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Sample records for granulocyte-macrophage colony-forming cells

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

    International Nuclear Information System (INIS)

    Yoshida, Hiroshi

    1994-01-01

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

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

    Science.gov (United States)

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

    2001-12-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-04-01

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

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

    Science.gov (United States)

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

    1984-04-01

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

  6. Exercise increases the frequency of circulating hematopoietic progenitor cells, but reduces hematopoietic colony-forming capacity.

    Science.gov (United States)

    Kroepfl, Julia Maria; Pekovits, Karin; Stelzer, Ingeborg; Fuchs, Robert; Zelzer, Sieglinde; Hofmann, Peter; Sedlmayr, Peter; Dohr, Gottfried; Wallner-Liebmann, Sandra; Domej, Wolfgang; Mueller, Wolfram

    2012-11-01

    Circulating hematopoietic progenitor cells (CPCs) may be triggered by physical exercise and/or normobaric hypoxia from the bone marrow. The aim of the study was to investigate the influence of physical exercise and normobaric hypoxia on CPC number and functionality in the peripheral blood as well as the involvement of oxidative stress parameters as possibly active agents. Ten healthy male subjects (25.3±4.4 years) underwent a standardized cycle incremental exercise test protocol (40 W+20 W/min) under either normoxic (FiO2 ∼0.21) or hypoxic conditions (FiO2exercise. The number of CPCs in the peripheral blood was analyzed by flow cytometry (CD34/CD45-positive cells). The functionality of cells present was addressed by secondary colony-forming unit-granulocyte macrophage (CFU-GM) assays. To determine a possible correlation between the mobilization of CPCs and reactive oxygen species, parameters for oxidative stress such as malondialdehyde (MDA) and myeloperoxidase (MPO) were obtained. Data showed a significant increase of CPC release under normoxic as well as hypoxic conditions after 10 min of recovery (Pexercise (Pexercise, possibly due to the influence of increased oxidative stress levels.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Patni Nidhi

    2005-01-01

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

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2007-10-01

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

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

    Science.gov (United States)

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

    2017-10-24

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

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

    International Nuclear Information System (INIS)

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

    1980-01-01

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

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

    Directory of Open Access Journals (Sweden)

    L Postiglione

    2009-06-01

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

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

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

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

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

    Directory of Open Access Journals (Sweden)

    Chang Rae Rho

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

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

    Science.gov (United States)

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

    2011-03-01

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

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

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    Magda Paula Pereira do Nascimento

    2011-09-01

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

  18. Responses of the Murine Myeloid Colony-Forming Cell to Ansamycin Antibiotics

    Science.gov (United States)

    Horoszewicz, Julius S.; Carter, William A.

    1974-01-01

    The in vitro susceptibility of murine myeloid colony-forming cells to the antiproliferative activities of three ansamycin antibiotics was determined. These cells were found to be 10- to 40-fold more susceptible than the corresponding human ones. PMID:4151701

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

    Science.gov (United States)

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

    1999-10-01

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

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

    OpenAIRE

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

    1998-01-01

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

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

    Science.gov (United States)

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

    2018-03-13

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

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

    Science.gov (United States)

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

    1992-06-01

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

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  4. Heterogeneity of the radiosensitivity and origins of tissue macrophage colony-forming cells

    Energy Technology Data Exchange (ETDEWEB)

    Oghiso, Yoichi; Yamada, Yutaka (National Inst. of Radiological Sciences, Chiba (Japan))

    1992-12-01

    Previous studies suggest that the radiosensitivity and origin of tissue macrophage precursors differ from those of hemopoietic macrophage colony-forming units (CFU-Ms) committed to macrophage-lineage cells. We assessed the origins of tissue macrophage colony-forming cells (M-CFCs) in mice by comparing their kinetics and radiosensitivities in the normal steady state and under the conditions of bone marrow depletion by [sup 89]Sr-administration and/or splenectomy. The results indicate that the radiosensitive peritoneal M-CFCs elicited by thioglycollate are derived from bone marrow macrophage precursors; where as alveolar M-CFCs, which are radioresistant, are self-sustained locally and independent of hemopoietic macrophage precursors. In contrast, highly radiosensitive liver M-CFCs are probably derived from CFU-Ms that appear to be propagated in the spleen in association with hemopoietic responses. (author).

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

    Science.gov (United States)

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

    2017-01-01

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

  6. Increased numbers of spleen colony forming units in B cell deficient CBA/N mice

    International Nuclear Information System (INIS)

    Wiktor-Jedrzejczak, W.; Krupienicz, A.; Scher, I.

    1986-01-01

    The formation of exogenous and endogenous spleen colonies was studied in immune-defective mice expressing the CBA/N X-linked xid gene. Bone marrow and spleen cells of immune deficient mice formed increased numbers of eight-day exogenous spleen colonies when transferred to either normal or B cell deficient lethally irradiated recipients. Moreover, defective mice showed increased formation of five-day endogenous spleen colonies (derived from transient endogenous colony forming units; T-CFU) and of ten-day endogenous spleen colonies (derived from CFU-S). Among the possible mechanisms responsible for the observed effects, the most probable appears the one in which decreased numbers of B cell precursors stimulate stem cell pools through a feedback mechanism. (orig.) [de

  7. Isolation and characterization of human umbilical cord-derived endothelial colony-forming cells

    Science.gov (United States)

    Zhang, Hao; Tao, Yanling; Ren, Saisai; Liu, Haihui; Zhou, Hui; Hu, Jiangwei; Tang, Yongyong; Zhang, Bin; Chen, Hu

    2017-01-01

    Endothelial colony-forming cells (ECFCs) are a population of endothelial progenitor cells (EPCs) that display robust proliferative potential and vessel-forming capability. Previous studies have demonstrated that a limited number of ECFCs may be obtained from adult bone marrow, peripheral blood and umbilical cord (UC) blood. The present study describes an effective method for isolating ECFCs from human UC. The ECFCs derived from human UC displayed the full properties of EPCs. Analysis of the growth kinetics, cell cycle and colony-forming ability of the isolated human UC-ECFCs indicated that the cells demonstrated properties of stem cells, including relative stability and rapid proliferation in vitro. Gene expression of Fms related tyrosine kinase 1, kinase insert domain receptor, vascular endothelial cadherin, cluster of differentiation (CD)31, CD34, epidermal growth factor homology domains-2, von Willebrand factor and endothelial nitric oxide synthase was assessed by reverse transcription-polymerase chain reaction. The cells were positive for CD34, CD31, CD73, CD105 and vascular endothelial growth factor receptor-2, and negative for CD45, CD90 and human leukocyte antigen-antigen D related protein according to flow cytometry. 1,1′-dioctadecyl-3,3,3′,3′-tetra-methyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein and fluorescein isothiocyanate-Ulex europaeus-l were used to verify the identity of the UC-ECFCs. Matrigel was used to investigate tube formation capability. The results demonstrated that the reported technique is a valuable method for isolating human UC-ECFCs, which have potential for use in vascular regeneration. PMID:29067104

  8. Functional Differences Between Placental Micro- and Macrovascular Endothelial Colony-Forming Cells

    Science.gov (United States)

    Solomon, Ioana; O’Reilly, Megan; Ionescu, Lavinia; Alphonse, Rajesh S.; Rajabali, Saima; Zhong, Shumei; Vadivel, Arul; Shelley, W. Chris; Yoder, Mervin C.

    2016-01-01

    Alterations in the development of the placental vasculature can lead to pregnancy complications, such as preeclampsia. Currently, the cause of preeclampsia is unknown, and there are no specific prevention or treatment strategies. Further insight into the placental vasculature may aid in identifying causal factors. Endothelial colony-forming cells (ECFCs) are a subset of endothelial progenitor cells capable of self-renewal and de novo vessel formation in vitro. We hypothesized that ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Human placentas were collected from term pregnancies delivered by cesarean section (n = 16). Placental micro- and macrovasculature was collected from the maternal and fetal side of the placenta, respectively, and ECFCs were isolated and characterized. ECFCs were CD31+, CD105+, CD144+, CD146+, CD14−, and CD45−, took up 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate-labeled acetylated low-density lipoprotein, and bound Ulex europaeus agglutinin 1. In vitro, macrovascular ECFCs had a greater potential to generate high-proliferative colonies and formed more complex capillary-like networks on Matrigel compared with microvascular ECFCs. In contrast, in vivo assessment demonstrated that microvascular ECFCs had a greater potential to form vessels. Macrovascular ECFCs were of fetal origin, whereas microvascular ECFCs were of maternal origin. ECFCs exist in the micro- and macrovasculature of the normal, term human placenta. Although macrovascular ECFCs demonstrated greater vessel and colony-forming potency in vitro, this did not translate in vivo, where microvascular ECFCs exhibited a greater vessel-forming ability. These important findings contribute to the current understanding of normal placental vascular development and may aid in identifying factors involved in preeclampsia and other pregnancy complications. Significance This research confirms that resident endothelial colony-forming

  9. Identification and enrichment of colony-forming cells from the adult murine pituitary

    International Nuclear Information System (INIS)

    Lepore, D.A.; Roeszler, K.; Wagner, J.; Ross, S.A.; Bauer, K.; Thomas, P.Q.

    2005-01-01

    Stem and progenitor cells have been identified in many adult tissues including bone marrow, the central nervous system, and skin. While there is direct evidence to indicate the activity of a progenitor cell population in the pituitary gland, this putative subpopulation has not yet been identified. Herein we describe the isolation and characterization of a novel clonogenic cell type in the adult murine pituitary, which we have termed Pituitary Colony-Forming Cells (PCFCs). PCFCs constitute 0.2% of pituitary cells, and generate heterogeneous colonies from single cells. PCFCs exhibit variable proliferative potential, and may exceed 11 population doublings in 14 days. Enrichment of PCFCs to 61.5-fold with 100% recovery can be obtained through the active uptake of the fluorescent dipeptide, β-Ala-Lys-Nε-AMCA. PCFCs are mostly contained within the large, agranular subpopulation of AMCA + cells, and constitute 28% of this fraction, corresponding to 140.5-fold enrichment. Interestingly, the AMCA + population contains rare cells that are GH + or PRL + . GH + cells were also identified in PCFC single cell colonies, suggesting that PCFCs have the potential to differentiate into GH + cells. Together, these data show that the pituitary contains a rare clonogenic population which may correspond to the somatotrope/lactotrope progenitors suggested by previous experiments

  10. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

    Science.gov (United States)

    Nakajima, Ryota; Takeda, Shizu

    2014-01-01

    The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Binucleate cell formation correlates to loss of colony-forming ability in X-irradiated cultured mammalian cells

    International Nuclear Information System (INIS)

    Sasaki, H.; Yoshinaga, H.; Kura, S.

    1986-01-01

    The relationship between binucleate cell formation and the loss of colony-forming ability was examined in several cultured mammalian cell lines irradiated with X rays. The maximum fraction of binucleate cells after X irradiation increased dose-dependently within the range in which reproductive cell death might predominate over interphase cell death. When the logarithm of percentage survival was plotted against the percentage binucleate cells, a similar correlation was found for all cell lines tested, with the exception of mouse leukemia L5178Y cells, the most radiosensitive cells used. These observations suggest that the fraction of binucleate cells in the cell population can serve as a measure of cellular radiation damage

  12. Matrix production and organization by endothelial colony forming cells in mechanically strained engineered tissue constructs.

    Directory of Open Access Journals (Sweden)

    Nicky de Jonge

    Full Text Available AIMS: Tissue engineering is an innovative method to restore cardiovascular tissue function by implanting either an in vitro cultured tissue or a degradable, mechanically functional scaffold that gradually transforms into a living neo-tissue by recruiting tissue forming cells at the site of implantation. Circulating endothelial colony forming cells (ECFCs are capable of differentiating into endothelial cells as well as a mesenchymal ECM-producing phenotype, undergoing Endothelial-to-Mesenchymal-transition (EndoMT. We investigated the potential of ECFCs to produce and organize ECM under the influence of static and cyclic mechanical strain, as well as stimulation with transforming growth factor β1 (TGFβ1. METHODS AND RESULTS: A fibrin-based 3D tissue model was used to simulate neo-tissue formation. Extracellular matrix organization was monitored using confocal laser-scanning microscopy. ECFCs produced collagen and also elastin, but did not form an organized matrix, except when cultured with TGFβ1 under static strain. Here, collagen was aligned more parallel to the strain direction, similar to Human Vena Saphena Cell-seeded controls. Priming ECFC with TGFβ1 before exposing them to strain led to more homogenous matrix production. CONCLUSIONS: Biochemical and mechanical cues can induce extracellular matrix formation by ECFCs in tissue models that mimic early tissue formation. Our findings suggest that priming with bioactives may be required to optimize neo-tissue development with ECFCs and has important consequences for the timing of stimuli applied to scaffold designs for both in vitro and in situ cardiovascular tissue engineering. The results obtained with ECFCs differ from those obtained with other cell sources, such as vena saphena-derived myofibroblasts, underlining the need for experimental models like ours to test novel cell sources for cardiovascular tissue engineering.

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

    Science.gov (United States)

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

    2004-03-01

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

  14. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Uyeki, E.M.; Wierzba, K.; Bisel, T.U.

    1981-01-01

    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  15. Heterogeneity within the spleen colony-forming cell population in rat bone marrow

    International Nuclear Information System (INIS)

    Martens, A.C.; van Bekkum, D.W.; Hagenbeek, A.

    1986-01-01

    The pluripotent hemopoietic stem cell (HSC) of the rat can be enumerated in a spleen colony assay (SCA) in rats as well as mice. After injection of rat bone marrow into lethally irradiated mice, macroscopically visible spleen colonies (CFU-S) are found from day 6 through 14, but the number varies on consecutive days. In normal bone marrow a constant ratio of day-8 to day-12 colony numbers is observed. However, this ratio is changed after in vivo treatment of rats with cyclophosphamide, as well as after in vitro treatment of rat bone marrow with cyclophosphamide derivatives. This indicates that the CFU-S that form colonies on day 8 react differently to this treatment than the CFU-S that form colonies on day 12, and suggests heterogeneity among the CFU-S population. Posttreatment regrowth of day-8 and day-12 CFU-S is characterized by differences in population-doubling times (Td = 0.85 days vs 1.65 days). Another argument in support of the postulate of heterogeneity within the rat CFU-S population is derived from the fact that (in contrast to normal rat spleen) the spleen of leukemic rats contains high numbers of CFU-S that show a ratio of day-8 to day-12 CFU-S of 4.5, which is different than that observed for a CFU-S population in normal bone marrow (a ratio of 2.4). It is concluded that, in rat hemopoiesis, two populations of spleen colony-forming cells can be distinguished using the rat-to-mouse SCA. This indicates that mouse and rat hemopoiesis are comparable in this respect and that heterogeneity in the stem cell compartment is a general phenomenon

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

    Science.gov (United States)

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

    1998-05-01

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

  17. Interleukin-3/granulocyte macrophage colony-stimulating factor receptor promotes stem cell expansion, monocytosis, and atheroma macrophage burden in mice with hematopoietic ApoE deficiency

    NARCIS (Netherlands)

    Wang, Mi; Subramanian, Manikandan; Abramowicz, Sandra; Murphy, Andrew J.; Gonen, Ayelet; Witztum, Joseph; Welch, Carrie; Tabas, Ira; Westerterp, Marit; Tall, Alan R.

    2014-01-01

    Coronary heart disease is associated with monocytosis. Studies using animal models of monocytosis and atherosclerosis such as ApoE(-/-) mice have shown bone marrow (BM) hematopoietic stem and multipotential progenitor cell (HSPC) expansion, associated with increased cell surface expression of the

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

    DEFF Research Database (Denmark)

    Agnholt, Jørgen; Kelsen, Jens; Brandsborg, Birgitte

    2004-01-01

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

  19. A role for granulocyte-macrophage colony-stimulating factor in the regulation of CD8{sup +} T cell responses to rabies virus

    Energy Technology Data Exchange (ETDEWEB)

    Wanjalla, Celestine N.; Goldstein, Elizabeth F.; Wirblich, Christoph [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Schnell, Matthias J., E-mail: matthias.schnell@jefferson.edu [Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States); Jefferson Vaccine Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107 (United States)

    2012-05-10

    Inflammatory cytokines have a significant role in altering the innate and adaptive arms of immune responses. Here, we analyzed the effect of GM-CSF on a RABV-vaccine vector co-expressing HIV-1 Gag. To this end, we immunized mice with RABV expressing HIV-1 Gag and GM-CSF and analyzed the primary and recall CD8{sup +} T cell responses. We observed a statistically significant increase in antigen presenting cells (APCs) in the spleen and draining lymph nodes in response to GM-CSF. Despite the increase in APCs, the primary and memory anti HIV-1 CD8{sup +} T cell response was significantly lower. This was partly likely due to lower levels of proliferation in the spleen. Animals treated with GM-CSF neutralizing antibodies restored the CD8{sup +} T cell response. These data define a role of GM-CSF expression, in the regulation of the CD8{sup +} T cell immune responses against RABV and has implications in the use of GM-CSF as a molecular adjuvant in vaccine development.

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

    Science.gov (United States)

    Ushach, Irina; Zlotnik, Albert

    2016-01-01

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

  1. A stimulator of proliferation of spleen colony-forming cells (CFU-S) in the bone marrow of irradiated rats

    Energy Technology Data Exchange (ETDEWEB)

    Ivanovic, Z.; Milenkovic, P.; Stojanovic, N.; Lukic, M.; Kataranovski, M.

    1993-07-01

    The presence and activity of a spleen colony - forming cell (CFU-S) proliferation stimulator was investigated in rat bone marrow after irradiation. The dose dependent increase in cytosine arabinoside induced cell dealth of normal mouse bone marrow. The results demonstrate the existence of a CFU-S proliferation stimulator in rat bone marrow similar to that originally found as a macrophage product in regenarating mouse bone marrow. The CFU-S proliferation stimulator activity was not associated with the presence of interleukin - 1,2, or 6 like activities in the material tested.

  2. Cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bootsma, A.L. (Rijksuniversiteit Utrecht (Netherlands). Academisch Ziekenhuis); Davids, J.A.G. (Netherlands Energy Research Foundation, Petten (Netherlands))

    1988-03-01

    In the CBA mouse testis, about 10% of the stem cell population is highly resistant to neutron irradiation (D/sub 0/, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea, it appeared that in this cycle the S-phase is less radiosensitive (D/sub 0/, 0.43 Gy) than the other phases of the cell cycle (D/sub 0/, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation, the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most. (author).

  3. Relationship of colony-stimulating activity to apparent kill of human colony-forming cells by irradiation and hydroxyurea

    International Nuclear Information System (INIS)

    Broxmeyer, H.E.; Galbraith, P.R.; Baker, F.L.

    1976-01-01

    Suspensions of human bone marrow cells were subjected to 137 Cs irradiation in vitro and then cultured in semisolid agar medium. Cultures of irradiated cells were stimulated with colony-stimulating activity (CSA) of different potencies, and it was found that the amount of stimulation applied to cultures influenced the apparent kill of colony-forming cells (CFC). It was also found that the effects of irradiation on colony formation were not confined to CFC kill since medium conditioned by cells during irradiation exhibited stimulatory and inhibitory properties after treatment by 600 and 1000 rads, respectively. Studies in which irradiated cells were pretreated with hydroxyurea indicated that CFC in the DNA synthetic phase of the cell cycle were particularly sensitive to low doses of irradiation. The proliferative capacity of CFC surviving 1000 rads was undiminished as judged by their ability to form large colonies. Estimates of CFC kill by hydroxyurea were also affected by the level of CSA

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  5. The hematopoietic chemokine CXCL12 promotes integration of human endothelial colony forming cell-derived cells into immature vessel networks.

    Science.gov (United States)

    Newey, Sarah E; Tsaknakis, Grigorios; Khoo, Cheen P; Athanassopoulos, Thanassi; Camicia, Rosalba; Zhang, Youyi; Grabowska, Rita; Harris, Adrian L; Roubelakis, Maria G; Watt, Suzanne M

    2014-11-15

    Proangiogenic factors, vascular endothelial growth factor (VEGF), and fibroblast growth factor-2 (FGF-2) prime endothelial cells to respond to "hematopoietic" chemokines and cytokines by inducing/upregulating expression of the respective chemokine/cytokine receptors. Coculture of human endothelial colony forming cell (ECFC)-derived cells with human stromal cells in the presence of VEGF and FGF-2 for 14 days resulted in upregulation of the "hematopoietic" chemokine CXCL12 and its CXCR4 receptor by day 3 of coculture. Chronic exposure to the CXCR4 antagonist AMD3100 in this vasculo/angiogenesis assay significantly reduced vascular tubule formation, an observation recapitulated by delayed AMD3100 addition. While AMD3100 did not affect ECFC-derived cell proliferation, it did demonstrate a dual action. First, over the later stages of the 14-day cocultures, AMD3100 delayed tubule organization into maturing vessel networks, resulting in enhanced endothelial cell retraction and loss of complexity as defined by live cell imaging. Second, at earlier stages of cocultures, we observed that AMD3100 significantly inhibited the integration of exogenous ECFC-derived cells into established, but immature, vascular networks. Comparative proteome profiler array analyses of ECFC-derived cells treated with AMD3100 identified changes in expression of potential candidate molecules involved in adhesion and/or migration. Blocking antibodies to CD31, but not CD146 or CD166, reduced the ECFC-derived cell integration into these extant vascular networks. Thus, CXCL12 plays a key role not only in endothelial cell sensing and guidance, but also in promoting the integration of ECFC-derived cells into developing vascular networks.

  6. Precursors (pre-CFCmulti) of multilineage hemopoietic colony-forming cells quantitated in vitro. Uniqueness of IL-1 requirement, partial separation from pluripotential colony-forming cells, and correlation with long term reconstituting cells in vivo

    International Nuclear Information System (INIS)

    Iscove, N.N.; Yan, X.Q.

    1990-01-01

    Pluripotential colony-forming cells (CFCmulti) from mouse marrow can expand significantly in number during 4 days of suspension culture with IL-1 and IL-3. In this study, the cells (pre-CFCmulti) which originate this response are characterized in terms of their frequency, progeny number, factor requirements, buoyant density, and extent of restoration following marrow transplantation. Parallel measurements of both CFCmulti and cells providing long term marrow reconstitution in vivo allowed direct comparisons to be made with pre-CFCmulti. The proliferative response of pre-CFCmulti was found to depend uniquely on the combination of IL-1 and IL-3, and neither of these regulators was replaceable by any of IL-4, IL-6, IL-7, GM-CSF, G-CSF, M-CSF or LIF. After separation on density gradients, pre-CFCmulti were recovered in fractions of lower density than most of the CFCmulti, but in the same fractions that contained most of the in vivo reconstituting cells. After irradiation and marrow transplantation, marrow CFCmulti were restored to near normal levels, while both pre-CFCmulti as well as reconstituting stem cells remained profoundly depressed. These results show pre-CFCmulti to be distinct from most CFCmulti and to represent the closest approach to quantitative detection of reconstituting stem cells so far achieved in vitro

  7. Effect of adrenalectomy on recipients of allogeneic lymphocytes on inactivation of endogenous colony-forming cells in mice

    International Nuclear Information System (INIS)

    Semenkov, V.F.

    1985-01-01

    This paper presents a study of the killer functions of lymph node cells directed against endogenous colony-forming cells in adrenalectomized recipients in a genetic system with one-way incompatibility: parental line - F 1 hybrid. Mice were irradiated with Co 60 gamma rays on the EGO-2 apparatus with dose rate from 200 to 250 R/min. The results were subjected to statistical analysis by Student's test. It can be tentatively suggested that the killer action of T lymphocytes on endogenous colonies was intensified in adrenal-ectomized recipients with endogenous hypocorticism, as a result of cooperation with the cortisol-sensitive subpopulation of T helper cells, of a change in the properties of the antigen-recognizing receptors, or an increase in the sensitivity of target cells to the killer action of T lymphocytes

  8. Reduced proliferation of endothelial colony-forming cells in unprovoked venous thromboembolic disease as a consequence of endothelial dysfunction

    Science.gov (United States)

    Hernandez-Lopez, Rubicel; Chavez-Gonzalez, Antonieta; Torres-Barrera, Patricia; Moreno-Lorenzana, Dafne; Lopez-DiazGuerrero, Norma; Santiago-German, David; Isordia-Salas, Irma; Smadja, David; C. Yoder, Mervin; Majluf-Cruz, Abraham

    2017-01-01

    Background Venous thromboembolic disease (VTD) is a public health problem. We recently reported that endothelial colony-forming cells (ECFCs) derived from endothelial cells (EC) (ECFC-ECs) from patients with VTD have a dysfunctional state. For this study, we proposed that a dysfunctional status of these cells generates a reduction of its proliferative ability, which is also associated with senescence and reactive oxygen species (ROS). Methods and results Human mononuclear cells (MNCs) were obtained from peripheral blood from 40 healthy human volunteers (controls) and 50 patients with VTD matched by age (20−50 years) and sex to obtain ECFCs. We assayed their proliferative ability with plasma of patients and controls and supernatants of cultures from ECFC-ECs, senescence-associated β-galactosidase (SA-β-gal), ROS, and expression of ephrin-B2/Eph-B4 receptor. Compared with cells from controls, cells from VTD patients showed an 8-fold increase of ECFCs that emerged 1 week earlier, reduced proliferation at long term (39%) and, in passages 4 and 10, a highly senescent rate (30±1.05% vs. 91.3±15.07%, respectively) with an increase of ROS and impaired expression of ephrin-B2/Eph-4 genes. Proliferation potential of cells from VTD patients was reduced in endothelial medium [1.4±0.22 doubling population (DP)], control plasma (1.18±0.31 DP), or plasma from VTD patients (1.65±0.27 DP). Conclusions As compared with controls, ECFC-ECs from individuals with VTD have higher oxidative stress, proliferation stress, cellular senescence, and low proliferative potential. These findings suggest that patients with a history of VTD are ECFC-ECs dysfunctional that could be associated to permanent risk for new thrombotic events. PMID:28910333

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

    Science.gov (United States)

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

    2002-01-01

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

  10. The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions

    NARCIS (Netherlands)

    Gremmels, Hendrik; De Jong, Olivier G.; Hazenbrink, Diënty H.; Fledderus, Joost O.; Verhaar, Marianne C.

    2017-01-01

    Background. Endothelial colony forming cells (ECFCs) have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS). The transcription factor Nrf2

  11. Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approach

    NARCIS (Netherlands)

    Zeisberger, Steffen M.; Zoller, Stefan; Riegel, Mariluce; Chen, Shuhua; Krenning, Guido; Harmsen, Martin C.; Sachinidis, Agapios; Zisch, Andreas H.

    Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and

  12. Manufacture of endothelial colony-forming progenitor cells from steady-state peripheral blood leukapheresis using pooled human platelet lysate.

    Science.gov (United States)

    Siegel, Georg; Fleck, Erika; Elser, Stefanie; Hermanutz-Klein, Ursula; Waidmann, Marc; Northoff, Hinnak; Seifried, Erhard; Schäfer, Richard

    2018-05-01

    Endothelial colony-forming progenitor cells (ECFCs) are promising candidates for cell therapies. However, ECFC translation to the clinic requires optimized isolation and manufacture technologies according to good manufacturing practice (GMP). ECFCs were manufactured from steady-state peripheral blood (PB) leukapheresis (11 donors), using GMP-compliant technologies including pooled human platelet (PLT) lysate, and compared to human umbilical cord endothelial cells, human aortic endothelial cells, and human cerebral microvascular endothelial cells. Specific variables assessed were growth kinetics, phenotype, trophic factors production, stimulation of tube formation, and Dil-AcLDL uptake. ECFCs could be isolated from PB leukapheresis units with mean processed volume of 5411 mL and mean white blood cell (WBC) concentration factor of 8.74. The mean frequency was 1.44 × 10 -8 ECFCs per WBC, corresponding to a mean of 177.8 ECFCs per apheresis unit. Expandable for up to 12 cumulative population doublings, calculated projection showed that approximately 730 × 10 3 ECFCs could be manufactured from 1 apheresis unit. ECFCs produced epidermal growth factor, hepatocyte growth factor, vascular endothelial growth factor (VEGF)-A, PLT-derived growth factor-B, interleukin-8, and monocyte chemoattractant protein-1, featured high potential for capillary-like tubes formation, and showed no telomerase activity. They were characterized by CD29, CD31, CD44, CD105, CD117, CD133, CD144, CD146, and VEGF-R2 expression, with the most common subpopulation CD34+CD117-CD133-. Compared to controls, ECFCs featured greater Dil-AcLDL uptake and higher expression of CD29, CD31, CD34, CD44, CD144, and VEGF-R2. Here we show that isolation of ECFCs with proangiogenic profile from steady-state PB leukapheresis is feasible, marking a first step toward ECFC product manufacture according to GMP. © 2018 AABB.

  13. Manipulation of the response of human endothelial colony-forming cells by focal adhesion assembly using gradient nanopattern plates.

    Science.gov (United States)

    Cui, Long-Hui; Joo, Hyung Joon; Kim, Dae Hwan; Seo, Ha-Rim; Kim, Jung Suk; Choi, Seung-Cheol; Huang, Li-Hua; Na, Ji Eun; Lim, I-Rang; Kim, Jong-Ho; Rhyu, Im Joo; Hong, Soon Jun; Lee, Kyu Back; Lim, Do-Sun

    2018-01-01

    Nanotopography plays a pivotal role in the regulation of cellular responses. Nonetheless, little is known about how the gradient size of nanostructural stimuli alters the responses of endothelial progenitor cells without chemical factors. Herein, the fabrication of gradient nanopattern plates intended to mimic microenvironment nanotopography is described. The gradient nanopattern plates consist of nanopillars of increasing diameter ranges [120-200 nm (GP 120/200), 200-280 nm (GP 200/280), and 280-360 nm (GP 280/360)] that were used to screen the responses of human endothelial colony-forming cells (hECFCs). Nanopillars with a smaller nanopillar diameter caused the cell area and perimeter of hECFCs to decrease and their filopodial outgrowth to increase. The structure of vinculin (a focal adhesion marker in hECFCs) was also modulated by nanostructural stimuli of the gradient nanopattern plates. Moreover, Rho-associated protein kinase (ROCK) gene expression was significantly higher in hECFCs cultured on GP 120/200 than in those on flat plates (no nanopillars), and ROCK suppression impaired the nanostructural-stimuli-induced vinculin assembly. These results suggest that the gradient nanopattern plates generate size-specific nanostructural stimuli suitable for manipulation of the response of hECFCs, in a process dependent on ROCK signaling. This is the first evidence of size-specific nanostructure-sensing behavior of hECFCs. Nano feature surfaces are of growing interest as materials for a controlled response of various cells. In this study, we successfully fabricated gradient nanopattern plates to manipulate the response of blood-derived hECFCs without any chemical stimulation. Interestingly, we find that the sensitive nanopillar size for manipulation of hECFCs is range between 120 nm and 200 nm, which decreased the area and increased the filopodial outgrowth of hECFCs. Furthermore, we only modulate the nanopillar size to increase ROCK expression can be an

  14. Effect of barbiturates on radiosensitivity of cells: a comparative study of electrophoretic mobility, colony forming ability and thymidine uptake on human amnion cells

    International Nuclear Information System (INIS)

    Lalwani, N.D.; Chaubal, K.A.

    1980-01-01

    Suspensions of human amnion cells were 60 Co γ-irradiated in the presence of phenobarbital or thiobarbital (50 μg/ml). The barbiturates protected the cells against the dose-dependent reduction in electrophoretic mobility (EPM) observed 4 hours after irradiation of untreated cells, although there was an initial decrease in the EPM of treated cells followed by recovery. Treated irradiated cells exhibited greater colony-forming ability than the untreated cells. Pentobarbital and phenobarbital had similar effects, but thiobarbital was not so effective. 3 H-TdR uptake increased within 4 hours of irradiation for the treated cells. The reproductive capacity of the cells was retained at doses as high as 500 rad. The results are discussed with reference to the effects of anaesthetics on cell membranes. (U.K.)

  15. Photoreactivation of ultraviolet light-induced damage in cultured fish cells as revealed by increased colony forming ability and decreased content of pyrimidine dimers

    International Nuclear Information System (INIS)

    Shima, A.; Ikenaga, M.; Egami, N.

    1981-01-01

    Cultured cells derived from a goldfish were irradiated with 254 nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m 2 ) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9(40 min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m 2 ) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120 min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light. (author)

  16. Photoreactivation of ultraviolet light-induced damage in cultured fish cells as revealed by increased colony forming ability and decreased content of pyrimidine dimers

    Energy Technology Data Exchange (ETDEWEB)

    Shima, A. (Shiga University of Medical Science, Otsu (Japan)); Ikenaga, M. (Osaka Univ. (Japan). Faculty of Medicine); Nikaido, O.; Takebe, H. (Kyoto Univ. (Japan)); Egami, N. (Tokyo Univ. (Japan). Faculty of Science)

    1981-03-01

    Cultured cells derived from a goldfish were irradiated with 254 nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m/sup 2/) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9(40 min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m/sup 2/) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120 min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.

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

    Science.gov (United States)

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

    2009-08-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  19. Increase in colony-forming efficiency in soft agar of thymus cells from radiation-induced thymomas of NIH Swiss mice

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Nobuko; Takamori, Yasuhiko; Hori, Yasuharu [Radiation Center of Osaka Prefecture, Sakai (Japan)

    1982-03-01

    Colony-forming efficiency in soft agar of radiation-induced thymoma in NIH Swiss mice was determined in the presence of cultured medium of reticulo-epitherial cells from normal thymus of NIH Swiss mouse as conditioned medium. A similar experiment was done with thymomas spontaneously developed in AKR mice. Most of colonies developed in soft agar were not composed of thymic lymphoma cells, but of macrophage-like cells. The ratio of the number of colonies to that of the seeded cells significantly increased in thymomas comparing with that in normal thymus. This result corresponded with the increased number of macrophages in thymoma, as determined by counting phagocytic cells of adherent cells.

  20. Reinfusion of autologous lymphocytes with granulocyte-macrophage colony-stimulating factor induces rapid recovery of CD4+ and CD8+ T cells after high-dose chemotherapy for metastatic breast cancer

    NARCIS (Netherlands)

    de Gast, G. C.; Vyth-Dreese, F. A.; Nooijen, W.; van den Bogaard, C. J. C.; Sein, J.; Holtkamp, M. M. J.; Linthorst, G. A. M.; Baars, J. W.; Schornagel, J. H.; Rodenhuis, S.

    2002-01-01

    PURPOSE: Repeated high-dose chemotherapy (HDCT) followed by peripheral-blood progenitor cell (PBPC) transplantation can induce a complete remission in patients with metastatic breast cancer sensitive to standard chemotherapy (CT), but the majority of patients relapse within 1 to 2 years. The immune

  1. Granulocyte-macrophage stimulating factor (GM-CSF increases circulating dendritic cells but does not abrogate suppression of adaptive cellular immunity in patients with metastatic colorectal cancer receiving chemotherapy

    Directory of Open Access Journals (Sweden)

    Martinez Micaela

    2012-01-01

    Full Text Available Abstract Background Advanced cancer and chemotherapy are both associated with immune system suppression. We initiated a clinical trial in patients receiving chemotherapy for metastatic colorectal cancer to determine if administration of GM-CSF in this setting was immunostimulatory. Methods Between June, 2003 and January, 2007, 20 patients were enrolled in a clinical trial (NCT00257322 in which they received 500 ug GM-CSF daily for 4 days starting 24 hours after each chemotherapy cycle. There were no toxicities or adverse events reported. Blood was obtained before chemotherapy/GM-CSF administration and 24 hours following the final dose of GM-CSF and evaluated for circulating dendritic cells and adaptive immune cellular subsets by flow cytometry. Peripheral blood mononuclear cell (PBMC expression of γ-interferon and T-bet transcription factor (Tbx21 by quantitative real-time PCR was performed as a measure of Th1 adaptive cellular immunity. Pre- and post-treatment (i.e., chemotherapy and GM-CSF samples were evaluable for 16 patients, ranging from 1 to 5 cycles (median 3 cycles, 6 biologic sample time points. Dendritic cells were defined as lineage (- and MHC class II high (+. Results 73% of patients had significant increases in circulating dendritic cells of ~3x for the overall group (5.8% to 13.6%, p = 0.02 and ~5x excluding non-responders (3.2% to 14.5%, p Tbx21 levels declined by 75% following each chemotherapy cycle despite administration of GM-CSF (p = 0.02. PBMC γ-interferon expression, however was unchanged. Conclusions This clinical trial confirms the suppressive effects of chemotherapy on Th1 cellular immunity in patients with metastatic colorectal cancer but demonstrates that mid-cycle administration of GM-CSF can significantly increase the proportion of circulating dendritic cells. As the role of dendritic cells in anti-tumor immunity becomes better defined, GM-CSF administration may provide a non-toxic intervention to augment this arm

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

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

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

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

    Science.gov (United States)

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

    1993-01-15

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

  4. Human endothelial colony-forming cells expanded with an improved protocol are a useful endothelial cell source for scaffold-based tissue engineering.

    Science.gov (United States)

    Denecke, Bernd; Horsch, Liska D; Radtke, Stefan; Fischer, Johannes C; Horn, Peter A; Giebel, Bernd

    2015-11-01

    One of the major challenges in tissue engineering is to supply larger three-dimensional (3D) bioengineered tissue transplants with sufficient amounts of nutrients and oxygen and to allow metabolite removal. Consequently, artificial vascularization strategies of such transplants are desired. One strategy focuses on endothelial cells capable of initiating new vessel formation, which are settled on scaffolds commonly used in tissue engineering. A bottleneck in this strategy is to obtain sufficient amounts of endothelial cells, as they can be harvested only in small quantities directly from human tissues. Thus, protocols are required to expand appropriate cells in sufficient amounts without interfering with their capability to settle on scaffold materials and to initiate vessel formation. Here, we analysed whether umbilical cord blood (CB)-derived endothelial colony-forming cells (ECFCs) fulfil these requirements. In a first set of experiments, we showed that marginally expanded ECFCs settle and survive on different scaffold biomaterials. Next, we improved ECFC culture conditions and developed a protocol for ECFC expansion compatible with 'Good Manufacturing Practice' (GMP) standards. We replaced animal sera with human platelet lysates and used a novel type of tissue-culture ware. ECFCs cultured under the new conditions revealed significantly lower apoptosis and increased proliferation rates. Simultaneously, their viability was increased. Since extensively expanded ECFCs could still settle on scaffold biomaterials and were able to form tubular structures in Matrigel assays, we conclude that these ex vivo-expanded ECFCs are a novel, very potent cell source for scaffold-based tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

  5. Characteristics of cells producing stimulator for the proliferation of colony forming unit-spleen (CFU-S)

    International Nuclear Information System (INIS)

    Abdul Manaf Ali; Wright, E.G.; Riches, A.C.

    1994-01-01

    The presence of stimulator for haemopoietic stem cell (CFU-S) proliferation in regenerating bone marrow was assayed by incubating the conditioned medium (CM) prepared from bone marrow with quiescent CFU-S from normal bone marrow. The percentage of CFU-S normal bone marrow in DNA synthesis increased more than 30 percent after incubating with CM of 4.5 Gy regenerating bone marrow. Stimulator was also present in bone marrow of mice at 9.0 Gy whole body X-irradiation. However the conditioned medium prepared from regenerating bone marrow without Fc and Ia-2k cells failed to increase the percentage of CFU-S in DNA synthesis. On the other hand, elimination of Thy 1.2 positive cells with complement cytolysis did not affect the ability of regenerating bone marrow to produce stimulator. These observations suggest that the stimulator producing cells are radio-resistant, Thy 1.2 negative, Fc and Ia-2k positive

  6. Culture on fibrin matrices maintains the colony-forming capacity and osteoblastic differentiation of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Colley, Helen; McArthur, Sally L; Stolzing, Alexandra; Scutt, Andy

    2012-01-01

    Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into a number of mesenchymal tissues including bone, cartilage, and tendon. Low numbers in vivo means exponential growth is needed in culture to enable therapeutic applications. MSC can expand rapidly in culture but usually lose their extensive capacity for differentiation that makes them therapeutically attractive. To try and maintain their capacity for differentiation and expansion in vitro, we cultured MSC on fibrin gels of different concentrations to create more physiological growth conditions for the cells. The cells were then re-plated onto tissue culture plastic and analysed. The cells that had been pre-cultured for seven days on fibrin, proliferated and maintained their differential potential to the osteogenic lineage better than tissue culture plastic expanded MSC. A concentration relationship between colony number and fibrin concentration was seen with decreasing numbers as fibrin concentration increased. These data support the concept that substrate signals significantly influence MSC growth and differentiation and that growth on a fibrin matrix could be used to maintain a stem cell phenotype during MSC expansion. (paper)

  7. Method of monolayer cultures of the guinea pig spleen used to evaluate the radioprotective action of mexamine on the survival of colony-forming cells. [Method of monolayer cultures, quantitative estimation of radioprotective action of drugs

    Energy Technology Data Exchange (ETDEWEB)

    Baisogolov, G D; Rudakova, S F; Rudakov, I A; Konoplyannikov, A G [Akademiya Meditsinskikh Nauk SSSR, Obninsk. Nauchno-Issledovatel' skij Inst. Meditsinskoj Radiologii

    1976-01-01

    Dose dependent survival of colony-forming splenic cells of guinea pigs has been studied after ..gamma..-irradiation in vitro. The average lethal dose (D/sub 0/) for colony-forming cells is 245 rads, the extrapolation number - 1.5. Radiosensitivity of the splenic cells is markedly lower than that of the bone marrow cells (128 rads). After intraperitoneal administration of mexamine (150 mg/kg) to guinea pigs 10-15 min before irradiation, D/sub 0/ increases up to 319 rads, extrapolation number is 1.4, and DRF is 1.3.

  8. Mechanism of suppression of normal hemopoietic activity by lymphokine-activated killer cells and their products

    International Nuclear Information System (INIS)

    Gibson, F.M.; Malkovska, V.; Myint, A.A.; Meager, A.; Gordon-Smith, E.C.

