by disrupting the TRAF2-cIAP2 ubiquitin ligase complex. This feed-forward kinase pathway is essential for the activation of non-canonical pathway. PMID:23543740

  8. 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.

  9. Dynamic Fluid Flow Stimulation on Cortical Bone and Alterations of the Gene Expressions of Osteogenic Growth Factors and Transcription Factors in a Rat Functional Disuse Model

    PubMed Central

    Hu, Minyi; Qin, Yi-Xian

    2014-01-01

    Recently we have developed a dynamic hydraulic stimulation (DHS) as a loading modality to induce anabolic responses in bone. To further study the functional process of DHS regulated bone metabolism, the objective of this study was to evaluate the effects of DHS on cortical bone and its alterations on gene expressions of osteogenic growth factors and transcription factors as a function of time. Using a model system of 5-month-old hindlimb suspended (HLS) female Sprague-Dawley rats, DHS was applied to the right tibiae of the stimulated rats with a loading frequency of 2Hz with 30mmHg (p-p) dynamic pressure, 5 days/week, for a total of 28 days. Midshafts of the tibiae were analyzed using μCT and histology. Total RNA was analyzed using RT-PCR on selected osteogenic genes (RUNX2, β-catenin, osteopontin, VEGF, BMP2, IGF-1, and TGF-β) on 3-,7-,14-, and 21-day. Results showed increased Cort.Th and Ct.BV/TV as well as a time-dependable fashion of gradual changes in mRNA levels upon DHS. While DHS-driven fold changes of the mRNA levels remained low before Day-7, its fold changes started to elevate by Day-14 and then dropped by Day-21. This study further delineates the underlying molecular mechanism of DHS-derived mechanical signals, and its time dependent optimization. PMID:24486201

  10. Vascular endothelial growth factor promotes macrophage apoptosis through stimulation of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT).

    PubMed

    Petreaca, Melissa L; Yao, Min; Ware, Carl; Martins-Green, Manuela M

    2008-01-01

    Resolution of inflammation is critical for normal wound healing. Inflammation is prolonged and fails to resolve properly in chronic wounds. We used in vivo and in vitro approaches to show that vascular endothelial growth factor (VEGF) induces macrophage apoptosis and to delineate mechanisms involved in this process. VEGF inhibition during wound healing leads to an increased number of macrophages remaining in wounds, suggesting the involvement of VEGF in removal of these cells from the wound. If this effect has physiological relevance, it likely occurs via apoptosis. We show that VEGF increases apoptosis of macrophages in vitro using Annexin V-FITC staining and caspase activation. Microarray analysis, reverse transcription-polymerase chain reaction, and immunoblotting showed that VEGF increases the expression of tumor necrosis factor superfamily member 14 (TNFSF14/LIGHT) in macrophages. We also show that in macrophages LIGHT promotes apoptosis through the lymphotoxin beta receptor. Moreover, inhibition of LIGHT prevents VEGF-induced death, suggesting that LIGHT mediates VEGF-induced macrophage apoptosis. Taken together, our results identify a novel role for VEGF and for LIGHT in macrophage apoptosis during wound healing, an event critical in the resolution of inflammation. This finding may lead to the development of new strategies to improve resolution of inflammation in problematic wounds. PMID:19128255

  11. 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

  12. Influence of perinatal factors on thyroid stimulating hormone level in cord blood

    PubMed Central

    Armanian, Amir-Mohammad; Hashemipour, Mahin; Esnaashari, Azadeh; Kelishadi, Roya; Farajzadegan, Ziba

    2013-01-01

    Background: The aim of the present study was to determine the effect of various perinatal factors on cord blood TSH among newborns in Isfahan, Iran. Materials and Methods: This was a descriptive–analytic cross sectional study which performed in Isfahan Iran. During a period of four months, since February to May 2012 a total number of 440 newborns delivered in Alzahra and Shahid beheshti hospitals were enrolled in the study. For all newborns one mL blood sample from umbilical vein was obtained by one of the project investigators and sent to laboratory for further examinations. Cord blood TSH and birth body weight (BBW), gestational age, history of gestational diabetes mellitus (GDM), apgar at one minute, apgar at five minute, newborn gender and the mother's age were documented. Differences considered statistically significant if P < 0.01. Results: 440 newborns enrolled in the study, 221 (50.2%) were male and 219 (49.8%) were female. Among study parameters, method of delivery had statistically significant relation with cord blood TSH (P < 0.001), and other factors such as BBW, gestational age, GDM, apgar at one minute, apgar at five minute, newborn gender and the mother's age didn’t have statistically significant relationship with cord TSH level. Conclusion: In conclusion we deduce that the only factor that can affect cord blood TSH was method of delivery. Infant with vaginal delivery has higher TSH level in cord blood. Other factors that were evaluated in this study didn’t have any statistically significant relationship. PMID:24516848

  13. The nuclear factor SPBP contains different functional domains and stimulates the activity of various transcriptional activators.

    PubMed

    Rekdal, C; Sjøttem, E; Johansen, T

    2000-12-22

    SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. PMID:10995766

  14. Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils.

    PubMed

    de Haas, M; Kerst, J M; van der Schoot, C E; Calafat, J; Hack, C E; Nuijens, J H; Roos, D; van Oers, R H; von dem Borne, A E

    1994-12-01

    In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1-antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.

  15. Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids.

    PubMed

    Rademakers, Rosa; Baker, Matt; Nicholson, Alexandra M; Rutherford, Nicola J; Finch, NiCole; Soto-Ortolaza, Alexandra; Lash, Jennifer; Wider, Christian; Wojtas, Aleksandra; DeJesus-Hernandez, Mariely; Adamson, Jennifer; Kouri, Naomi; Sundal, Christina; Shuster, Elizabeth A; Aasly, Jan; MacKenzie, James; Roeber, Sigrun; Kretzschmar, Hans A; Boeve, Bradley F; Knopman, David S; Petersen, Ronald C; Cairns, Nigel J; Ghetti, Bernardino; Spina, Salvatore; Garbern, James; Tselis, Alexandros C; Uitti, Ryan; Das, Pritam; Van Gerpen, Jay A; Meschia, James F; Levy, Shawn; Broderick, Daniel F; Graff-Radford, Neill; Ross, Owen A; Miller, Bradley B; Swerdlow, Russell H; Dickson, Dennis W; Wszolek, Zbigniew K

    2011-12-25

    Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.

  16. Identification of an isoform of colony-stimulating factor 1 receptor mRNA in the rat testis.

    PubMed

    Su, Hong; Wang, Yibiao; Söder, Olle; Hou, Mi

    2014-06-01

    Because alternative RNA splicing regulation in the testis is prevalent, we explored testes of Sprague-Dawley rats for existence of alternatively spliced colony-stimulating factor 1 receptor (CSF-1R) mRNA. Using RT-PCR and sequencing, we identified a variant of CSF-1R mRNA that was 284 bp shorter than the full-length CSF-1R transcript. This variant was present in the testis (late fetal stage to adult) and in other organs of rats (7 and 60 days old). The deletion of 284 bp disrupted the open reading frame, resulting in a noncoding mRNA product. When testicular macrophages were stimulated with CSF-1R ligand and lipopolysaccharide, proportionally increased expression of both short isoform and full-length CSF-1R mRNA was observed. Thus, the identified isoform of CSF-1R mRNA may interfere with the expression of full-length CSF-1R mRNA, thereby affecting the biological activity of the ligand/receptor signaling axis in Sprague-Dawley rats.

  17. 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.

  18. Epidermal growth factor-induced stimulation of proliferation and gene expression changes in the hypotrichous ciliate, Stylonychia lemnae.

    PubMed

    Mu, Weijie; Wang, Qi; Bourland, William A; Jiang, Chuanqi; Yuan, Dongxia; Pan, Xuming; Miao, Wei; Chen, Ying; Xiong, Jie

    2016-10-30

    Epidermal growth factor (EGF) induces proliferation of epidermal and epithelial tissues in mammals. However, the effect of EGF on the single-celled eukaryotes is not well characterized, especially in the protists. Ciliates, an important group of protists, are well characterized as both pollution indicators and model organisms for research. Stylonychia lemnae, is one of the most common free-living ciliates, widely distributed in ponds, rivers and marshes. Here, we report the role of EGF on cell proliferation stimulation in S. lemnae. The growth curve of S. lemnae was established, and the stimulation effect of EGF on the proliferation of S. lemnae was investigated. Based on the results, potential EGF receptors were identified in S. lemnae according to the conserved domains and gene expression. Differential gene expression revealed that EGF-induced genes in other organisms (e.g. antioxidant) also up-regulated in S. lemnae cells at propagation stages. In addition, our results showed that EGF could up-regulate the signal transduction-related processes in the decline stage of S. lemnae cells, indicating its potential function in apoptosis inhibition. In summary, this study reports findings of the first investigation of EGF effects in hypotrich ciliates, and establishes an additional system for the study of the molecular mechanisms of EGF actions in eukaryotic cell division and proliferation. PMID:27506312

  19. Spontaneous and stimulated release of tumor necrosis factor-alpha (TNF) from blood monocytes of miners with coal workers' pneumoconiosis

    SciTech Connect

    Borm, P.J.; Palmen, N.; Engelen, J.J.; Buurman, W.A.

    1988-12-01

    It is generally accepted that fibrotic lung diseases are mediated by macrophage-derived cytokines. We investigated the release of the monokine tumor necrosis factor-alpha (TNF) from blood monocytes in a group of 66 coal miners and 12 non-dust-exposed individuals. Twenty-seven miners had simple Coal Workers' Pneumoconiosis (CWP). Control miners (n = 39) were matched with respect to age, years underground, and smoking. Monocytes were assayed for TNF release, spontaneously or in response to soluble (endotoxin) or particulate (coal mine dust, silica) stimulation. TNF was measured with a TNF-specific ELISA. Monocytes of all subjects responded to stimulants by the release of TNF. Dust-exposed controls' monocytes revealed higher TNF release as compared to normal controls. The greatest discriminator between control miners and cases (CWP) was coal mine dust-induced TNF release. Interestingly, the largest difference was observed between controls and those cases with a small number of opacities (0/1, 1/0, 1/1, and 1/2), giving an odds ratio of 6.3 to find an individual with a high dust-induced TNF release in the patient group.

  20. 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. PMID:25366263

  1. 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.

  2. 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.

  3. Sodium butyrate stimulates expression of fibroblast growth factor 21 in liver by inhibition of histone deacetylase 3.

    PubMed

    Li, Huating; Gao, Zhanguo; Zhang, Jin; Ye, Xin; Xu, Aimin; Ye, Jianping; Jia, Weiping

    2012-04-01

    Fibroblast growth factor 21 (FGF21) stimulates fatty acid oxidation and ketone body production in animals. In this study, we investigated the role of FGF21 in the metabolic activity of sodium butyrate, a dietary histone deacetylase (HDAC) inhibitor. FGF21 expression was examined in serum and liver after injection of sodium butyrate into dietary obese C57BL/6J mice. The role of FGF21 was determined using antibody neutralization or knockout mice. FGF21 transcription was investigated in liver and HepG2 hepatocytes. Trichostatin A (TSA) was used in the control as an HDAC inhibitor. Butyrate was compared with bezafibrate and fenofibrate in the induction of FGF21 expression. Butyrate induced FGF21 in the serum, enhanced fatty acid oxidation in mice, and stimulated ketone body production in liver. The butyrate activity was significantly reduced by the FGF21 antibody or gene knockout. Butyrate induced FGF21 gene expression in liver and hepatocytes by inhibiting HDAC3, which suppresses peroxisome proliferator-activated receptor-α function. Butyrate enhanced bezafibrate activity in the induction of FGF21. TSA exhibited a similar set of activities to butyrate. FGF21 mediates the butyrate activity to increase fatty acid use and ketogenesis. Butyrate induces FGF21 transcription by inhibition of HDAC3.

  4. 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

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

    PubMed

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

    2016-07-27

    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.

  6. Epidermal growth factor-induced stimulation of proliferation and gene expression changes in the hypotrichous ciliate, Stylonychia lemnae.

    PubMed

    Mu, Weijie; Wang, Qi; Bourland, William A; Jiang, Chuanqi; Yuan, Dongxia; Pan, Xuming; Miao, Wei; Chen, Ying; Xiong, Jie

    2016-10-30

    Epidermal growth factor (EGF) induces proliferation of epidermal and epithelial tissues in mammals. However, the effect of EGF on the single-celled eukaryotes is not well characterized, especially in the protists. Ciliates, an important group of protists, are well characterized as both pollution indicators and model organisms for research. Stylonychia lemnae, is one of the most common free-living ciliates, widely distributed in ponds, rivers and marshes. Here, we report the role of EGF on cell proliferation stimulation in S. lemnae. The growth curve of S. lemnae was established, and the stimulation effect of EGF on the proliferation of S. lemnae was investigated. Based on the results, potential EGF receptors were identified in S. lemnae according to the conserved domains and gene expression. Differential gene expression revealed that EGF-induced genes in other organisms (e.g. antioxidant) also up-regulated in S. lemnae cells at propagation stages. In addition, our results showed that EGF could up-regulate the signal transduction-related processes in the decline stage of S. lemnae cells, indicating its potential function in apoptosis inhibition. In summary, this study reports findings of the first investigation of EGF effects in hypotrich ciliates, and establishes an additional system for the study of the molecular mechanisms of EGF actions in eukaryotic cell division and proliferation.

  7. Nerve growth factor stimulates axon outgrowth through negative regulation of growth cone actomyosin restraint of microtubule advance

    PubMed Central

    Turney, Stephen G.; Ahmed, Mostafa; Chandrasekar, Indra; Wysolmerski, Robert B.; Goeckeler, Zoe M.; Rioux, Robert M.; Whitesides, George M.; Bridgman, Paul C.

    2016-01-01

    Nerve growth factor (NGF) promotes growth, differentiation, and survival of sensory neurons in the mammalian nervous system. Little is known about how NGF elicits faster axon outgrowth or how growth cones integrate and transform signal input to motor output. Using cultured mouse dorsal root ganglion neurons, we found that myosin II (MII) is required for NGF to stimulate faster axon outgrowth. From experiments inducing loss or gain of function of MII, specific MII isoforms, and vinculin-dependent adhesion-cytoskeletal coupling, we determined that NGF causes decreased vinculin-dependent actomyosin restraint of microtubule advance. Inhibition of MII blocked NGF stimulation, indicating the central role of restraint in directed outgrowth. The restraint consists of myosin IIB- and IIA-dependent processes: retrograde actin network flow and transverse actin bundling, respectively. The processes differentially contribute on laminin-1 and fibronectin due to selective actin tethering to adhesions. On laminin-1, NGF induced greater vinculin-dependent adhesion–cytoskeletal coupling, which slowed retrograde actin network flow (i.e., it regulated the molecular clutch). On fibronectin, NGF caused inactivation of myosin IIA, which negatively regulated actin bundling. On both substrates, the result was the same: NGF-induced weakening of MII-dependent restraint led to dynamic microtubules entering the actin-rich periphery more frequently, giving rise to faster elongation. PMID:26631553

  8. 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.

  9. 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

  10. Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1.

    PubMed

    Xu, X X; Tessner, T G; Rock, C O; Jackowski, S

    1993-03-01

    Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle. Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated [3H]thymidine incorporation, although PC-PLC did not support continuous proliferation. PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor. The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta. Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester. PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells. PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased [3H]thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1. Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.

  11. 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.

  12. The transcriptional regulation of the Colony-Stimulating Factor 1 Receptor (csf1r) gene during hematopoiesis.

    PubMed

    Bonifer, Constanze; Hume, David A

    2008-01-01

    The colony-stimulating-factor 1 receptor (CSF-1 R) is a tyrosine kinase receptor that is absolutely required for macrophage differentiation and thus occupies a central role in hematopoiesis. Mice deficient for the csf1r gene show multiple defects in macrophage development, reproduction and tissue remodeling. Moreover, deregulation of this gene is a hallmark of many tumors. This includes repression of expression in acute myeloid leukemia and aberrant activation in certain solid tumors, such as breast cancer. Expression of this gene therefore needs to be tightly controlled. This review summarizes experiments providing a detailed picture of how transcription of csf1r gene expression is regulated. Aside from the direct relevance to hematopoiesis, studies of csf1r transcriptional regulation provide a model for understanding the molecular mechanisms that control mammalian cell fate.

  13. 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.

  14. Giant Cell Arteritis which Developed after the Administration of Granulocyte-colony Stimulating Factor for Cyclic Neutropenia.

    PubMed

    Umeda, Masataka; Ikenaga, Jin; Koga, Tomohiro; Michitsuji, Toru; Shimizu, Toshimasa; Fukui, Shoichi; Nishino, Ayako; Nakasima, Yoshikazu; Kawashiri, Sin-Ya; Iwamoto, Naoki; Ichinose, Kunihiro; Hirai, Yasuko; Tamai, Mami; Nakamura, Hideki; Origuchi, Tomoki; Kawakami, Atsushi

    2016-01-01

    A 78-year-old woman diagnosed with cyclic neutropenia 5 years previously had been treated with recombinant granulocyte-colony stimulating factor (G-CSF). She developed fever, tenderness and distension of temporal arteries after the treatment with G-CSF. Magnetic resonance imaging and ultrasonography revealed wall thickening of the temporal arteries. She was therefore diagnosed with giant cell arteritis (GCA). Small vessel vasculitis has been reported as a complication of G-CSF. However, the development of large vessel vasculitis after G-CSF treatment is quite rare. To our knowledge, the present case is the first report of GCA suspected to be associated with coexisting cyclic neutropenia and G-CSF treatment. PMID:27523011

  15. Evidence for a further stimulation of atrial natriuretic factor release by atrial pacing in patients with mitral stenosis.

    PubMed

    Malatino, L S; Stancanelli, B; Greco, G; Polizzi, G; Leonardi, C; Russo, G; Tamburino, C; Greco, G; Giuffrida, G; Tamburino, G

    1989-12-01

    To investigate the release of atrial natriuretic factor (ANF) in mitral stenosis and the effect of an increased atrial contraction frequency on atrial distension and ANF secretion, we studied 14 patients [New York Heart Association (NYHA) grades II-III] in sinus rhythm, undergoing cardiac catheterization for mitral stenosis. Echocardiographic tracings, repeat determinations of mean pulmonary artery wedge pressure and blood samples from the pulmonary artery for ANF measurements were taken at baseline, during atrial pacing (125 beats/min for 5 min) and 5 min after pacing. After pacing, ANF levels rose markedly with a parallel increase in mean pulmonary artery wedge pressure and left atrial size. These data indicate that atrial pacing is capable of further stimulating ANF release, even in patients with elevated baseline ANF and left atrial pressure and an increased left atrial dimension. PMID:2534411

  16. 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.

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

    PubMed Central

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

    1997-01-01

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

  18. Granulocyte-Macrophage Colony-Stimulating Factor- and Tumor Necrosis Factor Alpha-Mediated Matrix Metalloproteinase Production by Human Osteoblasts and Monocytes after Infection with Brucella abortus ▿

    PubMed Central

    Scian, Romina; Barrionuevo, Paula; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.; Delpino, M. Victoria

    2011-01-01

    Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. Since matrix metalloproteinases (MMPs) are involved in joint and bone damage in inflammatory and infectious diseases, we investigated the production of MMPs by human osteoblasts and monocytes, either upon Brucella abortus infection or upon reciprocal stimulation with factors produced by each infected cell type. B. abortus infection of the normal human osteoblastic cell line hFOB 1.19 triggered a significant release of MMP-2, which was mediated in part by granulocyte-macrophage colony-stimulating factor (GM-CSF) acting on these same cells. Supernatants from infected osteoblasts exhibited increased levels of monocyte chemoattractant protein 1 and induced the migration of human monocytes (THP-1 cell line). Infection with B. abortus induced a high MMP-9 secretion in monocytes, which was also induced by heat-killed B. abortus and by the Omp19 lipoprotein from B. abortus. These effects were mediated by Toll-like receptor 2 and by the action of tumor necrosis factor alpha (TNF-α) produced by these same cells. Supernatants from B. abortus-infected monocytes induced MMP-2 secretion in uninfected osteoblasts, and this effect was mediated by TNF-α. Similarly, supernatants from infected osteoblasts induced MMP-9 secretion in uninfected monocytes. This effect was mediated by GM-CSF, which induced TNF-α production by monocytes, which in turn induced MMP-9 in these cells. These results suggest that MMPs could be potentially involved in the tissue damage observed in osteoarticular brucellosis. PMID:20956574

  19. Microglia overexpressing the macrophage colony-stimulating factor receptor are neuroprotective in a microglial-hippocampal organotypic coculture system.

    PubMed

    Mitrasinovic, Olivera M; Grattan, Alicia; Robinson, Christopher C; Lapustea, Nicolae B; Poon, Clara; Ryan, Heather; Phong, Connie; Murphy, Greer M

    2005-04-27

    Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimer's disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury. Increased expression of M-CSFR causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. To determine whether M-CSFR-induced microglial activation affects neuronal survival, we assembled a coculture system consisting of BV-2 microglia transfected to overexpress the M-CSFR and hippocampal organotypic slices treated with NMDA. Twenty-four hours after assembly of the coculture, microglia overexpressing M-CSFR proliferated at a higher rate than nontransfected control cells and exhibited enhanced migration toward NMDA-injured hippocampal cultures. Surprisingly, coculture with c-fms-transfected microglia resulted in a dramatic reduction in NMDA-induced neurotoxicity. Similar results were observed when cocultures were treated with the teratogen cyclophosphamide. Biolistic overexpression of M-CSFR on microglia endogenous to the organotypic culture also rescued neurons from excitotoxicity. Furthermore, c-fms-transfected microglia increased neuronal expression of macrophage colony-stimulating factor (M-CSF), the M-CSFR, and neurotrophin receptors in the NMDA-treated slices, as determined with laser capture microdissection. In the coculture system, direct contact between the exogenous microglia and the slice was necessary for neuroprotection. Finally, blocking expression of the M-CSF ligand by exogenous c-fms-transfected microglia with a hammerhead ribozyme compromised their neuroprotective properties. These results demonstrate a protective role for microglia overexpressing M-CSFR in our coculture system and suggest under certain circumstances, activated microglia can help rather than harm neurons subjected to excitotoxic and teratogen-induced injury.

  20. A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells.

    PubMed

    Li, N F; Kocher, H M; Salako, M A; Obermueller, E; Sandle, J; Balkwill, F

    2009-02-01

    The receptor for macrophage colony-stimulating factor 1 receptor (CSF1R) is a product of the proto-oncogene c-fms and a member of the class III transmembrane tyrosine kinase receptor family. Earlier, we described increased mRNA expression of CSF1R in human telomerase reverse transcriptase (hTERT) immortalized human ovarian surface epithelial (IOSE) cell lines derived from a single donor. Here, we further describe that CSF1R is upregulated at both the mRNA and protein level in hTERT immortalized human normal OSE cells from two different donors and in hTERT immortalized human pancreatic ductal epithelial cells. CSF1R was not upregulated in hTERT immortalized epithelial clones that subsequently underwent senescence or in immortalized fibroblasts. Upon stimulation by the CSF1R ligand CSF1, the immortalized epithelial cell lines showed rapid internalization of CSF1R with concomitant down-modulation and colocalization of phosphorylated NFkappaBp65 with hTERT protein, hTERT translocation into the nucleus and the binding of c-Myc to the hTERT promoter region. Reducing the expression of CSF1R using short hairpin interfering RNA abolished these effects and also decreased cell survival and the number of population doublings under suboptimal culture conditions. The telomerase inhibitor GRN163L confirmed a role for telomerase in the cleavage of the intracellular domain of CSF1R. On the basis of these findings, we suggest that CSF1R may be a critical factor facilitating hTERT immortalization of epithelial cells.

  1. 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

  2. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  3. A novel combinatorial therapy with pulp stem cells and granulocyte colony-stimulating factor for total pulp regeneration.

    PubMed

    Iohara, Koichiro; Murakami, Masashi; Takeuchi, Norio; Osako, Yohei; Ito, Masataka; Ishizaka, Ryo; Utunomiya, Shinji; Nakamura, Hiroshi; Matsushita, Kenji; Nakashima, Misako

    2013-07-01

    Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.

  4. 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.

