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Sample records for factor zapb stimulates

  1. 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

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

    PubMed

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

    2013-03-01

    We report a case of 58 year old female diagnosed with Pulmonary Alveolar Proteinosis (PAP) with recurrence of PAP after 5 repeated whole lung lavage, responding to subcutaneous injections of Granulocyte Macrophage Colony Stimulating Factor therapy (GM-CSF). Thus indicating that GM-CSF therapy is a promising alternative in those requiring repeated whole lung lavage PMID:24475687

  3. Isolation of a murine osteoclast colony-stimulating factor.

    PubMed Central

    Lee, M Y; Eyre, D R; Osborne, W R

    1991-01-01

    Cultures of a cell line derived from a murine mammary carcinoma that induces hypercalcemia were examined for soluble products that could induce osteoclasts to differentiate from murine bone marrow cells. The serum-free culture supernatant of this cell line stimulated growth of colonies from bone marrow cells that exhibited tartrate-resistant acid phosphatase (TRAPase) activity. These TRAPase-positive cells demonstrated essential features of osteoclasts when cultured with mineralized bone or dentin. The culture period required for colony development and the frequency of colony-forming cells indicated that relatively primitive marrow progenitors were stimulated by a tumor-derived factor(s) to form immature osteoclasts. Other colony-stimulating factors (CSFs), including granulocyte CSF, macrophage CSF, granulocyte-macrophage CSF and interleukin 3, were ruled out as the source of the activity produced by the tumor cells. The biological activity was successfully purified by gel filtration chromatography and reverse-phase HPLC. By SDS/PAGE, the activity was traced to a protein of approximately 17 kDa. Functional and biochemical studies of the purified factor suggest that it is distinct from any known CSF of myeloid cells. This protein appears to be a CSF for the osteoclast lineage, osteoclast CSF (O-CSF). Images PMID:1924309

  4. 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

  5. Physical Factors Influencing Pleasant Touch during Passive Fingertip Stimulation

    PubMed Central

    Klöcker, Anne; Oddo, Calogero Maria; Camboni, Domenico; Penta, Massimo; Thonnard, Jean-Louis

    2014-01-01

    Objective Tactile explorations with the fingertips provide information regarding the physical properties of surfaces and their relative pleasantness. Previously, we performed an investigation in the active touch domain and linked several surface properties (i.e. frictional force fluctuations and net friction) with their pleasantness levels. The aim of the present study was to investigate physical factors being important for pleasantness perception during passive fingertip stimulation. Specifically we were interested to see whether factors, such as surfaces' topographies or their frictional characteristics could influence pleasantness. Furthermore, we ascertained how the stimulus pleasantness level was impacted by (i) the normal force of stimulus application (FN) and (ii) the stimulus temperature (TS). Methods and Results The right index fingertips of 22 blindfolded participants were stimulated using 27 different stimuli, which varied in average roughness (Ra) and TS. A 4-axis robot moved the stimuli horizontally under participants' fingertips with three levels of FN. The robot was equipped with force sensors, which recorded the FN and friction force (FT) during stimulation. Participants rated each stimulus according to a three-level pleasantness scale, as very pleasant (scored 0), pleasant (scored 1), or unpleasant (scored 2). These ordinal pleasantness ratings were logarithmically transformed into linear and unidimensional pleasantness measures with the Rasch model. Statistical analyses were conducted to investigate a possible link between the stimulus properties (i.e. Ra, FN, FT, and TS) and their respective pleasantness levels. Only the mean Ra and FT values were negatively correlated with pleasantness. No significant correlation was detected between FN or TS and pleasantness. Conclusion Pleasantness perception, resulting from passive fingertip stimulation, seems to be influenced by the surfaces' average roughness levels and average FT occurring during fingertip

  6. 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

  7. Tumor necrosis factor induced stimulation of granulopoiesis and radioprotection.

    PubMed

    Urbaschek, R; Männel, D N; Urbaschek, B

    1987-01-01

    Human recombinant tumor necrosis factor, TNF, was used to assess its ability to stimulate granulopoiesis and to protect mice against lethal irradiation, effects known to be inducable with TNF-rich postendotoxin serum from BCG infected mice (BCG/ET serum). Although the endotoxin contamination of this TNF preparation is extremely low its effects were compared in endotoxin low responder C3H/HeJ mice and susceptible NMRI mice. TNF is a potent inducer of serum colony stimulating activity, CSA, in both mouse strains. In peripheral blood a marked granulocytosis with a concomitant decrease in lymphocytes and monocytopenia occurs at 2 hours after injection of TNF. Moreover, TNF induces an increase in the number of splenic myelopoietic committed stem cells (GM-CFC, granulocyte-macrophage colony forming cells) determined five days after injection. The lethality rate, registered over 30 days after exposure to 660 cGy whole body X-irradiation is reduced to 40% in C3H/HeJ mice as compared to 75% in control animals. The reduction in lethality is observed both, when TNF was injected 24 hours before or after irradiation. In vitro, TNF significantly increases the number of colonies in the presence of CSA in bone marrow cultures. TNF per se does not effect colony growth. The studies reported here demonstrate that TNF is a myelopoiesis stimulating factor in mice which may be related to the reduction in lethality following whole body irradiation. PMID:3306175

  8. Granulocyte colony-stimulating factor induces in vitro lymphangiogenesis

    SciTech Connect

    Lee, Ae Sin; Kim, Dal; Wagle, Susbin Raj; Lee, Jung Eun; Jung, Yu Jin; Kang, Kyung Pyo; Lee, Sik; Park, Sung Kwang; Kim, Won

    2013-07-12

    Highlights: •G-CSF induces tube formation, migration and proliferation of lymphatic cells. •G-CSF increases phosphorylation of MAPK and Akt in lymphatic endothelial cells. •MAPK and Akt pathways are linked to G-CSF-induced in vitro lymphangiogenesis. •G-CSF increases sprouting of a lymphatic ring. •G-CSF produces peritoneal lymphangiogenesis. -- Abstract: Granulocyte-colony stimulating factor (G-CSF) is reported to induce differentiation in cells of the monocyte lineage and angiogenesis in vascular endothelial cells, but its effects on lymphangiogenesis is uncertain. Here we examined the effects and the mechanisms of G-CSF-induced lymphangiogenesis using human lymphatic endothelial cells (hLECs). Our results showed that G-CSF induced capillary-like tube formation, migration and proliferation of hLECs in a dose- and time-dependent manner and enhanced sprouting of thoracic duct. G-CSF increased phosphorylation of Akt and ERK1/2 in hLECs. Supporting the observations, specific inhibitors of phosphatidylinositol 3′-kinase and MAPK suppressed the G-CSF-induced in vitro lymphangiogenesis and sprouting. Intraperitoneal administration of G-CSF to mice also stimulated peritoneal lymphangiogenesis. These findings suggest that G-CSF is a lymphangiogenic factor.

  9. 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

  10. 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.

  11. 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

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

    PubMed Central

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

    2014-01-01

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

  13. Expression of granulocyte colony stimulating factor (GCSF) in Hansenula polymorpha

    PubMed Central

    Talebkhan, Yeganeh; Samadi, Tannaz; Samie, Armin; Barkhordari, Farzaneh; Azizi, Mohammad; Khalaj, Vahid; Mirabzadeh, Esmat

    2016-01-01

    Background and Objectives: During past decades Hansenula polymorpha has attracted global attention for the expression of recombinant proteins due to its high growth rate, minimal nutritional porequirements and use of methanol as a low cost inducer. Materials and Methods: The corresponding nucleotide sequences for the expression of heterologous genes in Hansenula poylmorpha were extracted and assembled in an E. coli vector. The constructed expression cassette included formate dehydrogenase promoter (pFMD), a secretory signal sequence, a multiple cloning site (MCS) and methanol oxidase (MOX) terminator. Zeocin resistance gene fragment and complete cDNA encoding granulocyte colony stimulating factor (GCSF) were cloned downstream of the expression cassette in-frame with signal sequence. Restriction mapping and sequence analysis confirmed the correct cloning procedures. Final vector was transformed into Hansenula and recombinant host was induced for the expression of GCSF protein by adding methanol. SDS-PAGE and immuno-blotting were performed to confirm the identity of r-GCSF. Results: The expression cassette containing gcsf gene (615bp) and zeocin resistance marker (sh-ble, 1200bp) was prepared and successfully transformed into competent Hansenula polymorpha cells via electroporation. Zeocin resistant colonies were selected and GCSF expression was induced in recombinant Hansenula transformants using 0.5% methanol and an approximately 19kDa protein was observed on SDS-PAGE. Western blot analysis using serum isolated from GCSF-treated rabbit confirmed the identity of the protein. Conclusions: Molecular studies confirmed the designed expression cassette containing gcsf gene along with pFMD and signal sequence. The expressed 19kDa protein also confirmed the ability of designed vector in expressing heterologous genes in Hansenula cells. PMID:27092221

  14. 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

  15. 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

  16. 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

  17. 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...

  18. Role of Stem Cell Factor and Granulocyte-Colony Stimulating Factor in Remodeling during Liver Regeneration

    PubMed Central

    Meng, Fanyin; Francis, Heather; Glaser, Shannon; Han, Yuyan; DeMorrow, Sharon; Stokes, Allison; Staloch, Dustin; Venter, Julie; White, Melanie; Ueno, Yoshiyuki; Reid, Lola M.; Alpini, Gianfranco

    2011-01-01

    Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte-macrophage colony stimulating factors (GM-CSF) and stem cell factor (SCF) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblots, and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time PCR. There was a significant and stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100A4 and miR-181b after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCF and GM-CSF in response to TGF-β. When SMCCs were incubated with TGF-β plus anti–SCF and GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF + GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) mRNA as well as miR-181b expression along with a reduction of metalloproteinase inhibitor 3 (TIMP-3). The levels of MMP-2, MMP-9 and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. CONCLUSION Our data suggest that altered expression of SCF and GM-CSF following PH can contribute to biliary remodeling (for example post-transplantation) by functional deregulation of activity of key signaling intermediates involved in cell expansion and multipotent differentiation. PMID:21932404

  19. Stimulation of monocytes, macrophages, and microglia by amphotericin B and macrophage colony-stimulating factor promotes remyelination.

    PubMed

    Döring, Axinia; Sloka, Scott; Lau, Lorraine; Mishra, Manoj; van Minnen, Jan; Zhang, Xu; Kinniburgh, David; Rivest, Serge; Yong, V Wee

    2015-01-21

    Approaches to stimulate remyelination may lead to recovery from demyelinating injuries and protect axons. One such strategy is the activation of immune cells with clinically used medications, since a properly directed inflammatory response can have healing properties through mechanisms such as the provision of growth factors and the removal of cellular debris. We previously reported that the antifungal medication amphotericin B is an activator of circulating monocytes, and their tissue-infiltrated counterparts and macrophages, and of microglia within the CNS. Here, we describe that amphotericin B activates these cells through engaging MyD88/TRIF signaling. When mice were subjected to lysolecithin-induced demyelination of the spinal cord, systemic injections of nontoxic doses of amphotericin B and another activator, macrophage colony-stimulating factor (MCSF), further elevated the representation of microglia/macrophages at the site of injury. Treatment with amphotericin B, particularly in combination with MCSF, increased the number of oligodendrocyte precursor cells and promoted remyelination within lesions; these pro-regenerative effects were mitigated in mice treated with clodronate liposomes to reduce circulating monocytes and tissue-infiltrated macrophages. Our results have identified candidates among currently used medications as potential therapies for the repair of myelin. PMID:25609628

  20. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    PubMed

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  1. 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

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

    SciTech Connect

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

    1987-10-01

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

  3. Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

    PubMed Central

    Pessin, M S; Altin, J G; Jarpe, M; Tansley, F; Bradshaw, R A; Raben, D M

    1991-01-01

    We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation. PMID:1892912

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

    SciTech Connect

    Ahren, B.

    1987-07-01

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

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

    PubMed

    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 24 hrs 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. PMID:25721667

  6. 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…

  7. Which Factors Obstruct or Stimulate Teacher Educators to Use ICT Innovatively?

    ERIC Educational Resources Information Center

    Drent, Marjolein; Meelissen, Martina

    2008-01-01

    This article discusses the factors which stimulate or limit the innovative use of ICT by teacher educators in the Netherlands. Innovative use of ICT is defined as the use of ICT applications that support the educational objectives based on the needs of the current knowledge society. Explorative path analysis and case studies were used to study the…

  8. Production of tumor necrosis factor alpha in human leukocytes stimulated by Cryptococcus neoformans.

    PubMed Central

    Levitz, S M; Tabuni, A; Kornfeld, H; Reardon, C C; Golenbock, D T

    1994-01-01

    Tumor necrosis factor alpha (TNF-alpha) is a key mediator of inflammation and may promote human immunodeficiency virus replication in latently infected cells. Since cryptococcosis often is associated with aberrations in the host inflammatory response and occurs preferentially in persons with AIDS, we defined the conditions under which human leukocytes produce TNF-alpha when stimulated by Cryptococcus neoformans. Peripheral blood mononuclear cells (PBMC) produced comparable amounts of TNF-alpha following stimulation with C. neoformans and lipopolysaccharide. Detectable TNF-alpha release in response to C. neoformans occurred only when fungi with small-sized capsules were used and complement-sufficient serum was added. Fractionation of PBMC established that monocytes were the predominant source of TNF-alpha. TNF-alpha gene expression and release occurred significantly later in PBMC stimulated with C. neoformans than in PBMC stimulated with LPS. C. neoformans was also a potent inducer of TNF-alpha from freshly isolated bronchoalveolar macrophages (BAM). Upon in vitro culture, BAM and monocytes bound greater numbers of fungal cells, yet their capacity to produce TNF-alpha following cryptococcal stimulation declined by 74 to 100%. However, this decline was reversed if the BAM and monocytes were cultured with gamma interferon. These data establish that C. neoformans can potently stimulate TNF-alpha release from human leukocytes. However, several variables profoundly affected the amount of TNF-alpha released, including the type of leukocyte and its state of activation, the size of the cryptococcal capsule, and the availability of opsonins. PMID:8168965

  9. Recombinant human granulocyte-macrophage colony-stimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival.

    PubMed Central

    Lopez, A F; Williamson, D J; Gamble, J R; Begley, C G; Harlan, J M; Klebanoff, S J; Waltersdorph, A; Wong, G; Clark, S C; Vadas, M A

    1986-01-01

    A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function. PMID:3021817

  10. 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.

  11. 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

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

    PubMed

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

    2015-07-01

    Although granulocyte-colony stimulating factor (G-CSF) is commonly used to support recovery from radiation-induced side-effects, the precise effects of G-CSF on colon cancer under radiotherapy remain poorly understood. In the present study, to investigate the effects of tumor growth following radiotherapy and G-CSF administration in a murine xenograft model of colon cancer, female BALB/c mice were injected with cells of a colon carcinoma cell line (CT26) with irradiation and G-CSF, alone or in combination. Mice received 2 Gy of focal radiation daily for 5 days and intraperitoneal injection of G-CSF (100 µg/kg/day) after irradiation for 7 days. Changes in the levels of myeloperoxidase (MPO), vascular endothelial growth factor (VEGF), matrix metalloproteinase type 9 (MMP-9) and CD31 were assessed in the mouse cancer induced by injection of colon cancer cells. We observed that G-CSF increased the number of circulating neutrophils, but facilitated tumor growth. However, G-CSF treatment did not affect radiation-induced cytotoxicity and cell viability in CT26 cells in vitro. Increased levels of myeloperoxidase, a neutrophil marker and those of vascular endothelial growth factor were observed in tumors with G-CSF supplementation. In addition, we found that increased levels of CD31 and matrix metalloproteinase-9 were correlated with the enhanced tumor growth after G-CSF treatment. Therefore, these data suggest that G-CSF may contribute to tumor growth and decrease the antitumor effect of radiotherapy, possibly by promoting vascularization in cancer lesions. PMID:25976379

  13. 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.

  14. Secretion of corticotrophin releasing factor from the adrenal during splanchnic nerve stimulation in conscious calves.

    PubMed Central

    Edwards, A V; Jones, C T

    1988-01-01

    1. The output of corticotrophin releasing factor-like immunoreactivity (CRF) from the adrenal gland has been investigated using the 'adrenal clamp' technique in conscious calves. 2. Stimulation of the peripheral end of the splanchnic nerve for 10 min increased the mean output of CRF progressively, so that it had risen by about twentyfold, to a peak incremental value of 24 +/- 4 pmol min-1 kg-1 at 10 min. This response was significantly increased by stimulating in bursts at 40 Hz for 1 s at 10 s intervals, which raised the mean CRF output by 44 +/- 7 pmol min-1 kg-1 at 10 min (P less than 0.05). 3. The mean output of adrenaline and noradrenaline rose more abruptly in response to splanchnic nerve stimulation with peak incremental values realized within 2.5 min. However, the ratios of adrenal CRF to catecholamine output were closely similar during the later stages of stimulation (7.5-10 min). There was a similarly abrupt rise in adrenal cortisol output in response to splanchnic nerve stimulation which was, nevertheless, linearly related to arterial plasma ACTH concentration throughout. 4. In hypophysectomized calves, administration of adrenocorticotrophic hormone (ACTH1-24) at a dose of 5 ng min-1 kg-1 reduced the output of adrenal CRF in response to splanchnic nerve stimulation by about 50% (P less than 0.05). 5. CRF isolated from adrenal venous effluent plasma, collected both at rest and during splanchnic nerve stimulation, was separated by reverse-phase high-pressure liquid chromatography and found to elute in a position identical to that of human 41CRF. This suggests that adrenal CRF is structurally closely similar to its pituitary counterpart. PMID:2843642

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

    PubMed Central

    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 methamphetamine users (N=544) in rural Arkansas and Kentucky were interviewed. Data were analyzed using both bivariate analyses and multiple logistic and log-linear regression models, with dependent variables being any substance abuse/dependence, stimulant abuse/dependence, total number of arrests since age 18 and days incarcerated since age 18. Findings One-third reported three or more conduct disorder problems prior to age 15; half reported initiation of substances (excluding alcohol) before age 15; and 60% reported family history of substance problems. All three variables were associated with adult substance abuse/dependence but only the latter two were associated with stimulant abuse/dependence. Conclusions This study highlights early risk factors for adult substance abuse/dependence among rural stimulant users. PMID:19166561

  16. Cohesion Establishment Factors Stimulate Endonuclease Activity of hFen1 Independently and Cooperatively.

    PubMed

    Kim, Do-Hyung; Kim, Jeong-Hoon; Park, Byoung Chul; Cho, Sayeon; Park, Sung Goo

    2015-10-01

    Human Fen1 protein (hFen1) plays an important role in Okazaki fragment processing by cleaving the flap structure at the junction between single-stranded (ss) DNA and doublestranded (ds) DNA, an intermediate formed during Okazaki fragment processing, resulting in ligatable nicked dsDNA. It was reported that hChlR1, a member of the cohesion establishment factor family, stimulates hFen1 nuclease activity regardless of its ATPase activity. In this study, we found that cohesion establishment factors cooperatively stimulate endonuclease activity of hFen1 in in vivo mimic condition, including replication protein-A-coated DNA and high salt. Our findings are helpful to explain how a DNA replication machinery larger than the cohesion complex goes through the cohesin ring structure on DNA during S phase in the cell cycle. PMID:26032365

  17. PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

    PubMed

    Shizu, Ryota; Abe, Taiki; Benoki, Satoshi; Takahashi, Miki; Kodama, Susumu; Miayata, Masaaki; Matsuzawa, Atsushi; Yoshinari, Kouichi

    2016-02-01

    Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression. PMID:26574435

  18. 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

  19. 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.

  20. Vasculitis complicating granulocyte colony stimulating factor treatment of leukopenia and infection in Felty's syndrome.

    PubMed

    Farhey, Y D; Herman, J H

    1995-06-01

    Recombinant myeloid growth factors have been increasingly used in recent years to combat induced and disease associated neutropenia. Their application in the management of Felty's syndrome with intercurrent infection has raised concern that resultant neutrophilia and activation of a diverse array of polymorphonuclear cell functions may have an adverse effect on the rheumatoid disease process. We describe a patient with Felty's syndrome receiving short term treatment with recombinant human granulocyte colony stimulating factor (GCSF), who then developed acute renal failure in conjunction with leukocytoclastic vasculitis and presumptive gout. We address the issue of "adding fuel to the fire" and review reported implications of GCSF in induction of vasculitis. PMID:7545756

  1. Cancer drug troglitazone stimulates the growth and response of renal cells to hypoxia inducible factors.

    PubMed

    Taub, Mary

    2016-03-11

    Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10(-5) M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia. PMID:26869517

  2. Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription.