    1991-01-01

    Interleukin 2 (IL-2)-activated lymphocytes (lymphokine-activated killer [LAK] cells) have been shown to inhibit the formation of autologous human granulocyte-macrophage hemopoietic progenitors (granulocyte-macrophage colony-forming units, CFU-GM) in vitro. Effects of LAK cells on these progenitors may include a number of different mechanisms. LAK cells are potent cytotoxic lymphocytes capable of lysing certain normal autologous cells. They also produce cytokines known to inhibit hemopoiesis (interferon gamma [IFN-gamma] and tumor necrosis factor alpha [TNF-alpha]) or enhance it (granulocyte-macrophage colony-stimulating factor, GM-CSF). In the authors' current study they analyzed the mechanism of suppression of autologous CFU-GM by LAK cells. Their results suggest that LAK cells are not directly cytotoxic to normal CFU-GM. They show that it is possible to abolish the hemopoiesis-inhibiting activity of LAK cells without abrogating their cytotoxicity against tumor cell lines using inhibitors of DNA synthesis, namely hydroxyurea or irradiation

  9. The Transcription Factor Nrf2 Protects Angiogenic Capacity of Endothelial Colony-Forming Cells in High-Oxygen Radical Stress Conditions

    Directory of Open Access Journals (Sweden)

    Hendrik Gremmels

    2017-01-01

    Full Text Available Background. Endothelial colony forming cells (ECFCs have shown a promise in tissue engineering of vascular constructs, where they act as endothelial progenitor cells. After implantation, ECFCs are likely to be subjected to elevated reactive oxygen species (ROS. The transcription factor Nrf2 regulates the expression of antioxidant enzymes in response to ROS. Methods. Stable knockdown of Nrf2 and Keap1 was achieved by transduction with lentiviral shRNAs; activation of Nrf2 was induced by incubation with sulforaphane (SFN. Expression of Nrf2 target genes was assessed by qPCR, oxidative stress was assessed using CM-DCFDA, and angiogenesis was quantified by scratch-wound and tubule-formation assays. Results. Nrf2 knockdown led to a reduction of antioxidant gene expression and increased ROS. Angiogenesis was disturbed after Nrf2 knockdown even in the absence of ROS. Conversely, angiogenesis was preserved in high ROS conditions after knockdown of Keap1. Preincubation of ECFCs with SFN reduced intracellular ROS in the presence of H2O2 and preserved scratch-wound closure and tubule-formation. Conclusion. The results of this study indicate that Nrf2 plays an important role in the angiogenic capacity of ECFCs, particularly under conditions of increased oxidative stress. Pretreatment of ECFCs with SFN prior to implantation may be a protective strategy for tissue-engineered constructs or cell therapies.

  10. Isolation of endothelial colony-forming cells from blood samples collected from the jugular and cephalic veins of healthy adult horses.

    Science.gov (United States)

    Sharpe, Ashley N; Seeto, Wen J; Winter, Randolph L; Zhong, Qiao; Lipke, Elizabeth A; Wooldridge, Anne A

    2016-10-01

    OBJECTIVE To evaluate optimal isolation of endothelial colony-forming cells (ECFCs) from peripheral blood of horses. SAMPLE Jugular and cephalic venous blood samples from 17 adult horses. PROCEDURES Each blood sample was divided; isolation was performed with whole blood adherence (WBA) and density gradient centrifugation (DGC). Isolated cells were characterized by uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-Ac-LDL), vascular tubule formation, and expression of endothelial (CD34, CD105, vascular endothelial growth factor receptor-2, and von Willebrand factor) and hematopoietic (CD14) cell markers by use of indirect immunofluorescence assay (IFA) and flow cytometry. RESULTS Colonies with cobblestone morphology were isolated from 15 of 17 horses. Blood collected from the cephalic vein yielded colonies significantly more often (14/17 horses) than did blood collected from the jugular vein (8/17 horses). Of 14 cephalic blood samples with colonies, 13 were obtained with DGC and 8 with WBA. Of 8 jugular blood samples with colonies, 8 were obtained with DGC and 4 with WBA. Colony frequency (colonies per milliliter of blood) was significantly higher for cephalic blood samples and samples isolated with DGC. Cells formed vascular tubules, had uptake of DiI-Ac-LDL, and expressed endothelial markers by use of IFA and flow cytometry, which confirmed their identity as ECFCs. CONCLUSIONS AND CLINICAL RELEVANCE Maximum yield of ECFCs was obtained for blood samples collected from both the jugular and cephalic veins and use of DGC to isolate cells. Consistent yield of ECFCs from peripheral blood of horses will enable studies to evaluate diagnostic and therapeutic uses.

  11. High frequency of endothelial colony forming cells marks a non-active myeloproliferative neoplasm with high risk of splanchnic vein thrombosis.

    Directory of Open Access Journals (Sweden)

    Vittorio Rosti

    Full Text Available Increased mobilization of circulating endothelial progenitor cells may represent a new biological hallmark of myeloproliferative neoplasms. We measured circulating endothelial colony forming cells (ECFCs in 106 patients with primary myelofibrosis, fibrotic stage, 49 with prefibrotic myelofibrosis, 59 with essential thrombocythemia or polycythemia vera, and 43 normal controls. Levels of ECFC frequency for patient's characteristics were estimated by using logistic regression in univariate and multivariate setting. The sensitivity, specificity, likelihood ratios, and positive predictive value of increased ECFC frequency were calculated for the significantly associated characteristics. Increased frequency of ECFCs resulted independently associated with history of splanchnic vein thrombosis (adjusted odds ratio = 6.61, 95% CI = 2.54-17.16, and a summary measure of non-active disease, i.e. hemoglobin of 13.8 g/dL or lower, white blood cells count of 7.8×10(9/L or lower, and platelet count of 400×10(9/L or lower (adjusted odds ratio = 4.43, 95% CI = 1.45-13.49 Thirteen patients with splanchnic vein thrombosis non associated with myeloproliferative neoplasms were recruited as controls. We excluded a causal role of splanchnic vein thrombosis in ECFCs increase, since no control had elevated ECFCs. We concluded that increased frequency of ECFCs represents the biological hallmark of a non-active myeloproliferative neoplasm with high risk of splanchnic vein thrombosis. The recognition of this disease category copes with the phenotypic mimicry of myeloproliferative neoplasms. Due to inherent performance limitations of ECFCs assay, there is an urgent need to arrive to an acceptable standardization of ECFC assessment.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    1989-04-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  16. Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

    Science.gov (United States)

    Mena, Hebe Agustina; Zubiry, Paula Romina; Dizier, Blandine; Schattner, Mirta; Boisson-Vidal, Catherine; Negrotto, Soledad

    2018-05-02

    We have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease. We now analyze whether this strategy is also effective under adverse conditions for vasculogenesis, such as the presence of ischemia-related toxic molecules or diabetes, one of the main target diseases for cell therapy due to their well-known healing impairments. Cord blood-derived CD34 + cells were seeded in endothelial growth culture medium (EGM2) and ECFC colonies were obtained after 14-21 days. ECFC were exposed at pH 6.6 (preconditioned) or pH 7.4 (nonpreconditioned) for 6 h, and then pH was restored at 7.4. A model of type 2 diabetes induced by a high-fat and high-sucrose diet was developed in nude mice and hind limb ischemia was induced in these animals by femoral artery ligation. A P value < 0.05 was considered statistically significant (by one-way analysis of variance). We found that acidic preconditioning increased ECFC adhesion and the release of pro-angiogenic molecules, and protected ECFC from the cytotoxic effects of monosodium urate crystals, histones, and tumor necrosis factor (TNF)α, which induced necrosis, pyroptosis, and apoptosis, respectively. Noncytotoxic concentrations of high glucose, TNFα, or their combination reduced ECFC proliferation, stromal cell-derived factor (SDF)1-driven migration, and tubule formation on a basement membrane matrix, whereas almost no inhibition was observed in preconditioned ECFC. In type 2 diabetic mice, intravenous administration of preconditioned ECFC significantly induced blood flow recovery at the ischemic limb as measured by Doppler, compared with the phosphate-buffered saline (PBS) and nonpreconditioned ECFC groups. Moreover, the histologic analysis of gastrocnemius muscles showed an increased vascular density and reduced

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  18. Dysregulation of endothelial colony-forming cell function by a negative feedback loop of circulating miR-146a and -146b in cardiovascular disease patients.

    Directory of Open Access Journals (Sweden)

    Ting-Yu Chang

    Full Text Available Functional impairment of endothelial colony-forming cells (ECFCs, a specific cell lineage of endothelial progenitor cells (EPCs is highly associated with the severity of coronary artery disease (CAD, the most common type of cardiovascular disease (CVD. Emerging evidence show that circulating microRNAs (miRNAs in CAD patients' body fluid hold a great potential as biomarkers. However, our knowledge of the role of circulating miRNA in regulating the function of ECFCs and the progression of CAD is still in its infancy. We showed that when ECFCs from healthy volunteers were incubated with conditioned medium or purified exosomes of cultured CAD ECFCs, the secretory factors from CAD ECFCs dysregulated migration and tube formation ability of healthy ECFCs. It is known that exosomes influence the physiology of recipient cells by introducing RNAs including miRNAs. By using small RNA sequencing (smRNA-seq, we deciphered the circulating miRNome in the plasma of healthy individual and CAD patients, and found that the plasma miRNA spectrum from CAD patients was significantly different from that of healthy control. Interestingly, smRNA-seq of both healthy and CAD ECFCs showed that twelve miRNAs that had a higher expression in the plasma of CAD patients also showed higher expression in CAD ECFCs when compared with healthy control. This result suggests that these miRNAs may be involved in the regulation of ECFC functions. For identification of potential mRNA targets of the differentially expressed miRNA in CAD patients, cDNA microarray analysis was performed to identify the angiogenesis-related genes that were down-regulated in CAD ECFCs and Pearson's correlation were used to identify miRNAs that were negatively correlated with the identified angiogenesis-related genes. RT-qPCR analysis of the five miRNAs that negatively correlated with the down-regulated angiogenesis-related genes in plasma and ECFC of CAD patients showed miR-146a-5p and miR-146b-5p up

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Gözde Isik

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

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

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

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

    International Nuclear Information System (INIS)

    Rovirosa, Angeles; Ferre, Jorge; Biete, Albert

    1998-01-01

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    1992-05-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

    Yanagita, Manabu; Kobayashi, Ryohei; Murakami, Shinya

    2009-01-01

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

  8. Observations of radiation-induced chromosome fragment loss in live mammalian cells in culture, and its effect on colony-forming ability

    International Nuclear Information System (INIS)

    Grote, S.J.; Joshi, G.P.; Revell, S.H.; Shaw, C.A.

    1981-01-01

    A preceding paper (Grote, Joshi, Revell and Shaw 1981) describe a method for the direct scrutiny of live cultured mammalian cells with a microscope, and reported that all diploid Syrian hamster cells (BHK 21 C13) of a sample given 1.4 Gy of 220 kV X-rays in G1 reached post-radiation mitosis without discernible abnormality, but then diverged in observed behaviour: descendant cells from some first mitoses continued to proliferate normally while cells from other first mitoses behaved abnormally and produced either slow-growth or stop-growth colonies. This paper completes the study of the same irradiated cell sample, and shows that these post-mitotic differences in clonogenic ability were related to acentric chromosome fragment losses at post-radiation mitosis, which were detected in live daughter-cell pairs as micronuclei. The proportion of live daughter-cell pairs scored as deficient was at least 80 per cent of the proportion of comparable fixed-and-stained mitoses with detected acentric fragments. (author)

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

    Directory of Open Access Journals (Sweden)

    Jae-Yol Lim

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

  10. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Science.gov (United States)

    Tasev, Dimitar; van Wijhe, Michiel H; Weijers, Ester M; van Hinsbergh, Victor W M; Koolwijk, Pieter

    2015-01-01

    Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. The presented isolation method and subsequent cell expansion in platelet lysate

  11. Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

    Directory of Open Access Journals (Sweden)

    Dimitar Tasev

    Full Text Available Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.The findings presented in this study indicate that human platelet lysate (PL as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS. Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator or uPAR (uPA receptor by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.The presented isolation method and subsequent cell expansion in platelet

  12. The influence of interleukin-2, feeder cells, and timing of irradiation on the radiosensitivity of human T lymphocytes assessed by the colony-forming assay

    International Nuclear Information System (INIS)

    Gerber, M.; Guichard, M.; Pioch, Y.; Dubois, J.B.

    1989-01-01

    The radiosensitivity of human lymphocytes was investigated by the method of colony formation in the absence of interleukin-2 (IL2) and feeder cells, both of which enhance growth of T-cell colonies. The shape of the survival curve and the radiosensitivity was shown to depend upon the ability of lymphocytes to produce IL2: the survival curve for lymphocytes that were the most competent producers of IL2 is the closest to linearity; the lymphocytes that were poor producers show biphasic survival curves. The radiosensitivity of the lymphocytes from the first group is less than that of the latter, when the comparison is based on the first part of the biphasic survival curve. This is more easily seen when cultures are irradiated 24 h after stimulation by phytohemagglutinin (the time of the peak IL2 production) than when cultures are irradiated 2 h before stimulation. This study demonstrates that growth conditions influence the response of lymphocytes to irradiation and that optimal growth conditions result in a linear survival curve

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

    Science.gov (United States)

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

    2015-06-01

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

  14. Peripheral blood stem cell harvest in patients with limited stage small-cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Katakami, Nobuyuki; Takakura, Shunji; Fujii, Hiroshi; Nishimura, Takashi; Umeda, Bunichi [Kobe City General Hospital (Japan)

    2000-06-01

    Chemotherapy plus granulocyte colony-stimulating factor (G-CSF) induced mobilization of peripheral blood stem cells (PBSC) was performed in patients with limited stage small-cell lung cancer. Chemotherapy consisted of cisplatin/etoposide or cisplatin/adriamycin/etoposide. The amounts of CD34 positive cells and granulocyte-macrophage colony forming units (CFU-GM) collected during 2-3 courses of apheresis were 3.1{+-}2.9 x 10{sup 6}/kg (n=10) and 3.1{+-}1.5 x 10{sup 5}/kg (n=8) , respectively. Adequate amounts of PBSC were also harvested even in patients treated with concurrent chemoradiotherapy. Eight patients were successfully treated with high-dose chemotherapy consisting of ifosfamide, carboplatin and etoposide with PBSC transfusion. The patients'-bone marrow reconstruction was rapid and no treatment-related death was observed. (author)

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

    Science.gov (United States)

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

    1998-06-01

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

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

    Science.gov (United States)

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

    2003-02-01

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

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

    Science.gov (United States)

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

    2017-04-04

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

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

    Science.gov (United States)

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

    2014-06-01

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

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  20. Quantitative assay for the number of leukemic spleen colony forming unit in radiation-induced murine myeloid leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Nara, N [Tokyo Medical and Dental Univ. (Japan). School of Medicine; Bessho, M

    1981-11-01

    In mice with myelogenous leukemia, leukemic spleen colony forming units were assayed quantitatively. When 5 x 10/sup 3/ - 2 x 10/sup 4/ leukemic cells were transplanted to other mice of the same strain, a rectilinear relationship (p < 0.01) was found between the number of the cells transplanted and that of the colonies formed on the surface of the spleen. From these results, the authors considered that myelogenous leukemia in mice is an adequate model for acute myelogenous leukemia in human adults, and that the quantitative assay of the leukemic colony forming units can be used for sensitivity tests of antileukemic agents.

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

    Directory of Open Access Journals (Sweden)

    Sonali Singh

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Frangou Matrona

    2010-07-01

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

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

    Science.gov (United States)

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

    2015-07-01

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

  5. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    International Nuclear Information System (INIS)

    Kanakura, Y.; Kuriu, A.; Waki, N.; Nakano, T.; Asai, H.; Yonezawa, T.; Kitamura, Y.

    1988-01-01

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-09

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

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

    Science.gov (United States)

    Jücker, M; Feldman, R A

    1995-11-17

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

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

    Science.gov (United States)

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

    2016-03-01

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

  9. The influence of gender- and age-related differences in the radiosensitivity of hematopoietic progenitor cells detected in steady-state human peripheral blood

    International Nuclear Information System (INIS)

    Kato, Kengo; Kashiwakura, Ikuo; Kuwabara, Mikinori

    2011-01-01

    To investigate the importance of gender and aging on the individual radiosensitivity of lineage-committed myeloid hematopoietic stem/progenitor cells (HSPCs) detected in mononuclear cells (MNCs) of steady-state human peripheral blood (PB), the clonogenic survival of HPCs, including colony-forming unit-granulocyte macrophage; burst-forming unit-erythroid; colony-forming unit-granulocyte-erythroid-macrophage-megakaryocyte cells derived from MNCs exposed to 0.5 Gy and 2 Gy X-irradiation were estimated. MNCs were prepared from the buffy-coats of 59 healthy individual blood donors. The results showed that large individual differences exist in the number of HSPCs, as well as in the surviving fraction of cells. Furthermore, the number of progenitor cells strongly correlated with their surviving fraction, suggesting that the radiosensitivity of hematopoietic progenitor cells decreases with the number of cells in the 10 5 cells population. A statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of an individual, however, none of these correlations were observed after 2 Gy irradiation. No statistically significant difference was observed in individual radiosensitivity between males and females at either radiation dose. The present results indicated a correlation between the individual responsiveness of HSPCs to ionizing irradiation, especially to low dose irradiation, and aging. (author)

  10. Isolation of hemopoietic stem cell subsets from murine bone marrow: II. Evidence for an early precursor of day-12 CFU-S and cells associated with radioprotective ability

    International Nuclear Information System (INIS)

    Ploemacher, R.E.; Brons, N.H.

    1988-01-01

    Counterflow centrifugal elutriation (CCE) in combination with plastic adherence and fluorescence-activated cell sorting were used consecutively to enrich functionally different subpopulations of pluripotent hemopoietic stem cells (HSC) from mouse bone marrow. The nonadherent CCE fractions were labeled with wheat germ agglutinin (WGA)-fluorescein isothiocyanate (FITC) and sorted according to differences in fluorescence within various windows on the basis of forward (FLS) and perpendicular (PLS) light scatter. The sorted cells were then assayed for their (1) in vivo colony-forming ability (day-7 and day-12 spleen colony-forming units [CFU-S]), (2) radioprotective ability (RPA; 30-day survival), and (3) their ability to repopulate the bone marrow or spleen over a 13-day period with day-12 CFU-S, granulocyte-macrophage colony-forming units (CFU-GM), nucleated cells, or cells associated with RPA. The highest incidence of day-12 CFU-S and cells with RPA was obtained by sorting the most WGA-positive cells with relatively high PLS (enrichment, 50- to 200-fold), lowering the effective dose (ED 50/30) to an average of 80 cells. The separative procedure enabled hemopoietic stem cells that repopulate both bone marrow and spleen with secondary RPA cells, CFU-S-12, and CFU-GM to be enriched and separated from part of the RPA cells, CFU-S-12, and cells that reconstitute the cellularity of bone marrow and spleen. These data suggest that cells generating both day-12 CFU-S and RPA cells differ from day-12 CFU-S and RPA cells themselves on the basis of PLS characteristics and affinity for WGA. Such early stem cells have also been detected in sorted fractions meeting the FLS/PLS characteristics of lymphocytes

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

    Science.gov (United States)

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

    2017-12-05

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

  12. Post-UV colony-forming ability of normal fibroblast strains and of the xeroderma pigmentosum group G strain

    International Nuclear Information System (INIS)

    Barrett, S.F.; Tarone, R.E.; Moshell, A.N.; Ganges, M.B.; Robbins, J.H.

    1981-01-01

    In xeroderma pigmentosum, an inherited disorder of defective DNA repair, post-uv colony-forming ability of fibroblasts from patients in complementation groups A through F correlates with the patients' neurological status. The first xeroderma pigmentosum patient assigned to the recently discovered group G had the neurological abnormalities of XP. Researchers have determined the post-uv colony-forming ability of cultured fibroblasts from this patient and from 5 more control donors. Log-phase fibroblasts were irradiated with 254 nm uv light from a germicidal lamp, trypsinized, and replated at known densities. After 2 to 4 weeks' incubation the cells were fixed, stained and scored for colony formation. The strains' post-uv colony-forming ability curves were obtained by plotting the log of the percent remaining post-uv colony-forming ability as a function of the uv dose. The post-uv colony-forming ability of 2 of the 5 new normal strains was in the previously defined control donor zone, but that of the other 3 extended down to the level of the most resistant xeroderma pigmentosum strain. The post-uv colony-forming ability curve of the group G fibroblasts was not significantly different from the curves of the group D fibroblast strains from patients with clinical histories similar to that of the group G patient

  13. Hematopoietic stem cells from NOD mice exhibit autonomous behavior and a competitive advantage in allogeneic recipients.

    Science.gov (United States)

    Chilton, Paula M; Rezzoug, Francine; Ratajczak, Mariusz Z; Fugier-Vivier, Isabelle; Ratajczak, Janina; Kucia, Magda; Huang, Yiming; Tanner, Michael K; Ildstad, Suzanne T

    2005-03-01

    Type 1 diabetes is a systemic autoimmune disease that can be cured by transplantation of hematopoietic stem cells (HSCs) from disease-resistant donors. Nonobese diabetic (NOD) mice have a number of features that distinguish them as bone marrow transplant recipients that must be understood prior to the clinical application of chimerism to induce tolerance. In the present studies, we characterized NOD HSCs, comparing their engraftment characteristics to HSCs from disease-resistant strains. Strikingly, NOD HSCs are significantly enhanced in engraftment potential compared with HSCs from disease-resistant donors. Unlike HSCs from disease-resistant strains, they do not require graft-facilitating cells to engraft in allogeneic recipients. Additionally, they exhibit a competitive advantage when coadministered with increasing numbers of syngeneic HSCs, produce significantly more spleen colony-forming units (CFU-Ss) in vivo in allogeneic recipients, and more granulocyte macrophage-colony-forming units (CFU-GMs) in vitro compared with HSCs from disease-resistant controls. NOD HSCs also exhibit significantly enhanced chemotaxis to a stromal cell-derived factor 1 (SDF-1) gradient and adhere significantly better on primary stroma. This enhanced engraftment potential maps to the insulin-dependent diabetes locus 9 (Idd9) locus, and as such the tumor necrosis factor (TNF) receptor family as well as ski/sno genes may be involved in the mechanism underlying the autonomy of NOD HSCs. These findings may have important implications to understand the evolution of autoimmune disease and impact on potential strategies for cure.

  14. Nitric oxide-induced murine hematopoietic stem cell fate involves multiple signaling proteins, gene expression, and redox modulation.

    Science.gov (United States)

    Nogueira-Pedro, Amanda; Dias, Carolina C; Regina, Helena; Segreto, C; Addios, Priscilla C; Lungato, Lisandro; D'Almeida, Vania; Barros, Carlos C; Higa, Elisa M S; Buri, Marcus V; Ferreira, Alice T; Paredes-Gamero, Edgar Julian

    2014-11-01

    There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO(•)), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO(•) , one produced by endothelial cells stimulated with carbachol in vitro and another using the NO(•)-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in vivo. Two main NO(•) effects were observed: proliferation of HSCs-especially of the short-term HSCs-and its commitment and terminal differentiation to the myeloid lineage. NO(•)-induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki-67, upregulation of Notch-1, Cx43, PECAM-1, CaR, ERK1/2, Akt, p38, PKC, and c-Myc. NO(•)-induced HSCs differentiation was characterized by the increase in granulocytic-macrophage progenitors, granulocyte-macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA-3 and Ikz-3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO(•) in inducing HSCs proliferation and myeloid differentiation involving multiple signaling. © 2014 AlphaMed Press.

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

    KAUST Repository

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

    2013-01-01

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

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

    KAUST Repository

    Sugumar, Thennarasu

    2013-06-25

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

  17. In vitro expansion of the murine pluripotent hemopoietic stem cell population in response to interleukin 3 and interleukin 6. Application to bone marrow transplantation

    International Nuclear Information System (INIS)

    Okano, A.; Suzuki, C.; Takatsuki, F.

    1989-01-01

    The synergistic action of interleukin 6 with interleukin 3 on the proliferation of a murine hemopoietic stem cell population in a short-term liquid culture system was examined by radioprotective assay. The numbers of colony-forming units in spleen (CFU-S), together with granulocyte/macrophage colony-forming units and viable nucleated cells, were found to increase markedly in culture in the presence of both IL-3 and IL-6, compared with the presence of IL-3 or IL-6 alone. The peak CFU-S value in response to the combination of IL-3 and IL-6 was obtained 6 days after culture initiation, exceeding 5-fold of the input value. Consistent with these data, marrow cells cultured with both IL-3 and IL-6 for 6 days were shown to have a much higher capability of rescuing lethally irradiated mice than did controls. The results may portend the potential clinical use of the combination of IL-3 and IL-6, in particular, in bone marrow transplantation

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

    International Nuclear Information System (INIS)

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

    1990-01-01

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

  19. Involvement of placental/umbilical cord blood acid-base status and gas values on the radiosensitivity of human fetal/neonatal hematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Yamaguchi, Masaru; Ebina, Satoko; Kashiwakura, Ikuo

    2013-01-01

    Arterial cord blood (CB) acid-base status and gas values, such as pH, PCO 2 , PO 2 , HCO 3 - and base excess, provide useful information on the fetal and neonatal condition. However, it remains unknown whether these values affect the radiosensitivity of fetal/neonatal hematopoiesis. The present study evaluated the relationship between arterial CB acid-base status, gas values, and the radiosensitivity of CB hematopoietic stem/progenitor cells (HSPCs). A total of 25 CB units were collected. The arterial CB acid-base status and gas values were measured within 30 min of delivery. The CD34 + HSPCs obtained from CB were exposed to 2 Gy X-irradiation, and then assayed for colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte erythroid, macrophage and megakaryocyte cells. Acid-base status and gas values for PCO 2 and HCO 3 - showed a statistically significant negative correlation with the surviving fraction of BFU-E. In addition, a significant positive correlation was observed between gestational age and PCO 2 . Moreover, the surviving fraction of BFU-E showed a significant negative correlation with gestational age. Thus, HSPCs obtained from CB with high PCO 2 /HCO 3 - levels were sensitive to X-irradiation, which suggests that the status of arterial PCO 2 /HCO 3 - influences the radiosensitivity of fetal/neonatal hematopoiesis, especially erythropoiesis. (author)

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

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

    Science.gov (United States)

    Grant, Susan M; Heel, Rennie C

    1992-04-01

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

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

    Science.gov (United States)

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

    1998-08-14

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

  3. Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

    Science.gov (United States)

    Carlo-Stella, Carmelo; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Stucchi, Claudio; Cleris, Loredana; Formelli, Franca; Gianni, Massimo A

    2002-11-01

    Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P Defibrotide plus rhG-CSF resulted in a significant increase (P Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

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

    Science.gov (United States)

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

    1997-04-15

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

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  6. Clonal evolution following chemotherapy-induced stem cell depletion in cats heterozygous for glucose-6-phosphate dehydrogenase

    International Nuclear Information System (INIS)

    Abkowitz, J.L.; Ott, R.M.; Holly, R.D.; Adamson, J.W.

    1988-01-01

    The number of hematopoietic stem cells necessary to support normal hematopoiesis is not known but may be small. If so, the depletion or damage of such cells could result in apparent clonal dominance. To test this hypothesis, dimethylbusulfan [2 to 4 mg/kg intravenously (IV) x 3] was given to cats heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD). These cats were the daughters of domestic X Geoffroy parents. After the initial drug-induced cytopenias (2 to 4 weeks), peripheral blood counts and the numbers of marrow progenitors detected in culture remained normal, although the percentages of erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (CFU-GM) in DNA synthesis increased, as determined by the tritiated thymidine suicide technique. In three of six cats treated, a dominance of Geoffroy-type G-6-PD emerged among the progenitor cells, granulocytes, and RBCs. These skewed ratios of domestic to Geoffroy-type G-6-PD have persisted greater than 3 years. No changes in cell cycle kinetics or G-6-PD phenotypes were noted in similar studies in six control cats. These data suggest that clonal evolution may reflect the depletion or damage of normal stem cells and not only the preferential growth and dominance of neoplastic cells

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

    Science.gov (United States)

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

    1999-01-01

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

  8. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Science.gov (United States)

    Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

    2016-01-01

    Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003

  9. In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

    Directory of Open Access Journals (Sweden)

    Erika Evangelina Coronado-Cerda

    2016-01-01

    Full Text Available Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE or IMMUNEPOTENT CRP® (ICRP is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM, cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

  10. Radiosensitivity of human haematopoietic stem/progenitor cells

    International Nuclear Information System (INIS)

    Kato, Kengo; Kashiwakura, Ikuo; Omori, Atsuko

    2013-01-01

    The haematopoietic system is regenerative tissue with a high proliferative potential; therefore, haematopoietic stem cells (HSCs) are sensitive to extracellular oxidative stress caused by radiation and chemotherapeutic agents. An understanding of this issue can help predict haematopoietic recovery from radiation exposure as well as the extent of radiation damage to the haematopoietic system. In the present study, the radiosensitivity of human lineage-committed myeloid haematopoietic stem/progenitor cells (HSPCs), including colony-forming unit–granulocyte macrophage, burst-forming unit–erythroid and colony-forming unit–granulocyte–erythroid–macrophage–megakaryocyte cells, which are contained in adult individual peripheral blood (PB) and fetus/neonate placental/umbilical cord blood (CB), were studied. The PB of 59 healthy individual blood donors and the CB of 42 neonates were investigated in the present study. HSPCs prepared from PB and CB were exposed to 0.5 or 2 Gy x-irradiation. The results showed that large individual differences exist in the surviving fraction of cells. In the case of adult PB, a statistically significant negative correlation was observed between the surviving fraction observed at a dose of 0.5 Gy and the age of the blood donors; however, none of these correlations were observed after 2 Gy x-irradiation. In addition, seasonal and gender variation were observed in the surviving fraction of CB HSPCs. The present results suggest that there are large individual differences in the surviving fraction of HSPCs contained in both adult PB and fetus/neonate CB. In addition, some factors, including the gender, age and season of birth, affect the radiosensitivity of HSPCs, especially with a relatively low-dose exposure. (paper)

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

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...... and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced...

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

    Science.gov (United States)

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

    1999-12-01

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

  13. Mitigating the Effects of Xuebijing Injection on Hematopoietic Cell Injury Induced by Total Body Irradiation with γ rays by Decreasing Reactive Oxygen Species Levels

    Directory of Open Access Journals (Sweden)

    Deguan Li

    2014-06-01

    Full Text Available Hematopoietic injury is the most common side effect of radiotherapy. However, the methods available for the mitigating of radiation injury remain limited. Xuebijing injection (XBJ is a traditional Chinese medicine used to treat sepsis in the clinic. In this study, we investigated the effects of XBJ on the survival rate in mice with hematopoietic injury induced by γ ray ionizing radiation (IR. Mice were intraperitoneally injected with XBJ daily for seven days after total body irradiation (TBI. Our results showed that XBJ (0.4 mL/kg significantly increased 30-day survival rates in mice exposed to 7.5 Gy TBI. This effect may be attributable to improved preservation of white blood cells (WBCs and hematopoietic cells, given that bone marrow (BM cells from XBJ-treated mice produced more granulocyte-macrophage colony forming units (CFU-GM than that in the 2 Gy/TBI group. XBJ also decreased the levels of reactive oxygen species (ROS by increasing glutathione (GSH and superoxide dismutase (SOD levels in serum and attenuated the increased BM cell apoptosis caused by 2 Gy/TBI. In conclusion, these findings suggest that XBJ enhances the survival rate of irradiated mice and attenuates the effects of radiation on hematopoietic injury by decreasing ROS production in BM cells, indicating that XBJ may be a promising therapeutic candidate for reducing hematopoietic radiation injury.

  14. Peripheral blood cells from children with RASopathies show enhanced spontaneous colonies growth in vitro and hyperactive RAS signaling

    International Nuclear Information System (INIS)

    Gaipa, G; Bugarin, C; Cianci, P; Sarno, J; Bonaccorso, P; Biondi, A; Selicorni, A

    2015-01-01

    Germline mutations in genes coding for molecules involved in the RAS/RAF/MEK/ERK pathway are the hallmarks of a newly classified family of autosomal dominant syndromes termed RASopathies. Myeloproliferative disorders (MPDs), in particular, juvenile myelomonocytic leukemia, can lead to potentially severe complications in children with Noonan syndrome (NS). We studied 27 children with NS or other RASopathies and 35 age-matched children as control subjects. Peripheral blood (PB) cells from these patients were studied for in vitro colony-forming units (CFUs) activity, as well as for intracellular phosphosignaling. Higher spontaneous growth of both burst-forming units-erythroid (BFU-E) and CFU-granulocyte/macrophage (CFU-GM) colonies from RAS-mutated patients were observed as compared with control subjects. We also observed a significantly higher amount of GM-colony-stimulating factor-induced p-ERK in children with RASopathies. Our findings demonstrate for the first time that PB cells isolated from children suffering from NS or other RASopathies without MPD display enhanced BFU-E and CFU-GM colony formation in vitro. The biological significance of these findings clearly awaits further studies. Collectively, our data provide a basis for further investigating of only partially characterized hematological alterations present in children suffering from RASopathies, and may provide new markers for progression toward malignant MPD in these patients

  15. Direct evaluation of radiation damage in human hematopoietic progenitor cells in vivo

    International Nuclear Information System (INIS)

    Kyoizumi, Seishi; McCune, J.M.; Namikawa, Reiko

    1994-01-01

    We have developed techniques by which normal functional elements of human bone marrow can be implanted into immunodeficient C.B-17 scid/scid (SCID) mice. Afterward, long-term multilineage human hematopoiesis is sustained in vivo. We evaluated the effect of irradiation on the function of human bone marrow with this in vivo model. After whole-body X irradiation of the engrafted animals, it was determined that the D 0 value of human committed progenitor cells within the human marrow was 1.00 ± 0.09 (SEM) Gy for granulocyte-macrophage colony-forming units (CFU-GM) and 0.74 ± 0.12 Gy for erythroidburst-forming units (BFU-E). The effects of irradiation on the hematopoietic elements were reduced when the radioprotective agent WR-2721 was administered prior to irradiation. After low-dose irradiation, recovery of human granulocyte colony-stimulating factor (G-CSF). This small animal model may prove amenable for the analysis of the risk of the exposure of humans to irradiation as well as for the development of new modalities for the prevention and treatment of radiation-induced hematopoietic damage. 41 refs., 5 figs., 1 tab

  16. A synthetic fragment of leptin increase hematopoietic stem cell population and improve its engraftment ability.

    Science.gov (United States)

    Dias, Carolina C; Nogueira-Pedro, Amanda; Tokuyama, Paula Yumi; Martins, Marta N C; Segreto, Helena Regina Comodo; Buri, Marcus V; Miranda, Antonio; Paredes-Gamero, Edgar J

    2015-07-01

    Several studies have shown the important actions of cytokine leptin that regulates food intake and energy expenditure. Additionally, the ability to modulate hematopoiesis has also been demonstrated. Previous reports have shown that some synthetic sequences of leptin molecules can activate leptin receptor. Herein, decapeptides encompassing amino acids from positions 98 to 122 of the leptin molecule were constructed to evaluate their effects on hematopoiesis. Among them, the synthetic peptide Lep(110-119)-NH2 (LEP F) was the only peptide that possessed the ability to increase the percentage of hematopoietic stem cells (HSC). Moreover, LEP F also produced an increase of granulocyte/macrophage colony-forming units and activated leptin receptor. Furthermore, LEP F also improves the grafting of HSC in bone marrow, but did not accelerate the recovery of bone marrow after ablation with 5-fluorouracil. These results show that LEP F is a positive modulator of the in vivo expansion of HSC and could be useful in bone marrow transplantation. © 2015 Wiley Periodicals, Inc.

  17. Colony form variation of Bacillus pumilus E601 after cultured and neutron irradiation

    International Nuclear Information System (INIS)

    Chen Xiaoming; Wei Baoli; Zhang Jianguo

    2008-01-01

    The distribution of two colony forms of Bacillus pumilus E601 and the effect of neutron irradiation on the colony form were reported. The translucent and opaque colonies were cultured several generations to observe the proportion of two form colonies. The spores of opaque colony were irradiated at 80, 800 and 2000 Gy of fast neutron from CFBR-II pulse pile, and the survivors of opaque colony were irradiated again at the same doses. The results showed that: (1) Bacillus pumilus E601 observed two types of colony form: translucent and opaque colony; (2) the translucent colony could produce both translucent and opaque colonies in equal, while the opaque colony couldn't produce translucent colony generally; (3) neutron irradiation could affect the colony form distribution. The ratio of survival translucent colony was increased with the increase of the first neutron irradiation doses, and the second neutron irradiation also increased the ratio of translucent colony. It was concluded that the instability of translucent colony was the main reason to produce two colony forms of Bacillus pumilus E601. The strain of translucent colony had a stronger ability to resist neutron irradiation than the opaque colony. (authors)

  18. L-Carnitine halts apoptosis and myelosuppression induced by carboplatin in rat bone marrow cell cultures (BMC).

    Science.gov (United States)

    Abd-Allah, Adel R A; Al-Majed, Abdulhakeem A; Al-Yahya, Abdulaziz A; Fouda, Soliman I; Al-Shabana, Othman A

    2005-07-01

    Carboplatin (CP), a second generation platinum compound, is effective against various types of cancers, producing less nephrotoxicity and ototoxicity but more myelotoxicity than cisplatinum. CP-myelosuppression is the rate-limiting step of its clinical use. Prevention of CP-myelosuppression is a major target in the field of chemotherapy. Therefore, the present study investigates the use of L-carnitine (LCR)-an antioxidant, cardioprotective, neuroprotective, and immunostimulant nontoxic natural compound-to protect against CP-induced myelosuppression. The viability of BMC was studied using a trypan blue exclusion technique following incubation with CP and/or LCR as a function of time and concentration. Apoptosis was tested for by detecting the amount of DNA fragmentation and the visualization of DNA ladders upon gel electrophoresis. Bone marrow progenitor cell function was examined by colony forming unit assay. Cellular contents of glutathione (GSH) and malondialdehyde (MDA) were also estimated. Results revealed that LC50 of CP is 4.7 mM and the highest safe concentration of LCR is 5 mM. Co-exposure of LCR+CP rescued BMC viability by 37% compared to the CP-treated cultures. The LCR halts CP-induced apoptosis and it significantly improves the function of the bone marrow progenitors by increasing the number of colony-forming units as a response to granulocyte/macrophage colony stimulating factors. Finally, LCR restores CP-induced GSH depletion and prevents MDA elevation in BMC. In summary, the results suggest that LCR is able to protect against CP-induced myelosuppression, which suggests its use as an adjuvant therapy. This finding merits further investigation into the mechanism(s) of such protection as well as its interaction with CP antitumor activity.

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

    International Nuclear Information System (INIS)

    Bueren, J.A.; Nieto, M.

    1983-01-01

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

  20. In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

    Directory of Open Access Journals (Sweden)

    P.B. Soares

    2013-01-01

    Full Text Available Imatinib mesylate (IM is used to treat chronic myeloid leukemia (CML because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM. The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM, using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells increased. At higher concentrations (15 µM, the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control. Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

  1. Effect of Fluoride Mouthrinse and Toothpaste on Number of Streptococcal Colony Forming Units of Dental Plaque

    Directory of Open Access Journals (Sweden)

    SE Jabbarifar

    2005-11-01

    Full Text Available Background: Frequent topical fluoride therapy through toothpaste, mouthrinse, professional gels and solutions causes decrease in incidence, pause and repair of dental caries in the enamel. These mechanisms are done through penetration of fluoride ions (F- and their replacement with hydroxyl ions (OH- of hydroxyappatite of enamel, interfere with microbial metabolism of dental plaque and bacteriostatic effect on some cariogenic bacterial strains such as streptococci. The aim of this study was to examine the effect of fluoride mouthrinse and toothpaste on the number of streptococcal colony forming units of dental plaque. Methods: 62 children with 6-7 years old were put in two groups. Samples of dental plaque from each group were collected both before and after use of the fluoride mouthrinse and or toothpaste. The samples were cultured on blood agar to find the number of streptococcal colony forming units (CFU. The mean colony forming unit was compared inter and intra groups before and after application of Fluoride products. Results: The streptococcal CFU of dental plaque before and after use of the mouthrinse and toothpaste respectively was (1240±1367, 1253±1341.5 and (551±716, 898±1151. Statistically, the streptococcal CFU in each group before and after use of the toothpaste and mouthrinse was significantly different. Conclusion: The findings of this study indicated that the fluoride toothpaste and mouthrinse reduce number of streptococcal colony forming units of dental plaque. Also this reduction was not depended on level of (F- Ions, sort of vehicle of fluoride and frequent application of the fluoride mouthrinse and toothpaste. Keywords: fluoride mouthrinse, fluoride toothpaste, colony forming unit (CFU, streptococcus

  2. Differential effect of L3T4+ cells on recovery from total-body irradiation

    International Nuclear Information System (INIS)

    Pantel, K.; Nakeff, A.

    1990-01-01

    We have examined the importance of L3T4+ (murine equivalent to CD4+) cells for hematopoietic regulation in vivo in unperturbed mice and mice recovering from total-body irradiation (TBI) using a cytotoxic monoclonal antibody (MoAb) raised with the GK 1.5 hybridoma. Ablating L3T4+ cells in normal (unperturbed) B6D2F1 mice substantially decreased the S-phase fraction (determined by in vivo hydroxyurea suicide) of erythroid progenitor cells (erythroid colony-forming units, CFU-E) as compared to the pretreatment level (10% +/- 14.1% [day 3 following depletion] vs 79.8% +/- 15.9%, respectively) with a corresponding decrease in the marrow content of CFU-E at this time to approximately 1% of the pretreatment value. Although the S-phase fraction of CFU-GM was decreased to 2.2% +/- 3.1% 3 days after L3T4+ cell ablation from the 21.3% +/- 8.3% pretreatment value, CFU-GM cellularity showed little change over the 3 days following anti-L3T4 treatment. Anti-L3T4 MoAb treatment had little or no effect on either the S-phase fraction or the marrow content of hematopoietic stem cells (spleen colony-forming units, CFU-S) committed to myeloerythroid differentiation. Ablating L3T4+ cells prior to a single dose of 2 Gy TBI resulted in significantly reduced marrow contents of CFU-S on day 3 and granulocyte-macrophage colony-forming units (CFU-GM) on day 6 following TBI, with little or no effect on the corresponding recovery of CFU-E. The present findings provide the first in vivo evidence that L3T4+ cells are involved in: (1) maintaining the proliferative activity of CFU-E and CFU-GM in unperturbed mice and (2) supporting the restoration of CFU-S and CFU-GM following TBI-induced myelosuppression

  3. Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

    Science.gov (United States)

    Sakurai, T; Fujiwara, K

    2001-01-01

    1. In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose-dependent manner during a 72-h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a microM level of AsBe. 3. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72-h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml(-1)) of recombinant murine granulocyte/macrophage colony-stimulating factor (rMu GM-CSF), significant additive-like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony-forming assay using a gelatinous medium. 4. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bueren, J A; Nieto, M

    1983-07-01

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

  5. Capacity of bone marrow colony forming unit-fibroblasts in vitro from mice with combined radiation-burn injury

    International Nuclear Information System (INIS)

    Chen Xinghua; Luo Chengji; Guo Chaohua; Wang Ping

    1999-01-01

    Objective: To investigate the capacity of bone marrow colony forming unit-fibroblasts (CFU-F) from mice with combined radiation-burn injury. Methods: Mice were treated with 5.0 Gy γ-ray radiation alone, 15% total body surface area (TBSA) III degree burn alone or combined radiation-burn. The numbers of CFU-Fs were assayed by Dexter's method. Results: The numbers of CFU-Fs from mice with radiation and combined radiation-burn injury were significantly decreased, compared with those of controls and mice with burn injury alone (P<0.05-0.01). conclusion: The results reveal that the repairing process of bone marrow stromal cells from mice with radiation injury and combined radiation-burn injury is slow, and the combined radiation-burn injury inflicted on the stromal cells possesses the characteristic of radiation injury

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

    Science.gov (United States)

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

    2014-10-01

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

  7. Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: implications for autologous bone marrow purging

    International Nuclear Information System (INIS)

    Gulliya, K.S.; Pervaiz, S.