  5. Isolated abdominal aortitis following administration of granulocyte colony stimulating factor (G-CSF).

    PubMed

    Miller, Edward B; Grosu, Roy; Landau, Zvi

    2016-06-01

    G-CSF is a myeloid growth factor produced by monocytes, macrophages, fibroblasts, and endothelial cells. Clinical uses of G-CSF includes mobilization of peripheral blood progenitor cells from healthy donors before hematopoietic stem cell transplantation, acceleration of neutrophil recovery following chemotherapy, and in the management of neutropenia due to other causes including AIDS and genetic disorders of granulocyte production. G-CSF is well tolerated and reports to be safe in healthy donors, although follow-up studies are limited in duration (D'Souza et al. in Transfus Med Rev 22(4):280-290, 2008).Isolated abdominal aortitis (IAA) is a rare disorder most commonly caused by the large-vessel vasculitides giant cell arteritis (GCA) and Takayasu arteritis, although it may also be associated with several other rheumatologic diseases and infections (Gornik and Creager in Circulation 117:3039-3051, 2008). To our knowledge, there only two cases have been published of IAA occurring in patients who had received G-CSF therapy (Dariea et al. in Rev Med Interne 25(3):225-229, 2004; Adiga et al. in Clin Drug Investig 29:821-825, 2009).We describe a case of a 55-year-old male, with peripheral vascular disease who after receiving Neupogen (G-CSF) developed a latent case of IAA. After further investigation and exclusion of other possible causative factors, we conclude that the most probable etiology is induction by G-CSF.

  6. The probiotic mixture VSL#3 accelerates gastric ulcer healing by stimulating vascular endothelial growth factor.

    PubMed

    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 × 10(9) bacteria) or high (1.2 × 10(10) 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

  7. Isolated abdominal aortitis following administration of granulocyte colony stimulating factor (G-CSF).

    PubMed

    Miller, Edward B; Grosu, Roy; Landau, Zvi

    2016-06-01

    G-CSF is a myeloid growth factor produced by monocytes, macrophages, fibroblasts, and endothelial cells. Clinical uses of G-CSF includes mobilization of peripheral blood progenitor cells from healthy donors before hematopoietic stem cell transplantation, acceleration of neutrophil recovery following chemotherapy, and in the management of neutropenia due to other causes including AIDS and genetic disorders of granulocyte production. G-CSF is well tolerated and reports to be safe in healthy donors, although follow-up studies are limited in duration (D'Souza et al. in Transfus Med Rev 22(4):280-290, 2008).Isolated abdominal aortitis (IAA) is a rare disorder most commonly caused by the large-vessel vasculitides giant cell arteritis (GCA) and Takayasu arteritis, although it may also be associated with several other rheumatologic diseases and infections (Gornik and Creager in Circulation 117:3039-3051, 2008). To our knowledge, there only two cases have been published of IAA occurring in patients who had received G-CSF therapy (Dariea et al. in Rev Med Interne 25(3):225-229, 2004; Adiga et al. in Clin Drug Investig 29:821-825, 2009).We describe a case of a 55-year-old male, with peripheral vascular disease who after receiving Neupogen (G-CSF) developed a latent case of IAA. After further investigation and exclusion of other possible causative factors, we conclude that the most probable etiology is induction by G-CSF. PMID:27094941

  8. Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

    SciTech Connect

    Clohisy, D.R.; Erdmann, J.M.; Wilner, G.D. )

    1990-05-15

    The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program Ligand suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and (3H)thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak (3H) thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin.

  9. Strontium-calcium coadministration stimulates bone matrix osteogenic factor expression and new bone formation in a large animal model.

    PubMed

    Li, Zhaoyang; Lu, William W; Chiu, Peter K Y; Lam, Raymond W M; Xu, Bing; Cheung, Kenneth M C; Leong, John C Y; Luk, Keith D K

    2009-06-01

    Strontium (Sr) has become increasingly attractive for use in the prevention and treatment of osteoporosis by concomitantly inhibiting bone resorption and enhancing bone formation. Strontium shares similar chemical, physical, and biological characteristics with calcium (Ca), which has been widely used as a dietary supplement in osteoporosis. However, the effects of Sr-Ca coadministration on bone growth and remodeling are yet to be extensively reported. In this study, 18 ovariectomized goats were divided into four groups: three groups of five goats each treated with 100 mg/kg/day Ca, Ca plus 24 mg/kg/day Sr (Ca + 24Sr), or Ca plus 40 mg/kg/day Sr (Ca + 40Sr), and three untreated goats fed low calcium feed. Serum Sr levels increased 6- and 10-fold in the Ca + 24Sr and Ca + 40Sr groups, respectively. Similarly, Sr in the bone increased four- and sixfold in these two groups. Sr-Ca coadministration considerably increased bone mineral apposition rate (MAR). The expression of insulin-like growth factor (IGF)-1 and runt-related transcription factor 2 (Runx2) was significantly upregulated within the Ca + 40Sr treatment group; tumor necrosis factor (TNF)-agr; expression was significantly downregulated in the Ca and Ca + 40Sr groups. The results indicate that Sr-Ca coadministration increases osteogenic gene expression and stimulates new bone formation. PMID:19025756

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. The role of colony-stimulating factor 1 and its receptor in the etiopathogenesis of endometrial adenocarcinoma.

    PubMed

    Smith, H O; Anderson, P S; Kuo, D Y; Goldberg, G L; DeVictoria, C L; Boocock, C A; Jones, J G; Runowicz, C D; Stanley, E R; Pollard, J W

    1995-03-01

    Colony-stimulating factor 1 (CSF-1) is a homodimeric growth factor that humorally regulates the growth and differentiation of mononuclear phagocytes, and locally regulates maternal-fetal interactions during pregnancy. It exerts these actions through a transmembrane tyrosine kinase receptor, colony-stimulating factor 1 receptor (CSF-1R), the product of the c-fms proto-oncogene. Recent studies have demonstrated overexpression of CSF-1 and its receptor in breast, ovarian, and endometrial adenocarcinomas. To further investigate the possible role of CSF-1 and its receptor in the pathogenesis of endometrial adenocarcinoma, a prospective study was undertaken to study CSF-1 expression in benign and neoplastic endometrial epithelium and to compare serum CSF-1 levels in endometrial adenocarcinoma patients with healthy perimenopausal women. The mean serum levels of CSF-1 in 71 patients with endometrial cancer (4.9 +/- 1.8 microgram/liter) were significantly elevated compared with levels found in the 32 controls (3.5 +/- 1.1 microgram/liter). Within the endometrial adenocarcinoma group, circulating CSF-1 levels were significantly elevated in patients with large tumor volume, high grade, myometrial invasion, residual disease, and circulating CA-125 levels. High serum levels of serum CSF-1 were associated with elevated serum CA19-9 and CA-125 levels. Immunohistochemistry results revealed in tumor epithelium intense staining for CSF-1R (27 of 54 cases, 50%) and elevated staining for CSF-1 (41 of 54 cases, 75.9%), with intense staining of CSF-1 in 16 of 54 cases (29.6%). Staining was significantly greater in intensity and number of cells involved in malignant compared with benign epithelium for CSF-1R and CSF-1 (P = 0.05 and <0.0001, respectively). A positive correlation between amount and intensity of CSF-1 and CSF-1R staining in endometrial adenocarcinoma tissue was also demonstrated (P = 0.007). CSF-1 and CSF-1R mRNA was also detected in the tumor samples, confirming the

  15. Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato.

    PubMed

    Goetz, Marc; Hooper, Lauren C; Johnson, Susan D; Rodrigues, Julio Carlyle Macedo; Vivian-Smith, Adam; Koltunow, Anna M

    2007-10-01

    Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:beta-glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato ('Monalbo') resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in

  16. Male engorgement factor: Role in stimulating engorgement to repletion in the ixodid tick, Dermacentor variabilis.

    PubMed

    Donohue, Kevin V; Khalil, Sayed M S; Ross, Elizabeth; Mitchell, Robert D; Roe, R Michael; Sonenshine, Daniel E

    2009-10-01

    Mating in ticks results in profound physiological changes that eventually results in egg production. In the American dog tick, Dermacentor variabilis, mating causes partially blood-fed female ticks to commence rapid engorgement to repletion and eventual detachment from the host and egg laying. The peptidic male pheromone (engorgement factor alpha/beta) transferred to the female during mating is known only from a single tick species, Amblyomma hebraeum, and was shown to consist of two peptides produced in the testis/vas deferens (TVD) and not in the male accessory gland (MAG). In the current study, we obtained 2704bp of sequence data for efalpha from D. variabilis, of 7kb as determined by Northern blot, and show that it is also present in the Southern cattle tick, Rhipicephalus microplus and the deer tick, Ixodes scapularis. Analysis of the male gonad transcriptome by pyrosequencing produced 563,093 reads of which 636 matched with efalpha; none matched with efbeta. No evidence of efbeta orthologs could be found in any publicly available database including the I. scapularis genome. Silencing efalpha in male ticks failed to significantly reduce the engorgement weight of females compared to controls. Injection of sephadex beads, replete female synganglia, fed male MAG, fed male TVD, or replete female vagina/seminal receptacle (VA/SR), separately, failed to initiate feeding to repletion like that found in normally mated females. However, a small percentage of females injected with VA/SR that fed beyond the arbitrary weight for repletion of 300mg, produced brown eggs (an indication of vitellogenin uptake by the oocytes). The greatest effect was observed in female ticks injected with a suspension of MAG and TVD combined; 50% fed to repletion and all of these dropped off from the host and laid brown eggs. The effect was abolished if the aqueous fraction of the MAG/TVD homogenate only was injected suggesting that EF in ticks is a non-secreted membrane-bound or intracellular

  17. Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

    PubMed Central

    Pietrzkowski, Z; Sell, C; Lammers, R; Ullrich, A; Baserga, R

    1992-01-01

    BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells. Images PMID:1324408

  18. The hematopoietic cytokine granulocyte-macrophage colony stimulating factor is important for cognitive functions

    PubMed Central

    Krieger, Markus; Both, Martin; Kranig, Simon A.; Pitzer, Claudia; Klugmann, Matthias; Vogt, Gerhard; Draguhn, Andreas; Schneider, Armin

    2012-01-01

    We recently reported expression of hematopoietic growth factor GM-CSF and its receptor (GM-CSFR) in CNS neurons. Here we evaluated this system in learning and memory formation using GM-CSF deficient mice. In complementation, GM-CSF signalling was manipulated specifically in adult murine hippocampus by adeno-associated virus (AAV)-mediated GM-CSFR alpha overexpression or knock-down. GM-CSF ablation caused various hippocampus and amygdala-dependent deficits in spatial and fear memory while rendering intact basic parameters like motor function, inherent anxiety, and pain threshold levels. Corroborating these data, spatial memory of AAV-injected mice was positively correlated with GM-CSFRα expression levels. Hippocampal neurons of knock-out mice showed markedly pruned dendritic trees, reduced spine densities, and lower percentages of mature spines. Despite such morphological alterations, long-term potentiation (LTP) was unimpaired in the knock-out hippocampus. Collectively, these results suggest that GM-CSF signalling plays a major role in structural plasticity relevant to learning and memory. PMID:23019518

  19. Maternal Factors and Complications of Preterm Birth Associated with Neonatal Thyroid Stimulating Hormone

    PubMed Central

    Ryckman, Kelli K; Spracklen, Cassandra N; Dagle, John M; Murray, Jeffrey C

    2014-01-01

    Thyroid hormones are important regulators of fetal neurodevelopment. Among preterm infants, TSH is highly variable. Understanding this variability will further improvements in screening for thyroid disorders in preterm infants. We examined 61 maternal and infant clinical and demographic factors for associations with neonatal TSH levels in 698 preterm neonates. TSH was measured as part of routine State-mandated newborn screening in Iowa. Of the maternal characteristics, nulliparous women (P=8x10−4), women with preeclampsia (P=2x10−3), and those with induced labor (P=3x10−3) had infants with higher TSH levels. TSH levels at the time of newborn screening were associated with respiratory distress syndrome (RDS) (P<0.0001) and sepsis (P=3x10−3). We replicated findings between parity and preeclampsia previously observed in primarily term infants. We also observed strong relationships between neonatal TSH and complications of prematurity including RDS and sepsis, which has implications for future studies examining this relationship both prenatally and longitudinally after birth. PMID:24854527

  20. Delivery of basic fibroblast growth factors from heparinized decellularized adipose tissue stimulates potent de novo adipogenesis.

    PubMed

    Lu, Qiqi; Li, Mingming; Zou, Yu; Cao, Tong

    2014-01-28

    Scaffolds based on decellularized adipose tissue (DAT) are gaining popularity in adipose tissue engineering due to their high biocompatibility and adipogenic inductive property. However, previous studies involving DAT-derived scaffolds have not fully revealed their potentials for in vivo adipose tissue construction. With the aim of developing a more efficient adipose tissue engineering technique based on DAT, in this study, we investigated the in vivo adipogenic potential of a basic fibroblast growth factor (bFGF) delivery system based on heparinized DAT (Hep-DAT). To generate this system, heparins were cross-linked to mouse DATs by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-Hydroxysuccinimide. The bFGF-binding Hep-DATs were first tested for controlled release ability in vitro and then transplanted subcutaneously. Highly vascularized adipose tissues were formed 6weeks after transplantation. Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues. Additionally, strong de novo adipogenesis could also be induced when bFGF-binding Hep-DATs were thoroughly minced and injected subcutaneously. In conclusion, our study demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.

  1. Induction of tumor necrosis factor production from monocytes stimulated with mannuronic acid polymers and involvement of lipopolysaccharide-binding protein, CD14, and bactericidal/permeability-increasing factor.

    PubMed Central

    Jahr, T G; Ryan, L; Sundan, A; Lichenstein, H S; Skjåk-Braek, G; Espevik, T

    1997-01-01

    Well-defined polysaccharides, such as beta1-4-linked D-mannuronic acid (poly[M]) derived from Pseudomonas aeruginosa, induce monocytes to produce tumor necrosis factor (TNF) through a pathway involving membrane CD14. In this study we have investigated the effects of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing factor (BPI) on poly(M) binding to monocytes and induction of TNF production. We show that LBP increased the TNF production from monocytes stimulated with poly(M). Addition of sCD14 alone had only minor effects, but when it was added together with LBP, a rise in TNF production was seen. BPI was found to inhibit TNF production from monocytes stimulated with poly(M) in the presence of LBP, LBP-sCD14, or 10% human serum. Binding studies showed that poly(M) bound to LBP- and BPI-coated immunowells, while no significant binding of poly(M) to sCD14-coated wells in the absence of serum was observed. Binding of poly(M) to monocytes was also examined by flow cytometry, and it was shown that the addition of LBP or 10% human serum clearly increased the binding of poly(M) to monocytes. BPI inhibited the binding of poly(M) to monocytes in the presence of LBP, LBP-sCD14, or 10% human serum. Our data demonstrate a role for LBP, LBP-sCD14, and BPI in modulating TNF responses of defined polysaccharides. PMID:8975896

  2. Invasive candidiasis stimulates hepatocyte and monocyte production of active transforming growth factor beta.

    PubMed

    Letterio, J J; Lehrnbecher, T; Pollack, G; Walsh, T J; Chanock, S J

    2001-08-01

    Candida albicans is an opportunistic fungal pathogen and a major cause of morbidity and mortality in patients with compromised immune function. The cytokine response to tissue invasion by C. albicans can influence the differentiation and function of lymphocytes and other mononuclear cells that are critical components of the host response. While the production of transforming growth factor beta (TGF-beta) has been documented in mice infected with C. albicans and is known to suppress phagocyte function, the cellular source and role of this cytokine in the pathogenesis of systemic candidiasis are not well understood. We have investigated the source of production of TGF-beta by immunohistochemical studies in tissue samples from patients with an uncommon complication of lymphoreticular malignancy, chronic disseminated candidiasis (CDC), and from a neutropenic-rabbit model of CDC. Liver biopsy specimens from patients with documented CDC demonstrated intense staining for extracellular matrix-associated TGF-beta1 within inflammatory granulomas, as well as staining for TGF-beta1 and TGF-beta3 within adjacent hepatocytes. These results correlate with the immunolocalization of TGF-beta observed in livers of infected neutropenic rabbits, using a neutralizing antibody that recognizes the mature TGF-beta protein. Human peripheral blood monocytes incubated with C. albicans in vitro release large amounts of biologically active TGF-beta1. The data demonstrate that local production of active TGF-betas by hepatocytes and by infected mononuclear cells is a component of the response to C. albicans infection that most probably contributes to disease progression in the immunocompromised host.

  3. Retinal injury, growth factors and cytokines converge on β-catenin and pStat3 signaling to stimulate retina regeneration

    PubMed Central

    Wan, Jin; Zhao, Xiao-Feng; Vojtek, Anne; Goldman, Daniel

    2014-01-01

    Summary Müller glia (MG) in the zebrafish retina respond to retinal injury by generating multipotent progenitors for retinal repair. Here we show that Insulin, Igf-1 and FGF signaling components are necessary for retina regeneration. Interestingly, these factors synergize with each other and with HB-EGF and cytokines to stimulate MG to generate multipotent progenitors in the uninjured retina. These factors act by stimulating a core set of signaling cascades (Mapk/Erk, PI3K, β-catenin and pStat3) that are also shared with retinal injury and exhibit a remarkable amount of crosstalk. Our studies suggest that MG are both the producers and responders of factors that stimulate MG reprogramming and proliferation following retinal injury. The identification of a core set of regeneration-associated signaling pathways required for MG reprogramming not only furthers our understanding of retina regeneration in fish, but also suggests new targets for enhancing regeneration in mammals. PMID:25263555

  4. Uptake and Economic Impact of First-Cycle Colony-Stimulating Factor Use During Adjuvant Treatment of Breast Cancer

    PubMed Central

    Hershman, Dawn L.; Wilde, Elizabeth T.; Wright, Jason D.; Buono, Donna L.; Kalinsky, Kevin; Malin, Jennifer L.; Neugut, Alfred I.

    2012-01-01

    Purpose In 2002, pegfilgrastim was approved by the US Food and Drug Administration and the benefits of dose-dense breast cancer chemotherapy, especially for hormone receptor (HR) –negative tumors, were reported. We examined first-cycle colony-stimulating factor use (FC-CSF) before and after 2002 and estimated US expenditures for dose-dense chemotherapy. Methods We identified patients in Surveillance, Epidemiology, and End Results–Medicare greater than 65 years old with stages I to III breast cancer who had greater than one chemotherapy claim within 6 months of diagnosis(1998 to 2005) and classified patients with an average cycle length less than 21 days as having received dose-dense chemotherapy. The associations of patient, tumor, and physician-related factors with the receipt of any colony-stimulating factor (CSF) and FC-CSF use were analyzed by using generalized estimating equations. CSF costs were estimated for patients who were undergoing dose-dense chemotherapy. Results Among the 10,773 patients identified, 5,266 patients (48.9%) had a CSF claim. CSF use was stable between 1998 and 2002 and increased from 36.8% to 73.7% between 2002 and 2005, FC-CSF use increased from 13.2% to 67.9%, and pegfilgrastim use increased from 4.1% to 83.6%. In a multivariable analysis, CSF use was associated with age and chemotherapy type and negatively associated with black/Hispanic race, rural residence, and shorter chemotherapy duration. FC-CSF use was associated with high socioeconomic status but not with age or race/ethnicity. The US annual CSF expenditure for women with HR-positive tumors treated with dose-dense chemotherapy is estimated to be $38.8 million. Conclusion A rapid increase in FC-CSF use occurred over a short period of time, which was likely a result of the reported benefits of dose-dense chemotherapy and the ease of pegfilgrastim administration. Because of the increasing evidence that elderly HR-positive patients do not benefit from dose-dense chemotherapy

  5. 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

  6. Brain-Derived Neurotrophic Factor Stimulates Production of Prostacyclin in Cerebral Arteries

    PubMed Central

    Santhanam, Anantha Vijay R.; Smith, Leslie A.; Katusic, Zvonimir S.

    2009-01-01

    Background The role of Brain Derived Neurotrophic Factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), in control of cerebral circulation is poorly understood. The present study was designed to investigate the cerebral vascular effects of BDNF in vivo. Methods Replication incompetent adenovirus encoding either rat BDNF (AdBDNF) or green fluorescent protein (AdGFP) was injected intracisternally into rabbits. Forty eight hours later, animals were euthanized. Plasma and cerebrospinal fluid (CSF) levels of BDNF were measured by ELISA, vasomotor function of isolated basilar arteries was studied in organ chambers, protein expression in the basilar arteries was studied by Western blotting, prostanoid levels measured by ELISA and cyclic adenosine 3′,5′-monophosphate (cyclic AMP) levels were measured by radioimmunoassay. Results The levels of BDNF in the CSF were significantly elevated in AdBDNF-treated rabbits as compared to AdGFP-treated rabbits (37 ± 5 ng/ml vs. 0.006 ± 0.003 ng/ml, respectively, P<0.05, n=14). Western blotting studies revealed that in basilar arteries AdBDNF increased protein expression of prostacyclin (PGI2) synthase, while expression of endothelial nitric oxide synthase (eNOS) and phosphorylated (Ser 1177) eNOS remained unchanged. During incubation with arachidonic acid (1 μmol/L), PGI2 production and levels of cyclic AMP were significantly elevated only in AdBDNF-treated rabbit basilar arteries (P<0.05, n=6). Relaxations to acetylcholine (10−9 to 10−5 mol/L) and arachidonic acid (10−9 to 10−5 mol/L) were significantly potentiated in basilar arteries from rabbits injected with AdBDNF. Potentiation of relaxations to acetylcholine in AdBDNF-treated basilar arteries was inhibited by the non-selective cyclooxygenase inhibitor, indomethacin (10−5 mol/l, P<0.05, n=6) and constitutive phospholipase A2 inhibitor, AACOCF3 (2 × 10−5 mol/L, P<0.05, n=5). Conclusion Our results demonstrate that in cerebral arteries, BDNF

  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. Granulocyte Macrophage-Colony Stimulating Factor-induced Zn Sequestration Enhances Macrophage Superoxide and Limits Intracellular Pathogen Survival

    PubMed Central

    Vignesh, Kavitha Subramanian; Landero Figueroa, Julio A.; Porollo, Aleksey; Caruso, Joseph A.; Deepe, George S.

    2013-01-01

    SUMMARY Macrophages possess numerous mechanisms to combat microbial invasion, including sequestration of essential nutrients, like Zn. The pleiotropic cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) enhances antimicrobial defenses against intracellular pathogens such as Histoplasma capsulatum, but its mode of action remains elusive. We have found that GM-CSF activated infected macrophages sequestered labile Zn by inducing binding to metallothioneins (MTs) in a STAT3 and STAT5 transcription factor-dependent manner. GM-CSF upregulated expression of Zn exporters, Slc30a4 and Slc30a7 and the metal was shuttled away from phagosomes and into the Golgi apparatus. This distinctive Zn sequestration strategy elevated phagosomal H+ channel function and triggered reactive oxygen species (ROS) generation by NADPH oxidase. Consequently, H. capsulatum was selectively deprived of Zn, thereby halting replication and fostering fungal clearance. GM-CSF mediated Zn sequestration via MTs in vitro and in vivo in mice and in human macrophages. These findings illuminate a GM-CSF-induced Zn-sequestration network that drives phagocyte antimicrobial effector function. PMID:24138881

  9. Expression of colony-stimulating factor 1 is associated with occurrence of osteochondral change in pigmented villonodular synovitis.