    PubMed Central

    Dale, T C; Rosen, J M; Guille, M J; Lewin, A R; Porter, A G; Kerr, I M; Stark, G R

    1989-01-01

    A 14 bp interferon (IFN)-stimulated response element (ISRE) from 6-16, a human gene regulated by alpha-IFN, confers IFN inducibility on a heterologous thymidine kinase promoter. A 39 bp double-stranded oligonucleotide corresponding to a 5' region of 6-16 which includes the ISRE competes for factors required for gene expression by alpha-IFN in transfected cells and a single base change (A-11 to C) within the ISRE (GGGAAAATGAAACT) abolishes this competition. Band-shift assays performed with whole-cell extracts and the 39 bp oligonucleotide reveal specific complexes formed by rapidly induced and constitutive factors, both of which fail to bind to the A-11 to C oligonucleotide. A detailed footprinting analysis reveals that these two types of factors bind to overlapping sites within the ISRE, but in very different ways. These data were used to design oligonucleotides which decreased the formation of the inducible complex without affecting the constitutive one. Changes at the 5' margin of the ISRE and upstream of it markedly decrease formation of the induced but not the constitutive complex and also abolish the ability of the 39 bp sequence to function as an inducible enhancer with the thymidine kinase promoter. Thus, induction of 6-16 transcription in IFN-treated cells is likely to be stimulated by binding of the induced factor to the ISRE and upstream sequences, while the subsequent suppression of transcription may involve competition for the ISRE by the other class of factors. Images PMID:2721502

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

    PubMed

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

    2016-06-01

    Neurite outgrowth and axon regeneration are known to benefit from electrical stimulation. However, how neuritis and their surroundings react to electrical field is difficult to replicate by monolayer cell culture. In this work freshly harvested rat sciatic nerves were cultured and exposed to two types of electrical field, after which time the nerve tissues were immunohistologically stained and the expression of neurotrophic factors and cytokines were evaluated. ELISA assay was used to confirm the production of specific proteins. All cell populations survived the 48 h culture with little necrosis. Electrical stimulation was found to accelerate Wallerian degeneration and help Schwann cells to switch into migratory phenotype. Inductive electrical stimulation was shown to upregulate the secretion of multiple neurotrophic factors. Cellular distribution in nerve tissue was altered upon the application of an electrical field. This work thus presents an ex vivo model to study denervated axon in well controlled electrical field, bridging monolayer cell culture and animal experiment. It also demonstrated the critical role of electrical field distribution in regulating cellular activities. PMID:26516696

  4. 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

  5. Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

    PubMed

    Wang, J C; Strömstedt, P E; O'Brien, R M; Granner, D K

    1996-07-01

    Transcription of the hepatic phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids and inhibited by insulin. The glucocorticoid response is mediated by a complex glucocorticoid response unit that consists of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and two accessory factor-binding sites (AF1 and AF2). The complete unit is required for the full glucocorticoid response. The dominant insulin effect is mediated in part through an insulin response sequence that is coincident with the AF2 element. Members of the hepatic nuclear factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families bind to the AF2 element; however, there is no correlation between binding of these factors and the ability of the AF2 element to mediate an insulin response. We show here that binding of HNF3 does correlate with the stimulation of the glucocorticoid response by the AF2 element and that C/EBP is apparently not involved in this effect. This requirement for HNF3 is quite specific since the substitution of elements known to enhance the action of the GR in other promoters fails to recapitulate AF2 accessory factor activity. By contrast, an HNF3-binding site from the transthyretin gene is able to substitute for the wild type AF2 sequence and elicit a maximal glucocorticoid response. Based on current and previous observations, the glucocorticoid response unit consists of four DNA elements that bind four different proteins. These are: AF1 (hepatic nuclear factor 4/chicken ovalbumin upstream promoter transcription factor), AF2 (HNF3), GR1 (GR), and GR2 (GR). PMID:8813720

  6. 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

  7. Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

    SciTech Connect

    Ellsworth, J.L.; Brown, C.; Cooper, A.D.

    1988-05-01

    The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by (3H)thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; (14C)acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column.

  8. 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

  9. Enhanced jun gene expression is an early genomic response to transforming growth factor beta stimulation.

    PubMed Central

    Pertovaara, L; Sistonen, L; Bos, T J; Vogt, P K; Keski-Oja, J; Alitalo, K

    1989-01-01

    Transforming growth factor beta (TGF beta) is a multifunctional polypeptide that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF beta signal transduction in cells is unknown. We report here that an early effect of TGF beta is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF beta, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF beta, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TGF beta. The increase in jun mRNA occurred with picomolar TGF beta concentrations within 1 h of TGF beta stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-line levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF beta, evidently in a protein synthesis-independent fashion. The junB response to TGF beta was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF beta may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF beta. Images PMID:2725496

  10. 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

  11. Domains of ERRgamma that mediate homodimerization and interaction with factors stimulating DNA binding.

    PubMed

    Hentschke, Moritz; Süsens, Ute; Borgmeyer, Uwe

    2002-08-01

    The estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is an orphan member of the nuclear receptor superfamily closely related to the estrogen receptors. To explore the DNA binding characteristics, the protein-DNA interaction was studied in electrophoretic mobility shift assays (EMSAs). In vitro translated ERRgamma binds as a homodimer to direct repeats (DR) without spacing of the nuclear receptor half-site 5'-AGGTCA-3' (DR-0), to extended half-sites, and to the inverted estrogen response element. Using ERRgamma deletion constructs, binding was found to be dependent on the presence of sequences in the ligand binding domain (LBD). A far-Western analysis revealed that ERRgamma forms dimers even in the absence of DNA. Two elements, located in the hinge region and in the LBD, respectively, are necessary for DNA-independent dimerization. DNA binding of bacterial expressed ERRgamma requires additional factors present in the serum and in cellular extracts. Fusion proteins of the germ cell nuclear factor (GCNF/NR6A1) with ERRgamma showed that the characteristic feature to be stimulated by additional factors can be transferred to a heterologous protein. The stimulating activity was further characterized and its target sequence narrowed down to a small element in the hinge region. PMID:12180985

  12. 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.

  13. Transforming growth factor-beta stimulates epithelial-mesenchymal transformation in the proepicardium.

    PubMed

    Olivey, Harold E; Mundell, Nathan A; Austin, Anita F; Barnett, Joey V

    2006-01-01

    The proepicardium (PE) migrates over the heart and forms the epicardium. A subset of these PE-derived cells undergoes epithelial-mesenchymal transformation (EMT) and gives rise to cardiac fibroblasts and components of the coronary vasculature. We report that transforming growth factor-beta (TGFbeta) 1 and TGFbeta2 increase EMT in PE explants as measured by invasion into a collagen gel, loss of cytokeratin expression, and redistribution of ZO1. The type I TGFbeta receptors ALK2 and ALK5 are both expressed in the PE. However, only constitutively active (ca) ALK2 stimulates PE-derived epithelial cell activation, the first step in transformation, whereas caALK5 stimulates neither activation nor transformation in PE explants. Overexpression of Smad6, an inhibitor of ALK2 signaling, inhibits epithelial cell activation, whereas BMP7, a known ligand for ALK2, has no effect. These data demonstrate that TGFbeta stimulates transformation in the PE and suggest that ALK2 partially mediates this effect. PMID:16245329

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

    SciTech Connect

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

    1986-05-01

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

  15. Colony Stimulating Factor-1 exerts direct effects on the proliferation and invasiveness of endometrial epithelial cells

    PubMed Central

    Aligeti, Sabitha; Kirma, Nameer B.; Binkely, Peter A; Schenken, Robert S.; Tekmal, Rajeshwar Rao

    2011-01-01

    Although macrophage colony stimulating factor (CSF-1) has been suggested to play a role in part in maintaining the chronic inflammatory response in endometriosis, our previous data suggest that CSF-1 may also play a role in early endometriosis lesion formation. In the current studies, we have examined this possibility and have shown that CSF-1, in an autocrine fashion, has a direct effect on endometrial epithelial cell proliferation and attachment to peritoneal mesothelial cells, early steps in endometriosis lesion formation on the peritoneum. PMID:21481374

  16. Potentiation of photodynamic therapy by granulocyte-macrophage colony stimulating factor immunotherapy

    NASA Astrophysics Data System (ADS)

    Krosl, Gorazd; Korbelik, Mladen; Krosl, Jana; Dougherty, Graeme J.

    1995-03-01

    The murine squamous carcinoma cell line (SCCVII) was genetically engineered to produce high levels of granulocyte-macrophage colony stimulating factor (GM-CSF). Lethally irradiated GM-CSF producing cells were injected under the subcutaneously growing parental SCCVII tumor at various times before and/or after PDT. Even a single treatment with GM- CSF producing cells injected two days before PDT markedly enhanced the tumor cure rate when compared to the PDT treatment alone. Effective potentiation was observed with PDT mediated either by Photofrin or by benzoporphyrin derivative.

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

    PubMed Central

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

    2014-01-01

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

  18. 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

  19. Transcriptional mechanisms underlying sensitization of peripheral sensory neurons by Granulocyte-/Granulocyte-macrophage colony stimulating factors

    PubMed Central

    2013-01-01

    Background Cancer-associated pain is a major cause of poor quality of life in cancer patients and is frequently resistant to conventional therapy. Recent studies indicate that some hematopoietic growth factors, namely granulocyte macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), are abundantly released in the tumor microenvironment and play a key role in regulating tumor-nerve interactions and tumor-associated pain by activating receptors on dorsal root ganglion (DRG) neurons. Moreover, these hematopoietic factors have been highly implicated in postsurgical pain, inflammatory pain and osteoarthritic pain. However, the molecular mechanisms via which G-/GMCSF bring about nociceptive sensitization and elicit pain are not known. Results In order to elucidate G-/GMCSF mediated transcriptional changes in the sensory neurons, we performed a comprehensive, genome-wide analysis of changes in the transcriptome of DRG neurons brought about by exposure to GMCSF or GCSF. We present complete information on regulated genes and validated profiling analyses and report novel regulatory networks and interaction maps revealed by detailed bioinformatics analyses. Amongst these, we validate calpain 2, matrix metalloproteinase 9 (MMP9) and a RhoGTPase Rac1 as well as Tumor necrosis factor alpha (TNFα) as transcriptional targets of G-/GMCSF and demonstrate the importance of MMP9 and Rac1 in GMCSF-induced nociceptor sensitization. Conclusion With integrative approach of bioinformatics, in vivo pharmacology and behavioral analyses, our results not only indicate that transcriptional control by G-/GMCSF signaling regulates a variety of established pain modulators, but also uncover a large number of novel targets, paving the way for translational analyses in the context of pain disorders. PMID:24067145

  20. 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

  1. The synergistic therapeutic effect of hepatocyte growth factor and granulocyte colony-stimulating factor on pulmonary hypertension in rats.

    PubMed

    Guo, Yinghua; Su, Longxiang; Li, Yinghui; Guo, Na; Xie, Lixin; Zhang, Dong; Zhang, Xiaojun; Li, Hongxia; Zhang, Guizhi; Wang, Yajuan; Liu, Changting

    2014-07-01

    Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary arterial pressure and vascular resistance. Despite advances in therapy for PAH, its treatment and prognosis remain poor. We aimed to investigate whether the transplantation of bone marrow mesenchymal stem cells (MSCs) overexpressing hepatocyte growth factor (HGF), alone or in combination with granulocyte colony-stimulating factor (G-CSF), attenuates the development of experimental monocrotaline (MCT)-induced PAH. Three weeks after MCT administration, rats were divided into the following groups: (1) untreated (PAH); (2) HGF treated; (3) MSCs administered; (4) HGF-MSCs treated; and (5) HGF-MSCs plus G-CSF treated. After 3 weeks, hemodynamic changes, histomorphology, and angiogenesis were evaluated. To elucidate the molecular mechanisms of vascular remodeling and angiogenesis, serum levels of transforming growth factor (TGF)-β and endothelin-1 (ET-1) were measured, and the gene and protein expression levels of vascular cell adhesion molecule-1 (VCAM-1) and matrix metalloproteinase-9 (MMP-9) were determined. Compared with the PAH, MSC, and G-CSF groups, the HGF and HGF+G-CSF groups exhibited significantly reduced right ventricular hypertrophy and mean pulmonary arterial pressure (P < 0.05). Histologically, vessel muscularization or thickening and collagen deposition were also significantly decreased (P < 0.05). The number of vessels in the HGF+G-CSF group was higher than that in the other groups (P < 0.05). The TGF-β and ET-1 concentrations in the plasma of pulmonary hypertensive rats were markedly lower in the HGF and HGF+G-CSF groups (P < 0.05). Furthermore, HGF induced the expression of VCAM-1, and HGF treatment together with G-CSF synergistically stimulated MMP-9 expression. Transplanted HGF-MSCs combined with G-CSF potentially offer synergistic therapeutic benefit for the treatment of PAH. PMID:23933910

  2. Hepatoma-derived growth factor stimulates smooth muscle cell growth and is expressed in vascular development

    PubMed Central

    Everett, Allen D.; Lobe, David R.; Matsumura, Martin E.; Nakamura, Hideji; McNamara, Coleen A.

    2000-01-01

    Hepatoma-derived growth factor (HDGF) is the first member identified of a new family of secreted heparin-binding growth factors highly expressed in the fetal aorta. The biologic role of HDGF in vascular growth is unknown. Here, we demonstrate that HDGF mRNA is expressed in smooth muscle cells (SMCs), most prominently in proliferating SMCs, 8–24 hours after serum stimulation. Exogenous HDGF and endogenous overexpression of HDGF stimulated a significant increase in SMC number and DNA synthesis. Rat aortic SMCs transfected with a hemagglutinin-epitope–tagged rat HDGF cDNA contain HA-HDGF in their nuclei during S-phase. We also detected native HDGF in nuclei of cultured SMCs, of SMCs and endothelial cells from 19-day fetal (but not in the adult) rat aorta, of SMCs proximal to abdominal aortic constriction in adult rats, and of SMCs in the neointima formed after endothelial denudation of the rat common carotid artery. Moreover, HDGF colocalizes with the proliferating cell nuclear antigen (PCNA) in SMCs in human atherosclerotic carotid arteries, suggesting that HDGF helps regulate SMC growth during development and in response to vascular injury. PMID:10712428

  3. Immunosuppressive drugs prevent a rapid dephosphorylation of transcription factor NFAT1 in stimulated immune cells.

    PubMed Central

    Shaw, K T; Ho, A M; Raghavan, A; Kim, J; Jain, J; Park, J; Sharma, S; Rao, A; Hogan, P G

    1995-01-01

    The immunosuppressive drugs cyclosporin A and FK506 interfere with the inducible transcription of cytokine genes in T cells and in other immune cells, in part by preventing the activation of NF-AT (nuclear factor of activated T cells). We show that transcription factor NFAT1 in T cells is rapidly dephosphorylated on stimulation, that dephosphorylation occurs before translocation of NFAT1 into the cell nucleus, and that dephosphorylation increases the affinity of NFAT1 for its specific sites in DNA. Cyclosporin A prevents the dephosphorylation and the nuclear translocation of NFAT1 in T cells, B cells, macrophages, and mast cells, delineating at least one mechanism that contributes to the profound immunosuppressive effects of this compound. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7479966

  4. 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.

  5. 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

  6. 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

  7. Palmitoleate is a mitogen, formed upon stimulation with growth factors, and converted to palmitoleoyl-phosphatidylinositol.

    PubMed

    Koeberle, Andreas; Shindou, Hideo; Harayama, Takeshi; Shimizu, Takao

    2012-08-01

    Controversial correlations between biological activity and concentration of the novel lipokine palmitoleate (9Z-hexadecenoate, 16:1) might depend on the formation of an active 16:1 metabolite. For its identification, we analyzed the glycerophospholipid composition of mouse Swiss 3T3 fibroblasts in response to 16:1 using LC-MS/MS. 16:1 was either supplemented to the cell culture medium or endogenously formed when cells were stimulated with insulin or growth factors as suggested by the enhanced mRNA expression of 16:1-biosynthetic enzymes. The proportion of 1-acyl-2-16:1-sn-phosphatidylinositol (16:1-PI) was time-dependently and specifically increased relative to other glycerophospholipids under both conditions and correlated with the proliferation of fatty acid (16:1, palmitate, oleate, or arachidonate)-supplemented cells. Accordingly, cell proliferation was impaired by blocking 16:1 biosynthesis using the selective stearoyl-CoA desaturase-1 inhibitor CAY10566 and restored by supplementation of 16:1. The accumulation of 16:1-PI occurred throughout cellular compartments and within diverse mouse cell lines (Swiss 3T3, NIH-3T3, and 3T3-L1 cells). To elucidate further whether 16:1-PI is formed through the de novo or remodeling pathway of PI biosynthesis, phosphatidate levels and lyso-PI-acyltransferase activities were analyzed as respective markers. The proportion of 16:1-phosphatidate was significantly increased by insulin and growth factors, whereas lyso-PI-acyltransferases showed negligible activity for 16:1-coenzyme A. The relevance of the de novo pathway for 16:1-PI biosynthesis is supported further by the comparable incorporation rate of deuterium-labeled 16:1 and tritium-labeled inositol into PI for growth factor-stimulated cells. In conclusion, we identified 16:1 or 16:1-PI as mitogen whose biosynthesis is induced by growth factors. PMID:22700983

  8. Angiotensin II stimulated expression of transforming growth factor-beta1 in cardiac fibroblasts and myofibroblasts.

    PubMed

    Campbell, S E; Katwa, L C

    1997-07-01

    Angiotensin II (Ang II) stimulates pathologic myocardial fibrosis. Cardiac fibroblasts (CFb) and myofibroblasts mediate this response, perhaps in part by indirect production of specific cytokines. We sought to determine if Ang II could stimulate transforming growth factor-beta1 (TGF-beta1) gene expression and protein production in adult rat CFb and two cardiac myofibroblast cell types, scar myofibroblasts (MyoFb) and valvular interstitial cells (VIC). Confluent CFb, MyoFb, and VIC in serum-deprived (0.4% FCS) media were treated with Ang II (10(-7) m for CFb; 10(-9) m for MyoFb, VIC) for 24 h. Untreated cells served as controls. Culture media was collected and TGF-beta1 levels determined in triplicate using a sandwich ELISA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed to determine TGF-beta1 mRNA expression. Ang II increased CFb (P<0.02) and VIC (P<0.04) TGF-beta1 mRNA expression, while the increase in MyoFb was not statistically significant. MyoFb produced the highest TGF-beta1 levels under control conditions compared to VIC and CFb. Ang II stimulated further TGF-beta1 secretion in VIC and CFb, but not MyoFb. The AT1 receptor antagonist Losartan (10(-7) m) greatly attenuated Ang II-stimulated TGF-B1 secretion and decreased TGF-beta1 immunostaining in VIC. The AT2 receptor antagonist PD123177 (10(-7) m) also decreased secretion and immunostaining of TGF-beta1 in VIC, but to a lesser extent than Losartan. TGF-beta1 secretion by MyoFb was unaffected by Losartan and PD123177, although TGF-B1 immunostaining was absent or greatly decreased, respectively, compared to Ang II-treated MyoFb. Ang II stimulates TGF-beta1 gene expression and/or protein production in cardiac fibroblast-like cells which may act as an autocrine/paracrine stimulus to collagen formation. Furthermore, TGF-beta1 production and secretion in these cells can be modulated by specific Ang II receptor antagonists, suggesting a potential benefit in preventing

  9. Granulocyte colony-stimulating factor promotes behavioral recovery in a mouse model of traumatic brain injury.

    PubMed

    Song, Shijie; Kong, Xiaoyuan; Acosta, Sandra; Sava, Vasyl; Borlongan, Cesar; Sanchez-Ramos, Juan

    2016-05-01

    Hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) represent a novel approach for treatment of traumatic brain injury (TBI). After mild controlled cortical impact (CCI), mice were treated with G-CSF (100 μg/kg) for 3 consecutive days. The primary behavioral endpoint was performance on the radial arm water maze (RAWM), assessed 7 and 14 days after CCI. Secondary endpoints included 1) motor performance on a rotating cylinder (rotarod), 2) measurement of microglial and astroglial response, 3) hippocampal neurogenesis, and 4) measures of neurotrophic factors (brain-derived neurotrophic factor [BDNF] and glial cell line-derived neurotrophic factor [GDNF]) and cytokines in brain homogenates. G-CSF-treated animals performed significantly better than vehicle-treated mice in the RAWM at 1 and 2 weeks but not on the rotarod. Cellular changes found in the G-CSF group included increased hippocampal neurogenesis as well as astrocytosis and microgliosis in both the striatum and the hippocampus. Neurotrophic factors GDNF and BDNF, elaborated by activated microglia and astrocytes, were increased in G-CSF-treated mice. These factors along with G-CSF itself are known to promote hippocampal neurogenesis and inhibit apoptosis and likely contributed to improvement in the hippocampal-dependent learning task. Six cytokines that were modulated by G-CSF treatment following CCI were elevated on day 3, but only one of them remained altered by day 7, and all of them were no different from vehicle controls by day 14. The pro- and anti-inflammatory cytokines modulated by G-CSF administration interact in a complex and incompletely understood network involving both damage and recovery processes, underscoring the dual role of inflammation after TBI. PMID:26822127

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

    PubMed

    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

  11. 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

  12. 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.