    1989-01-01

    Laser photoradiation therapy was tested in an in vitro model for its efficacy in the elimination of non-Hodgkin's lymphoma cells. Results show that at 31.2 J/cm2 of laser light in the presence of 20 micrograms/mL of merocyanine 540 (MC540) there was greater than 5 log reduction in Burkitt's lymphoma (Daudi) cells. Similar tumor cell kill was obtained for leukemia (HL-60) cells at a laser light dose of 93.6 J/cm2. However, to obtain the same efficiency of killing for histiocytic lymphoma (U-937) cells, a higher dose of MC540 (25 micrograms/mL) was required. Clonogenic tumor stem cell colony formation was reduced by greater than 5 logs after laser photoradiation therapy. Under identical conditions for each cell line the percent survival for granulocyte-macrophage colony-forming units (CFU-GM, 45.9%, 40%, 17.5%), granulocyte/erythroid/macrophage/megakaryocyte (GEMM, 40.1%, 20.1%, 11.5%), colony-forming units (CFU-C, 16.2%, 9.1%, 1.8%), and erythroid burst-forming units (BFU-E, 33.4%, 17.8%, 3.9%) was significantly higher than the tumor cells. Mixing of gamma ray-irradiated normal marrow cells with tumor cells (1:1 and 10:1 ratio) did not interfere with the elimination of tumor cells. The effect of highly purified recombinant interferon alpha (rIFN) on laser photoradiation therapy of tumor cells was also investigated. In the presence of rIFN (30 to 3,000 U/mL), the viability of leukemic cells was observed to increase from 0% to 1.5% with a concurrent decrease in membrane polarization, suggesting an increase in fluidity of cell membrane in response to rIFN. However, at higher doses of rIFN (6,000 to 12,000 U/mL) this phenomenon was not observed. The viability of lymphoma cells remained unaffected at all doses of rIFN tested

  8. Activation of adenosine A3 receptors potentiates stimulatory effects of IL-3, SCF, and GM-CSF on mouse granulocyte-macrophage hematopoietic progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Vacek, Antonín; Pospíšil, Milan; Holá, Jiřina; Štreitová, Denisa; Znojil, V.

    2009-01-01

    Roč. 58, č. 2 (2009), s. 247-252 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/06/0015; GA ČR(CZ) GA305/08/0158 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hematopoiesis * adenosine A3 receptor agonist * hematopoietic growth factors Subject RIV: BO - Biophysics Impact factor: 1.430, year: 2009

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

    NARCIS (Netherlands)

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

    1997-01-01

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

  10. Plasma levels of stromal cell-derived factor-1 (CXCL12) and circulating endothelial progenitor cells in women with idiopathic heavy menstrual bleeding.

    Science.gov (United States)

    Elsheikh, E; Andersson, E; Sylvén, C; Ericzon, B-G; Palmblad, J; Mints, M

    2014-01-01

    Do plasma levels of stromal cell-derived factor-1 (CXCL12, sometimes termed SDF-1) and the numbers of circulating endothelial progenitor cells (EPCs), EPC colony-forming units (EPC-CFU) and mature endothelial cells (ECs) differ between women with idiopathic heavy menstrual bleeding of endometrial origin (HMB-E) and controls and are they related to plasma levels of other angiogenic growth factors? Angiogenesis is altered in women with HMB-E, characterized by a reduction in mean plasma levels of CXCL12, a low number of EPCs-CFUs and a high level of circulating ECs. Plasma levels of CXCL12 are significantly higher during the proliferative than the secretory phase of the menstrual cycle in healthy women and exhibit a negative correlation with blood EPC-CFUs. A prospective cohort study in a university hospital setting. Between 2008 and 2009 10 HMB-E patients were recruited from Karolinska University Hospital. Ten healthy women were also included in the analysis. Ten healthy control women and 10 HMB-E patients, all with regular menstrual cycles, provided 4 blood samples during a single menstrual cycle: 2 in the proliferative phase, 1 at ovulation and 1 in the secretory phase. We assessed plasma levels of CXCL12, vascular endothelial growth factor A(165) (VEGFA), basic fibroblast growth factor (bFGF) and granulocyte and granulocyte-macrophage colony-stimulating factors by ELISA. We counted circulating EPC-CFUs by culture, and ECs and EPCs by flow cytometry and immunostaining for cell surface markers. Plasma levels of CXCL12 were significantly lower in HMB-E patients compared with control women (P Market Insurance. The authors have no conflict of interest to declare.

  11. Hemopoietic stem-cell compartment of the SCID mouse: Double-exponential survival curve after γ irradiation

    International Nuclear Information System (INIS)

    Taniguchi, Satoshi; Hirabayashi, Yoko; Inoue, Tohru; Kanisawa, Masayoshi; Sasaki, Hideki; Komatsu, Kenshi; Mori, K.J.

    1993-01-01

    It has been reported that SCID (severe combined immunodeficiency, scid/scid) mice are more radiosensitive than normal mice. In the present studies, graded doses of radiation were given to bone marrow cells from SCID mice, and double-exponential survival curves were observed for day-9 and day-12 colony-forming units in the spleen (CFU-S). Single-exponential curves were found for SCID CFU in in vitro assays for granulocyte/macrophage-CFUs and erythroid burst-forming units, as reported elsewhere. Since the size of this more resistant fraction seems to decrease with stem-cell maturation, the finding implies that this fraction is a primitive subpopulation of the stem-cell compartment. The mean lethal dose (D 0 ), however, of this less sensitive SCID CFU-S is much less than the D 0 of regular CFU-S in normal littermates. Spleen colonies produced by SCID bone marrow were relatively small and abortive. The size of these colonies decreased nearly exponentially with increasing doses of radiation. These colonies produced by the sensitive fraction have disappeared, being killed by a relatively low dose of radiation. This observation might account for the high lymphomagenesis arising from primitive hemopoietic stem cells in SCID mice, because the smallness of the colonies suggests that there is unrepaired or misrepaired damage. Furthermore, this less sensitive fraction might be a source of the open-quotes leakyclose quotes change of T and B cells, possibly due to the induction of an equivocal repair system which appears in the later stages of life in the SCID mice. 34 refs., 5 figs., 3 tabs

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

    International Nuclear Information System (INIS)

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

    1998-01-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  14. Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

    Energy Technology Data Exchange (ETDEWEB)

    Holbrook, S.T.; Ohls, R.K.; Schibler, K.R.; Yang, Y.C.; Christensen, R.D. (Univ. of Utah School of Medicine, Salt Lake City (USA))

    1991-05-15

    We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6.

  15. Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors

    International Nuclear Information System (INIS)

    Holbrook, S.T.; Ohls, R.K.; Schibler, K.R.; Yang, Y.C.; Christensen, R.D.

    1991-01-01

    We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst-forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6

  16. Hemopoietic regeneration in murine spleen following transfusion of normal and irradiated marrow: different response of granulocyte/macrophage and erythroid precursors

    International Nuclear Information System (INIS)

    Wangenheim, K.-H. von; Peterson, H.-P.; Huebner, G.E.; Feinendegen, L.E.

    1987-01-01

    To investigate cell proliferation in regenerating spleen, bone marrow of normal and gamma-irradiated donor mice (3 weeks after 5 Gy) was transfused into lethally irradiated recipients. In the donors and in the recipient spleens numbers of CFU-S and progenitor cells were determined. In the irradiated donors the progenitors were at control level after 3 weeks of recovery although CFU-S were still at 50% of control. Recipients of the irradiated marrow received therefore an increased proportion of progenitors. CFU-C appeared to be self-renewing and/or increased in number due to enhanced CFU-S differentiation, but not the erythroid progenitors. CFU-S self-renewal was reduced after 5 Gy. The data suggest that cell differentiation and maturation proceed during early splenic regeneration. The quantity of CFU-C does not necessarily mirror the situation in the stem cell compartment. (author)

  17. Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia

    NARCIS (Netherlands)

    Lowenberg, B; Boogaerts, MA; Daenen, SMGJ; Verhoef, GEG; Hagenbeek, A; Vellenga, E; Ossenkoppele, GJ; Huijgens, PC; Verdonck, LF; vanderLelie, J; Wielenga, JJ; Gmur, J; Gratwohl, A; Hess, U; Fey, MF; vanPutten, WLJ

    1997-01-01

    Purpose: The hematopoietic growth factors (HGFs) introduced into induction chemotherapy (CT) of acute myeloid leukemia (AML) might be of benefit to treatment outcome by at least two mechanisms. HGFs given on days simultaneously with CT might sensitize the leukemic cells and enhance their

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

    Science.gov (United States)

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

    1996-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Harrington KJ

    2016-11-01

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

  20. Granulocyte macrophage-colony stimulating factor (GM-CSF) and sucralfate in prevention of radiation-induced mucositis: a prospective randomized study

    International Nuclear Information System (INIS)

    Makkonen, Tuula A.; Minn, Heikki; Jekunen, Antti; Vilja, Pekka; Tuominen, Juhani; Joensuu, Heikki

    2000-01-01

    Purpose: To compare subcutaneously given molgramostim (GM-CSF) and sucralfate mouth washings to sucralfate mouth washings in prevention of radiation-induced mucositis. Methods and Materials: Forty head and neck cancer patients were randomly assigned to use either GM-CSF and sucralfate (n = 20) or sucralfate alone (n = 20) during radiotherapy. Sucralfate was used as 1.0 g mouth washing 6 times daily after the first 10 Gy of radiotherapy, and 150-300 μg GM-CSF was given subcutaneously. The grade of radiation mucositis and blood cell counts were monitored weekly. Salivary lactoferrin was measured as a surrogate marker for oral mucositis. Results: We found no significant difference between the molgramostim and the control groups in the oral mucositis grade, oral pain, use of analgesic drugs, weight loss, or survival. The median maximum neutrophil counts (median, 9.2 x 10 9 /L vs. 5.9 x 10 9 /L, p = 0.0005), eosinophil counts (median, 1.3 x 10 9 /L vs. 0.2 x 10 9 /L, p = 0.0004), and salivary lactoferrin concentrations were higher in patients who received GM-CSF. The most common toxicities in the GM-CSF plus sucralfate group were skin reactions at the GM-CSF injection site (65%), fever (30%), bone pain (25%), and nausea (15%), whereas the toxicity of sucralfate given alone was minimal. Conclusion: We found no evidence indicating that subcutaneously given GM-CSF reduces the severity of radiation-induced mucositis

  1. Efficacy and safety of granulocyte macrophage-colony stimulating factor (GM-CSF) on the frequency and severity of radiation mucositis in patients with head and neck carcinoma

    International Nuclear Information System (INIS)

    Kannan, V.; Bapsy, Poonamallee P.; Anantha, Naranappa; Doval, Dinesh Chandra; Vaithianathan, Hema; Banumathy, G.; Reddy, Krishnamurthy B.; Kumaraswamy, Saklaspur Veerappaiah; Shenoy, Ashok Mohan

    1997-01-01

    Purpose: Based on the clinical evidence of mucosal protection by GM-CSF during cytotoxic chemotherapy, a pilot study was undertaken to determine the safety and mucosal reaction of patients receiving GM-CSF while undergoing definitive conventional fractionated radiotherapy in head and neck carcinoma. Methods and Materials: Patients were considered eligible if buccal mucosa and oropharynx were included in the teleradiation field. Ten adult patients with squamous cell carcinoma of head and neck (buccal mucosa--8 and posterior (1(3)) tongue--2) were entered into the trial. Radiation therapy was delivered with telecobalt machine at conventional 2 Gy fraction and 5 fractions/week. The radiation portals consisted of two parallel opposing lateral fields. GM-CSF was given subcutaneously at a dose of 1 μg/kg body weight, daily, after 20 Gy until the completion of radiation therapy. Patients were evaluated daily for mucosal reaction, pain, and functional impairment. Results: The median radiation dose was 66 Gy. Eight patients received ≥60 Gy. The tolerance to GM-CSF was good. All 10 patients completed the planned daily dose of GM-CSF without interruption. Mucosal toxicity was Grade I in four patients till the completion of radiotherapy (dose range 50-66 Gy). Six patients developed Grade II reaction, fibrinous mucosal lesions of maximum size 1.0-1.5 cm, during radiotherapy. None developed Grade III mucositis. The maximum mucosal pain was Grade I during GM-CSF therapy. In two patients after starting GM-CSF the pain reduced in intensity. Functional impairment was mild to moderate. All patients were able to maintain adequate oral intake during the treatment period. Total regression of mucosal reaction occurred within 8 days following completion of radiotherapy. Conclusions: GM-CSF administration concurrently with conventional fractionated radiotherapy was feasible without significant toxicity. The acute side effects of radiotherapy namely mucositis, pain, and functional

  2. Lymphocyte colony forming units and its application to the study of radiosensitivity

    International Nuclear Information System (INIS)

    Ma Xiangrui; Wang Tao; Wang Hongyun

    1991-07-01

    Kinetics and radiosensitivity of human lymphocytes were studied by the techniques of monolayer agar culture and liquid culture in vitro. In the experiments of lymphocyte kinetics, PHA was designated as a motogen for T lymphocyte. LPS, MEBC and BSA were chosen as mitogens for B lymphocyte. The data from thses experiments showed that under the alone or combination stimulation of LPS, MRBC and BSA, B lymphocytes developed to form colonies in agar culture (0.3%) with the same manner. The stimulation of LPS to B lymphocytes was most significant. By the day 6 after seeding, the numbers of colonies in agar culture were maximal. Whereas the numbers decreased significantly by the day 8. The number of T lymphocyte colonies increased with culture time within 12 days. The peak of 3 H-TdR incorporation into T lymphocytes in liquid culture occured at 5th day after seeding. The data above-mentioned demonstrated that the kinetics of lymphocytes cultured in two kinds of environments were different. The studies of the radiosensitivity of T lymphocytes showed that the decreasing in the number of colonies and rate of 3 H-TdR incorporation varied in different dose ranges. In the range of 0∼1.0 Gy, r = -0.96, D 0 value was 1.71 Gy for TL-CFC in agar culture, r = -.96, D 0 value was 4.34 Gy for the proliferation T lymphocytes in liquid culture. In the range of 1.0∼6.0 Gy, r were -0.99 and -0.98, the D 0 were 5.88 and 7.36 Gy respectively. The declining tendency in colonies formed by BL-CFC was the same as that of TL-CFC, r = -0.97, for the range of 0∼1.0 Gy, r = -0.97, for the range of 1.0∼3.0, the D 0 values were 1.35 and 4.36 Gy respectively. The results from these experiments shown that the colony technique was a good method for the study in radiosensitivity

  3. Substitution of Soybean Meal and Cornmeal to Moisture, PH, Bacterial Colony Forming and Shelf Life of Rejected Duck Meatballs

    OpenAIRE

    Deni, Novia; Juliyarsi, Indri; Melia, S; Vermalida, W.A

    2013-01-01

    This study aimed to determine the effect of substitution of soybean meal with cornmeal to moisture, pH, bacterial colony forming and the shelf life of rejected duck meatballs. This research material using duck meat Coast (Indian Runner) salvage as much as 4000 grams were obtained from the Livestock Anduring Padang and soybean meal with Mungbean trademarks and cornmeal with cornstarch trademarks respectively of 600 grams were obtained at Raya Padang market. The research method used was experim...

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

    International Nuclear Information System (INIS)

    Bueren, J. A.; Nieto, M.

    1983-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Bueren, J A; Nieto, M

    1983-07-01

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

  6. Influence of radiation fractionation on survival of mice and spleen colony-forming units

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Bullis, J.E.

    1994-01-01

    C57B1/6 mice were given Gy X-rays fractionated in several ways. There was a cyclical pattern of animal survival which was correlated to the fractionation interval and which indicated a periodicity of 6 h. Ten grays given a single dose is fatal to 100% of the mice and depresses the CFU-S to about one per leg with no evidence of proliferation during the remaining life. Ten grays given in 2.5-Gy increments at 24-h intervals causes no fatalities and results in a similar CFU-S depression but is followed by an exponential increase in CFU-S over the ensuing 12 days. Although bone marrow from survivors of such treatment was comparable to control marrow in its capacity for short-term rescue, it was clearly, inferior in its capability for long-term rescue. The periodicity of 6 h suggests that the cells responsible for survival of the mice have been synchronized into more or less radiosensitive and radioresistant stages of the cell cycle as a result of the time between the 25-Gy increments. Implications for the CFU-S and long-term repopulating cells are discussed. 22 refs., 5 figs., 1 tab

  7. Production of humoral factors that stimulate spleen colony-forming units in mice irradiated with moderate doses of X rays

    International Nuclear Information System (INIS)

    Grande, T.; Gonzalez, J.; Tejero, C.; Maganto, G.; Bueren, J.A.

    1990-01-01

    The production of humoral factors that stimulate spleen colony-forming units (CFU-S) has been studied in irradiated mice using an in vivo diffusion chamber assay. The experiments show that a significant release of factors that stimulate CFU-S takes place in the first few days after irradiation with moderate doses of 1.5 or 5 Gy. In contrast, the release of significant amounts of these humoral factors was not seen in animals irradiated with either low (0.75 Gy) or high (10 Gy) doses of X rays. The correlation observed between the production of factors that stimulate the CFU-S and the hemopoietic regeneration kinetics of the irradiated mice suggests that these factors represent part of the physiological regulators controlling the proliferation of CFU-S

  8. 1. modification of the proliferative capacity of transplanted bone marrow colony forming units by changes in the host environment

    International Nuclear Information System (INIS)

    Beran, M.; Tribukait, B.

    1974-01-01

    Regulation of the proliferation of transplanted colony forming units (CFUs) was investigated in lethally irradiated mice, pretreated by methods known to accelerate hemopoietic recovery after sublethal irradiation. Prospective recipients were exposed to either hypoxia, vinblastine or priming irradiation and at different intervals thereafter lethally irradiated and transplanted with bone marrow. Repopulation of CFUs was determined by counting the number of splenic colonies in primary recipients or by retransplantation. Regeneration of grafted CFUs was greatly accelerated and their self-renewal capacity increased in mice grafted within 2 days after hypoxia. Also the number of splenic colonies formed by grafted syngeneic CFUs as well as by C57B1 parent CFUs growing in BC3F1 hosts was significantly increased. The effect was not dependent on the seeding efficiency of CFUs and apparently resulted from hypoxia induced changes in the hosts' physiological environment. Proliferative capacity of grafted CFUs increased remarkably in hosts receiving vinblastine 2 or 4 days prior to irradiation. Priming irradiation given 6 days before main irradiation accelerated, given 2 days before impaired regeneration of CFUs. The increased rate of regeneration was not related to the cellularity of hemopoietic organs at the time of transplantation. The growth of CFUs in diffusion chambers implanted into posthypoxic mice was only slightly improved which does indicate that the accelerated regeneration of CFUs in posthypoxic mice is mainly due to the changes in the hemopoietic microenvironment. A short conditioning of transplanted CFUs by host factor(s) was sufficient to improve regeneration. The results might suggest that the speed of hemopoietic regeneration depends on the number of CFUs being induced to proliferate shortly after irradiation, rather than on the absolute numbers of CFUs available to the organism. (author)

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

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Vacek, Antonín; Pospíšil, Milan; Weiterová, Lenka; Holá, Jiřina; Štreitová, Denisa; Znojil, V.

    2006-01-01

    Roč. 55, č. 5 (2006), s. 591-596 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/06/0015 Institutional research plan: CEZ:AV0Z50040507 Keywords : hematopoiesis * adenosine * cytokines Subject RIV: BO - Biophysics Impact factor: 2.093, year: 2006

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

    Science.gov (United States)

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

    2015-10-01

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

  11. SIZE FRACTIONS OF AMBIENT PARTICULATE MATTER INDUCE GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR IN HUMAN BRONCHIAL EPITHELIAL CELLS BY MITOGEN-ACTIVATED PROTEIN KINASE PATHWAYS. (R827351C004)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  12. Characterization of monocyte-derived dendritic cells maturated with IFN-alpha

    DEFF Research Database (Denmark)

    Svane, I M; Nikolajsen, K; Walter, M R

    2006-01-01

    Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). In order to obtain full functional capacity, maturation is required......, maturation with IFN-alpha has only a small effect on induction of autologous T-cell stimulatory capacity of the DC. However, an increase in DC allogeneic T-cell stimulatory capacity was observed. These data suggest that IFN-alpha has a potential as a maturation agent used in DC-based cancer vaccine trials...

  13. Tumor regression induced by intratumor therapy with a disabled infectious single cycle (DISC) herpes simplex virus (HSV) vector, DISC/HSV/murine granulocyte-macrophage colony-stimulating factor, correlates with antigen-specific adaptive immunity.

    Science.gov (United States)

    Ali, Selman A; Lynam, June; McLean, Cornelia S; Entwisle, Claire; Loudon, Peter; Rojas, José M; McArdle, Stephanie E B; Li, Geng; Mian, Shahid; Rees, Robert C

    2002-04-01

    Direct intratumor injection of a disabled infectious single cycle HSV-2 virus encoding the murine GM-CSF gene (DISC/mGM-CSF) into established murine colon carcinoma CT26 tumors induced a significant delay in tumor growth and complete tumor regression in up to 70% of animals. Pre-existing immunity to HSV did not reduce the therapeutic efficacy of DISC/mGM-CSF, and, when administered in combination with syngeneic dendritic cells, further decreased tumor growth and increased the incidence of complete tumor regression. Direct intratumor injection of DISC/mGM-CSF also inhibited the growth of CT26 tumor cells implanted on the contralateral flank or seeded into the lungs following i.v. injection of tumor cells (experimental lung metastasis). Proliferation of splenocytes in response to Con A was impaired in progressor and tumor-bearer, but not regressor, mice. A potent tumor-specific CTL response was generated from splenocytes of all mice with regressing, but not progressing tumors following in vitro peptide stimulation; this response was specific for the gp70 AH-1 peptide SPSYVYHQF and correlated with IFN-gamma, but not IL-4 cytokine production. Depletion of CD8(+) T cells from regressor splenocytes before in vitro stimulation with the relevant peptide abolished their cytolytic activity, while depletion of CD4(+) T cells only partially inhibited CTL generation. Tumor regression induced by DISC/mGM-CSF virus immunotherapy provides a unique model for evaluating the immune mechanism(s) involved in tumor rejection, upon which tumor immunotherapy regimes may be based.

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

    Science.gov (United States)

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

    2015-06-01

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

  15. Production of two hemopoietic growth factors is differentially regulated in single T lymphocytes activated with an anti-T cell receptor antibody

    DEFF Research Database (Denmark)

    Kelso, A; Owens, T

    1988-01-01

    A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred by microma......A method has been developed to measure the production by single activated T lymphocytes of two hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and multipotential CSF (multi-CSF or IL-3). When individual cells of the L3T4 (CD4)+ F23.1+ T cell clone E9.D4 were transferred...... by micromanipulation into wells coated with the monoclonal anti-T cell receptor antibody F23.1, up to 90% of cells produced CSF as detected by CSF-dependent hemopoietic cell lines. Production occurred in the absence of proliferation and did not require the addition of accessory cells or IL-2. Both the frequency of CSF......-producing cells and the average production per positive cell depended on the density of the immobilized stimulating ligand, indicating that the response of each cell is not an all-or-none phenomenon but varies with the strength of stimulation. Individual cells of the clone varied over a 100-fold range...

  16. [In vitro activity of human bone marrow cells after cryopreservation in liquid nitrogen for 21 - 25 years].

    Science.gov (United States)

    Huang, You-Zhang; Shen, Jian-Liang; Gong, Li-Zhong; Zheng, Pei-Hao; Liu, Yi; Yin, Wen-Jie; Cen, Jian; Wang, Ning; Zhao, De-Feng

    2010-02-01

    The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at -80 degrees C. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80 degrees C refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP (10% dimethylsulfonamide, 10% autologous plasma) or DMSO-HES-HuA (5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin) as cryoprotectant for 21 to 25 years. They were thawed in 38 degrees C. The cell sample frozen in -80 degrees C refrigerator was frozen at a low frozen speed of 1 degrees C/min which was the same as the programmed freezer before -30 degrees C. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38 degrees C, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and platelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stem cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly [(3.5 +/- 1.5)% versus (12.6 +/- 4.8)%], the hemolysis rate was lower [(3.3 +/- 1.6)% versus (23.1 +/- 5.1)%]. Osmotic fragility of erythrocytes in the former was not changed, but was dropped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophage colony forming units and long term culture-initiating cells were higher in the former than that in the latter [(96.1 +/- 1.8)%, (70.0 +/- 9.5)%, (49.2 +/- 10.9)%, (54.2 +/- 13.8)% versus (76.3 +/- 5.6)%, (52.7 +/- 8.1)%, (43.5 +/- 12.3)%, (47.2 +/- 13.6)% respectively]. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The

  17. In vitro repopulation of haemopoietic stem cells after irradiation

    International Nuclear Information System (INIS)

    Mori, K.J.; Kumagai, Keiko; Seto, Akira; Ito, Yohei

    1981-01-01

    A culture system was designed in which proliferation of the haemopoietic stem cells was supported by adherent 'stromal' cell colonies. Application of the culture system to studies on kinetic behaviour of the haemopoietic stem cells after irradiation revealed; i) bone marrow stromal cells were radiosensitive with D 0 = 95R, when measured as the capability to proliferate and form adherent cell colonies in vitro, ii) radiosensitivity of the pluripotent stem cells (CFUs) in vitro was within the range of the in vivo sensitivity, iii) irradiated bone marrow cells under in vitro condition could repopulate at the same rate as those under in vivo condition, thereby suggesting that the function related to the support of haemopoiesis was radioresistant, iv) concentrations of both CFUs and granulocyte-macrophage precursor cells (CFUc) were higher in the irradiated cultures than those in unirradiated control culture at 3 weeks after irradiation. (author)

  18. [The effects of an aroma candy on oral Candida albicans colony-forming units (CFU) and oral hygiene states in healthy elderly carrying Candida albicans].

    Science.gov (United States)

    Suzuki, Motofumi; Hayama, Kazumi; Takahashi, Miki; Ezawa, Kunio; Yamazaki, Masatoshi; Matsukawa, Taiji; Kishi, Akinobu; Satou, Nobuya; Abe, Shigeru

    2015-01-01

    In a preceding paper, we showed that aroma candy containing oligonol, capric acid, and cinnamon (cassia) powder had potent inhibitory activity against mycelial growth of Candida albicans in vitro and protective activity against murine oral candidiasis. In order to assess the effects of this candy (the test candy) on oral C. albicans colony-forming units (CFU) and oral hygiene states, a placebo-controlled double-blind crossover comparative study was performed. Twenty subjects were divided into two groups. One group ingested the test candy in the first 7 days followed by 2 weeks washing-off period, then ingested the placebo candy (control candy) for 7 days. The other group was vice versa. C. albicans CFU in all oral rinse samples from the subjects before and after 7 days ingestion of candy was measured. The degree of oral malodor in all subjects was monitored using a portable measuring instrument. The results showed no statistically significant difference between test-candy group and placebo group for C. albicans CFU. However, C. albicans CFU in test-candy group with>4,000 CFUs was significantly decreased after 7 days ingestion of test-candy (poral malodor in the test-candy group was significantly decreased after 7 days ingestion of test-candy (poral hygiene states indicated that in the test-candy group, oral malodor, glutinous feeling, and refreshing feeling significantly improved in comparison with control-candy group (poral health care of elderly carrying C. albicans.

  19. Comparative study of 5-fluorouracil and its derivative, N1-(2'-tetrahydrofuryl)-5-fluorouracil on endogenous colony forming units in spleens of mice

    International Nuclear Information System (INIS)

    Ueno, Yori

    1976-01-01

    The effect of intraperitoneal injection of N 1 -(2'-tetrahydrofuryl)-5-fluorouracil (FT-207) which is metabolized to 5-fluorouracil (5-FU) in vivo, on endogeneous colony forming units in the spleen (endogeneous CFU-s) was observed and compared with that of 5-FU. The 8 x 10 -4 m eqv of FT-207 injected in mice 20 minutes before x-rays irradiation of 450 rads suppressed the endogeneous CFU-s count to about 60% and about 5.5 x 10 -5 m eqv of 5-FU had the same effect. The suppression of FT-207 continued over 120 minutes and increased gradually. The effect of 5-FU was strong shortly after the injection but faded out for 120 minutes. FT-207 had almost no effect on the recovery constant (Blair) of endogeneous CFU-s for 3 days, whereas 5-FU reduced seriously, especially for the first 2 days. The differences in the effects of both compounds could not be explained on the base of the only differences in the metabolism of both compounds. (auth.)

  20. Enhanced radiosensitivity of cultured fibroblasts from ataxia telangiectasia heterozygotes manifested by defective colony-forming ability and reduced DNA repair replication after hypoxic γ-irradiation

    International Nuclear Information System (INIS)

    Paterson, M.C.; Anderson, A.K.; Smith, B.P.; Smith, P.J.

    1979-01-01

    We have measured the sensitivity to γ-ray inactivation of diploid skin fibroblasts cultured from 10 persons in four families with ataxia telangiectasia (AT). Persons heterozygous for AT, including parents of afflicted patients, are not as yet detectable by any specific clinical or laboratory marker but are believed to constitute a substantial portion of the middle-aged cancer population. In one AT family, fibroblast strains from both parents exhibited a colony-forming ability after hypoxic irradiation which was intermediate between that displayed by five control strains from normal children and that from the affected child. In the remaining three families, cultures from only one parent were available; one parental strain displayed an intermediate survival capacity as above, whereas the other two responded normally. The homozygous recessive strains from the five afflicted children in the four families were all equally hypersensitive to hypoxic γ-ray inactivation. The three presumed AT heterozygous strains that displayed intermediate rayiosensitivity also carried out γ-rad-induced DNA repair replication to an extent intermediate between those in normals and AT homozygotes. These findings suggest that a numerically significant, cancer-prone subpopulation of humans carrying one normal and one abnormal AT gene may also be moderately sensitive to lethal effects of hypoxic γ-rays due to a defect in the enzymatic repair of DNA

  1. Do different standard plate counting (IDF/ISSO or AOAC) methods interfere in the conversion of individual bacteria counts to colony forming units in raw milk?

    Science.gov (United States)

    Cassoli, L D; Lima, W J F; Esguerra, J C; Da Silva, J; Machado, P F; Mourão, G B

    2016-10-01

    This study aimed to establish the correlation between individual bacterial count (IBC) obtained by flow cytometry and the number of colony forming units (CFU) determined by standard plate count (SPC) in raw milk using two different reference methodologies: the methodology of the International Dairy Federation (IDF) - International Organization for Standardization (ISO) 4833, incubation for 72 h at 30°C and the methodology of the Association of Official Analytical Chemists (AOAC), incubation for 48 h at 35°C. For this, 100 bovine milk samples (80 ml) from different farms were collected in a sterile bottle and maintained refrigerated at 4°C and were delivered to the laboratory. In the laboratory, the samples were divided into two vials of 40 ml each. Then, half of the vials were forwarded for the SPC analysis, and the other half were analysed using the equipment BactoScan FC. The analyses by flow cytometry and SPC were performed at the same time (maximum deviation of +/- 1 h). To transform the data from IBC ml(-1) to CFU ml(-1) (IDF or AOAC methodology), a standard linear regression equation was used, as recommended by IDF/ISO-196. The difference between the reference methodologies affects the equation that transforms IBC into CFU and therefore the accuracy of the results. The results estimated by the equation using the ISO 4833 methodology were on average 0·18 log units higher than the results estimated using the equation using the AOAC methodology. After the comparison of the methodologies, it was concluded that there is an impact of the reference methodologies on the conversion of the results from IBC to CFU. Depending on the methodology adopted by each laboratory or country, there may not be equivalence in the results. Hence, the laboratories specialized in milk quality analysis that have changed their methodology for analysis, passing from the MAPA (AOAC) methodology to the IDF standard, need to develop new conversion equations to make their

  2. Generation of blood-derived dendritic cells in dogs with oral malignant melanoma.

    Science.gov (United States)

    Catchpole, B; Stell, A J; Dobson, J M

    2002-01-01

    Advances in treatment of human melanoma indicate that immunotherapy, particularly dendritic cell (DC) immunization, may prove useful. The aim of this study was to investigate whether blood-derived DCs could be generated from canine melanoma patients. Peripheral blood mononuclear cells were isolated from three such dogs and cultured with recombinant canine granulocyte-macrophage colony stimulating factor (GM-CSF), canine interleukin 4 and human Flt3-ligand for 7 days. The resulting cells demonstrated a typical dendritic morphology, and were enriched for cells expressing CD1a, CD11c and MHC II by flow cytometric analysis. Thus, canine blood-derived DCs can be generated in vitro and DC immunization should be feasible in dogs. Copyright Harcourt Publishers Ltd.

  3. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block

    DEFF Research Database (Denmark)

    Kirstetter, Peggy; Anderson, Kristina; Porse, Bo T

    2006-01-01

    Wnt signaling increases hematopoietic stem cell self-renewal and is activated in both myeloid and lymphoid malignancies, indicating involvement in both normal and malignant hematopoiesis. We report here activated canonical Wnt signaling in the hematopoietic system through conditional expression...... of hematopoietic stem cell function was associated with decreased expression of Cdkn1a (encoding the cell cycle inhibitor p21(cdk)), Sfpi1, Hoxb4 and Bmi1 (encoding the transcription factors PU.1, HoxB4 and Bmi-1, respectively) and altered integrin expression in Lin(-)Sca-1(+)c-Kit(+) cells, whereas PU.1...... of a stable form of beta-catenin. This enforced expression led to hematopoietic failure associated with loss of myeloid lineage commitment at the granulocyte-macrophage progenitor stage; blocked erythrocyte differentiation; disruption of lymphoid development; and loss of repopulating stem cell activity. Loss...

  4. Induction of various immune modulatory molecules in CD34(+) hematopoietic cells

    DEFF Research Database (Denmark)

    Umland, Oliver; Heine, Holger; Miehe, Michaela

    2004-01-01

    ), and intercellular adhesion molecule-1 (ICAM-1) in SUP(LPS)-stimulated KG-1a cells and up-regulation of interferon (IFN)-inducible T cell-chemoattractant, interleukin (IL)-8, macrophage-inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, RANTES, CD70, granulocyte macrophage-colony stimulating factor, and IL-1beta......, and IL-18 receptor was only detectable in CD34(+) BMCs. More importantly, CD34(+) BMCs stimulated by TNF-alpha also showed enhanced secretion of MCP-1, MIP-1alpha, MIP-1beta, and IL-8, and increased ICAM-1 protein expression could be detected in stimulated KG-1a cells and CD34(+) BMCs. Furthermore, we...

  5. Inhibitory effects of Piper betle on production of allergic mediators by bone marrow-derived mast cells and lung epithelial cells.

    Science.gov (United States)

    Wirotesangthong, Mali; Inagaki, Naoki; Tanaka, Hiroyuki; Thanakijcharoenpath, Witchuda; Nagai, Hiroichi

    2008-03-01

    The leaves of the Piper betle Linn. (Piperaceae) are used in traditional medicine and possess anti-oxidant, anti-bacterial, anti-fungal, anti-diabetic and radioprotective activities. However, little is known about their anti-allergic activity. Therefore, the effects of P. betle ethanolic extract (PE) on the production of histamine and granulocyte macrophage-colony-stimulating factor (GM-CSF) by murine bone marrow mast cells (BMMCs) and on the secretion of eotaxin and IL-8 by the human lung epithelial cell line, BEAS-2B, were investigated in vitro. PE significantly decreased histamine and GM-CSF produced by an IgE-mediated hypersensitive reaction, and inhibited eotaxin and IL-8 secretion in a TNF-alpha and IL-4-induced allergic reaction. The results suggest that P. betle may offer a new therapeutic approach for the control of allergic diseases through inhibition of production of allergic mediators.

  6. Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures

    International Nuclear Information System (INIS)

    Cashman, J.; Eaves, A.C.; Eaves, C.J.

    1985-01-01

    We have examined the cycling status of various classes of erythroid and granulopoietic progenitor populations maintained for many weeks in standard normal long-term human marrow cultures. These were initiated with a single inoculum of marrow aspirate and were routinely fed by weekly removal of half of the nonadherent cells and replacement of half of the growth medium. Progenitors of large erythroid colonies (more than eight erythroblast clusters) present in the nonadherent fraction and progenitors of small granulocyte/macrophage colonies (fewer than 500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at all times examined (28% to 63% kill following a 20-minute exposure to 20 microCi/mL of high specific activity 3 H-thymidine). In contrast, progenitors of large granulocyte/macrophage colonies (more than 500 cells) and progenitors of large erythroid colonies (more than eight erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the time of each weekly medium change and a proliferating state two to three days later (0% to 13% kill and 21% to 49% kill, respectively). Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hematopoietic progenitor cell types. Further, they indicate that the proliferative activity of the most primitive of these progenitors is regulated by stage-specific cell-cell interactions that are subject to manipulation

  7. Comparative evaluation of bone marrow cells morpho-functional activity in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors of the first and second generation

    Directory of Open Access Journals (Sweden)

    I. O. Zhaleyko

    2014-07-01

    Full Text Available The efficiency of using the culture techniques of research for monitoring the patient’s response to the treatment by tyrosine kinase inhibitors of the first and second generation is shown. Thus, the functional activity of bone marrow cells in patients having the optimal treatment response to inhibitors of tyrosine kinases was significantly lower compared with patients with the acquired resistance to the drug, and patients who had CML diagnosed for first time. Furthermore, for patients with the optimal response to the nilotinib therapy, numbers of colonies in semi-solid agar in vitro was lower, than in patients with the optimal response to imatinib. When the leukaemic cell clone becomes resistant to tyrosine kinase inhibitors, the prevalence of early cells of granulocyte-macrophage hematopoietic stem cells is observed in CFU culture which can be an important prognostic factor for choosing the appropriate treatment strategy.

  8. Effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors in murine bone marrow cells

    Energy Technology Data Exchange (ETDEWEB)

    Saitou, Mikio; Sirata, Katsutoshi; Yanai, Takanori; Tanaka, Satoshi; Onodera, Junichi; Otsu, Hiroshi; Sato, Fumiaki [Institute for Environmental Sciences, Department of Radiobiology, Rokkasho, Aomori (Japan)

    1999-07-01

    To evaluate effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors of cells, the dose dependency of the expression of cytokines, interleukin-6 (IL-6) and granulocyte-macrophage colony stimulating factor (GM-CSF), of mice is being measured at accumulated doses between 1 and 8 Gy, with the dose interval of 1 Gy. In the present work, specific-pathogen-free (SPF) C3H-HeN female mice were irradiated by {sup 137}Cs {gamma}-rays with the doses of 5-8 Gy at the dose rate of 20 mGy (22 h-day){sup -1}, and the expression of IL-6 and GM-CSF in bone marrow and spleen cells from the mice were measured semiquantitatively by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. (author)

  9. Effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors in murine bone marrow cells

    Energy Technology Data Exchange (ETDEWEB)

    Saitou, Mikio; Yamada, Yutaka; Shirata, Katsutoshi; Yanai, Takanori; Izumi, Jun; Tanaka, Satoshi; Onodera, Jun' ichi; Otsu, Hiroshi; Sato, Fumiaki [Institute for Environmental Sciences, Rokkasho, Aomori (Japan)

    2000-07-01

    To evaluate effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors of cells, the expression of cytokines, interleukin-6 (IL-6) and granulocyte-macrophage colony stimulating factor (GM-CSF), of mice is being measured at accumulated doses between 1 and 8 Gy, with the dose interval of 1 Gy. In the present work, ten specific-pathogen-free (SPF) C3H/HeN female mice per experimental group were irradiated with {sup 137}Cs {gamma}-rays with the doses of 1-4 Gy at the dose rate of 20 mGy/(22 h-day), and the expression of IL-6 and GM-CSF in bone marrow and spleen cells from the mice was measured semiquantitatively by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. (author)

  10. Effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors in murine bone marrow cells

    International Nuclear Information System (INIS)

    Saitou, Mikio; Yamada, Yutaka; Shirata, Katsutoshi; Yanai, Takanori; Izumi, Jun; Tanaka, Satoshi; Onodera, Jun'ichi; Otsu, Hiroshi; Sato, Fumiaki

    2000-01-01

    To evaluate effects of prolonged irradiation by low dose-rate ionizing radiation on the production of growth factors of cells, the expression of cytokines, interleukin-6 (IL-6) and granulocyte-macrophage colony stimulating factor (GM-CSF), of mice is being measured at accumulated doses between 1 and 8 Gy, with the dose interval of 1 Gy. In the present work, ten specific-pathogen-free (SPF) C3H/HeN female mice per experimental group were irradiated with 137 Cs γ-rays with the doses of 1-4 Gy at the dose rate of 20 mGy/(22 h-day), and the expression of IL-6 and GM-CSF in bone marrow and spleen cells from the mice was measured semiquantitatively by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. (author)

  11. Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine Fator estimulante de colônias de granu-lócitos e macrófagos (GM-CSF não aumenta a eficácia ou potência da vacina de subunidades da febre aftosa em suínos

    Directory of Open Access Journals (Sweden)

    Luizinho Caron

    2005-09-01

    Full Text Available Foot-and-mouth disease (FMD is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5 vector containing the FMDV capsid (P1-2A and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24. An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C, however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF. However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.A febre aftosa é uma das doenças mais temidas nos rebanhos em todo o mundo. A vacinação tem sido uma arma eficiente no controle da doença, no entanto há preocupações com as vacinas atualmente utilizadas incluindo a necessidade de instalações de alta segurança para a produção dessas vacinas e a falta de um teste de diagnóstico aprovado que faça distinção precisa entre animais vacinados dos infectados. Várias vacinas têm sido testadas contra a febre aftosa e uma dessas

  12. Effect of poly-A:U, dextran sulfate and yeast RNA on the bone marrow colony-forming ability in irradiated mice

    International Nuclear Information System (INIS)

    Chernyavskij, V.I.; Lysenko, A.I.; Kulakova, G.S.