    PubMed

    Ota, Takehiro; Urakawa, Hiroshi; Kozawa, Eiji; Ikuta, Kunihiro; Hamada, Shunsuke; Tsukushi, Satoshi; Shimoyama, Yoshie; Ishiguro, Naoki; Nishida, Yoshihiro

    2015-07-01

    Pigmented villonodular synovitis (PVNS) is a benign, translocation-derived neoplasm. Because of its high local recurrence rate after surgery and occurrence of osteochondral destruction, a novel therapeutic target is required. The present study aimed to evaluate the significance of protein expression possibly associated with the pathogenesis during the clinical course of PVNS. In 40 cases of PVNS, positivity of colony-stimulated factor 1 (CSF1), its receptor (CSF1R), and receptor activator of nuclear factor kappa-B ligand (RANKL) were immunohistochemically determined. The relationship between the positivity and clinical outcomes was investigated. High positivity of CSF1 staining intensity was associated with an increased incidence of osteochondral lesions (bone erosion and osteoarthritis) (p = 0.009), but not with the rate of local recurrence. Positivity of CSF1R and RANKL staining was not associated with any clinical variables. The number of giant cells was not correlated with positivity of any of the three proteins, or with the clinical outcome. Focusing on knee cases, CSF1 positivity was also associated with the incidence of osteochondal change (p = 0.02). CSF1R positivity was high in cases which had local recurrence, but not significantly so (p = 0.129). Determination of CSF1 and CSF1R expression may be useful as a prognosticator of the clinical course and/or outcomes of PVNS.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    1996-08-01

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

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

    PubMed

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

    2014-11-01

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

  13. Sequences of elongation factors-1 alpha and -1 gamma and stimulation by juvenile hormone in Locusta migratoria.

    PubMed

    Zhou, S; Zhang, J; Fam, M D; Wyatt, G R; Walker, V K

    2002-11-01

    Two cDNAs encoding the alpha and gamma subunits of translation elongation factor-1 (EF-1) have been cloned and sequenced from the African migratory locust, Locusta migratoria. Southern blotting and real-time PCR analyses indicated that these sequences represent single copy genes. Comparison with sequences from other species indicated greater conservation for EF-1 alpha than for EF-1 gamma. The developmental profiles for EF-1 alpha and -1 gamma mRNA expression in the fat body paralleled reported changes in the hemolymph juvenile hormone (JH) titer in the fifth instar and were elevated during early reproductive maturation in the female adult. In maturing adults, there was a greater accumulation of EF-1 alpha and -1 gamma transcripts in females than in males. The levels of both transcripts were greatly increased by an enriched diet, previously shown to elevate JH titers and accelerate vitellogenin production. Treating JH-deprived adult females with the JH analog, methoprene, resulted in more than doubling of transcript levels of both genes, supporting the hypothesis that JH could stimulate the accumulation of LmEF-1 alpha and -1 gamma transcripts. We suggest that production of elongation factors, increased by JH, may contribute to the massive protein synthesis required for egg production. PMID:12530224

  14. 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.

  15. Pharmacological stimulation of Hypoxia Inducible Factor-1α facilitates the corticosterone response to a mild acute stressor

    PubMed Central

    Harrell, Constance S.; Rowson, Sydney A.; Neigh, Gretchen N.

    2015-01-01

    While both glucocorticoids (the principal output of the hypothalamic-pituitary-adrenal axis) and oxidative stress have been implicated in outcomes due to an excessive or prolonged stress response, the precise mechanisms linking these two systems remain poorly elucidated. One potential mediator between the hypothalamic-pituitary-adrenal axis and oxidative stress is the Hypoxia Inducible Factor-1 (HIF-1) pathway. HIF-1 is an oxygen-responsive transcription factor with diverse effects including changes in cellular metabolism. The experiments in this manuscript sought to determine if pharmacological stimulation of HIF-1α via administration of dimethyloxalylglycine (DMOG) would facilitate the corticosterone response to a mild acute stressor. DMOG administration significantly increased plasma corticosterone five minutes after an acute airpuff without changing baseline plasma corticosterone or plasma corticosterone level two hours post-startle. DMOG administration also reduced hippocampal gene expression of the pro-translocation co-chaperone for the glucocorticoid receptor, FKBP4, two hours after airpuff startle. At this same two-hour time point, hippocampal expression of FKBP5, an anti-translocation co-chaperone of glucocorticoid receptor, in the DMOG-treated group was also positively correlated with plasma corticosterone levels. These data indicate that there is significant crosstalk between the hypothalamic-pituitary-axis and the HIF-1 pathway and extend the current knowledge of glucocorticoid and hypoxia interactions in an ethologically relevant stress model. PMID:26037418

  16. Transforming growth factor-beta 1 stimulates glomerular mesangial cell synthesis of the 72-kd type IV collagenase.

    PubMed Central

    Marti, H. P.; Lee, L.; Kashgarian, M.; Lovett, D. H.

    1994-01-01

    Transforming growth factor-beta 1 (TGF-beta 1) is generally considered to exert positive effects on the accumulation of extracellular matrices. These occur as the net result of enhanced matrix protein synthesis, diminished matrix metalloproteinase (MMP) synthesis, and augmented production of specific inhibitors, including the tissue inhibitor of metalloproteinases (TIMP-1). Given that glomerular TGF-beta 1 synthesis is induced by inflammation, the effects of this cytokine on synthesis of the 72-kd type IV collagenase and TIMP-1 by cultured human mesangial cells were evaluated. Concentrations of TGF-beta 1 of 5 ng/ml and above specifically stimulated the synthesis of the 72-kd type IV collagenase. This effect was independent of the stimulatory effect of TGF-beta 1 on TIMP-1 synthesis, which was maximal in a lower concentration range (0.1 to 1 ng/ml). Most significantly, the net effect at the higher concentrations of TGF-beta 1 was an excess of enzyme over the TIMP-1 inhibitor. Northern blot analysis of TGF-beta 1-stimulated human mesangial cells demonstrated a specific increase in the abundance of the 3.1 kb mRNA transcript encoding the 72-kd type IV collagenase, presumably mediated by a direct stimulation of 72-kd type IV collagenase mRNA transcription observed as early as 3 hours after exposure to TGF-beta 1. These studies were extended to an analysis of the expression of TGF-beta 1 and 72-kd type IV collagenase mRNAs in normal and nephritic rats. In normal animals, basal TGF-beta 1 and 72-kd type IV collagenase mRNA expression was observed in a strictly mesangial distribution. After induction of acute immune complex-mediated glomerulonephritis, there was a major increase in TGF-beta 1 and 72-kd type IV collagenase mRNA expression, which was strictly limited to the expanded, hypercellular mesangial compartment. Enhanced synthesis of the mesangial type IV collagenase in response to TGF-beta 1 released during glomerular inflammatory processes could have an important

  17. Up-regulation of transferrin receptor gene expression by granulocyte colony-stimulating factor in human myeloid leukemia cells.

    PubMed

    Morishita, Y; Kataoka, T; Towatari, M; Ito, T; Inoue, H; Ogura, M; Morishima, Y; Saito, H

    1990-12-15

    Granulocyte colony-stimulating factor (G-CSF) enhanced surface transferrin receptor (TfR) expression in two human myeloid leukemia cell lines, NKM-1 and NOMO-1, which possess G-CSF receptors. Radioligand-binding assay revealed that 10 ng/ml G-CSF significantly increased TfR to 186 +/- 20 and 276 +/- 38% of control for NKM-1 cells and NOMO-1 cells, respectively, in a 24-h culture. Scatchard analysis showed the increase of transferrin (Tf)-binding sites but no change in the receptor affinity. The enhanced TfR expression was not mediated either by the kinetic change of receptor cycling or by cellular iron content. Immunoprecipitation with anti-TfR antibody was used, and the increased biosynthesis of the receptor was demonstrated in G-CSF-stimulated cells. Northern blot analysis showed a 2- to 3-fold increase of TfR mRNA of NKM-1 cells cultured in medium containing Tf and G-CSF, whereas the mRNA declined without G-CSF. The effect of G-CSF on the TfR mRNA was observed within 2 h, which preceded the increase of surface TfR and the transition to the S phase of the cell cycle. G-CSF also potentiated TfR expression in freshly obtained myeloid leukemia cells. The present study shows up-regulation of TfR expression by G-CSF in myeloid leukemia cells and provides evidence that the regulation is mediated by controlling the steady-state level of the mRNA. PMID:1701357

  18. Evidence for a subventricular zone neural stem cell phagocytic activity stimulated by the vitamin K-dependent factor protein S.

    PubMed

    Ginisty, Aurélie; Gély-Pernot, Aurore; Abaamrane, Loubna; Morel, Franck; Arnault, Patricia; Coronas, Valérie; Benzakour, Omar

    2015-02-01

    Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology.

  19. Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1*

    PubMed Central

    Pan, Dingxin; Barber, Mark A.; Hornigold, Kirsti; Baker, Martin J.; Toth, Judit M.; Oxley, David; Welch, Heidi C. E.

    2016-01-01

    P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1. PMID:26792863

  20. 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

  1. Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21.

    PubMed

    Caescu, Cristina I; Guo, Xingyi; Tesfa, Lydia; Bhagat, Tushar D; Verma, Amit; Zheng, Deyou; Stanley, E Richard

    2015-02-19

    Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721-regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721-regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21-regulated messenger RNAs revealed that 80% of the CSF-1-regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R-mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R-regulated miR-21 network that modulates macrophage polarization.

  2. Colony stimulating factor-1 receptor signaling networks inhibit mouse macrophage inflammatory responses by induction of microRNA-21

    PubMed Central

    Caescu, Cristina I.; Guo, Xingyi; Tesfa, Lydia; Bhagat, Tushar D.; Verma, Amit; Zheng, Deyou

    2015-01-01

    Macrophage polarization between the M2 (repair, protumorigenic) and M1 (inflammatory) phenotypes is seen as a continuum of states. The detailed transcriptional events and signals downstream of colony-stimulating factor 1 receptor (CSF-1R) that contributes to amplification of the M2 phenotype and suppression of the M1 phenotype are largely unknown. Macrophage CSF-1R pTyr-721 signaling promotes cell motility and enhancement of tumor cell invasion in vitro. Combining analysis of cellular systems for CSF-1R gain of function and loss of function with bioinformatic analysis of the macrophage CSF-1R pTyr-721–regulated transcriptome, we uncovered microRNA-21 (miR-21) as a downstream molecular switch controlling macrophage activation and identified extracellular signal-regulated kinase1/2 and nuclear factor-κB as CSF-1R pTyr-721–regulated signaling nodes. We show that CSF-1R pTyr-721 signaling suppresses the inflammatory phenotype, predominantly by induction of miR-21. Profiling of the miR-21–regulated messenger RNAs revealed that 80% of the CSF-1–regulated canonical miR-21 targets are proinflammatory molecules. Additionally, miR-21 positively regulates M2 marker expression. Moreover, miR-21 feeds back to positively regulate its own expression and to limit CSF-1R–mediated activation of extracellular signal-regulated kinase1/2 and nuclear factor-κB. Consistent with an anti-inflammatory role of miRNA-21, intraperitoneal injection of mice with a miRNA-21 inhibitor increases the recruitment of inflammatory monocytes and enhances the peritoneal monocyte/macrophage response to lipopolysaccharide. These results identify the CSF-1R–regulated miR-21 network that modulates macrophage polarization. PMID:25573988

  3. Expression of E-Cadherin, Leukemia Inhibitory Factor and Progesterone Receptor in Mouse Blastocysts after Ovarian Stimulation

    PubMed Central

    Movaghar, Bahar; Askarian, Saeedeh

    2012-01-01

    Objective: The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles. Materials and Methods: In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test). Results: Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05). Conclusion: Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from

  4. [Effect of ascorbic acid, epidermal growth factor and follicle stimulating hormone on in vitro culture of sheep ovarian cortical tissue].

    PubMed

    Peng, Xiayu; Wang, Liqin; Yang, Mei; Chen, Tong; Guo, Zhiqin

    2010-06-01

    In this study, we evaluated the effects of ascorbic acid (VC), epidermal growth factor (EGF) and follicle stimulating hormone (FSH) on in vitro culture of sheep ovarian cortical tissue. Using 2 x 2 x 2 factor experimental design, we cultured sheep ovarian cortex fragments in 8 media with MEM (control), MEM+VC (50 microg/mL), MEM +EGF (100 ng/mL), MEM+FSH (50 ng/mL), MEM+VC+EGF, MEM+VC+FSH, MEM+EGF+FSH, MEM+VC+EGF+FSH. After 0 (non-cultured control), 2, 6, 12 days of culture, the pieces of ovarian cortex were proceed to histological and proliferating cell nuclear antigen (PCNA) examination, or observed by transmission electron microscopy (TEM). The percentages of developing follicles were increased (P < 0.05) and the percentages of healthy follicles were reduced (P < 0.05). When compared to the MEM group, the addition of FSH with VC or EGF promoted a significant increase of follicles diameter and follicles survival rate (P < 0.05), and stimulated the proliferation of granulosa cells. After 12 days of culture, medium supplemented with MEM+VC+EGF resulted the lowest proportion of developing follicles (49.3% +/- 3.2%), follicles diameter((32.3 +/- 2.3) microm), follicles survival rate (41.6% +/- 3.1%) and the proportion of PCNA stained follicles (26.4% +/- 1.2%, P < 0.05). In contrast, MEM+VC+EGF+FSH resulted the highest follicles diameter ((42.5 +/- 5.1) microm), follicles survival rate (59.7% +/- 6.1%) and proportion of PCNA stained follicles (43.5% +/- 4.1%, P < 0.05). Ultrastructural analysis confirmed the integrity of follicles cultured in VC+EGF+FSH group, while follicles cultured in MEM+VC+EGF groups showed more degeneration characters. In conclusion, the addition of VC and EGF to culture medium inhibited follicular development, VC+EGF+FSH was the most effective treatment to maintain follicular integrity and promote sheep primordial follicular activation and growth during in vitro culture.

  5. The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

    PubMed Central

    Bridges, Rebecca G.; Sohn, Sook-Young; Wright, Jordan

    2016-01-01

    ABSTRACT Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection. PMID:26814176

  6. A new set of regulatory molecules in plants: A plant phospholipid similar to platelet-activating factor stimulates protein kinase and proton-translocating ATPase in membrane vesicles.

    PubMed

    Scherer, G F; Martiny-Baron, G; Stoffel, B

    1988-08-01

    1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, an ether phospholipid from mammals known as platelet-activating factor (PAF), specifically stimulates proton transport in zucchini (Cucurbita pepo L.) microsomes (G.F.E. Scherer, 1985, Biochem. Biophys. Res. Commm. 133, 1160-1167). When plant lipids were analyzed by two-dimensional thin-layer chromatography a lipid was found with chromatographic properties very similar to the PAF (G.F.E. Scherer and B. Stoffel, 1987, Planta, 172, 127-130). This lipid was isolated from zucchini hypocotyls, red beet root, lupin root, maize seedlings and crude soybean phospholipids. It had biological activity similar to that of the PAF, based on phosphorus content, and stimulated the steady-state ΔpH in zucchini hypocotyl microsomes about twofold. Other phospholipids, monoglyceride, diglyceride, triglyceride, oleic acid, phorbol ester, and 1-O-alkylglycerol did not stimulate proton transport. When microsomes were washed the PAF was ineffective but when soluble protein was added the PAF stimulation of H(+) transport was reconstituted. The soluble protein responsible for the PAF-dependent stimulation of transport activity could be partially purified by diethylaminoethyl Sephacel column chromatography. In the same fractions where the PAF-dependent transport-stimulatory protien was found, a protein kinase was active. This protein kinase was stimulated twofold either by the PAF or by Ca(2+). When Ca(2+) was present the PAF did not stimulate protein-kinase activity. When either the PAF, protein kinase, or both were added to membranes isolated on a linear sucrose gradient, ATPase activity was stimulated up to 30%. Comparison with marker enzymes indicated the possibility that tonoplast and plasma-membrane H(+)-ATPase might be stimulated by the PAF and protein kinase. We speculate that a PAF-dependent protein kinase is involved in the regulation of proton transport in plants in vitro and in vivo.

  7. 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

  8. 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.

  9. 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.

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

    PubMed

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

    2000-11-01

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

  11. Randomized Trial of Two Dosages of Prophylactic Granulocyte Colony-Stimulating Factor after Induction Chemotherapy in Pediatric Acute Myeloid Leukemia

    PubMed Central

    Inaba, Hiroto; Cao, Xueyuan; Pounds, Stanley; Pui, Ching-Hon; Rubnit, Jeffrey E.; Ribeiro, Raul C.; Razzouk, Bassem I.

    2010-01-01

    Background Granulocyte colony-stimulating factor (G-CSF) is effective in accelerating neutrophil recovery after intensive chemotherapy for acute myeloid leukemia (AML). However, the optimal G-CSF dosage for patients with AML has not been determined. To our knowledge, G-CSF dosages have not been compared in a randomized AML study. Methods Patients enrolled on the St. Jude AML97 protocol who remained on study after window therapy were eligible to participate. The effect of the dosage of G-CSF given after induction chemotherapy courses 1 and 2 was analyzed in 46 patients randomly assigned in a double-blinded manner to receive 5 or 10 μg/kg/day of G-CSF. The number of days of G-CSF treatment, neutropenia (absolute neutrophil count < 0.5 × 109/L), and hospitalization; the number of episodes of febrile neutropenia, grade 2-4 infection, and antimicrobial therapy; transfusion requirements; the cost of supportive care; and survival were compared between the two study arms. Results We found no statistically significant difference between the two arms in any of the endpoints measured. Conclusions The higher G-CSF dosage (10 μg/kg/day) offers no greater benefit than the lower dosage (5 μg/kg/day) in patients undergoing intensive chemotherapy for AML. PMID:21381017

  12. Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

    PubMed

    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.

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

    PubMed

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

    2009-08-13

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

  14. Accelerated phagocytosis of amyloid-beta by mouse and human microglia overexpressing the macrophage colony-stimulating factor receptor.

    PubMed

    Mitrasinovic, Olivera M; Murphy, Greer M

    2002-08-16

    Microglia surrounding A beta plaques in Alzheimer's disease and in the APPV717F transgenic mouse model of Alzheimer's disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated A beta. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of A beta in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-microm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of A beta was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of A beta. M-CSFR antibody blocking experiments demonstrated that increased A beta uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of A beta after immunization.

  15. Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor.

    PubMed

    Mulero, Iván; Pilar Sepulcre, M; Roca, Francisco J; Meseguer, José; García-Ayala, Alfonsa; Mulero, Victoriano

    2008-01-01

    Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr(+)) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr(+) cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

  16. Colony-stimulating factor 1 receptor (CSF1R) signaling in injured neurons facilitates protection and survival.

    PubMed

    Luo, Jian; Elwood, Fiona; Britschgi, Markus; Villeda, Saul; Zhang, Hui; Ding, Zhaoqing; Zhu, Liyin; Alabsi, Haitham; Getachew, Ruth; Narasimhan, Ramya; Wabl, Rafael; Fainberg, Nina; James, Michelle L; Wong, Gordon; Relton, Jane; Gambhir, Sanjiv S; Pollard, Jeffrey W; Wyss-Coray, Tony

    2013-01-14

    Colony-stimulating factor 1 (CSF1) and interleukin-34 (IL-34) are functional ligands of the CSF1 receptor (CSF1R) and thus are key regulators of the monocyte/macrophage lineage. We discovered that systemic administration of human recombinant CSF1 ameliorates memory deficits in a transgenic mouse model of Alzheimer's disease. CSF1 and IL-34 strongly reduced excitotoxin-induced neuronal cell loss and gliosis in wild-type mice when administered systemically before or up to 6 h after injury. These effects were accompanied by maintenance of cAMP responsive element-binding protein (CREB) signaling in neurons rather than in microglia. Using lineage-tracing experiments, we discovered that a small number of neurons in the hippocampus and cortex express CSF1R under physiological conditions and that kainic acid-induced excitotoxic injury results in a profound increase in neuronal receptor expression. Selective deletion of CSF1R in forebrain neurons in mice exacerbated excitotoxin-induced death and neurodegeneration. We conclude that CSF1 and IL-34 provide powerful neuroprotective and survival signals in brain injury and neurodegeneration involving CSF1R expression on neurons.

  17. Augmentation of Human Macrophage Candidacidal Capacity by Recombinant Human Myeloperoxidase and GranulocyteMacrophage Colony-Stimulating Factor

    PubMed Central

    Maródi, László; Tournay, Christophe; Káposzta, Rita; Johnston, Richard B.; Moguilevsky, Nicole

    1998-01-01

    Phagocyte myeloperoxidase (MPO) is believed to be particularly important in defense against candida infection. We reported earlier that monocytes, rich in MPO, killed Candida albicans at a significantly higher rate and extent than did monocyte-derived macrophages, known to lack MPO, and that C. albicans is less resistant to MPO-dependent oxidants than less pathogenic Candida species. We hypothesized, therefore, that the capacity of macrophages to kill C. albicans might be improved in the presence of MPO. In this study, we evaluated the ability of recombinant human MPO (rhMPO) to augment the killing of C. albicans by resident macrophages and macrophages activated by recombinant human granulocyte-macrophage colony-stimulating factor. Addition of rhMPO (concentration range, 0.8 to 6.4 U/ml) to suspensions of resident and activated macrophages and opsonized C. albicans resulted in concentration-dependent and significant increases in candida killing. This enhancement was particularly pronounced with activated macrophages, whether C. albicans was opsonized or unopsonized and ingested through the macrophage mannose receptor. rhMPO did not affect the killing of C. albicans by monocytes, nor did it affect phagocytosis of opsonized or unopsonized C. albicans. These results indicate that exogenous rhMPO can augment the candidacidal capacity of both resident and activated macrophages, with a more profound effect on activated cells. We suggest that rhMPO may be effective in the treatment of invasive candidiasis. PMID:9596743

  18. GRANULOCYTE COLONY-STIMULATING FACTOR: MOLECULAR MECHANISMS OF ACTION DURING STEADY STATE AND ‘EMERGENCY’ HEMATOPOIESIS

    PubMed Central

    Panopoulos, Athanasia D.; Watowich, Stephanie S.

    2008-01-01

    Neutrophils are phagocytes whose principal function is to maintain anti-bacterial immunity. Neutrophils ingest and kill invading bacteria, releasing cytotoxic, chemotactic and inflammatory mediators at sites of infection. This serves to control the immediate host immune response and attract other cells, such as macrophages and dendritic cells, which are important for establishing long-term adaptive immunity. Neutrophils thus contribute to both the initiation and the maintenance of inflammation at sites of infection. Aberrant neutrophil activity is deleterious; suppressed responses can cause extreme susceptibility to infection while overactivation can lead to excessive inflammation and tissue damage. This review will focus on neutrophil regulation by granulocyte colony-stimulating factor (G-CSF), the principal cytokine controlling neutrophil development and function. The review will emphasize the molecular aspects of G-CSF-driven granulopoiesis in steady state (healthy) conditions and during demand-driven or ‘emergency’ conditions elicited by infection or clinical administration of G-CSF. Understanding the molecular control of granulopoiesis will aid in the development of new approaches designed to treat disorders of neutrophil production and function. PMID:18400509

  19. 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

  20. Factor for inversion stimulation-dependent growth rate regulation of individual tRNA species in Escherichia coli.

    PubMed

    Nilsson, L; Emilsson, V

    1994-04-01

    We have studied the involvement of the factor for inversion stimulation (FIS) in the growth rate-dependent expression of the arginine, leucine, and methionine acceptor tRNA species. The concentration of individual tRNA species relative to 16 S rRNA was determined by blot hybridization using RNA preparations from bacteria with the fis gene deleted and from isogenic wild type bacteria. The RNA preparations were obtained from bacteria growing under steady state conditions in different media. The levels of tRNA(1Leu), tRNA(2Arg), tRNA(4Arg), and tRNA(5Arg decreased in the fis bacteria, relative to the wild type. The difference in levels increased with increasing growth rate. Surprisingly, tRNA(3Leu), tRNA(rMet), and tRNA(eMet) showed the opposite response, with an increase of the tRNA/16 S ratio in the fis bacteria. The tRNA(2Leu, tRNA(4Leu), tRNA(5Leu), and tRNA(3 Arg) had unaffected tRNA/16 S ratios in fis cells. We conclude that FIS, directly or indirectly, is involved in growth rate regulation of some tRNA species and that it affects the composition of the cellular tRNA pool.

  1. Granulocyte-colony stimulating factor decreases the extent of ovarian damage caused by cisplatin in an experimental rat model

    PubMed Central

    Akdemir, Ali; Akman, Levent; Ergenoglu, Ahment Mete; Yeniel, Ahmet Ozgur; Erbas, Oytun; Yavasoglu, Altug; Terek, Mustafa Cosan; Taskiran, Dilek

    2014-01-01

    Objective To investigate whether granulocyte-colony stimulating factor (G-CSF) can decrease the extent of ovarian follicle loss caused by cisplatin treatment. Methods Twenty-one adult female Sprague-Dawley rats were used. Fourteen rats were administered 2 mg/kg/day cisplatin by intraperitoneal injection twice per week for five weeks (total of 20 mg/kg). Half of the rats (n=7) were treated with 1 mL/kg/day physiological saline, and the other half (n=7) were treated with 100 µg/kg/day G-CSF. The remaining rats (n=7, control group) received no therapy. The animals were then euthanized, and both ovaries were obtained from all animals, fixed in 10% formalin, and stored at 4℃ for paraffin sectioning. Blood samples were collected by cardiac puncture and stored at -30℃ for hormone assays. Results All follicle counts (primordial, primary, secondary, and tertiary) and serum anti-Müllerian hormone levels were significantly increased in the cisplatin+G-CSF group compared to the cisplatin+physiological saline group. Conclusion G-CSF was beneficial in decreasing the severity of follicle loss in an experimental rat model of cisplatin chemotherapy. PMID:25142624

  2. Characterization of Stress-Exposed Granulocyte Colony Stimulating Factor Using ELISA and Hydrogen/Deuterium Exchange Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Tsuchida, Daisuke; Yamazaki, Katsuyoshi; Akashi, Satoko

    2014-10-01

    Information on the higher-order structure is important in the development of biopharmaceutical drugs. Recently, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) has been widely used as a tool to evaluate protein conformation, and unique automated systems for HDX-MS are now commercially available. To investigate the potential of this technique for the prediction of the activity of biopharmaceuticals, granulocyte colony stimulating factor (G-CSF), which had been subjected to three different stress types, was analyzed using HDX-MS and through comparison with receptor-binding activity. It was found that HDX-MS, in combination with ion mobility separation, was able to identify conformational changes in G-CSF induced by stress, and a good correlation with the receptor-binding activity was demonstrated, which cannot be completely determined by conventional peptide mapping alone. The direct evaluation of biological activity using bioassay is absolutely imperative in biopharmaceutical development, but HDX-MS can provide the alternative information in a short time on the extent and location of the structural damage caused by stresses. Furthermore, the present study suggests the possibility of this system being a versatile evaluation method for the preservation stability of biopharmaceuticals.