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

    PubMed

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

    2013-01-01

    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 over-expression 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

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

    SciTech Connect

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

    1990-06-01

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

  15. Hepatic transforming growth factor-β 1 stimulated clone-22 D1 controls systemic cholesterol metabolism☆

    PubMed Central

    Jäger, Julia; Greiner, Vera; Strzoda, Daniela; Seibert, Oksana; Niopek, Katharina; Sijmonsma, Tjeerd P.; Schäfer, Michaela; Jones, Allan; De Guia, Roldan; Martignoni, Marc; Dallinga-Thie, Geesje M.; Diaz, Mauricio B.; Hofmann, Thomas G.; Herzig, Stephan

    2014-01-01

    Disturbances in lipid homeostasis are hallmarks of severe metabolic disorders and their long-term complications, including obesity, diabetes, and atherosclerosis. Whereas elevation of triglyceride (TG)-rich very-low-density lipoproteins (VLDL) has been identified as a risk factor for cardiovascular complications, high-density lipoprotein (HDL)-associated cholesterol confers atheroprotection under obese and/or diabetic conditions. Here we show that hepatocyte-specific deficiency of transcription factor transforming growth factor β 1-stimulated clone (TSC) 22 D1 led to a substantial reduction in HDL levels in both wild-type and obese mice, mediated through the transcriptional down-regulation of the HDL formation pathway in liver. Indeed, overexpression of TSC22D1 promoted high levels of HDL cholesterol in healthy animals, and hepatic expression of TSC22D1 was found to be aberrantly regulated in disease models of opposing energy availability. The hepatic TSC22D1 transcription factor complex may thus represent an attractive target in HDL raising strategies in obesity/diabetes-related dyslipidemia and atheroprotection. PMID:24634828

  16. Enhanced jun gene expression is an early genomic response to transforming growth factor. beta. stimulation

    SciTech Connect

    Pertovaara, L.; Sistonen, L.; Keski-Oja, J.; Alitalo, K. ); Bos, T.J.; Vogt, P.K. . Dept. of Microbiology)

    1989-03-01

    Transforming growth factor {beta} (TGF{beta}) is a multifunctional polypeptide4 that regulates proliferation, differentiation, and other functions of many cell types. The pathway of TGF{beta} signal transduction in cells is unknown. The authors report here that an early effect of TGF{beta} is an enhancement of the expression of two genes encoding serum- and phorbol ester tumor promoter-regulated transcription factors: the junB gene and the c-jun proto-oncogene, respectively. This stimulation was observed in human lung adenocarcinoma A549 cells which were growth inhibited by TGF{beta}, AKR-2B mouse embryo fibroblasts which were growth stimulated by TGF{beta}, and K562 human erythroleukemia cells, which were not appreciably affected in their growth by TFG{beta}. The increase in jun mRNA occurred with picomolar TGF{beta} concentrations within 1 h of TGF{beta} stimulation, reached a peak between 1 and 5 h in different cells, and declined gradually to base-fine levels. This mRNA response was followed by a large increase in the biosynthesis of the c-jun protein (AP-1), as shown by metabolic labeling and immunoprecipitation analysis. However, differential and cell type-specific regulation appeared to determine the timing and magnitude of the response of each jun gene in a given cell. In AKR-2B and NIH 3T3 cells, only junB was induced by TGF{beta}, evidently in a protein synthesis-independent fashion. The junB response to TGF{beta} was maintained in c-Ha-ras and neu oncogene-transformed cells. Thus, one of the earliest genomic responses to TGF{beta} may involve nuclear signal transduction and amplification by the junB and c-jun transcription factors in concert with c-fos, which is also induced. The differential activation of the jun genes may explain some of the pleiotropic effects of TGF{beta}.

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

    PubMed Central

    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

  18. 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

  19. Insulin-like growth factor-1 stimulates regulatory T cells and suppresses autoimmune disease

    PubMed Central

    Bilbao, Daniel; Luciani, Luisa; Johannesson, Bjarki; Piszczek, Agnieszka; Rosenthal, Nadia

    2014-01-01

    The recent precipitous rise in autoimmune diseases is placing an increasing clinical and economic burden on health systems worldwide. Current therapies are only moderately efficacious, often coupled with adverse side effects. Here, we show that recombinant human insulin-like growth factor-1 (rhIGF-1) stimulates proliferation of both human and mouse regulatory T (Treg) cells in vitro and when delivered systemically via continuous minipump, it halts autoimmune disease progression in mouse models of type 1 diabetes (STZ and NOD) and multiple sclerosis (EAE) in vivo. rhIGF-1 administration increased Treg cells in affected tissues, maintaining their suppressive properties. Genetically, ablation of the IGF-1 receptor specifically on Treg cell populations abrogated the beneficial effects of rhIGF-1 administration on the progression of multiple sclerotic symptoms in the EAE model, establishing a direct effect of IGF-1 on Treg cell proliferation. These results establish systemically delivered rhIGF-1 as a specific, effective stimulator of Treg cell action, underscoring the clinical feasibility of manipulating natural tolerance mechanisms to suppress autoimmune disease. PMID:25339185

  20. 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

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

    PubMed

    Kang, Tae Heung; Kim, Young Seob; Kim, Seokho; 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

    2015-09-29

    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

  2. Granulocyte-macrophage colony-stimulating factor: pleiotropic cytokine with potential clinical usefulness.

    PubMed

    Ruef, C; Coleman, D L

    1990-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a 23-kDa glycoprotein with remarkably diverse effects on immune and nonimmune cells. GM-CSF induces differentiation of granulocyte, macrophage, and eosinophil precursor cells. Proliferation of monocyte-macrophages, T lymphocytes, keratinocytes, and endothelial cells is also stimulated by GM-CSF. In addition, GM-CSF alters the functional properties of mature granulocytes, macrophages, eosinophils, and basophils. GM-CSF is produced by T lymphocytes, macrophages, and several cell types in extramedullary sites, where it may act in a paracrine manner to regulate the local response to antigenic challenge. Clinical trials of GM-CSF have been conducted in patients with AIDS, aplastic anemia, myelodysplastic syndromes, and sarcoma and following bone marrow transplantation and accidental radiation exposure. GM-CSF significantly increased circulating numbers of several myeloid cells and produced dose-dependent toxicity consisting primarily of myalgias, fever, fluid retention, and serosal effusions. Additional studies are needed to define the role of GM-CSF in treatment of patients with qualitative and quantitative dysfunction of immune cells. PMID:2405468

  3. Nicotine Stimulates Nerve Growth Factor in Lung Fibroblasts through an NFκB-Dependent Mechanism

    PubMed Central

    Wongtrakool, Cherry; Grooms, Kora; Bijli, Kaiser M.; Crothers, Kristina; Fitzpatrick, Anne M.; Hart, C. Michael

    2014-01-01

    Rationale Airway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment. Methods Primary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid. Results NGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells. Conclusion Nicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways

  4. 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

  5. Role of granulocyte‐macrophage colony‐stimulating factor in pulmonary fibrosis following pulmonary alveolar proteinosis

    PubMed Central

    Sha, Joy

    2016-01-01

    Abstract Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by accumulation of lipoproteinacious material in alveoli, with distinct features on high resolution computed tomography and biopsy. Its association with pulmonary fibrosis is infrequently encountered, and a clear understanding of the underlying pathogenesis is yet to be established. We report the case of a 48‐year‐old woman with known autoimmune PAP (aPAP) first diagnosed 20 years ago, who presented with worsening hypoxemia and radiological features consistent with pulmonary fibrosis, after many years of stable disease. We present a review of previously considered mechanisms of causation behind such changes, and in particular, postulate the role of granulocyte‐macrophage colony‐stimulating factor deficiency in pulmonary fibrosis seen in aPAP. PMID:27512562

  6. 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

  7. Functional interaction between mutations in the granulocyte colony-stimulating factor receptor in severe congenital neutropenia.

    PubMed

    Ward, Alister C; Gits, Judith; Majeed, Fidel; Aprikyan, Andrew A; Lewis, Rowena S; O'Sullivan, Lynda A; Freedman, Melvin; Shigdar, Sarah; Touw, Ivo P; Dale, David C; Dror, Yigal

    2008-08-01

    Most severe congenital neutropenia (SCN) cases possess constitutive neutrophil elastase mutations; a smaller cohort has acquired mutations truncating the granulocyte colony-stimulating factor receptor (G-CSF-R). We have described a case with constitutive extracellular G-CSF-R mutation hyporesponsive to ligand. Here we report two independent acquired G-CSF-R truncation mutations and a novel constitutive neutrophil elastase mutation in this patient. Co-expression of a truncated receptor chain restored STAT5 signalling responses of the extracellular G-CSF-R mutant, while constitutively-active STAT5 enhanced its proliferative capacity. These data add to our knowledge of SCN and further highlight the importance of STAT5 in mediating proliferative responses to G-CSF. PMID:18513286

  8. Applications of recombinant DNA technology in the production of glycosylated recombinant human granulocyte colony stimulating factor.

    PubMed

    Holloway, C J

    1994-01-01

    Lenograstim has been developed by recombinant DNA technology and is expressed in large-scale mammalian cell culture. It has been shown that lenograstim is indistinguishable in its physicochemical, structural and biological properties with respect to native granulocyte colony stimulating factor isolated from a human cell line. In particular, both the recombinant and natural proteins have identical amino acid sequences, contain the same intra-polypeptide chain disulphide bridges and exhibit the same posttranslational carbohydrate structures. Lenograstim is manufactured by expanding inoculum from vials of the Manufacturer's Working Cell Bank (from molecular cloning) followed by culture in a large bioreactor. Purification of lenograstim involves a four-step chromatographic process. The active ingredient is monitored by in-process controls at all stages of manufacture and routinely as purified bulk. The finished product is formulated into excipients reflecting conditions close to the natural environment of the protein with respect to pH, osmolarity and the presence of human serum albumin. PMID:7535067

  9. 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.

  10. 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…

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

    SciTech Connect

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

    1987-07-13

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

  12. Prostacyclin synthesis stimulating plasma factor in patients with peripheral vascular disease.

    PubMed

    Strobl-Jäger, E; Fitscha, P; Kaliman, J; Sinzinger, H; Peskar, B A

    1987-08-01

    Human plasma contains a factor capable of stimulating vascular prostacyclin generation even in atherosclerotic vessels with minimal in-vitro capacity for PGI2-synthesis. The activity of this prostacyclin stimulating plasma factor (PSPF) has been reported to be elevated in renal failure and hepatic coma. We are not aware of any data as to whether this PSPF plays a role in maintaining hemostatic balance in patients with peripheral vascular lesions. Therefore, we examined 62 patients with peripheral vascular disease (PVD). This study group was subdivided into normo- and hyperlipemic subjects, patients with and without maturity onset diabetes, and plasma beta-thromboglobulin levels higher and lower than 50 ng/ml. 10 healthy sex and age matched persons served as controls. Vascular prostacyclin formation was studied in vitro after incubation of the patients' plasma and a buffer control with various tissue samples (human femoral artery, rat abdominal and thoracic aorta of healthy and of streptozotocin induced diabetic animals, swine endothelial layer and remaining tissue (media and adventitia) and cultured endothelial (EC) and smooth muscle cells (SMC) of minipig arota. In addition, 6-oxo-PFG1 alpha formation by cultured EC and SMC (minipig aorta source) after incubation with tris HCl-buffer or plasma were estimated by means of specific radioimmunoassays. In general, tissue samples and cells incubated in plasma exhibit a marked increase of in-vitro PGI2-formation as compared to buffer. No difference could be found between PSPF of CHD-patients and healthy controls. Similar findings were obtained using incubated vascular tissue and cultured cells by means of the bioassay and specific RIA, respectively. These findings indicate that the PSPF does not seem to be of any clinical relevance in hemostatic regulation in patients with advanced atherosclerosis. PMID:2958884

  13. Subthalamic Nucleus Stimulation Increases Brain Derived Neurotrophic Factor in the Nigrostriatal System and Primary Motor Cortex

    PubMed Central

    Spieles-Engemann, Anne L.; Steece-Collier, Kathy; Behbehani, Michael M.; Collier, Timothy J.; Wohlgenant, Susan L.; Kemp, Christopher J.; Cole-Strauss, Allyson; Levine, Nathan D.; Gombash, Sara E.; Thompson, Valerie B.; Lipton, Jack W.; Sortwell, Caryl E.

    2011-01-01

    The mechanisms underlying the effects of long-term deep brain stimulation of the subthalamic nucleus (STN DBS) as a therapy for Parkinson’s disease (PD) remain poorly understood. The present study examined whether functionally effective, long-term STN DBS modulates glial cell line-derived neurotrophic factor (GDNF) and/or brain-derived neurotrophic factor (BDNF) in both unlesioned and unilateral 6-hydroxydopamine lesioned rats. Lesioned rats that received two weeks of continuous unilateral STN DBS exhibited significant improvements in parkinsonian motor behaviors in tests of forelimb akinesia and rearing activity. Unilateral STN DBS did not increase GDNF in the nigrostriatal system, primary motor cortex (M1), or hippocampus of unlesioned rats. In contrast, unilateral STN DBS increased BDNF protein 2–3 fold bilaterally in the nigrostriatal system with the location (substantia nigra vs. striatum) dependent upon lesion status. Further, BDNF protein was bilaterally increased in M1 cortex by as much as 2 fold regardless of lesion status. STN DBS did not impact cortical regions that receive less input from the STN. STN DBS also was associated with bilateral increases in BDNF mRNA in the substantia nigra (SN) and internal globus pallidus (GPi). The increase observed in GPi was completely blocked by pretreatment with 5-Methyl-10,11-dihydro-5 H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), suggesting that the activation of N-methyl-D-aspartate (NMDA) receptors was involved in this phenomenon. The upregulation of BDNF associated with long term STN DBS suggest that this therapy may exert pronounced and underappreciated effects on plasticity in the basal ganglia circuitry that may play a role in the symptomatic effects of this therapy as well as support the neuroprotective effect of stimulation documented in this rat model. PMID:22328911

  14. 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

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

    SciTech Connect

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

    1989-08-15

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

  16. FGF2 stimulates SDF-1 expression through the Erm transcription factor in Sertoli cells.

    PubMed

    Yoon, Kyung-Ae; Chae, Young-Mi; Cho, Je-Yoel

    2009-07-01

    Ets-related molecule (Erm) is a member of the Ets transcription factor family. Erm is known to be an important factor for the self-renewal of Spermatogonial stem cells (SSCs) and the maintenance of spermatogenesis. We investigated the molecular mechanism of Erm regulation on SDF-1 in TM4 Sertoli cells. Erm and Sdf-1 levels were up-regulated after FGF2 treatment in TM4 cells, whereas these levels were significantly decreased by FGF2 in ST2 bone marrow stromal cells. Knockdown of Erm by siRNA in the presence of FGF2 decreased the Sdf-1 levels in TM4 cells. The expression levels of Erm were similar and Erm overexpression increased the Sdf-1 in both TM4 and ST2 cells. FGFR subtype analysis revealed that FGFR4 was expressed in TM4 cells but not in ST2 cells. A blocking experiment also confirmed that FGFR4 is partly responsible for the up-regulation of Erm and SDF-1 induced by FGF2 stimulation in TM4 cells. FGF2 and ERM increased the activity of Sdf-1 gene promoter region in a dose-dependent manner. EMSA revealed that ERM strongly binds to the -846 to -851 nucleotide region of the potential Ets binding site (EBS) in the Sdf-1 promoter. In addition, CXCR4, the SDF-1 receptor, was expressed in spermatogonia and Sertoli cells in the seminiferous tubules of the mouse testis. Our results indicate that ERM directly regulates Sdf-1 gene expression by interacting with its cis-acting element in response to FGF2 stimulation in TM4 cells. PMID:19301256

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

    NASA Astrophysics Data System (ADS)

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

    1993-04-01

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

  18. 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.

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

    PubMed

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

    2015-01-01

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

  20. Granulocyte colony-stimulating factor improves alternative activation of microglia under microenvironment of spinal cord injury.

    PubMed

    Guo, Y; Zhang, H; Yang, J; Liu, S; Bing, L; Gao, J; Hao, A

    2013-05-15

    Granulocyte colony-stimulating factor (G-CSF) was investigated in the present study to examine whether it could affect the activation status of microglia under microenvironment of spinal cord injury and provide a potential therapeutic treatment for spinal cord injury. We established mouse spinal cord hemisection model and injected recombinant human G-CSF (rhG-CSF) subcutaneously. The results demonstrated that G-CSF could recruit microglia to the injury site in the first 72h after spinal cord injury. Moreover, G-CSF inhibits the expression of pro-inflammatory factors and promotes the expression of neurotrophic factors. Additionally, G-CSF also increases the expression of markers of M2 macrophage and inhibits the expression of markers of M1 macrophage in BV2 microglia in vitro model, favoring the M2 polarization of microglia under the microenvironment of spinal cord hemisection. NFκB signal pathway was involved in G-CSF-induced polarization of BV2 microglia. As a conclusion, we suggested that administration of G-CSF within the first 72h after spinal cord injury might reduce early inflammation-induced detrimental effect and promote an anti-inflammatory response that favors repair via improving alternative activation of microglia. Administration of G-CSF in the acute phase of spinal cord injury may be a promising strategy in restorative therapy after spinal cord injury. PMID:23419550

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

    PubMed Central

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

    2015-01-01

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

  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. Factors Associated with the Success of Trial Spinal Cord Stimulation in Patients with Chronic Pain from Failed Back Surgery Syndrome

    PubMed Central

    Son, Byung-chul; Kim, Deok-ryeong; Lee, Sang-won

    2013-01-01

    Objective Spinal cord stimulation (SCS) is an effective means of treatment of chronic neuropathic pain from failed back surgery syndrome (FBSS). Because the success of trial stimulation is an essential part of SCS, we investigated factors associated with success of trial stimulation. Methods Successful trial stimulation was possible in 26 of 44 patients (63.6%) who underwent insertion of electrodes for the treatment of chronic pain from FBSS. To investigate factors associated with successful trial stimulation, patients were classified into two groups (success and failure in trial). We investigated the following factors : age, sex, predominant pain areas (axial, limb, axial combined with limbs), number of operations, duration of preoperative pain, type of electrode (cylindrical/paddle), predominant type of pain (nociceptive, neuropathic, mixed), degree of sensory loss in painful areas, presence of motor weakness, and preoperative Visual Analogue Scale. Results There were no significant differences between the two groups in terms of age, degree of pain, number of operations, and duration of pain (p>0.05). Univariate analysis revealed that the type of electrode and presence of severe sensory deficits were significantly associated with the success of trial stimulation (p<0.05). However, the remaining variable, sex, type of pain, main location of pain, degree of pain duration, degree of sensory loss, and presence of motor weakness, were not associated with the trial success of SCS for FBSS. Conclusion Trial stimulation with paddle leads was more successful. If severe sensory deficits occur in the painful dermatomes in FBSS, trial stimulation were less effective. PMID:24527193

  4. Granulocyte-colony stimulating factor as a treatment for diabetic neuropathy in rat.

    PubMed

    Kim, Kyung-Soo; Song, Yi-Sun; Jin, Jiyong; Joe, Jun-Ho; So, Byung-Im; Park, Jun-Young; Fang, Cheng-Hu; Kim, Mi Jung; Cho, Youl-Hee; Hwang, Sejin; Ro, Young-Suck; Kim, Hyuck; Ahn, You-Hern; Sung, Hak-Joon; Sung, Jung-Joon; Park, Sung-Hye; Lipton, Stuart A

    2015-10-15

    Effective treatment of diabetic neuropathy (DN) remains unsolved. We serendipitously observed dramatic relief of pain in several patients with painful DN receiving granulocyte-colony stimulating factor (G-CSF). The aim of this study was to determine if G-CSF could treat DN in an animal model and to ascertain its mechanism of action. In a rodent model of DN, G-CSF dramatically recovered nerve function, retarded histological nerve changes and increased the expression of neurotrophic factors within nerve. A sex-mismatched bone marrow transplantation (BMT) study revealed that G-CSF treatment increased the abundance of bone marrow (BM)-derived cells in nerves damaged by DN. However, we did not observe evidence of transdifferentiation or cell fusion of BM-derived cells. The beneficial effects of G-CSF were dependent on the integrity of BM. In conclusion, G-CSF produced a therapeutic effect in a rodent model of DN, which was attributed, at least in part, to the actions of BM-derived cells. PMID:26190836

  5. Granulocyte macrophage colony-stimulating factor and the intestinal innate immune cell homeostasis in Crohn's disease.

    PubMed

    Däbritz, Jan

    2014-03-01

    Current literature consolidates the view of Crohn's disease (CD) as a form of immunodeficiency highlighting dysregulation of intestinal innate immunity in the pathogenesis of CD. Intestinal macrophages derived from blood monocytes play a key role in sustaining the innate immune homeostasis in the intestine, suggesting that the monocyte/macrophage compartment might be an attractive therapeutic target for the management of CD. Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that also promotes myeloid cell activation, proliferation, and differentiation. GM-CSF has a protective effect in human CD and mouse models of colitis. However, the role of GM-CSF in immune and inflammatory reactions in the intestine is not well defined. Beneficial effects exerted by GM-CSF during intestinal inflammation could relate to modulation of the mucosal barrier function in the intestine, including epithelial cell proliferation, survival, restitution, and immunomodulatory actions. The aim of this review is to summarize potential mechanistic roles of GM-CSF in intestinal innate immune cell homeostasis and to highlight its central role in maintenance of the intestinal immune barrier in the context of immunodeficiency in CD. PMID:24503766

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

    SciTech Connect

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

    1986-05-15

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

  7. Upregulation of glucose metabolism by granulocyte-monocyte colony-stimulating factor

    SciTech Connect

    Schuler, A.; Spolarics, Z.; Lang, C.H.; Bagby, G.J.; Nelson, S.; Spitzer, J.J. )

    1991-01-01

    Alterations of glucose metabolism were investigated for 6 hours following an intraarterial injection of murine recombinant granulocyte-monocyte colony-stimulating factor (GM-CSF). GM-CSF resulted in a transient elevation of plasma glucose. The rate of whole body glucose appearance, as measured by infusion of (6-{sup 3}H)glucose, was increased by about 10% between 0.5 and 3 hours following GM-CSF injection. In vivo glucose utilization of individual tissues was investigated by the tracer 2-deoxyglucose technique. At 30 min, GM-CSF increased glucose utilization by 80-90% in liver and lung, and 50-60% in skin and spleen. At 3 and 6 hours, glucose utilization by these tissues returned toward control levels except for lung. There was a 40-50% increase in glucose utilization by skeletal muscle 30 min after GM-CSF which was sustained for 6 hours. Glucose utilization of testis, ileum and kidney did not change significantly. Plasma concentrations of insulin, glucagon and tumor necrosis factor were not altered in response to GM-CSF. These findings indicate that some of the acute metabolic effects of a short-term administration of GM-CSF are observed in macrophage-rich tissues, and suggest that GM-CSF may be involved in the metabolic upregulation of immunologically active tissues.