    1977-01-01

    It has been shown, that poly-A:U, dextran sulfate and yeast RNA increased a number of endogenic colonies (COE) in the mouse spleen sublethally irradiated, as a result of, apparently, their mitogenic effect on proliferous COE. The preparations did not affect the number of exogenic colonies when introducting them together with transfer of syngenic cells of bone marrow, taken from the intact donors. Dextran sulfate increased 2.7 times the number of the endogenic colonies in the spleens of nonuniformly irradiated mice mainly due to the COE migration from protected bone marrow areas. The complex of poly-A:U and yest RNA in such experiment type were ineffective. One of the most important factors in the mechanism of the dextran sulfate adjuvant activity possibly is its ability to increase migration potencies of the stem blood-forming cells

  13. Immune responses of dendritic cells after acquiring antigen from apoptotic hepatocholangioma cells caused by γ-ray

    International Nuclear Information System (INIS)

    Wu Gang; Gu Hongguang; Han Benli; Pei Xuetao

    2002-01-01

    Objective: To investigate the induction of cytotoxic T lymphocytes (CTLs) in antitumor responsiveness and therapeutic effects after dendritic cells (DCs) acquired antigen from apoptotic hepatocholangioma cells. Methods: DCs from blood mononuclear cells that maintain the characteristics of immaturity-anti-gen-capturing and-processing capacity were established in vitro by using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4. Then, apoptosis in hepatocholangioma cells was induced with γ-radiation. The experimental groups included (1) co-culture of DCs, and apoptotic cancer cells and T cells; (2) co-culture of DCs necrotic cancer cells and T cells; (3) co-culture of DCs-cultured cancer cell and T cells. These cells were co-cultured for 7 days. DCs and T cell were enriched separately. Finally, antitumor response test was carried out. Results: These cells had typical dendritic morphology, expressed high levels of CD1a, B7 and acquired antigen from apoptotic cells caused by γ-rays and induced an increased T cell-stimulatory capacity in MLR. Conclusions: DCs obtained from blood mononuclear cells using GM-CSF and IL-4 and DCs can efficiently present antigen driven from apoptotic cells caused by γ-rays and induce T cells increasing obviously. It can probably become an effective approach of DC transduction with antigen

  14. Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells

    DEFF Research Database (Denmark)

    Kirstetter, Peggy; Schuster, Mikkel B; Bereshchenko, Oksana

    2008-01-01

    Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p...... penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.......42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete...

  15. Chemical structure of carbamoylating groups and their relationship to bone marrow toxicity and antiglioma activity of bifunctionally alkylating and carbamoylating nitrosoureas.

    Science.gov (United States)

    Ali-Osman, F; Giblin, J; Berger, M; Murphy, M J; Rosenblum, M L

    1985-09-01

    Although the antitumor effects of chloroethylnitrosoureas have been shown to be due primarily to DNA-DNA cross-linking by the alkylating moieties of these agents, the basis of the often accompanying bone marrow toxicity has been more controversial. We report on the relative bone marrow toxicity of four model nitrosoureas with different alkylating and carbamoylating activities: 1,3-bis(2-chloroethyl)-1-nitrosourea; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea; chlorozotozin, (2-[3-(2-chloroethyl)-3 -nitrosoureido]-2-deoxy-D-glucopyranose); and -3-(beta-D-glucopyranosyl)-1-nitrosourea. Inhibitions of DNA, RNA, and protein synthesis in murine bone marrow cells and of colony growth of myeloid precursor cells (granulocyte-macrophage colony-forming units) were used as in vitro end points of myelotoxicity. Further, we determined the antiglioma activity of the four nitrosoureas on two human gliomas in a clonogenic tumor cell assay and studied the effect of the non-nitrosourea carbamoylators potassium cyanate, chloroethyl isocyanate, cyclohexyl isocyanate, ethyl isocyanate, and ethyl isothiocyanate on granulocyte-macrophage colony-forming units. The results show that, at equivalent drug exposures, clonogenic glioma cell kill was significant and comparative for 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea, and chlorozotocin; 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea showed little activity. In contrast, granulocyte-macrophage colony-forming unit toxicity was low with chlorozotocin and 1-(2-chloroethyl)-3-(beta-D-glucopyranosyl)-1-nitrosourea and very high with 1,3-bis(2-chloroethyl)-1-nitrosourea and 1,3-bis(trans-4-hydroxycyclohexyl)-1-nitrosourea. Of the isocyanates, bone marrow toxicity was highest with chloroethyl isocyanate and cyclohexyl isocyanate, intermediate with ethyl isocyanate, and lowest with KOCN and ethyl isothiocyanate. Our results indicate that (a) bifunctional alkylation is essential for

  16. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    Directory of Open Access Journals (Sweden)

    David M Harris

    Full Text Available Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza and the growth factors (GF granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  17. Induction and identification of rabbit peripheral blood derived dendritic cells

    Science.gov (United States)

    Zhou, Jing; Yang, FuYuan; Chen, WenLi

    2012-03-01

    Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

  18. Granulocyte-Macrophage Colony-Stimulating Factor Gene-Modified Vaccines for Immunotherapy of Cancer

    Czech Academy of Sciences Publication Activity Database

    Bubeník, Jan

    1999-01-01

    Roč. 45, č. 4 (1999), s. 115-119 ISSN 0015-5500 R&D Projects: GA MZd NC45011; GA MZd NC5526; GA ČR GA312/98/0826; GA ČR GA312/99/0542 Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.493, year: 1999

  19. Comparative evaluation of rotary ProTaper, Profile, and conventional stepback technique on reduction in Enterococcus faecalis colony-forming units and vertical root fracture resistance of root canals.

    Science.gov (United States)

    Singla, Mamta; Aggarwal, Vivek; Logani, Ajay; Shah, Naseem

    2010-03-01

    The purpose of this in vitro study was to evaluate the effect of various root canal instrumentation techniques with different instrument tapers on cleaning efficacy and resultant vertical root fracture (VRF) strength of the roots. Fifty human mandibular first premolar roots were enlarged to ISO size 20, inoculated with Enterococcus faecalis [ATCC2912] for 72 hours and divided into 5 groups: group I: prepared with .02 taper hand instruments ISO size 40; group II: Profile .04 taper size 40; group III: Profile .06 taper size 40; group IV: ProTaper size F4; and group V (control group) further divided into: Va: with bacterial inoculation and no mechanical instrumentation; and Group Vb: neither bacterial inoculation nor mechanical instrumentation. Cleaning efficacy was evaluated in terms of reduction of colony forming units (CFUs). The VRF strength was evaluated using D11 spreader as wedge in an Instron testing machine. Root canals instrumented with ProTaper and 6% Profile instruments showed maximum reduction in CFUs, with statistically insignificant difference between them. The VRF resistance decreased in all instrumented groups. The difference of VRF between 2% and 4% taper Profile groups was statistically insignificant (P = .195). One-way analysis of variance showed that canals instrumented with ProTaper F4 showed maximum reduction in VRF resistance compared with control uninstrumented group. Profile 6% taper instruments offer the advantage of maximum debridement without significant reduction in root fracture resistance. Copyright 2010 Mosby, Inc. All rights reserved.

  20. Mast cell-deficient Kit(W-sh) "Sash" mutant mice display aberrant myelopoiesis leading to the accumulation of splenocytes that act as myeloid-derived suppressor cells.

    Science.gov (United States)

    Michel, Anastasija; Schüler, Andrea; Friedrich, Pamela; Döner, Fatma; Bopp, Tobias; Radsak, Markus; Hoffmann, Markus; Relle, Manfred; Distler, Ute; Kuharev, Jörg; Tenzer, Stefan; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Schild, Hansjörg; Schmitt, Edgar; Becker, Marc; Stassen, Michael

    2013-06-01

    Mast cell-deficient Kit(W-sh) "sash" mice are widely used to investigate mast cell functions. However, mutations of c-Kit also affect additional cells of hematopoietic and nonimmune origin. In this study, we demonstrate that Kit(W-sh) causes aberrant extramedullary myelopoiesis characterized by the expansion of immature lineage-negative cells, common myeloid progenitors, and granulocyte/macrophage progenitors in the spleen. A consistent feature shared by these cell types is the reduced expression of c-Kit. Populations expressing intermediate and high levels of Ly6G, a component of the myeloid differentiation Ag Gr-1, are also highly expanded in the spleen of sash mice. These cells are able to suppress T cell responses in vitro and phenotypically and functionally resemble myeloid-derived suppressor cells (MDSC). MDSC typically accumulate in tumor-bearing hosts and are able to dampen immune responses. Consequently, transfer of MDSC from naive sash mice into line 1 alveolar cell carcinoma tumor-bearing wild-type littermates leads to enhanced tumor progression. However, although it can also be observed in sash mice, accelerated growth of transplanted line 1 alveolar cell carcinoma tumors is a mast cell-independent phenomenon. Thus, the Kit(W-sh) mutation broadly affects key steps in myelopoiesis that may have an impact on mast cell research.

  1. Protection from radiation injury through oral administration of PF4 gene carried by attenuated salmonella

    International Nuclear Information System (INIS)

    Zhao Lihua; Liu Bin; Yu Xiaofei; Zhang Lei; Han Zhongchao

    2005-01-01

    Objective: To investigate the in vivo radiation protection effect of PF4 by oral administration of attenuated salmonella as the carrier in mice. Methods: The eukaryotic vector pIRES2-EGFP-carried PF4 gene was transferred into an aroA-autotrophic mutant of salmonella typhimurium (SL3261), which was administered orally to BALBPc mice at 1x10 8 PFu once every interval three days. At 12 hours after the third oral administration the mice were subjected to a total body irradiation (TBI) of 700 cGy by a 60 Co source. The protective effect of SL3261/PF4 was determined by detection GFP ( green fluorescence protein) expression in tissues, peripheral blood count, culture of bone marrow colony-forming cells and survival time of mice. Results: Expression of GFP could be detected in the liver, spleen, intestine, kidney, peripheral blood and bone marrow. On days 7 and 14 after irradiation, Compared to controls, there were obvious differences in number of bone marrow mononuclear cells, CFU-GM (granulocyte-macrophage colony-forming unit ) and HPP-CFC (high proliferating potential-colony-forming cells) of mice treated with SL3261/PF4 (P<0.05) as well as prolongation of the survival time. Conclusion: These data demonstrate for the first time that PF4 protects mice from TBI injury and accelerates recovery of hematopoiesis by oral administration of attenuated salmonella carrying PF4 gene. (authors)

  2. T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

    Science.gov (United States)

    van Langelaar, Jamie; van der Vuurst de Vries, Roos M; Janssen, Malou; Wierenga-Wolf, Annet F; Spilt, Isis M; Siepman, Theodora A; Dankers, Wendy; Verjans, Georges M G M; de Vries, Helga E; Lubberts, Erik; Hintzen, Rogier Q; van Luijn, Marvin M

    2018-05-01

    Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4

  3. In vitro effects of recombinant human stem cell factor on hematopoietic cells from patients with acute radiation sickness

    International Nuclear Information System (INIS)

    Li Chuansheng; Cheng Tao; Xu Yanqun

    1994-01-01

    The effects of rhSCF, rhPIXY 321, rhGM-CSF and rhIL-3 on clonal proliferation of hematopoietic cells from five cases of acute radiation sickness were studied. The results showed that rhSCF could stimulate clonal proliferation of normal hematopoietic cells and the best results were obtained when the concentration of rhSCF was 5 x 10 4 ng/L. Clonal proliferation of hematopoietic cells from four cases of acute radiation sickness was stimulated while that from one case was inhibited. Moreover, the responsiveness of cells to rhSCF was correlated with the doses of radiation. Analysis of cell surface antigen, cell morphology and histochemistry revealed that rhSCF promoted predominantly the proliferation of granulocyte-macrophage lineage. rhSCF in combination with other three factors could further enhance the clonal proliferation of hematopoietic cells. The effects of rhPIXY 321, a fusion protein of GM-CSF and IL-3, were also analysed and found it to be a novel valuable hematopoietic growth factor

  4. CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

    Science.gov (United States)

    Ramberger, Melanie; Högl, Birgit; Stefani, Ambra; Mitterling, Thomas; Reindl, Markus; Lutterotti, Andreas

    2017-03-01

    Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients. © Sleep Research Society 2016. Published by Oxford University Press [on behalf of the Sleep Research Society].

  5. Production of Multiple Growth Factors by a Newly Established Human Thyroid Carcinoma Cell Line

    Science.gov (United States)

    Yoshida, Yataro; Ohashi, Kensaku; Sano, Emiko; Kobayashi, Hisataka; Endo, Keigo; Naruto, Masanobu; Nakamura, Toru

    1992-01-01

    A multiple growth factor‐producing tumor cell line (NIM‐1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM‐1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM‐1‐conditioned medium (NIM‐1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony‐stimulating factor (CSF)‐dependent cell lines, NFS60‐KX and TF‐1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte‐CSF (G‐CSF), granulocyte/macrophage‐CSF (GM‐CSF) and interleukin(IL)‐6 mRNAs in NIM‐1 cells. Enzyme‐linked immunosorbent assays (ELISA) using NIM‐1CM also confirmed the production of IL‐la and a small amount of IL‐1β besides G‐CSF, GM‐CSF and IL‐6 in NIM‐1 cells. In addition, unexpected production of IL‐11 in NIM‐1 cells was detected by northern blot hybridization analysis and by bioassay using an IL‐11‐dependent cell line. Therefore, NIM‐1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM‐CSF, IL‐6 and IL‐11. PMID:1372885

  6. Play the Immune System Defender Game

    Science.gov (United States)

    ... Questionnaire The Immune System Play the Immune System Game About the game Granulocytes, macrophages and dendritic cells are immune cells ... last will in Paris. Play the Blood Typing Game Try to save some patients and learn about ...

  7. Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity.

    Science.gov (United States)

    Westerterp, Marit; Gautier, Emmanuel L; Ganda, Anjali; Molusky, Matthew M; Wang, Wei; Fotakis, Panagiotis; Wang, Nan; Randolph, Gwendalyn J; D'Agati, Vivette D; Yvan-Charvet, Laurent; Tall, Alan R

    2017-06-06

    Autoimmune diseases such as systemic lupus erythematosus (SLE) are associated with increased cardiovascular disease and reduced plasma high-density lipoprotein (HDL) levels. HDL mediates cholesterol efflux from immune cells via the ATP binding cassette transporters A1 and G1 (ABCA1/G1). The significance of impaired cholesterol efflux pathways in autoimmunity is unknown. We observed that Abca1/g1-deficient mice develop enlarged lymph nodes (LNs) and glomerulonephritis suggestive of SLE. This lupus-like phenotype was recapitulated in mice with knockouts of Abca1/g1 in dendritic cells (DCs), but not in macrophages or T cells. DC-Abca1/g1 deficiency increased LN and splenic CD11b + DCs, which displayed cholesterol accumulation and inflammasome activation, increased cell surface levels of the granulocyte macrophage-colony stimulating factor receptor, and enhanced inflammatory cytokine secretion. Consequently, DC-Abca1/g1 deficiency enhanced T cell activation and T h 1 and T h 17 cell polarization. Nlrp3 inflammasome deficiency diminished the enlarged LNs and enhanced T h 1 cell polarization. These findings identify an essential role of DC cholesterol efflux pathways in maintaining immune tolerance. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  9. Enhancers of Polycomb EPC1 and EPC2 sustain the oncogenic potential of MLL leukemia stem cells

    Science.gov (United States)

    Huang, Xu; Spencer, Gary J; Lynch, James T; Ciceri, Filippo; Somerville, Tim D D; Somervaille, Tim C P

    2013-01-01

    Through a targeted knockdown (KD) screen of chromatin regulatory genes we identified the EP400 complex components EPC1 and EPC2 as critical oncogenic co-factors in acute myeloid leukemia (AML). EPC1 and EPC2 were required for the clonogenic potential of human AML cells of multiple molecular subtypes. Focusing on MLL-mutated AML as an exemplar, Epc1 or Epc2 KD induced apoptosis of murine MLL-AF9 AML cells and abolished leukemia stem cell potential. By contrast, normal hematopoietic stem and progenitor cells (HSPC) were spared. Similar selectivity was observed for human primary AML cells versus normal CD34+ HSPC. In keeping with these distinct functional consequences, Epc1 or Epc2 KD induced divergent transcriptional consequences in murine MLL-AF9 granulocyte-macrophage progenitor-like (GMP) cells versus normal GMP, with a signature of increased MYC activity in leukemic but not normal cells. This was caused by accumulation of MYC protein and was also observed following KD of other EP400 complex genes. Pharmacological inhibition of MYC:MAX dimerization, or concomitant MYC KD, reduced apoptosis following EPC1 KD, linking the accumulation of MYC to cell death. Therefore EPC1 and EPC2 are components of a complex which directly or indirectly serves to prevent MYC accumulation and AML cell apoptosis, thus sustaining oncogenic potential. PMID:24166297

  10. Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation.

    LENUS (Irish Health Repository)

    Rishi, Loveena

    2014-04-10

    The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein α (C\\/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop for E2F1, C\\/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C\\/EBPα-p42, and in normal granulocyte\\/macrophage progenitor cells, we detect C\\/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle or Trib2 knockdown resulted in a block in AML cell proliferation. Our work proposes a novel paradigm whereby E2F1 plays a key role in the regulation of Trib2 expression important for AML cell proliferation control. Importantly, we identify the contribution of dysregulated C\\/EBPα and E2F1 to elevated Trib2 expression and leukemic cell survival, which likely contributes to the initiation and maintenance of AML and may have significant implications for normal and malignant hematopoiesis.

  11. Optimization of Ex Vivo Murine Bone Marrow Derived Immature Dendritic Cells: A Comparative Analysis of Flask Culture Method and Mouse CD11c Positive Selection Kit Method

    Directory of Open Access Journals (Sweden)

    Rahul Ashok Gosavi

    2018-01-01

    Full Text Available 12–14 days of culturing of bone marrow (BM cells containing various growth factors is widely used method for generating dendritic cells (DCs from suspended cell population. Here we compared flask culture method and commercially available CD11c Positive Selection kit method. Immature BMDCs’ purity of adherent as well as suspended cell population was generated in the decreasing concentration of recombinant-murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF in nontreated tissue culture flasks. The expression of CD11c, MHCII, CD40, and CD86 was measured by flow cytometry. We found significant difference (P<0.05 between the two methods in the adherent cells population but no significant difference was observed between the suspended cell populations with respect to CD11c+ count. However, CD11c+ was significantly higher in both adhered and suspended cell population by culture method but kit method gave more CD11c+ from suspended cells population only. On the other hand, using both methods, immature DC expressed moderate level of MHC class II molecules as well as low levels of CD40 and CD86. Our findings suggest that widely used culture method gives the best results in terms of yield, viability, and purity of BMDCs from both adherent and suspended cell population whereas kit method works well for suspended cell population.

  12. Mobilizing stem cells from normal donors: is it possible to improve upon G-CSF?

    Science.gov (United States)

    Cashen, A F; Lazarus, H M; Devine, S M

    2007-05-01

    Currently, granulocyte colony stimulating factor (G-CSF) remains the standard mobilizing agent for peripheral blood stem cell (PBSC) donors, allowing the safe collection of adequate PBSCs from the vast majority of donors. However, G-CSF mobilization can be associated with some significant side effects and requires a multi-day dosing regimen. The other cytokine approved for stem cell mobilization, granulocyte-macrophage colony stimulating factor (GM-CSF), alters graft composition and may reduce the development of graft-versus-host disease, but a significant minority of donors fails to provide sufficient CD34+ cells with GM-CSF and some experience unacceptable toxicity. AMD3100 is a promising new mobilizing agent, which may have several advantages over G-CSF for donor mobilization. As it is a direct antagonist of the interaction between the chemokine stromal-derived factor-1 and its receptor CXCR4, AMD3100 mobilizes PBSCs within hours rather than days. It is also well tolerated, with no significant side effects reported in any of the clinical trials to date. Studies of autologous and allogeneic transplantation of AMD3100 mobilized grafts have demonstrated prompt and stable engraftment. Here, we review the current state of stem cell mobilization in normal donors and discuss novel strategies for donor stem cell mobilization.

  13. NF-kappa B activity in T cells stably expressing the Tax protein of human T cell lymphotropic virus type I

    International Nuclear Information System (INIS)

    Lacoste, J.; Cohen, L.; Hiscott, J.

    1991-01-01

    The effect of constitutive Tax expression on the interaction of NF-κ B with its recognition sequence and on NF-κ B-dependent gene expression was examined in T lymphoid Jurkat cell lines (19D and 9J) stably transformed with a Tax expression vector. Tax expressing T cell lines contained a constitutive level of NF-κ B binding activity, detectable by mobility shift assay and uv cross-linking using a palindromic NF-κ B probe homologous to the interferon beta PRDII site. In Jurkat and NC2.10 induction with phorbol esters resulted in the appearance of new DNA binding proteins of 85, 75, and 54 kDa, whereas in Tax expressing cells the 85-kDa protein and a 92-kDa DNA binding protein were constitutively induced. Expression of Tax protein in 19D and 9J resulted in transcription of the endogenous NF-kappa B-dependent granulocyte-macrophage colony stimulating factor gene and increased basal level expression of transfected NF-kappa B-regulated promoters. Nonetheless transcription of both the endogenous and the transfected gene was inducible by PMA treatment. Tax expression in Jurkat T cells may alter the stoichiometry of NF-kappa B DNA binding proteins and thus change the expression of NF-kappa B-regulated promoters

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

    Science.gov (United States)

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

    2014-01-01

    Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2. Safety, mechanism, immunoeffectiveness, and suggested benefit were previously shown [Senzer et al.: Mol Ther 2012;20:679-689; Senzer et al.: J Vaccines Vaccin 2013;4:209]. We now provide further follow-up of a subset of 8 HCC patients. FANG manufacturing was successful in 7 of 8 attempts (one failure due to insufficient cell yield). Median GM-CSF expression was 144 pg/10(6) cells, TGFβ1 knockdown was 100%, and TGFβ2 knockdown was 93% of the vector-transported cells. Five patients were vaccinated (1 or 2.5×10(7) cells/intradermal injection, 6-11 vaccinations). No FANG toxicity was observed. Three of these patients demonstrated evidence of an immune response to the autologous tumor cell sample. Long-term follow-up demonstrated survival of 319, 729, 784, 931+, and 1,043+ days of the FANG-treated patients. In conclusion, evidence supports further assessment of the FANG immunotherapy in HCC. © 2014 S. Karger AG, Basel.

  15. Karyotype of cryopreserved bone marrow cells

    Directory of Open Access Journals (Sweden)

    M.L.L.F. Chauffaille

    2003-07-01

    Full Text Available The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis. Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05. Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05. GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

  16. Karyotype of cryopreserved bone marrow cells.

    Science.gov (United States)

    Chauffaille, M L L F; Pinheiro, R F; Stefano, J T; Kerbauy, J

    2003-07-01

    The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

  17. Cordyceps sinensis health supplement enhances recovery from taxol-induced leukopenia.

    Science.gov (United States)

    Liu, Wei-Chung; Chuang, Wei-Ling; Tsai, Min-Lung; Hong, Ji-Hong; McBride, William H; Chiang, Chi-Shiun

    2008-04-01

    This study aimed to evaluate the ability of the health food supplement Cordyceps sinensis (CS) to ameliorate suppressive effects of chemotherapy on bone marrow function as a model for cancer treatment. Mice were treated with Taxol (17 mg/kg body wt) one day before oral administration of a hot-water extract of CS (50 mg/kg daily) that was given daily for 3 weeks. White blood cell counts in peripheral blood of mice receiving Taxol were at 50% of normal levels on day 28 but had recovered completely in mice treated with CS. In vitro assays showed that CS enhanced the colony-forming ability of both granulocyte macrophage colony forming unit (GM-CFU) and osteogenic cells from bone marrow preparations and promoted the differentiation of bone marrow mesenchymal stromal cells into adipocytes, alkaline phosphatase-positive osteoblasts, and bone tissue. This result could be attributed to enhanced expression of Cbfa1 (core binding factor a) and BMP-2 (bone morphogenetic protein) with concurrent suppression of ODF (osteoclast differentiation factor/RANK [receptor activator of NF-kappaB]) ligand. In summary, CS enhances recovery of mice from leukopenia caused by Taxol treatment. It appears to do so by protecting both hematopoietic progenitor cells directly and the bone marrow stem cell niche through its effects on osteoblast differentiation.

  18. Squalene Selectively Protects Mouse Bone Marrow Progenitors Against Cisplatin and Carboplatin-Induced Cytotoxicity In Vivo Without Protecting Tumor Growth

    Directory of Open Access Journals (Sweden)

    Bikul Das

    2008-10-01

    Full Text Available Squalene, an isoprenoid antioxidant is a potential cytoprotective agent against chemotherapy-induced toxicity. We have previously published that squalene protects light-density bone marrow cells against cis-diamminedichloroplatinum( II (cisplatin-induced toxicity without protecting tumor cells in vitro. Here, we developed an in vivo mouse model of cisplatin and cis-diammine (cyclobutane-1,1-dicarboxylato platinum(II (carboplatin-induced toxicity to further investigate squalene-mediated LD-BM cytoprotection including the molecular mechanism behind selective cytoprotection. We found that squalene significantly reduced the body weight loss of cisplatin and carboplatin-treated mice. Light-density bone marrow cells from squalene-treated mice exhibited improved formation of hematopoietic colonies (colony-forming unit-granulocyte macrophage. Furthermore, squalene also protected mesenchymal stem cell colonies (colony-forming unit-fibroblast from cisplatin and carboplatin-induced toxicity. Squalene-induced protection was associated with decreased reactive oxygen species and increased levels of glutathione and glutathione peroxidase/glutathione-S-transferase. Importantly, squalene did not protect neuroblastoma, small cell carcinoma, or medulloblastoma xenografts against cisplatin-induced toxicity. These results suggest that squalene is a potential candidate for future development as a cytoprotective agent against chemotherapeutic toxicity.

  19. Regulation of Trib2 by an E2F1-C/EBPα feedback loop in AML cell proliferation

    DEFF Research Database (Denmark)

    Rishi, Loveena; Hannon, Maura; Salomè, Mara

    2014-01-01

    α (C/EBPα)-p42, occurs in acute myeloid leukemia (AML), resulting in the perturbation of cell cycle and apoptosis, emphasizing its importance in the molecular pathogenesis of AML. Here we show that E2F family members directly regulate Trib2 in leukemic cells and identify a feedback regulatory loop......The loss of regulation of cell proliferation is a key event in leukemic transformation, and the oncogene tribbles (Trib)2 is emerging as a pivotal target of transcription factors in acute leukemias. Deregulation of the transcription factor E2F1, normally repressed by CCAAT enhancer-binding protein...... for E2F1, C/EBPα, and Trib2 in AML cell proliferation and survival. Further analyses revealed that E2F1-mediated Trib2 expression was repressed by C/EBPα-p42, and in normal granulocyte/macrophage progenitor cells, we detect C/EBPα bound to the Trib2 promoter. Pharmacological inhibition of the cell cycle...

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

    Science.gov (United States)

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

    2015-01-01

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

  1. Epidermal Langerhans' cell induction of immunity against an ultraviolet-induced skin tumour

    International Nuclear Information System (INIS)

    Cavanagh, L.L.; Sluyter, R.; Henderson, K.G.; Barnetson, R.St.C.; Halliday, G.M.

    1996-01-01

    Lanerghans' cells (LC) have been shown experimentally to induce immune response against many antigens; however, their role in the initiation of anti-tumour immunity has received little attention. This study examined the ability of murine epidermal LC to induce immunity to an ultraviolet radiation (UV)-induced skin tumour. Freshly prepared epidermal cells (EC) were cultured for 2 or 20 hr with granulocyte-macrophage colony-stimulating factor (GM-CSF), pulsed with an extract of the UV-13-1 tumour, then used to immunize naive syngeneic mice. Delayed type hypersensitivity (DTH) was elicited 10 days after immunization by injection of UV-13-1 tumour cells into the ear pinna, and measured 24 hr later. EC cultured with GM-CSF for 2 hr induced anti-tumour DTH, as did EC cultured for 20 hr without GM-CSF. Conversely, EC cultured for 2 hr without GM-CSF, or EC cultured for 20 hr with GM-CSF were unable to induce a DTH. Induction of immunity required active presentation of tumour antigens by Ia + EC and was tumour specific. Thus Ia + epidermal cells are capable of inducing anti-tumour immunity to UV-induced skin tumours, but only when they contact antigen in particular states of maturation. (author)

  2. Effects of ultrasound, cysteamine, and x-rays on V79 Chinese hamster cells

    International Nuclear Information System (INIS)

    Fu, Y.K.

    1978-01-01

    Chinese hamster V79 cells were exposed to different intensities and durations of 1 MHz ultrasound and were studied for cell lysis and colony-forming ability. The threshold for cell lysis and loss of viability (reduction in colony-forming ability) were found to be ultrasonic intensity, sonication duration, and energy dependent. Above the threshold there was an initial correlation between ultrasound intensity and biological effect: the higher the intensity, the greater the amount of cell lysis and reduction in colony-forming ability. These effects were maximal at an intensity of 10-20 W/cm 2 , and were less at 30 W/cm 2 . Cells were also exposed to ultrasound in the presence of cysteamine and results are compared with the effects of x radiation on cells exposed in the presence of cysteamine or methionine

  3. RNA interference-mediated silencing of speckle-type POZ protein promotes apoptosis of renal cell cancer cells.

    Science.gov (United States)

    Liu, Xiaoxia; Sun, Guiling; Sun, Xiuju

    2016-01-01

    This study aimed to investigate the effects of silencing the speckle-type POZ protein (SPOP) gene on renal cell cancer (RCC) cells and to explore its possible mechanism. The A498 and ACHN RCC cells were transfected with small interference RNA (siRNA)-SPOP by lipofection methods. The silencing efficiency was monitored by quantitative real-time polymerase chain reaction and Western blot. The effects of SPOP silencing on cell apoptosis, cell viability, colony formation ability, cell migration ability, and chemosensitivity to Sorafenib were assessed by flow cytometry, an MTT assay, a colony formation assay, a trans-well migration assay, and a CCK-8 assay, respectively. Its effects on the expression of several cytokines were determined by a protein microarray. Relevant signaling pathways were also analyzed. Compared with the control group, the cell apoptosis rate was significantly higher; the cell viability, the colony formation, and migration ability were significantly decreased in the siRNA-SPOP group. The protein microarray screening showed that the expression of vascular endothelial growth factor receptor, matrix metallopeptidase-9, vascular cell adhesion molecule-1, and stromal cell-derived factor-1 in the siRNA group was significantly decreased and that the expression of granulocyte-macrophage colony-stimulating factor and E-cadherin was significantly increased (Pmatrix organization signal pathway. SPOP gene silencing induced cell apoptosis, decreased cell viability, colony formation, and migration ability, and elevated the drug sensitivity in the RCC cells. A possible mechanism is that silencing SPOP induces the differential expression of E-cadherin, vascular endothelial growth factor receptor, matrix metallopeptidase-9, and vascular cell adhesion molecule, which are related to the integrin-mediated cell surface interactions and extracellular matrix organization signaling pathway.

  4. Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

    Science.gov (United States)

    Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

    2012-07-01

    Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

  5. Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

    Science.gov (United States)

    Lazzeri, N; Belvisi, M G; Patel, H J; Yacoub, M H; Chung, K F; Mitchell, J A

    2001-01-01

    Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

  6. Distinct pattern of lesion distribution in multiple sclerosis is associated with different circulating T-helper and helper-like innate lymphoid cell subsets.

    Science.gov (United States)

    Gross, Catharina C; Schulte-Mecklenbeck, Andreas; Hanning, Uta; Posevitz-Fejfár, Anita; Korsukewitz, Catharina; Schwab, Nicholas; Meuth, Sven G; Wiendl, Heinz; Klotz, Luisa

    2017-06-01

    Distinct lesion topography in relapsing-remitting multiple sclerosis (RRMS) might be due to different antigen presentation and/or trafficking routes of immune cells into the central nervous system (CNS). To investigate whether distinct lesion patterns in multiple sclerosis (MS) might be associated with a predominance of distinct circulating T-helper cell subset as well as their innate counterparts. Flow cytometric analysis of lymphocytes derived from the peripheral blood of patients with exclusively cerebral (n = 20) or predominantly spinal (n = 12) disease manifestation. Patients with exclusively cerebral or preferential spinal lesion manifestation were associated with increased proportions of circulating granulocyte-macrophage colony-stimulating factor (GM-CSF) producing T H 1 cells or interleukin (IL)-17-producing T H 17 cells, respectively. In contrast, proportions of peripheral IL-17/IL-22-producing lymphoid tissue inducer (LTi), the innate counterpart of T H 17 cells, were enhanced in RRMS patients with exclusively cerebral lesion topography. Distinct T-helper and T-helper-like innate lymphoid cell (ILC) subsets are associated with different lesion topography in RRMS.

  7. The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

    Science.gov (United States)

    Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

    2014-01-01

    Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

  8. The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

    Directory of Open Access Journals (Sweden)

    Laura Pitzonka

    Full Text Available Co-transcriptionally assembled ribonucleoprotein (RNP complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

  9. Signaling profiling at the single-cell level identifies a distinct signaling signature in murine hematopoietic stem cells.

    Science.gov (United States)

    Du, Juan; Wang, Jinyong; Kong, Guangyao; Jiang, Jing; Zhang, Jingfang; Liu, Yangang; Tong, Wei; Zhang, Jing

    2012-07-01

    Hematopoietic stem cell (HSC) function is tightly regulated by cytokine signaling. Although phospho-flow cytometry allows us to study signaling in defined populations of cells, there has been tremendous hurdle to carry out this study in rare HSCs due to unrecoverable critical HSC markers, low HSC number, and poor cell recovery rate. Here, we overcame these difficulties and developed a "HSC phospho-flow" method to analyze cytokine signaling in murine HSCs at the single-cell level and compare HSC signaling profile to that of multipotent progenitors (MPPs), a cell type immediately downstream of HSCs, and commonly used Lin(-) cKit(+) cells (LK cells, enriched for myeloid progenitors). We chose to study signaling evoked from three representative cytokines, stem cell factor (SCF) and thrombopoietin (TPO) that are essential for HSC function and granulocyte macrophage-colony-stimulating factor (GM-CSF) that is dispensable for HSCs. HSCs display a distinct TPO and GM-CSF signaling signature from MPPs and LK cells, which highly correlates with receptor surface expression. In contrast, although majority of LK cells express lower levels of cKit than HSCs and MPPs, SCF-evoked ERK1/2 activation in LK cells shows a significantly increased magnitude for a prolonged period. These results suggest that specific cellular context plays a more important role than receptor surface expression in SCF signaling. Our study of HSC signaling at the homeostasis stage paves the way to investigate signaling changes in HSCs under conditions of stress, aging, and hematopoietic diseases. Copyright © 2012 AlphaMed Press.

  10. Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo.

    Science.gov (United States)

    Vasvari, Gergely P; Dyckhoff, Gerhard; Kashfi, Farzaneh; Lemke, Britt; Lohr, Jennifer; Helmke, Burkhard M; Schirrmacher, Volker; Plinkert, Peter K; Beckhove, Philipp; Herold-Mende, Christel C

    2007-10-15

    Thalidomide is an immunomodulatory, antiangiogenic drug. Although there is evidence that it might be more effective in combination with chemotherapy the exact mechanism of action is unclear. Therefore, we investigated its effect in combination with metronomically applied cisplatin in a xenotransplant mouse model characteristic for advanced head and neck squamous cell carcinomas, its possible synergistic action in vitro, and which tumor-derived factors might be targeted by thalidomide. Although thalidomide alone was ineffective, a combined treatment with low-dose cisplatin inhibited significant tumor growth, proliferation and angiogenesis in vivo as well as migration and tube formation of endothelial cells in vitro. Noteworthy, the latter effect was enhanced after coapplication of cisplatin in nontoxic doses. An inhibitory effect on tumor cell migration was also observed suggesting a direct antitumor effect. Although thalidomide alone did not influence cell proliferation, it augmented antiproliferative response after cisplatin application emphasizing the idea of a potentiated effect when both drugs are combined. Furthermore, we could show that antiangiogenic effects of thalidomide are related to tumor-cell derived factors including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and Il-8 some known and with, granulocyte colony stimulating growth factor and granulocyte macrophage colony stimulating growth factor, some new target molecules of thalidomide. Altogether, our findings reveal new insights into thalidomide-mediated antitumor and antiangiogenic effects and its interaction with cytostatic drugs. (c) 2007 Wiley-Liss, Inc.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  12. Sphingosine 1 Phosphate at the Blood Brain Barrier: Can the Modulation of S1P Receptor 1 Influence the Response of Endothelial Cells and Astrocytes to Inflammatory Stimuli?

    Directory of Open Access Journals (Sweden)

    Simona F Spampinato

    Full Text Available The ability of the Blood Brain Barrier (BBB to maintain proper barrier functions, keeping an optimal environment for central nervous system (CNS activity and regulating leukocytes' access, can be affected in CNS diseases. Endothelial cells and astrocytes are the principal BBB cellular constituents and their interaction is essential to maintain its function. Both endothelial cells and astrocytes express the receptors for the bioactive sphingolipid S1P. Fingolimod, an immune modulatory drug whose structure is similar to S1P, has been approved for treatment in multiple sclerosis (MS: fingolimod reduces the rate of MS relapses by preventing leukocyte egress from the lymph nodes. Here, we examined the ability of S1P and fingolimod to act on the BBB, using an in vitro co-culture model that allowed us to investigate the effects of S1P on endothelial cells, astrocytes, and interactions between the two. Acting selectively on endothelial cells, S1P receptor signaling reduced cell death induced by inflammatory cytokines. When acting on astrocytes, fingolimod treatment induced the release of a factor, granulocyte macrophage colony-stimulating factor (GM-CSF that reduced the effects of cytokines on endothelium. In an in vitro BBB model incorporating shear stress, S1P receptor modulation reduced leukocyte migration across the endothelial barrier, indicating a novel mechanism that might contribute to fingolimod efficacy in MS treatment.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Shu-Yi, E-mail: in_shuyi@hotmail.com [Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC (China); Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC (China); Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC (China); Wang, Chien-Yu, E-mail: sallywang1973@hotmail.com [Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC (China); Yang, Ning-Sun, E-mail: nsyang@gate.sinica.edu.tw [Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC (China); Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC (China); Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC (China)

    2011-09-10

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

  14. Opposite cytokine synthesis by fibroblasts in contact co-culture with osteosarcoma cells compared with transwell co-cultures.

    Science.gov (United States)

    David, Manu S; Kelly, Elizabeth; Zoellner, Hans

    2013-04-01

    We recently reported exchange of membrane and cytoplasm during contact co-culture between human Gingival Fibroblasts (h-GF) and SAOS-2 osteosarcoma cells, a process we termed 'cellular sipping' to reflect the manner in which cells become morphologically diverse through uptake of material from the opposing cell type, independent of genetic change. Cellular sipping is increased by Tumor Necrosis Factor-α (TNF-α), and we here show for the first time altered cytokine synthesis in contact co-culture supporting cellular sipping compared with co-culture where h-GF and SAOS-2 were separated in transwells. SAOS-2 had often undetectably low cytokine levels, while Interleukin-6 (IL-6), Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) were secreted primarily by TNF-α stimulated h-GF and basic Fibroblast Growth Factor (FGF) was prominent in h-GF lysates (p cultures permitting cellular sipping had lower IL-6, G-CSF and GM-CSF levels, as well as higher lysate FGF levels compared with TNF-α treated h-GF alone (p cultures in transwells, with increased IL-6, G-CSF and GM-CSF levels (p cultures where cellular sipping occurs, potentially contributing to tumor inflammatory responses. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  15. Effect of Egg White Combined with Chalcanthite on Lipopolysaccharide induced Inflammatory Cytokine Expression in RAW 264.7 cells

    Directory of Open Access Journals (Sweden)

    Choi Eun-A

    2012-03-01

    Full Text Available Historically, mineral compound herbal medicines have long been used in treatments of immune-related diseases in Korea, China and other Asian countries. In this study, we inv-estigated the anti-inflammatory effect of egg white combined with chalcanthite (IS4 on lipopolysaccharide (LPS-stimulated RAW 264.7 cells. RAW 264.7 cells cultured with LPS and various con-centrations of IS4 were analyzed to determine the production of pro-inflammatory cytokines and mediators by using enzyme-linked immune sorbent assays (ELISAs. IS4 concentration inhibited the production of interleukin-1beta (IL-1β, interleukin-6 (IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF induced by LPS. IS4 at high concentrations (25 and 50`㎍/ml inhibited, in concentration-dependent manner, the expression of tumor necrosis factor-alpha (TNF–α stimulated by LPS. IS4 has shown an anti-inflammatory effect in RAW 264.7 cells.

  16. Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use.

    Science.gov (United States)

    Gülen, D; Maas, S; Julius, H; Warkentin, P; Britton, H; Younos, I; Senesac, J; Pirruccello, Samuel M; Talmadge, J E

    2012-05-01

    In this study, we examined the effects of cryoprotectant, freezing and thawing, and adenovirus (Adv) transduction on the viability, transgene expression, phenotype, and function of human dendritic cells (DCs). DCs were differentiated from cultured peripheral blood (PB) monocytes following Elutra isolation using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 6 days and then transduced using an Adv vector with an IL-12 transgene. Fresh, cryopreserved, and thawed transduced immature DCs were examined for their: 1) cellular concentration and viability; 2) antigenicity using an allogeneic mixed lymphocyte reaction (MLR); 3) phenotype (HLA-DR and CD11c) and activation (CD83); and 4) transgene expression based on IL-12 secretion. Stability studies revealed that transduced DCs could be held in cryoprotectant for as long as 75 min at 2-8°C prior to freezing with little effect on their viability and cellularity. Further, cryopreservation, storage, and thawing reduced the viability of the transduced DCs by an average of 7.7%; and had no significant impact on DC phenotype and activation. In summary, cryopreservation, storage, and thawing had no significant effect on DC viability, function, and transgene expression by Adv-transduced DCs. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Impaired T-lymphocyte colony formation by cord blood mononuclear cells

    International Nuclear Information System (INIS)

    Herrod, H.G.; Valenski, W.R.