  3. Carcinoembryonic antigen-related cell adhesion molecule-1 regulates granulopoiesis by inhibition of granulocyte colony-stimulating factor receptor.

    PubMed

    Pan, Hao; Shively, John E

    2010-10-29

    Although carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is an activation marker for neutrophils and delays neutrophil apoptosis, the role of CEACAM1 in granulopoiesis and neutrophil-dependent host immune responses has not been investigated. CEACAM1 expression correlated with granulocytic differentiation, and Ceacam1(-/-) mice developed neutrophilia because of loss of the Src-homology-phosphatase-1 (SHP-1)-dependent inhibition of granulocyte colony-stimulating factor receptor (G-CSFR) signal transducer and activator of transcription (Stat3) pathway provided by CEACAM1. Moreover, Ceacam1(-/-) mice were hypersensitive to Listeria Monocytogenes (LM) infection with an accelerated mortality. Reintroduction of CEACAM1 into Ceacam1(-/-) bone marrow restored normal granulopoiesis and host sensitivity to LM infection, while mutation of its immunoreceptor tyrosine-based inhibitory motifs (ITIMs) abrogated this restoration. shRNA-mediated reduction of Stat3 amounts rescued normal granulopoiesis, attenuating host sensitivity to LM infection in Ceacam1(-/-) mice. Thus, CEACAM1 acted as a coinhibitory receptor for G-CSFR regulating granulopoiesis and host innate immune response to bacterial infections.

  4. Using hydrogen/deuterium exchange mass spectrometry to study conformational changes in granulocyte colony stimulating factor upon PEGylation.

    PubMed

    Wei, Hui; Ahn, Joomi; Yu, Ying Qing; Tymiak, Adrienne; Engen, John R; Chen, Guodong

    2012-03-01

    PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring, and protein therapeutic characterization in the biopharmaceutical industry.

  5. Treatment of methimazole-induced severe aplastic anemia with recombinant human granulocyte-monocyte colony-stimulating factor and glucocorticosteroids.

    PubMed

    López-Karpovitch, X; Ulloa-Aguirre, A; von Eiff, C; Hurtado-Monroy, R; Alanis, A

    1992-01-01

    The in vivo response to recombinant human granulocyte-monocyte colony-stimulating factor (rHu GM-CSF) in facilitating the reconstitution of granulomonopoiesis was evaluated in a patient with Graves' disease who developed severe aplastic anemia during methimazole therapy. After 10 days of treatment with rHu GM-CSF, the neutrophil and monocyte counts rose to 1.65 x 10(9)/l and 0.41 x 10(9)/l, respectively. However, the patient was still dependent on erythrocyte and platelet transfusions. Two days after rHu GM-CSF withdrawal, the neutrophil count dropped off to 0.41 x 10(9)/l.rHu GM-CSF was reinitiated for 2 days along with glucocorticosteroids. With this combined therapeutic approach, the neutrophil count returned to normal and remained stable, and both Hb and platelet values began to improve. It is concluded that the combination of rHu GM-CSF and glucocorticosteroids can be used as a therapeutic option that may lead to beneficial results in drug-induced aplastic anemia.

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

    PubMed Central

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

    2006-01-01

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

  7. Protective effect of recombinant murine granulocyte-macrophage colony-stimulating factor against Pseudomonas aeruginosa infection in leukocytopenic mice.

    PubMed Central

    Tanaka, T.; Okamura, S.; Okada, K.; Suga, A.; Shimono, N.; Ohhara, N.; Hirota, Y.; Sawae, Y.; Niho, Y.

    1989-01-01

    The effects of recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) against Pseudomonas aeruginosa infection in ICR mice were investigated. Mice were treated with cyclophosphamide (CPA) and were then injected intraperitoneally with rmGM-CSF three times daily, beginning on the day after CPA treatment, for 7 days. The number of peripheral blood leukocytes in both CPA- and rmGM-CSF-treated mice and control CPA-treated mice reached a nadir on day 4, when P. aeruginosa was injected intraperitoneally. The administration of rmGM-CSF significantly increased the proportion of survivors among mice infected with a lethal dose of P. aeruginosa. This effect was further analyzed by monitoring sequential changes in leukocyte count and bacterial growth in various organs. The number of bacteria in the peritoneal cavities, peripheral blood samples, and livers of GM-CSF-treated mice decreased to an undetectable level after a transient increase, and the number was significantly lower than that in control mice. In GM-CSF-treated mice, the neutrophil levels in peripheral blood started to increase 5 days after CPA administration and were consistently higher than those in controls. Furthermore, the neutrophils in GM-CSF-treated mice were more mature morphologically. Thus, the prophylactic effect of rmGM-CSF against P. aeruginosa infection may result from a rapid recovery of myelopoiesis and a partial enhancement of mature neutrophil function. PMID:2656523

  8. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation

    PubMed Central

    Bordeleau, Marie-Eve; Matthews, James; Wojnar, Joanna M.; Lindqvist, Lisa; Novac, Olivia; Jankowsky, Eckhard; Sonenberg, Nahum; Northcote, Peter; Teesdale-Spittle, Paul; Pelletier, Jerry

    2005-01-01

    RNA helicases are the largest group of enzymes in eukaryotic RNA metabolism. The DEXD/H-box putative RNA helicases form the helicase superfamily II, whose members are defined by seven highly conserved amino acid motifs, making specific targeting of selected members a challenging pharmacological problem. The translation initiation factor eIF4A is the prototypical DEAD-box RNA helicase that works in conjunction with eIF4B and eIF4H and as a subunit of eIF4F to prepare the mRNA template for ribosome binding, possibly by unwinding the secondary structure proximal to the 5′ m7GpppN cap structure. We report the identification and characterization of a small molecule inhibitor of eukaryotic translation initiation that acts in an unusual manner by stimulating eIF4A-associated activities. Our results suggest that proper control of eIF4A helicase activity is necessary for efficient ribosome binding and demonstrate the feasibility of selectively targeting DEAD-box RNA helicases with small molecules. PMID:16030146

  9. Macrophage-Colony Stimulating Factor Derived from Injured Primary Afferent Induces Proliferation of Spinal Microglia and Neuropathic Pain in Rats.

    PubMed

    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

  10. 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

  11. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

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

    SciTech Connect

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

    1990-11-15

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

  13. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

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

    PubMed

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

    1991-04-01

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

  15. Allograft inflammatory factor-1 stimulates hemocyte immune activation by enhancing phagocytosis and expression of inflammatory cytokines in Crassostrea gigas.

    PubMed

    Zhang, Yang; Li, Jun; Yu, Feng; He, Xiaocui; Yu, Ziniu

    2013-05-01

    Allograft inflammatory factor-1 (AIF-1) is a calcium-binding cytokine associated with immune cell activation and inflammatory response. Presently, we have identified and characterized an AIF-1 in a marine bivalve mollusk, Crassostrea gigas, and designated it as CgAIF-1. The full-length CgAIF-1 cDNA is 794 bp, encoding a protein of 149 amino acids with two conserved EF hand Ca(2+)-binding motifs. CgAIF-1 is constitutively expressed in various tissues with enriched expression in hemocytes. Moreover, CgAIF-1 transcription is induced by multiple Pathogen-Associated Molecular Patterns (PAMPs), including poly (I: C), LPS, PGN, HKLM and HKVA, but is limited by 1,3-β-glucan. Furthermore, recombinant CgAIF-1 can specifically stimulate phagocytic ability of granulocytes, but not of intermediate cells and hyalinocytes. CgAIF-1 also enhances mRNA levels of MIF, TNF and IL-17. These results provide the first functional evidence that CgAIF-1 is involved in hemocyte activation in C. gigas, revealing conserved functions of AIF-1 in host defense from mollusks to mammals.

  16. 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

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

    PubMed

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

    2013-04-12

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

  18. 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.

  19. 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

  20. 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.

  1. Factors Associated With Upper Extremity Functional Recovery Following Low-Frequency Repetitive Transcranial Magnetic Stimulation in Stroke Patients

    PubMed Central

    2016-01-01

    Objective To investigate the factors related to upper extremity functional improvement following inhibitory repetitive transcranial magnetic stimulation (rTMS) in stroke patients. Methods Forty-one stroke patients received low-frequency rTMS over the contralesional hemisphere according to a standard protocol, in addition to conventional physical and occupational therapy. The rTMS-treated patients were divided into two groups according to their responsiveness to rTMS measured by the self-care score of the Korean version of Modified Barthel Index (K-MBI): responded group (n=19) and non-responded group (n=22). Forty-one age-matched stroke patients who had not received rTMS served as controls. Neurological, cognitive and functional assessments were performed before rTMS and 4 weeks after rTMS treatment. Results Among the rTMS-treated patients, the responded group was significantly younger than the non-responded group (51.6±10.5 years and 65.5±13.7 years, respectively; p=0.001). Four weeks after rTMS, the National Institutes of Health Stroke Scale, the Brunnstrom recovery stage and upper extremity muscle power scores were significantly more improved in the responded group than in the control group. Besides the self-care score, the mobility score of the K-MBI was also more improved in the responded group than in the non-responded group or controls. Conclusion Age is the most obvious factor determining upper extremity functional responsiveness to low-frequency rTMS in stroke patients. PMID:27446773

  2. Clinical practice in febrile neutropenia risk assessment and granulocyte colony-stimulating factor primary prophylaxis of febrile neutropenia in Poland

    PubMed Central

    Chmielowska, Ewa; Filipczyk-Cisarż, Emilia; Krzemieniecki, Krzysztof; Leśniewski-Kmak, Krzysztof; Litwiniuk, Maria M.; Wieruszewska-Kowalczyk, Karolina; Kosno-Kruszewska, Elżbieta

    2014-01-01

    Aim of the study The first aim was to investigate the knowledge and awareness of oncologists concerning febrile neutropenia (FN) risk assessment and indications for granulocyte colony-stimulating factor (G-CSF) primary prophylaxis (PP), based on current therapeutic guidelines (PTOK and EORTC). The second aim was to educate the oncologists on best practices for risk assessment and neutropenia management. Material and methods The project participants included 169 oncologists from 7 regions working in large specialist oncological centres, university hospitals, regional and city hospitals, specialist outpatient clinics, and oncological wards in small local hospitals. The participants completed a questionnaire based on seven prepared clinical cases of patients with different tumour types and patient characteristics, receiving chemotherapy (CT), and with different levels of FN risk. Participants answered questions related to FN risk assessment and G-CSF use. After completing the questionnaire, the participants proceeded to an educational module in which they were provided with an analysis of correct diagnostic and therapeutic procedures according to the PTOK and EORTC guidelines. Results and Conclusions Febrile neutropenia risk assessment was found to be a routine procedure performed for over 90% of the clinical cases by the participant oncologists. However, the FN risk assessment of clinical cases was correct and consistent with therapeutic guidelines in only 65% of responses. Indications for G-CSF PP were properly identified in 76% of responses and it appeared that indications for G-CSF PP were more likely to be correctly identified in patients receiving high-risk or low-risk regimens than in those receiving intermediate-risk regimens, where the decision to give G-CSF PP is based on additional assessment of patient risk factors. The vast majority of participants who correctly identified the need for PP administered G-CSF in accordance with the dose and schedule

  3. 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. PMID:26375397

  4. 1,25-dihydroxycholecalciferol-induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters

    SciTech Connect

    Bettens, F.; Schlick, E.; Farrar, W.; Ruscetti, F.

    1986-12-01

    The murine myelomonocytic leukemia cell line WEHI-3B D/sup +/, which differentiates in response to granulocyte colony stimulating factor (G-CSF), can also be induced to differentiate into monocyte-macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI-3B D/sup -/ subline, which is unresponsive to G-CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by 1,25-dihydroxycholecalciferol (1,25-(OH)/sub 2/ D3), the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI-3B D/sup +/ line, named WEHI-3B D/sup +/G, which was responsive to G-CSF but not to PMA, was also differentiated to granulocytes by 1,25-(OH)/sub 2/ D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25-(OH)/sub 2/ D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25-(OH)/sub 2/ D3, which has cytosolic/nuclear receptors. These results suggest that low doses of 1,25-(OH)/sub 2/ D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.

  5. Excision of the Shigella Resistance Locus Pathogenicity Island in Shigella flexneri Is Stimulated by a Member of a New Subgroup of Recombination Directionality Factors

    PubMed Central

    Luck, Shelley N.; Turner, Sally A.; Rajakumar, Kumar; Adler, Ben; Sakellaris, Harry

    2004-01-01

    Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis. PMID:15292162

  6. Stimulation of microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis factor alpha-induced neurotoxicity in concert with microglial-derived Fas ligand.

    PubMed

    Taylor, Deanna L; Jones, Fleur; Kubota, Eva S F Chen Seho; Pocock, Jennifer M

    2005-03-16

    Activated microglia may be detrimental to neuronal survival in a number of neurodegenerative diseases. Thus, strategies that reduce microglial neurotoxicity may have therapeutic benefit. Stimulation of group II metabotropic glutamate (mGlu) receptors on rat primary microglia with the specific group II agonist 2S,2'R,3'R-2-(2',3'-dicarboxy-cyclopropyl)glycine for 24 h induced microglial activation and resulted in a neurotoxic microglial phenotype. These effects were attributable to preferential mGlu2 stimulation, because N-acetyl-L-aspartyl-L-glutamate, a specific mGlu3 agonist, did not induce microglial activation or neurotoxicity. Stimulation of microglial mGlu2 but not mGlu3 induced caspase-3 activation in cerebellar granule neurons in culture, using microglial-conditioned media as well as cocultures. Stimulation of microglial mGlu2 induced tumor necrosis factor-alpha (TNFalpha) release, which contributed to microglial neurotoxicity mediated via neuronal TNF receptor 1 and caspase-3 activation. Stimulation of microglial group I or III mGlu receptors did not induce TNFalpha release. TNFalpha was only neurotoxic in the presence of microglia or microglial-conditioned medium. The toxicity of TNFalpha could be prevented by coexposure of neurons to conditioned medium from microglia stimulated by the specific group III agonist L-2-amino-4-phosphono-butyric acid. The neurotoxicity of TNFalpha derived from mGlu2-stimulated microglia was potentiated by microglial-derived Fas ligand (FasL), the death receptor ligand. FasL was constitutively expressed in microglia and shed after mGlu2 stimulation. Our data suggest that selective and inverse modulation of microglial mGlu2 and mGlu3 may prove a therapeutic target in neuroinflammatory diseases such as Alzheimer's disease and multiple sclerosis. PMID:15772355

  7. Prostaglandin E2 transactivates the colony-stimulating factor-1 receptor and synergizes with colony-stimulating factor-1 in the induction of macrophage migration via the mitogen-activated protein kinase ERK1/2.

    PubMed

    Digiacomo, Graziana; Ziche, Marina; Dello Sbarba, Persio; Donnini, Sandra; Rovida, Elisabetta

    2015-06-01

    Prostaglandin E2 (PGE2), a key mediator of immunity, inflammation, and cancer, acts through 4 G-protein-coupled E-prostanoid receptors (EPs 1-4). Crosstalk between EPs and receptor tyrosine kinases also occurs. Colony-stimulating factor-1 receptor (CSF-1R) is an RTK that sustains the survival, proliferation, and motility of monocytes/macrophages, which are an essential component of innate immunity and cancer development. The aim of this study was to investigate on a possible crosstalk between EP and CSF-1R. In BAC1.2F5 and RAW264.7 murine macrophages, CSF-1 (EC₅₀ = 18.1 and 10.2 ng/ml, respectively) and PGE2 (EC₅₀ = 1.5 and 5.5 nM, respectively) promoted migration. PGE2 induced rapid CSF-1R phosphorylation that was dependent on Src family kinases (SFKs). CSF-1R inhibition reduced PGE2-elicited ERK1/2 phosphorylation and macrophage migration, indicating that CSF-1R plays a role in PGE2-mediated immunoregulation. EP4 appeared responsible for functional PGE2/CSF-1R crosstalk. Furthermore, PGE2 synergized with CSF-1 in inducing ERK1/2 phosphorylation and macrophage migration. ERK1/2 inhibition completely blocked migration induced by the combination CSF-1/PGE2. CSF-1/PGE2 functional interaction with respect to migration also occurred in bone marrow-derived murine macrophages (EC₅₀ CSF-1, 6.7 ng/ml; EC₅₀ PGE2, 16.7 nM). These results indicated that PGE2 transactivates CSF-1R and synergizes with its signaling at ERK1/2 level in promoting macrophage migration.

  8. 1,25-dihydroxyvitamin D3 stimulates transforming growth factor-beta1 synthesis by mouse renal proximal tubular cells.

    PubMed

    Weinreich, T; Landolt, M; Booy, C; Wüthrich, R; Binswanger, U

    1999-01-01

    1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] is a secosteroid hormone with effects on cell growth, differentiation and immunoregulatory functions in a number of tissues not primarily involved in mineral metabolism. We recently demonstrated growth-regulating effects of 1, 25-(OH)2 D3 on human mesangial cells and proximal tubular cells. To investigate whether 1,25-(OH)2 D3 might also affect the synthesis of cytokines and growth factors in proximal tubular cells, we assessed in the present study the expression and secretion of transforming growth factor-beta1 (TGF-beta1) in a mouse proximal tubular cell line (MCT) in vitro. TGF-beta1 synthesis was measured by a monospecific ELISA in culture supernatant. The secreted TGF-beta1 was proven to be biologically active by means of a bioassay system (CCL-64 mink lung epithelial cell proliferation assay). TGF-beta1 gene expression was assessed by RT-PCR. To analyze whether TGF-beta1 expression mediates the 1,25-(OH)2 D3-induced antiproliferative actions in MCT, proliferation studies in the absence or presence of a blocking monoclonal anti TGF-beta1-3 antibody were performed. 1, 25-(OH)2 D3 (10(-11) to 10(-7) M) specifically increased the TGF-beta1 protein secretion in MCT with a maximum at 10(-8) M. No detectable effect was found with 25 D3 at 10 times higher concentrations. A synthetic 20-epi analogue, MC 1288, increased TGF-beta1 secretion up to similar amounts at equimolar concentrations as the natural hormone 1,25-(OH)2 D3. Steady-state TGF-beta1 mRNA concentration in MCT was transiently increased by 1, 25-(OH)2 D3 between 12 and 24 h, returning to control values at 48 h. Blocking TGF-beta1 did not reduce or abrogate the antiproliferative effect of 1,25-(OH)2 D3. In conclusion, 1,25-(OH)2 D3 stimulates TGF-beta1 expression in renal proximal tubular cells, a growth factor with anti-inflammatory and profibrotic actions which plays an important role in the development and progression of nephrosclerosis. PMID:10394107

  9. Direct Demonstration of Separate Receptors for Growth and Metabolic Activities of Insulin and Multiplication-stimulating Activity (an Insulinlike Growth Factor) Using Antibodies to the Insulin Receptor

    PubMed Central

    King, George L.; Kahn, C. Ronald; Rechler, Matthew M.; Nissley, S. Peter

    1980-01-01

    Insulin and such insulinlike growth factors as multiplication stimulating activity (MSA) are related polypeptides that have common biological activities. Both insulin and MSA produce acute metabolic responses (stimulation of glucose oxidation in isolated fat cells) as well as growth effects (stimulation of [3H]thymidine incorporation into DNA in cultured fibroblasts). In addition, most cells have separate receptors for insulin and insulinlike growth factors, and both peptides have weaker affinity for each other's specific receptors than for their own. To determine, therefore, whether these effects are mediated by receptors for insulin, insulinlike growth factors, or both, we have selectively blocked insulin receptors with a specific antagonist, namely Fab fragments derived from naturally occurring antibodies to the insulin receptor. In rat adipocytes, 10 μg/ml of antireceptor Fab inhibited insulin binding by 90%, whereas it inhibited MSA binding <5%. The anti-insulin receptor Fab is without intrinsic biological activity, but acts as a competitive inhibitor of insulin receptors. Blockade of insulin receptors with Fab fragments produced a 30-fold rightward shift in the dose response for stimulation of glucose oxidation by both insulin and MSA. The dose-response curves for stimulation of oxidation by vitamin K5 and spermine, agents that stimulate glucose oxidation through noninsulin receptor pathways, were not affected by the blockade of insulin receptors with Fab antibody fragments. These data suggest that this acute metabolic effect of both insulin and MSA is mediated via the insulin receptor. In cultured human fibroblasts, 10 μg/ml of Fab inhibited insulin binding by 90% and MSA binding by 15%. In fibroblasts, however, blockade of the insulin receptor did not alter the dose response for stimulation of thymidine incorporation into DNA by either insulin or MSA. Furthermore, intact antireceptor antibody immunoglobulin (Ig)G, which produces multiple other insulinlike

  10. 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.

  11. Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis.

    PubMed

    Nijaguna, Mamatha Bangalore; Patil, Vikas; Urbach, Serge; Shwetha, Shivayogi D; Sravani, Kotha; Hegde, Alangar S; Chandramouli, Bangalore A; Arivazhagan, Arimappamagan; Marin, Philippe; Santosh, Vani; Somasundaram, Kumaravel

    2015-09-18

    Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NFκB pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NFκB-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

  12. Tumour necrosis factor-alpha-induced glucose-stimulated insulin secretion inhibition in INS-1 cells is ascribed to a reduction of the glucose-stimulated Ca2+ influx.

    PubMed

    Kim, Hyo-Eun; Choi, Sung-E; Lee, Soo-Jin; Lee, Ji-Hyun; Lee, Youn-Jung; Kang, Sang Sun; Chun, Jaesun; Kang, Yup

    2008-09-01

    The present study was undertaken to determine how tumour necrosis factor-alpha (TNF-alpha) elicits the inhibition of glucose-stimulated insulin secretion (GSIS) in rat insulinoma cells (INS)-1 beta-cells. TNF-alpha pretreatment did not change the expression levels of insulin, PDX-1, glucose transporter 2, glucokinase, K(ATP) channels, Ca(2)(+) channels, and exocytotic molecules and, furthermore, did not reduce the glucose-stimulated ATP level. On the other hand, TNF-alpha reduced the glucose-stimulated influx of Ca(2)(+). The TNF-alpha treatment was thought to activate c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and NF-kappaB inflammatory signals, since TNF-alpha increased phospho-JNK and phospho-p38 and reduced I kappaB levels. Inhibitors of these signaling pathways prevented the TNF-alpha-induced reduction of the Ca(2)(+) influx and GSIS. Overexpression of MEKK3, a possible mediator from the TNF-alpha receptor to the JNK/p38 and NK-kappaB signaling cascade, increased the levels of phospho-JNK, phospho-p38, and NF-kappaB, and reduced the glucose-stimulated Ca(2)(+) influx and GSIS. The reduction of the Ca(2)(+) influx and GSIS in MEKK3-overexpressing INS-1 cells was also prevented by inhibitors of JNK, p38, and NF-kappaB. These data demonstrate that TNF-alpha inhibits GSIS by reducing the glucose-stimulated Ca(2)(+) influx, possibly through the activation of JNK and p38 MAPK and NF-kappaB inflammatory signals. Thus, our findings suggest that the activation of stress and inflammatory signals can contribute to the inhibition of GSIS in the development of diabetes.

  13. Retinal injury, growth factors, and cytokines converge on β-catenin and pStat3 signaling to stimulate retina regeneration.