  8. Sulfur mustard-induced neutropenia: treatment with granulocyte colony-stimulating factor.

    PubMed

    Anderson, Dana R; Holmes, Wesley W; Lee, Robyn B; Dalal, Stephen J; Hurst, Charles G; Maliner, Beverly I; Newmark, Jonathan; Smith, William J

    2006-05-01

    Although best known as a blistering agent, sulfur mustard (HD) can also induce neutropenia in exposed individuals, increasing their susceptibility to infection. Granulocyte colony-stimulating factor (G-CSF) and pegylated G-CSF (peg-G-CSF) have been approved by the U.S. Food and Drug Administration as hematopoietic growth factors to treat chemotherapy-induced neutropenia. The goal of this study was to determine the effectiveness of G-CSF and peg-G-CSF in ameliorating HD-induced neutropenia. African green monkeys (Chlorocebus aethiops) were challenged with HD and, at 1, 3, 5, or 7 days after exposure, G-CSF therapy (10 microg/kg per day for 21 days) was initiated. Peg-G-CSF (300 microg/kg, single treatment) was similarly tested, with treatment given at 3 days after exposure. Untreated HD-exposed animals recovered from neutropenia 28 days after exposure, whereas G-CSF- or peg-G-CSF-treated animals recovered 8 to 19 days after exposure (p < 0.05). These results indicate that G-CSF or peg-G-CSF may provide Food and Drug Administration-approved treatments that will reduce the duration of HD-induced neutropenia. PMID:16761898

  9. 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

  10. Hypoxia-induced fibroblast growth factor 11 stimulates capillary-like endothelial tube formation.

    PubMed

    Yang, Jimin; Kim, Woo Jean; Jun, Hyoung Oh; Lee, Eun Ju; Lee, Kyeong Won; Jeong, Jae-Yeon; Lee, Sae-Won

    2015-11-01

    Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor 11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis. PMID:26323829

  11. 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

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

    PubMed Central

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

    2014-01-01

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

  13. Effect of hepatocyte-stimulating factor and glucocorticoids on plasma fibronectin levels.

    PubMed Central

    Amrani, D L; Mauzy-Melitz, D; Mosesson, M W

    1986-01-01

    We evaluated the effects of hepatocyte-stimulating factor (HSF) and a glucocorticoid (dexamethasone) on changes in the levels, in vivo and in vitro, of plasma fibronectin (Fn), a glycoprotein that is synthesized and secreted by hepatocytes. In turpentine-treated chickens, plasma levels of Fn, which peaked at 48 h (whereas fibrinogen levels were maximum at 72 h) rose 200-250% over basal levels, whereas albumin levels decreased by 20-40%. Corticosterone levels in serum samples taken between 5 and 48 h after injection revealed a 124% increase in hormone levels at 24 h in turpentine-treated chickens. We also showed that circulating HSF levels were maximal 8 to 12 h after injection and that HSF activity, as assessed by molecular-exclusion chromatography, was eluted in the 30-45 kDa range. Addition of either serum-derived HSF or dexamethasone (2 nM) to chick hepatocyte cultures resulted in a 130-150% increase in secreted Fn as well as in fibrinogen. When HSF and dexamethasone were added together, a 360-489% increase in the secreted levels of both proteins was found. Chicken mononuclear phagocytic cells treated with lipopolysaccharide secreted an HSF activity that was eluted in two peaks, a minor peak at approximately 70 kDa and a major peak in the 25-40 kDa range. Addition of mononuclear-cell-derived HSF resulted in a greater increase in Fn levels than did the addition of serum HSF. These findings indicate that Fn, like fibrinogen, is an acute-phase protein, the production of which, at least in chickens, is stimulated by HSF and glucocorticoids in an additive manner. PMID:3099768

  14. Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

    PubMed Central

    Tanaka, S; Takahashi, N; Udagawa, N; Tamura, T; Akatsu, T; Stanley, E R; Kurokawa, T; Suda, T

    1993-01-01

    The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts. Images PMID:8423223

  15. 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

  16. 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

  17. Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa.

    PubMed Central

    Baldi, E; Falsetti, C; Krausz, C; Gervasi, G; Carloni, V; Casano, R; Forti, G

    1993-01-01

    The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa. Images Figure 2 Figure 4 Figure 7 PMID:8503848

  18. 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

  19. [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. PMID:27412938

  20. Cardiopulmonary effects of granulocyte colony-stimulating factor in a canine model of bacterial sepsis.

    PubMed

    Eichacker, P Q; Waisman, Y; Natanson, C; Farese, A; Hoffman, W D; Banks, S M; MacVittie, T J

    1994-11-01

    We investigated the effects of recombinant granulocyte colony-stimulating factor (G-CSF) in a canine model of septic shock. Awake 2-yr-old beagles were studied before and after intraperitoneal placement of an Escherichia coli-infected clot. Nine days before and until 3 days after clot placement, animals received daily high-dose (G-CSF (5 microgram/kg body wt; n = 17), low-dose G-CSF (0.1 microgram/kg body wt; n = 17), or a control protein (5 micrograms/kg body wt; n = 20). Survival rate was greater (P < 0.04, Wilcoxon test) in the high-dose G-CSF group (14/17) than in the low-dose G-CSF (10/17) and control (12/20) groups. High-dose G-CSF improved cardiovascular function, as evidenced by increased left ventricular ejection fraction (day 1 after clot; P < 0.001) and mean arterial pressure (day 2; P < 0.02) compared with low-dose G-CSF and control groups. High-dose G-CSF increased (P < 0.001) mean peripheral neutrophils before (-3 days) and after (2 h to 4 days) clot and produced a more rapid (P < 0.001) rise (day 2) and fall (day 4) in mean alveolar neutrophil numbers compared with the low-dose G-CSF and control groups. High-dose G-CSF decreased mean serum endotoxin (2-8 h; P < 0.002) and tumor necrosis factor (2 h; P < 0.02) levels and lowered blood bacteria counts (2-6 h; P < 0.04) compared with the low-dose G-CSF and control groups. Thus, in this canine model, G-CSF sufficient to increase peripheral neutrophils before and during peritonitis and septic shock enhances host defense, reduces cytokine (tumor necrosis factor) levels, and improves cardiovascular function and survival. PMID:7532649

  1. Improved granulocyte colony-stimulating factor mobilization of hemopoietic progenitors using cytokine combinations in primates.

    PubMed

    Larsen, Stephen R; Chng, Keefe; Battah, Fiona; Martiniello-Wilks, Rosetta; Rasko, John E J

    2008-11-01

    Peripheral blood stem cells (PBSCs), usually mobilized with granulocyte colony-stimulating factor (G-CSF) alone or in combination with chemotherapy, are the preferred source of cells for hemopoietic stem cell transplantation. Up to 25% of otherwise eligible transplant recipients fail to harvest adequate PBSCs. Therefore it is important to investigate existing and novel reagents to improve PBSC mobilization. Because of marked interindividual variation in humans, we developed a robust nonhuman primate model that allows the direct comparison of the efficacy of two PBSC mobilization regimens within the same animal. Using this model, we compared pegylated G-CSF (pegG-CSF) with standard G-CSF and compared the combination of G-CSF and pegylated megakaryocyte growth and development factor (pegMGDF) with G-CSF plus stem cell factor (SCF) by measuring the levels of CD34(+) cells, colony-forming cells (CFCs), and SCID repopulating cells (SRCs) before and after cytokine administration. Mobilization of CD34(+) cells, CFCs and SRCs using pegG-CSF achieved similar levels to those resulting from 5 days of standard G-CSF. The combination of G-CSF+pegMGDF mobilized progenitors to levels similar to G-CSF+SCF but greater than standard G-CSF for CD34(+) cells and CFC. This first direct comparison of PBSC mobilization in individual primates demonstrates that peg-G-CSF is equivalent to daily G-CSF and that the addition of pegMGDF to G-CSF improves mobilization. In light of the development of new thrombopoietin agonists, these data offer the potential for improved stem cell mobilization strategies. Disclosure of potential conflicts of interest is found at the end of this article. PMID:18719223

  2. 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.

  3. 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

  4. Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization.

    PubMed Central

    Wiedłocha, A; Falnes, P O; Rapak, A; Muñoz, R; Klingenberg, O; Olsnes, S

    1996-01-01

    U2OS Dr1 cells, originating from a human osteosarcoma, are resistant to the intracellular action of diphtheria toxin but contain toxin receptors on their surfaces. These cells do not have detectable amounts of fibroblast growth factor receptors. When these cells were transfected with fibroblast growth factor receptor 4, the addition of acidic fibroblast growth factor to the medium induced tyrosine phosphorylation, DNA synthesis, and cell proliferation. A considerable fraction of the cell-associated growth factor was found in the nuclear fraction. When the growth factor was fused to the diphtheria toxin A fragment, it was still bound to the growth factor receptor and induced tyrosine phosphorylation but did not induce DNA synthesis or cell proliferation, nor was any fusion protein recovered in the nuclear fraction. On the other hand, when the fusion protein was associated with the diphtheria toxin B fragment to allow translocation to the cytosol by the toxin pathway, the fusion protein was targeted to the nucleus and stimulated both DNA synthesis and cell proliferation. In untransfected cells containing toxin receptors but not fibroblast growth factor receptors, the fusion protein was translocated to the cytosol and targeted to the nucleus, but in this case, it stimulated only DNA synthesis. These data indicate that the following two signals are required to stimulate cell proliferation in transfected U2OS Dr1 cells: the tyrosine kinase signal from the activated fibroblast growth factor receptor and translocation of the growth factor into the cell. PMID:8524304

  5. Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor beta and epidermal growth factor.

    PubMed Central

    Boerner, P; Resnick, R J; Racker, E

    1985-01-01

    Glycolysis in normal resting rat kidney cells (NRK-49F) was stimulated by a 2-hr exposure to transforming growth factors prior to assay. Transforming growth factor beta (TGF-beta) was effective when added alone, and further addition of epidermal growth factor (EGF) had little effect. The stimulation by TGF-beta was abolished when cycloheximide was present during the incubation, suggesting that protein synthesis is required for the effect. Incubation of the cells with 25 mM methionine abolished the stimulation of glycolysis by TGF-beta. The uptake of methylaminoisobutyrate via system A was stimulated by either TGF-beta or EGF. The greater than 3-fold stimulation of uptake by 1 ng of pure TGF-beta per ml was usually somewhat enhanced on addition of 0.5 ng of EGF per ml. Moreover, an antiserum against EGF receptor partially depressed the response to TGF-beta, suggesting some overlapping interactions of EGF and TGF-beta. Images PMID:3871948

  6. Enhancement of human granulocyte-colony stimulating factor production in recombinant E. coli using batch cultivation.

    PubMed

    Babaeipour, Valiollah; Abbas, Mahdi Pesaran Haji; Sahebnazar, Zahra; Alizadeh, Reza

    2010-06-01

    Development of inexpensive and simple culture media is always favorable for recombinant protein over-expression in E. coli. The effects of medium composition on the production of recombinant human granulocyte-colony stimulating factor (rh-GCSF) were investigated in batch culture of E. coli BL21 (DE3) [pET23a-hgcsf]. First, the optimum medium for production of rh-GCSF was determined; and, then it was shown that mixture of amino acid addition at induction time, which was determined on the basis of amino acids frequency in the recombinant protein, increases recombinant protein expression level significantly. Furthermore, the effect of glucose concentration on productivity of rh-GCSF was investigated; 20 g/l of glucose will result in maximum attainable biomass and rh-GCSF in this process. At optimum conditions, a cell dry weight of 10.5 g/l, an expression level of about 35% of total cellular protein, rh-GCSF concentration of 1.75 +/- 0.1 g/l, and overall rh-GCSF yield of 165 +/- 5 mg/g were obtained. PMID:19859744

  7. Mechanisms of endothelial cell-dependent leukocyte adhesion stimulated by platelet-activating factor.

    PubMed

    Ding, Z; Li, S; Wu, Z

    1992-04-01

    Platelet-activating factor (PAF) stimulates leukocyte-endothelial cell (EC) adhesion through its effects either on leukocytes or on ECs. ECs may be injured, synthesize, or express new adhesive proteins to increase leukocyte adhesion. Intermediary mediators produced by activated ECs are also likely involved in promoting leukocyte adhesion. Our experiments demonstrated that PAF induced no obvious damage to bovine pulmonary artery ECs evaluated by lactic dehydrogenase release rate, angiotensin-converting enzyme activity, and cellular malondialdehyde content. Treatment of EC monolayers with 10(-9) M PAF increased polymorphonuclear leukocyte (PMN) adhesion. Increasing PAF concentration did not induce more PMN adherence. PAF elicited both a rapid and prolonged increment of PMN adherence to EC monolayers. The rapid adherence was greatly attenuated by pretreatment of ECs with PAF receptor antagonist SRI 63-441 but was not affected by pretreatment of PMNs with SRI 63-441, suggesting that PAF increases PMN adherence rapidly through its effects on specific receptors on ECs. Increased PMN adherence lasted if PAF treatment of ECs was sustained for 3 or 6 h. Pretreatment of ECs with actinomycin D, a protein synthesis inhibitor, significantly decreased PAF-induced sustained PMN adherence, but the inhibition is incomplete, suggesting that other mechanisms than protein synthesis also participated in the prolonged PMN adherence. We concluded from the results that PAF may induce both rapid and prolonged PMN adhesion to ECs. The effects are receptor mediated. The prolonged PMN adhesion is partly the result of protein synthesis. PMID:1592489

  8. Granulocyte-macrophage colony-stimulating factor and pulmonary surfactant homeostasis.

    PubMed

    Reed, J A; Whitsett, J A

    1998-01-01

    Pulmonary surfactant lining the alveolus of the lung is critical to postnatal adaptation to air breathing. Precise concentrations of surfactant proteins and lipids are maintained in the alveolar space by a careful balance among synthesis, recycling, and catabolism. Pulmonary alveolar proteinosis is a rare pulmonary disease associated with accumulation of surfactant lipids and proteins in the alveolar spaces. Recent work with transgenic mice demonstrated that disruption of the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) or the common beta-subunit of the GM-CSF receptor caused alveolar proteinosis that was histologically similar to that seen in human patients. The defect in surfactant homeostasis is caused by decreased surfactant clearance, mediated (at least in part) by dysfunction of the alveolar macrophage. Local production of GM-CSF corrects the alveolar proteinosis in the GM-CSF knockout mouse. Likewise, transplantation of wild-type bone marrow cells expressing the common beta-chain of the GM-CSF receptor restores surfactant homeostasis in the GM-CSF receptor knockout mouse. These studies demonstrate the previously unanticipated role of GM-CSF signaling in surfactant homeostasis, mediated (at least in part) by its actions on the clearance of surfactant lipids and proteins by the alveolar macrophage. These findings may have important implications for the diagnosis and treatment of pulmonary alveolar proteinosis syndromes in humans. PMID:9686680

  9. Factors Associated With Upper Extremity Motor Recovery After Repetitive Transcranial Magnetic Stimulation in Stroke Patients

    PubMed Central

    Lee, Jong Hwa; Kim, Sang Beom; Lee, Kyeong Woo; Kim, Min Ah; Lee, Sook Joung

    2015-01-01

    Objective To determine factors associated with motor recovery of the upper extremity after repetitive transcranial magnetic stimulation (rTMS) treatment in stroke patients. Methods Twenty-nine patients with subacute stroke participated in this study. rTMS was applied to the hand motor cortex for 10 minutes at a 110% resting motor threshold and 10 Hz frequency for two weeks. We evaluated the biographical, neurological, clinical, and functional variables, in addition to the motor-evoked potential (MEP) response. The Manual Function Test (MFT) was performed before, immediately after, and two weeks after, the treatment. Patients were divided into a responder and non-responder group according to their respective improvements on the MFT. Data were compared between the two groups. Results Patients with exclusively subcortical stroke, absence of aphasia, the presence of a MEP response, high scores on the Mini-Mental Status Examination, Motricity Index arm score, Functional Independence Measure, and Functional Ambulatory Classification; and a shorter period from stroke onset to rTMS were found to be significantly associated with a response to rTMS. Conclusion The results of this study suggest that rTMS may have a greater effect on upper extremity motor recovery in stroke patients who have a MEP response, suffer an exclusively subcortical stroke, mild paresis, and have good functional status. Applying rTMS early would have additional positive effects in the patients with the identified characteristics. PMID:25932424

  10. Effect of granulocyte-colony stimulating factor on spinal cord tissue after experimental contusion injury.

    PubMed

    Sanli, A Metin; Serbes, Gökhan; Calişkan, Murat; Kaptanoğlu, Erkan; Sargon, Mustafa F; Kilinç, Kamer; Beşalti, Omer; Sekerci, Zeki

    2010-12-01

    The purpose of this study was to investigate the early effects of granulocyte-colony stimulating factor (G-CSF) on myeloperoxidase (MPO) activity, lipid peroxidation (LPO) and ultrastructural findings in rats after spinal cord injury (SCI). We also compared the effects of G-CSF and methylprednisolone sodium succinate (MPSS). Wistar rats were divided into four groups: control, SCI alone (50 g/cm weight drop trauma), SCI+MPSS (30 mg/kg), and SCI+G-CSF (50 μg/kg). Administration of G-CSF and MPSS significantly decreased LPO (p < 0.05) and MPO activity (p < 0.05) in the first 24 hours. MPSS was more effective than G-CSF in reducing LPO (p < 0.05) and in minimizing ultrastructure changes. The results of this study indicate that G-CSF exerts a beneficial effect by decreasing MPO activity and LPO and may reduce tissue damage in the first 24 hours after SCI. Our findings do not exclude the possibility that G-CSF has a protective effect on spinal cord ultrastructure after the first 24 hours following SCI. PMID:20801040

  11. Immunocytochemical Localization of Latent Transforming Growth Factor-B1 Activation by Stimulated Macrophages

    SciTech Connect

    Chong, Hyonkyong; Vodovotz, Yoram; Cox, G.W.; Barcellos-Hoff, M.H.

    1998-09-22

    Transforming growth factor-{beta}1 (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.

  12. Capillary electrophoretic separation of poly(ethylene glycol)-modified granulocyte-colony stimulating factor.

    PubMed

    Lee, Kyung Soo; Na, Dong Hee

    2010-03-01

    We evaluated the utility of capillary electrophoretic methods for analyzing poly(ethylene glycol) (PEG)-modified granulocyte-colony stimulating factor (G-CSF), a long-acting form of GCSF for the treatment of cancer therapy-induced neutropenia. Low- and high-molecularweight PEG-G-CSF conjugates prepared with aldehyde-activated PEG-5K and PEG-20K were separated by high-performance size-exclusion chromatography (HP-SEC), capillary zone electrophoresis (CZE), and sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). HPSEC showed low resolution for separating mono- and di-PEG-G-CSFs. SDS-CGE had higher resolution, but required a long analysis and had low peak efficiency. CZE could successfully separate both PEG-5K- and PEG-20K-conjugated G-CSFs with a running time of 20 min and high peak efficiency. In conclusion, CZE was better than SDS-CGE for separating PEG-G-CSF conjugates and will be useful for PEGylation studies, such as reaction monitoring for optimization of the PEGylation reaction, and purity and stability tests of PEG-G-CSF. PMID:20361316

  13. 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.

  14. 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

  15. 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.

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. The Effects of Granulocyte-Colony Stimulating Factor on Regeneration in Nerve Crush Injuries in Rats.