    1982-01-01

    When compared to adult mononuclear cells, cord blood mononuclear cells demonstrated significantly decreased T-lymphocyte colony formation (1351 +/- 643 vs 592 +/- 862, P less than 0.01). This diminished colony-forming activity did not appear to be associated with impaired responsiveness to the stimulant phytohemagglutinin or with excessive suppressor-cell activity. Irradiation reduced the colony-forming capacity of cord blood mononuclear cells more than it did that of adult mononuclear cells. Depletion of adherent cells reduced cord blood mononuclear-cell colony-forming capacity by 40%, while similar treatment reduced adult colony formation by 10%. Lymphocyte proliferation in liquid culture of cord and adult cells was minimally affected by these procedures. The colony-forming capacity of cord blood could be enhanced by the addition of irradiated adult cells (284 +/- 72 vs 752 +/- 78, P less than 0.01). This enhancement was demonstrated to be due to a soluble factor produced by a population of irradiated adult cells depleted of the OKT8+ subpopulation of lymphocytes. These results indicate that the progenitor cells of T-lymphocyte colonies in cord blood have distinct biologic characteristics when compared to colony progenitors present in adult blood. This assay may prove to be useful in our efforts to understand the differentiation of T-cell function in man

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

    International Nuclear Information System (INIS)

    Morrissey, P.J.; Ireland, R.

    1991-01-01

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

  19. Increased levels of inflammatory cytokines in the female reproductive tract are associated with altered expression of proteases, mucosal barrier proteins, and an influx of HIV-susceptible target cells.

    Science.gov (United States)

    Arnold, Kelly B; Burgener, Adam; Birse, Kenzie; Romas, Laura; Dunphy, Laura J; Shahabi, Kamnoosh; Abou, Max; Westmacott, Garrett R; McCorrister, Stuart; Kwatampora, Jessie; Nyanga, Billy; Kimani, Joshua; Masson, Lindi; Liebenberg, Lenine J; Abdool Karim, Salim S; Passmore, Jo-Ann S; Lauffenburger, Douglas A; Kaul, Rupert; McKinnon, Lyle R

    2016-01-01

    Elevated inflammatory cytokines (EMCs) at mucosal surfaces have been associated with HIV susceptibility, but the underlying mechanisms remain unclear. We characterized the soluble mucosal proteome associated with elevated cytokine expression in the female reproductive tract. A scoring system was devised based on the elevation (upper quartile) of at least three of seven inflammatory cytokines in cervicovaginal lavage. Using this score, HIV-uninfected Kenyan women were classified as either having EMC (n=28) or not (n=68). Of 455 proteins quantified in proteomic analyses, 53 were associated with EMC (5% false discovery rate threshold). EMCs were associated with proteases, cell motility, and actin cytoskeletal pathways, whereas protease inhibitor, epidermal cell differentiation, and cornified envelope pathways were decreased. Multivariate analysis identified an optimal signature of 16 proteins that distinguished the EMC group with 88% accuracy. Three proteins in this signature were neutrophil-associated proteases that correlated with many cytokines, especially GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-1β (interleukin-1β), MIP-3α (macrophage inflammatory protein-3α), IL-17, and IL-8. Gene set enrichment analyses implicated activated immune cells; we verified experimentally that EMC women had an increased frequency of endocervical CD4(+) T cells. These data reveal strong linkages between mucosal cytokines, barrier function, proteases, and immune cell movement, and propose these as potential mechanisms that increase risk of HIV acquisition.

  20. Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines.

    Science.gov (United States)

    Zawada, James F; Yin, Gang; Steiner, Alexander R; Yang, Junhao; Naresh, Alpana; Roy, Sushmita M; Gold, Daniel S; Heinsohn, Henry G; Murray, Christopher J

    2011-07-01

    Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Copyright © 2011 Wiley Periodicals, Inc.

  1. Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice - role of regulatory T cells.

    Science.gov (United States)

    Singpiel, Alena; Kramer, Julia; Maus, Regina; Stolper, Jennifer; Bittersohl, Lara Friederike; Gauldie, Jack; Kolb, Martin; Welte, Tobias; Sparwasser, Tim; Maus, Ulrich A

    2018-03-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγ pos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans. Copyright © 2017 Elsevier GmbH. All rights reserved.

  2. The production of lymphokines by primary alloreactive T-cell clones: a co-ordinate analysis of 233 clones in seven lymphokine assays.

    Science.gov (United States)

    Sanderson, C J; Strath, M; Warren, D J; O'Garra, A; Kirkwood, T B

    1985-01-01

    A total of 233 primary alloreactive T-cell clones have been tested for the production of interleukin-2 (IL-2), interleukin-3 (IL-3), immune(gamma) interferon (IFN) and granulocyte-macrophage colony-stimulating factor (CSF-2), B-cell growth factor I and II (BCGFI, BCGFII), and eosinophil differentiation factor (EDF). EDF was assayed by means of the eosinophil differentiation assay (EDA). Two principal correlations were observed: IL-3 was shown to be the major lymphokine detected in the bone marrow proliferation assay (BMPA) used to detect CSF-2, and there was a high correlation between the EDA and BCGFII. Subsequent work has suggested that this latter correlation is because a single factor is responsible for both activities. Apart from these two exceptions, and low level correlations probably due to the fact that different assays detect more than one lymphokine, there was no evidence for co-ordinate expression of lymphokines. There was a large variation in amounts of individual lymphokines produced. More clones produced multiple lymphokines than would be expected from independent control. Taken together, this pattern of regulation is consistent with the hypothesis that antigen stimulation of T cells results in the activation of all the lymphokine genes, but the amount of each produced is determined by secondary controlling mechanisms. PMID:3935571

  3. Listeria monocytogenes alters mast cell phenotype, mediator and osteopontin secretion in a listeriolysin-dependent manner.

    Directory of Open Access Journals (Sweden)

    Catherine E Jobbings

    Full Text Available Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF, and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection.

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

    DEFF Research Database (Denmark)

    Agerholm, Inge; Loft, Anne; Hald, Finn

    2010-01-01

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

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

    DEFF Research Database (Denmark)

    Agerholm, Inge; Loft, Anne; Hald, Finn

    2010-01-01

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

  6. Chemoimmunotherapy of cancer: potentiated effectiveness of granulocyte-macrophage colony-stimulating factor and ifosfamide derivative CBM-4A

    Czech Academy of Sciences Publication Activity Database

    Indrová, Marie; Bubeník, Jan; Šímová, Jana; Bieblová, Jana; Jandlová, Táňa; Šmahel, M.; Vonka, V.; Glazman-Kusnierczyk, H.; Pajtasz-Piasecka, E.; Radzikowski, C.; Mikyšková, Romana

    2001-01-01

    Roč. 8, č. 6 (2001), s. 1371-1374 ISSN 1021-335X R&D Projects: GA MZd NC5526; GA MZd NC5900; GA ČR GA312/99/0542; GA ČR GA301/01/0985; GA ČR GA301/00/0114; GA AV ČR IAA7052002 Institutional research plan: CEZ:AV0Z5052915 Keywords : chemoimmunotherapy * murine * neoplasms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.224, year: 2001

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

    NARCIS (Netherlands)

    Isik, Gözde; van Montfort, Thijs; Boot, Maikel; Cobos Jiménez, Viviana; Kootstra, Neeltje A.; Sanders, Rogier W.

    2013-01-01

    HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  9. In vitro studies of the sensitivity of canine bone-marrow erythroid burst-forming units (BFU-E) and fibroblast colony-forming units (CFU-F) to X-irradiation

    International Nuclear Information System (INIS)

    Kreja, Ludwika; Baltschukat, Klaus; Nothdurft, Wilhelm

    1989-01-01

    The radiosensitivity of the early erythroid progenitor cells (BFU-E) and the progenitor cells of the stroma (CFU-F) in canine bone marrow was studied under steady-state conditions by in vitro irradiation with 280 kV X-rays. The dose-effect relationship for colony formation was determined for BFU-E obtained from the iliac crest marrow, and for CFU-F in bone marrow collected from the iliac crest and the humerus of adult beagles. The BFU-E were adequately stimulated with serum from lethally irradiated dogs to obtain a source of BPA (burst-promoting activity). The BFU-E proved to be extremely radiosensitive, (the survival curve was exponential (D o 15.3 ± 1.8 cGY)). Buffy-coat leukocytes separated from bone marrow leukocytes obtained by aspiration were an optimum source of CFU-F. A curve was fitted to data obtained for CFU-F obtained from iliac crest or humerus, resulting in D o = 241 ± 38 cGY and an extrapolation number n = 1.38 ± 0.62 or D o = 261 ± 40 cGY and n = 1.04 ± 0.42, respectively. (author)

  10. Effect of histones on hematopoietic stem cells-precursor in normal and irradiated organism

    International Nuclear Information System (INIS)

    Semina, O.V.; Semenets, T.N.; Zeppezauer, M.; Cebecauer, L.; Poverenny, A.M.

    1994-01-01

    Radiotherapeutic activity of histone fractions H 1 and H 2A /H 2B were studied. It was demonstrated that both fractions are able to reduce the damaging effect of ionizing radiation on spleen colony forming unit (CFU-S) population. Histone preparations stimulated colony-forming activity of bone marrow cells exposed to dose of 0.5-3.0 Gy both in the case of incubation with preparations and intravenous or intraperitoneal administration into recipients of irradiated cells. The effect of histones and accessory thymocytes on CFU-S population is compared

  11. Reduction of erythroid progenitors in protein-energy malnutrition.

    Science.gov (United States)

    Borelli, Primavera; Blatt, Solange; Pereira, Juliana; de Maurino, Beatriz Beutler; Tsujita, Maristela; de Souza, Ana Cristina; Xavier, José Guilherme; Fock, Ricardo Ambrósio

    2007-02-01

    Protein-energy malnutrition is a syndrome in which anaemia together with multivitamin and mineral deficiency may be present. The pathophysiological mechanisms involved have not, however, yet been completely elucidated. The aim of the present study was to evaluate the pathophysiological processes that occur in this anaemia in animals that were submitted to protein-energy malnutrition, in particular with respect to Fe concentration and the proliferative activity of haemopoietic cells. For this, histological, histochemical, cell culture and immunophenotyping techniques were used. Two-month-old male Swiss mice were submitted to protein-energy malnutrition with a low-protein diet (20 g/kg) compared with control diet (400 g/kg). When the experimental group had attained a 20 % loss of their original body weight, the animals from both groups received, intravenously, 20 IU erythropoietin every other day for 14 d. Malnourished animals showed a decrease in red blood cells, Hb concentration and reticulocytopenia, as well as severe bone marrow and splenic atrophy. The results for serum Fe, total Fe-binding capacity, transferrin and erythropoietin in malnourished animals were no different from those of the control animals. Fe reserves in the spleen, liver and bone marrow were found to be greater in the malnourished animals. The mixed colony-forming unit assays revealed a smaller production of granulocyte-macrophage colony-forming units, erythroid burst-forming units, erythroid colony-forming units and CD45, CD117, CD119 and CD71 expression in the bone marrow and spleen cells of malnourished animals. These findings suggest that, in this protein-energy malnutrition model, anaemia is not caused by Fe deficiency or erythropoietin deficiency, but is a result of ineffective erythropoiesis.

  12. ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

    Science.gov (United States)

    Zhang, Hu; Zhang, Shuhong; Zhang, Jilin; Liu, Dongxin; Wei, Jiayi; Fang, Wengang; Zhao, Weidong; Chen, Yuhua; Shang, Deshu

    2018-05-01

    The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

  13. Assessment of perioperative stress in colorectal cancer by use of in vitro cell models: a systematic review

    Directory of Open Access Journals (Sweden)

    Tove Kirkegaard

    2017-11-01

    Full Text Available Background The perioperative period is important for patient outcome. Colorectal cancer surgery can lead to metastatic disease due to release of disseminated tumor cells and the induction of surgical stress response. To explore the overall effects on surgically-induced changes in serum composition, in vitro model systems are useful. Methods A systematic search in PubMed and EMBASE was performed to identify studies describing in vitro models used to investigate cancer cell growth/proliferation, cell migration, cell invasion and cell death of serum taken pre- and postoperatively from patients undergoing colorectal tumor resection. Results Two authors (MG and TK independently reviewed 984 studies and identified five studies, which fulfilled the inclusion criteria. Disagreements were solved by discussion. All studies investigated cell proliferation and cell invasion, whereas three studies investigated cell migration, and only one study investigated cell death/apoptosis. One study investigated postoperative peritoneal infection due to anastomotic leak, one study investigated mode of anesthesia (general anesthesia with volatile or intravenous anesthetics, and one study investigated preoperative intervention with granulocyte macrophage colony stimulating factor (GMCSF. In all studies an increased proliferation, cell migration and invasion was demonstrated after surgery. Anesthetics with propofol and intervention with GMCSF significantly reduced postoperative cell proliferation, whereas peritoneal infection enhanced the invasive capability of tumor cells. Conclusion This study suggests that in vitro cell models are useful and reliable tools to explore the effect of surgery on colorectal cancer cell proliferation and metastatic ability. The models should therefore be considered as additional tests to investigate the effects of perioperative interventions.

  14. STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

    Science.gov (United States)

    Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

    2014-01-01

    Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

  15. Mannan-MUC1-pulsed dendritic cell immunotherapy: a phase I trial in patients with adenocarcinoma.

    Science.gov (United States)

    Loveland, Bruce E; Zhao, Anne; White, Shane; Gan, Hui; Hamilton, Kate; Xing, Pei-Xiang; Pietersz, Geoffrey A; Apostolopoulos, Vasso; Vaughan, Hilary; Karanikas, Vaios; Kyriakou, Peter; McKenzie, Ian F C; Mitchell, Paul L R

    2006-02-01

    Tumor antigen-loaded dendritic cells show promise for cancer immunotherapy. This phase I study evaluated immunization with autologous dendritic cells pulsed with mannan-MUC1 fusion protein (MFP) to treat patients with advanced malignancy. Eligible patients had adenocarcinoma expressing MUC1, were of performance status 0 to 1, with no autoimmune disease. Patients underwent leukapheresis to generate dendritic cells by culture ex vivo with granulocyte macrophage colony-stimulating factor and interleukin 4 for 5 days. Dendritic cells were then pulsed overnight with MFP and harvested for reinjection. Patients underwent three cycles of leukapheresis and reinjection at monthly intervals. Patients with clinical benefit were able to continue with dendritic cell-MFP immunotherapy. Ten patients with a range of tumor types were enrolled, with median age of 60 years (range, 33-70 years); eight patients were of performance status 0 and two of performance status 1. Dendritic cell-MFP therapy led to strong T-cell IFNgamma Elispot responses to the vaccine and delayed-type hypersensitivity responses at injection sites in nine patients who completed treatments. Immune responses were sustained at 1 year in monitored patients. Antibody responses were seen in three patients only and were of low titer. Side effects were grade 1 only. Two patients with clearly progressive disease (ovarian and renal carcinoma) at entry were stable after initial therapy and went on to further leukapheresis and dendritic cell-MFP immunotherapy. These two patients have now each completed over 3 years of treatment. Immunization produced T-cell responses in all patients with evidence of tumor stabilization in 2 of the 10 advanced cancer patients treated. These data support further clinical evaluation of this dendritic cell-MFP immunotherapy.

  16. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  17. Effect of low-dose irradiation on pregnant mouse haemopoiesis

    International Nuclear Information System (INIS)

    Weinberg, S.R.; McCarthy, E.G.; MacVittie, T.J.; Baum, S.J.

    1981-01-01

    The effects of low-dose gamma radiation to haemopoietic progenitor cell compartments of the marrow and spleen of virgin female mice and pregnant mice were studied. Microplasma clot cultures were used to assess burst-forming uniterythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) activity, and double-layer agar cultures were established to evaluate granulocyte-macrophage colony-forming cell (GM-CFC) and macrophage colony-forming cell (M-CFC). The apparent shift in maternal erythropoiesis from the bone marrow to the enlarged spleen was reflected by an increase in CFU-E and BFU-E per spleen and a concomitant decrease in CFU-E and BFU-E per femur. Whereas maternal GM-CFC values per femur increased 36%, maternal GM-CFC per spleen increased by 172% compared to virgin values. Total-body irradiation to the day-10.5 pregnant mouse caused a further suppression of day-14.5 medullary erythropoiesis (i.e. decreased CFU-E values) compared to the virgin female mouse. An ability of the maternal spleen to support further compensatory erythropoiesis following increasing doses of radiation was demonstrated. Four days after 1.0 Gy exposure, maternal values for GM-CFC per femur or spleen decreased to nonirradiated virgin mice values. M-CFC per maternal femur decreased to nonirradiated virgin mice values. M-CFC per maternal femur decreased following 1.5 Gy, but M-CFC per spleen appeared to be unaffected with doses from 0.5 to 2.0 Gy. (author)

  18. Reactivation of neutron killed mammalian cells by gamma irradiation: The observations, possible mechanism and implication

    International Nuclear Information System (INIS)

    Calkins, J.; Harrison, W.; Einspenner, M.

    1990-01-01

    We have observed that combinations of neutron plus gamma ray exposure can significantly increase the colony forming ability of monkey and human cell cultures over the neutron dose alone. The 'reactivation' of neutron killed mammalian cells by gamma rays is analogous to observations made in lower eukaryotic organisms and fits the pattern termed 'T repair' previously postulated for yeast and protozoans. (orig.)

  19. In vitro assays for cobblestone area-forming cells, LTC-IC, and CFU-C

    NARCIS (Netherlands)

    van Os, Ronald P; Dethmers-Ausema, Bertien; de Haan, Gerald; Bunting, Kevin

    2008-01-01

    Various assays exist that measure the function of hematopoietic stemcells (HSCs). In this chapter, in vitro assays are described that measure the frequency of progenitors (colony-forming unit in culture; CFU-C), stem cells (long-term culture-initiating cell; LTC-IC), or both (cobblestone

  20. Cytotoxic activity of Justicia spicigera is inhibited by bcl-2 proto-oncogene and induces apoptosis in a cell cycle dependent fashion.

    Science.gov (United States)

    Cáceres-Cortés, J R; Cantú-Garza, F A; Mendoza-Mata, M T; Chavez-González, M A; Ramos-Mandujano, G; Zambrano-Ramírez, I R

    2001-12-01

    Identification of organic compounds from plants is of clinical significance because of the effect that they might have in patients with haematopoietic disorders. We studied the effect of the plant extract Justicia spicigera (Acanthaceae) in different haematopoietic cells: human leukaemic cell lines, umbilical cord blood cells, and mouse bone marrow cells. By examining colony formation and performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay it was shown that the plant extract of Justicia spicigera contains cytotoxic factors for leukaemic cells and has no proliferative activity on normal haematopoietic progenitor cells. Our results show that this plant extract induces apoptosis in the human leukaemia cell line TF-1, but not in the bcl-2 transfectant cell line TB-1. Similar results were obtained using a haemopoietic cell line 32D and 32DBcl2. The cultures of umbilical cord blood cells and mouse bone marrow that contain granulocyte-macrophage colony-stimulating factor (GM-CSF) do not proliferate or become terminally differentiated in the presence of the infusion of Justicia spicigera. GM-CSF that acts by abrogating programmed cell death is not sufficient to inhibit the apoptotic stimulus in TF-1 and 32D cells. Moreover mouse fibroblasts (3T3) and two cervical carcinoma cell lines CALO and INBL, undergo apoptosis in the presence of different concentrations of an infusion from the plant. Our data show that there is a strong correlation between the cytotoxic effect and cell proliferation. Together, these results indicate that the plant infusion of Justicia spicigera does not contain any haematopoietic activity, induces apoptosis inhibited by bcl-2 and is linked to cell proliferation. Copyright 2001 John Wiley & Sons, Ltd.

  1. Improved viability and activity of neutrophils differentiated from HL-60 cells by co-culture with adipose tissue-derived mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yoon Shin; Lim, Goh-Woon [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Cho, Kyung-Ah; Woo, So-Youn; Shin, Meeyoung [Department of Microbiology, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Yoo, Eun-Sun [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of); Chan Ra, Jeong [Stem Cell Research Center, RNL BIO, Seoul 153-768 (Korea, Republic of); Ryu, Kyung-Ha, E-mail: ykh@ewha.ac.kr [Department of Pediatrics, Ewha Womans University, School of Medicine, Ewha Medical Research Center, Seoul (Korea, Republic of)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer Neutropenia is a principal complication of cancer treatment. Black-Right-Pointing-Pointer Co-culture of neutrophils with AD-MSC retained cell survival and proliferation and inhibited neutrophil apoptosis under serum starved conditions. Black-Right-Pointing-Pointer AD-MSC increased functions of neutrophil. Black-Right-Pointing-Pointer AD-MSC promoted the viability of neutrophils by enhancing respiratory burst through the expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Black-Right-Pointing-Pointer AD-MSC can be used to improve immunity for neutropenia treatment. -- Abstract: Neutropenia is a principal complication of cancer treatment. We investigated the supportive effect of adipose tissue-derived mesenchymal stem cells (AD-MSCs) on the viability and function of neutrophils. Neutrophils were derived from HL-60 cells by dimethylformamide stimulation and cultured with or without AD-MSCs under serum-starved conditions to evaluate neutrophil survival, proliferation, and function. Serum starvation resulted in the apoptosis of neutrophils and decreased cell survival. The co-culture of neutrophils and AD-MSCs resulted in cell survival and inhibited neutrophil apoptosis under serum-starved conditions. The survival rate of neutrophils was prolonged up to 72 h, and the expression levels of interferon (IFN)-{alpha}, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, and transforming growth factor (TGF)-{beta} in AD-MSCs were increased after co-culture with neutrophils. AD-MSCs promoted the viability of neutrophils by inhibiting apoptosis as well as enhancing respiratory burst, which could potentially be mediated by the increased expression of IFN-{alpha}, G-CSF, and TGF-{beta}. Thus, we conclude that the use of AD-MSCs may be a promising cell-based therapy for increasing immunity by accelerating neutrophil function.

  2. Toll-like receptors 2 and 4 mediate the capacity of mesenchymal stromal cells to support the proliferation and differentiation of CD34{sup +} cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xingbing, E-mail: wangxingbing91@hotmail.com [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Cheng, Qiansong; Li, Lailing; Wang, Jian; Xia, Liang [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Xu, Xiucai [The Center Laboratory of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China); Sun, Zimin [Department of Hematology of Anhui Provincial Hospital, Anhui Medical University, Hefei, Anhui (China)

    2012-02-01

    Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1-10 by reverse transcription-polymerase chain reaction. TLR1-6, but not TLR7-10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 and TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34{sup +} hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34{sup +} cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM{sub 3}CSK{sub 4}) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM{sub 3}CSK{sub 4} and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.

  3. Toll-like receptors 2 and 4 mediate the capacity of mesenchymal stromal cells to support the proliferation and differentiation of CD34+ cells

    International Nuclear Information System (INIS)

    Wang, Xingbing; Cheng, Qiansong; Li, Lailing; Wang, Jian; Xia, Liang; Xu, Xiucai; Sun, Zimin

    2012-01-01

    Bone marrow derived-mesenchymal stromal cells (BM-MSCs) are multipotent, nonhematopoietic progenitors in a hematopoietic microenvironment and indispensable for regulating hematopoiesis. Several studies have reported that toll-like receptors (TLRs) are expressed in mesenchymal stromal cells (MSCs) to modulate their biological functions. In this study, we investigated the possible role(s) of TLRs in mediating the hematopoiesis-supporting role of human BM-MSCs. Human BM-MSCs were analyzed for mRNA expression of TLR1–10 by reverse transcription-polymerase chain reaction. TLR1–6, but not TLR7–10 were expressed by BM-MSCs. The protein expression of TLR2 and TLR4 was also confirmed by flow cytometry. We further explored the role of TLR2 and TLR4 in mediating the capacity of BM-MSCs to support the proliferation and differentiation of CD34 + hematopoietic stem/progenitor cells obtained from cord blood. BM-MSCs increased proliferation of CD34 + cells and promoted the differentiation towards the myeloid lineage 7 or 14 days after co-culture, as well as colony formation by those cells and the production of interleukin 1 (IL-1), IL-8, IL-11, stem cell factor (SCF), granulocyte colony-stimulating factor (CSF), macrophage CSF and granulocyte-macrophage CSF, if MSCs had been stimulated with TLR2 agonist (PAM 3 CSK 4 ) or TLR4 agonist (LPS). Interestingly, although these effects were elevated in a different degree, a synergistic effect was not observed in BM-MSCs co-stimulated with PAM 3 CSK 4 and LPS. Together, our findings suggest that TLR2 and TLR4 signaling may indirectly regulate hematopoiesis by modulating BM-MSCs' functions. The increased hematopoietic proliferation and differentiation could be mediated, at least in part, by augmented hematopoiesis-related cytokine production of BM-MSCs.

  4. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

    Directory of Open Access Journals (Sweden)

    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  5. Preventive effect of ganoderma lucidum (Reishi) on the suppression of hematopoietic system by X-ray irradiation

    International Nuclear Information System (INIS)

    Itoh, Hiroko; Nakata, Fukuyoshi; Sasaki, Hiroyuki; Ito, Hitoshi

    2013-01-01

    Reishi has been used for a roborant or the elixir of life from ancient times. In the present study, the preventive effects of Reishi on hematopoietic suppression from X-ray irradiation in mice were investigated. 5.0 Gy-irradiated mice induced the decrease of red blood cell, platelet, white blood cell, granulocytes, the index of spleen and thymus, and the number of spleen cells. Oral administration of Reishi tended to decrease these damage on hematopoiesis, and reticuloendothelial system. Reishi enhanced the degree of spleen cells-mediated sheep red blood cells (SRBC) hemolysis (quantitative hemolysis of SRBC). Reishi augmented the level of erythroid burst-forming cell (BFU-E), and accelerated the recovery of the number of BFU-E in X-irradiated mice, although Reishi did not influence red blood cell counts and colony-forming unit-erythropoetin dependent (CFU-E) number. A significant elevation in the CFU-GM (granulocytes-macrophages) level was observed. Histological examinations revealed that Reishi accerelated the hematopoietic recovery and decrease on damage of spleen and thymus. (author)

  6. Autologous blood stem-cell transplantation in patients with advanced Hodgkin's disease and prior radiation to the pelvic site

    International Nuclear Information System (INIS)

    Koerbling, M.H.; Holle, R.; Haas, R.; Knauf, W.; Doerken, B.H.; Ho, A.D.; Kuse, R.; Pralle, H.; Fliedner, T.M.; Hunstein, W.

    1990-01-01

    Patients with relapsed Hodgkin's disease who respond to salvage therapy are successfully treated with cyclophosphamide, carmustine (BCNU), and etoposide (VP-16) (CBV) followed by autologus bone marrow transplantation (ABMT). Because of heavy pretreatment including radiation to the pelvic site, marrow harvest was not feasible in those patients. We therefore used blood-derived hemopoietic precursor cells as an alternative stem-cell source to rescue them after superdose chemotherapy. Hemopoietic precursor cells were mobilized into the peripheral blood either by chemotherapeutic induction of transient myelosuppression followed by an overshooting of blood stem-cell concentration, or by continuous intravenous (IV) granulocyte-macrophage colony-stimulating factor (GM-CSF) administration. The median time to reach 1,000 WBC per microliter, 500 polymorphonuclear cells (PMN) per microliter, or 20,000 platelets per microliter was 10, 20.5, and 38 days, respectively, for 50% of all patients. The platelet counts of two patients never dropped below 20,000/microL following autologous blood stem-cell transplantation (ABSCT), whereas two other patients had to be supported with platelets for 75 and 86 days posttransplant until a stable peripheral platelet count of 20,000/microL was attained. Among the 11 assessable patients, seven are in unmaintained complete remission (CR) at a median follow-up of 318 days. This is a first report on a series of ABSCTs in patients with advanced Hodgkin's disease proving that, despite prior damage to the marrow site, the circulating stem-cell pool is still a sufficient source of hemopoietic precursor cells for stem-cell rescue

  7. Cellular interaction of a layer-by-layer based drug delivery system depending on material properties and cell types.

    Science.gov (United States)

    Brueckner, Mandy; Jankuhn, Steffen; Jülke, Eva-Maria; Reibetanz, Uta

    2018-01-01

    Drug delivery systems (DDS) and their interaction with cells are a controversial topic in the development of therapeutic concepts and approaches. On one hand, DDS are very useful for protected and targeted transport of defined dosages of active agents. On the other hand, their physicochemical properties such as material, size, shape, charge, or stiffness have a huge impact on cellular uptake and intracellular processing. Additionally, even identical DDS can undergo a completely diverse interaction with different cell types. However, quite often in in vitro DDS/cell interaction experiments, those aspects are not considered and DDS and cells are randomly chosen. Hence, our investigations provide an insight into layer-by-layer designed microcarriers with modifications of only some of the most important parameters (surface charge, stiffness, and applied microcarrier/cell ratio) and their influence on cellular uptake and viability. We also considered the interaction of these differently equipped DDS with several cell types and investigated professional phagocytes (neutrophil granulocytes; macrophages) as well as non-professional phagocytes (epithelial cells) under comparable conditions. We found that even small modifications such as layer-by-layer (LbL)-microcarriers with positive or negative surface charge, or LbL-microcarriers with solid core or as hollow capsules but equipped with the same surface properties, show significant differences in interaction and viability, and several cell types react very differently to the offered DDS. As a consequence, the properties of the DDS have to be carefully chosen with respect to the addressed cell type with the aim to efficiently transport a desired agent.

  8. CD38 gene-modified dendritic cells inhibit murine asthma development by increasing IL-12 production and promoting Th1 cell differentiation.

    Science.gov (United States)

    Wang, Jiaoli; Zhu, Weiguo; Chen, Yinghu; Lin, Zhendong; Ma, Shenglin

    2016-11-01

    Predominant T helper (Th)2 and impaired Th1 cell polarization has a crucial role in the development of asthma. Cluster of differentiation (CD)38 is associated with the increased release of interleukin (IL)‑12 from dendritic cells (DCs) and DC‑induced Th1 cell polarization. However, whether CD38 expression affects DC function in asthma development remains unknown. In the current study, adenoviruses were constructed containing the murine CD38 gene. Overexpression of CD38 protein level in DCs induced from bone‑marrow derived DCs (BMDCs) by recombinant mouse granulocyte macrophage colony‑stimulating factor and IL‑4 was achieved through 24 h adenovirus infection. The results demonstrated that BMDCs with CD38 overexpression exhibited no phenotypic change; however, following stimulation with lipopolysaccharide (LPS), maturation and IL‑12 secretion were increased. In addition, CD38‑overexpressing BMDCs stimulated with LPS exhibited more effective Th1 cell differentiation. Mice that were administered CD38‑overexpressing BMDCs exhibited milder symptoms of asthma. Furthermore, decreased IL‑4, IL‑5 and IL‑13 levels were detected in bronchoalveolar lavage fluid (BALF), reduced immunoglobulin E levels were measured in the sera, and increased interferon‑γ was detected in BALF from the recipients of CD38‑overexpressing BMDCs. Increased phosphorylated‑p38 expression was also detected in LPS-stimulated CD38-overexpressing BMDCs, whereas pretreatment with a p38‑specific inhibitor was able to abolish the effects of LPS stimulation and CD38 overexpression on IL‑12 release and Th1 cell differentiation in BMDCs. These results suggested that CD38 may be involved in the DC function of alleviating asthma via restoration of the Th1/Th2 balance, thus providing a novel strategy for asthma therapy.

  9. Cell growth, intracellular calcium concentration and metabolic cooperation measured in cells exposed to 50 Hz electromagnetic fields

    International Nuclear Information System (INIS)

    Skauli, K.S.

    1996-08-01

    Colony-forming efficiency, DNA/protein and DNA/cell were measured in cells exposed to magnetic fields of 0.2 and 1 mT at a frequency of 50 Hz. Intracellular calcium concentrations were measured in cells exposed to 0.3 and 1 mT at 50 Hz. Metabolic cooperation was measured in cells exposed to 1 mT at 50 Hz. No significant effects of the fields were observed. 20 refs., 10 figs

  10. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    International Nuclear Information System (INIS)

    Kawahara, Takeshi

    2012-01-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine–aminopterin–thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D 2 and leukotriene C 4 in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.

  11. Cadmium modulates hematopoietic stem and progenitor cells and skews toward myelopoiesis in mice

    International Nuclear Information System (INIS)

    Zhang, Yandong; Yu, Xinchun; Sun, Shuhui; Li, Qian; Xie, Yunli; Li, Qiang; Zhao, Yifan; Pei, Jianfeng; Zhang, Wenmin; Xue, Peng; Zhou, Zhijun; Zhang, Yubin

    2016-01-01

    The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3 months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors, the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis. - Highlights: • Cd increases the number of LT-HSCs but impairs their development. • Cd-treated hosts have compromised ability to support LT-HSCs. • Cd promotes myelopoiesis at the expense of lymphopoiesis at the MPP level.

  12. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, Takeshi, E-mail: tkawafb@shinshu-u.ac.jp [Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Nagano (Japan)

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  13. Cadmium modulates hematopoietic stem and progenitor cells and skews toward myelopoiesis in mice

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yandong; Yu, Xinchun [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Sun, Shuhui [Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032 (China); Li, Qian [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Xie, Yunli [Insititute of Brain Sciences, Fudan University, Shanghai 200032 (China); Li, Qiang [Putuo District Center for Disease Control and Prevention, Shanghai 200062 (China); Zhao, Yifan; Pei, Jianfeng; Zhang, Wenmin; Xue, Peng; Zhou, Zhijun [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China); Zhang, Yubin, E-mail: yz001@fudan.edu.cn [School of Public Health and Key Laboratory of Public Health, MOE, Fudan University, Shanghai 200032 (China)

    2016-12-15

    The heavy metal cadmium (Cd) is known to modulate immunity and cause osteoporosis. However, how Cd influences on hematopoiesis remain largely unknown. Herein, we show that wild-type C57BL/6 (B6) mice exposed to Cd for 3 months had expanded bone marrow (BM) populations of long-term hematopoietic stem cells (LT-HSCs), common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs), while having reduced populations of multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). A competitive mixed BM transplantation assay indicates that BM from Cd-treated mice had impaired LT-HSC ability to differentiate into mature cells. In accordance with increased myeloid progenitors and decreased lymphoid progenitors, the BM and spleens of Cd-treated mice had more monocytes and/or neutrophils and fewer B cells and T cells. Cd impaired the ability of the non-hematopoietic system to support LT-HSCs, in that lethally irradiated Cd-treated recipients transplanted with normal BM cells had reduced LT-HSCs after the hematopoietic system was fully reconstituted. This is consistent with reduced osteoblasts, a known critical component for HSC niche, observed in Cd-treated mice. Conversely, lethally irradiated control recipients transplanted with BM cells from Cd-treated mice had normal LT-HSC reconstitution. Furthermore, both control mice and Cd-treated mice that received Alendronate, a clinical drug used for treating osteoporosis, had BM increases of LT-HSCs. Thus, the results suggest Cd increase of LT-HSCs is due to effects on HSCs and not on osteoblasts, although, Cd causes osteoblast reduction and impaired niche function for maintaining HSCs. Furthermore, Cd skews HSCs toward myelopoiesis. - Highlights: • Cd increases the number of LT-HSCs but impairs their development. • Cd-treated hosts have compromised ability to support LT-HSCs. • Cd promotes myelopoiesis at the expense of lymphopoiesis at the MPP level.

  14. Radioprotective effects of ginsan through the stimulation hematopoiesis and antioxidant enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Song, Jie Young; Son, Soo Jung; Ahn, Ji Yeon; Shim, Ji Young; Han, Young Soo; Yun, Yeon Sook [Korea Institute of Radiological and Medical Sciences, Taejon (Korea, Republic of)

    2003-07-01

    An immunomodulator ginsan which was isolated from Panax ginseng showed the mitogenic activity, generation of LAK cells, and secretion of several cytokines. In the extended effort to search other immunostimulatory effects, we evaluated protective effects of in vivo injected Ginsan against irradiation, by measuring the recovery of CFU-S, and the functioning of bone marrow (BM) and spleen cells. Ginsan was found to significantly increase the number of BM cells, spleen cells, granulocytes macrophage-colony forming cells (GM-CFC), and the number of circulating neutrophils, lymphocytes and platelets in irradiated mice. In addition, it induced endogenous production of cytokines such as IL-1, IL-6, IFN-g and IL-12, which are required for hematopoietic recovery, and was able to enhance Th1 function while interfering with the Th2 response in irradiated mice. We demonstrated that the pretreatment with Ginsan protected mice from lethal effects of ionizing radiation more effectively than given immediately or after the irradiation. A significant increase of the survival of Ginsan-treated group (100mg/kg) from LD50/30 7.54 Gy of PBS-injection to 10.93 Gy was observed. Moreover, the levels of the antioxidant enzymes such as SODs, catalase and gluthathion peroxidase were increased 1.5-2 fold in ginsan treated mice compared to the irradiated mice. These findings indicate that ginsan may be a promising agent to be used in reducing the time needed for reconstituting of hematopoietic cells after irradiation treatment.

  15. A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

    Science.gov (United States)

    Mesel-Lemoine, Mariana; Millet, Jean; Vidalain, Pierre-Olivier; Law, Helen; Vabret, Astrid; Lorin, Valérie; Escriou, Nicolas; Albert, Matthew L; Nal, Béatrice; Tangy, Frédéric

    2012-07-01

    Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

  16. Effect of intramammary infusion of recombinant bovine GM-CSF and IL-8 on CMT score, somatic cell count, and milk mononuclear cell populations in Holstein cows with Staphylococcus aureus subclinical mastitis.

    Science.gov (United States)

    Kiku, Yoshio; Ozawa, Tomomi; Takahashi, Hideyuki; Kushibiki, Shiro; Inumaru, Shigeki; Shingu, Hiroyuki; Nagasawa, Yuya; Watanabe, Atsushi; Hata, Eiji; Hayashi, Tomohito

    2017-09-01

    The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.

  17. The effect of cytosolic extract of Alternaria aternata fungus on Monocyte-derived dendritic cell maturation and T-lymphocyte polarization in the presence of myelin basic protein

    Directory of Open Access Journals (Sweden)

    Loghmanni A

    2013-03-01

    Full Text Available Background: Multiple Sclerosis (MS is an autoimmune disease with impairment in function of central nervous system. Macrophages and dendritic cells play important roles in alleviating or progression of the disease. These cells can cause inflammation and damage to the myelin of nerve cells by realizing of harmful substances when these cells get matured. We studied the effect of Alternaria alternata extract on maturation of monocyte- derived dendritic cell (modc and T-cell responses in the presence of Myelin Basic Protein (MBP as a laboratory model of multiple sclerosis (MS. The purpose of this study is suitable dendritic cells production for usage in MS immunotherapy.Methods: For this study plastic adherent monocytes were cultured with granulocyte/ macrophage- colony stimulating factor (GM-CSF and interleukin -4 for converting these cells to modc and pulsed with MBP and matured in the presence of monocyte-conditioned medium (MCM in control group and MCM + Alternaria alternata extract in treatment groups. Anti-CD14, anti-CD83, anti-human leukocyte antigen-DR (anti HLA-DR monoclonal antibody were carried out for phenotyping. Autologos T cell responses and cytokine production were evaluated.Results: The results showed that the expression of CD14 decreased and CD83, HLA-DR increased in treatment groups in comparison with control groups. The production amount of IL-10 overcame IL-12 and in T cell the production of cytokines, IL-17 and Interferon-γ (IFN-γ decreased and IL-4 was increased (P<0.05. These effects escalated with increasing of dosage from 50 to 100 (mg/ml (P<0.001.Conclusion: Alternaria alternata extract can cause maturation of MBP-pulsed modc and skewing of T- lymphocyte toward Th2 and thereby can evolve into a new strategy in immunotherapy of MS.

  18. Prednisolone phosphate-containing TRX-20 liposomes inhibit cytokine and chemokine production in human fibroblast-like synovial cells: a novel approach to rheumatoid arthritis therapy.

    Science.gov (United States)

    Harigai, Takashi; Hagiwara, Hitomi; Ogawa, Yumi; Ishizuka, Takanobu; Kaneda, Shinichi; Kimura, Junji

    2007-01-01

    To evaluate the potential of using prednisolone phosphate (PSLP)-containing 3,5-dipentadecyloxybenzamidine hydrochloride (TRX-20) liposomes to treat rheumatoid arthritis (RA), we examined their ability to bind human fibroblast-like synovial (HFLS) cells and their effects in these cells. To test for binding, Lissamine rhodamine B-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine)-labelled PSLP-containing TRX-20 liposomes were added to HFLS cells, and the fluorescence intensity of the rhodamine bound to the cells was evaluated. Rhodamine-labelled PSLP-containing liposomes without TRX-20 were used as a negative control. To evaluate the uptake of liposomes by the HFLS cells, we used TRX-20 liposomes containing 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) and p-xylene-bis-pyridinium bromide (DPX), and observed the cells by fluorescence microscopy. The effects of the PSLP in TRX-20 liposomes on HFLS cells were assessed by the inhibition of the production of two inflammatory cytokines (interleukin 6 and granulocyte macrophage colony-stimulating factor) and one inflammatory chemokine (interleukin 8). The interaction of the PSLP-containing TRX-20 liposomes with HFLS cells was approximately 40 times greater than that of PSLP-containing liposomes without TRX-20. PSLP-containing TRX-20 liposomes bound to HFLS cells primarily via chondroitin sulfate. TRX-20 liposomes taken up by the cell were localized to acidic compartments. Furthermore, the PSLP-containing TRX-20 liposomes inhibited the production of the inflammatory cytokines and the chemokine more effectively than did the PSLP-containing liposomes without TRX-20. These results indicate that PSLP-containing TRX-20 liposomes show promise as a novel drug delivery system that could enhance the clinical use of glucocorticoids for treating RA.

  19. Effects of high glucose on mesenchymal stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Li, Yu-Ming; Schilling, Tatjana; Benisch, Peggy

    2007-01-01

    High glucose (HG) concentrations impair cellular functions and induce apoptosis. Exposition of mesenchymal stem cells (MSC) to HG was reported to reduce colony forming activity and induce premature senescence. We characterized the effects of HG on human MSC in vitro using telomerase-immortalized...

  20. The fungal quorum-sensing molecule farnesol activates innate immune cells but suppresses cellular adaptive immunity.

    Science.gov (United States)

    Leonhardt, Ines; Spielberg, Steffi; Weber, Michael; Albrecht-Eckardt, Daniela; Bläss, Markus; Claus, Ralf; Barz, Dagmar; Scherlach, Kirstin; Hertweck, Christian; Löffler, Jürgen; Hünniger, Kerstin; Kurzai, Oliver

    2015-03-17

    Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-α] and macrophage inflammatory protein 1 alpha [MIP-1α]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be

  1. Comparison of the Ex Vivo Expansion of UCB-Derived CD34+ in 3D DBM/MBA Scaffolds with USSC as a Feeder Layer.

    Science.gov (United States)

    Sadat Hashemi, Zahra; Forouzandeh Moghadam, Mahdi; Soleimani, Masoud

    2013-10-01

    Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34(+) were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.