    PubMed

    Wan, Jin; Zhao, Xiao-Feng; Vojtek, Anne; Goldman, Daniel

    2014-10-01

    Müller glia (MG) in the zebrafish retina respond to retinal injury by generating multipotent progenitors for retinal repair. Here, we show that Insulin, Igf-1, and fibroblast growth factor (FGF) signaling components are necessary for retina regeneration. Interestingly, these factors synergize with each other and with heparin-binding EGF-like growth factor (HB-EGF) and cytokines to stimulate MG to generate multipotent progenitors in the uninjured retina. These factors act by stimulating a core set of signaling cascades (Mapk/Erk, phosphatidylinositol 3-kinase [PI3K], β-catenin, and pStat3) that are also shared with retinal injury and exhibit a remarkable amount of crosstalk. Our studies suggest that MG both produce and respond to factors that stimulate MG reprogramming and proliferation following retinal injury. The identification of a core set of regeneration-associated signaling pathways required for MG reprogramming not only furthers our understanding of retina regeneration in fish but also suggests targets for enhancing regeneration in mammals.

  14. Improved Production and Characterization of Recombinant Human Granulocyte Colony Stimulating Factor from E. coli under Optimized Downstream Processes.

    PubMed

    Vemula, Sandeep; Thunuguntla, Rahul; Dedaniya, Akshay; Kokkiligadda, Sujana; Palle, Chaitanya; Ronda, Srinivasa Reddy

    2015-04-01

    This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coli BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4g/L) and purified rhG-CSF (136mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8M urea at pH 9.5. Refolding efficiency studies showed maximum refolding ⩾86% and ⩾84% at 20°C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (⩾95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with ⩾98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF.

  15. A novel glycobiomarker, Wisteria floribunda agglutinin macrophage colony-stimulating factor receptor, for predicting carcinogenesis of liver cirrhosis.

    PubMed

    Iio, Etsuko; Ocho, Makoto; Togayachi, Akira; Nojima, Masanori; Kuno, Atsushi; Ikehara, Yuzuru; Hasegawa, Izumi; Yatsuhashi, Hiroshi; Yamasaki, Kazumi; Shimada, Noritomo; Ide, Tatsuya; Shinkai, Noboru; Nojiri, Shunske; Fujiwara, Kei; Joh, Takashi; Mizokami, Masashi; Narimatsu, Hisashi; Tanaka, Yasuhito

    2016-03-15

    Recently, we identified a novel liver fibrosis glycobiomarker, Wisteria floribunda agglutinin (WFA)-reactive colony stimulating factor 1 receptor (WFA(+) -CSF1R), using a glycoproteomics-based strategy. The aim of this study was to assess the value of measuring WFA(+) -CSF1R levels for the prognosis of carcinogenesis and outcome in liver cirrhosis (LC) patients with hepatitis C virus (HCV). WFA(+) -CSF1R and Total-CSF1R levels were measured in serum samples from 214 consecutive HCV-infected patients to evaluate their impact on carcinogenesis and the survival of LC patients. Serum WFA(+) -CSF1R levels were significantly higher in LC patients than chronic hepatitis (CH) patients (p < 0.001). The AUC of WFA(+) -CSF1R for predicting overall survival, calculated by time-dependent ROC analysis, was 0.691 and the HR (per 1-SD increase) was 1.80 (95% CI, 1.23-2.62, p < 0.001). Furthermore, the survival rate of LC patients with high WFA(+) -CSF1R levels (≥ 310 ng/ml) was significantly worse than those with lower levels (p < 0.01). The AUC of WFA(+) /total-CSF1R percentage (WFA(+) -CSF1R%) for predicting the cumulative carcinogenesis rate was 0.760, with an HR of 1.66 (95% CI 1.26-2.20, p < 0.001). In fact, the carcinogenesis rate was significantly higher in LC patients with a high WFA(+) -CSF1R% (≥ 35%, p = 0.006). Assessing serum levels of WFA(+) -CSF1R has diagnostic value for predicting carcinogenesis and the survival of LC patients.

  16. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

  17. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

    PubMed Central

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C. A.; Woll, Petter S.; Jacobsen, Sten Eirik W.

    2016-01-01

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  18. Effect of recombinant human granulocyte colony-stimulating factor on efficacy of radiation therapy in tumor-bearing rats

    SciTech Connect

    Koji Kabaya; Masahiko Watanabe; Masaru Kusaka; Hiromichi Akahori; Masatoshi Seki; Masato Fushiki

    1994-07-01

    The effect of recombinant human granulocyte colony-stimulating factor on radiation-induced neutropenia and on growth of transplanted tumors treated by irradiation was investigated using tumor-bearing rats as a model for radiation therapy. In a preliminary study using normal rats, neutropenia induced by upper hemi-body irradiation at 3 Gy/day 5 times a week for 3 weeks was prevented by consecutive subcutaneous injections of rhG-CSF at 100 {mu}g/kg/day. Rats bearing Walker-256, a mammary tumor, were scheduled to receive upper hemibody irradiation at 3 Gy/day for 15 times in 3 weeks if white blood cell (WBC) counts were maintained above 3,000/{mu}l. In control tumor-bearing rats not receiving rhG-CSF, irradiation was often withheld because of the decrease in WBC counts below 3,000/{mu}l. In contrast, a decrease in WBC counts below 3,000/{mu}l was rarely found in tumor-bearing rats injected daily with rhG-CSF. The average number of radiation treatments in control rats and rats treated with rhG-CSF was about 8 and 14, respectively, out of the scheduled 15 treatments in 3 weeks. Treatment with rgG-CSF made it possible to complete the radiation therapy regimen and thus inhibit the growth of the transplanted tumor more effectively. These results suggest that rgG-CSF may be useful to ensure radiation therapy on schedule in cancer patients. 20 refs., 4 figs., 1 tab.

  19. 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.

  20. Tumor necrosis factor-α suppresses the protein fractional synthesis rate of the small intestine stimulated by glutamine in rats

    PubMed Central

    ZHOU, JIHONG; FAN, SHENGXIAN; CAO, YACHENG; ZHU, MINGFANG; HAN, YONG; CAO, XUEYING; LI, YOUSHENG

    2015-01-01

    The objective of this study was to examine whether and how TNF-α affects glutamine-enhanced protein synthesis and the expression of the amino acid transporter ASCT2 in the small intestine at the mRNA and protein levels. A total of 30 male Sprague-Dawley rats were randomly assigned into three groups, namely the total parenteral nutrition (TPN; control), glutamine-treated (Gln), and glutamine- and tumor necrosis factor-α (TNF-α)-treated (TNF-α) groups. At 30 min prior to examination, all rats were mainlined with [L-15N]leucine. The concentration of TNF-α in plasma and of glutamine in plasma and the small intestine was measured. The fractional synthesis rate (FSR) of protein and the mRNA and protein expression levels of ASCT2 in the small intestine were assessed. The level of TNF-α was highest in the TNF-α group and the glutamine concentration was elevated to a greater extent in the TNF-α group than in the other two groups. However, the FSR of protein in the small intestine was significantly higher in the Gln group compared with that in the TNF-α group. The mRNA and protein expression levels of ASCT2 in the experimental groups were significantly higher that those in the control group, but did not differ significantly between the Gln and TNF-α groups. These results indicate that TNF-α may attenuate glutamine-stimulated protein synthesis in the small intestine in the early stage of sepsis in rats. The mechanism may be that TNF-α inhibits the function of the glutamine transporter in the uptake the glutamine into target cells for protein synthesis. This inhibition may occur at or following protein translation. PMID:25574232

  1. 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

  2. MicroRNA-181b stimulates inflammation via the nuclear factor-κB signaling pathway in vitro

    PubMed Central

    WANG, YAZHEN; MAO, GENXIANG; LV, YUANDONG; HUANG, QINGDONG; WANG, GUOFU

    2015-01-01

    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. PMID:26622531

  3. Granulocyte-colony stimulating factor for acute-on-chronic liver failure: systematic review and meta-analysis.

    PubMed

    Chavez-Tapia, Norberto C; Mendiola-Pastrana, Indira; Ornelas-Arroyo, Victoria J; Noreña-Herrera, Camilo; Vidaña-Perez, Desiree; Delgado-Sanchez, Guadalupe; Uribe, Misael; Barrientos-Gutierrez, Tonatiuh

    2015-01-01

    Acute-on-chronic liver failure (ACLF) is associated with increased short and long-term mortality. Animal models of liver failure have demonstrated that granulocyte-colony stimulating factor (G-CSF) accelerates the liver regeneration process and improves survival. However, clinical evidence regarding the use of G-CSF in ACLF remains scarce. The aim of this study was to assess the benefits and harms of G-CSF in patients with acute-on-chronic liver failure. An electronic search was made in The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE and LILACS up to November 2013. Randomized clinical trials comparing the use of any regimen of G-CSF against placebo or no intervention in patients with ACLF were included. Primary outcomes included overal mortality, mortality due multi-organ failure, and adverse events. Relative risk (RR) and mean difference (MD) were used. Two trials involving 102 patients were included. A significant reduction in short-term overall mortality was observed in patients receiving G-CSF compared to controls (RR 0.56; 95%CI 0.39,0.80). G-CSF failed to reduce mortality secondary to gastrointestinal bleeding (RR 1.45; 95%CI 0.50, 4.27). Adverse effects reported included: fever, rash, herpes zoster, headache and nausea. In conclusion, the use of G-CSF for the treatment of patients with ACLF significantly reduced short-term mortality. While the evidence is still limited, the apparent benefit observed on short-term mortality, mild adverse effects and lack of an alternative therapy make the use of G-CSF in ACLF patients a reasonable alternative when liver transplantation is contraindicated or unavailable.

  4. Granulocyte colony-stimulating factor (G-CSF) production in hemorrhagic shock requires both the ischemic and resuscitation phase.

    PubMed

    Hierholzer, C; Kelly, E; Billiar, T R; Tweardy, D J

    1997-01-01

    Granulocyte colony-stimulating factor (G-CSF) is the cytokine that is critical for polymorphonuclear neutrophilic granulocyte (PMN) production as well as being a potent agonist of PMN activation. We have recently reported that in the lung and the liver of rats resuscitated after hemorrhagic shock (HS) G-CSF mRNA expression is induced. It is not known if both phases of HS, the ischemic and the reperfusion phase, are required for G-CSF mRNA induction. The present study was designed to test the hypothesis that the upregulation of G-CSF mRNA expression is the consequence of HS followed by resuscitation and that ischemia alone is insufficient to induce G-CSF mRNA expression in the affected organs. Male Sprague-Dawley rats were subjected to resuscitated and unresuscitated shock protocols of varying severity. Control animals were subjected to anesthesia and all surgical preparations except for hemorrhage. Lungs and livers were isolated and their RNA extracted. Using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we demonstrated that G-CSF mRNA was induced in the lung and liver of shock animals above the level observed in control animals. Upregulation of G-CSF mRNA relative to controls occurred only in animals undergoing resuscitated HS and not in ones subjected to unresuscitated HS. These results indicate that G-CSF production specific for the hemorrhage component of shock is dependent on resuscitation. As a consequence, the production of this cytokine may be decreased through modifications in the resuscitation protocols.

  5. 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.

  6. Identification of a novel Stat3 recruitment and activation motif within the granulocyte colony-stimulating factor receptor.

    PubMed

    Chakraborty, A; Dyer, K F; Cascio, M; Mietzner, T A; Tweardy, D J

    1999-01-01

    Stat3 is essential for early embryonic development and for myeloid differentiation induced by the cytokines granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6). Two isoforms of Stat3 have been identified, (p92) and beta (p83), which have distinct transcriptional and biological functions. Activation of both Stat3 and Stat3beta requires the distal cytoplasmic domain of the G-CSFR, which contains four Tyr at positions 704, 729, 744, and 764. The studies reported here were undertaken to determine which, if any, of these tyrosine residues participated in Stat3/beta recruitment and activation. We showed that Stat3 and Stat3beta were affinity purified using phosphopeptides containing Y704 and Y744 but not by nonphosphorylated peptide analogues or by phosphopeptides containing Y729 and Y764. Complementary results were obtained in studies examining the ability of these peptides to destabilize and inhibit DNA binding of activated Stat3. Both Y704 and Y744 contributed to optimal activation of Stat3/beta in M1 murine myeloid leukemia cells containing wild-type and Y-to-F mutant G-CSFR constructs. Carboxy-terminal to Y704 at the +3 position is Gln; YXXQ represents a consensus Stat3 recruitment and activation motif. Y744 is followed at the +3 position by Cys (C); YXXC, represents a novel motif implicated in the recruitment and activation of Stat3. Modeling of the SH2 domain of Stat3 based on homologous SH2 domains of known structure revealed polar residues whose side chains contact the +3 position. This substitution may confer specificity for the Y704- and Y744-based ligands by allowing H-bond formation between the binding surface and the Gln or Cys found at the respective +3 position.

  7. Granulocyte colony-stimulating factor administration to HIV-infected subjects augments reduced leukotriene synthesis and anticryptococcal activity in neutrophils.

    PubMed Central

    Coffey, M J; Phare, S M; George, S; Peters-Golden, M; Kazanjian, P H

    1998-01-01

    Neutrophil (PMN) dysfunction occurs in HIV infection. Leukotrienes (LT) are mediators derived from the 5-lipoxygenase (5-LO) pathway that play a role in host defense and are synthesized by PMN. We investigated the synthesis of LT by PMN from HIV-infected subjects. There was a reduction (4.0+/-1.3% of control) in LT synthesis in PMN from HIV-infected compared with normal subjects. This was associated with reduced expression of 5-LO-activating protein (31.2+/-9.6% of normal), but not of 5-LO itself. Since HIV does not directly infect PMN, we considered that these effects were due to reduced release of cytokines, such as granulocyte colony-stimulating factor (G-CSF). We examined the effect of G-CSF treatment (300 microgram daily for 5 d) on eight HIV-infected subjects. PMN were studied in vitro before therapy (day 1) and on days 4 and 7. LTB4 synthesis was increased on day 4 of G-CSF treatment, and returned toward day 1 levels on day 7. 5-LO and 5-LO-activating protein expression were increased in parallel. As a functional correlate to this increase in PMN LT synthesis by G-CSF, we examined the effects on killing of Cryptococcus neoformans. Anticryptococcal activity of PMN from HIV-infected subjects was less than that of PMN from normal subjects. G-CSF treatment improved fungistatic activity of PMN. This increase in antifungal activity was attenuated by in vitro treatment with the LT synthesis inhibitor, MK-886. In conclusion, PMN from HIV-infected subjects demonstrate reduced 5-LO metabolism and antifungal activity in vitro, which was reversed by in vivo G-CSF therapy. PMID:9710433

  8. 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

  9. Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice.

    PubMed

    Zriwil, Alya; Böiers, Charlotta; Wittmann, Lilian; Green, Joanna C A; Woll, Petter S; Jacobsen, Sten Eirik W; Sitnicka, Ewa

    2016-07-14

    Although it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19(+) B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R(+)CD19(+) ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R(+) myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R(+) myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported. PMID:27207794

  10. Functional granulocyte/macrophage colony stimulating factor receptor is constitutively expressed on neoplastic plasma cells and mediates tumour cell longevity.

    PubMed

    Villunger, A; Egle, A; Kos, M; Egle, D; Tinhofer, I; Henn, T; Uberall, F; Maly, K; Greil, R

    1998-09-01

    It has been shown that granulocyte/macrophage colony stimulating factor (GM-CSF) is able to support myeloma cell propagation in cooperation with interleukin (IL)-6, the major growth factor for malignant plasma cells, although the biological mechanisms involved remain unknown. Therefore we investigated (i) the expression levels of the GM-CSF receptor (GM-CSFR) constituents in three malignant plasma cell lines and in native malignant plasma cells, (ii) the ability of the receptor to mediate common signalling pathways regulating proliferation and cell survival in malignant plasma cell lines, and (iii) the effects of GM-CSF on tumour cell biology. The GM-CSFRalpha subunit was detected in the malignant plasma cell lines RPMI-8226, MC/CAR, IM-9 as well as 6/6 native myeloma cell samples derived from the bone marrow of patients with overt disease. Furthermore, GM-CSFR expression was also detected in the CD19+ fraction from 2/3 bone marrow samples and 5/8 peripheral blood samples derived from patients with malignant plasma cell disorders, but not in the CD19+ fraction of peripheral blood from healthy donors. The expressed cytokine receptor alpha-subunit was able to constitute a functional signalling complex with the ubiquitously expressed GM-CSFRbeta subunit, as demonstrated by the fact that GM-CSF induced the p21-ras/mitogen-activated protein kinase (MAPK) signalling cascade in malignant plasma cell lines. Since this signalling cascade plays an essential role in the mediation of both proliferation and cell survival, we investigated the impact of GM-CSF on these two events. Application of GM-CSF led to an increase of DNA-synthesis in MC/CAR, IM-9 and RPMI-8226 cells. Furthermore, it increased longevity of these malignant plasma cell lines by reducing the rates of spontaneous apoptosis. We conclude that (i) the functional GM-CSFR is commonly expressed on malignant plasma cells and that (ii) GM-CSF promotes the clonal expansion of myeloma cells by inhibiting spontaneous

  11. A Physiological Stimulating Factor of Water Intake during and after Dry Forage Feeding in Large-type Goats

    PubMed Central

    Van Thang, Tran; Sunagawa, Katsunori; Nagamine, Itsuki; Kishi, Tetsuya; Ogura, Go

    2012-01-01

    plasma osmolality in the second hour of the 2 h feeding period is the main physiological stimulating factor of water intake during and after dry forage feeding in large-type goats. PMID:25049591

  12. Interleukin 1 alpha and tumor necrosis factor alpha stimulate autocrine amphiregulin expression and proliferation of human papillomavirus-immortalized and carcinoma-derived cervical epithelial cells.

    PubMed Central

    Woodworth, C D; McMullin, E; Iglesias, M; Plowman, G D

    1995-01-01

    Infection with multiple sexually transmitted agents has been associated with inflammation of the cervix and an increased risk of cervical cancer in women infected with human papillomaviruses (HPVs). Two proinflammatory cytokines, interleukin 1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha), inhibited proliferation of normal epithelial cells cultured from human cervix. In contrast, both cytokines significantly stimulated proliferation of cervical cell lines (5 of 7) immortalized by transfection with HPV-16 or -18 DNAs or lines derived from cervical carcinomas (7 of 11). Stimulation was dose dependent from 0.01 to 1.0 nM and was blocked by specific inhibitors, such as the IL-1 receptor antagonist or the TNF type 1 or 2 soluble receptors. Growth stimulation by IL-1 alpha or TNF-alpha was accompanied by a 6- to 10-fold increase in RNA encoding amphiregulin, an epidermal growth factor (EGF) receptor ligand. Recombinant human amphiregulin (0.1 nM) was as effective as IL-1 alpha or TNF-alpha in promoting proliferation. Monoclonal antibodies that blocked signal transduction by the EGF receptor or that neutralized amphiregulin activity prevented mitogenic stimulation by IL-1 alpha or TNF-alpha. These studies indicate that IL-1 alpha and TNF-alpha stimulate proliferation of immortal and malignant cervical epithelial cells by an EGF receptor-dependent pathway requiring autocrine stimulation by amphiregulin. Furthermore, they suggest that chronic inflammation and release of proinflammatory cytokines might provide a selective growth advantage for abnormal cervical cells in vivo. Images Fig. 1 Fig. 4 PMID:7708734

  13. Variable region sequences of murine IgM anti-IgG monoclonal autoantibodies (rheumatoid factors). II. Comparison of hybridomas derived by lipopolysaccharide stimulation and secondary protein immunization

    PubMed Central

    1987-01-01

    We have obtained the complete variable region mRNA sequences of 11 LPS- derived and 14 secondary immunization-derived monoclonal IgM anti-IgG antibodies (rheumatoid factors, RFs). A comparative analysis of these sequences showed that monoclonal RFs derived after polyclonal activation are structurally very similar to RFs derived after secondary protein immunization. This study was undertaken to evaluate the potential relationship between two previously described phenomena: (a) during a secondary response to a protein antigen, RF is produced in quantities that equal or exceed the immunogen-specific antibody; and (b) the frequency of B cells that make RF after polyclonal activation is quite high; 3-10%. It has been unclear whether LPS-stimulated cells that produce IgM anti-IgG that is detected by an in vitro assay are related to the cells that produce RF after in vivo stimulation. The similarity of the antigen receptors found in the two types of RF, however, suggests that most or all of the RF-producing B cells detected after LPS stimulation would also be stimulated during the secondary immune response. Thus, the presence of relatively large number of B cells that can make RF after nonspecific stimulation provides an explanation for the magnitude of RF production accompanying the secondary immune response. PMID:3494096

  14. Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells.

    PubMed

    Kosheverova, Vera V; Kamentseva, Rimma S; Gonchar, Ilya V; Kharchenko, Marianna V; Kornilova, Elena S

    2016-04-22

    Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering.

  15. 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.

  16. Growth hormone and insulin-like growth factor-1 differentially stimulate the expression of preprosomatostatin mRNAs in the Brockmann bodies of rainbow trout, Oncorhynchus mykiss.

    PubMed

    Melroe, Gregory T; Ehrman, Melissa M; Kittilson, Jeffrey D; Sheridan, Mark A

    2004-05-01

    We previously characterized three cDNAs obtained from the endocrine pancreas (Brockmann body) of rainbow trout that encode for distinct preprosomatostatin (PPSS) molecules: PPSS I containing somatostain-14 (SS-14) at its C-terminus and two separate PPSS IIs, PPSS II' and PPSS II'', containing [Tyr7,Gly10]-SS-14 at their C-termini. In this study, we examined the control of PPSS I, PPSS II', and PPSS II'' mRNA expression by growth hormone (GH) and insulin-like growth factor-1 (IGF-1). Rainbow trout implanted with GH for 21 days displayed elevated pancreatic expression of all PPSS mRNAs compared to control animals. Growth hormone directly stimulated the expression of all pancreatic PPSS mRNAs in vitro in a dose-dependent manner; however, GH was a more potent stimulator of PPSS II' expression than of PPSS I or PPSS II'' expression. Insulin-like growth factor-1 also directly stimulated the expression of PPSS mRNAs in a dose-dependent manner in Brockmann bodies incubated in vitro; IGF-1 was a more potent stimulator of PPSS I and PPSS II' expression than of PPSS II'' expression. These results indicate that the expression of PPSS mRNAs in the Brockmann body of trout is differentially regulated by GH and IGF-1 and suggest that SS mediate the feedback regulation of GH and IGF-1. PMID:15081835

  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. 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

  19. The Role of Granulocyte Colony-Stimulating Factor in the Neutrophilia Observed in the Fetal Inflammatory Response Syndrome

    PubMed Central

    Chaiworapongsa, Tinnakorn; Romero, Roberto; Berry, Stanley M.; Hassan, Sonia S.; Yoon, Bo Hyun; Edwin, Samuel; Mazor, Moshe

    2012-01-01

    OBJECTIVE Fetal neutrophilia is present in two-thirds of cases with the fetal inflammatory response syndrome (FIRS). The mechanisms responsible for this finding have not been elucidated. Granulocyte Colony-Stimulating Factor (G-CSF) is the primary physiologic regulator of neutrophil production and plays a key role in the rapid generation and release of neutrophils in stressful conditions (i.e., infection). The objective of this study was to determine: 1) whether FIRS was associated with changes in fetal plasma G-CSF concentrations; and 2) if fetal plasma G-CSF concentrations correlated with fetal neutrophil counts, chorioamnionitis, neonatal morbidity/mortality and cordocentesis-to-delivery interval. STUDY DESIGN Percutaneous umbilical cord blood sampling was performed in a population of patients with preterm labor (n=107). A fetal plasma interleukin-6 (IL-6) concentration >11 pg/mL was used to define FIRS. Cord blood G-CSF was measured by a sensitive and specific immunoassay. An absolute neutrophil count was determined and corrected for gestational age. Receiver operating characteristic (ROC) curve, survival analysis and Cox proportional hazard model were employed. RESULTS 1) G-CSF was detected in all fetal blood samples; 2) fetuses with FIRS had a higher median fetal plasma G-CSF concentration than those without FIRS (p<0.001); 3) a fetal plasma G-CSF concentration ≥134 pg/mL (derived from an ROC curve) was associated with a shorter cordocentesis-to-delivery interval, a higher frequency of chorioamnionitis (clinical and histological), intra-amniotic infection, and composite neonatal morbidity/mortality than a fetal plasma concentration below this cut-off; and 4) a fetal plasma G-CSF concentration ≥134 pg/mL was associated with a shorter cordocentesis-to-delivery interval (hazard ratio 3.2; 95% confidence interval 1.8-5.8) after adjusting for confounders. CONCLUSIONS 1) G-CSF concentrations are higher in the peripheral blood of fetuses with FIRS than in

  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. Myelodysplastic syndrome and acute myeloid leukemia following adjuvant chemotherapy with and without granulocyte colony-stimulating factors for breast cancer.