    PubMed

    Song, Yi-Sun; Joe, Jun-Ho; Joo, Hyun-Woo; Park, In-Hwa; Shen, Guang-Yin; Kim, Ki-Jun; Lee, Yonggu; Shin, Jeong Hun; Kim, Hyuck; Kim, Kyung-Soo

    2016-07-01

    Granulocyte-colony stimulating factor (G-CSF) is widely known to have a neuroprotective effect, but its effects on function and morphology in mechanical nerve injury are not well understood. The aim of this study was to confirm the time course of the functional changes and morphological effects of G-CSF in a rat model of nerve crush injury. Twelve-eight rats were divided into three group: sham-operated control group, G-CSF-treated group, and saline treated group. 2 weeks after the nerve crush injury, G-CSF was injected for 5 days. After 4 weeks, functional tests such as motor nerve conduction velocity (MNCV), mechanical and cold allodynia tests, and morphological studies were performed. G-CSF-treated rats had significantly improved nerve function including MNCV and mechanical and cold allodynia. In addition, G-CSF-treated rats had significantly higher the density of myelinated fibers than saline-treated rats. In conclusion, we found that 100 μg/kg administration of G-CSF promoted long-term functional recovery in a rat model of nerve crush injury. PMID:26980007

  19. 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

  20. Granulocyte-Macrophage Colony-Stimulating Factor in Staphylococcus aureus-Induced Arthritis

    PubMed Central

    Verdrengh, Margareta; Tarkowski, Andrej

    1998-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that is able to increase not only the production of phagocytic cells but also their efficacy with respect to, e.g., bactericidal properties. In this study, we wanted to analyze the impact of GM-CSF on experimental Staphylococcus aureus-induced arthritis. For that purpose, mice were administered GM-CSF before and after bacterial inoculation. Although there was an increase in the total number of leukocytes as well as in the granulocyte fraction, there was no favorable effect on the severity of arthritis or on survival rates. There were no obvious differences between the GM-CSF-pretreated animals and controls with regard to growth of staphylococci in joints and kidneys 4 days after the bacterial inoculation. In contrast, mice that had been pretreated with GM-CSF prior to bacterial inoculation showed approximately four times lower numbers of bacteria in their blood 24 h later. These results, along with those of our previous studies, suggest that on the one hand the granulocyte is the main protective cell during the course of S. aureus infection but that on the other hand, upregulation of granulocyte-macrophage production will not exert any additional protective effects with respect to tissue injury. PMID:9453655

  1. Vascular Endothelial Growth Factor Release Following Electrical Stimulation in Human Subjects

    PubMed Central

    Liebano, Richard Eloin; Machado, Aline Fernanda Perez

    2014-01-01

    Significance: Angiogenesis is an important phenomenon involved in the healing of chronic wounds, and it is mainly mediated by the release of vascular endothelial growth factor (VEGF) from endothelial cells. Electrical stimulation (ES) is a well-documented treatment used to assist the healing of chronic wounds. Due to the importance of VEGF in the healing process, and the need to know the mechanisms of action of ES involved in the process, this report aimed to determine by a literature review whether the VEGF release occurs following ES in human subjects. Recent Advances: The findings of this literature review suggest that ES releases VEGF, and this effect may be responsible for promoting angiogenesis after ES. Critical Issues: Despite the findings of this literature review on the release of VEGF by ES on wound healing are promising, a large number of studies are needed to confirm such effects. Future Directions: Further studies should be conducted to identify the best parameters and treatment schedule of ES to be used for the VEGF release. PMID:24761350

  2. Establishment of a cell line of gallbladder carcinoma (GBK-1) producing human colony stimulating factor.

    PubMed

    Egami, H; Sakamoto, K; Yoshimura, R; Kikuchi, H; Akagi, M

    1986-02-01

    A cell line designated GBK-1 was established from a patient with anaplastic carcinoma of the gallbladder, marked neutrophilia and fever, and has been propagated for the past 18 months. The cells grew as a monolayer sheet with a doubling time of 43 hr. The GBK-1 cells were of a pleomorphic polygonal epitheloid shape and they were transplantable into nude mice. Mice bearing the tumor developed marked granulocytosis. In the culture supernatant of GBK-1 cells, high colony stimulating factor (CSF) activity was evident with both human bone marrow cells and C57BL mouse bone marrow cells, and granulocytic colonies, macrophage colonies or granulocyte-macrophage mixed colonies were produced. The CSF activity was distributed in the molecular weight range of 25,000 to 60,000 with two distinct peaks at molecular weights of approximately 50,000 and 30,000. CSF activity was inactivated by heat treatment at 70 degrees for 30 min. GBK-1 is a new human cell line that produces heat-labile human GM-CSF. PMID:3082828

  3. Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris.

    PubMed

    Maity, Nitu; Thawani, Ankita; Sharma, Anshul; Gautam, Ashwani; Mishra, Saroj; Sahai, Vikram

    2016-01-01

    Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask. PMID:26410223

  4. [Emergency therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF)].

    PubMed

    Gratwohl, A; Dazzi, H; Tichelli, A; Stebler, C; Wernli, M; Thomssen, C; Kim, I; Dieterle, A; Obrist, R; Stern, A

    1991-03-23

    Granulocyte-macrophage colony stimulating factor (GM-CSF) has been tested for tolerability and efficacy on a compassionate need case basis in 17 patients (5 females, 12 males aged 4-72 years, median 35 years). GM-CSF was given at the rate of 3.5-32 micrograms/kg for 2-64 days as a continuous infusion for the following indications: impending rejection following bone marrow transplantation (5 patients), severe neutropenia secondary to chemotherapy in tumor patients (5), severe aplastic anemia (3), immune granulocytopenia (2) and accidental overdose with cytostatic agents (2 patients). Tolerance of GM-CSF was good in regard to doses of up to 16 micrograms/kg. Fever, myalgia and eosinophilia were the most frequent side effects. The patient treated with 32 micrograms/kg developed thrombosis of the vena cava. Efficacy is more difficult to assess in this heterogenous population, but 11 of 17 patients showed increased granulocyte counts and 3 patients clearly recovered from severe neutropenia. The role of GM-CSF in this recovery, however, cannot be proven. The results further indicate that GM-CSF cannot reverse ongoing rejection following allogenic BMT and cannot correct immune neutropenia. The value of GM-CSF therapy in patients with severe aplastic anemia and in the context of chemotherapy still needs to be defined. It is certainly indicated in patients with an accidental overdose of chemotherapeutic agents. PMID:2028244

  5. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion.

    PubMed

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E; Bornfeldt, Karin E; Duffield, Jeremy S; Heinecke, Jay W

    2015-06-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  6. 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.

  7. 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.

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

    PubMed Central

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

    2013-01-01

    Background: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. Methods: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. Results: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. Conclusions: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment. PMID:23748895

  9. Biomimetic scaffold combined with electrical stimulation and growth factor promotes tissue engineered cardiac development.

    PubMed

    Park, Hyoungshin; Larson, Benjamin L; Kolewe, Martin E; Vunjak-Novakovic, Gordana; Freed, Lisa E

    2014-02-15

    Toward developing biologically sound models for the study of heart regeneration and disease, we cultured heart cells on a biodegradable, microfabricated poly(glycerol sebacate) (PGS) scaffold designed with micro-structural features and anisotropic mechanical properties to promote cardiac-like tissue architecture. Using this biomimetic system, we studied individual and combined effects of supplemental insulin-like growth factor-1 (IGF-1) and electrical stimulation (ES). On culture day 8, all tissue constructs could be paced and expressed the cardiac protein troponin-T. IGF-1 reduced apoptosis, promoted cell-to-cell connectivity, and lowered excitation threshold, an index of electrophysiological activity. ES promoted formation of tissue-like bundles oriented in parallel to the electrical field and a more than ten-fold increase in matrix metalloprotease-2 (MMP-2) gene expression. The combination of IGF-1 and ES increased 2D projection length, an index of overall contraction strength, and enhanced expression of the gap junction protein connexin-43 and sarcomere development. This culture environment, designed to combine cardiac-like scaffold architecture and biomechanics with molecular and biophysical signals, enabled functional assembly of engineered heart muscle from dissociated cells and could serve as a template for future studies on the hierarchy of various signaling domains relative to cardiac tissue development. PMID:24240126

  10. 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.

  11. Granulocyte colony-stimulating factor delays neutrophil apoptosis by inhibition of calpains upstream of caspase-3

    PubMed Central

    Drewniak, Agata; Groenewold, Vincent; van den Berg, Timo K.; Kuijpers, Taco W.

    2008-01-01

    Neutrophils have a very short life span and undergo apoptosis within 24 hours after leaving the bone marrow. Granulocyte colony-stimulating factor (G-CSF) is essential for the recruitment of fresh neutrophils from the bone marrow but also delays apoptosis of mature neutrophils. To determine the mechanism by which G-CSF inhibits neutrophil apoptosis, the kinetics of neutrophil apoptosis during 24 hours in the absence or presence of G-CSF were analyzed in vitro. G-CSF delayed neutrophil apoptosis for approximately 12 hours and inhibited caspase-9 and -3 activation, but had virtually no effect on caspase-8 and little effect on the release of proapoptotic proteins from the mitochondria. However, G-CSF strongly inhibited the activation of calcium-dependent cysteine proteases calpains, upstream of caspase-3, via apparent control of Ca2+-influx. Calpain inhibition resulted in the stabilization of the X-linked inhibitor of apoptosis (XIAP) and hence inhibited caspase-9 and -3 in human neutrophils. Thus, neutrophil apoptosis is controlled by G-CSF after initial activation of caspase-8 and mitochondrial permeabilization by the control of postmitochondrial calpain activity. PMID:18524991

  12. Colony Stimulating Factors 1, 2, 3 and early pregnancy steps: from bench to bedside.

    PubMed

    Rahmati, Mona; Petitbarat, Marie; Dubanchet, Sylvie; Bensussan, Armand; Chaouat, Gerard; Ledee, Nathalie

    2015-06-01

    Reproductive immunology applies general immunology principles to specialised targets, reproduction and development. The involvement of colony-stimulating factors (CSFs) in reproduction illustrates this. The CSF family includes CSF-1 or macrophage CSF (M-CSF), CSF-2 or granulocyte macrophage CSF (GM-CSF), and CSF-3 or granulocyte CSF (G-CSF). Each member has a specific localisation and timed expression in the reproductive tract with specific functions involving them in ovulation, embryo implantation, placentation and further embryonic development. They are used in reproductive medicine, either as biomarkers of oocyte quality and competence (follicular G-CSF), or to supplement embryo culture media with human recombinant GM-CSF, or they are used as an innovative therapy by using human recombinant G-CSF for infertile patients. Given fundamental considerations on CSFs and their strong implication in reproduction, this review aimed to detail the current knowledge for each member of the family to improve our understanding of their implication in the maternal-foetal cytokinic dialogue and in possibly preventing reproductive disorders. PMID:25721620

  13. 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…

  14. PROLINE IS REQUIRED FOR THE STIMULATION OF DNA SYNTHESIS IN HEPATOCYTE CULTURES BY EGF (EPIDERMAL GROWTH FACTOR)

    EPA Science Inventory

    Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). The authors report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has th...

  15. The cutaneous epidermal growth factor network: Can it be translated clinically to stimulate hair growth?

    PubMed

    Alexandrescu, Doru T; Kauffman, C Lisa; Dasanu, Constantin A

    2009-01-01

    The influences exerted by the epidermal growth factor receptor (EGFR) on the skin act at multiple levels, which involve compartments that normally express EGFR. These include the basal and suprabasal layers of the epidermis, sebaceous glands, and the outer root sheath of the hair follicles. The physiological roles of EGFR ensure epidermal renewal and integrity, along with a gatekeeping and function and hair growth stimulation functions. Important cellular functions that are altered during EGF receptor blocking therapy consist of epidermal differentiation, proliferation, apoptosis, and migration, with an overall dominating effect of inducing growth arrest and terminal differentiation of the keratinocytes in the basal layers. The effects of EGFR blockage on the hair cycle include terminal differentiation of the hair follicle, which in certain cases may be associated with trichomegaly. Trichomegaly of the eyelashes may occur as an isolated occurrence or, frequently, as part of a generalized phenomenon that may be associated with the use of the EGFR inhibitors. Molecular changes associated with EGFR blockage are discussed, relevant to their association with hair growth. Modulation of Akt, AP2alpha, CDK4, Notch-1, p27KIP1, and Hedgehog expression are involved in the initiation of the hair cycle and inducement of the anagen phase, followed by proliferation and differentiation of the hair follicles. Epidermal growth factor receptor inhibitors have been developed as therapeutic molecules directed against cancer; in these regimens the knowledge of EGF receptor signaling functions has been translated into significant clinical results. However, among their various collateral effects on the skin, hair growth is observed to occur in certain patients. A particular "wavy" hair phenotype is observed during the pharmacological EGFR receptor blockade, just as in murine transgenic models that carry loss of function of TGF-alpha or EGFR genes. A better characterization of the

  16. 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

  17. Platelet-Derived Growth Factor Gene Delivery Stimulates ex Vivo Gingival Repair

    PubMed Central

    ANUSAKSATHIEN, ORASA; WEBB, SARAH A.; JIN, QI-MING; GIANNOBILE, WILLIAM V.

    2008-01-01

    Destruction of tooth support due to the chronic inflammatory disease periodontitis is a major cause of tooth loss. There are limitations with available treatment options to tissue engineer soft tissue periodontal defects. The exogenous application of growth factors (GFs) such as platelet-derived growth factor (PDGF) has shown promise to enhance oral and periodontal tissue regeneration. However, the topical administration of GFs has not led to clinically significant improvements in tissue regeneration because of problems in maintaining therapeutic protein levels at the defect site. The utilization of PDGF gene transfer may circumvent many of the limitations with protein delivery to soft tissue wounds. The objective of this study was to test the effect of PDGF-A and PDGF-B gene transfer to human gingival fibroblasts (HGFs) on ex vivo repair in three-dimensional collagen lattices. HGFs were transduced with adenovirus encoding PDGF-A and PDGF-B genes. Defect fill of bilayer collagen gels was measured by image analysis of cell repopulation into the gingival defects. The modulation of gene expression at the defect site and periphery was measured by RT-PCR during a 10-day time course after gene delivery. The results demonstrated that PDGF-B gene transfer stimulated potent (>4-fold) increases in cell repopulation and defect fill above that of PDGF-A and corresponding controls. PDGF-A and PDGF-B gene expression was maintained for at least 10 days. PDGF gene transfer upregulated the expression of phosphatidylinosital 3-kinase and integrin α5 subunit at 5 days after adenovirus transduction. These results suggest that PDGF gene transfer has potential for periodontal soft tissue-engineering applications. PMID:13678451

  18. Changes in the proteomic profile during differentiation and maturation of human monocyte-derived dendritic cells stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 and lipopolysaccharide.

    PubMed

    Pereira, Sandra Rodrigues; Faça, Vitor Marcel; Gomes, Glauce Gaspar; Chammas, Roger; Fontes, Aparecida Maria; Covas, Dimas Tadeu; Greene, Lewis Joel

    2005-04-01

    Dendritic cells (DCs) are highly specialized antigen-presenting cells that play an essential role in the immune response. We used the proteomic approach based on two-dimensional gel electrophoresis and mass spectrometry to identify the protein changes that occur during differentiation of DCs from monocytes (Mo) stimulated with granulocyte macrophage colony stimulating factor/interleukin-4 (GM-CSF/IL-4) and during the maturation of immature DCs stimulated with lipopolysaccharide. Sixty-three differentially expressed proteins (+/- two-fold) were unambiguously identified with sequence coverage greater than 20%. They corresponded to only 36 different proteins, because 11 were present as 38 electrophoretic forms. Some proteins such as tropomyosin 4 and heat shock protein 71 presented differentially expressed electrophoretic forms, suggesting that many of the changes in protein expression that accompany differentiation and maturation of DCs occur in post-translationally modified proteins. The largest differences in expression were observed for actin (21-fold in Mo), Rho GDP-dissociation inhibitor 2 (20-fold in Mo), vimentin (eight-fold in immature DCs), lymphocyte-specific protein 1 (12-fold in mature DCs) and thioredoxin (14-fold in mature DCs). Several proteins are directly related to functional and morphological characteristics of DCs, such as cytoskeletal proteins (cytoskeleton rearrangement) and chaperones (antigen processing and presentation), but other proteins have not been assigned specific functions in DCs. Only a few proteins identified here were the same as those reported in proteomic studies of DCs, which used different stimuli to produce the cells (GM-CSF/IL-4 and tumor necrosis factor-alpha). These data suggest that the DC protein profile depends on the stimuli used for differentiation and especially for maturation. PMID:15800872

  19. NOREPINEPHRINE AND EPIDERMAL GROWTH FACTOR: DYNAMICS OF THEIR INTERACTION IN THE STIMULATION OF HEPATOCYTE DNA SYNTHESIS

    EPA Science Inventory

    Primary cultures of adult rat hepatocytes are stimulated to enter DNA synthesis by norepinephrine (NE). This stimulation is maximal if the hepatocytes are incubated with NE for more than 12 hr, beginning no later than 2-4 hr after the cells are first plated. After 24 hr in cultur...

  20. Thyrotropin (TSH)-induced production of vascular endothelial growth factor in thyroid cancer cells in vitro: evaluation of TSH signal transduction and of angiogenesis-stimulating growth factors.

    PubMed

    Hoffmann, Sebastian; Hofbauer, Lorenz C; Scharrenbach, Vera; Wunderlich, Anette; Hassan, Iyad; Lingelbach, Susanne; Zielke, Andreas

    2004-12-01

    Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not

  1. Granulocyte-Colony Stimulating Factor Related Pathways Tested on an Endometrial Ex-Vivo Model

    PubMed Central

    Rahmati, Mona; Petitbarat, Marie; Dubanchet, Sylvie; Bensussan, Armand; Chaouat, Gerard; Ledee, Nathalie

    2014-01-01

    Introduction Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. Materials and Methods Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. Results At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. Conclusion RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early

  2. 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

  3. 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.

  4. Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis.

    PubMed

    Frost, R A; Lang, C H; Gelato, M C

    1997-10-01

    Tumor necrosis factor-alpha (TNF-alpha) induces cachexia and is postulated to be responsible for muscle wasting in several pathophysiological conditions. The purpose of the present study was to investigate whether exposure of human myoblasts to TNF-alpha could directly inhibit the ability of serum or insulin-like growth factor I (IGF-I) to stimulate protein synthesis as assessed by the incorporation of [3H]phenylalanine into protein. Serum and IGF-I stimulated protein synthesis dose dependently. Half-maximal stimulation of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I, respectively. TNF-alpha inhibited IGF-I-stimulated protein synthesis in a dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-alpha impaired the ability of IGF-I to stimulate protein synthesis by 33% and, at a dose of 100 ng/ml, TNF-alpha completely prevented the increase in protein synthesis induced by either serum or a maximally stimulating dose of IGF-I. Inhibition of protein synthesis was independent of whether TNF-alpha and growth factors were added to cells simultaneously or if the cells were pretreated with growth factors. Exposure ofmyoblasts to TNF-alpha for 10 min completely inhibited serum-induced stimulation of protein synthesis. TNF-alpha inhibited protein synthesis up to 48 h after addition of the cytokine. TNF-alpha also inhibited serum-stimulated protein synthesis in human myoblasts that were differentiated into myotubes. In contrast, exposure of myoblasts to TNF-alpha had no effect on IGF-I binding and failed to alter the ability of either IGF-I or serum to stimulate [3H]thymidine uptake. These data indicate that transient exposure of myoblasts or myotubes to TNF-alpha inhibits protein synthesis. Thus, the anabolic actions of IGF-I on muscle protein synthesis may be impaired during catabolic conditions in which TNF-alpha is over expressed. PMID:9322924

  5. 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

  6. 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...

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

    PubMed Central

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

    2016-01-01

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

  8. Granulocyte-colony stimulating factor as treatment option in patients with recurrent miscarriage.

    PubMed

    Santjohanser, Claudia; Knieper, Catherine; Franz, Cordula; Hirv, Kaino; Meri, Osama; Schleyer, Manfred; Würfel, Wolfgang; Toth, Bettina

    2013-04-01

    In 1-5% of patients during childbearing years recurrent miscarriages (RM) occur. There are established risk factors like anatomical, endocrine and hemostatic disorders as well as immunological changes in the maternal immune system. Nevertheless, further elucidation of the pathogenesis remains a matter of debate. In addition, there are no standardized immunological treatment strategies. Recent studies indicate possible effects of tumor necrosis factor α blocker and granulocyte-colony stimulating factor (G-CSF) concerning live birth rate (LBR) in RM patients. Therefore, we performed a retrospective cohort study in patients undergoing assisted reproductive treatment (ART) with known RM analysing the possible benefits of G-CSF application. From January 2002 to December 2010, 127 patients (199 cylces) with RM (at least 2 early miscarriages) 49 (72 cycles) receiving G-CSF and 78 (127 cycles) controls receiving either no medication (subgroup 1) or Cortisone, intravenous immunoglobulins or low molecular weight heparin (subgroup 2) undergoing ART for in vitro fertilisation/intracytoplasmic sperm injection were analysed. G-CSF was administered weekly once (34 Mill) in 11 patients, 38 patients received 2 × 13 Mill G-CSF per week until the 12th week of gestation. Statistical analysis was performed with SPSS for Windows (19.0), p < 0.05 significant. The mean age of the study population was 37.3 ± 4.4 years (mean ± standard deviation) and differed not significantly between patients and subgroups. However, the number of early miscarriages was significantly higher in the G-CSF group as compared to the subgroups (G-CSF 2.67 ± 1.27, subgroup 1 0.85 ± 0.91, subgroup 2 0.64 ± 0.74) and RM patients receiving G-CSF had significantly more often a late embryo transfer (day 5) (G-CSF 36.7%, subgroup 1 12.1%, subgroup 2 8.9%). The LBR of patients and the subgroups differed significantly (G-CSF 32%, subgroup 1 13%, subgroup 2 14%). Side effects were present in less than 10% of

  9. Modulation of the Endocannabinoid System: Vulnerability Factor and New Treatment Target for Stimulant Addiction

    PubMed Central

    Olière, Stéphanie; Jolette-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

  10. 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

  11. 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

  12. Does granulocyte-colony stimulating factor administration induce damage or repair response in schistosomiasis?

    PubMed Central

    Ghanem, Lobna Y; Dahmen, Uta; Dirsch, Olaf; Nosseir, Mona MF; Mahmoud, Soheir S; Mansour, Wafaa AF

    2010-01-01

    AIM: To introduce Granulocyte-colony stimulating factor (G-CSF) as a new therapeutic modality for schistosomiasis through stem cell mobilization, immunomodulation or fibrosis remodeling. METHODS: In this study, a 5 d course of human recombinant G-CSF (100 μg/kg sc) was applied to Schistosoma mansoni-infected mice at different stages of disease (5 d before infection as well as 3, 5 and 7 wk post-infection). The animals were sacrificed at 10 d as well as 4, 6 and 8 wk post infection. Mice were examined for: (1) Total leukocyte count which is an accepted surrogate marker for the stem cell mobilization into the circulation; (2) Egg count in intestine and liver tissue to assess the parasitic load; and (3) Histopathological changes in Hx/E and Masson trichrome stained sections as well as collagen content in Sirius red-stained liver sections to determine the severity of liver fibrosis. RESULTS: Mice developed leukocytosis. The egg load and the number of granulomas were not affected by the G-CSF treatment but there was an obvious change in the composition of granulomas towards an increased cellularity. Moreover, fibrosis was significantly decreased in treated groups compared to untreated animals (collagen content either preinfection or at 3 and 5 wk post infection: 5.8 ± 0.5, 4.7 ± 0.5, 4.0 ± 0.7 vs 8.2 ± 0.9; P ≤ 0.01). CONCLUSION: Although G-CSF did not cause direct elimination of the parasite, it enhanced granulomatous reaction and reduced the fibrosis. Further investigation of the underlying mechanisms of these two actions is warranted. PMID:21191519