  2. Di-(2-ethylhexyl) phthalate affects immune cells from atopic prone mice in vitro

    International Nuclear Information System (INIS)

    Koike, Eiko; Inoue, Ken-ichiro; Yanagisawa, Rie; Takano, Hirohisa

    2009-01-01

    Phthalate esters as plasticizers have been widespread in the environment and may be associated with development of allergic diseases such as asthma and atopic dermatitis. However, the underlying mechanisms have not been fully elucidated. The present study investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) on immune cells from atopic prone NC/Nga mice in vitro. Bone marrow-derived dendritic cells (BMDC) as a professional antigen-presenting cell and splenocytes as mixture of immune cells were used. BMDC were differentiated by culture with granulocyte macrophage-colony stimulating factor (GM-CSF) in the presence of DEHP (0.1-10 μM) for 6 days. In another experiments, BMDC were differentiated by culture with GM-CSF for 8 days then these BMDC were exposed to DEHP (0.1-100 μM) for 24 h. Splenocytes were exposed to DEHP for 24 h (0.1-100 μM) or 72 h (0.1-1000 nM). After the culture, the phenotypic markers and the function of BMDC and splenocytes were evaluated. BMDC differentiated in the presence of DEHP showed enhancement in the expression of MHC class II, CD86, CD11c and DEC205, and in their antigen-presenting activity. On the other hand, the function of the differentiated BMDC was not activated by DEHP although DEHP partly enhanced their expression of DEC205. DEHP-exposed splenocytes showed increases in their TCR and CD3 expression, interleukin-4 production, and antigen-stimulated proliferation. These results demonstrate that DEHP enhances BMDC differentiation but not activation and also enhances Th2 response in splenocytes from atopic prone mice. The enhancement might contribute to the aggravating effect of DEHP on allergic disorders.

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

    Science.gov (United States)

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

    2010-04-01

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

  4. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  5. Functional Defects in Type 3 Innate Lymphoid Cells and Classical Monocytes in a Patient with Hyper-IgE Syndrome.

    Science.gov (United States)

    Chang, Yuna; Kang, Sung-Yoon; Kim, Jihyun; Kang, Hye-Ryun; Kim, Hye Young

    2017-10-01

    Hyper-IgE syndrome (HIES) is a very rare primary immune deficiency characterized by elevated serum IgE levels, recurrent bacterial infections, chronic dermatitis, and connective tissue abnormalities. Autosomal dominant (AD) HIES involves a mutation in signal transducer and activator of transcription 3 (STAT3) that leads to an impaired T H 17 response. STAT3 signaling is also involved in the function of RORγt + type 3 innate lymphoid cells (ILC3s) and RORγt + T H 17 cells. The aim of this study was to investigate the role of innate immune cells such as innate lymphoid cells (ILCs), granulocytes, and monocytes in a patient with HIES. Peripheral blood mononuclear cells (PBMCs) from a patient with HIES and three age-matched healthy controls were obtained for the analysis of the innate and adaptive immune cells. The frequencies of ILCs in PBMCs were lower in the patient with HIES than in the controls. Moreover, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A produced by ILC3s in PBMCs were lower in the patient with HIES than the controls. Compared with the controls, classical monocytes (CD14 + CD16 low ), which have a high antimicrobial capability, were also lower in the patient with HIES, while non-classical monocytes (CD14 low CD16 + ) as well as intermediate monocytes (CD14 + CD16 intermediate ) were higher. Taken together, these results indicate that the impaired immune defense against pathogenic microbes in the patient with HIES might be partially explained by functional defects in ILC3s and inflammatory monocytes.

  6. Chromosome aberrations and cell survival in irradiated mammalian cells

    International Nuclear Information System (INIS)

    Tremp, J.

    1981-01-01

    A possible correlation between chromosome aberrations and reduced proliferation capacity or cell death was investigated. Synchronized Chinese hamster fibroblast cells were irradiated with 300 rad of x rays in early G 1 . Despite synchronization the cells reached the subsequent mitosis at different times. The frequency of chromosome aberrations was determined in the postirradiation division at 2-h intervals. The highest frequency occurred in cells with a first cell cycle of medium length. The colony-forming ability of mitotic cells was measured in parallel samples by following the progress of individual mitoses. The proportion of cells forming macrocolonies decreased with increasing cell cycle length, and the number of non-colony-forming cells increased. Irrespective of various first cell cycle lengths and different frequencies of chromosome aberrations, the number of cells forming microcolonies remained constant. A correlation was found between the absence of chromosome aberrations and the ability of cells to form macrocolonies. However, cells with a long first cell cycle formed fewer macrocolonies than expected

  7. VEGF and IHH rescue definitive hematopoiesis in Gata-4 and Gata-6-deficient murine embryoid bodies.

    Science.gov (United States)

    Pierre, Monique; Yoshimoto, Momoko; Huang, Lan; Richardson, Matthew; Yoder, Mervin C

    2009-09-01

    Murine embryonic stem cells can be differentiated into embryoid bodies (EBs), which serve as an in vitro model recapitulating many aspects of embryonic yolk sac hematopoiesis. Differentiation of embryonic stem cells deficient in either Gata-4 or Gata-6 results in EBs with disrupted visceral endoderm (VE). While lack of VE has detrimental effects on hematopoiesis in vivo, it is unclear whether lack of VE affects hematopoiesis in EBs. Therefore, we compared Gata-4 null (G4N) and Gata-6 null (G6N) EBs with wild-type EBs to assess their ability to commit to hematopoietic cells. EB VE formation was examined using cell-sorting techniques and analysis visceral endoderm gene expression. Hematopoietic progenitor potential of EBs cultured under various conditions was assessed using colony-forming assays. Definitive erythroid, granulocyte-macrophage, and mixed colonies were significantly reduced in G4N and G6N EBs compared to wild-type EBs. Vascular endothelial growth factor (VEGF) expression and secretion were also reduced in both G4N and G6N EBs, consistent with VE serving as a site of VEGF production. Addition of exogenous VEGF(165), to EB cultures completely rescued definitive colony-forming cells in G4N and G6N EBs. This rescue response could be blocked by addition of soluble Flk-1 Fc to EB cultures. Similarly, addition of exogenous Indian hedgehog to EB cultures also recovers the diminishment in definitive hematopoiesis in a reversible manner. These results suggest that the absence of VE in G4N and G6N EBs does not prevent emergence of definitive progenitors from EBs. However, the decreased level of VEGF and Indian hedgehog production in VE devoid G4N and G6N EBs attenuates definitive hematopoietic progenitor cell expansion.

  8. Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

    Science.gov (United States)

    Papadopoulos, Alexia; Gagnaire, Aurélie; Degos, Clara; de Chastellier, Chantal; Gorvel, Jean-Pierre

    2016-01-01

    Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

  9. Role of endothelial progenitor cells and inflammatory cytokines in healing of diabetic foot ulcers.

    Directory of Open Access Journals (Sweden)

    Francesco Tecilazich

    Full Text Available To evaluate changes in endothelial progenitor cells (EPCs and cytokines in patients with diabetic foot ulceration (DFU in association with wound healing.We studied healthy subjects, diabetic patients not at risk of DFU, at risk of DFU and with active DFU. We prospectively followed the DFU patients over a 12-week period. We also investigated similar changes in diabetic rabbit and mouse models of wound healing.All EPC phenotypes except the kinase insert domain receptor (KDR(+CD133(+ were reduced in the at risk and the DFU groups compared to the controls. There were no major EPC differences between the control and not at risk group, and between the at risk and DFU groups. Serum stromal-cell derived factor-1 (SDF-1 and stem cell factor (SCF were increased in DFU patients. DFU patients who healed their ulcers had lower CD34(+KDR(+ count at visits 3 and 4, serum c-reactive protein (CRP and granulocyte-macrophage colony-stimulating factor (GM-CSF at visit 1, interleukin-1 (IL-1 at visits 1 and 4. EPCs tended to be higher in both diabetic animal models when compared to their non-diabetic counterparts both before and ten days after wounding.Uncomplicated diabetes does not affect EPCs. EPCs are reduced in patients at risk or with DFU while complete wound healing is associated with CD34(+KDR(+ reduction, suggesting possible increased homing. Low baseline CRP, IL-1α and GM-CSF serum levels were associated with complete wound healing and may potentially serve as prognostic markers of DFU healing. No animal model alone is representative of the human condition, indicating the need for multiple experimental models.

  10. Combined measurement of growth and differentiation in suspension cultures of purified human CD34-positive cells enables a detailed analysis of myelopoiesis

    NARCIS (Netherlands)

    Kerst, J. M.; Slaper-Cortenbach, I. C.; von dem Borne, A. E.; van der Schoot, C. E.; van Oers, R. H.

    1992-01-01

    In this study we have made a detailed analysis of growth factor (granulocyte-macrophage colony-stimulating factor [GM-CSF], granulocyte colony-stimulating factor [G-CSF], and macrophage colony-stimulating factor [M-CSF])-induced proliferation and differentiation of highly purified CD34+ committed

  11. Normal and sublethally irradiated stem and granulocyte progenitor cell regeneration in an in vivo culture system. The cellular response to humoral factors released through the action of cyclophosphamide

    International Nuclear Information System (INIS)

    MacVittie, T.

    1977-01-01

    The in vivo diffusion chamber (DC) method of marrow culture was used to determine if the injection of host mice with cyclophosphamide (CY) caused, through its cytoxic action, the release of a humoral factor(s) capable of initiating stem cell (CFU-s) and granulocyte-macrophage progenitor cell (CFU-c) proliferation. Host mice were injected with CY 1-4 days prior to 800 rad of 60 Co WBI and implantation of DCs containing normal or 400 rad sublethally irradiated (SLI) marrow cells. The greatest proliferative response within CFU-s and CFU-c populations occurred in those mice injected with CY 3 days prior to implant. The marked CFU-s and CFU-c regeneration was initiated during the initial 24 hr of culture in both normal and SLI marrow cells. Thereafter growth rates were approximately the same. SLI marrow, however, showed a greater response to the humoral effects of CY injection than did normal marrow. These data provided evidence that CY induced the release of a diffusible factor(s) capable of accelerating regeneration of normal and sublethally irradiated CFU-s and CFU-c, the magnitude of which was dependent upon the time elapsed between CY injected and implantation of DCs. The marked proliferative response of the SLI stem and progenitor cells to the humoral stimulation may be indicative of the heterogeneity of both CFU-s and CFU-c populations surviving sublethal radiation exposure. The target cells may have possessed a differential sensitivity to the factor(s) initiating cell proliferation

  12. Stimulated human fibroblast cell survival

    International Nuclear Information System (INIS)

    Smith, B.P.; Gale, K.L.; Einspenner, M.; Greenstock, C.L.; Gentner, N.E.

    1992-01-01

    Techniques for cloning cultured mammalian cells have supported the most universally-accepted method for measuring the induction of lethality by geno-toxicants such as ionizing radiation: the 'survival of colony-forming ability (CFA)' assay. Since most cultured human cell lines exhibit plating efficiency (i.e. the percentage of cells that are capable of reproductively surviving and dividing to form visible colonies) well below 100%, such assays are in essence 'survival of plating efficiency' assays, since they are referred to the plating (or cloning) efficiency of control (i.e. unirradiated) cells. (author). 8 refs., 2 figs

  13. Number and proliferative capacity of osteogenic stem cells are maintained during aging and in patients with osteoporosis

    DEFF Research Database (Denmark)

    Stenderup, Karin; Justesen, J.; Eriksen, E.F.

    2001-01-01

    Decreased bone formation is an important pathophysiological mechanism responsible for bone loss associated with aging and osteoporosis. Osteoblasts (OBs), originate from mesenchymal stem cells (MSCs) that are present in the bone marrow and form colonies (termed colony-forming units-fibroblastic [...

  14. Anti-proliferative effects of T cells expressing a ligand-based chimeric antigen receptor against CD116 on CD34+ cells of juvenile myelomonocytic leukemia

    Directory of Open Access Journals (Sweden)

    Yozo Nakazawa

    2016-03-01

    Full Text Available Abstract Background Juvenile myelomonocytic leukemia (JMML is a fatal, myelodysplastic/myeloproliferative neoplasm of early childhood. Patients with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF receptor (GMR, CD116 signaling pathway. Allogeneic hematopoietic stem cell transplantation is currently the only curative treatment option for JMML; however, disease recurrence is a major cause of treatment failure. We investigated adoptive immunotherapy using GMR-targeted chimeric antigen receptor (CAR for JMML. Methods We constructed a novel CAR capable of binding to GMR via its ligand, GM-CSF, and generated piggyBac transposon-based GMR CAR-modified T cells from three healthy donors and two patients with JMML. We further evaluated the anti-proliferative potential of GMR CAR T cells on leukemic CD34+ cells from six patients with JMML (two NRAS mutations, three PTPN11 mutations, and one monosomy 7, and normal CD34+ cells. Results GMR CAR T cells from healthy donors suppressed the cytokine-dependent growth of MO7e cells, but not the growth of K562 and Daudi cells. Co-culture of healthy GMR CAR T cells with CD34+ cells of five patients with JMML at effector to target ratios of 1:1 and 1:4 for 2 days significantly decreased total colony growth, regardless of genetic abnormality. Furthermore, GMR CAR T cells from a non-transplanted patient and a transplanted patient inhibited the proliferation of respective JMML CD34+ cells at onset to a degree comparable to healthy GMR CAR T cells. Seven-day co-culture of GMR CAR T cells resulted in a marked suppression of JMML CD34+ cell proliferation, particularly CD34+CD38− cell proliferation stimulated with stem cell factor and thrombopoietin on AGM-S3 cells. Meanwhile, GMR CAR T cells exerted no effects on normal CD34+ cell colony growth. Conclusions Ligand-based GMR CAR T cells may have anti-proliferative effects on stem and progenitor cells in JMML.

  15. Differential transfection efficiency rates of the GM-CSF gene into human renal cell carcinoma lines by lipofection.

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    Hernández, A; Zöller, K; Enczmann, J; Ebert, T; Schmitz-Draeger, B; Ackermann, R; Wernet, P

    1997-01-01

    One of the major questions in any gene therapy approach is the selection of the appropriate vector system. Here, the optimization of a gene transfer protocol for renal cell carcinoma using lipofection as a nonviral gene transduction system was evaluated. To select the promoter which gives the highest expression, different plasmids which are able to express Escherichia coli beta-galactosidase gene as a reporter gene under the control of different promoters were tested: human cytomegalovirus promoter (pCMVbeta), simian virus 40 promoter (pSVbeta), adenovirus promoter (ADbeta), and herpes simplex virus thymidine kinase promoter (TKbeta). The pCMVbeta revealed the highest expression of the beta-gal gene in the renal cell carcinoma (RCC) lines. Thus this CMV promoter was selected for the expression of the granulocyte-macrophage colony stimulator factor (GM-CSF) gene. Three different lipids (LipofectAmine, LipofectAce, and Lipofectin) were compared for their transduction efficiency, and the optimal conditions for quantitatively high lipofection rates were established. The consistently best results regarding gene expression as well as viability of the RCC lines were obtained when Lipofectin was used. Gene expression was monitored by a specific enzyme-linked immunosorbent assay and functionally validated by a cell proliferation test. The GM-CSF expression profile showed a peak at 48 hours after transfection and was still detectable after 5 days. Here the feasibility of efficient lipofection of the GM-CSF gene into RCC lines is demonstrated. Most importantly, considerable differences in the relative quantity of GM-CSF gene transfer into the different RCC lines was observed here. This may be of critical relevance for the design of any clinical gene transduction protocol in tumor cell vaccination attempts.

  16. Isolation of monocytes from leukapheretic products for large-scale GMP-grade generation of cytomegalovirus-specific T-cell lines by means of an automated elutriation device.

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    Perseghin, Paolo; D'Amico, Giovanna; Dander, Erica; Gaipa, Giuseppe; Dassi, Maria; Biagi, Ettore; Biondi, Andrea

    2008-08-01

    Dendritic cells (DC) act as antigen-presenting cells in immune response-mediated mechanisms against malignant cells and/or viral or fungal pathogens. CD14+ monocytes have been so far isolated by techniques of plastic adherence or by using immunomagnetic methods. Here the effectiveness of a commercially available cell separation system (Elutra, Gambro BCT) in the separation of monocytes and the large-scale production of cytomegalovirus (CMV)-specific T-cell lines were investigated. Six mononuclear cell (MNC) collections were processed with the Elutra system. Monocyte-enriched fraction was differentiated into DCs by addition of granulocyte-macrophage-colony-stimulating factor and interleukin (IL)-4. After 6 days of culture, DCs were matured in the presence of interferon (IFN)-gamma, IFN-alpha, IL-1beta, tumor necrosis factor-alpha, and poly(I:C) and pulsed with a pool of 48 MHC Class I and II-binding CMV peptides. Lymphocytes were then stimulated with mature autologous CMV peptide-pulsed DCs. After elutriation, the mean monocyte yield was 0.89 x 10(9) +/- 0.65 x 10(9), with a 51.0 +/- 31.6 percent recovery and a 51.1 +/- 35.4 percent purity. A significant correlation was observed when basal monocyte content was related to the postelutriation recovery (p < 0.0116). More than 60 percent of plated monocytes were differentiated into DCs, which after pulsing with CMV peptides, were able to stimulate a robust enrichment in CMV antigen-specific T cells in all tested samples (mean percentage of pentamer-positive CD8+ cells, 35% compared to the initial 2%). Our findings might be helpful for an appropriate MNC collection, to maximize the efficiency of the elutriation system and subsequently obtain an optimal monocyte-enriched yield for further DC generation and T-cell stimulation.

  17. Radioprotective effects of combination broncho-vaxom, a macrophage activator, and indomethacin, an inhibitor of prostaglandin production. Relationship to myelopoiesis

    Energy Technology Data Exchange (ETDEWEB)

    Fedorocko, P.; Mackova, N.O. [Safarik Univ., Faculty ofSciences, Dept. of Cellular and Molecular Biology, Kosice (Slovakia)

    1996-01-01

    The effects of the bacterial extract broncho-vaxom (BV; radioprotective immunomodulator; 500 {mu}g/mouse i.p., -24 h) and indomethacin (INDO; inhibitor of prostaglandin production; 2x40 {mu}g/mouse i.m., - 24 h and - 3 h) on the post-irradiation recovery of hemopoietic functions in mice were investigated. Both agents were administered either alone or in combination. Endogenous spleen colony formation was increased in all treatment groups, with combination-treated mice exhibiting the greatest effects. Similarly, 24 h after combined administration of BV and INDO (i.e. at the time of presumed irradiation) to the non-irradiated mice granulocyte-macrophage colony-forming cell (GM-CFC) numbers were greater in the bone marrow and spleen. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in the S-phase of the cell cycle in the bone marrow. However, GM-CFC in the spleen of combination pretreated mice was not stimulated to significant proliferation as compared to GM-CFC in the spleen of mice injected with BV alone. Combined modality treatment was also more effective than single agent treatments in accelerating bone marrow cellularity and GM-CFC regeneration, but not in accelerating GM-CFC regeneration in the spleen. Combined administration of BV and INDO to mice prior to lethal irradiation exerted and additional radioprotective effect and protected 95% of the C57B1/6 mice. (au) 42 refs.

  18. The protective effect of Royal Jelly against the hemopoiesis dysfunction in X-irradiated mice

    Energy Technology Data Exchange (ETDEWEB)

    Emori, Yutaka; Oka, Hideki; Ohya, Osamu; Tamaki, Hajime; Hayashi, Yoshiro [Zeria Pharmaceutical Co., Ltd., Konan, Saitama (Japan). Central Research Laboratories; Nomoto, Kikuo

    1998-02-01

    The protective effect of Royal Jelly (RJ) against the hemopoietic dysfunction in whole body X-irradiated C57BL/6 mice was investigated. When RJ (1.0 g/kg, po or 0.5 g/kg, ip) was administered every day beginning two weeks before X-irradiation (10 Gy), a significant increase in the number of leukocytes and erythrocytes was observed in mice treated with RJ, as compared with X-irradiated control. In addition, the number of colony forming units in culture (CFU-C) of bone marrow cells or splenocytes was significantly increased in mice treated with RJ. Therefore, when granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) in peripheral blood was measured by ELISA kit, a significant increase in the amount of GM-CSF and IL-3 was observed. These results suggest that the protective effect of RJ against hemopoietic dysfunction could be expressed through an increase in the number of hemopoietic stem cells by the induction of hemopoietic factor such as GM-CSF and IL-3. (author)

  19. Apigenin inhibits TNFα/IL-1α-induced CCL2 release through IKBK-epsilon signaling in MDA-MB-231 human breast cancer cells.

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    David Bauer

    Full Text Available Mortality associated with breast cancer is attributable to aggressive metastasis, to which TNFα plays a central orchestrating role. TNFα acts on breast tumor TNF receptors evoking the release of chemotactic proteins (e.g. MCP-1/CCL2. These proteins direct inward infiltration/migration of tumor-associated macrophages (TAMs, tumor-associated neutrophils (TANs, myeloid-derived suppressor cells (MDSCs, T-regulatory cells (Tregs, T helper IL-17-producing cells (Th17s, metastasis-associated macrophages (MAMs and cancer-associated fibroblasts (CAFs. Tumor embedded infiltrates collectively enable immune evasion, tumor growth, angiogenesis, and metastasis. In the current study, we investigate the potential of apigenin, a known anti-inflammatory constituent of parsley, to downregulate TNFα mediated release of chemokines from human triple-negative cells (MDA-MB-231 cells. The results show that TNFα stimulation leads to large rise of CCL2, granulocyte macrophage colony-stimulating factor (GMCSF, IL-1α and IL-6, all suppressed by apigenin. While many aspects of the transcriptome for NFkB signaling were evaluated, the data show signaling patterns associated with CCL2 were blocked by apigenin and mediated through suppressed mRNA and protein synthesis of IKBKe. Moreover, the data show that the attenuation of CCL2 by apigenin in the presence TNFα paralleled the suppression of phosphorylated extracellular signal-regulated kinase 1 (ERK 1/ 2. In summary, the obtained findings suggest that there exists a TNFα evoked release of CCL2 and other LSP recruiting cytokines from human breast cancer cells, which can be attenuated by apigenin.

  20. Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

    Science.gov (United States)

    Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

    2017-01-06

    Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

  1. Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

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    Mukae Hiroshi

    2005-08-01

    Full Text Available Abstract Background Studies from our laboratory have shown that human alveolar macrophages (AM and bronchial epithelial cells (HBEC exposed to ambient particles (PM10 in vitro increase their production of inflammatory mediators and that supernatants from PM10-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation. Methods The present study concerns co-culture of AM and HBEC exposed to PM10 (EHC-93 and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM-1 in the production of these mediators. Results AM/HBEC co-cultures exposed to 100 μg/ml of PM10 for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF, M-CSF, macrophage inflammatory protein (MIP-1β, monocyte chemotactic protein (MCP-1, interleukin (IL-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p 10-induced increase in co-culture mRNA expression. Conclusion We conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM10-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.

  2. Hematopoietic stem/progenitor cell proliferation and differentiation is differentially regulated by high-density and low-density lipoproteins in mice.

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    Yingmei Feng

    Full Text Available RATIONALE: Hematopoietic stem/progenitor cells (HSPC are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation. OBJECTIVES: We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells. METHODS AND RESULTS: HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr(-/- mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB. In addition, an increased proportion of BM HSPC was in G(2M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP increased in BM of LDLr(-/- mice. When BM Lin-Sca-1+cKit+ (i.e. "LSK" cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin- cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL or reconstituted HDL (rHDL, the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro. CONCLUSION: Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.

  3. Dexamethasone-Induced Myeloid-Derived Suppressor Cells Prolong Allo Cardiac Graft Survival through iNOS- and Glucocorticoid Receptor-Dependent Mechanism

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    Yang Zhao

    2018-02-01

    Full Text Available How to induce immune tolerance without long-term need for immunosuppressive drugs has always been a central problem in solid organ transplantation. Modulating immunoregulatory cells represents a potential target to resolve this problem. Myeloid-derived suppressor cells (MDSCs are novel key immunoregulatory cells in the context of tumor development or transplantation, and can be generated in vitro. However, none of current systems for in vitro differentiation of MDSCs have successfully achieved long-term immune tolerance. Herein, we combined dexamethasone (Dex, which is a classic immune regulatory drug in the clinic, with common MDSCs inducing cytokine granulocyte macrophage colony stimulating factor (GM-CSF to generate MDSCs in vitro. Addition of Dex into GM-CSF system specifically increased the number of CD11b+ Gr-1int/low MDSCs with an enhanced immunosuppressive function in vitro. Adoptive transfer of these MDSCs significantly prolonged heart allograft survival and also favored the expansion of regulatory T cells in vivo. Mechanistic studies showed that inducible nitric oxide sythase (iNOS signaling was required for MDSCs in the control of T-cell response and glucocorticoid receptor (GR signaling played a critical role in the recruitment of transferred MDSCs into allograft through upregulating CXCR2 expression on MDSCs. Blockade of GR signaling with its specific inhibitor or genetic deletion of iNOS reversed the protective effect of Dex-induced MDSCs on allograft rejection. Together, our results indicated that co-application of Dex and GM-CSF may be a new and important strategy for the induction of potent MDSCs to achieve immune tolerance in organ transplantation.

  4. [Imiquimod combined with dendritic cell vaccine decreases Treg proportion and enhances anti-tumor responses in mice bearing melanoma].

    Science.gov (United States)

    Ren, Shurong; Wang, Qiubo; Zhang, Yanli; Lu, Cuixiu; Li, Ping; Li, Yumei

    2017-02-01

    Objective To investigate the therapeutic effect of Toll-like receptor 7 (TLR7) agonist imiquimod combined with dendritic cell (DC)-based tumor vaccine on melanoma in mice and the potential mechanism. Methods Melanoma-bearing mouse models were established by subcutanous injection of B16-OVA cells into C57BL/6 mice. DCs were isolated from mouse bone marrow and propagated in culture medium with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant mouse interleukin-4 (rmIL-4). DC vaccine (OVA-DC) was prepared by overnight incubation of DCs added with chicken ovalbumin. C57BL/6 mice were separated into four groups which were treated with PBS, topical imiquimod application, OVA-DC intradermal injection and imiquimod plus OVA-DC, respectively. The tumor size was calculated by digital vernier caliper. Peripheral blood CD4 + FOXP3 + Tregs of the tumor-bearing mice was detected by flow cytometry. The cytotoxicity of splenic lymphocyte against B16-OVA was assessed in vitro by CCK-8 assay. Results Compared with the other three groups, B16-OVA-bearing mice treated with imiquimod plus DC vaccine had the smallest tumor volume. The percentage of CD4 + FOXP3 + Tregs decreased significantly in the combined treated mice. The combined treatment enhanced significantly cytotoxicity of splenic lymphocytes against B16-OVA cells. Conclusion Imiquimod combined with antigen-pulsed-DC vaccine could reduce CD4 + FOXP3 + Treg proportion and promote anti-tumor effect in mice with melanoma.

  5. Proliferation-stimulating effect of colony stimulating factor 2 on porcine trophectoderm cells is mediated by activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase.

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    Wooyoung Jeong

    Full Text Available Colony-stimulating factor 2 (CSF2, also known as granulocyte macrophage colony-stimulating factor, facilitates mammalian embryonic development and implantation. However, biological functions and regulatory mechanisms of action of porcine endometrial CSF2 in peri-implantation events have not been elucidated. The aim of present study was to determine changes in cellular activities induced by CSFs and to access CSF2-induced intracellular signaling in porcine primary trophectoderm (pTr cells. Differences in expression of CSF2 mRNA in endometrium from cyclic and pregnant gilts were evaluated. Endometrial CSF2 mRNA expression increases during the peri-implantation period, Days 10 to 14 of pregnancy, as compared to the estrous cycle. pTr cells obtained in Day 12 of pregnancy were cultured in the presence or absence of CSF2 (20 ng/ml and LY294002 (20 µM, U0126 (20 µM, rapamycin (20 nM, and SB203580 (20 µM. CSF2 in pTr cell culture medium at 20 ng/ml significantly induced phosphorylation of AKT1, ERK1/2, MTOR, p70RSK and RPS6 protein, but not STAT3 protein. Also, the PI3K specific inhibitor (LY294002 abolished CSF2-induced increases in p-ERK1/2 and p-MTOR proteins, as well as CSF2-induced phosphorylation of AKT1. Changes in proliferation and migration of pTr cells in response to CSF2 were examined in dose- and time-response experiments. CSF2 significantly stimulated pTr cell proliferation and, U0126, rapamycin and LY294002 blocked this CSF2-induced proliferation of pTr cells. Collectively, during the peri-implantation phase of pregnancy in pigs, endometrial CSF2 stimulates proliferation of trophectoderm cells by activation of the PI3K-and ERK1/2 MAPK-dependent MTOR signal transduction cascades.

  6. Growth-inhibiting effect of tumor necrosis factor on human umbilical vein endothelial cells is enhanced with advancing age in vitro

    International Nuclear Information System (INIS)

    Shimada, Y.; Kaji, K.; Ito, H.; Noda, K.; Matsuo, M.

    1990-01-01

    We have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon-gamma (IFN-gamma). The growth and colony-forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70-80% of life span completed). It was found that treatment with TNF inhibited growth and colony-forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony-forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF-binding sites per cell-surface area remained rather constant. The effect of TNF was augmented by IFN-gamma at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN-gamma was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony-forming ability was observed only in aged cells. These results suggest that the intrinsic responsiveness of HUVECs to growth-inhibiting factors, as well as to growth-stimulating factors, changes during aging in vitro

  7. Comparison of 864 and 935 MHz microwave radiation effects on cell culture

    International Nuclear Information System (INIS)

    Pavicic, I.; Trosic, I.; Sarolic, A.

    2005-01-01

    The aim of our study was to evaluate and compare the effect of 864 and 935 MHz microwave radiation on proliferation, colony forming and viability of Chinese hamster lung cells, cell line V79. Cell cultures were exposed both to the 864 MHz microwave field in transversal electromagnetic mode cell (TEM-cell) and to the 935 MHz field in Gigahertz transversal electromagnetic mode cell (GTEM-cell) for 1, 2 and 3 hours. Philips PM 5508 generator connected with a signal amplifier generated the frequency of 864 MHz, whereas Hewlett Packard HP8657A signal generator was used to generate the frequency of 935 MHz. The average specific absorption rate (SAR) was 0.08 W/kg for 864 MHz and 0.12 W/kg for 935 MHz. To determine the cell growth, V79 cells were plated in the concentration of 1x10 4 cells per milliliter of nutrient medium. Cells were cultured in a humidified atmosphere at 37 degrees of C in 5% CO 2 . Cell proliferation was determined by cell counts for each hour of exposure during the five post-exposure days. To identify colony-forming ability, cells were cultivated in the concentration of 40 cells/mL of medium and incubated as described above. Colony-forming ability was assessed for each exposure time by colony count on post-exposure day 7. Trypan blue exclusion test was used to determine cell viability. On post-exposure day 3, the growth curve of 864 MHz irradiated cells showed a significant decrease (p less than 0.05) after 2 and 3 hours of exposure in comparison with control cells. Cells exposed to 935 MHz radiation showed a significant decrease (p less than 0.05) after 3 hours of exposure on post-exposure day 3. Both the colony-forming ability and viability of 864 MHz and 935 MHz exposed cells did not significantly differ from matched control cells. In conclusion, both applied RF/MW fields have shown similar effects on cell culture growth, colony forming and cell viability of the V79 cell line.(author)

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Changes in some pro-and anti-inflammatory cytokines produced by bovine peripheral blood mononuclear cells following foot and mouth disease vaccination

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    N. Delirezh

    2016-09-01

    Full Text Available Interleukin (IL-17 is exclusively produced by CD4 helper T-cells upon activation. It most often acts as a pro-inflammatory cytokine, which stimulates the release of pro-inflammatory cytokines IL-6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF. In this study, we studied the in-vitro IL-17 response to specific antigens and a variety of mitogens and compared the IL-17 response to IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-γ responses. We used a foot and mouth disease (FMD vaccine as specific antigens and mitogens (phytohemagglutinin [PHA], pokeweed mitogen [PWM], and concanavalin A [Con A] to stimulate peripheral blood mononuclear cells (PBMCs of vaccinated calves. Cell culture supernatant was harvested and analyzed for cytokines, using commercially available bovine ELISA kits. The mitogens induced a significant increase in IL-17 production. IL-17 was produced at high levels in response to the T cell-stimulated mitogens, PHA, and Con A, and at low levels in response to PWM mitogens. In contrast, level of the produced IL-17 cytokines in response to the FMDV antigens was lower as compared to those produced by mitogens. The FMDV antigens and mitogens significantly increased IL-17 production. There was not a correlation between IL-17 production and type-1 cytokine, IFN-γ, and IL-2, while there was a correlation between type-2 cytokine, IL-4, and IL-5 at either cytokine level produced by PBMCs stimulated by FMDV antigens. Moreover, there was an interaction between IL-17 and IL-6, that is, as IL-6 cytokine level elevated or diminished, IL-17 cytokine level increased or decreased, as well.

  10. Effects of abdominal lavage fluid from rats with radiation injury and combined radiation-burn injury on growth of hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Su, Y.-P.; Cheng, T.-M.; Guo, C.-H.; Liu, X.-H.; Qu, J.-F.

    2003-01-01

    Full text: Objective: To observe the effects of abdominal lavage fluid from rats with radiation injury, burn injury and combined radiation-burn injury on growth of hematopoietic progenitor cells. Methods Rats were irradiated with a single dose of 12 Gy γ-ray of 60Co, combined with 30% of total body surface area (TBSA) generated under a 5 KW bromo-tungsten lamp for 25 s. Lavage fluid from the peritoneum was collected 3, 12, 24, 48 and 72 hours after injury. Then the lavage fluid was added to the culture media of erythrocyte progenitor cells (CFU-E, BFE-E) or of granulocyte-macrophage progenitor cells (CFU-GM) at 40 mg/ml final concentration. Results The formed clones of CFU-E, BFU-E and CFU-GM of the lavage fluid from rats with radiation injury or combined radiation-burn injury at 3h, 12h, 24h, 48h and 72h time points were significantly higher than those from normal. They reached their peaks at 24h after injury (215.7%, 202.3%, or 241.2% from burned rats and 188.1%, 202.3% or 204.6% from rats inflected with combined radiation-burn injury as compared with those from normal rats). However, few CFU-E, BFU-E or CFU-GM clones were found after addition of lavage fluid from irradiated rats. Conclusion Peritoneal lavage fluid from rats with burn injury or combined radiation-burn injury enhances the growth of erythrocytes and granulocyte progenitor cells. On the contrary, the lavage fluid from irradiated rats shows inhibitory effects

  11. Regulation of CTL responses to MHC-restricted class I peptide of the gp70 tumour antigen by splenic parenchymal CD4+ T cells in mice failing immunotherapy with DISC-mGM-CSF.

    Science.gov (United States)

    Ahmad, Murrium; Rees, Robert C; McArdle, Stephanie E; Li, Geng; Mian, Shahid; Entwisle, Claire; Loudon, Peter; Ali, Selman A

    2005-07-20

    Direct intratumour injection of the disabled infectious single-cycle-herpes simplex virus-encoding murine granulocyte/macrophage colony-stimulating factor (DISC-HSV-mGM-CSF) into established colon carcinoma CT26 tumours induced complete tumour rejection in up to 70% of treated animals (regressors), while the remaining mice developed progressive tumours (progressors). This murine Balb/c model was used to dissect the cellular mechanisms involved in tumour regression or progression following immunotherapy. CTLs were generated by coculturing lymphocytes and parenchymal cells from the same spleens of individual regressor or progressor animals in the presence of the relevant AH-1 peptide derived from the gp70 tumour-associated antigens expressed by CT26 tumours. Tumour regression was correlated with potent CTL responses, spleen weight and cytokine (IFN-gamma) production. Conversely, progressor splenocytes exhibited weak to no CTL activity and poor IFN-gamma production, concomitant with the presence of a suppressor cell population in the progressor splenic parenchymal cell fraction. Further fractionation of this parenchymal subpopulation demonstrated that cells inhibitory to the activation of AH-1-specific CTLs, restimulated in vitro with peptide, were present in the nonadherent parenchymal fraction. In vitro depletion of progressor parenchymal CD3+/CD4+ T cells restored the CTL response of the cocultured splenocytes (regressor lymphocytes and progressor parenchymal cells) and decreased the production of IL-10, suggesting that CD3+CD4+ T lymphocytes present in the parenchymal fraction regulated the CTL response to AH-1. We examined the cellular responses associated with tumour rejection and progression, identifying regulatory pathways associated with failure to respond to immunotherapy. Copyright 2005 Wiley-Liss, Inc.

  12. Clonal analysis of the progeny of UV-irradiated cells of Bacillus subtilis (uvr+ and uvr)

    International Nuclear Information System (INIS)

    Lotareva, O.V.; Filippov, V.D.

    1975-01-01

    The revertants to adenine prototrophy or mutants to auxotrophy can be easily identified on synthetic media which are pathly enriched with caseine hydrolyzate and yeast extract. It is shown with the use of these media that 1.5% colonies formed by Bacillus subtilis cells of the original type (ade6 met5) have mutant clones which are initiated by spontaneous revertants to adenine prototrophy. These revertants arise in the time of division of cells in macrocolonies. After plating diluted suspension of irradiated cells those colonies which contain mutant clones formed by spontaneous revertants can be erroneously taken for mixed colonies formed by induced revertants. About 40% mutants to auxotrophy induced by high dose of UV-light in uvr + cells form pure mutant colonies. The same mutants, induced in uvr cells by a five-times as-low UV-dose, usually form mixed colonies

  13. In vitro expansion of Lin+ and Lin− mononuclear cells from human peripheral blood

    International Nuclear Information System (INIS)

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul; Rohaya, M. A. W.

    2013-01-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin − ) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin + ) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin − cell population. The ability of Lin + and Lin − to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin + and Lin − were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin + mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin − stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation

  14. In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

    Science.gov (United States)

    Norhaiza, H. Siti; Rohaya, M. A. W.; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul

    2013-11-01

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin+) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin- to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin

  15. [Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

    Science.gov (United States)

    Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

    2013-08-01

    This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

  16. Langerhans cell-like dendritic cells stimulated with an adjuvant direct the development of Th1 and Th2 cells in vivo.

    Science.gov (United States)

    Matsui, K; Mori, A; Ikeda, R

    2015-10-01

    It is well known that Langerhans cells (LCs) work as the primary orchestrators in the polarization of immune responses towards a T helper type 1 (Th1) or Th2 milieu. In this study, we attempted to generate LCs from murine bone marrow cells and elicit a Th1- or Th2-prone immune response through the LCs after stimulation with Th1 or Th2 adjuvant. LCs were generated from murine bone marrow cells using granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and transforming growth factor (TGF)-β, and were obtained as I-A(d) positive cells. Mice were primed with Th1/Th2 adjuvant- and ovalbumin (OVA)-pulsed LCs and then given a booster injection of OVA 2 days later via the hind footpad. Five days after the OVA injection, the cytokine response in the draining popliteal lymph nodes was investigated by reverse transcription-polymerase chain reaction (RT-PCR) flow cytometry and enzyme-linked immunosorbent assay (ELISA). The generated LCs expressed typical LC surface markers, E-cadherin and Langerin, and were classified accordingly as LC-like dendritic cells (LDCs). Administration of Th1 adjuvant, cytosine-phosphate-guanosine (CpG)-DNA- and OVA-pulsed LDCs into the hind footpads of mice induced a Th1-prone immune response, as represented by up-regulation of IFN-γ production and down-regulation of IL-4 production in the lymph node cells. Conversely, Th2 adjuvant, histamine-pulsed LDCs induced a Th2-prone immune response, as represented by up-regulation of IL-4 production and down-regulation of IFN-γ production. These results suggest that LDCs may be used as a substitute for LCs and have the ability to induce the development of Th1 and Th2 cells in vivo. Our experimental system would therefore be useful for screening of inhibitors of Th1/Th2 differentiation in order to control allergic disease. © 2015 British Society for Immunology.

  17. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  18. Differential effect of procaine on irradiated mammalian cells in culture

    International Nuclear Information System (INIS)

    Djordjevic, B.

    1979-01-01

    HeLa and V-79 Chinese hamster cells temporarily stored in ampoules were treated with the local anesthetic procaine. Postirradiation treatment increased lethality in HeLa cells depending on drug concentration, duration of treatment, and cell density, as measured by colony-forming ability upon plating. If present during irradiation only, procaine protected from irradiation. In V-79 cells, procaine potentiated radiation lethality only in freshly trypsinized cells. Procaine effect was thus cell type specific and most likely involved the cell membrane

  19. Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

    Science.gov (United States)

    Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

    2008-09-01

    In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

  20. Phase 1/2 study of immunotherapy with dendritic cells pulsed with autologous tumor lysate in patients with refractory bone and soft tissue sarcoma.

    Science.gov (United States)

    Miwa, Shinji; Nishida, Hideji; Tanzawa, Yoshikazu; Takeuchi, Akihiko; Hayashi, Katsuhiro; Yamamoto, Norio; Mizukoshi, Eishiro; Nakamoto, Yasunari; Kaneko, Shuichi; Tsuchiya, Hiroyuki

    2017-05-01

    There are limited options for the curative treatment of refractory bone and soft tissue sarcomas. The purpose of this phase 1/2 study was to assess the immunological and clinical effects of dendritic cells (DCs) pulsed with autologous tumor lysate (TL) in patients with advanced bone and soft tissue sarcomas. Thirty-seven patients with metastatic or recurrent sarcomas were enrolled in this study. Peripheral blood mononuclear cells obtained from the patients were suspended in media containing interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor. Subsequently, these cells were treated with TL, tumor necrosis factor α, and OK-432. The DCs were injected into the inguinal or axillary region. One treatment course comprised 6 weekly DC injections. The toxicity, clinical response (tumor volume, serum interferon-γ [IFN-γ], and serum IL-12), and oncological outcomes were observed. In total, 47 courses of DC therapy were performed in 37 patients. No severe adverse events or deaths associated with the DC injections were observed in the study patients. Increased serum IFN-γ and IL-12 levels were observed 1 month after the DC injection. Among the 37 patients, 35 patients were assessed for clinical responses: 28 patients showed tumor progression, 6 patients had stable disease, and 1 patient showed a partial response 8 weeks after the DC injection. The 3-year overall and progression-free survival rates of the patients were 42.3% and 2.9%, respectively. Although DC therapy appears safe and resulted in an immunological response in patients with refractory sarcoma, it resulted in an improvement of the clinical outcome in only a small number of patients. Cancer 2017;123:1576-1584. © 2017 American Cancer Society. © 2017 American Cancer Society.