    PubMed

    Calip, Gregory S; Malmgren, Judith A; Lee, Wan-Ju; Schwartz, Stephen M; Kaplan, Henry G

    2015-11-01

    Risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) post-breast cancer treatment with adjuvant chemotherapy and granulocyte colony-stimulating factors (G-CSF) is not fully characterized. Our objective was to estimate MDS/AML risk associated with specific breast cancer treatments. We conducted a retrospective cohort study of women aged ≥66 years with stage I-III breast cancer between 2001 and 2009 using the Surveillance, Epidemiology, and End Results-Medicare database. Women were classified as receiving treatment with radiation, chemotherapy, and/or G-CSF. We used multivariable Cox proportional hazards models to estimate adjusted hazard ratios (HR) and 95 % confidence intervals (CI) for MDS/AML risk. Among 56,251 breast cancer cases, 1.2 % developed MDS/AML during median follow-up of 3.2 years. 47.1 % of women received radiation and 14.3 % received chemotherapy. Compared to breast cancer cases treated with surgery alone, those treated with chemotherapy (HR = 1.38, 95 %-CI 0.98-1.93) and chemotherapy/radiation (HR = 1.77, 95 %-CI 1.25-2.51) had increased risk of MDS/AML, but not radiation alone (HR = 1.08, 95 % CI 0.86-1.36). Among chemotherapy regimens and G-CSF, MDS/AML risk was differentially associated with anthracycline/cyclophosphamide-containing regimens (HR = 1.86, 95 %-CI 1.33-2.61) and filgrastim (HR = 1.47, 95 %-CI 1.05-2.06), but not pegfilgrastim (HR = 1.10, 95 %-CI 0.73-1.66). We observed increased MDS/AML risk among older breast cancer survivors treated with anthracycline/cyclophosphamide chemotherapy that was enhanced by G-CSF. Although small, this risk warrants consideration when determining adjuvant chemotherapy and neutropenia prophylaxis for breast cancer patients.

  3. Thiotepa cyclophosphamide followed by granulocyte colony-stimulating factor mobilized allogeneic peripheral blood cells in adults with advanced leukemia.

    PubMed

    Bacigalupo, A; Van Lint, M T; Valbonesi, M; Lercari, G; Carlier, P; Lamparelli, T; Gualandi, F; Occhini, D; Bregante, S; Valeriani, A; Piaggio, G; Pitto, A; Benvenuto, F; Figari, O; De Stefano, G; Caimo, A; Sessarego, M

    1996-07-01

    Thirty-one patients (median age, 44 years) with advanced hematologic malignancies were given thiotepa 15 mg/kg, and cyclophosphamide 120 (n = 14) or 150 (n = 17) mg/kg followed by unfractionated peripheral blood stem cell transplants (PBSCT) from genotypically identical siblings (n = 28) or one antigen mismatched family donor (n = 3). Donors were mobilized with granulocyte colony-stimulating factor 5 to 10 microgram/kg/d for 6 days and underwent two to three leukapheresis on days +5, +6, +7. The median cell yield per donor expressed/kg of recipients body weight was as follows: nucleated cells 13 x 10(8)/kg; CD34+ cells 6 x 10(6)/kg; colony-forming unit-granulocyte macrophage 38 x 10(4)/kg, and CD3+ cells 449 x 10(6)/kg. The diagnoses were chronic myeloid leukemia (n = 4), acute myeloid (n = 9) or lymphoid leukemia (n = 2), acute myelofibrosis (n = 2), multiple myeloma (n = 1), lymphoma (n = 6), chronic lymphocytic leukemia (n = 1) myelodysplasia (n = 6). Twenty-eight patients had advanced disease, 29 patients were first grafts, and 2 were second transplants 3 and 9 years after the first. Neutrophil counts of 0.5 x 10(9)/L and platelet counts of 30 x 10(9)/L platelets were both achieved on day +14 (median). Engraftment could be proven by sex markers or DNA polymorphism in 29 of 31 patients: one had early leukemia relapse and one patient was unevaluable because of early death. Acute graft-versus-host disease (GVHD) was scored as minimal or absent (grade 0 to 1) in 14 patients, moderate (grade II) in 13, and severe (grade III to IV) in four. Causes of death were leukemia (n = 4), acute GVHD (n = 4, with associated cytomegalovirus infections in three), sepsis (n = 1), liver failure (n = 1), multiorgan failure (n = 1), and hemorrhage (n = 1). The actuarial transplant mortality is 29%, the actuarial relapse rate 22%. Nineteen patients survive with a median follow up of 288 days (100-690). The actuarial 2-year survival is 57%. Three patients received PBSCT from family

  4. Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation.

    PubMed

    Boswell, Sarah; Mathew, John; Beach, Michael; Osuna, Robert; Colón, Wilfredo

    2004-03-16

    The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond-salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71-98) and the dimer core (residues 26-70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond-salt bridge network. These results highlight the importance of

  5. Effect of soluble factors derived from oral cancer cells on the production of interferon-γ from peripheral blood mononuclear cells following stimulation with OK-432.

    PubMed

    Ohe, Go; Sasai, Akiko; Uchida, Daisuke; Tamatani, Tetsuya; Nagai, Hirokazu; Miyamoto, Youji

    2013-08-01

    The streptococcal antitumor agent OK-432 is commonly used as an immunopotentiator for immunotherapy in various types of malignant tumors including oral cancer. It has been demonstrated that OK-432 elicits an antitumor effect by stimulating immunocompetent cells, thereby inducing multiple cytokines including interferon (IFN)-γ, interleukin (IL)-2 and IL-12. Serum concentrations of IFN-γ in patients with oral cancer were examined 24 h after administration of OK-432. Serum concentrations of IFN-γ in patients with advanced cancer were significantly lower than those in patients with early cancer. These results suggested that some soluble factors produced by cancer cells may inhibit IFN-γ production with OK-432. Thus, in the present study, an in vitro simulation model was established for the immune status of patients with oral cancer by adding conditioned medium (CM) derived from oral cancer cell lines into a culture of peripheral blood mononuclear cells (PBMCs) derived from a healthy volunteer. We investigated whether soluble factors derived from oral cancer cells affected IFN-γ production from PBMCs following stimulation with OK-432. PBMCs stimulated with OK-432 produced a large amount of IFN-γ; however, both IFN-γ production and cytotoxic activity from PBMCs induced by OK-432 were inhibited by the addition of CM in a dose-dependent manner. In order to examine these inhibitory effects against IFN-γ production, the contribution of inhibitory cytokines such as IL-4, IL-6, IL-10, transforming growth factor-β and vascular endothelial growth factor was investigated. However, neutralization of these inhibitory cytokines did not recover IFN-γ production inhibited by CM. These results indicated that unknown molecules may inhibit IFN-γ production from PBMCs following stimulation with OK-432.

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

    PubMed Central

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

    2014-01-01

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

  7. Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

    PubMed

    Cuellar, Araceli; Reddi, A Hari

    2015-07-01

    Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS.

  8. Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-β in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans

    PubMed Central

    Cuellar, Araceli

    2015-01-01

    Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-β. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-β1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-β1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-β1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-β1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-β1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-β1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-β1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-β1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-β1 and exogenous HS. PMID:25398329

  9. Peripheral noxious stimulation reduces withdrawal threshold to mechanical stimuli after spinal cord injury: Role of tumor necrosis factor alpha and apoptosis

    PubMed Central

    Woller, Sarah A.; Huie, J. Russell; Hartman, John J.; Hook, Michelle A.; Miranda, Rajesh C.; Huang, Yung-Jen; Ferguson, Adam R.; Grau, James W.

    2014-01-01

    We previously showed that peripheral noxious input after spinal cord injury (SCI) inhibits beneficial spinal plasticity and impairs recovery of locomotor and bladder functions. These observations suggest that noxious input may similarly affect the development and maintenance of chronic neuropathic pain, an important consequence of SCI. In adult rats with a moderate contusion SCI, we investigated the effect of noxious tail stimulation, administered one day after SCI, on mechanical withdrawal responses to von Frey stimuli from 1 to 28 days, post-treatment. In addition, because the pro-inflammatory cytokine tumor necrosis factor α (TNFα) is implicated in numerous injury-induced processes including pain hypersensitivity, we assessed the temporal and spatial expression of TNFα, TNF receptors, and several downstream signaling targets after stimulation. Our results showed that unlike sham surgery or SCI only, nociceptive stimulation following SCI induced mechanical sensitivity by 24 hours. These behavioral changes were accompanied by increased expression of TNFα. Cellular assessments of downstream targets of TNFα revealed that nociceptive stimulation increased the expression of caspase 8 and the active subunit (12 kDa) of caspase 3 at a time point consistent with the onset of mechanical allodynia, indicative of active apoptosis. In addition, immunohistochemical analysis revealed distinct morphological signs of apoptosis in neurons and microglia at 24 hours post-stimulation. Interestingly, expression of the inflammatory mediator NFκB was unaltered by nociceptive stimulation. These results suggest that noxious input caudal to the level of SCI can increase the onset and expression of behavioral responses indicative of pain, potentially involving TNFα signaling. PMID:25180012

  10. Colony-Stimulating Factor-1-Responsive Macrophage Precursors Reside in the Amphibian (Xenopus laevis) Bone Marrow Rather than the Hematopoietic Sub-Capsular Liver

    PubMed Central

    Grayfer, Leon; Robert, Jacques

    2013-01-01

    Macrophage precursors originate from, and undergo lineage commitment within designated sites of hematopoiesis, such as the mammalian bone marrow. These cells subsequently differentiate in response to stimulation with macrophage colony-stimulating factor (CSF-1). The amphibian bone marrow, unlike that of mammals, has been overlooked as a source of leukocyte precursors in favor of the liver sub-capsular region, where hematopoiesis occurs in anurans. Here we report that the bone marrow rather than the liver periphery provides macrophage progenitors to the amphibian Xenopus laevis. We identified the amphibian CSF-1, examined its gene expression in developing and virally infected X. laevis and produce it in recombinant form (rXlCSF-1). This rXlCSF-1 did not bind or elicit proliferation/differentiation of sub-cortical liver cells. Surprisingly, a sub-population of bone marrow cells engaged this growth factor and formed rXlCSF-1-concentration-dependant colonies in semi-solid medium. Furthermore, rXlCSF-1-treated bone marrow (but not liver) cultures comprised of cells with characteristic macrophage morphology and high gene expression of the macrophage marker, colony stimulating factor-1 receptor (CSF-1R). Together, our findings indicate that in contrast to all other vertebrates studied to date, Xenopus committed macrophage precursors populations are not present in the central site of hematopoiesis, but reside in the bone marrow. PMID:23485675

  11. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters.

    PubMed Central

    Conaway, R C; Conaway, J W

    1989-01-01

    A transcription factor required for synthesis of accurately initiated run-off transcripts by RNA polymerase II has been purified and shown to have an associated DNA-dependent ATPase (dATPase) activity that is strongly stimulated by the TATA region of promoters. This transcription factor, designated delta, was purified more than 3000-fold from extracts of crude rat liver nuclei and has a native molecular mass of approximately 230 kDa. DNA-dependent ATPase (dATPase) and transcription activities copurify when delta is analyzed by hydrophobic interaction and ion-exchange HPLC, arguing that transcription factor delta possesses an ATPase (dATPase) activity. ATPase (dATPase) is specific for adenine nucleotides; ATP and dATP, but not CTP, UTP, or GTP, are hydrolyzed. ATPase (dATPase) is stimulated by both double-stranded and single-stranded DNAs, including pUC18, ssM13, and poly(dT); however, DNA fragments containing the TATA region of either the adenovirus 2 major late or mouse interleukin 3 promoters stimulate ATPase as much as 10-fold more effectively than DNA fragments containing nonpromoter sequences. These data suggest the intriguing possibility that delta plays a critical role in the ATP (dATP)-dependent activation of run-off transcription through a direct interaction with the TATA region of promoters. Images PMID:2552440

  12. Angiotensin II inhibits insulin-stimulated phosphorylation of eukaryotic initiation factor 4E-binding protein-1 in proximal tubular epithelial cells.

    PubMed Central

    Senthil, D; Faulkner, J L; Choudhury, G G; Abboud, H E; Kasinath, B S

    2001-01-01

    Interaction between angiotensin II, which binds a G-protein-coupled receptor, and insulin, a ligand for receptor tyrosine kinase, was examined in renal proximal tubular epithelial cells. Augmented protein translation by insulin involves activation of eukaryotic initiation factor 4E (eIF4E) which follows the release of the factor from a heterodimeric complex by phosphorylation of its binding protein, 4E-BP1. Angiotensin II (1 nM) or insulin (1 nM) individually stimulated 4E-BP1 phosphorylation. However, pre-incubation with angiotensin II abrogated insulin-induced phosphorylation of 4E-BP1, resulting in persistent binding to eIF4E. Although angiotensin II and insulin individually activated phosphoinositide 3-kinase and extracellular signal-regulated kinase (ERK)-1/-2-type mitogen-activated protein (MAP) kinase, pre-incubation with angiotensin II abolished insulin-induced stimulation of these kinases, suggesting more proximal events in insulin signalling may be intercepted. Pretreatment with angiotensin II markedly inhibited insulin-stimulated tyrosine phosphorylation of insulin-receptor beta-chain and insulin-receptor substrate 1. Losartan prevented angiotensin II inhibition of insulin-induced ERK-1/-2-type MAP kinase activation and 4E-BP1 phosphorylation, suggesting mediation of the effect of angiotensin II by its type 1 receptor. Insulin-stimulated de novo protein synthesis was also abolished by pre-incubation with angiotensin II. These data show that angiotensin II inhibits 4E-BP1 phosphorylation and stimulation of protein synthesis induced by insulin by interfering with proximal events in insulin signalling. Our data provide a mechanistic basis for insulin insensitivity induced by angiotensin II. PMID:11695995

  13. Temporal and Geographic Variations in the Receipt of Colony-Stimulating Factors and Erythropoiesis-Stimulating Agents in a Large Retrospective Cohort of Older Women With Breast Cancer From 2000 to 2009.

    PubMed

    Du, Xianglin L; Zhang, Yefei; Hardy, Dale

    2016-01-01

    The purpose of this study was to use the most recent national data for a large cohort of patients diagnosed with breast cancer to evaluate temporal trend of receiving hematopoietic growth factors from 2000 to 2009 and to examine significant factors associated with increasing trends and geographic variations. We identified 26,130 women aged 65-89 years who were diagnosed with breast cancer and received chemotherapy in 2000-2009 from the Surveillance, Epidemiology, and End Results (SEER)-Medicare data. Colony-stimulating factors (CSFs) were identified if there was a claim from the following procedure codes: filgrastim, pegfilgrastim, or sargramostim. Erythropoiesis-stimulating agents (ESAs) were identified if there was a claim from the following procedure codes: epoetin or darbepoetin. Overall, 51.7% of patients with breast cancer received CSFs, which increased from 21.7% in 2000 to 63.2% in 2009. The percentage of patients receiving pegfilgrastim increased from 2.7% in 2000 to 19.5% in 2003 and then continuously to 49.7% in 2009. The overall percentage of patients receiving ESAs was 39.3%, which increased from 26.4% in 2000 to 60.8% in 2006, and then decreased significantly from 40.7% in 2007 to 12.9% in 2009. The receipt of both CSFs and ESAs differed significantly across different geographic areas. The receipt of CSFs continued to increase from 2000 to 2009, and pegfilgrastim started to replace filgrastim since 2003. The receipt of ESAs increased until 2006 and then declined substantially due to the black box warning. There were substantial geographic variations in the use of these hematopoietic growth factors.

  14. Effects of nitric oxide (NO) on platelet-activating factor (PAF)- and. alpha. -adrenergic-stimulated vasoconstriction and glycogenolysis in the perfused rat liver

    SciTech Connect

    Moy, J.A.; Bates, J.N.; Fisher, R.A. )

    1991-03-11

    Effects of NO on hemodynamic and glycogenolytic responses to platelet-activating factor (PAF) and phenylephrine were investigated in perfused livers derived from fed rats. Infusion of NO into perfused livers inhibited PAF-induced increases in hepatic glucose output and portal pressure approximately 90% and 85%, respectively, and abolished effects of PAF on hepatic oxygen consumption. NO attenuated PAF-stimulated increases in glucose output and portal pressure, the latter indicative of hepatic vasoconstriction, with a similar dose-dependence with an IC{sub 50} of approximately 8 {mu}M. In contrast to its effects on PAF-induced responses in the perfused liver, NO inhibited increases in hepatic portal pressure in response to phenylephrine approximately 75% without altering phenylephrine-stimulated glucose output and oxygen consumption. Similarly, infusion of NO into perfused livers inhibited significantly increases in hepatic portal pressure but not increases in glucose output in response to a submaximal concentration of phenylephrine. Like NO, sodium nitroprusside significantly inhibited hemodynamic but not glycogenolytic responses to phenylephrine in perfused livers. However, PAF-stimulated alterations in hepatic portal pressure, glucose output and oxygen consumption were unaffected by infusion of sodium nitroprusside into perfused livers. These results provide the first evidence for regulatory effects of NO in the perfused liver and support the contention that PAF, unlike phenylephrine, stimulates glycogenolysis by mechanisms secondary to hepatic vasoconstriction. These observations raise the intriguing possibility that NO may act in liver to regulate hemodynamic responses to vasoactive mediators.

  15. Expression of nuclear Methyl-CpG binding protein 2 (Mecp2) is dependent on neuronal stimulation and application of Insulin-like growth factor 1.

    PubMed

    Tropea, Daniela; Mortimer, Niall; Bellini, Stefania; Molinos, Ines; Sanfeliu, Albert; Shovlin, Stephen; McAllister, Donna; Gill, Michael; Mitchell, Kevin; Corvin, Aiden

    2016-05-16

    Methyl-CpG binding protein 2 (MECP2) is a chromosome-binding protein that regulates the development and maintenance of brain circuits. Altered function of the protein product of MECP2 plays an important role in the etiology of many neurodevelopmental disorders. Mutations involving a loss of function are implicated in the etiology of Rett syndrome, intellectual disability, psychosis and severe encephalopathy. Conversely, MECP2 duplications have been identified in autism and intellectual disability. MECP2 action is dependent on neuronal function, as the DNA binding is modulated by activity, and it is phosphorylated in response to stimulation. Although MECP2 is considered a major risk factor for neurodevelopmental disorders, and it is a mediator of activity-dependent mechanisms, the expression levels in response to neuronal activity have never been measured. We studied the expression of Mecp2 protein and RNA in mice neuronal cultures in response to different stimulation conditions and in the presence of insulin-like growth factor1 (IGF1): a growth factor involved in brain development and plasticity. The stimulation protocols were selected according to their ability to induce different forms of synaptic plasticity: rapid depolarization, feed-forward plasticity (LTP, LTD) and feedback forms of plasticity (TTX, KCl). We find a significant reduction of Mecp2 protein nuclear expression in neurons in response to stimuli that induce a potentiation of neuronal response, suggesting that Mecp2 protein expression is modulated by neuronal activation. Application of IGF1 to the cultures induces an increase in the expression of Mecp2 transcript and nuclear Mecp2 protein in neurons. These results show that Mecp2 is responsive to neuronal stimulation and IGF1, and different stimuli have different effects on Mecp2 expression; this differential response may have downstream effects on functional mechanisms regulating brain development and plasticity.

  16. Trefoil Factor-3 (TFF3) Stimulates De Novo Angiogenesis in Mammary Carcinoma both Directly and Indirectly via IL-8/CXCR2

    PubMed Central

    Lau, Wai-Hoe; Pandey, Vijay; Kong, Xiangjun; Wang, Xiao-Nan; Wu, ZhengSheng; Zhu, Tao; Lobie, Peter E

    2015-01-01

    Mammary carcinoma cells produce pro-angiogenic factors to stimulate angiogenesis and tumor growth. Trefoil factor-3 (TFF3) is an oncogene secreted from mammary carcinoma cells and associated with poor prognosis. Herein, we demonstrate that TFF3 produced in mammary carcinoma cells functions as a promoter of tumor angiogenesis. Forced expression of TFF3 in mammary carcinoma cells promoted proliferation, survival, invasion and in vitro tubule formation of human umbilical vein endothelial cells (HUVEC). MCF7-TFF3 cells with forced expression of TFF3 generated tumors with enhanced microvessel density as compared to tumors formed by vector control cells. Depletion of TFF3 in mammary carcinoma cells by siRNA concordantly decreased the angiogenic behavior of HUVEC. Forced expression of TFF3 in mammary carcinoma cells stimulated IL-8 transcription and subsequently enhanced IL-8 expression in both mammary carcinoma cells and HUVEC. Depletion of IL-8 in mammary carcinoma cells with forced expression of TFF3, or antibody inhibition of IL-8, partially abrogated mammary carcinoma cell TFF3-stimulated HUVEC angiogenic behavior in vitro, as did inhibition of the IL-8 receptor, CXCR2. Depletion of STAT3 by siRNA in MCF-7 cells with forced expression of TFF3 partially diminished the angiogenic capability of TFF3 on stimulation of cellular processes of HUVEC. Exogenous recombinant hTFF3 also directly promoted the angiogenic behavior of HUVEC. Hence, TFF3 is a potent angiogenic factor and functions as a promoter of de novo angiogenesis in mammary carcinoma, which may co-coordinate with the growth promoting and metastatic actions of TFF3 in mammary carcinoma to enhance tumor progression. PMID:26559818

  17. Trefoil Factor-3 (TFF3) Stimulates De Novo Angiogenesis in Mammary Carcinoma both Directly and Indirectly via IL-8/CXCR2.

    PubMed

    Lau, Wai-Hoe; Pandey, Vijay; Kong, Xiangjun; Wang, Xiao-Nan; Wu, ZhengSheng; Zhu, Tao; Lobie, Peter E

    2015-01-01

    Mammary carcinoma cells produce pro-angiogenic factors to stimulate angiogenesis and tumor growth. Trefoil factor-3 (TFF3) is an oncogene secreted from mammary carcinoma cells and associated with poor prognosis. Herein, we demonstrate that TFF3 produced in mammary carcinoma cells functions as a promoter of tumor angiogenesis. Forced expression of TFF3 in mammary carcinoma cells promoted proliferation, survival, invasion and in vitro tubule formation of human umbilical vein endothelial cells (HUVEC). MCF7-TFF3 cells with forced expression of TFF3 generated tumors with enhanced microvessel density as compared to tumors formed by vector control cells. Depletion of TFF3 in mammary carcinoma cells by siRNA concordantly decreased the angiogenic behavior of HUVEC. Forced expression of TFF3 in mammary carcinoma cells stimulated IL-8 transcription and subsequently enhanced IL-8 expression in both mammary carcinoma cells and HUVEC. Depletion of IL-8 in mammary carcinoma cells with forced expression of TFF3, or antibody inhibition of IL-8, partially abrogated mammary carcinoma cell TFF3-stimulated HUVEC angiogenic behavior in vitro, as did inhibition of the IL-8 receptor, CXCR2. Depletion of STAT3 by siRNA in MCF-7 cells with forced expression of TFF3 partially diminished the angiogenic capability of TFF3 on stimulation of cellular processes of HUVEC. Exogenous recombinant hTFF3 also directly promoted the angiogenic behavior of HUVEC. Hence, TFF3 is a potent angiogenic factor and functions as a promoter of de novo angiogenesis in mammary carcinoma, which may co-coordinate with the growth promoting and metastatic actions of TFF3 in mammary carcinoma to enhance tumor progression.

  18. Evidence for a factor required for transcriptional stimulation by the chimeric acidic activator GAL-VP16 in HeLa cell extracts.