  13. Molecular Mechanism of Regulation of MTA1 Expression by Granulocyte Colony-stimulating Factor.

    PubMed

    Kumar, Arathy S; Jagadeeshan, Sankar; Subramanian, Anirudh; Chidambaram, Saravana Babu; Surabhi, Rohan Prasad; Singhal, Mahak; Bhoopalan, Hemadev; Sekar, Sathiya; Pitani, Ravi Shankar; Duvuru, Prathiba; Venkatraman, Ganesh; Rayala, Suresh K

    2016-06-01

    Parkinson disease (PD) is a neurodegenerative disorder with loss of dopaminergic neurons of the brain, which results in insufficient synthesis and action of dopamine. Metastasis-associated protein 1 (MTA1) is an upstream modulator of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and hence MTA1 plays a significant role in PD pathogenesis. To impart functional and clinical significance to MTA1, we analyzed MTA1 and TH levels in the substantia nigra region of a large cohort of human brain tissue samples by Western blotting, quantitative PCR, and immunohistochemistry. Our results showed that MTA1 and TH levels were significantly down-regulated in PD samples as compared with normal brain tissue. Correspondingly, immunohistochemistry analysis for MTA1 in substantia nigra sections revealed that 74.1% of the samples had a staining intensity of <6 in the PD samples as compared with controls, 25.9%, with an odds ratio of 8.54. Because of the clinical importance of MTA1 established in PD, we looked at agents to modulate MTA1 expression in neuronal cells, and granulocyte colony-stimulating factor (G-CSF) was chosen, due to its clinically proven neurogenic effects. Treatment of the human neuronal cell line KELLY and acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model with G-CSF showed significant induction of MTA1 and TH with rescue of phenotype in the mouse model. Interestingly, the observed induction of TH was compromised on silencing of MTA1. The underlying molecular mechanism of MTA1 induction by G-CSF was proved to be through induction of c-Fos and its recruitment to the MTA1 promoter. PMID:27044752

  14. Safety of recombinant human granulocyte-macrophage colony-stimulating factor in healing pediatric severe burns.

    PubMed

    Chi, Y F; Chai, J K; Luo, H M; Zhang, Q X; Feng, R

    2015-01-01

    We explored the safety of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for healing burns in children. Subjects were randomly assigned to two groups: the experimental group received external rhGM-CSF gel, and the control group received rhGM-CSF gel matrix components, applied to the burn surface. Neither group was given any other drugs that promote wound healing. Each day we recorded the pulse, body temperature, and respiration status in the two groups. We detected the blood routine, urine routine, and hepatic and renal function before the patients received drug treatment and after 72 h. The wound scab and healing states in the two groups were recorded every 4 days to evaluate wound healing rate and time taken for complete healing. Adverse reactions and their rate of occurrence were also recorded. The median time of healing was 15 days in the experimental group and 19 days in the control group (log-rank χ(2) = 5.139, P < 0.05). After 10 days, the experimental group healing rate was consistently higher than that of the control group (significantly different using intuitive analysis), suggesting the experimental group method was more effective. There were no obvious adverse reactions. There was no significant difference between the blood routine, urine routine, and liver and kidney function in the two groups before the treatment and after 3 days (P > 0.05). Compared with saline treatment of severe burns, rhGM-CSF can effectively shorten the healing time without significant adverse reactions, and is an effective and safe treatment for burns in children. PMID:25867422

  15. The effect of granulocyte-colony stimulating factor in global cerebral ischemia in rats

    PubMed Central

    Matchett, Gerald A.; Calinisan, Jason B.; Matchett, Genoveve C.; Martin, Robert D.; Zhang, John H.

    2007-01-01

    Granulocyte-colony stimulating factor (G-CSF) is an endogenous peptide hormone of the hematopoietic system that has entered Phase I/II clinical trials for treatment of ischemic stroke. Severe intraoperative hypotension can lead to global cerebral ischemia and apoptotic neuron loss within the hippocampus. We tested G-CSF in a rat model of global cerebral ischemia. Global cerebral ischemia was induced in male Sprague-Dawley rats (280–330g) with the 2-vessel occlusion model (hemorrhagic hypotension to a mean arterial pressure of 30–35 mmHg and bilateral common carotid artery occlusion for 8 minutes). Three groups of animals were used: global ischemia without treatment (GI, n=49), global ischemia with G-CSF treatment (GI+G-CSF, n=42), and sham surgery (Sham, n = 26). Rats in the treatment group received G-CSF (50 μg / kg, subcutaneously) 12 hours before surgery, on the day of surgery, and on post-operative Day 1, and were euthanized on Day 2, 3, and 14. Mild hyperglycemia was observed in all groups. T-maze testing for spontaneous alternation demonstrated initial improvement in the G-CSF treatment group but no long-term benefit. Measurement of daily body weight demonstrated an initial trend toward improvement in the G-CSF group. Quantitative Nissl histology of the hippocampus demonstrated equivalent outcomes on Day 3 and 14, which was supported by quantitative TUNEL stain. Immunohistochemistry and Western blot demonstrated an initial increase in phosphorylated-AKT in the GI+G-CSF group on Day 2. We conclude that G-CSF treatment is associated with transient early improvement in neurobehavioral outcomes after global ischemia complicated by mild hyperglycemia, but no long-term protection. PMID:17210148

  16. Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.

    PubMed

    Jin, Hongfan; Cantin, Greg T; Maki, Steven; Chew, Lawrence C; Resnick, Sol M; Ngai, Jerry; Retallack, Diane M

    2011-07-01

    Cost-effective production of soluble recombinant protein in a bacterial system remains problematic with respect to expression levels and quality of the expressed target protein. These constraints have particular meaning today as "biosimilar" versions of innovator protein drugs are entering the clinic and the marketplace. A high throughput, parallel processing approach to expression strain engineering was used to evaluate soluble expression of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens. The human g-csf gene was optimized for expression in P. fluorescens and cloned into a set of periplasmic expression vectors. These plasmids were transformed into a variety of P. fluorescens host strains each having a unique phenotype, to evaluate soluble expression in a 96-well growth and protein expression format. To identify a strain producing high levels of intact, soluble Met-G-CSF product, more than 150 protease defective host strains from the Pfēnex Expression Technology™ toolbox were screened in parallel using biolayer interferometry (BLI) to quantify active G-CSF binding to its receptor. A subset of these strains was screened by LC-MS analysis to assess the quality of the expressed G-CSF protein. A single strain with an antibiotic resistance marker insertion in the pfaI gene was identified that produced>99% Met-GCSF. A host with a complete deletion of the autotransporter-coding gene pfaI from the genome was constructed, and expression of soluble, active Met-GSCF in this strain was observed to be 350mg/L at the 1 liter fermentation scale. PMID:21396452

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

    PubMed

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

    1990-06-01

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

  18. Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

    PubMed

    Carreira, Bruno Pereira; Morte, Maria Inês; Inácio, Angela; Costa, Gabriel; Rosmaninho-Salgado, Joana; Agasse, Fabienne; Carmo, Anália; Couceiro, Patrícia; Brundin, Patrik; Ambrósio, António Francisco; Carvalho, Caetana Monteiro; Araújo, Inês Maria

    2010-07-01

    Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 microM) increased cell proliferation, whereas higher concentrations (100 microM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. PMID:20506358

  19. Effects of Granulocyte Colony-Stimulating Factor on Patients with Liver Failure: a Meta-Analysis

    PubMed Central

    Yang, Qiao; Yang, Ying; Shi, Yu; Lv, Fangfang; He, Jiliang; Chen, Zhi

    2016-01-01

    Abstract Background and Aims: It remains controversial whether granulocyte colony-stimulating factor (G-CSF) prolongs survival in liver failure (LF) patients. This meta-analysis was performed to evaluate the effect of G-CSF on patients with LF. Methods: PubMed, EMBASE, and Web of Science databases were searched to identify English language randomized controlled trials comparing G-CSF with control therapy published before14 February 2015. A meta-analysis was performed to examine changes in liver function and patient survival. The association was tested using odds ratio (OR) or risk ratio (RR) with 95% confidence intervals (CI). Results: Five randomized controlled trials were eligible for the meta-analysis. Significant amelioration of prothrombin time and total bilirubin in LF patients was attributed to G-CSF therapy (OR, −0.064; 95% CI,−0.481 to 0.353; p< 0.001; and OR, −0.803; 95% CI, −1.177 to −0.430; p = 0.000, respectively). Treatment with G-CSF resulted in improved Model for End-Stage Liver Disease and Child-Turcotte-Pugh scores (OR, −1.741; 95% CI, −2.234 to −1.250; p = 0.000; and OR, −0.830, 95% CI, −1.194 to −0.465; p = 0.000, respectively). A lower incidence of sepsis was found in patients treated with G-CSF (RR, 0.367; 95% CI, 0.158 to 0.854; p = 0.020). G-CSF therapy significantly increased survival rate in LF patients (RR, 2.25; 95% CI, 1.517 to 3.338; p = 0.000). Conclusions: The results of this meta-analysis indicate that G-CSF treatment in patients with LF significantly improved liver function, reduced the incidence of sepsis, and prolonged short-term survival. PMID:27350939

  20. Granulocyte/Macrophage Colony-stimulating Factor-dependent Dendritic Cells Restrain Lean Adipose Tissue Expansion*

    PubMed Central

    Pamir, Nathalie; Liu, Ning-Chun; Irwin, Angela; Becker, Lev; Peng, YuFeng; Ronsein, Graziella E.; Bornfeldt, Karin E.; Duffield, Jeremy S.; Heinecke, Jay W.

    2015-01-01

    The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2−/−) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2−/− mice had 75% fewer CD45+Cd11b+Cd11c+MHCII+ F4/80− DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2−/− mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2−/− mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45+Cd11b+Cd11c+MHCII+F4/80− DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion. PMID:25931125

  1. Granulocyte colony stimulating factor attenuates inflammation in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    2011-01-01

    Background Granulocyte colony stimulating factor (GCSF) is protective in animal models of various neurodegenerative diseases. We investigated whether pegfilgrastim, GCSF with sustained action, is protective in a mouse model of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease with manifestations of upper and lower motoneuron death and muscle atrophy accompanied by inflammation in the CNS and periphery. Methods Human mutant G93A superoxide dismutase (SOD1) ALS mice were treated with pegfilgrastim starting at the presymptomatic stage and continued until the end stage. After long-term pegfilgrastim treatment, the inflammation status was defined in the spinal cord and peripheral tissues including hematopoietic organs and muscle. The effect of GCSF on spinal cord neuron survival and microglia, bone marrow and spleen monocyte activation was assessed in vitro. Results Long-term pegfilgrastim treatment prolonged mutant SOD1 mice survival and attenuated both astro- and microgliosis in the spinal cord. Pegfilgrastim in SOD1 mice modulated the inflammatory cell populations in the bone marrow and spleen and reduced the production of pro-inflammatory cytokine in monocytes and microglia. The mobilization of hematopoietic stem cells into the circulation was restored back to basal level after long-term pegfilgrastim treatment in SOD1 mice while the storage of Ly6C expressing monocytes in the bone marrow and spleen remained elevated. After pegfilgrastim treatment, an increased proportion of these cells in the degenerative muscle was detected at the end stage of ALS. Conclusions GCSF attenuated inflammation in the CNS and the periphery in a mouse model of ALS and thereby delayed the progression of the disease. This mechanism of action targeting inflammation provides a new perspective of the usage of GCSF in the treatment of ALS. PMID:21711557

  2. 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

  3. 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

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

    PubMed Central

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

    2015-01-01

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

  5. Neurotoxic factors released by stimulated human monocytes and THP-1 cells.

    PubMed

    Lee, Moonhee; Suk, Kyoungho; Kang, Yunhee; McGeer, Edith; McGeer, Patrick L

    2011-07-11

    Activated monocytes/macrophages are known to release toxic materials. Identification of these materials is important for developing more effective treatments for inflammatory disorders where self attack occurs. We stimulated human monocytes and THP-1 cells with LPS/IFNγ and measured the toxic effects of their conditioned media against differentiated human NT-2 cells. Their cytotoxicity, as measured by LDH release, was reduced by half when their conditioned media was passed through a 3kDa cutoff filter, indicating an equal division between high and low molecular weight materials. When the high molecular weight components tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and IL-6 were removed from the conditioned medium by specific antibodies, the toxicity was reduced by 37-38%. When prostaglandin production was blocked by treatment with the COX inhibitors acetylsalicylic acid and ibuprofen, toxicity was reduced by 15-16%. When oxygen free radical production was blocked by the NADPH inhibitor diphenylene iodonium (DPI) the toxicity was reduced by 17-18%. Treatment with the nitric oxide scavenger carboxy-phenyl-tetramethylimidazolineoxyl-oxide, or the NOS inhibitor N(G)-monomethylene-l-arginine, attenuated the toxicity by about 20%. Removal of released glutamate by glutamate decarboxylase also attenuated the toxicity by 12-13%. In combination, these treatments reduced the toxicity by approximately 50% accounting for the low molecular weight component toxicity. About 10% of the overall toxicity, which was associated with the high molecular weight component, was not identified. Optimal antiinflammatory therapy may require combined suppression of these identified toxin-generating pathways as well as relatively minor pathways yet to be identified. PMID:21640980

  6. 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.

  7. E-cadherin Surface Levels in Epithelial Growth Factor-stimulated Cells Depend on Adherens Junction Protein Shrew-1

    PubMed Central

    Gross, Julia Christina; Schreiner, Alexander; Engels, Knut

    2009-01-01

    Gain- and loss-of-function studies indicate that the adherens junction protein shrew-1 acts as a novel modulator of E-cadherin internalization induced by epithelial growth factor (EGF) or E-cadherin function-blocking antibody during epithelial cell dynamics. Knocking down shrew-1 in MCF-7 carcinoma cells preserves E-cadherin surface levels upon EGF stimulation. Overexpression of shrew-1 leads to preformation of an E-cadherin/EGF receptor (EGFR) HER2/src-kinase/shrew-1 signaling complex and accelerated E-cadherin internalization. Shrew-1 is not sufficient to stimulate E-cadherin internalization, but facilitates the actions of EGFR and thus may promote malignant progression in breast cancer cells with constitutive EGFR stimulation by reducing surface E-cadherin expression. PMID:19515834

  8. mTOR Complexes Repress Hypertrophic Agonist-Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells.

    PubMed

    Sundararaj, Kamala; Pleasant, Dorea L; Moschella, Phillip C; Panneerselvam, Kavin; Balasubramanian, Sundaravadivel; Kuppuswamy, Dhandapani

    2016-02-01

    Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium. PMID:26371948

  9. Early Systemic Granulocyte-Colony Stimulating Factor Treatment Attenuates Neuropathic Pain after Peripheral Nerve Injury

    PubMed Central

    Lee, Yun-Lin; Chen, Jin-Chung; Wang, Hung-Li; Yang, Yi-Ling; Cheng, Mei-Yun; Liao, Ming-Feng; Ro, Long-Sun

    2012-01-01

    Recent studies have shown that opioid treatment can reduce pro-inflammatory cytokine production and counteract various neuropathic pain syndromes. Granulocyte colony-stimulating factor (G-CSF) can promote immune cell differentiation by increasing leukocytes (mainly opioid-containing polymorphonuclear (PMN) cells), suggesting a potential beneficial role in treating chronic pain. This study shows the effectiveness of exogenous G-CSF treatment (200 µg/kg) for alleviating thermal hyperalgesia and mechanical allodynia in rats with chronic constriction injury (CCI), during post-operative days 1–25, compared to that of vehicle treatment. G-CSF also increases the recruitment of opioid-containing PMN cells into the injured nerve. After CCI, single administration of G-CSF on days 0, 1, and 2, but not on day 3, relieved thermal hyperalgesia, which indicated that its effect on neuropathic pain had a therapeutic window of 0–48 h after nerve injury. CCI led to an increase in the levels of interleukin-6 (IL-6) mRNA and tumor necrosis factor-α (TNF-α) protein in the dorsal root ganglia (DRG). These high levels of IL-6 mRNA and TNF-α were suppressed by a single administration of G-CSF 48–144 h and 72–144 h after CCI, respectively. Furthermore, G-CSF administered 72–144 h after CCI suppressed the CCI-induced upregulation of microglial activation in the ipsilateral spinal dorsal horn, which is essential for sensing neuropathic pain. Moreover, the opioid receptor antagonist naloxone methiodide (NLXM) reversed G-CSF-induced antinociception 3 days after CCI, suggesting that G-CSF alleviates hyperalgesia via opioid/opioid receptor interactions. These results suggest that an early single systemic injection of G-CSF alleviates neuropathic pain via activation of PMN cell-derived endogenous opioid secretion to activate opioid receptors in the injured nerve, downregulate IL-6 and TNF-α inflammatory cytokines, and attenuate microglial activation in the spinal dorsal horn. This

  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. Adjuvant granulocyte colony-stimulating factor therapy results in improved spatial learning and stimulates hippocampal neurogenesis in a mouse model of pneumococcal meningitis.

    PubMed

    Schmidt, Anna Kathrin; Reich, Arno; Falkenburger, Björn; Schulz, Jörg B; Brandenburg, Lars Ove; Ribes, Sandra; Tauber, Simone C

    2015-01-01

    Despite the development of new antibiotic agents, mortality of pneumococcal meningitis remains high. In addition, meningitis results in severe long-term morbidity, most prominently cognitive deficits. Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic progenitor cells and increases the number of circulating neutrophil granulocytes. This study investigated the effect of adjuvant G-CSF treatment on cognitive function after pneumococcal meningitis. C57BL/6 mice were infected by subarachnoid injection of Streptococcus pneumoniae serotype 3 and treated with ceftriaxone and G-CSF subcutaneously or ceftriaxone alone for 5 days. Clinical scores, motor performance, and mortality during bacterial meningitis were unaffected by adjuvant G-CSF treatment. No effect of G-CSF treatment on production of proinflammatory cytokines or activation of microglia or astrocytes was observed. The G-CSF treatment did, however, result in hippocampal neurogenesis and improved spatial learning performance 6 weeks after meningitis. These results suggest that G-CSF might offer a new adjuvant therapeutic approach in bacterial meningitis to reduce long-term cognitive deficits. PMID:25470346

  12. 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

  13. 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

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

    PubMed

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

    1995-10-01

    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. PMID:7575481

  15. I. Evidence that lymphotoxin activity involves both cytotoxic and stimulating factors.

    PubMed

    Ryzewska, A G; Darbrowska, B K

    1976-11-01

    This paper describes experiments undertaken to determine more uniform conditions for the generation of cytotoxic lymphokine--'lymphotoxin' (LT) in differently stimulated rat lymphocyte cultures, and to compare the sensitivity of different test systems for detecting rat LT activity in vitro. Wistar rat lymphoid cells were activated by culture with phytohaemagglutinin (PHA) and mixed lymphocyte cultures (MLC) with semi-allogeneic (Wistar x August) F1 lymphoid cells. Lymphocyte supernatants harvested between 1 and 8 days were tested for their effect on metabolism and viability of cultured mouse fibroblasts (L-929 cells) by four methods: (i) inhibition of [14C]leucine incorporation; (ii) total and viable cell counts in test tube cultures ('macro test'); (iii) viable cell counts in microtitre plates ('microtest'); and (iv) chromium (51Cr) release from chromated L-cell monolayers. Cytotoxic effects on target cells of lymphocyte supernatants were evident after 3 days of PHA stimulation and 6 days of mixed lymphocyte culture, and the most sensitive indication of cytotoxic activity provided by inhibition of amino-acid incorporation and by loss of viable L cells from monolayers in tube cultures. In dilutions greater than 1:16-1:32 both cytotoxic supernatants exhibited a stimulating effect on target-cell proliferation. Stimulation of L cells growth was also observed when monolayers were exposed for 24 h to 'early' (24 h) PHA undiluted supernatants. At a later time of exposure to these supernatants a considerable loss of total and viable cells in the monolayers was evident. The results indicated that both cytotoxic and growth-stimulating lymphokines could be generated during activation of rat lymphocytes. A hypothesis is suggested whereby 'lymphotoxin' activity in vitro arises from the sequential effects of stimulating and cytotoxic lymphokine, and whereby the balance of these effects in vivo might determine the response of fibroblasts involved in reactions of chronic allergic

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

    PubMed

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

    2013-10-01

    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

  17. Over-the-counter drugs: factors in adult use of sedatives, tranquilizers, and stimulants.

    PubMed Central

    Bell, R

    1984-01-01

    Despite a growing research interest in over-the-counter (OTC) drug use, little information has been available about the determinants of use for this category of medications. The researcher examined the effects of demographic, need, and physician utilization measures on the use of 10 OTC drugs that were categorized as sedatives, tranquilizers, or stimulants. A statewide survey in 1975 of drug-using behavior in the previous year by Illinois adults ages 18-59 resulted in 2,738 questionnaires that could be analyzed. Thirteen variables, representing the demographic, need, and physician utilization characteristics of the respondents, were entered as predictors into logistic multiple regression models to estimate their effects on drug use. Only 10.37 percent of the respondents indicated that they had used any of the OTC drugs in the previous year. Sedative use was found to be increased in persons who were tense or were having trouble sleeping. Having trouble sleeping also increased the probability of using OTC tranquilizers and stimulants. Women had a much higher probability of using OTC tranquilizers than men, and men had a higher probability of using stimulants. Non-whites had a higher probability of using tranquilizers than did whites. Stimulants were more likely to be used by younger adults and unmarried adults. Physician utilization, measured by the number of visits to physicians, did not significantly affect OTC drug use. PMID:6429733

  18. 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

  19. 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.