  1. β-Galactomannan and Saccharomyces cerevisiae var. boulardii modulate the immune response against Salmonella enterica serovar Typhimurium in porcine intestinal epithelial and dendritic cells.

    Science.gov (United States)

    Badia, Roger; Brufau, M Teresa; Guerrero-Zamora, Ana Maria; Lizardo, Rosil; Dobrescu, Irina; Martin-Venegas, Raquel; Ferrer, Ruth; Salmon, Henri; Martínez, Paz; Brufau, Joaquim

    2012-03-01

    Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes inflammation, necrosis, and diarrhea in pigs, as well as being an important source of food-borne diseases in humans. Probiotics and prebiotics are promising alternatives to antibiotics to control and prevent intestinal infections. The present work investigated a recently developed β-galactomannan (βGM) prebiotic compared to the proven probiotic Saccharomyces cerevisiae var. boulardii on porcine ileum intestinal epithelial cells (IECs) of the IPI-2I line and monocyte-derived dendritic cells (DCs) cocultured in vitro with Salmonella. We observed that both S. cerevisiae var. boulardii and βGM inhibited the association of Salmonella with IECs in vitro. Our data indicated that βGM has a higher ability than S. cerevisiae var. boulardii to inhibit Salmonella-induced proinflammatory mRNA (cytokines tumor necrosis factor alpha [TNF-α], interleukin-1α [IL-1α], IL-6, and granulocyte-macrophage colony-stimulating factor [GM-CSF] and chemokines CCL2, CCL20, and CXCL8) and at protein levels (IL-6 and CXCL8). Additionally, βGM and S. cerevisiae var. boulardii induced some effects on DCs that were not observed on IECs: βGM and S. cerevisiae var. boulardii showed slight upregulation of mRNA for TNF-α, GM-CSF, and CCR7 receptor on porcine monocyte-derived dendritic cells (DCs). Indeed, the addition of βGM or S. cerevisiae var. boulardii on DCs cocultured with Salmonella showed higher gene expression (mRNA) for TNF-α, GM-CSF, and CXCL8 compared to that of the control with Salmonella. In conclusion, the addition of βGM inhibits Salmonella-induced proinflammatory profiles in IECs but may promote DC activation, although associated molecular mechanisms remain to be elucidated.

  2. Recovery of the proliferative and functional integrity of mouse bone marrow in long-term cultures established after whole-body irradiation at different doses and dose rates

    International Nuclear Information System (INIS)

    Bierkens, J.G.; Hendry, J.H.; Testa, N.G.

    1991-01-01

    Injury inflicted upon the bone marrow stroma following whole-body irradiation and its repair over a 1-year period has been assessed in murine long-term bone marrow cultures established at increasing time intervals after irradiation. Different doses at different dose rates (10 Gy at 0.05 cGy/min, 4.5 Gy and 10 Gy at 1.6 cGy/min, and 4 x 4.5 Gy [3 weeks between doses] at 60 cGy/min) were chosen so as to maximize differences in effect in the stroma. The cellularity of the adherent layer in long-term cultures established 1 month after irradiation was reduced by 40%-90% depending on the dose and dose rate. Simultaneous with the poor ability of the marrow to form adherent layers, the cumulative spleen colony-forming unit (CFU-S) and granulocyte-macrophage colony-forming cell (GM-CFC) production over a 7-week period was reduced to 0% and 30% of control cultures, respectively. The slow recovery of the adherent layer was paralleled by an increase in the numbers of CFU-S and GM-CFC in the supernatant. Cultures established from repeatedly irradiated mice performed poorly over the entire 1-year period. Whereas the regeneration of the stroma was near complete 1 year after irradiation, the CFU-S and GM-CFC levels reached only between 50% and 80% of control cultures, respectively. Also, the concentration of CFU-S and GM-CFC in the supernatant remained persistently lower in cultures established from irradiated mice as compared to control cultures. The levels of sulfated glycosaminoglycans, which have been implicated in the establishment of the functional integrity of the microenvironment, were not reduced in the adherent layers at any time after irradiation. These results indicate that the regeneration of the stroma is accompanied by an incomplete recovery of active hemopoiesis in vitro

  3. Cell cycle-dependent regulation of kainate-induced inward currents in microglia

    International Nuclear Information System (INIS)

    Yamada, Jun; Sawada, Makoto; Nakanishi, Hiroshi

    2006-01-01

    Microglia are reported to have α-amino-hydroxy-5-methyl-isoxazole-4-propionate/kainate (KA) types. However, only small population of primary cultured rat microglia (approximately 20%) responded to KA. In the present study, we have attempted to elucidate the regulatory mechanism of responsiveness to KA in GMIR1 rat microglial cell line. When the GMIR1 cells were plated at a low density in the presence of granulocyte macrophage colony-stimulating factor, the proliferation rate increased and reached the peak after 2 days in culture and then gradually decreased because of density-dependent inhibition. At cell proliferation stage, approximately 80% of the GMIR1 cells exhibited glutamate (Glu)- and KA-induced inward currents at cell proliferation stage, whereas only 22.5% of the cells showed responsiveness to Glu and KA at cell quiescent stage. Furthermore, the mean amplitudes of inward currents induced by Glu and KA at cell proliferation stage (13.8 ± 3.0 and 8.4 ± 0.6 pA) were significantly larger than those obtained at cell quiescent stage (4.7 ± 0.8 and 6.2 ± 1.2 pA). In the GMIR1 cells, KA-induced inward currents were markedly inhibited by (RS)-3-(2-carboxybenzyl) willardiine (UBP296), a selective antagonist for KA receptors. The KA-responsive cells also responded to (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective agonist for GluR5, in both GMIR1 cells and primary cultured rat microglia. Furthermore, mRNA levels of the KA receptor subunits, GluR5 and GluR6, at the cell proliferation stage were significantly higher than those at the cell quiescent stage. Furthermore, the immunoreactivity for GluR6/7 was found to increase in activated microglia in the post-ischemic hippocampus. These results strongly suggest that microglia have functional KA receptors mainly consisting of GluR5 and GluR6, and the expression levels of these subunits are closely regulated by the cell cycle mechanism

  4. Endothelial progenitor cell subsets and preeclampsia: Findings and controversies

    Directory of Open Access Journals (Sweden)

    Armin Attar

    2017-10-01

    Full Text Available Vascular remodeling is an essential component of gestation. Endothelial progenitor cells (EPCs play an important role in the regulation of vascular homeostasis. The results of studies measuring the number of EPCs in normal pregnancies and in preeclampsia have been highly controversial or even contradictory because of some variations in technical issues and different methodologies enumerating three distinct subsets of EPCs: circulating angiogenic cells (CAC, colony forming unit endothelial cells (CFU-ECs, and endothelial colony-forming cells (ECFCs. In general, most studies have shown an increase in the number of CACs in the maternal circulation with a progression in the gestational age in normal pregnancies, while functional capacities measured by CFU-ECs and ECFCs remain intact. In the case of preeclampsia, mobilization of CACs and ECFCs occurs in the peripheral blood of pregnant women, but the functional capacities shown by culture of the derived colony-forming assays (CFU-EC and ECFC assays are altered. Furthermore, the number of all EPC subsets will be reduced in umbilical cord blood in the case of preeclampsia. As EPCs play an important role in the homeostasis of vascular networks, the difference in their frequency and functionality in normal pregnancies and those with preeclampsia can be expected. In this review, there was an attempt to provide a justification for these controversies.

  5. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Soares, Carlos Roberto Jorge

    2000-01-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU10 6 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU10 6 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  6. Characteristic cytokine generation patterns in cancer cells and infiltrating lymphocytes in oral squamous cell carcinomas and the influence of chemoradiation combined with immunotherapy on these patterns.

    Science.gov (United States)

    Yamamoto, Tetsuya; Kimura, Tsuyoshi; Ueta, Eisaku; Tatemoto, Yukihiro; Osaki, Tokio

    2003-01-01

    Cytokines produced by tumor cells and tumor-infiltrating lymphocytes (TIL) appear to regulate tumor cell growth and the cytotoxic activity of TIL. The objectives of the present study were to investigate cytokine generation patterns in tumor cells and TIL and to examine the influence of cancer therapy on this cytokine production and the cytotoxic activity of TIL. We determined the levels of cytokines produced by tumor cells and TIL in vitro and measured the cytotoxic activity of TIL against Daudi cells in patients with oral squamous cell carcinoma (OSC) before and 1 week after the start of concomitant chemo-radio-immunotherapy. Before the therapy, OSC cells generated higher levels of granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) than did oral keratinocytes isolated from the noninflamed gingivae of healthy individuals, but both kinds of cells generated similar levels of interleukin (IL)-1beta and IL-6. Compared with peripheral blood mononuclear cells (PBMC) of the patients, TIL produced higher levels of IL-1beta, IL-6, IL-10, TNF-alpha and TGF-beta, whereas their production of IL-12 and interferon-gamma (IFN-gamma) was only slightly higher than that in PBMC. After 1 week of therapy, the cytokine production by OSC cells had largely decreased, while the production of TNF-alpha, IFN-gamma, TGF-beta and IL-12 by TIL had increased greatly, although other cytokine levels were almost constant during the investigations. The cytotoxic activity of TIL was higher than that of PBMC before the therapy, and this activity was strongly increased by 1 week of therapy. These results suggest that the cytokine productivities of TIL and tumor cells differ from those of PBMC and normal keratinocytes, respectively, and that chemo-radio-immunotherapy modulates in situ cytokine generation, which is advantageous for inhibition of tumor cell growth and activation of TIL. Copyright 2003 S. Karger AG

  7. Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

    Science.gov (United States)

    Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

    2017-10-01

    We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

  8. GM-CSF-Producing Th Cells in Rats Sensitive and Resistant to Experimental Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Stojić-Vukanić, Zorica; Pilipović, Ivan; Vujnović, Ivana; Nacka-Aleksić, Mirjana; Petrović, Raisa; Arsenović-Ranin, Nevena; Dimitrijević, Mirjana; Leposavić, Gordana

    2016-01-01

    Given that granulocyte macrophage colony-stimulating factor (GM-CSF) is identified as the key factor to endow auto-reactive Th cells with the potential to induce neuroinflammation in experimental autoimmune encephalomyelitis (EAE) models, the frequency and phenotype of GM-CSF-producing (GM-CSF+) Th cells in draining lymph nodes (dLNs) and spinal cord (SC) of Albino Oxford (AO) and Dark Agouti (DA) rats immunized for EAE were examined. The generation of neuroantigen-specific GM-CSF+ Th lymphocytes was impaired in dLNs of AO rats (relatively resistant to EAE induction) compared with their DA counterparts (susceptible to EAE) reflecting impaired CD4+ lymphocyte proliferation and less supportive of GM-CSF+ Th cell differentiation dLN cytokine microenvironment. Immunophenotyping of GM-CSF+ Th cells showed their phenotypic heterogeneity in both strains and revealed lower frequency of IL-17+IFN-γ+, IL-17+IFN-γ-, and IL-17-IFN-γ+ cells accompanied by higher frequency of IL-17-IFN-γ- cells among them in AO than in DA rats. Compared with DA, in AO rats was also found (i) slightly lower surface density of CCR2 (drives accumulation of highly pathogenic GM-CSF+IFN-γ+ Th17 cells in SC) on GM-CSF+IFN-γ+ Th17 lymphocytes from dLNs, and (ii) diminished CCL2 mRNA expression in SC tissue, suggesting their impaired migration into the SC. Moreover, dLN and SC cytokine environments in AO rats were shown to be less supportive of GM-CSF+IFN-γ+ Th17 cell differentiation (judging by lower expression of mRNAs for IL-1β, IL-6 and IL-23/p19). In accordance with the (i) lower frequency of GM-CSF+ Th cells in dLNs and SC of AO rats and their lower GM-CSF production, and (ii) impaired CCL2 expression in the SC tissue, the proportion of proinflammatory monocytes among peripheral blood cells and their progeny (CD45hi cells) among the SC CD11b+ cells were reduced in AO compared with DA rats. Collectively, the results indicate that the strain specificities in efficacy of several mechanisms

  9. In vitro expansion of Lin{sup +} and Lin{sup −} mononuclear cells from human peripheral blood

    Energy Technology Data Exchange (ETDEWEB)

    Norhaiza, H. Siti; Zarina, Z. A. Intan; Hisham, Z. A. Shahrul [School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Selangor (Malaysia); Rohaya, M. A. W. [Department of Orthodontics, Faculty of Dentistry, Universiti Kebangsaan Malaysia, 50300, Kuala Lumpur (Malaysia)

    2013-11-27

    Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin{sup −}) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin{sup +}) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin{sup −} cell population. The ability of Lin{sup +} and Lin{sup −} to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin{sup +} and Lin{sup −} were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin{sup +} mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin{sup −} stem cells were not able to survive in proliferation medium however

  10. Radiosensitivity of spermatogenous epithelium stem cells of mice of different strains and age

    International Nuclear Information System (INIS)

    Konoplyannikova, O.A.; Konoplyannikov, A.G.

    1988-01-01

    In experiments on CBA and BALB/c male mices (3 months of age) and F 1 (CBAxC57BL/6) hybrides (at the age of 3, 12, and 24 months) a difference was noted in the radiosensitivity of spermatogenous epithelium stem cells displayed by the changes in their colony-forming ability to testicular tubules 42 days following local 60 Co-γ-irradiation. The older the hybrid mice the smaller was the number of spermatogenous epithelium stem cells

  11. Results of a Phase 1/2 Study in Metastatic Renal Cell Carcinoma Patients Treated with a Patient-specific Adjuvant Multi-peptide Vaccine after Resection of Metastases.

    Science.gov (United States)

    Rausch, Steffen; Gouttefangeas, Cécile; Hennenlotter, Jörg; Laske, Karoline; Walter, Kerstin; Feyerabend, Susan; Chandran, Premachandran Anoop; Kruck, Stephan; Singh-Jasuja, Harpreet; Frick, Annemarie; Kröger, Nils; Stevanović, Stefan; Stenzl, Arnulf; Rammensee, Hans-Georg; Bedke, Jens

    2017-10-04

    Treatment of metastatic renal cell carcinoma comprises metastasectomy±systemic medical treatment. Specific immunotherapy after metastasectomy could be a complementary option. In this phase 1/2 study, safety and tolerability of an adjuvant multi-peptide vaccine (UroRCC) after metastasectomy was evaluated together with immune response and efficacy, compared with a contemporary cohort of patients (n=44) treated with metastasectomy only. Nineteen metastatic renal cell carcinoma patients received UroRCC via intradermal or subcutaneous application randomized to immunoadjuvants (granulocyte-macrophage colony-stimulating factor or Montanide). Adverse events of UroRCC were mainly grade I and II; frequency of immune response was higher for major histocompatibility complex class II peptides (17/19, 89.5%) than for major histocompatibility complex class I peptides (8/19, 42.1%). Median overall survival was not reached in the UroRCC group (mean: 112.6 mo, 95% confidence interval [CI]: 92.1-133.1) and 58.0 mo (95% CI: 32.7-83.2) in the control cohort (p=0.015). UroRCC was an independent prognosticator of overall survival (hazard ratio=0.19, 95% CI: 0.05-0.69, p=0.012). Adjuvant UroRCC multi-peptide vaccine after metastasectomy was well tolerated, immunogenic, and indicates potential clinical benefit when compared with a contemporary control cohort (NCT02429440). The application of a patient-specific peptide vaccine after complete resection of metastases in metastatic renal cell carcinoma patients resulted in favorable tolerability and outcome. Copyright © 2017 European Association of Urology. Published by Elsevier B.V. All rights reserved.

  12. Molecular signatures of maturing dendritic cells: implications for testing the quality of dendritic cell therapies

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2010-01-01

    Full Text Available Abstract Background Dendritic cells (DCs are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin-4 (IL-4 stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS and interferon-γ (IFN-γ induction in order to characterize the usefulness of mature DCs (mDCs for immune therapy and to identify biomarkers for assessing the quality of mDCs. Methods Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-γ and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA expression analysis. Results After 24 hours of LPS and IFN-γ stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs. Conclusion DCs, matured with LPS and IFN-γ, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

  13. Immunogenicity of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma in vitro model: studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic T lymphocytes

    Directory of Open Access Journals (Sweden)

    Heinrich B

    2017-05-01

    Full Text Available B Heinrich,1 J Klein,1 M Delic,1 K Goepfert,1 V Engel,1 L Geberzahn,1 M Lusky,2 P Erbs,2 X Preville,3 M Moehler1 1First Department of Internal Medicine, University Medical Center Mainz, Mainz, Germany; 2Transgene SA, Illkirch-Graffenstaden, 3Amoneta Diagnostics, Huningue, France Abstract: Oncolytic virotherapy is an emerging immunotherapeutic modality for cancer treatment. Oncolytic viruses with genetic modifications can further enhance the oncolytic effects on tumor cells and stimulate antitumor immunity. The oncolytic vaccinia viruses JX-594-GFP+/hGM-CSF (JX-GFP and TG6002 are genetically modified by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF or transforming 5-fluorocytosine (5-FC into 5-fluorouracil (5-FU. We compared their properties to kill tumor cells and induce an immunogenic type of cell death in a human melanoma cell model using SK29-MEL melanoma cells. Their influence on human immune cells, specifically regarding the activation of dendritic cells (DCs and the interaction with the autologous cytotoxic T lymphocyte (CTL clone, was investigated. Melanoma cells were infected with either JX-GFP or TG6002 alone or in combination with 5-FC and 5-FU. The influence of viral infection on cell viability followed a time- and multiplicity of infection dependent manner. Combination of virus treatment with 5-FU resulted in stronger reduction of cell viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction of cell viability in this setting. Expression of calreticulin and high mobility group 1 protein (HMGB1, markers of immunogenic cell death (ICD, could be detected after viral infection. Accordingly, DC maturation was noted after viral oncolysis. DCs presented stronger expression of activation and maturation markers. The autologous CTL clone IVSB expressed the activation marker CD69, but viral treatment failed to enhance cytotoxicity marker. In summary, vaccinia viruses JX-GFP and TG6002 lyse

  14. Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

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    Chiang Cheryl L-L

    2011-11-01

    Full Text Available Abstract Background Dendritic cells (DCs are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. Methods Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1 culture media; 2 culture surface; 3 duration of activating DCs with lipopolysaccharide (LPS and interferon (IFN-gamma; 4 method of DC harvest; and 5 cryomedia and final DC product formulation. Results DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs, and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and

  15. Correlation of proliferative and clonogenic tumor cells in multiple myeloma

    International Nuclear Information System (INIS)

    Karp, J.E.; Burke, P.J.; Saylor, P.L.; Humphrey, R.L.

    1984-01-01

    To expand on the findings from previous clinical trials that the growth of residual tumor is increased at a predictable time following initial drug administration, malignant plasma cells from bone marrows of patients with multiple myeloma (MM) were examined for changes in proliferation and clonogenicity induced in vivo by cyclophosphamide and in vitro by drug-induced humoral stimulatory activity. Peak plasma cell [ 3 H]thymidine labeling index (LI) occurred predictably following drug and paralleled changes in agar colony formation by marrow cells obtained during therapy. Colony-forming capacity of pretreatment MM marrow populations was enhanced when those cells were cultured with humoral stimulatory activity, similar to the increased colony formation detected in Day 9 postcyclophosphamide marrows at the time of peak plasma cell LI. To further define a relationship between proliferative plasma cells and colony-forming tumor cells, MM marrows were fractionated by sedimentation on an isokinetic gradient. Enrichment of a proliferative tumor cell cohort was achieved, evidenced by [ 3 H]thymidine LI. Colony-forming cells were also enriched by isokinetic gradient sedimentation, and agar colony formation by MM marrow cell fractions correlated with the kinetic characteristics of the isolated subpopulations. These studies of whole and fractionated human MM marrow cell populations suggest that the kinetically active cells which are induced to proliferate in vivo and in vitro are closely related to the clonogenic tumor cells which produce colonies in agar and which, like those cells measured by [ 3 H]thymidine LI, respond to growth stimulation by drug-induced humoral stimulatory activity

  16. Phase I study utilizing a novel antigen-presenting cell-targeted vaccine with Toll-like receptor stimulation to induce immunity to self-antigens in cancer patients.

    Science.gov (United States)

    Morse, Michael A; Chapman, Robert; Powderly, John; Blackwell, Kimberly; Keler, Tibor; Green, Jennifer; Riggs, Renee; He, Li-Zhen; Ramakrishna, Venky; Vitale, Laura; Zhao, Biwei; Butler, Stephen A; Hobeika, Amy; Osada, Takuya; Davis, Thomas; Clay, Timothy; Lyerly, H Kim

    2011-07-15

    The use of tumor-derived proteins as cancer vaccines is complicated by tolerance to these self-antigens. Tolerance may be broken by immunization with activated, autologous, ex vivo generated and antigen-loaded, antigen-presenting cells (APC); however, targeting tumor antigen directly to APC in vivo would be a less complicated strategy. We wished to test whether targeted delivery of an otherwise poorly immunogenic, soluble antigen to APC through their mannose receptors (MR) would induce clinically relevant immunity. Two phase I studies were conducted with CDX-1307, a vaccine composed of human chorionic gonadotropin beta-chain (hCG-β) fused to an MR-specific monoclonal antibody, administered either locally (intradermally) or systemically (intravenously) in patients with advanced epithelial malignancies. An initial dose escalation of single-agent CDX-1307 was followed by additional cohorts of CDX-1307 combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) and the Toll-like receptor (TLR) 3 agonist polyinosinic-polycytidylic acid (poly-ICLC) and TLR7/8 agonist resiquimod to activate the APC. CDX-1307 induced consistent humoral and T-cell responses to hCG-β when coadministered with TLR agonists. Greater immune responses and clinical benefit, including the longest duration of stable disease, were observed with immunization combined with local TLR agonists. Immune responses were induced equally efficiently in patients with elevated and nonelevated levels of serum hCG-β. Antibodies within the serum of vaccinated participants had tumor suppressive function in vitro. Toxicity consisted chiefly of mild injection site reactions. APC targeting and activation induce adaptive immunity against poorly immunogenic self-antigens which has implications for enhancing the efficacy of cancer immunotherapy.

  17. A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer.

    Science.gov (United States)

    Morse, Michael A; Niedzwiecki, Donna; Marshall, John L; Garrett, Christopher; Chang, David Z; Aklilu, Mebea; Crocenzi, Todd S; Cole, David J; Dessureault, Sophie; Hobeika, Amy C; Osada, Takuya; Onaitis, Mark; Clary, Bryan M; Hsu, David; Devi, Gayathri R; Bulusu, Anuradha; Annechiarico, Robert P; Chadaram, Vijaya; Clay, Timothy M; Lyerly, H Kim

    2013-12-01

    To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).

  18. Stem-Cell Inactivation on Transplantation of Haemopoietic Cell Suspensions from Genetically Different Donors

    Energy Technology Data Exchange (ETDEWEB)

    Petrov, R. V. [Institute of Biophysics, Ministry of Public Health of the USSR, Moscow, USSR (Russian Federation)

    1969-07-15

    The transplantation of a mixture of haemopoietic or lymphoid cells from two genetically different mice into lethally irradiated F{sub 1} recipients results in marked or total inactivation of the colony-forming units of the graft. This phenomenon is observed following transplantation of mixtures of spleen cells or bone-marrow cells from animals of different genotypes: CBA + C57BL, A + CBA, A + C57BL, C3H + C57BL, CBA + (CBA x C57BL) F{sub 1}. Maximum inactivation is observed when lymph-node cells of one genotype are transplanted with spleen or bone-marrow cells of another genotype. Use of non-syngenic kidney cells or lymphoid cells inactivated by irradiation as one component of the mixture shows that inactivation of genetically heterogeneous stem cells requires the participation of viable lymphoid cells. The inactivation phenomenon is also observed with Jerne's method. This shows that inactivation affects not only colony-forming cells but also the immunologically competent precursors of antibody-producing cells. (author)

  19. Hematologic status of mice submitted to sublethal total body irradiation with mixed neutron-gamma radiation

    International Nuclear Information System (INIS)

    Herodin, F.; Court, L.

    1989-01-01

    The hematologic status of mice exposed to sublethal whole body irradiation with mixed neutron-gamma radiation (mainly neutrons) is studied. A slight decrease of the blood cell count is still observed below 1 Gy. The recovery of bone marrow granulocyte-macrophage progenitors seems to require more time than after pure gamma irradiation [fr

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

    NARCIS (Netherlands)

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

    1988-01-01

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

  1. Lack of the p42 form of C/EBPα leads to spontaneous immortalization and lineage infidelity of committed myeloid progenitors

    DEFF Research Database (Denmark)

    Schuster, Mikkel B; Frank, Anne-Katrine; Bagger, Frederik O

    2013-01-01

    transforming events. In this study, we use premalignant cells from a Cebpa mutant AML model, in which the LIC population resembles granulocyte-macrophage progenitors (GMPs), to show that premalignant GMPs undergo spontaneous immortalization with a high clonal frequency when cultured in vitro, suggesting...

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

    Directory of Open Access Journals (Sweden)

    Lea Vojta

    2015-09-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  4. Lead effects on development and function of bone marrow-derived dendritic cells promote Th2 immune responses

    International Nuclear Information System (INIS)

    Gao Donghong; Mondal, Tapan K.; Lawrence, David A.

    2007-01-01

    Although lead (Pb) has significant effects on the development and function of macrophages, B cells, and T cells and has been suggested to promote allergic asthma in mice and humans, Pb modulation of bone marrow (BM)-derived dendritic cells (DCs) and the resultant DC effects on Th1 and Th2 development have not been examined. Accordingly, we cultured BM cells with murine granulocyte macrophage-colony stimulating factor (mGM-CSF) ± PbCl 2 . At day 10, culture supernatant (SN) and non-adherent cells were harvested for analysis. Additionally, day 10 non-adherent BM-DCs were harvested and recultured with mGM-CSF + LPS ± Pb for 2 days. The day 10 Pb exposure significantly inhibited BM-DC generation, based on CD11c expression. Although fewer DCs were generated with Pb, the existing Pb-exposed DCs had significantly greater MHC-II expression than did the non-Pb-exposed DCs. However, these differences diminished upon LPS stimulation. After LPS stimulation, CD80, CD86, CD40, CD54, and MHC-II were all up-regulated on both Pb-DCs and DCs, but Pb-DCs expressed significantly less CD80 than did DCs. The CD86:CD80 ratio suggests a Pb-DC potential for Th2 cell development. After LPS stimulation, IL-6, IL-10, IL-12 (p70), and TNF-α levels significantly increased with both Pb-DCs and DCs, but Pb-DCs produced significantly less cytokines than did DCs, except for IL-10, which further supports Pb-DC preferential skewing toward type-2 immunity. In vitro studies confirm that Pb-DCs have the ability to polarize antigen-specific T cells to Th2 cells. Pb-DCs also enhanced allogeneic and autologous T cell proliferation in vitro, and in vivo studies suggested that Pb-DCs inhibited Th1 effects on humoral and cell-mediated immunity. The Pb effect was mainly on DCs, rather than on T cells, and Pb's modification of DC function appears to be the main cause of Pb's promotion of type-2-related immunity, which may relate to Pb's enhanced activation of the Erk/MAP kinase pathway

  5. Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models

    Directory of Open Access Journals (Sweden)

    Hammami Ines

    2012-07-01

    Full Text Available Abstract Background The tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs. Incubating bone marrow (BM precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF and interleukin-6 (IL-6 generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression. Results Exposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK was activated during MDSC maturation in GM-CSF and IL-6–treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was

  6. The fps/fes proto-oncogene regulates hematopoietic lineage output.

    Science.gov (United States)

    Sangrar, Waheed; Gao, Yan; Zirngibl, Ralph A; Scott, Michelle L; Greer, Peter A

    2003-12-01

    The fps/fes proto-oncogene is abundantly expressed in myeloid cells, and the Fps/Fes cytoplasmic protein-tyrosine kinase is implicated in signaling downstream from hematopoietic cytokines, including interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). Studies using leukemic cell lines have previously suggested that Fps/Fes contributes to granulomonocytic differentiation, and that it might play a more selective role in promoting survival and differentiation along the monocytic pathway. In this study we have used a genetic approach to explore the role of Fps/Fes in hematopoiesis. We used transgenic mice that tissue-specifically express a mutant human fps/fes transgene (fps(MF)) that was engineered to encode Fps/Fes kinase that is activated through N-terminal myristoylation (MFps). Hematopoietic function was assessed using lineage analysis, hematopoietic progenitor cell colony-forming assays, and biochemical approaches. fps(MF) transgenic mice displayed a skewed hematopoietic output reflected by increased numbers of circulating granulocytic and monocytic cells and a corresponding decrease in lymphoid cells. Bone marrow colony assays of progenitor cells revealed a significant increase in the number of both granulomonocytic and multi-lineage progenitors. A molecular analysis of signaling in mature monocytic cells showed that MFps promoted GM-CSF-induced STAT3, STAT5, and ERK1/2 activation. These observations support a role for Fps/Fes in signaling pathways that contribute to lineage determination at the level of multi-lineage hematopoietic progenitors as well as the more committed granulomonocytic progenitors.

  7. Evaluation of drug-induced hematotoxicity using novel in vitro monkey CFU-GM and BFU-E colony assays.

    Science.gov (United States)

    Goto, Koichi; Goto, Mayumi; Ando-Imaoka, Masako; Kai, Kiyonori; Mori, Kazuhiko

    2017-01-01

    In order to evaluate drug-induced hematotoxicity in monkey cells in vitro, colony-forming unit-granulocyte, macrophage (CFU-GM), and burst-forming unit-erythroid (BFU-E) colony assays were established using mononuclear cells in the bone marrow collected from male cynomolgus monkeys. Furthermore, the effects of doxorubicin, chloramphenicol, and linezolid on CFU-GM and BFU-E colony formation were investigated using established monkey CFU-GM and BFU-E colony assays in comparison with those on human CFU-GM and BFU-E colonies acquired from human umbilical cord blood cells. Bone marrow mononuclear cells were collected from the ischial or iliac bone of male cynomolgus monkeys. The cells were subsequently processed by density gradient separation at 1.067, 1.070, or 1.077 g/mL for CFU-GM or 1.077 g/mL for BFU-E, and then cultured in methylcellulose medium for 9 or 13 days, respectively. A sufficient number of CFU-GM colonies were formed from mononuclear cells processed at a density of 1.070 g/mL. Moreover, the number of BFU-E colonies from the cells processed at a density of 1.077 g/mL was sufficient for the colony assay. The number of CFU-GM or BFU-E colonies decreased after treatment with the drugs of interest in a concentration-dependent manner. Compared with human CFU-GM, monkey CFU-GM were more sensitive to chloramphenicol and resistant to doxorubicin, whereas monkey BFU-E were more sensitive to all compounds in comparison to the sensitivity of human BFU-E. In conclusion, monkey CFU-GM and BFU-E colony assays were established and considered useful tools to evaluate the differences in drug-induced hematotoxicity between species.

  8. In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice.

    Science.gov (United States)

    Río, Paula; Segovia, José Carlos; Hanenberg, Helmut; Casado, José Antonio; Martínez, Jesús; Göttsche, Kerstin; Cheng, Ngan Ching; Van de Vrugt, Henri J; Arwert, Fré; Joenje, Hans; Bueren, Juan A

    2002-09-15

    Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca(-/-) mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect of Fanca(-/-) progenitors, either when total BM or when purified Lin(-)Sca-1(+) cells were subjected to in vitro stimulation. Liquid cultures of Fanca(-/-) BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca(-/-) progenitors, purified Lin(-)Sca-1(+) cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCA genes. Lin(-)Sca-1(+) cells from Fanca(-/-) mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors of Fanca(-/-) mice.

  9. Isolation and In Vitro Characterization of Epidermal Stem Cells

    DEFF Research Database (Denmark)

    Moestrup, Kasper S; Andersen, Marianne Stemann; Jensen, Kim Bak

    2017-01-01

    flow cytometry. Using markers that define the spatial origin of epidermal cells, it is possible to interrogate the specific characteristics of subpopulations of cells based on their in vivo credentials. Here, we describe how to isolate, culture, and characterize keratinocytes from murine back and tail......Colony-forming assays represent prospective methods, where cells isolated from enzymatically dissociated tissues or from tissue cultures are assessed for their proliferative capacity in vitro. Complex tissues such as the epithelial component of the skin (the epidermis) are characterized...

  10. Nature of leukemic stem cells in murine myelogenous leukemia

    International Nuclear Information System (INIS)

    Yoshida, K.; Nemoto, K.; Nishimura, M.; Hayata, I.; Inoue, T.; Seki, M.

    1986-01-01

    We investigated the nature of myelogenous leukemic stem cells in mice. L-8057, a megakaryoblastic leukemia cell line used in this study, produces in vivo and in vitro colonies. By means of typical chromosomal aberrations in L-8057, one can conveniently detect the origin of the cells in each colony derived from a leukemic stem cell. Direct evidence of whether cells from each colony had leukemogenicity in recipient mice was successfully obtained by the colony transplantation assay. Both leukemic colony-forming unit-spleen (L-CFU-s) and leukemic colony-forming unit-culture (L-CFU-c) in L-8057 may have belonged to the same differentiating stage in the stem cells because of their similar radiosensitivity, although some parts of the L-CFU of L-8057 seemed to have lost their capability to regenerate L-CFU-s when the cells were plated in dishes. This leukemic stem cell preserves high self-renewal ability in vitro after 10 passages. In addition, in vitro colony formation by this leukemic cell during the above course of serial passages did not require any additional exogenous stimulators. The same sort of trials have been made on other types of leukemias. Leukemic stem cells showed remarkable variety in their response to stimulating factors and in their self-renewal activity, which suggests that they may have consisted of heterogeneous populations

  11. Effect of the administration time of HS6101 on hematopoietic recovery in ICR mice injured by cyclophosphamide

    Directory of Open Access Journals (Sweden)

    Shuang XING

    2015-06-01

    Full Text Available Objective To explore the effect of the administration time of HS6101 on hematopoietic recovery in ICR mice injured by cyclophosphamide (CTX. Methods One hundred and three male ICR mice were divided into 4 groups: CTX control, HS6101 prevention, HS6101 treatment, and HS6101 prevention+treatment groups. CTX was intraperitoneally injected into the ICR mice at a dose of 100mg/(kg.d for three consecutive days to establish a chemotherapeutics-injured model. HS6101 at a dose of 27μg/mouse in 0.2ml was subcutaneously injected into the mice 1h before the first administration of CTX in HS6101-prevention group, 1h after the last administration of CTX in HS6101 treatment group, and both at 1h before the first administration and 1h after the last administration of CTX in HS6101 prevention + treatment group. Physiological saline was subcutaneously injected into the mice in CTX control group (0.2ml/mouse. 10μl peripheral blood was collected from the caudal vein for WBC, neutrophil lymphocyte, RBC and platelet counts on day -1, 3, 5, 7, 9, 11, 13, 15, 17 with the MEK-7222K cell analyzer, and the cell count was compared between HS6101 treatment mice and CTX control mice. Another 30 male ICR mice were used for bone marrow colony forming unit (CFU assay and bone marrow histopathological examination, and they were assigned into normal control, CTX control, HS6101 prevention, HS6101treatment and HS6101 prevention + treatment groups (each n=6. On the day 4 and day 9 after CTX injection, mice were sacrificed and bone marrow cells were collected from the left femur for mononuclear cell (MNC isolation. 1×104 MNCs were planted in 1.0ml mouse CFU culture medium M3434 and cultured in incubator with the temperature of 37℃, and 5% CO2 for 7 days. After that, granulocyte macrophage-colony-forming unit (GM-CFU, megakaryocyte colony forming unit (MK-CFU, mixture-colony-forming unit (Mix-CFU, burst-forming unit-erythroid (BFU-E and colony-forming unit-erythroid (CFU

  12. Radiation and thermal characteristics of mouse lymphoma cells and their radiation-sensitive mutant

    International Nuclear Information System (INIS)

    Baba, Yuji; Yasunaga, Tadamasa; Uozumi, Hideaki; Takahashi, Mutsumasa; Sawada, Shozo.

    1988-01-01

    Radiation and thermal characteristics of L5178Y cells and their radiation-sensitive mutant M10 cells were studied by the colony-forming method and the dye-exclusion method using eosin-Y. Although M10 cells were remarkably radiation-sensitive compared with L5178Y cells, it was diffcult to cause interphase death of M10 after a large dose of irradiation. After heat treatments, L5178Y cells revealed more cell destruction and were stained well by eosin-Y, but it was relatively difficult to produce cell destruction of M10 cells, which showed poor staining by eosin-Y. When assayed by the colony-forming method, M10 cells were also heat-resistant compared to L5178Y. The dye-exclusion rate was closely correlated with cell survival after hyperthermia of L5178Y cells, suggesting that this is a simple method of detecting the thermosensitivity and thermotolerance of cancer cells. The difference in survival of L5178Y cells and M10 cells after combined treatment with gamma irradiation and hyperthermia was smaller than with gamma irradiation alone. It was also found that there was a relationship between radiation-induced interphase death and hyperthermia-induced interphase death, and that interphase death accounted for a major part of cell death caused by hyperthermia in mouse leukemia cells. (author)

  13. Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells

    International Nuclear Information System (INIS)

    Kapp, L.N.; Painter, R.B.

    1989-01-01

    SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid containing a normal human DNA library and selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one stably resistant to radiation. Resistance to ionizing radiation of both primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G 2 chromatic aberrations; both cell lines retained AT-like radioresistant DNA synthesis. Results suggest that, because radioresistance in transfected cells was not as great as in normal human cells, two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene. (author)

  14. Cytokinetics and Regulation of Progenitor Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lajtha, L. G. [Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (United Kingdom)

    1967-07-15

    Full text: In spite of great differences in the life-span of fully differentiated haemic cells, the cellular kinetics of their production appears to be similar. Recent evidence indicates a common ultimate stem cell for most of the cells in the peripheral blood. The various pathways of differentiation, however, result in transient dividing and differentiating cell populations which differ from each other not only in their specific biochemical processes but also in the manner of control and kinetic pattern of their proliferation. The population best understood is the erythroid progenitor series of cells, primarily because it has the greatest number of experimentally measurable parameters at the present. This will be discussed in detail and comparisons will be made with the myeloid and lymphoid progenitor populations. The fine structure of the bone-marrow stem cell population will be examined in particular, with regard to the suitability or otherwise of the current stem cell models to explain the kinetic pattern of all the peripheral blood elements after perturbations of their steady-state values. Four different assay methods of bone-marrow stem cells have been examined with regard to the kinetic pattern following perturbation of the steady-state system, e.g. by irradiation. Basically, the stem cell assays fall into two categories: those depending on grafting haemopoietic cells into suitably treated recipients, and those in which recovery of the population is allowed in the animal in which the perturbation was produced, without handling the cells. Evidence is accumulating which indicates that in the grafting techniques, a selective loss of stem cells may occur, . especially stem cells in cell cycle, hence in early stages of recovery of the population unduly low numerical values might be noted. In view of this observation, the concept of the colony-forming cell may have to be revised and instead the colony-forming property of the stem cell introduced. (author)

  15. Cytokinetics and Regulation of Progenitor Cells

    International Nuclear Information System (INIS)

    Lajtha, L.G.

    1967-01-01

    Full text: In spite of great differences in the life-span of fully differentiated haemic cells, the cellular kinetics of their production appears to be similar. Recent evidence indicates a common ultimate stem cell for most of the cells in the peripheral blood. The various pathways of differentiation, however, result in transient dividing and differentiating cell populations which differ from each other not only in their specific biochemical processes but also in the manner of control and kinetic pattern of their proliferation. The population best understood is the erythroid progenitor series of cells, primarily because it has the greatest number of experimentally measurable parameters at the present. This will be discussed in detail and comparisons will be made with the myeloid and lymphoid progenitor populations. The fine structure of the bone-marrow stem cell population will be examined in particular, with regard to the suitability or otherwise of the current stem cell models to explain the kinetic pattern of all the peripheral blood elements after perturbations of their steady-state values. Four different assay methods of bone-marrow stem cells have been examined with regard to the kinetic pattern following perturbation of the steady-state system, e.g. by irradiation. Basically, the stem cell assays fall into two categories: those depending on grafting haemopoietic cells into suitably treated recipients, and those in which recovery of the population is allowed in the animal in which the perturbation was produced, without handling the cells. Evidence is accumulating which indicates that in the grafting techniques, a selective loss of stem cells may occur, . especially stem cells in cell cycle, hence in early stages of recovery of the population unduly low numerical values might be noted. In view of this observation, the concept of the colony-forming cell may have to be revised and instead the colony-forming property of the stem cell introduced. (author)

  16. The role of the DNA repair system in increasing the viability of E.coli cells under the action of small UV doses

    International Nuclear Information System (INIS)

    Kuzin, A.M.; Vilenchik, M.M.; Isakov, B.K.; AN Kazakhskoj SSR, Alma-Ata. Inst. Botaniki)

    1976-01-01

    The authors studied the action of the ultraviolet light (UV) on the colony-forming ability of E.coli K12-HCR + cultured in a meat infusion broth in the presence of glucose. An unusual shape of the curve indicates that the number of viable cells increases under the action of low UV doses. The experiment was repeated seven times, and each time the phenomenon was fully asserted (p 0.01). So it was suggested that low UV doses (about 140 erg/mm 2 ) activate the system of dark DNA repair (induction of the synthesis of repair enzymes) which repairs 'spontaneous' DNA defects and increases the number of colony-forming cells. (orig.) [de

  17. Role of the DNA repair system in increasing the viability of E. coli cells under the action of small UV doses

    Energy Technology Data Exchange (ETDEWEB)

    Kuzin, A M; Vilenchik, M M; Isakov, B K [AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki; AN Kazakhskoj SSR, Alma-Ata. Inst. Botaniki)

    1976-12-01

    The authors studied the action of the ultraviolet light (UV) on the colony-forming ability of E.coli K12-HCR/sup +/ cultured in a meat infusion broth in the presence of glucose. An unusual shape of the curve indicates that the number of viable cells increases under the action of low UV doses. The experiment was repeated seven times, and each time the phenomenon was fully asserted (p 0.01). So it was suggested that low UV doses (about 140 erg/mm/sup 2/) activate the system of dark DNA repair (induction of the synthesis of repair enzymes) which repairs 'spontaneous' DNA defects and increases the number of colony-forming cells.

  18. Dietary restriction ameliorates haematopoietic ageing independent of telomerase, whilst lack of telomerase and short telomeres exacerbates the ageing phenotype.