    PubMed Central

    White, J H; Brou, C; Wu, J; Burton, N; Egly, J M; Chambon, P

    1991-01-01

    We provide biochemical evidence for the existence of a transcriptional intermediary factor (TIF) in HeLa whole-cell extracts (WCE) that is distinct from the basic transcription factors and that is required for transcriptional stimulation by the chimeric acidic activator GAL-VP16. We have fractionated HeLa WCE by heparin-agarose chromatography. Of transcriptionally active fractions eluting in a step between 0.24 and 0.6 M KCl, the initial fractions are refractory to GAL-VP16 stimulation, whereas subsequent fractions are strongly stimulated by the activator. Aliquots of GAL-VP16-responsive fractions efficiently complement refractory fractions for transcriptional stimulation. Aliquots of responsive fractions are also far more efficient than those of refractory fractions in overcoming transcriptional inhibition that is brought about by high concentrations of GAL-VP16. Experiments performed with heat-treated WCE support the idea that HeLa cells contain a TIF that is essential for GAL-VP16 stimulation, but that is not required for basal transcription. Addition of recombinant yeast or human transcription factor TFIID (rTFIIDY and rTFIIDH, respectively) to a WCE heated at 48 degrees C for 15 min restores basal transcription, but in neither case is the reconstituted system activated by GAL-VP16. However, a 45 degrees C heat-treated WCE reconstituted with either rTFIIDH or rTFIIDY is stimulated by GAL-VP16, suggesting that a HeLa TIF can be selectively inactivated by heating at 48 degrees C, but not at 45 degrees C. Interestingly, a TFIID fraction partially purified from HeLa cell extracts, but not rTFIIDH, efficiently relieves transcriptional inhibition by GAL-VP16, suggesting that there may be an association between TIF(s) and TFIID and, moreover, that TIF(s) may be the direct target of the acidic domain of GAL-VP16. In summary, our results support the existence of a TIF that is not essential for basal transcription but that is required to mediate the stimulatory activity

  19. Modulation of Decidual Macrophage Polarization by Macrophage Colony-Stimulating Factor Derived from First-Trimester Decidual Cells: Implication in Preeclampsia.

    PubMed

    Li, Min; Piao, Longzhu; Chen, Chie-Pein; Wu, Xianqing; Yeh, Chang-Ching; Masch, Rachel; Chang, Chi-Chang; Huang, S Joseph

    2016-05-01

    During human pregnancy, immune tolerance of the fetal semiallograft occurs in the presence of abundant maternal leukocytes. At the implantation site, macrophages comprise approximately 20% of the leukocyte population and act as primary mediators of tissue remodeling. Decidual macrophages display a balance between anti-inflammatory and proinflammatory phenotypes. However, a shift to an M1 subtype is reported in preeclampsia. Granulocyte-macrophage colony-stimulating-factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) are major differentiating factors that mediate M1 and M2 polarization, respectively. Previously, we observed the following: i) the preeclamptic decidua contains an excess of both macrophages and GM-CSF, ii) the preeclampsia-associated proinflammatory cytokines, IL-1β and tumor necrosis factor-α, markedly enhance GM-CSF and M-CSF expression in cultured leukocyte-free first-trimester decidual cells (FTDCs), iii) FTDC-secreted GM-CSF polarizes macrophages toward an M1 subtype. The microenvironment is a key determinant of macrophage phenotype. Thus, we examined proinflammatory stimulation of FTDC-secreted M-CSF and its role in macrophage development. Immunofluorescence staining demonstrated elevated M-CSF-positive decidual cell numbers in preeclamptic decidua. In FTDCs, IL-1β and tumor necrosis factor-α signal through the NF-κB pathway to induce M-CSF production, which does the following: i) enhances differentiation of and elevates CD163 expression in macrophages, ii) increases macrophage phagocytic capacity, and iii) inhibits signal-regulatory protein α expression by macrophages. These findings suggest that FTDC-secreted M-CSF modulates the decidual immune balance by inducing M2 macrophage polarization and phagocytic capacity in response to proinflammatory stimuli.

  20. Preventive effect of bis-eugenol, a eugenol ortho dimer, on lipopolysaccharide-stimulated nuclear factor kappa B activation and inflammatory cytokine expression in macrophages.

    PubMed

    Murakami, Yukio; Shoji, Masao; Hanazawa, Shigemasa; Tanaka, Shoji; Fujisawa, Seiichiro

    2003-09-15

    Eugenol exhibits antioxidant and anti-inflammatory activities, but at higher concentrations acts as an oxidant and potent allergen. It was earlier shown that bis-eugenol synthesized by the oxidation of eugenol was less cytotoxic and more highly antioxidative than eugenol. But its anti-inflammatory mechanism remains yet unclear. Since nuclear factor-kappa B (NF-kappa B) is a key transcriptional factor in the expression of inflammatory cytokines, we examined whether eugenol and bis-eugenol are inhibitors of NF-kappa B activation. We observed that bis-eugenol, but not eugenol, clearly inhibited the degradation of inhibitory kappa B-alpha in RAW264.7 murine macrophages stimulated with lipopolysaccharide and, consequently, the transcriptional activity of the stimulated NF-kappa B in the cells. In addition, bis-eugenol actually inhibited LPS-stimulated expression of inflammatory cytokines at both gene and protein levels. These findings suggest that bis-eugenol acts as a potent inhibitor of NF-kappa B.

  1. 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.

  2. Effect of immobilized granulocyte colony-stimulating factor on hemopoietic precursors of various classes during cytostatic-induced myelosuppression.

    PubMed

    Dygai, A M; Skurikhin, E G; Andreeva, T V; Madonov, P G; Vereshagin, E I; Kinsht, D N; Pershina, O V; Khmelevskaya, E S

    2010-09-01

    Experiments were performed on the model of cytostatic myelosuppression induced by cyclophosphamide. We compared the effect of immobilized granulocyte CSF (the preparation was created in Russia) and reference standard preparation of granulocyte CSF on the development of neutrophilic leukopenia and hemopoietic precursors of various classes. It was found that preparations of granulocyte CSF decreased the duration and degree of peripheral blood neutropenia. The granulocytopoiesis-stimulating effect was related to stimulation of multipotent hemopoietic precursors, granulocyte-erythroid-macrophage-megakaryocyte precursors, and granulocyte precursors. Induction of division and maturation of multipotent hemopoietic precursors, granulocyte-erythroid-macrophage-megakaryocyte precursors, and granulocyte precursors and recovery of cellularity of the granulocytic hemopoietic stem after administration of immobilized granulocyte CSF were observed at later terms compared to treatment with the reference preparation of granulocyte CSF.

  3. 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.

  4. Heterogeneous profiles of a factor that renders neutrophils cytotoxic obtained from a concanavalin A-stimulated spleen cell culture in partial purification process

    SciTech Connect

    Fujii, Y.; Inoue, T.; Ito, M.; Kimura, S.; Fuyama, S.; Arai, S.; Naiki, M.; Sendo, F.

    1986-05-15

    Concanavalin A (Con A)-stimulated rat spleen cells were cultured in a serum-free conditioned medium. This culture supernatant contained a certain factor(s) that renders neutrophil cytotoxic for various tumor cells. The factor was tentatively termed neutrophil-activating factor (NAF). NAF activity was eluted in broad fractions by the ion exchange chromatography and the gel filtration. Moreover, on the Con A column, some NAF activities were bound to the column, but other activities passed through the column. These results showed the heterogeneity or polydispersity of NAF activity in both molecular size and charge-based separation properties. Monoclonal antibodies were produced by fusing BALB/c myeloma cells (P3-X63 Ag8.653) with spleen cells from syngeneic mice immunized with partially purified NAF (pNAF) obtained from the gel filtration. Absorbent beads which were linked with one monoclonal antibody (ANAF-10) partially absorbed NAF activity from supernatants of a Con A-stimulated spleen cell culture. By further purification of pNAF the NAF activity was concentrated about 10,000-fold. Heterogeneity of NAF activity, however, did not disappear in even this affinity chromatography. On the other hand, /sup 125/I-labeled material of the final product migrated to one major band corresponding with an m.w. of about 20,000 as determined by SDS-PAGE analysis, and NAF activity was detected in the same band.

  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. Influence of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), macrophage-colony stimulating factor (M-CSF) and fetal calf serum on human osteoclast formation and activity.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schmidt, Carla; Claes, Lutz; Ignatius, Anita

    2007-10-01

    Human osteoclast (OC) formation and activity was studied in cultures of peripheral blood mononuclear cells (PBMNC) from six healthy donors after stimulation with fetal calf serum (FCS), under the influence of the receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and the macrophage-colony stimulating factor (M-CSF). The results showed that selected FCS could stimulate OC formation without any medium supplementation with osteoclastogenic factors. The OC formation, investigated by quantification of multinucleated tartrate-resistant acid phosphatase-positive cells (TRAP+ cells), and the sensitivity of OC progenitors to RANKL and M-CSF, varied widely between individual donors. The OC resorption activity, measured in the "pit-assay" on dentine, was strictly dependent on the presence of RANKL and M-CSF in the medium and was also donor dependent. The considerable donor variability should be considered in culture studies investigating, e.g. the interactions of OC with biomaterials or the influence of cytokines, growth factors and drugs on osteoclastogenesis. PMID:18161075

  7. Transcutaneous auricular vagus nerve stimulation regulates expression of growth differentiation factor 11 and activin-like kinase 5 in cerebral ischemia/reperfusion rats.

    PubMed

    Ma, Jingxi; Zhang, Lina; He, Guoqian; Tan, Xiaodan; Jin, Xinhao; Li, Changqing

    2016-10-15

    Growth differentiation factor 11 (GDF11), as a rejuvenation factor in heterochronic parabiosis, can increase proliferation of primary brain capillary endothelial cells (ECs). However, the angiogenic role of GDF11 in ischemia-induced brain injury is still unclear. There are no previous reports on the spatiotemporal expression of GDF11 in cerebral ischemia/reperfusion (I/R) rats. Our recent work has strongly suggested that transcutaneous auricular vagus nerve stimulation (ta-VNS) reduces infarct size and induces angiogenesis in focal cerebral I/R rats. This study focused on expression of GDF11 and activin-like kinase 5 (ALK5) and the effects of ta-VNS in a rat cerebral I/R model. For ta-VNS, electrical stimulation of the left cavum concha (1h duration) using percutaneous needles was initiated 30min after induction of ischemia. Expression of GDF11 was analyzed by enzyme-linked immunosorbent assay, immunohistochemistry, real-time polymerase chain reaction, and western blot 24h, 3d, and 7d after reperfusion. In addition, neurobehavioral function, EC proliferation, and expression of ALK5 in ECs in the peri-infarct cortex were measured. Results showed that levels of GDF11 were significantly elevated after cerebral I/R, both in plasma and the peri-infarct cerebral cortex. Interestingly, splenic GDF11 levels decreased after ischemia. ALK5 was expressed in ECs in the peri-infarct cerebral cortex where active vessel remodeling was noted. ta-VNS improved neurobehavioral recovery, upregulated cerebral GDF11 and downregulated splenic GDF11, indicating a brain-spleen communication during stroke. ta-VNS also increased expression of ALK5 in ECs and stimulated proliferation of ECs. These results suggest that, after cerebral ischemia, GDF11 redistributes and participates in angiogenesis as an angiogenic factor that acts at least in part through ALK5. GDF11/ALK5 may represent a new potential therapy target for stroke. PMID:27653860

  8. Nerve Growth Factor Stimulates Interaction of Cayman Ataxia Protein BNIP-H/Caytaxin with Peptidyl-Prolyl Isomerase Pin1 in Differentiating Neurons

    PubMed Central

    Buschdorf, Jan Paul; Chew, Li Li; Soh, Unice Jim Kim; Liou, Yih-Cherng; Low, Boon Chuan

    2008-01-01

    Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation. (213 words) PMID:18628984

  9. Posttranscriptional Suppression of Lipopolysaccharide-Stimulated Inflammatory Responses by Macrophages in Middle-Aged Mice: A Possible Role for Eukaryotic Initiation Factor 2 α.

    PubMed

    Shirato, Ken; Imaizumi, Kazuhiko

    2014-01-01

    The intensities of macrophage inflammatory responses to bacterial components gradually decrease with age. Given that a reduced rate of protein synthesis is a common age-related biochemical change, which is partially mediated by increased phosphorylation of eukaryotic initiation factor-2 α (eIF-2 α ), we investigated the mechanism responsible for the deterioration of macrophage inflammatory responses, focusing specifically on the age-related biochemical changes in middle-aged mice. Peritoneal macrophages isolated from 2-month-old (young) and 12-month-old (middle-aged) male BALB/c mice were stimulated with lipopolysaccharide (LPS). Although LPS-stimulated secretion of tumor necrosis factor- α (TNF- α ) by the macrophages from middle-aged mice was significantly lower than that from young mice, LPS caused marked increases in levels of TNF- α mRNA in macrophages from middle-aged as well as young mice. Moreover, LPS evoked similar levels of phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor- κ B (NF- κ B) in young and middle-aged mice. In contrast, the basal level of phosphorylated eIF-2 α in macrophages from middle-aged mice was higher than that in macrophages from young mice. Salubrinal, an inhibitor of the phosphatase activity that dephosphorylates eIF-2 α , suppressed the LPS-stimulated inflammatory responses in a murine macrophage cell line RAW264.7. These results suggest that posttranscriptional suppression of macrophage inflammatory responses during middle age requires phosphorylation of eIF-2 α . PMID:24808968

  10. Tensile stress stimulates the expression of osteogenic cytokines/growth factors and matricellular proteins in the mouse cranial suture at the site of osteoblast differentiation.

    PubMed

    Ikegame, Mika; Tabuchi, Yoshiaki; Furusawa, Yukihiro; Kawai, Mariko; Hattori, Atsuhiko; Kondo, Takashi; Yamamoto, Toshio

    2016-01-01

    Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

  11. Tumor necrosis factor alpha and interleukin-1 stimulate bone resorption in vivo as measured by urinary ( sup 3 H)tetracycline excretion from prelabeled mice

    SciTech Connect

    Koenig, A.M.; Muehlbauer, R.C.F.; Fleisch, H. )

    1988-12-01

    Tumor necrosis factor alpha (TNF-alpha) and interleukin-1 (IL-1) have been shown to stimulate bone resorption in vitro. We have now investigated whether these cytokines also cause a similar action when administered in vivo. This was made possible by the adaptation of a newly developed technique that enables the continual assessment of bone resorption in vivo in mice by measuring urinary excretion of {sup 3}H from ({sup 3}H)tetracycline-prelabeled animals. Experiments using maneuvers known to influence bone resorption, such as a change in dietary calcium or administration of parathyroid hormone or dichloromethylenebisphosphonate, indicate that the technique is reliable and sensitive in mice. Daily intravenous administration of either recombinant human or recombinant murine TNF-alpha, as well as subcutaneous administration of recombinant human IL-1 alpha, were found to stimulate bone resorption in a dose-dependent manner. The effect was maximal within 2 days. Thus, exogenous TNF-alpha and IL-1 alpha can stimulate bone resorption in vivo, suggesting that these cytokines may also exert a systemic effect on bone.

  12. An activator of protein kinase C (phorbol dibutyrate) attenuates atrial-natriuretic-factor-stimulated cyclic GMP accumulation in smooth-muscle cells.

    PubMed Central

    Nambi, P; Whitman, M; Aiyar, N; Stassen, F; Crooke, S T

    1987-01-01

    Rat thoracic aortic smooth-muscle cells (A-10; A.T.C.C. CRL 1476) displays a high density of vasopressin and atrial-natriuretic-factor (ANF) receptors and a low density of beta-adrenergic receptors. ANF stimulates cGMP (cyclic GMP) accumulation in a time- and dose-dependent fashion. Pretreatment of these cells with phorbol dibutyrate (PDBu), a known activator of protein kinase C, attenuated ANF-stimulated cGMP accumulation without affecting basal cGMP concentrations. This effect was concentration-dependent and was observed as early as 2 min after treatment. 4 alpha-Phorbol 12, 13-didecanoate (alpha PDD), which does not activate protein kinase C, did not inhibit the cGMP accumulation. PDBu pretreatment did not affect the density and affinity of ANF receptors. These data suggest that PDBu, presumably via activation of protein kinase C, might stimulate phosphorylation of a key regulatory protein in the ANF/cGMP pathway. PMID:2822009

  13. (-)-Epicatechin gallate (ECG) stimulates osteoblast differentiation via Runt-related transcription factor 2 (RUNX2) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated transcriptional activation.

    PubMed

    Byun, Mi Ran; Sung, Mi Kyung; Kim, A Rum; Lee, Cham Han; Jang, Eun Jung; Jeong, Mi Gyeong; Noh, Minsoo; Hwang, Eun Sook; Hong, Jeong-Ho

    2014-04-01

    Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (-)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

  14. Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein.

    PubMed

    Demont, Yohann; Corbet, Cyril; Page, Adeline; Ataman-Önal, Yasemin; Choquet-Kastylevsky, Genevieve; Fliniaux, Ingrid; Le Bourhis, Xuefen; Toillon, Robert-Alain; Bradshaw, Ralph A; Hondermarck, Hubert

    2012-01-13

    The precursor of nerve growth factor (proNGF) has been described as a biologically active polypeptide able to induce apoptosis in neuronal cells, via the neurotrophin receptor p75(NTR) and the sortilin receptor. Herein, it is shown that proNGF is produced and secreted by breast cancer cells, stimulating their invasion. Using Western blotting and mass spectrometry, proNGF was detected in a panel of breast cancer cells as well as in their conditioned media. Immunohistochemical analysis indicated an overproduction of proNGF in breast tumors, when compared with benign and normal breast biopsies, and a relationship to lymph node invasion in ductal carcinomas. Interestingly, siRNA against proNGF induced a decrease of breast cancer cell invasion that was restored by the addition of non-cleavable proNGF. The activation of TrkA, Akt, and Src, but not the MAP kinases, was observed. In addition, the proNGF invasive effect was inhibited by the Trk pharmacological inhibitor K252a, a kinase-dead TrkA, and siRNA against TrkA sortilin, neurotensin, whereas siRNA against p75(NTR) and the MAP kinase inhibitor PD98059 had no impact. These data reveal the existence of an autocrine loop stimulated by proNGF and mediated by TrkA and sortilin, with the activation of Akt and Src, for the stimulation of breast cancer cell invasion.

  15. Rac1 augments Wnt signaling by stimulating β-catenin–lymphoid enhancer factor-1 complex assembly independent of β-catenin nuclear import

    PubMed Central

    Jamieson, Cara; Lui, Christina; Brocardo, Mariana G.; Martino-Echarri, Estefania; Henderson, Beric R.

    2015-01-01

    ABSTRACT β-Catenin transduces the Wnt signaling pathway and its nuclear accumulation leads to gene transactivation and cancer. Rac1 GTPase is known to stimulate β-catenin-dependent transcription of Wnt target genes and we confirmed this activity. Here we tested the recent hypothesis that Rac1 augments Wnt signaling by enhancing β-catenin nuclear import; however, we found that silencing/inhibition or up-regulation of Rac1 had no influence on nuclear accumulation of β-catenin. To better define the role of Rac1, we employed proximity ligation assays (PLA) and discovered that a significant pool of Rac1–β-catenin protein complexes redistribute from the plasma membrane to the nucleus upon Wnt or Rac1 activation. More importantly, active Rac1 was shown to stimulate the formation of nuclear β-catenin–lymphoid enhancer factor 1 (LEF-1) complexes. This regulation required Rac1-dependent phosphorylation of β-catenin at specific serines, which when mutated (S191A and S605A) reduced β-catenin binding to LEF-1 by up to 50%, as revealed by PLA and immunoprecipitation experiments. We propose that Rac1-mediated phosphorylation of β-catenin stimulates Wnt-dependent gene transactivation by enhancing β-catenin–LEF-1 complex assembly, providing new insight into the mechanism of cross-talk between Rac1 and canonical Wnt/β-catenin signaling. PMID:26403202

  16. Reductions in the Cardiac Transient Outward K+ Current Ito Caused by Chronic β-Adrenergic Receptor Stimulation Are Partly Rescued by Inhibition of Nuclear Factor κB.

    PubMed

    Panama, Brian K; Korogyi, Adam S; Aschar-Sobbi, Roozbeh; Oh, Yena; Gray, Charles B B; Gang, Hongying; Brown, Joan Heller; Kirshenbaum, Lorrie A; Backx, Peter H

    2016-02-19

    The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation. PMID:26742842

  17. Promotion of insulin-like growth factor-I production by sensory neuron stimulation; molecular mechanism(s) and therapeutic implications.

    PubMed

    Okajima, Kenji; Harada, Naoaki

    2008-01-01

    Insulin-like growth factor-I (IGF-I) plays various important roles in cellular proliferation, differentiation, survival and functions of the cell, thereby contributing to the maintenance of tissue integrity. Although it is well known that growth hormone (GH) increases serum IGF-I levels by stimulating the hepatic production, little is known about the mechanism by which local production of IGF-I in individual tissues is regulated. Stimulation of sensory neurons by capsaicin increases tissue levels of IGF-I and IGF-I mRNA in various organs via increased calcitonin gene-related peptide (CGRP) release in mice. This sensory neuron-mediated IGF-I production contributes to reducing reperfusion-induced liver injury through prevention of apoptosis in mice. Isoflavone, a phytoestrogen, increases CGRP production by increasing its transcription in sensory neurons. Administration of capsaicin and isoflavone increases IGF-I production in hair follicles, thereby promoting hair growth in mice and in volunteers with alopecia. Topical application of capsaicin increases dermal levels of IGF-I by stimulating sensory neurons in mice and increases facial skin elasticity in humans. Plasma and tissue levels of CGRP and IGF-I in spontaneously hypertensive rats (SHR) are lower than those in normotensive Wistar Kyoto rats (WKY), contributing to the development of hypertension, heart failure and insulin resistance in SHR. Administration of capsaicin increases CGRP and IGF-I levels in plasma, kidneys and the heart in SHR to WKY levels, and normalizes mean arterial blood pressure in SHR. Since administration of GH or IGF-I has some deleterious effects, pharmacological stimulation of sensory neurons leading to increased tissue IGF-I levels might be a novel therapeutic strategy for various pathologic conditions.

  18. Transforming growth factor-beta 1 stimulates vascular smooth muscle cell L-proline transport by inducing system A amino acid transporter 2 (SAT2) gene expression.

    PubMed Central

    Ensenat, D; Hassan, S; Reyna, S V; Schafer, A I; Durante, W

    2001-01-01

    Transforming growth factor-beta1 (TGF-beta 1) is a multifunctional cytokine that contributes to arterial remodelling by stimulating vascular smooth muscle cell (SMC) growth and collagen synthesis at sites of vascular injury. Since l-proline is essential for the synthesis of collagen, we examined whether TGF-beta 1 regulates the transcellular transport of l-proline by vascular SMCs. l-Proline uptake by vascular SMCs was primarily sodium-dependent, pH-sensitive, blocked by neutral amino acids and alpha-(methylamino)isobutyric acid, and exhibited trans-inhibition. Treatment of SMCs with TGF-beta 1 stimulated l-proline transport in a concentration- and time-dependent manner. The TGF-beta 1-mediated l-proline uptake was inhibited by cycloheximide or actinomycin D. Kinetic studies indicated that TGF-beta 1-induced l-proline transport was mediated by an increase in transport capacity independent of any changes in the affinity for l-proline. TGF-beta 1 stimulated the expression of system A amino acid transporter 2 (SAT2) mRNA in a time-dependent fashion that paralleled the increase in l-proline transport. Reverse transcriptase PCR failed to detect the presence of SAT1 or amino acid transporter 3 (ATA3) in either untreated or TGF-beta 1-treated SMCs. These results demonstrate that l-proline transport by vascular SMCs is mediated predominantly by the SAT and that TGF-beta 1 stimulates SMC l-proline uptake by inducing the expression of the SAT2 gene. The ability of TGF-beta 1 to induce SAT2 expression may function to provide SMCs with the necessary levels of l-proline required for collagen synthesis and cell growth. PMID:11716780

  19. Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors.

    PubMed

    Nishida, Tsutomu; Tsuji, Shingo; Kimura, Arata; Tsujii, Masahiko; Ishii, Syuji; Yoshio, Toshiyuki; Shinzaki, Shinichiro; Egawa, Satoshi; Irie, Takanobu; Yasumaru, Masakazu; Iijima, Hideki; Murata, Hiroaki; Kawano, Sunao; Hayashi, Norio

    2006-05-01

    Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.

  20. Leptin stimulates endothelin-1 expression via extracellular signal-regulated kinase by epidermal growth factor receptor transactivation in rat aortic smooth muscle cells.