  20. Zinc deprivation impairs growth factor-stimulated calcium influx into murine 3T3 cells associated with decreased cell proliferation.

    PubMed

    O'Dell, Boyd L; Browning, Jimmy D

    2011-06-01

    Zinc plays a critical role in growth, a process that depends primarily on cell proliferation. Murine fibroblasts, Swiss 3T3 cells, were used to explore the hypothesis that a critical role of zinc in cell proliferation relates to its function in calcium influx. Cells were deprived of zinc by an impermeant chelator, diethylenetriaminepentaacetate (0.6 mmol/L), and low-calcium status was achieved by using a low- (<5 μmol/L) calcium medium. Cells were stimulated by a composite of growth factors (GF): platelet-derived GF, insulin-like GF-I, and epidermal GF. GF stimulation of cell proliferation was assessed by the incorporation of tritiated thymidine and calcium influx by the increase in fluorescence of cells loaded with Fluo-4. Proliferation was dependent on both zinc and calcium and they interacted in this process. GF stimulated an immediate sharp increase in intracellular calcium, indicative of internal calcium release, which peaked within 1 min and decreased to an elevated plateau, a pattern typical of a store-operated calcium channel. The sustained calcium influx of zinc-deprived cells was markedly lower than that of supplemented cells. Verapamil, a calcium channel blocker, also depressed both cell proliferation and calcium influx. In summary, zinc deficiency impaired GF-stimulated calcium influx into murine fibroblasts in association with decreased cell proliferation. PMID:21508206

  1. 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

  2. Activity of matrix metalloproteinases 2 and 9 in cultured rabbit corneal epithelium cells stimulated by tumor necrosis factor alpha.

    PubMed

    Wu, Z-Q; Zhang, Z-L; Nie, S-W; Yuan, J; Yang, Y-N

    2015-01-01

    We studied the activity of matrix metalloproteinases (MMP) 2 and 9 generated by cultured rabbit corneal epithelium cells that had been stimulated with tumor necrosis factor alpha (TNF-α), to investigate the possible regulative mechanisms of MMP-2/9 and their potential effect on corneal inflammatory diseases. The rabbit corneal epithelium cells were cultured in vitro and incubated with different concentrations of TNF-α (0, 1, 10, and 100 ng/mL) for 24 h. The activity of MMP-2/9 was examined using gelatin zymography. The results were analyzed by computer image analysis and statistical tests. TNF-α stimulated the secretion of MMP-2/9 in a dose-dependent manner, and MMP-2 was activated by TNF-α. Inflammatory factors such as TNF-α can stimulate MMP-2/9 activity in corneal epithelium cells. This may be a potential manipulating mechanism of MMP expression in the pathogenesis of corneal diseases, and could play an important role in the prevention and treatment of corneal inflammatory diseases. PMID:26125840

  3. Production of IgM rheumatoid factor by normal lymphocytes after stimulation with preparations containing IgM rheumatoid factor from patients with juvenile arthritis.

    PubMed Central

    Nesher, G; Moore, T L; Osborn, T G; Dorner, R W

    1991-01-01

    Preparations containing IgM rheumatoid factor (RF) and hidden IgM RF were isolated from the serum samples of nine patients with juvenile rheumatoid arthritis. Six of these preparations stimulated lymphocytes from normal donors to produce IgG and IgM, of which up to 11% had IgM RF activity. In contrast, the polyclonal activator pokeweed mitogen also stimulated IgM production, but only 1% had IgM RF activity. A relation between the activator and IgM RF or hidden IgM RF is suggested. This is based on the positive correlation between IgM RF concentration in these preparations and their ability to stimulate lymphocytes to produce IgG, IgM, and IgM RF. These data indicate that preparations from patients with juvenile rheumatoid arthritis containing IgM RF and hidden IgM RF are potent stimulants of lymphocytes from normal donors, with specific production of IgM RF. PMID:2015007

  4. 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.

  5. Ovarian stimulation medications and patients’ responses as prognostic factors in IUI-treated infertile Saudi patients

    PubMed Central

    M. Isa, Ahmed; Abu-Rafea, Basim; Al-Asiri, Sahel; Al-Motawa, Johara; Almady, Khalid; Alwaznah, Raheek

    2014-01-01

    Background: Intrauterine Insemination (IUI) remains the first thought of infertility treatment. Objective: To compare the stimulation effects and Pregnancy rate (PR) outcomes of two ovulation induction (OI) medications, human-derived menopausal gonadotrophins (hMGH), Merional (MER), and recombinant follicular stimulating hormone (rFSH), Puregon (PUR), in a cohort of Saudi infertile patients, for better predictability of treatment results. Materials and Methods: During a 24-month period, 296 women underwent IUI single treatments. PR’s were correlated with the type of stimulation medication that were prospectively and randomly assigned to each patient, and with the number and size of maturing follicles detected on the hCG injection day. Results: MER and PUR needed comparable number of days (9.26±4.74 and 9.73±6.27 respectively) before follicles were ready for IUI, although the average amount used from MER, 1199.90 IU, was about double that was used from PUR, 621.08 IU. The overall PR in case of PUR however was nearly double that of MER, 13.28% and 7.14% respectively. The best PR, 16.22%, occurred when the follicles matured within 12-13 days. Three follicles of at least 15-mm diameter on the hCG day had better PR’s than one or two, however when the follicles’ diameters were at least 18-mm, PR was significantly higher, (p=0.013). Conclusion: MER and PUR had comparable stimulation effects; however PUR had noticeably higher PR. The best PR occurred when the follicles matured within 12-13 days. PR in case of three maturing follicles on the hCG day was better than only one or two, and significantly better when their diameters were at least 18 mm. PMID:25114672

  6. Kunbi-Boshin-Hangam-Tang stimulates nitic oxide production through activation of nuclear factor-kappaB.

    PubMed

    Koo, H N; Jeong, H J; Park, J H; Moon, G; Chae, H J; Kim, H R; Kim, C H; Seo, S B; An, N H; Kim, H M

    2001-05-01

    The objective of the currently study was to determine the effect of Kunbi-Boshin-Hangam-Tang (KBH-Tang) on the production of nitric oxide (NO). Stimulation of RAW 264.7 cells with KBH-Tang after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in increased NO synthesis. KBH-Tang partially increased NO synthesis by itself. When KBH-Tang was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) protein. NO production was inhibited by NG-monomethyl-L-arginine (NGMMA). Furthermore, activation of nuclear factor (NF)-kappaB was increased by KBH-Tang. These results suggest that KBH-Tang may stimulate the NO production through the activation of the NF-kappaB. PMID:11417846

  7. Modulation of basal and corticotropin-releasing factor-stimulated proopiomelanocortin gene expression by vasopressin in rat anterior pituitary.

    PubMed

    Levin, N; Blum, M; Roberts, J L

    1989-12-01

    In several anterior pituitary hormone systems, factors that stimulate hormone release also stimulate hormone biosynthesis. In the corticotropes of the anterior pituitary, CRF stimulates ACTH release as well as cAMP accumulation and transcription of the POMC gene. Arginine vasopressin (AVP) is a well documented coregulator of ACTH release and potentiates CRF-stimulated cAMP accumulation. The present studies were undertaken to determine if AVP also potentiates the early effects of CRF on POMC gene expression in rat anterior pituitary primary cultures. We have measured the levels of the POMC primary transcript, processing intermediate, and mature mRNA in nuclear RNA samples and POMC mRNA in cytoplasmic RNA samples as well as ACTH release after treatment with CRF and/or AVP. After a 30-min treatment with 0.5 nM CRF, POMC primary transcript levels were increased by 200-400%. Thirty-minute or 1-h treatments with AVP alone (10 or 100 nM) did not affect primary transcript levels, but increased the amount of the processing intermediate, while a 2-h incubation with 100 nM AVP significantly decreased POMC primary transcript levels. The effects of CRF on the POMC primary transcript and nuclear processing intermediate were not potentiated by cotreatment with AVP, although a potentiation of CRF-stimulated ACTH release was observed. POMC nuclear and cytoplasmic mRNA levels were not affected by these 2-h or less treatments, while an 18-h incubation with 0.5 nM CRF alone or in combination with 100 nM AVP increased POMC cytoplasmic mRNA levels to about 140% of vehicle-treated control values. When a 1-h AVP treatment (100 nM) preceded presentation of CRF, we observed an attenuation of the stimulation of POMC primary transcript and processing intermediate levels by the 1-h CRF treatment. The effects of CRF on POMC primary transcript and processing intermediate levels in these experiments are consistent with a CRF-induced increase in POMC gene transcription. Our findings that AVP

  8. Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

    PubMed Central

    Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

    2000-01-01

    Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640

  9. Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells

    SciTech Connect

    Cannistra, S.A.; Groshek, P.; Griffin, J.D. ); Garlick, R.; Miller, J. )

    1990-01-01

    Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors. By using radiolabeled GM-CSF with high specific activity, the authors have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, they also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

  10. 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

  11. 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

  12. Circadian variation of tissue plasminogen activator and its inhibitor, von Willebrand factor antigen, and prostacyclin stimulating factor in men with ischaemic heart disease.

    PubMed Central

    Bridges, A B; McLaren, M; Scott, N A; Pringle, T H; McNeill, G P; Belch, J J

    1993-01-01

    OBJECTIVES--To determine whether plasma concentrations of tissue plasminogen activator antigen, von Willebrand factor antigen, and prostacyclin stimulating factor and plasminogen activator inhibitor activity show circadian variation in men with ischaemic heart disease. DESIGN--Blood samples were obtained every four hours for 24 hours from 10 men with ischaemic heart disease. The men were ambulant from 08:10 until 00:00 when they went to bed and they remained in bed until 08:00 the following morning. PATIENTS--Ten men with positive diagnostic exercise tolerance tests with no significant past history, who were not regularly taking any medical treatment except for glyceryl trinitrate. RESULTS--There was significant circadian variation in plasminogen activator inhibitor activity (p = 0.001) (peak value 04:00 and trough value 20:00), but not in plasma concentrations of tissue plasminogen activator antigen, von Willebrand factor, or prostacyclin stimulating factor. CONCLUSION--Men with ischaemic heart disease showed a significant circadian variation in fibrinolysis. The combination of peak values of plasminogen activator inhibitor activity and failure of plasma concentrations of tissue plasminogen activator antigen to increase in the early morning must predispose to thrombosis at this time. The circadian variation in fibrinolysis may contribute to the increased incidence of myocardial infarction in the morning. PMID:8435236

  13. Insulin-like growth factor I stimulates telomerase activity in prostate cancer cells.

    PubMed

    Wetterau, Lawrence A; Francis, Malik J; Ma, Liqun; Cohen, Pinchas

    2003-07-01

    IGF-I has been implicated in the pathogenesis of human cancer. We sought to establish a role for IGF-I in the regulation of telomerase, an enzyme critically involved in cancer cell immortalization. Telomerase activity was assayed in LAPC-4, PC-3, and DU-145 prostate cancer cell lines treated with and without IGF-I/IGF-I analogs. Relative expression of human telomerase reverse transcriptase (hTERT) mRNA and protein was determined by quantitative RT-PCR and Western immunoblot, respectively. IGF-I stimulated baseline telomerase activity in all three cell lines, ranging from 2- to 10-fold (P < 0.05). Enhancement was noted at IGF concentrations as low as 10 ng/ml and was maximal at 100 ng/ml. Stimulation was noted by 0.5 h, was maximal by 8 h, and persisted to 48 h. A similar 3-fold enhancement (P < 0.01) was noted in response to Long-R3 IGF-I, but not in response to [Ala(31),Leu(60)]IGF-I. Pretreatment with the Akt kinase inhibitor wortmannin abolished the stimulatory IGF effect, whereas blockade of MAPK activity did not. Lastly, IGF-I provoked a 2-fold increase in hTERT mRNA and protein expression (P < 0.01). In summary, IGF-I clearly stimulates telomerase activity in prostate cancer cells through a dual mode of action, including early rapid effects probably involving phosphorylation of hTERT by Akt and later up-regulation of hTERT expression. PMID:12843187

  14. Restoration of nucleic acid biosynthesis after clinical death and factors stimulating the process in vivo.

    PubMed

    Konikova, A S; Petukhova, L M; Pogossova, A V; Vinarskaya, A A; Nikulin, V I

    1975-01-01

    The biosynthesis of RNA and DNA falls almost to zero in 60 min after the death of rabbits from anoxia, in all the organs of the body. Rapid artificial cooling of the rabbits to 20 degrees C undertaken within 10 min after death preserved nucleic acid biosynthesis and permitted restoration of life 3-4 h after death, with recovery beginning in 60 min. During the reanimation the addition of ATP to the blood stimulated the restoration of RNA biosynthesis in the spinal cord to a considerable extent; the addition of cocarboxylase to the blood promoted cardiac RNA biosynthesis as well as cardiac and pancreatic DNA biosynthesis during recovery. PMID:1197938

  15. Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice.

    PubMed

    Zhang, D E; Zhang, P; Wang, N D; Hetherington, C J; Darlington, G J; Tenen, D G

    1997-01-21

    Transcription factors are master regulatory switches of differentiation, including the development of specific hematopoietic lineages from stem cells. Here we show that mice with targeted disruption of the CCAAT enhancer binding protein alpha gene (C/EBP alpha) demonstrate a selective block in differentiation of neutrophils. Mature neutrophils and eosinophils are not observed in the blood or fetal liver of mutant animals, while other hematopoietic lineages, including monocytes, are not affected. Instead, most of the white cells in the peripheral blood of mutant mice had the appearance of myeloid blasts. We also observed a selective loss of expression of a critical gene target of CCAAT enhancer binding protein alpha, the granulocyte colony-stimulating factor receptor. As a result, multipotential myeloid progenitors from the mutant fetal liver are unable to respond to granulocyte colony-stimulating factor signaling, although they are capable of forming granulocyte-macrophage and macrophage colonies in methylcellulose in response to other growth factors. Finally, we demonstrate that the lack of granulocyte development results from a defect intrinsic to the hematopoietic system; transplanted fetal liver from mutant mice can reconstitute lymphoid but not neutrophilic cells in irradiated recipients. These studies suggest a model by which transcription factors can direct the differentiation of multipotential precursors through activation of expression of a specific growth factor receptor, allowing proliferation and differentiation in response to a specific extracellular signal. In addition, the c/ebp alpha -/- mice may be useful in understanding the mechanisms involved in acute myelogenous leukemia, in which a block in differentiation of myeloid precursors is a key feature of the disease. PMID:9012825

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

    PubMed

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

    2016-03-01

    Therapeutic use of activated platelet-rich plasma (PRP) has been explored for wound healing, hemostasis and antimicrobial wound applications. Pulse electric field (PEF) stimulation may provide more consistent platelet activation and avoid complications associated with the addition of bovine thrombin, the current state of the art ex vivo activator of therapeutic PRP. The aim of this study was to compare the ability of PEF, bovine thrombin and thrombin receptor activating peptide (TRAP) to activate human PRP, release growth factors and induce cell proliferation in vitro. Human PRP was prepared in the Harvest SmartPreP2 System and treated with vehicle, PEF, bovine thrombin, TRAP or Triton X-100. Platelet activation and procoagulant markers and microparticle generation were measured by flow cytometry. Released growth factors were measured by ELISA. The releasates were tested for their ability to stimulate proliferation of human epithelial cells in culture. PEF produced more platelet-derived microparticles, P-selectin-positive particles and procoagulant annexin V-positive particles than bovine thrombin or TRAP. These differences were associated with higher levels of released epidermal growth factor after PEF than after bovine thrombin or TRAP but similar levels of platelet-derived, vascular-endothelial, and basic fibroblast growth factors, and platelet factor 4. Supernatant from PEF-treated platelets significantly increased cell proliferation compared to plasma. In conclusion, PEF treatment of fresh PRP results in generation of microparticles, exposure of prothrombotic platelet surfaces, differential release of growth factors compared to bovine thrombin and TRAP and significant cell proliferation. These results, together with PEF's inherent advantages, suggest that PEF may be a superior alternative to bovine thrombin activation of PRP for therapeutic applications. PMID:26030682

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

    PubMed Central

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

    2016-01-01

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

  18. Expression of human granulocyte colony stimulating factor (hG-CSF) in colon adenocarcinoma cell line (Caco-2).

    PubMed

    Jana, Snehasis; Patel, Hitesh

    2012-10-01

    Growth and progression of many cancer cells are mediated by alterations in the microenvironment often caused by an aberrant expression of growth factors and receptors. There is no report on expression of growth factor granulocyte colony-stimulating factor (G-CSF) in the experimental model, colon adenocarcinoma cell line (Caco2), that is commonly used in drug permeability assays. We hypothesize that in vitro, the Caco2 model is associated with a constitutive neo-expression of the hematopoietic G-CSF thereby causing an autocrine stimulation of Caco2 growth and proliferation in vitro. To test our hypothesis, we analyzed mRNA and protein expression of G-CSF in Caco2 cells using reverse transcriptase-PCR and SDS-PAGE. G-CSF mRNA and protein were detected in Caco2 cells. Expression of G-CSF protein was similar at different passages of this cell line. The expression of G-CSF has a significant role in the autocrine regulation of Caco2 cell growth and proliferation. PMID:22714276

  19. 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

  20. Successful Granulocyte Colony-stimulating Factor Treatment of Relapsing Candida albicans Meningoencephalitis Caused by CARD9 Deficiency.

    PubMed

    Celmeli, Fatih; Oztoprak, Nefise; Turkkahraman, Doga; Seyman, Derya; Mutlu, Esvet; Frede, Natalie; Köksoy, Sadi; Grimbacher, Bodo

    2016-04-01

    Caspase-associated recruitment domain-9 (CARD9) deficiency is an autosomal-recessive primary immunodeficiency with genetic defects in Th17 immunity marked by susceptibility to recurrent and invasive Candida infections. We present a case of relapsing Candida albicans meningoencephalitis over 1-year period despite appropriate antifungal therapy. We detected a homozygous p.Q295X mutation in CARD9 as well as a defective interleukin-17 and interferon gamma synthesis in Enzyme-Linked ImmunoSpot tests. We achieved complete clinical remission, and improvement of interleukin-17 secretion with subcutaneous granulocyte colony-stimulating factor) treatment. PMID:26658378

  1. Insulin and insulin-like growth factors (IGFs) stimulate production of IGF-binding proteins by ovarian granulosa cells.

    PubMed

    Grimes, R W; Hammond, J M

    1992-08-01

    Ligand blot analysis of granulosa cell (GC)-conditioned culture medium revealed several easily measurable insulin-like growth factor (IGF)-binding proteins (IGFBPs), including IGFBP-3 [40-44 kilodaltons (kDa)] and IGFBP-2 (34 kDa). In the present study, IGF-I, in a dose-dependent manner, significantly stimulated the production of these IGFBPs. Insulin, but not IGF-II, mimicked IGF-I's action on IGFBP-3 and -2 production, but was less potent. The synthetic IGF, long R3-IGF-I, which has very low affinity for IGFBPs and only slightly reduced affinity for the IGF-I (type I) receptor, had significantly greater potency in stimulating IGFBP-3 and -2 production compared to IGF-I. Des-(1-3)-IGF-I had similar effects. IGF-I, IGF-II, and the IGF-I analogs, but not insulin, also induced production of an unidentified 30-kDa IGFBP not normally detectable in these cultures. However, in the presence of epidermal growth factor (which was without independent effect on the 30-kDa IGFBP), insulin also induced this 30-kDa IGFBP. By Northern analysis the expression of IGFBP-3 mRNA was found to be significantly stimulated by IGF-I. In summary, insulin stimulated IGFBP-3 and -2 production in a manner that mimics that of IGF-I and the more potent long R3-IGF-I. However, its low potency suggested that IGFBP production is regulated via the IGF-I (type I) receptor. The much higher potency of long R3-IGF-I compared to that of IGF-I suggests that the IGFBPs themselves modulate the action of IGFs by sequestering exogenous IGFs. Thus, one cellular response to IGF stimulation is the production of IGFBPs, which, in turn, reduce or negate the biological activity of the IGFs. The effects of insulin-like peptides are exerted at least in part by increasing levels of mRNA for specific BPs. PMID:1379161

  2. 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.

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

    SciTech Connect

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

    1987-07-01

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

  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. Combined Stimulation with the Tumor Necrosis Factor α and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices.

    PubMed

    Finot, Francis; Masson, Régis; Desmots, Fabienne; Ribault, Catherine; Bichet, Nicole; Vericat, Joan A; Lafouge, Patricia; Guguen-Guillouzo, Christiane; Loyer, Pascal

    2012-01-01

    The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration. PMID:23119170

  6. Combined Stimulation with the Tumor Necrosis Factor α and the Epidermal Growth Factor Promotes the Proliferation of Hepatocytes in Rat Liver Cultured Slices

    PubMed Central

    Finot, Francis; Masson, Régis; Desmots, Fabienne; Ribault, Catherine; Bichet, Nicole; Vericat, Joan A.; Lafouge, Patricia; Guguen-Guillouzo, Christiane; Loyer, Pascal

    2012-01-01

    The culture liver slices are mainly used to investigate drug metabolism and xenobiotic-mediated liver injuries while apoptosis and proliferation remain unexplored in this culture model. Here, we show a transient increase in LDH release and caspase activities indicating an ischemic injury during the slicing procedure. Then, caspase activities decrease and remain low in cultured slices demonstrating a low level of apoptosis. The slicing procedure is also associated with the G0/G1 transition of hepatocytes demonstrated by the activation of stress and proliferation signalling pathways including the ERK1/2 and JNK1/2/3 MAPKinases and the transient upregulation of c-fos. The cells further progress up to mid-G1 phase as indicated by the sequential induction of c-myc and p53 mRNA levels after the slicing procedure and at 24 h of culture, respectively. The stimulation by epidermal growth factor induces the ERK1/2 phosphorylation but fails to activate expression of late G1 and S phase markers such as cyclin D1 and Cdk1 indicating that hepatocytes are arrested in mid-G1 phase of the cell cycle. However, we found that combined stimulation by the proinflammatory cytokine tumor necrosis factor α and the epidermal growth factor promotes the commitment to DNA replication as observed in vivo during the liver regeneration. PMID:23119170

  7. Treatment with recombinant granulocyte colony-stimulating factor (Filgrastin) stimulates neutrophils and tissue macrophages and induces an effective non-specific response against Mycobacterium avium in mice.