    PubMed

    Chao, Hung-Hsing; Hong, Hong-Jye; Liu, Ju-Chi; Lin, Jia-Wei; Chen, Yen-Ling; Chiu, Wen-Ta; Wu, Chieh-Hsi; Shyu, Kou-Gi; Cheng, Tzu-Hurng

    2007-11-14

    Obesity is a major risk factor for the development of hypertension. Recent studies have suggested that leptin, a 167-amino acid peptide hormone produced by white adipose tissue, is related to the pathogenesis of obesity-related hypertension. However, the signaling mechanisms underlying the effects of leptin remain to be extensively examined. In this study, we found that leptin induced extracellular signal-regulated kinase phosphorylation and endothelin-1 expression in rat aortic smooth muscle cells. Both PD98059 and U0126, inhibitors of the upstream activator of mitogen-activated protein kinase kinase, inhibited augmentation of endothelin-1 expression stimulated with leptin. Leptin induced significant tyrosine phosphorylation of epidermal growth factor receptor, which was significantly attenuated by two inhibitors, an epidermal growth factor receptor tyrosine kinase inhibitor, AG1478, and a broad-spectrum matrix metalloproteinase inhibitor, GM6001. This indicates that the pathway of epidermal growth factor receptor transactivation induced by leptin is dependent on proteolytically released epidermal growth factor receptor ligands. Pretreatment of cells with AG1478 significantly reduced the degree of phosphorylation of extracellular signal-regulated kinase and endothelin-1 expression. Our results reveal that epidermal growth factor receptor transactivation is involved in the leptin signaling pathway in vascular smooth muscle cells, which may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects. PMID:17678888

  1. Identification and mutagenesis of the TACE and γ-secretase cleavage sites in the colony-stimulating factor 1 receptor.

    PubMed

    Vahidi, Arrash; Glenn, Gary; van der Geer, Peter

    2014-07-18

    Stimulation of macrophages with phorbolesters, bacterial DNA, or lipopolysaccharides causes regulated intramembrane proteolysis or RIPping of the CSF-1 receptor. This process involves TACE-mediated cleavage in the extracellular domain, followed by γ-secretase-mediated cleavage within the transmembrane region. In the current study, we have identified the TACE cleavage site, which is present twelve residues from the carboxy-terminal end of the extracellular domain. Replacement of fourteen residues at the end of the extracellular domain blocked TACE cleavage. In addition, we identified the γ-secretase cleavage site, which is present four residues from the carboxy-terminal end of the transmembrane region. Replacement of six residues surrounding this site strongly reduced intramembrane cleavage. Our results provide new insights into the molecular physiology of the CSF-1 receptor and contribute to our understanding of substrate selection by TACE and γ-secretase.

  2. Treatment of adjuvant arthritis with granulocyte-colony stimulating factor and peptide derived from heat shock protein 65.

    PubMed

    Brendolan, Andrea; Higuchi, Masanori; Sibley, Richard; Strober, Samuel

    2003-01-01

    Adjuvant arthritis in Lewis rats is induced by the subcutaneous injection of Mycobacterium tuberculosis in mineral oil, and the predominant T cell immune reactivity is against the heat shock protein 65 derived peptide 176-190. We treated Lewis rats with human recombinant G-CSF followed by (i.v) administration of peptide 176-190 after induction of adjuvant arthritis (AA), and observed decreased disease severity, joint destruction, new bone formation and joint ankylosis. Treatment with G-CSF alone was also effective, but to a lesser extent. In addition, we found that splenocytes from rats treated with G-CSF had reduced antigen presenting capacity compared with splenocytes from vehicle treated rats. Primed lymph node cells from G-CSF plus peptide treated rats showed a marked reduction in proliferation and secretion of IFN-gamma after stimulation with the heat shock protein peptide in vitro as compared to controls.

  3. The role of insulin-like growth factor I in clenbuterol-stimulated growth in growing lambs.

    PubMed

    Young, O A; Watkins, S; Oldham, J M; Bass, J J

    1995-10-01

    We examined the role of IGF-I in muscle growth stimulated by a beta-adrenergic agonist, clenbuterol. Ewe lambs (90 d old, 20.4 kg mean live weight) were allotted to five groups. A pretreatment control group of five lambs was slaughtered immediately (0 d). The other four groups of six ewes ate freely for 38 or 80 d and were then slaughtered. Half those lambs received clenbuterol (400 micrograms.kg live weight-1.d-1) as a dietary supplement. Blood was collected at intervals from 19 d before supplementation began (0 d) until slaughter. Prerigor muscle samples were sectioned for detection of IGF-I receptors and myofibrillar ATPase activity. Carcass weights were slightly increased by treatment, whereas muscle weights (semimembranosus, gastrocnemius, and biceps femoris) were greatly increased (P < .001), up to 48% at 80 d for semimembranosus. Clenbuterol significantly decreased collagen concentration because myofibrillar proteins were preferentially produced. Collagen solubility was unaffected. Total RNA:total DNA in semimembranosus and gastrocnemius showed transcription was still stimulated between 38 and 80 d. Fiber type area analysis indicated a shift toward glycolytic metabolism, confirmed by iron measurements. However, clenbuterol did not change the portion of muscle occupied by each ATPase class, and the data indicated that type I fibers, though smaller, became relatively more numerous. In spite of significant muscle changes, plasma IGF-I was unaffected by clenbuterol. Similarly, there was no difference in the specific binding of [125I]IGF-I at slaughter between treated and control lambs. However, a response in the first few days of treatment, preceding visible hypertrophy, cannot be excluded.

  4. Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein.

    PubMed

    Wang, Lihong; Cao, Hailong; Liu, Liping; Wang, Bangmao; Walker, W Allan; Acra, Sari A; Yan, Fang

    2014-07-18

    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 Egfr(wa5) 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.

  5. Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.

    PubMed

    Coulonval, K; Vandeput, F; Stein, R C; Kozma, S C; Lamy, F; Dumont, J E

    2000-06-01

    The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action. PMID:10816429

  6. Stimulation of neuropeptide Y gene expression by brain-derived neurotrophic factor requires both the phospholipase Cgamma and Shc binding sites on its receptor, TrkB.

    PubMed Central

    Williams, A G; Hargreaves, A C; Gunn-Moore, F J; Tavaré, J M

    1998-01-01

    In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cgamma (PLCgamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCgamma. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCgamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified. PMID:9677306

  7. Anti-invasive effects of Celastrus Orbiculatus extract on interleukin-1 beta and tumour necrosis factor-alpha combination-stimulated fibroblast-like synoviocytes

    PubMed Central

    2014-01-01

    Background Invasion of fibroblast-like synoviocytes (FLSs) is critical in the pathogenesis of rheumatoid arthritis (RA). The metalloproteinases (MMPs) and activator of nuclear factor-kappa B (NF-κB) pathway play a critical role in RA-FLS invasion induced by interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). The present study aimed to explore the anti-invasive activity and mechanism of Celastrus orbiculatus extract (COE) on IL-1β and TNF-α combination-stimulated human RA-FLSs. Methods We investigated the effect of COE on IL-1β and TNF-α combination-induced FLS invasion as well as MMP expression and explored upstream signal transduction. Results COE suppressed IL-1β and TNF-α combination-stimulated FLSs invasion by inhibiting MMP-9 expression and activity. Furthermore, our results revealed that COE inhibited the transcriptional activity of MMP-9 by suppression of the binding activity of NF-κB in the MMP-9 promoter, and inhibited IκBα phosphorylation and nuclear translocation of NF-κB. Conclusions COE inhibits IL-1β and TNF-α combination-induced FLSs invasion by suppressing NF-κB-mediated MMP-9 expression. PMID:24552146

  8. 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

  9. 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

  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. 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.

  12. CYP51A1 induced by growth differentiation factor 9 and follicle-stimulating hormone in granulosa cells is a possible predictor for unfertilization.

    PubMed

    Nakamura, Tomoko; Iwase, Akira; Bayasula, B; Nagatomo, Yoshinari; Kondo, Mika; Nakahara, Tatsuo; Takikawa, Sachiko; Goto, Maki; Kotani, Tomomi; Kiyono, Tohru; Kikkawa, Fumitaka

    2015-03-01

    Growth differentiation factor 9 (GDF9), an oocyte-secreted factor, whose receptors exist in granulosa cells, is involved in follicle progression. Therefore, GDF9 is considered to potentially mediate signals necessary for follicular growth. However, the effect of GDF9 on human granulosa cells is not fully understood. Human immortalized nonluteinized granulosa cell line (HGrC1) which we have previously reported was stimulated with GDF9 and/or follicle-stimulating hormone (FSH). Granulosa cells obtained from in vitro fertilization (IVF) patients were also evaluated with quantitative reverse transcription polymerase chain reaction (RT-PCR). Real-time RT-PCR showed that GDF9 increased messenger RNA (mRNA) levels of enzymes required for cholesterol biosynthesis, such as 3-hydroxy-3-methylglutanyl-CoA synthase 1 (HMGCS1), farnesyl-diphosphate farnesyltransferase 1, squalene epoxidase, lanosterol synthase, and cytochrome P450, family 51, subfamily A, polypeptide 1 (CYP51A1). A greater increase in mRNA levels of HMGCS1 and CYP51A1 was observed by combined treatment with GDF9 and FSH. Clinical samples showed a significant increase in CYP51A1 mRNA in the group of granulosa cells connected with unfertilized oocytes. Our results suggest that GDF9, possibly with FSH, may play significant roles in the regulation of cholesterol biosynthesis and the expression of CYP51A1 which might be a predictor for unfertilization.

  13. Defective production of interleukin-1 and tumour necrosis factor-alpha by stimulated monocytes from patients with systemic lupus erythematosus.

    PubMed

    Mũzes, G; Vien, C V; González-Cabello, R; Fehér, J; Gergely, P

    1989-01-01

    Interleukin-1 and tumour necrosis factor-alpha activity by E. coli lipopolysaccharide-triggered monocytes was studied in patients with systemic lupus erythematosus in various stages of activity. Monocytes from both groups of SLE patients produced significantly less tumour necrosis factor-alpha activity than those of age and sex matched healthy controls. However, interleukin-1 activity was only significantly reduced in patients with active stage of the disease. These findings indicate further immunoregulatory disturbances in monocyte function concerning SLE. PMID:2636360

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

    PubMed

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

    2015-01-01

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

  15. 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.

  16. Co-stimulation of gastrointestinal tumour cell growth by gastrin, transforming growth factor alpha and insulin like growth factor-I.

    PubMed Central

    Durrant, L. G.; Watson, S. A.; Hall, A.; Morris, D. L.

    1991-01-01

    Epidermal growth factor receptors and insulin like growth factor-I receptors were co-expressed on two gastric and three colorectal tumour cell lines. Previous studies have shown that gastrin receptors were also expressed at a low level or two of these cell lines. Both TGF alpha and IGF-I promoted cell growth in all of the cell lines tested. The cell doubling time of a colorectal cell line was reduced from 48 to 30-34 h. Furthermore the effects of the growth factors were additive. Each growth factor also increased the response of the cells to gastrin, but a combination of both growth factors and gastrin did not further increase growth. PMID:1846553

  17. Regulation of Granulocyte Colony-Stimulating Factor and Its Receptor in Skeletal Muscle is Dependent Upon the Type of Inflammatory Stimulus.

    PubMed

    Wright, Craig Robert; Brown, Erin Louise; Della Gatta, Paul A; Fatouros, Ioannis G; Karagounis, Leonidas G; Terzis, Gerasimos; Mastorakos, Georgios; Michailidis, Yannis; Mandalidis, Dimitris; Spengos, Kontantinos; Chatzinikolaou, Athanasios; Methenitis, Spiros; Draganidis, Dimitrios; Jamurtas, Athanasios Z; Russell, Aaron Paul

    2015-09-01

    The cytokine granulocyte colony-stimulating factor (G-CSF) binds to its receptor (G-CSFR) to stimulate hematopoietic stem cell mobilization, myelopoiesis, and the production and activation of neutrophils. In response to exercise-induced muscle damage, G-CSF is increased in circulation and G-CSFR has recently been identified in skeletal muscle cells. While G-CSF/G-CSFR activation mediates pro- and anti-inflammatory responses, our understanding of the role and regulation in the muscle is limited. The aim of this study was to investigate, in vitro and in vivo, the role and regulation of G-CSF and G-CSFR in skeletal muscle under conditions of muscle inflammation and damage. First, C2C12 myotubes were treated with lipopolysaccharide (LPS) with and without G-CSF to determine if G-CSF modulates the inflammatory response. Second, the regulation of G-CSF and its receptor was measured following eccentric exercise-induced muscle damage and the expression levels we investigated for redox sensitivity by administering the antioxidant N-acetylcysteine (NAC). LPS stimulation of C2C12 myotubes resulted in increases in G-CSF, interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNFα) messenger RNA (mRNA) and an increase in G-CSF, IL-6, and MCP-1 release from C2C12 myotubes. The addition of G-CSF following LPS stimulation of C2C12 myotubes increased IL-6 mRNA and cytokine release into the media, however it did not affect MCP-1 or TNFα. Following eccentric exercise-induced muscle damage in humans, G-CSF levels were either marginally increased in circulation or remain unaltered in skeletal muscle. Similarly, G-CSFR levels remained unchanged in response to damaging exercise and G-CSF/G-CSFR did not change in response to NAC. Collectively, these findings suggest that G-CSF may cooperate with IL-6 and potentially promote muscle regeneration in vitro, whereas in vivo aseptic inflammation induced by exercise did not change G-CSF and G

  18. The epidemiology of iodine-deficiency disorders in relation to goitrogenic factors and thyroid-stimulating-hormone regulation.

    PubMed

    Thilly, C H; Swennen, B; Bourdoux, P; Ntambue, K; Moreno-Reyes, R; Gillies, J; Vanderpas, J B

    1993-02-01

    In children aged 5-7 y from goiter-endemic areas in Ubangi, Zaire, and Ntcheu, Malawi, mean serum thyroxin (T4) concentrations were 53 +/- 49 vs 81 +/- 33 nmol/L (P < 0.05), and thyroid-stimulating hormone (TSH) values were 24.3 +/- 9.6 vs 4.5 +/- 3.3 mU/L respectively (P < 0.01); mean urinary iodine concentrations were 0.14 +/- 0.02 vs 0.09 +/- 0.02 mumol/L, and mean thiocyanate concentrations were 0.33 +/- 0.05 vs 0.17 +/- 0.05 nmol/L, respectively (P < 0.05). Mean serum selenium concentrations were 0.343 +/- 0.176 mumol/L in Ubangi and 0.437 +/- 0.178 mumol/L in Ntcheu (P < 0.05). In two groups of 11 adolescent girls from Ubangi, the mean values for excretion of urinary iodine were 1.31 +/- 0.14 and 0.58 +/- 0.17 mumol/L (P < 0.05) after a meal of cassava or a control meal of rice, respectively. In euthyroid subjects from Ubangi, mean serum TSH for a given serum T4 was approximately twice as high for children aged < 15 y than for those aged 16-25 y. The high frequency of myxedematous cretins observed in Ubangi very probably result from both severe iodine and selenium deficiency together with thiocyanate overload. PMID:8427202

  19. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  20. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

  1. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  2. Combined suicide and granulocyte-macrophage colony-stimulating factor gene therapy induces complete tumor regression and generates antitumor immunity.

    PubMed

    Jones, R K; Pope, I M; Kinsella, A R; Watson, A J; Christmas, S E

    2000-12-01

    The use of prodrug-activated ("suicide") gene therapy has been shown to be effective in inducing tumor regression when only a small proportion of tumor cells contains the suicide gene. These experiments were designed to test whether additional therapeutic benefit may be obtained by stimulating the immune response. Murine MC26 colon carcinoma cells, either untransduced or transduced with genes for herpes simplex virus-1 thymidine kinase (HSV1-TK) or human GM-CSF, were injected subcutaneously into syngeneic BALB/c mice in various combinations. Inoculation of equal numbers of untransduced and HSV1-TK-containing cells followed by ganciclovir (GCV) treatment resulted in almost complete tumor regression, but by 7 weeks, tumors had recurred in all mice. A similar initial regression was obtained using equal numbers of cells containing HSV1-TK and GM-CSF genes, but >80% of these mice remained tumor-free after 3 months. Groups of tumor-free mice that had received GM-CSF-containing cells were left for different periods of time and rechallenged with unmodified MC26 cells on the opposite flank. Of the mice rechallenged 14, 28, and 108 days later, 100%, 88%, and 57%, respectively, showed complete resistance to unmodified tumor cells. In mice that showed tumor regrowth, tumor volume was much less than in control mice. Adoptive transfer of spleen cells from resistant mice to naïve syngeneic mice resulted in partial resistance to challenge with unmodified tumor cells. Specific cytotoxicity against MC26 cells was only demonstrable in mice receiving GM-CSF- and HSV1-TK-containing tumor cells. These experiments show that the presence of cells secreting GM-CSF in HSV1-TK-containing, regressing tumor is able to induce complete or partial resistance to tumor rechallenge. This indicates the potential usefulness of GM-CSF in enhancing other antitumor therapies.

  3. Residues 39-56 of Stem Cell Factor Protein Sequence Are Capable of Stimulating the Expansion of Cord Blood CD34+ Cells

    PubMed Central

    Shen, Bin; Jiang, Wenhong; Fan, Jie; Dai, Wei; Ding, Xinxin; Jiang, Yongping

    2015-01-01

    Background Stem cell factor (SCF) can stimulate hematopoietic stem cell (HSC) expansion; however, the specific structural region(s) of SCF protein that are critical for this function are still unknown. A novel monoclonal antibody (named 23C8) against recombinant human SCF (rhSCF) was previously found to inhibit the ability of rhSCF to enhance HSC expansion, making it possible to identify the relevant active region to HSC. Methods Eleven polypeptides were synthesized, which were designed to cover the full-length of rhSCF, with overlaps that are at least 3 amino acids long. ELISA was used to identify the polypeptide(s) that specifically react with the anti-SCF. The effects of the synthetic polypeptides on human HSC expansion, or on the ability of the full-length rhSCF to stimulate cell proliferation, were evaluated ex vivo. Total cell number was monitored using hemocytometer whereas CD34+ cell number was calculated based on the proportion determined via flow cytometry on day 6 of culture. Results Of all polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 39–56, was recognized by 23C8 mAb during ELISA. P0 stimulated the expansion of CD34+ cells derived from human umbilical cord blood (UCB). Addition of P0 increased the numbers of total mononucleated cells and CD34+ cells, by ~2 fold on day 6. P0 also showed partial competition against full-length rhSCF in the ex vivo cell expansion assay. Conclusion Residues 39–56 of rhSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells. The peptide P0 is a potential candidate for further development as a synthetic substitute for rhSCF in laboratory and clinical applications. PMID:26505626

  4. Stimulation of Synthesis and Release of Brain-Derived Neurotropic Factor (BDNF) from Intestinal Smooth Muscle Cells by Substance P and Pituitary Adenylate Cyclase-Activating Peptide (PACAP)

    PubMed Central

    Al-Qudah, M.; Alkahtani, R.; Akbarali, H.I.; Murthy, K.S.; Grider, J.R.

    2015-01-01

    Background Brain-derived neurotrophic factor (BDNF) is a neurotrophin present in the intestine where it participates in survival and growth of enteric neurons, augmentation of enteric circuits, and stimulation of intestinal peristalsis and propulsion. Previous studies largely focused on the role of neural and mucosal BDNF. The expression and release of BDNF from intestinal smooth muscle and the interaction with enteric neuropeptides has not been studied in gut. Methods The expression and secretion of BDNF from smooth muscle cultured from rabbit longitudinal intestinal muscle in response to substance P and pituitary adenylate cyclase activating peptide (PACAP) was measured by western blot and ELISA. BDNF mRNA was measured by rt-PCR. Key Results The expression of BNDF protein and mRNA was greater in smooth muscle cells from the longitudinal muscle than from circular muscle layer. PACAP and substance P increased the expression of BDNF protein and mRNA in cultured longitudinal smooth muscle cells. PACAP and substance P also stimulated the secretion of BDNF from cultured longitudinal smooth muscle cells. Chelation of intracellular calcium with BAPTA prevented substance P-induced increase in BDNF mRNA and protein expression as well as substance P-induced secretion of BDNF. Conclusions & Inferences Neuropeptides known to be present in enteric neurons innervating the longitudinal layer increase the expression of BDNF mRNA and protein in smooth muscle cells and stimulate the release of BDNF. Considering the ability of BDNF to enhance smooth muscle contraction, this autocrine loop may partially explain the characteristic hypercontractility of longitudinal muscle in inflammatory bowel disease. PMID:26088546

  5. Effects of recombinant human insulin-like growth factor I administration on spontaneous and growth hormone (GH)-releasing hormone-stimulated GH secretion in anorexia nervosa.

    PubMed

    Gianotti, L; Pincelli, A I; Scacchi, M; Rolla, M; Bellitti, D; Arvat, E; Lanfranco, F; Torsello, A; Ghigo, E; Cavagnini, F; Müller, E E

    2000-08-01

    Exaggerated GH and reduced insulin-like growth factor I (IGF-I) levels are common features in anorexia nervosa (AN). A reduction of the negative IGF-I feedback could account, in part, for GH hypersecretion. To ascertain this, we studied the effects of recombinant human (rh)IGF-I on spontaneous and GH-releasing hormone (GHRH)-stimulated GH secretion in nine women with AN [body mass index, 14.1 +/- 0.6 kg/m2] and in weight matched controls (normal weight). Mean basal GH concentrations (mGHc) and GHRH (2.0 microg/kg, iv) stimulation were significantly higher in AN. rhIGF-I administration (20 microg/kg, sc) significantly reduced mGHc in AN (P < 0.01), but not normal weight, and inhibited peak GH response to GHRH in both groups; mGHc and peak GH, however, persisted at a significantly higher level in AN. Insulin, glucose, and IGFBP-1 basal levels were similar in both groups. rhIGF-I inhibited insulin in AN, whereas glucose remained unaffected in both groups. IGFBP-1 increased in both groups (P < 0.05), with significantly higher levels in AN. IGFBP-3 was under basal conditions at a lower level in AN (P < 0.05) and remained unaffected by rhIGF-I. This study demonstrates that a low rhIGF-I dose inhibits, but does not normalize, spontaneous and GHRH-stimulated GH secretion in AN, pointing also to the existence of a defective hypothalamic control of GH release. Moreover, the increased IGFBP-1 levels might curtail the negative IGF-I feedback in AN.

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

    PubMed Central

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

    1988-01-01

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

  7. 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

  8. Prenatal Exposure to Dietary Fat Induces Changes in the Transcriptional Factors,TEF and YAP, Which May Stimulate Differentiation of Peptide Neurons in Rat Hypothalamus

    PubMed Central

    Poon, Kinning; Mandava, Sushma; Chen, Karen; Barson, Jessica R.; Buschlen, Sylvie; Leibowitz, Sarah F.

    2013-01-01

    Gestational exposure to a high-fat diet (HFD) stimulates the differentiation of orexigenic peptide-expressing neurons in the hypothalamus of offspring. To examine possible mechanisms that mediate this phenomenon, this study investigated the transcriptional factor, transcription enhancer factor-1 (TEF), and co-activator, Yes-associated protein (YAP), which when inactivated stimulate neuronal differentiation. In rat embryos and postnatal offspring prenatally exposed to a HFD compared to chow, changes in hypothalamic TEF and YAP and their relationship to the orexigenic peptide, enkephalin (ENK), were measured. The HFD offspring at postnatal day 15 (P15) exhibited in the hypothalamic paraventricular nucleus a significant reduction in YAP mRNA and protein, and increased levels of inactive and total TEF protein, with no change in mRNA. Similarly, HFD-exposed embryos at embryonic day 19 (E19) showed in whole hypothalamus significantly decreased levels of YAP mRNA and protein and TEF mRNA, and increased levels of inactive TEF protein, suggesting that HFD inactivates TEF and YAP. This was accompanied by increased density and fluorescence intensity of ENK neurons. A close relationship between TEF and ENK was suggested by the finding that TEF co-localizes with this peptide in hypothalamic neurons and HFD reduced the density of TEF/ENK co-labeled neurons, even while the number and fluorescence intensity of single-labeled TEF neurons were increased. Increased YAP inactivity by HFD was further evidenced by a decrease in number and fluorescence intensity of YAP-containing neurons, although the density of YAP/ENK co-labeled neurons was unaltered. Genetic knockdown of TEF or YAP stimulated ENK expression in hypothalamic neurons, supporting a close relationship between these transcription factors and neuropeptide. These findings suggest that prenatal HFD exposure inactivates both hypothalamic TEF and YAP, by either decreasing their levels or increasing their inactive form, and that

  9. 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 Grou