    PubMed Central

    Bermudez, L E; Petrofsky, M; Stevens, P

    1998-01-01

    A role of neutrophils in the host response against Mycobacterium avium (MAC) has recently been suggested. To investigate this matter further, we determined the effect of granulocyte colony-stimulating factor (G-CSF) on the outcome of MAC infection in mice. C57BL/6bg+/bg- black mice were intravenously infected with 1 x 10(7) MAC and then divided into four experimental groups to receive G-CSF as follows: (i) 10 micrograms/kg/day; (ii) 50 micrograms/kg/day; (iii) 100 micrograms/kg/day; (iv) placebo control. Mice were killed at 2 and 4 weeks of treatment to determine the bacterial load of liver and spleen. Treatment with G-CSF at both 10 and 50 micrograms/kg/day doses significantly decreased the number of viable bacteria in liver and spleen after 2 weeks (approximately 70.5% and 69.0%, respectively), and after 4 weeks (approximately 53% and 52%, respectively, P < 0.05 compared with placebo control). Treatment with 100 micrograms/kg/day did not result in decrease of bacterial colony-forming units in the liver and spleen after 4 weeks. Administration of G-CSF induced interleukin-10 (IL-10) and IL-12 production by splenocytes. To examine if the protective effect of G-CSF was accompanied by the activation of phagocytic cells, blood neutrophils and splenic macrophages were purified from mice receiving G-CSF and their ability to kill MAC was examined ex vivo. Neutrophils and macrophages from G-CSF-treated mice were able to inhibit the growth of or to kill MAC ex vivo, while phagocytic cells from untreated control mice had no anti-MAC effect. These results suggest that activation of neutrophils appears to induce an effective non-specific host defence against MAC, and further studies should aim for better understanding of the mechanisms of protection. Images Figure 3 Figure 4 Figure 5 PMID:9767410

  8. Expression and function of hematopoiesis-stimulating factor receptors on the GPI− and GPI+ hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria/aplastic anemia syndrome

    PubMed Central

    FU, RONG; DING, SHAO-XUE; LIU, YI; LI, LI-JUAN; LIU, HUI; WANG, HONG-LEI; ZHANG, TIAN; SHAO, ZONG-HONG

    2016-01-01

    Paroxysmal nocturnal hemoglobinuria/aplastic anemia (PNH/AA) syndrome presents a markedly increased population of cells deficient in glycophosphatidylinositol (GPI− cells) and signs of bone marrow failure, which requires treatment with hematopoiesis-stimulating factors, such as granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). However, little is known about the effects of these stimulating factors on GPI− cells. In order to explore the effects of stimulating factors in PNH/AA, G-CSF receptor (CD114) and SCF receptor (CD117) expression levels on GPI+ and GPI− hematopoietic stem cells (HSCs) were measured by flow cytometry (FCM). The mean fluorescence intensity (MFI) values of signal transducer and activator of transcription 5 (STAT5) and phosphorylated (P)-STAT5 were measured in GPI+ and GPI− HSCs by FCM following stimulation with G-CSF or SCF in vitro. The expression levels of CD114 and CD117 on GPI− HSCs were significantly lower (P<0.01) than those on GPI+ HSCs in PNH/AA patients and normal controls. The MFI values of STAT5 in the GPI− and GPI+ HSCs of PNH/AA patients and normal controls were not significantly different. However, the MFI values of P-STAT5 in the GPI− HSCs of PNH/AA patients were significantly lower than those in the GPI+ HSCs of PNH/AA patients and normal controls prior to and following stimulation with G-CSF or SCF (P<0.01). The GPI− HSCs of PNH/AA patients responded poorly to stimulation by hematopoiesis-stimulating factors, which indicates that these factors can be used safely in patients with PNH/AA. PMID:27168787

  9. Granulocyte colony-stimulating factor enhances bone tumor growth in mice in an osteoclast-dependent manner

    PubMed Central

    Hirbe, Angela C.; Uluçkan, Özge; Morgan, Elizabeth A.; Eagleton, Mark C.; Prior, Julie L.; Piwnica-Worms, David; Trinkaus, Kathryn; Apicelli, Anthony

    2007-01-01

    Inhibition of osteoclast (OC) activity has been associated with decreased tumor growth in bone in animal models. Increased recognition of factors that promote osteoclastic bone resorption in cancer patients led us to investigate whether increased OC activation could enhance tumor growth in bone. Granulocyte colony-stimulating factor (G-CSF) is used to treat chemotherapy-induced neutropenia, but is also associated with increased markers of OC activity and decreased bone mineral density (BMD). We used G-CSF as a tool to investigate the impact of increased OC activity on tumor growth in 2 murine osteolytic tumor models. An 8-day course of G-CSF alone (without chemotherapy) significantly decreased BMD and increased OC perimeter along bone in mice. Mice administered G-CSF alone demonstrated significantly increased tumor growth in bone as quantitated by in vivo bioluminescence imaging and histologic bone marrow tumor analysis. Short-term administration of AMD3100, a CXCR4 inhibitor that mobilizes neutrophils with little effect on bone resorption, did not lead to increased tumor burden. However, OC-defective osteoprotegerin transgenic (OPGTg) mice and bisphosphonate-treated mice were resistant to the effects of G-CSF administration upon bone tumor growth. These data demonstrate a G-CSF–induced stimulation of tumor growth in bone that is OC dependent. PMID:17192391

  10. 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

  11. Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis.

    PubMed Central

    Shanafelt, A B; Kastelein, R A

    1989-01-01

    Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule. Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene. The mutant proteins were expressed in Escherichia coli and assayed for biological activity. This procedure identified four regions critical to activity. These critical regions were further delineated by additional three-amino acid deletion mutants. Larger deletions at each terminus were also made, as well as changes of specific amino acid residues. The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115. The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity. The possible structural and/or functional roles of the critical regions are discussed. Images PMID:2662186

  12. 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.

  13. The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta.

    PubMed

    Kanzaki, H; Yui, J; Iwai, M; Imai, K; Kariya, M; Hatayama, H; Mori, T; Guilbert, L J; Wegmann, T G

    1992-04-01

    A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts. PMID:1522204

  14. 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

  15. Crystal Structure of Thermus Aquaticus Gfh1, a Gre-factor Paralog that Inhibits rather than Stimulates transcript Cleavage

    SciTech Connect

    Lamour,V.; Hogan, B.; Erie, D.; Darst, S.

    2006-01-01

    Transcription elongation in bacteria is promoted by Gre-factors, which stimulate an endogenous, endonucleolytic transcript cleavage activity of the RNA polymerase. A GreA paralog, Gfh1, present in Thermus aquaticus and Thermus thermophilus, has the opposite effect on elongation complexes, inhibiting rather than stimulating transcript cleavage. We have determined the 3.3 Angstroms-resolution X-ray crystal structure of T. aquaticus Gfh1. The structure reveals an N-terminal and a C-terminal domain with close structural similarity to the domains of GreA, but with an unexpected conformational change in terms of the orientation of the domains with respect to each other. However, structural and functional analysis suggests that when complexed with RNA polymerase, Gfh1 adopts a conformation similar to that of GreA. These results reveal considerable structural flexibility for Gfh1, and for Gre-factors in general, as suggested by structural modeling, and point to a possible role for the conformational switch in Gre-factor and Gfh1 regulation. The opposite functional effect of Gfh1 compared with GreA may be determined by three structural characteristics. First, Gfh1 lacks the basic patch present in Gre-factors that likely plays a role in anchoring the 3'-fragment of the backtracked RNA. Second, the loop at the tip of the N-terminal coiled-coil is highly flexible and contains extra acidic residues compared with GreA. Third, the N-terminal coiled-coil finger lacks a kink in the first a-helix, resulting in a straight coiled-coil compared with GreA. The latter two characteristics suggest that Gfh1 chelates a magnesium ion in the RNA polymerase active site (like GreA) but in a catalytically inactive configuration.

  16. Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.

    PubMed

    Collins, S J; Ulmer, J; Purton, L E; Darlington, G

    2001-10-15

    The transcription factor C/EBPalpha is an important mediator of granulocyte differentiation and regulates the expression of multiple granulocyte-specific genes including the granulocyte-colony-stimulating factor (G-CSF) receptor, neutrophil elastase, and myeloperoxidase. Indeed C/EBPalpha knockout mice display a profound block in granulocyte differentiation. To study this block in granulocytic differentiation in more detail, retroviral vector-mediated transduction of a dominant-negative retinoic acid receptor was used to establish hematopoietic growth factor-dependent, lympho-myeloid progenitor cell lines from the fetal livers of both the C/EBPalpha knockout animals (C/EBPalpha(-/-)) and their heterozygous littermates (C/EBPalpha(+/-)). Surprisingly, the C/EBPalpha(-/-) cell lines displayed significant spontaneous granulocytic differentiation, and this differentiation was markedly enhanced when the cells were stimulated with granulocyte macrophage (GM)-CSF. This GM-CSF-mediated differentiation was associated with the up-regulation of G-CSF receptor mRNA, and the combination of GM-CSF and G-CSF generated more than 95% mature neutrophils in the C/EBPalpha(-/-) cultures. The addition of all-trans retinoic acid also enhanced this granulocytic differentiation of the cultured C/EBPalpha(-/-) cells, indicating that the activated retinoic acid receptors can enhance granulocytic differentiation through a molecular pathway that is independent of C/EBPalpha. These studies clearly indicate that terminal granulocytic differentiation associated with the up-regulation of C/EBPalpha-responsive genes can occur in the absence of C/EBPalpha, and they indicate the existence of multiple independent molecular pathways potentially used by primitive hematopoietic precursors that can lead to the development of mature granulocytes. PMID:11588034

  17. 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

  18. 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. PMID:25027706

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. Unilamellar liposomes modulate secretion of tumor necrosis factor by lipopolysaccharide-stimulated macrophages.

    PubMed Central

    Brisseau, G F; Kresta, A; Schouten, D; Bohnen, J M; Shek, P N; Fok, E; Rotstein, O D

    1994-01-01

    Liposomal encapsulation of antimicrobial agents has been used to improve drug delivery, particularly against intracellular pathogens. The effect of unilamellar liposomes on macrophage activation in response to Escherichia coli lipopolysaccharide was examined. Liposomes caused a dose- and time-dependent inhibition of tumor necrosis factor release by lipopolysaccharide-treated cells. The accumulation of tumor necrosis factor mRNA transcripts was unaffected, suggesting a posttranscriptional mechanism for this effect. However, induction of macrophage procoagulant activity was unaffected by liposomes, indicating a selective rather than a global inhibition. These data suggest that liposomes used for drug delivery may modulate the host response to infection. Images PMID:7872768

  4. Prolactin, growth hormone, erythropoietin and granulocyte-macrophage colony stimulating factor induce MGF-Stat5 DNA binding activity.

    PubMed Central

    Gouilleux, F; Pallard, C; Dusanter-Fourt, I; Wakao, H; Haldosen, L A; Norstedt, G; Levy, D; Groner, B

    1995-01-01

    The molecular components which mediate cytokine signaling from the cell membrane to the nucleus were studied. Upon the interaction of cytokines with their receptors, members of the janus kinase (Jak) family of cytoplasmic protein tyrosine kinases and of the signal transducers and activators of transcription (Stat) family of transcription factors are activated through tyrosine phosphorylation. It has been suggested that the Stat proteins are substrates of the Jak protein tyrosine kinases. MGF-Stat5 is a member of the Stat family which has been found to confer the prolactin response. MGF-Stat5 can be phosphorylated and activated in its DNA binding activity by Jak2. The activation of MGF-Stat5 is not restricted to prolactin. Erythropoietin (EPO) and growth hormone (GH) stimulate the DNA binding activity of MGF-Stat5 in COS cells transfected with vectors encoding EPO receptor and MGF-Stat5 or vectors encoding GH receptor and MGF-Stat5. The activation of DNA binding by prolactin, EPO and GH requires the phosphorylation of tyrosine residue 694 of MGF-Stat5. The transcriptional induction of a beta-casein promoter luciferase construct in transiently transfected COS cells is specific for the prolactin activation of MGF-Stat5; it is not observed in EPO- and GH-treated cells. In the UT7 human hematopoietic cell line, EPO and granulocyte-macrophage colony stimulating factor activate the DNA binding activity of a factor closely related to MGF-Stat5 with respect to its immunological reactivity, DNA binding specificity and molecular weight. These results suggest that MGF-Stat5 regulates physiological processes in mammary epithelial cells, as well as in hematopoietic cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7744007

  5. Transient supplementation of anabolic growth factors rapidly stimulates matrix synthesis in engineered cartilage

    PubMed Central

    Ng, Kenneth W.; O’Conor, Christopher J.; Kugler, Lindsay E.; Cook, James L.; Ateshian, Gerard A.; Hung, Clark T.

    2012-01-01

    The purpose of the presented work is to examine the response of engineered cartilage to a transient, 2-week application of anabolic growth factors compared to continuous exposure in in vitro culture. Immature bovine chondrocytes were suspended in agarose hydrogel and cultured for 28 days (Study 1) or 42 days (Study 2) in chondrogenic media with TGF-β1, TGF-β3, or IGF-I either added for only the first 14 days in culture or added to the media for the entire study period. In both studies, there were no statistical differences in tissue mechanical or biochemical properties between the growth factors on day 14. In Study 1, growth factor removal led to a significant and drastic increase in Young’s modulus and GAG content compared to continuously exposed controls on day 28. In Study 2, both TGF-β1 and β3 led to significantly higher mechanical properties and collagen content versus IGF-I on day 42. These results indicate that the rapid rise in tissue properties (previously observed with TGF-β3 only) is not dependent on the type but rather the temporal application of the anabolic growth factor. These findings shed light on possible techniques to rapidly develop engineered cartilage tissue for the future treatment of osteoarthritis. PMID:21833681

  6. 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

  7. Production of tumor necrosis factors alpha and beta by human mononuclear leukocytes stimulated with mitogens, bacteria, and malarial parasites.

    PubMed Central

    Ferrante, A; Staugas, R E; Rowan-Kelly, B; Bresatz, S; Kumaratilake, L M; Rzepczyk, C M; Adolf, G R

    1990-01-01

    Tumor necrosis factors alpha and beta (TNF-alpha and TNF-beta) are multifaceted polypeptide cytokines which may mediate some of the significant changes in cellular homeostasis which accompany the invasion of the mammalian host by viruses, bacteria, and parasites. Although it is well established that bacterial lipopolysaccharide is a potent inducer of TNF-alpha, there is still very little known of the types of agents which can trigger the production of TNFs in mononuclear leukocytes. Using an enzyme-linked immunosorbent assay for measuring TNF-alpha and TNF-beta, we examined the capacity of various T-lymphocyte and beta-lymphocyte mitogens as well as microbial components to stimulate production of these cytokines in culture. The mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen induced production of both TNF-alpha and TNF-beta, while whole-killed Staphylococcus aureus and Bordetella pertussis, like lipopolysaccharide, were potent inducers of TNF-alpha but failed to stimulate TNF-beta production. TNF-alpha production was detectable within 1 h after stimulation, while TNF-beta production was not detected until after 8 h of culture. The bacterial products tetanus toxoid, purified protein derivative, pertussis filamentous hemagglutinin, and pertussis toxin were all able to induce TNF-alpha and TNF-beta production. Disrupted (frozen-thawed) Plasmodium falciparum-infected erythrocytes were also potent inducers of TNF-alpha and TNF-beta. The results demonstrated that a wide variety of microbial components are inducers of TNF-alpha. Some may not only be more effective than lipopolysaccharide but can also induce TNF-beta production. Furthermore, evidence is presented showing that TNF-beta but not TNF-alpha production correlates with lymphoproliferation. PMID:2254024

  8. Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor

    PubMed Central

    Burstein, David E.; Greene, Lloyd A.

    1978-01-01

    Studies on the mechanism of action of nerve growth factor (NGF) were carried out with PC12 rat pheochromocytoma cells. PC12 cells are uniquely useful for such studies because they respond to, but (unlike normal neurons) do not require, NGF and may undergo either generation or regeneration of neurites in response to NGF. Regeneration is defined here as NGF-dependent regrowth of neurites within 24 hr after subculture of NGF-treated PC12 cells. As in cultures of normal NGF-responsive neurons, neurite regeneration by PC12 cells occurs even in the presence of high concentrations of RNA synthesis inhibitors. Generation of neurites is defined as the de novo initiation of outgrowth when PC12 cells are exposed to NGF for the first time. In contrast to regeneration, neurite generation takes place with a lag of at least 24 hr and is blocked by low concentrations of RNA synthesis inhibitors. Such findings suggest that there are both RNA synthesis-dependent and -independent pathways in the mechanism whereby NGF stimulates neurite outgrowth. In addition, NGF-treated PC12 cells undergo a time-dependent loss of the capacity for neurite regeneration after pretreatment with RNA synthesis inhibitors or withdrawal of NGF. Such findings suggest that (i) initiation of neurite outgrowth requires NGF-stimulated, RNA synthesis-dependent accumulation of intracellular material(s), (ii) once such accumulation occurs, RNA synthesis-independent regeneration can occur (but only in the presence of NGF), and (iii) the turnover of such material(s) in the absence of their replacement leads to loss of the capacity for regeneration. A tentative sequence is presented for the events whereby NGF may stimulate neurite outgrowth. PMID:310552

  9. Ciliary Neurotrophic Factor Stimulates Muscle Glucose Uptake by a PI3-Kinase–Dependent Pathway That Is Impaired With Obesity

    PubMed Central

    Steinberg, Gregory R.; Watt, Matthew J.; Ernst, Matthias; Birnbaum, Morris J.; Kemp, Bruce E.; Jørgensen, Sebastian Beck

    2009-01-01

    OBJECTIVE Ciliary neurotrophic factor (CNTF) reverses muscle insulin resistance by increasing fatty acid oxidation through gp130-LIF receptor signaling to the AMP-activated protein kinase (AMPK). CNTF also increases Akt signaling in neurons and adipocytes. Because both Akt and AMPK regulate glucose uptake, we investigated muscle glucose uptake in response to CNTF signaling in lean and obese mice. RESEARCH DESIGN AND METHODS Mice were injected intraperitoneally with saline or CNTF, and blood glucose was monitored. The effects of CNTF on skeletal muscle glucose uptake and AMPK/Akt signaling were investigated in incubated soleus and extensor digitorum longus (EDL) muscles from muscle-specific AMPKα2 kinase-dead, gp130ΔSTAT, and lean and obese ob/ob and high-fat–fed mice. The effect of C2-ceramide on glucose uptake and gp130 signaling was also examined. RESULTS CNTF reduced blood glucose and increased glucose uptake in isolated muscles in a time- and dose-dependent manner with maximal effects after 30 min with 100 ng/ml. CNTF increased Akt-S473 phosphorylation in soleus and EDL; however, AMPK-T172 phosphorylation was only increased in soleus. Incubation of muscles from AMPK kinase dead (KD) and wild-type littermates with the PI3-kinase inhibitor LY-294002 demonstrated that PI3-kinase, but not AMPK, was essential for CNTF-stimulated glucose uptake. CNTF-stimulated glucose uptake and Akt phosphorylation were substantially reduced in obesity (high-fat diet and ob/ob) despite normal induction of gp130/AMPK signaling—effects also observed when treating myotubes with C2-ceramide. CONCLUSIONS CNTF acutely increases muscle glucose uptake by a mechanism involving the PI3-kinase/Akt pathway that does not require AMPK. CNTF-stimulated glucose uptake is impaired in obesity-induced insulin resistance and by ceramide. PMID:19136654

  10. Granulocyte colony-stimulating factor reprograms bone marrow stromal cells to actively suppress B lymphopoiesis in mice

    PubMed Central

    Day, Ryan B.; Bhattacharya, Deepta; Nagasawa, Takashi

    2015-01-01

    The mechanisms that mediate the shift from lymphopoiesis to myelopoiesis in response to infectious stress are largely unknown. We show that treatment with granulocyte colony-stimulating factor (G-CSF), which is often induced during infection, results in marked suppression of B lymphopoiesis at multiple stages of B-cell development. Mesenchymal-lineage stromal cells in the bone marrow, including CXCL12-abundant reticular (CAR) cells and osteoblasts, constitutively support B lymphopoiesis through the production of multiple B trophic factors. G-CSF acting through a monocytic cell intermediate reprograms these stromal cells, altering their capacity to support B lymphopoiesis. G-CSF treatment is associated with an expansion of CAR cells and a shift toward osteogenic lineage commitment. It markedly suppresses the production of multiple B-cell trophic factors by CAR cells and osteoblasts, including CXCL12, kit ligand, interleukin-6, interleukin-7, and insulin-like growth factor-1. Targeting bone marrow stromal cells is one mechanism by which inflammatory cytokines such as G-CSF actively suppress lymphopoiesis. PMID:25814527