    Science.gov (United States)

    Al-Ajmi, Nouf; Saretzki, Gabriele; Miles, Colin; Spyridopoulos, Ioakim

    2014-10-01

    Ageing is associated with an overall decline in the functional capacity of tissues and stem cells, including haematopoietic stem and progenitor cells (HSPCs), as well as telomere dysfunction. Dietary restriction (DR) is a recognised anti-ageing intervention that extends lifespan and improves health in several organisms. To investigate the role of telomeres and telomerase in haematopoietic ageing, we compared the HSPC profile and clonogenic capacity of bone marrow cells from wild type with telomerase-deficient mice and the effect of DR on these parameters. Compared with young mice, aged wild type mice demonstrated a significant accumulation of HSPCs (1.3% vs 0.2%, P=0.002) and elevated numbers of granulocyte/macrophage colony forming units (CFU-GM, 26.4 vs 17.3, P=0.0037) consistent with myeloid "skewing" of haematopoiesis. DR was able to restrict the increase in HSPC number as well as the myeloid "skewing" in aged wild type mice. In order to analyse the influence of short telomeres on the ageing phenotype we examined mice lacking the RNA template for telomerase, TERC(-/-). Telomere shortening resulted in a similar bone marrow phenotype to that seen in aged mice, with significantly increased HSPC numbers and an increased formation of all myeloid colony types but at a younger age than wild type mice. However, an additional increase in erythroid colonies (BFU-E) was also evident. Mice lacking telomerase reverse transcriptase without shortened telomeres, TERT(-/-), also presented with augmented haematopoietic ageing which was ameliorated by DR, demonstrating that the effect of DR was not dependent on the presence of telomerase in HSPCs. We conclude that whilst shortened telomeres mimic some aspects of haematopoietic ageing, both shortened telomeres and the lack of telomerase produce specific phenotypes, some of which can be prevented by dietary restriction. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Silencing of Foxp3 enhances the antitumor efficacy of GM-CSF genetically modified tumor cell vaccine against B16 melanoma

    Directory of Open Access Journals (Sweden)

    Miguel A

    2017-01-01

    Full Text Available Antonio Miguel,1 Luis Sendra,1 Verónica Noé,2 Carles J Ciudad,2 Francisco Dasí,3,4 David Hervas,5 María José Herrero,1,6 Salvador F Aliño17 1Department of Pharmacology, Faculty of Medicine, University of Valencia, 2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, 3Research University Hospital of Valencia, INCLIVA Health Research Institute, 4Department of Physiology, Faculty of Medicine, University of Valencia Foundation, 5Biostatistics Unit, 6Pharmacogenetics Unit, Instituto de Investigación Sanitaria La Fe (IIS La Fe, 7Clinical Pharmacology Unit, ACM Hospital Universitario y Politécnico La Fe, Valencia, Spain Abstract: The antitumor response after therapeutic vaccination has a limited effect and seems to be related to the presence of T regulatory cells (Treg, which express the immunoregulatory molecules CTLA4 and Foxp3. The blockage of CTLA4 using antibodies has shown an effective antitumor response conducing to the approval of the human anti-CTLA4 antibody ipilimumab by the US Food and Drug Administration. On the other hand, Foxp3 is crucial for Treg development. For this reason, it is an attractive target for cancer treatment. This study aims to evaluate whether combining therapeutic vaccination with CTLA4 or Foxp3 gene silencing enhances the antitumor response. First, the “in vitro” cell entrance and gene silencing efficacy of two tools, 2'-O-methyl phosphorotioate-modified oligonucleotides (2'-OMe-PS-ASOs and polypurine reverse Hoogsteen hairpins (PPRHs, were evaluated in EL4 cells and cultured primary lymphocytes. Following B16 tumor transplant, C57BL6 mice were vaccinated with irradiated B16 tumor cells engineered to produce granulocyte-macrophage colony-stimulating factor (GM-CSF and were intraperitoneally treated with CTLA4 and Foxp3 2'-OMe-PS-ASO before and after vaccination. Tumor growth, mice survival, and CTLA4 and Foxp3 expression in blood cells were measured. The following

  20. Endothelial Cells Promote Expansion of Long-Term Engrafting Marrow Hematopoietic Stem and Progenitor Cells in Primates.

    Science.gov (United States)

    Gori, Jennifer L; Butler, Jason M; Kunar, Balvir; Poulos, Michael G; Ginsberg, Michael; Nolan, Daniel J; Norgaard, Zachary K; Adair, Jennifer E; Rafii, Shahin; Kiem, Hans-Peter

    2017-03-01

    Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34 + cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34 + C38 - HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34 + cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34 + cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs. Stem Cells Translational Medicine 2017;6:864-876. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  1. Bone marrow cells other than stem cells seed the bone marrow after rescue transfusion of fatally irradiated mice

    International Nuclear Information System (INIS)

    Cronkite, E.P.; Inoue, T.; Bullis, J.E.

    1987-01-01

    In a previous publication, iodinated deoxyuridine ( 125 IUdR) incorporation data were interpreted as indicating that spleen colony-forming units (CFU-S) in DNA synthesis preferentially seeded bone marrow. In the present studies, the CFU-S content of marrow from irradiated, bone-marrow transfused mice was directly determined. Pretreatment of the transfused cells with cytocidal tritiated thymidine resulted in an insignificant diminution in CFU-S content when compared with nontritiated thymidine pretreatment, implying that there is no preferential seeding. The 125 IUdR incorporation data have been reinterpreted as being a result of the proliferation of other progenitor cells present that have seeded the bone marrow

  2. Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature.

    Science.gov (United States)

    Psaltis, Peter J; Puranik, Amrutesh S; Spoon, Daniel B; Chue, Colin D; Hoffman, Scott J; Witt, Tyra A; Delacroix, Sinny; Kleppe, Laurel S; Mueske, Cheryl S; Pan, Shuchong; Gulati, Rajiv; Simari, Robert D

    2014-07-18

    Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1(+)CD45(+) cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67(+)) Lin(-)c-Kit(+)CD135(-)CD115(+)CX3CR1(+)Ly6C(+)CD11b(-) subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1(+)CD45(+) adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE(-/-) and LDL-R(-/-) mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage

  3. Amifostine (WR-2721, a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

    Directory of Open Access Journals (Sweden)

    C.A. De Souza

    2000-07-01

    Full Text Available Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY (7 g/m2, used to mobilize peripheral blood progenitor cells (PBPC and to reduce tumor burden. We enrolled 29 patients, 22 (75.9% affected by aggressive and 7 (24.1% by indolent non-Hodgkin's lymphoma (NHL, who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04. None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively. Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

  4. Granulocyte-colony stimulating factor for hematopoietic stem cell donation from healthy female donors during pregnancy and lactation: what do we know?

    Science.gov (United States)

    Pessach, Ilias; Shimoni, Avichai; Nagler, Arnon

    2013-01-01

    BACKGROUND Hematopoietic growth factors (HGFs) are mostly used as supportive measures to reduce infectious complications associated with neutropenia. Over the past decade, the use of HGFs became a common method for mobilizing human CD34+ stem cells, either for autologous or allogeneic transplantation. However, since their introduction the long-term safety of the procedure has become a major focus of discussion and research. Most information refers to healthy normal donors and data concerning pregnant and lactating women are scarce. The clinical question, which is the core of this review, is whether stem cell donation, preceded by administration of granulocyte-colony stimulating factor (G-CSF) for mobilization, is a safe procedure for pregnant donors. METHODS Literature searches were performed in Pubmed for English language articles published before the end of May 2012, focusing on G-CSF administration during pregnancy, lactation and hematopoietic stem cell donation. Searches included animal and human studies. RESULTS Data from animals (n = 15 studies) and women (n = 46 studies) indicate that G-CSF crosses the placenta, stimulates fetal granulopoiesis, improves neonatal survival mostly for very immature infants, promotes trophoblast growth and placental metabolism and has an anti-abortive role. Granulocyte macrophage-CSF is a key cytokine in the maternal immune tolerance towards the implanted embryo and exerts protective long-term programming effects to preimplantation embryos. The available data suggest that probably CSFs should not be administered during the time of most active organogenesis (first trimester), except perhaps for the first week during which implantation takes place. Provided CSF is administered during the second and third trimesters, it appears to be safe, and pregnant women receiving the CSF treatment can become hematopoietic stem cell donors. There are also risks related to the anesthesia, which is required for the bone marrow aspiration. During

  5. Normal Hematopoietic Progenitor Subsets Have Distinct Reactive Oxygen Species, BCL2 and Cell-Cycle Profiles That Are Decoupled from Maturation in Acute Myeloid Leukemia.

    Directory of Open Access Journals (Sweden)

    Naeem Khan

    Full Text Available In acute myeloid leukemia (AML quiescence and low oxidative state, linked to BCL2 mitochondrial regulation, endow leukemic stem cells (LSC with treatment-resistance. LSC in CD34+ and more mature CD34- AML have heterogeneous immunophenotypes overlapping with normal stem/progenitor cells (SPC but may be differentiated by functional markers. We therefore investigated the oxidative/reactive oxygen species (ROS profile, its relationship with cell-cycle/BCL2 for normal SPC, and whether altered in AML and myelodysplasia (MDS. In control BM (n = 24, ROS levels were highest in granulocyte-macrophage progenitors (GMP and CD34- myeloid precursors but megakaryocyte-erythroid progenitors had equivalent levels to CD34+CD38low immature-SPC although they were ki67high. BCL2 upregulation was specific to GMPs. This profile was also observed for CD34+SPC in MDS-without-excess-blasts (MDS-noEB, n = 12. Erythroid CD34- precursors were, however, abnormally ROS-high in MDS-noEB, potentially linking oxidative stress to cell loss. In pre-treatment AML (n = 93 and MDS-with-excess-blasts (MDS-RAEB (n = 14, immunophenotypic mature-SPC had similar ROS levels to co-existing immature-SPC. However ROS levels varied between AMLs; Flt3ITD+/NPM1wild-type CD34+SPC had higher ROS than NPM1mutated CD34+ or CD34- SPC. An aberrant ki67lowBCL2high immunophenotype was observed in CD34+AML (most prominent in Flt3ITD AMLs but also in CD34- AMLs and MDS-RAEB, suggesting a shared redox/pro-survival adaptation. Some patients had BCL2 overexpression in CD34+ ROS-high as well as ROS-low fractions which may be indicative of poor early response to standard chemotherapy. Thus normal SPC subsets have distinct ROS, cell-cycle, BCL2 profiles that in AML /MDS-RAEB are decoupled from maturation. The combined profile of these functional properties in AML subpopulations may be relevant to differential treatment resistance.

  6. Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Chenxi [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Chen, Mouying [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); You, Huihui [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Qiu, Feng [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Wen, Huaxiao; Yuan, Junlin [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China); Xiang, Shuanglin, E-mail: xshlin@hunnu.edu.cn [Key Laboratory of Ecological Safety Monitoring and Evaluation, School of Life Sciences, Hunan Normal University, Changsha 410081, Hunan (China); Yang, Xu, E-mail: yangxu@mail.ccnu.edu.cn [Section of Environmental Biomedicine, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University, Wuhan 430079, Hubei (China)

    2017-06-01

    Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3 mg/m{sup 3} FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk. - Highlights: • Nucleated bone marrow cell count recovered after 7 days post-FA and/or BZ exposure. • CFU-GM showed an increase in colonies and 8-OHdG after 7 days post-FA + BZ exposure. • Levels of ROS in CFU-GM and BFU-E were increased by FA or FA + BZ during recovery. • Levels of

  7. Formaldehyde and co-exposure with benzene induce compensation of bone marrow and hematopoietic stem/progenitor cells in BALB/c mice during post-exposure period

    International Nuclear Information System (INIS)

    Wei, Chenxi; Chen, Mouying; You, Huihui; Qiu, Feng; Wen, Huaxiao; Yuan, Junlin; Xiang, Shuanglin; Yang, Xu

    2017-01-01

    Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3 mg/m 3 FA for 2 weeks, mimicking occupational exposure, and were monitored for another 7 days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk. - Highlights: • Nucleated bone marrow cell count recovered after 7 days post-FA and/or BZ exposure. • CFU-GM showed an increase in colonies and 8-OHdG after 7 days post-FA + BZ exposure. • Levels of ROS in CFU-GM and BFU-E were increased by FA or FA + BZ during recovery. • Levels of GM

  8. ANTITUMOR AND APOPTOTIC EFFECTS OF CUCURBITACIN A IN A-549 LUNG CARCINOMA CELLS IS MEDIATED VIA G2/M CELL CYCLE ARREST AND M-TOR/PI3K/AKT SIGNALLING PATHWAY.

    Science.gov (United States)

    Wang, Wen-Dong; Liu, Yan; Su, Yuan; Xiong, Xian-Zhi; Shang, Dan; Xu, Juan-Juan; Liu, Hong-Ju

    2017-01-01

    The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study. MTT assay and clonogenic assay were carried out to study effects of this compound on cell cytotoxicity and colony forming tendency in A-549 cells. Moreover, phase and fluorescence microscopic techniques were used to examine the effects on cell morphology and induction of apoptosis. The effects on cell cycle phase distribution were investigated by flow cytometry and effects on m-TOR/PI3K/Akt signalling proteins were assessed by western blot analysis. Results showed that cucurbitacin A induced dose-dependent cytotoxic effects along with suppressing the colony forming tendency in these cells. Cucurbitacin A also induced morphological changes in these cells featuring chromatin condensation, cell shrinkage and apoptotic body formation. G2/M phase cell cycle collapse was also induced by Cucurbitacin A along with inhibition of expression levels of m-TOR/PI3K/Akt proteins. In conclusion, cucurbitacin A inhibits cancer growth in A-549 NSCLC cells by inducing apoptosis, targeting m-TOR/PI3K/Akt signalling pathway and G2/M cell cycle.

  9. Mutagenic effects of 3-carbethoxypsoralen and 8-methoxypsoralen plus 365-nm irradiation in mammalian cells

    International Nuclear Information System (INIS)

    Papadopoulo, D.; Sagliocco, F.; Averbeck, D.

    1983-01-01

    Cell survival, i.e. colony-forming ability, and the induction of 6-thioguanine-resistant (6-TGsup(r)) mutants were determined in Chinese hamster V79 cells by using two photoreactive furocoumarins of photochemotherapeutic interest: the bifunctional compound 8-methoxypsoralen (8-MOP) and the monofunctional compound 3-carbethoxypsoralen (3-CPs). To quantify the mutation induction in V79 cells mutants deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected with the purine analogue 6-thioguanine (6-TG). Both compounds exhibited lethal and mutagenic activities but the monofunctional compound 3-CPs was less lethal and mutagenic than the bifunctional compound 8-MOP. (Auth.)

  10. Hoxa9 and Hoxa10 induce CML myeloid blast crisis development through activation of Myb expression.

    Science.gov (United States)

    Negi, Vijay; Vishwakarma, Bandana A; Chu, Su; Oakley, Kevin; Han, Yufen; Bhatia, Ravi; Du, Yang

    2017-11-17

    Mechanisms underlying the progression of Chronic Myeloid Leukemia (CML) from chronic phase to myeloid blast crisis are poorly understood. Our previous studies have suggested that overexpression of SETBP1 can drive this progression by conferring unlimited self-renewal capability to granulocyte macrophage progenitors (GMPs). Here we show that overexpression of Hoxa9 or Hoxa10 , both transcriptional targets of Setbp1 , is also sufficient to induce self-renewal of primary myeloid progenitors, causing their immortalization in culture. More importantly, both are able to cooperate with BCR/ABL to consistently induce transformation of mouse GMPs and development of aggressive leukemias resembling CML myeloid blast crisis, suggesting that either gene can drive CML progression by promoting the self-renewal of GMPs. We further identify Myb as a common critical target for Hoxa9 and Hoxa10 in inducing self-renewal of myeloid progenitors as Myb knockdown significantly reduced colony-forming potential of myeloid progenitors immortalized by the expression of either gene. Interestingly, Myb is also capable of immortalizing primary myeloid progenitors in culture and cooperating with BCR/ABL to induce leukemic transformation of mouse GMPs. Significantly increased levels of MYB transcript also were detected in all human CML blast crisis samples examined over chronic phase samples, further suggesting the possibility that MYB overexpression may play a prevalent role in driving human CML myeloid blast crisis development. In summary, our results identify overexpression of HOXA9 , HOXA10 , and MYB as critical drivers of CML progression, and suggest MYB as a key therapeutic target for inhibiting the self-renewal of leukemia-initiating cells in CML myeloid blast crisis patients.

  11. Highly active antiretroviral therapy normalizes the function of progenitor cells in human immunodeficiency virus-infected patients

    DEFF Research Database (Denmark)

    Dam Nielsen, S.; Ersbøll, A. K.; Mathiesen, L.

    1998-01-01

    -infected patients were determined prior to HAART and after 2, 4, 8, and 12 weeks of therapy. The mean number of colony-forming units (cells) per milliliter (cfu/mL) was 15.0 prior to HAART vs. 109.8 in healthy controls (P.../mL eliminated the differences between HIV-infected patients and controls. Significant increases in numbers of CD34 cells were not detected. Of importance, the cloning efficiency of CD34 cells increased from 1.7% prior to therapy to a peak at 18.7% (P=.003). In conclusion, HAART normalized CD34 cell function...

  12. Comparison of Preterm and Term Wharton's Jelly-Derived Mesenchymal Stem Cell Properties in Different Oxygen Tensions.

    Science.gov (United States)

    Balgi-Agarwal, Saloni; Winter, Caitlyn; Corral, Alexis; Mustafa, Shamimunisa B; Hornsby, Peter; Moreira, Alvaro

    2018-06-27

    Mesenchymal stem cells (MSCs) have shown promise as therapeutic agents in treating morbidities associated with premature birth. MSCs derived from the human umbilical cord are easy to isolate and have low immunogenicity and a robust ability to secrete paracrine factors. To date, there are no studies evaluating preterm versus term umbilical cord tissue-derived MSCs. Therefore, our aim was twofold: (1) to compare stem cell properties in preterm versus term MSCs and (2) to examine the impact of oxygen tension on stem cell behavior. Umbilical cord tissue was obtained from 5 preterm and 5 term neonates. The cells were isolated and characterized as MSCs in accordance with the International Society for Cellular Therapy. We exposed MSCs to different oxygen tensions to examine the impact of environmental factors on cell performance. We studied the following stem cell properties: (i) motility, (ii) proliferation, (iii) senescence, (iv) cell viability, (v) colony-forming unit efficiency, and (vi) inflammatory cytokine expression. Under normoxia (21% O2), cells from preterm and term infants had similar properties. Under hypoxic conditions (1% O2), term MSCs had better cell proliferation; however, cells exposed to hyperoxia (90% O2) had the slowest motility and lowest cell viability (p cytokine expression between the groups. The term cells demonstrated more colony-forming efficiency than the preterm cells. In sum, our preliminary findings suggest that MSCs derived from term and preterm umbilical cords have similar characteristics, offering the potential of future autologous/allogeneic MSC transplants in neonates. © 2018 S. Karger AG, Basel.

  13. Suicide of EMT-6 tumor cells by decays from radioactively-labelled sensitizer adducts

    International Nuclear Information System (INIS)

    Roa, W.H.Y.; Chapman, J.D.

    1987-01-01

    Nitroaromatic radiosensitizers become metabolically bound preferentially to hypoxic cells and at least 10/sup 9/ adducts/cell can be tolerated as non-toxic. EMT-6 tumor cells have been incubated in hypoxia in the presence of /sup 3/H-Misonidazole and /sup 125/I-Azomycin Riboside for various times and the amount of /sup 3/H or /sup 125/I bound/cell was determined. Cells were stored as monolayers at 25 0 C for up to 96 hr to accumulate radioactive decays and transferred at various times to 37 0 C for colony-forming assays. No radiation inactivation was measured in cells which had incorporated at least 10/sup 6/ /sup 3/H or 10/sup 5/ /sup 125/I atoms. Previous studies had shown that -- 1% of MISO adducts to EMT-6 cells was associated with cellular DNA. These data indicate that the radiation-induced damage produced by these quantities of bound /sup 3/H or /sup 125/I causes little or not cell inactivation. The results of current studies to measure the colony-forming ability of sensitizer-labelled cells which have been stored in liquid nitrogen to facilitate the accumulation of more decays will be reported. These data suggest that a ''sensitizer-adduct suicide technique'' as a hypoxic cell selective adjunct to other cancer therapies is not feasible. These data are also instructive for those who attempt to develop radiolabelled ''tumor specific'' antibodies for therapeutic purposes

  14. The effect of cilengitide in combination with irradiation and chemotherapy in head and neck squamous cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Heiduschka, G.; Lill, C.; Schneider, S.; Kotowski, U.; Thurnher, D.; Seemann, R.; Kornek, G.; Schmid, R.

    2014-01-01

    Integrins are highly attractive targets in oncology due to their involvement in angiogenesis in a wide spectrum of cancer entities. Among several integrin inhibitors under clinical evaluation, cilengitide is the most promising compound. However, little is known about the cellular processes induced during cilengitide therapy in combination with irradiation and cisplatin in head and neck squamous cell carcinoma (HNSCC). The cytostatic effect of cilengitide was assessed by proliferation assay in the three HNSCC cell lines SCC25, FaDu and CAL27. Combination experiments with cisplatin and irradiation were performed. Possible synergistic effects were calculated in combination index (CI) analyses. Colony forming inhibition was investigated in clonogenic assays. Real-time PCR arrays were used to evaluate target protein gene expression patterns. Flow cytometry was used to detect apoptosis. Used alone, cilengitide has only minor cytotoxic effects in HNSCC cell lines. However, combination with cisplatin resulted in synergistic growth inhibition in all three cell lines. Irradiation showed synergism in short-term experiments and in colony forming assays, an additive effect was detected. Real-time PCR assay detected downregulation of the antiapoptotic protein Bcl-2 after exposure of cells to cilengitide. Cilengitide in combination with cisplatin and irradiation may be a feasible option for the treatment of patients with head and neck cancer. However, further investigations are required to understand the exact mechanism that leads to synergistic cytotoxicity. (orig.) [de

  15. Genome-wide immunity studies in the rabbit: transcriptome variations in peripheral blood mononuclear cells after in vitro stimulation by LPS or PMA-Ionomycin.

    Science.gov (United States)

    Jacquier, Vincent; Estellé, Jordi; Schmaltz-Panneau, Barbara; Lecardonnel, Jérôme; Moroldo, Marco; Lemonnier, Gaëtan; Turner-Maier, Jason; Duranthon, Véronique; Oswald, Isabelle P; Gidenne, Thierry; Rogel-Gaillard, Claire

    2015-01-23

    Our purpose was to obtain genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptome profiling was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome. The LPS affected 15 to 20 times fewer genes than PMA-Ionomycin after both 4 hours (T4) and 24 hours (T24), of in vitro stimulation, in comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, and CCL4 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24, and detected a down-regulation of DEFB1 and BPI at T24. A concerted up-regulation of SAA1, S100A12 and F3 was found upon stimulation by LPS. PMA-Ionomycin induced a very early expression of Th1, Th2, Treg, and Th17 responses by PBMCs at T4. The Th1 response increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines which significantly decreased from T4 to T24 (IL2, IL4, IL10, IL13, IL17A, CD69) by comparison to mock-stimulation. The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA-Ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in PBMC apoptosis. We report new data on the responses of PBMCs to LPS and PMA-Ionomycin in the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with high throughput genomic tools should pave the way for more

  16. Haemopoietic progenitor cells in human peripheral blood

    International Nuclear Information System (INIS)

    Zwaan, F.E.

    1980-01-01

    The purpose of the investigation reported is to purify haemopoietic progenitor cells from human peripheral blood using density gradient centrifugation in order to isolate a progenitor cell fraction without immunocompetent cells. The purification technique of peripheral blood flow colony forming unit culture (CFU-c) by means of density gradient centrifugation and a combined depletion of various rosettes is described. The results of several 'in vitro' characteristics of purified CFU-c suspensions and of the plasma clot diffusion chamber culture technique are presented. Irradiation studies revealed that for both human bone marrow and peripheral blood the CFU-c were less radioresistant than clusters. Elimination of monocytes (and granulocytes) from the test suspensions induced an alteration in radiosensitivity pararmeters. The results obtained with the different techniques are described by analysing peripheral progenitor cell activity in myeloproliferative disorders. (Auth.)

  17. Dairy cows produce cytokine and cytotoxic T cell responses following vaccination with an antigenic fraction from Streptococcus uberis.

    Science.gov (United States)

    Wedlock, D Neil; Buddle, Bryce M; Williamson, John; Lacy-Hulbert, S Jane; Turner, Sally-Anne; Subharat, Supatsak; Heiser, Axel

    2014-07-15

    Streptococcus uberis is a major cause of mastitis in dairy cows worldwide and currently, there is no vaccine commercially available against this form of mastitis. In the current study, cell-free extracts (CFE) were prepared from each of three different S. uberis strains, designated as #3, #24 and #363 representative of the three main sequence types of S. uberis that cause mastitis in New Zealand. These proteins were formulated into vaccines with Emulsigen-D and the immunogenicity of the vaccines was determined in both calves and dairy cows. Two groups of calves (n=5/group) were vaccinated subcutaneously with CFE from strain #24 or strains #3, #24 and #363 formulated with Emulsigen-D, respectively. A third group (n=5) was vaccinated with CFE from the three strains formulated with Emulsigen-D and also containing recombinant bovine granulocyte macrophage colony-stimulating factor while, a control group (n=5) was not vaccinated. Vaccinated animals produced strong antibody responses to the S. uberis antigens and an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, with no significant differences in responses observed between the three vaccinated groups. In a second trial, the safety and immunogenicity of the vaccine containing CFE from all three strains of S. uberis and Emulsigen-D was determined in dairy cows. A group of six cows were vaccinated subcutaneously at 3 and 1 week prior to dry off and revaccinated 2-3 weeks before calving. Immune responses in blood and mammary gland secretions (MGS) were monitored during the dry period and in the subsequent lactation. The vaccine was well tolerated with no adverse effect from vaccination observed in any of the cows. Vaccination induced an antigen-specific cytotoxic effect against blood monocytes/macrophages that had phagocytosed S. uberis, moderate antigen-specific IFN-γ responses in blood and strong antibody responses in both blood and MGS. In conclusion, the results

  18. Characterization of the response of a human breast carcinoma cell line (T-47D) to radiation and chemotherapeutic agents

    International Nuclear Information System (INIS)

    Prager, A.; Ben-Hur, E.; Riklis, E.

    1981-01-01

    The response of a human breast carcinoma cell line (T-47D) to various antitumour agents, gamma irradiation, UV light and heat was studied, using the colony-forming ability technique. Combinations of radiation with drugs and heat were also tested. The resulting survival curves corresponded to one of five patterns: simple exponential, biphasic exponential, threshold exponential, exponential plateau and ineffectual. Whereas the cells were particularly sensitive to gamma irradiation, the response to UV light was normal. The patient from whom this cell line originated did not respond to METHOTREXATEsup(R) therapy. The in vitro results correlated with this observation. (author)

  19. Heterogeneity of clonogenic cells in acute myeloblastic leukemia.

    Science.gov (United States)

    Sabbath, K D; Ball, E D; Larcom, P; Davis, R B; Griffin, J D

    1985-02-01

    The expression of differentiation-associated surface antigens by the clonogenic leukemic cells from 20 patients with acute myeloblastic leukemia (AML) was studied with a panel of seven cytotoxic monoclonal antibodies (anti-Ia, -MY9, -PM-81, -AML-2-23, -Mol, -Mo2, and -MY3). The surface antigen phenotypes of the clonogenic cells were compared with the phenotypes of the whole leukemic cell population, and with the phenotypes of normal hematopoietic progenitor cells. In each case the clonogenic leukemic cells were found within a distinct subpopulation that was less "differentiated" than the total cell population. Clonogenic leukemic cells from different patients could be divided into three phenotype groups. In the first group (7 of 20 cases), the clonogenic cells expressed surface antigens characteristic of the normal multipotent colony-forming cell (Ia, MY9). These cases tended to have "undifferentiated" (FAB M1) morphology, and the total cell population generally lacked expression of "late" monocyte antigens such as MY3 and Mo2. A second group (seven cases) of clonogenic cells expressed surface antigens characteristic of an "early" (day 14) colony-forming unit granulocyte-monocyte (CFU-GM), and a third group (six cases) was characteristic of a "late" (day 7) CFU-GM. The cases in these latter two groups tended to have myelomonocytic (FAB M4) morphology and to express monocyte surface antigens. These results suggest that the clonogenic cells are a distinct subpopulation in all cases of AML, and may be derived from normal hematopoietic progenitor cells at multiple points in the differentiation pathway. The results further support the possibility that selected monoclonal antibodies have the potential to purge leukemic clonogenic cells from bone marrow in some AML patients without eliminating critical normal progenitor cells.

  20. Radioresistant canine hematopoietic cells

    International Nuclear Information System (INIS)

    Kawakami, T.G.; Shimizu, J.; Rosenblatt, L.S.; Goldman, M.

    1987-01-01

    Survival of dogs that are continuously exposed to a moderate dose-rate of gamma radiation (10 cGy/day) is dependent on the age of the dog at the time of exposure. Most dogs exposed postpartum to gamma radiation suffered from suppressed hematopoiesis and died of aplasia. On the other hand, none of the in utero-exposed dogs suffered from suppressed hematopoiesis and most became long-term survivors, tolerating 10-fold greater total dose, but dying of myeloproliferative disease (MPD). Using acute gamma irradiation of hematopoietic cells and colony forming unit cell assay (CFU), they observed that a canine hematopoietic cell line established from a myeloid leukemic dog that was a long-term survivor of continuous irradiation was approximately 4-fold more radioresistant than a hematopoietic cell line established from a dog with nonradiation-induced myeloid leukemia or hematopoietic cells from normal canine bone marrow. In utero dogs that are long-term survivors of continuous irradiation have radioresistant hematopoietic cells, and radioresistance that is a constitutive property of the cells

  1. DNA repair capacity and rate of excision repair in UV-irradiated mammalian cells

    International Nuclear Information System (INIS)

    Inoue, Masao; Takebe, Hiraku.

    1978-01-01

    Repair capacities of five mammalian cell strains were measured by colony-forming ability, HCR of UV-irradiated virus, UDS, pyrimidine dimer excision, and semi-conservative DNA replication. Colony-forming ability of UV-irradiated cells was high for human amnion FL cells and mouse L cells, slightly low for African green monkey CV-1 cells, and extremely low for xeroderma pigmentosum cells. HCR of UV-irradiated Herpes simplex virus was high in CV-1 cells, FL and normal human fibroblast cells, low in both XP and L cells. The amount of UDS was high in FL and normal human fibroblast cells, considerably low in CV-1 cells, and essentially no UDS was observed in XP cells. Rate of UDS after UV-irradiation was slower for CV-1 cells than FL and human fibroblast cells. Rate of the excision of thymine-containing dimers from the acid-insoluble fraction during post-irradiation incubation of the cells was rapid in FL and normal human cells and slow in CV-1 cells, and no excision took place in XP cells. Semi-conservative DNA synthesis was reduced after UV-irradiation in all cell lines, but subsequently recovered in FL, normal human and CV-1 cells. The onset of recovery was 4 h after UV-irradiation for FL and normal human cells, but about 6 h for CV-1 cells. The apparent intermediate repair of CV-1 cells except for HCR may be related to the slow rate of excision repair. ''Patch and cut'' model is more favorable than ''cut and patch'' model to elucidate these results. (auth.)

  2. Diffusive boundary layers, photosynthesis, and respiration of the colony-forming plankton algae, Phaeocystis sp

    DEFF Research Database (Denmark)

    Ploug, Helle; Stolte, W.; Epping, E.H.G.

    1999-01-01

    H increased up to 0.4 units when measured in light at saturating intensities (>90 mu mol photons m(-2) s(-1)). The respiration in the dark was low, resulting in a 6% lowering in oxygen concentration and 0.04 units lowering in pH inside colonies, compared to the bulk water phase. Such colonies were net...... heterotrophic communities at light intensities up to 10 mu mol photons m(-2) s(-1). A week later, colonies were net heterotrophic at light intensities up to 80 mu mol photons m(-2) s(-1). The effective diffusion coefficient for oxygen in the gelatinous colonies was not significantly different from that in sea......Diffusive boundary layers, photosynthesis, and respiration in Phaeocystis colonies were studied by the use of microelectrodes for oxygen and pH during a bloom in the Barents Sea, 1993, and in the Marsdiep, Dutch North Sea, 1994. The oxygen microenvironment of a Phaeocystis colony with a mean...

  3. Competitive interactions between corals and turf algae depend on coral colony form.

    Science.gov (United States)

    Swierts, Thomas; Vermeij, Mark Ja

    2016-01-01

    Turf algae are becoming more abundant on coral reefs worldwide, but their effects on other benthic organisms remain poorly described. To describe the general characteristics of competitive interactions between corals and turf algae, we determined the occurrence and outcomes of coral-turf algal interactions among different coral growth forms (branching, upright, massive, encrusting, plating, and solitary) on a shallow reef in Vietnam. In total, the amount of turf algal interaction, i.e., the proportion of the coral boundary directly bordering turf algae, was quantified for 1,276 coral colonies belonging to 27 genera and the putative outcome of each interaction was noted. The amount of turf algal interaction and the outcome of these interactions differed predictably among the six growth forms. Encrusting corals interacted most often with turf algae, but also competed most successfully against turf algae. The opposite was observed for branching corals, which rarely interacted with turf algae and rarely won these competitive interactions. Including all other growth forms, a positive relationship was found between the amount of competitive interactions with neighboring turf algae and the percentage of such interaction won by the coral. This growth form dependent ability to outcompete turf algae was not only observed among coral species, but also among different growth forms in morphologically plastic coral genera (Acropora, Favia, Favites, Montastrea, Montipora, Porites) illustrating the general nature of this relationship.

  4. The effect of mixotrophic chrysophyte on toxic and colony-forming cyanobacteria

    NARCIS (Netherlands)

    Van Donk, E.; Cerbin, S.; Wilken, S.; Helmsing, N.R.; Ptacnik, R.; Verschoor, A.M.

    2009-01-01

    1. In order to test the effect of Ochromonas sp., a mixotrophic chrysophyte, on cyanobacteria, grazing experiments were performed under controlled conditions. We studied grazing on three Microcystis aeruginosa strains, varying in toxicity and morphology, as well as on one filamentous cyanobacterium,

  5. Effect of Fluoride Mouthrinse and Toothpaste on Number of Streptococcal Colony Forming Units of Dental Plaque

    OpenAIRE

    SE Jabbarifar; SA Tabibian; F Poursina

    2005-01-01

    Background: Frequent topical fluoride therapy through toothpaste, mouthrinse, professional gels and solutions causes decrease in incidence, pause and repair of dental caries in the enamel. These mechanisms are done through penetration of fluoride ions (F-) and their replacement with hydroxyl ions (OH-) of hydroxyappatite of enamel, interfere with microbial metabolism of dental plaque and bacteriostatic effect on some cariogenic bacterial strains such as streptococci. The aim of this study was...

  6. The genetics of colony form and function in Caribbean Acropora corals.

    Science.gov (United States)

    Hemond, Elizabeth M; Kaluziak, Stefan T; Vollmer, Steven V

    2014-12-17

    Colonial reef-building corals have evolved a broad spectrum of colony morphologies based on coordinated asexual reproduction of polyps on a secreted calcium carbonate skeleton. Though cnidarians have been shown to possess and use similar developmental genes to bilaterians during larval development and polyp formation, little is known about genetic regulation of colony morphology in hard corals. We used RNA-seq to evaluate transcriptomic differences between functionally distinct regions of the coral (apical branch tips and branch bases) in two species of Caribbean Acropora, the staghorn coral, A. cervicornis, and the elkhorn coral, A. palmata. Transcriptome-wide gene profiles differed significantly between different parts of the coral colony as well as between species. Genes showing differential expression between branch tips and bases were involved in developmental signaling pathways, such as Wnt, Notch, and BMP, as well as pH regulation, ion transport, extracellular matrix production and other processes. Differences both within colonies and between species identify a relatively small number of genes that may contribute to the distinct "staghorn" versus "elkhorn" morphologies of these two sister species. The large number of differentially expressed genes supports a strong division of labor between coral branch tips and branch bases. Genes involved in growth of mature Acropora colonies include the classical signaling pathways associated with development of cnidarian larvae and polyps as well as morphological determination in higher metazoans.

  7. First report on the colony-forming freshwater ciliate Ophrydium versatile in an African river

    CSIR Research Space (South Africa)

    Oberholster, Paul J

    2010-04-01

    Full Text Available Ophrydium versatile (Müller 1786) Ehrenberg 1830 – a symbiotic ciliate that forms gelatinous colonies – is widely distributed in temperate lakes in Europe and America, but has not previously been recorded from rivers. In this paper we report...

  8. Response to high LET radiation 12C (LET, 295 keV/microm) in M5 cells, a radio resistant cell strain derived from Chinese hamster V79 cells.

    Science.gov (United States)

    Pathak, R; Sarma, A; Sengupta, B; Dey, S K; Khuda-Bukhsh, A R

    2007-01-01

    To study the effects of 12C-beam of 295 keV/microm (57.24 MeV) on M5 and Chinese hamster V79 cells by using cytogenetic assays like micronuclei (MN) induction, chromosomal aberrations (CA) and apoptosis. Additionally, the relative survival of these two cell lines was tested by the colony forming ability of the cells, with a view to understanding the mechanism of cellular damages that lead to difference in cell survival. Confluent cells were irradiated with 12C-beam at various doses using 15UD Pelletron accelerator. Cell survival was studied by the colony forming ability of cells. MN assay was done by fluorescent staining. Different types of chromosomal aberrations in metaphase cells were scored at 12 h after irradiation. Apoptosis was measured at different post irradiation times as detected by nuclear fragmentation and DNA ladder was prepared after 48 h of incubation. Dose-dependent decrease in surviving fractions was found in both the cell lines. However, the surviving fractions were higher in M5 cells in comparison to V79 cells when exposed to the same radiation doses. On the other hand, induced MN frequencies, CA frequencies and apoptosis percentages were less in M5 cells than V79 cells. Very good correlations between surviving fractions and induced MN frequencies or induced total CA or induced apoptosis percentages were obtained in this study. The cell strain M5 showed relatively more radio-resistance to 12C-beam compared to Chinese hamster V79 cells in this study. As the MN formation, CA and apoptosis induction were less in M5 cells as compared to parental V79 cells, the higher cell survival in the former could possibly be attributed to their better repairing ability leading to higher cell survival.

  9. Octanoate in Human Albumin Preparations Is Detrimental to Mesenchymal Stromal Cell Culture

    Directory of Open Access Journals (Sweden)

    Way-Wua Wong

    2015-01-01

    Full Text Available Cell therapies hold great promise as the next major advance in medical treatment. To enable safe, effective ex vivo culture whilst maintaining cell phenotype, growth media constituents must be carefully controlled. We have used a chemically defined mesenchymal stromal cell culture medium to investigate the influence of different preparations of human serum albumin. We examined two aspects of cell culture, growth rate as measured by population doubling time and colony forming ability which is a representative measure of the stemness of the cell population. Albumin preparations showed comparative differences in both of these criteria. Analysis of the albumin bound fatty acids also showed differences depending on the manufacturing procedure used. We demonstrated that octanoate, an additive used to stabilize albumin during pasteurization, slows growth and lowers colony forming ability during ex vivo culture. Further to this we also found the level of Na+/K+ ATPase, a membrane bound cation pump inhibited by octanoate, is increased in cells exposed to this compound. We conclude that the inclusion of human serum albumin in ex vivo growth media requires careful consideration of not only the source of albumin, but also the associated molecular cargo, for optimal cell growth and behavior.

  10. Use of Limiting Dilution Method for Isolation of Nucleus Pulposus Mesenchymal Stem/Progenitor Cells and Effects of Plating Density on Biological Characteristics and Plasticity

    Directory of Open Access Journals (Sweden)

    Linghan Lin

    2017-01-01

    Full Text Available Objectives. To evaluate the effects of the limiting dilution method and plating density in rat nucleus pulposus mesenchymal stem/progenitor cells (NPMSCs. Materials and Methods. Nucleus pulposus tissues were isolated from 12-week-old male Sprague-Dawley rats and NPMSCs were isolated using limiting dilution method. Cells were then classified into 3 groups according to plating density. Cell morphologies were observed, and colony-forming units, migration abilities, proliferative capacities, cell cycle percentages, multilineage differentiation capacities, stem cell biomarker expression levels, and immunophenotyping were also examined in each group. Results. Low density group (LD had higher morphological homogeneity, stronger colony-forming ability, higher cell proliferation capacity, and enhanced cell migration ability relative to the other two groups (p<0.05. Moreover, LD had more cells entering S phase, with fewer cells arrested in G0/G1 phase (p<0.05. While all three density groups showed a multilineage differentiation potential, LD showed a higher degree of observed and semiquantified lineage specific staining (p<0.05. Furthermore, LD displayed higher expression levels of stem cell biomarkers (Nanog, Oct4, and Sox2 and showed higher percentages of CD29+, CD44+, and CD90+ cells (p<0.05 following flow cytometry analysis. Conclusions. Limiting dilution method is suggested when isolating NPMSCs as a means of improving cell activity and plasticity.

  11. Reconstitution of the myeloid and lymphoid compartments after the transplantation of autologous and genetically modified CD34+ bone marrow cells, following gamma irradiation in cynomolgus macaques

    Directory of Open Access Journals (Sweden)

    Auregan Gwenaelle

    2008-06-01

    Full Text Available Abstract Background Prolonged, altered hematopoietic reconstitution is commonly observed in patients undergoing myeloablative conditioning and bone marrow and/or mobilized peripheral blood-derived stem cell transplantation. We studied the reconstitution of myeloid and lymphoid compartments after the transplantation of autologous CD34+ bone marrow cells following gamma irradiation in cynomolgus macaques. Results The bone marrow cells were first transduced ex vivo with a lentiviral vector encoding eGFP, with a mean efficiency of 72% ± 4%. The vector used was derived from the simian immunodeficiency lentivirus SIVmac251, VSV-g pseudotyped and encoded eGFP under the control of the phosphoglycerate kinase promoter. After myeloid differentiation, GFP was detected in colony-forming cells (37% ± 10%. A previous study showed that transduction rates did not differ significantly between colony-forming cells and immature cells capable of initiating long-term cultures, indicating that progenitor cells and highly immature hematopoietic cells were transduced with similar efficiency. Blood cells producingeGFP were detected as early as three days after transplantation, and eGFP-producing granulocyte and mononuclear cells persisted for more than one year in the periphery. Conclusion The transplantation of CD34+ bone marrow cells had beneficial effects for the ex vivo proliferation and differentiation of hematopoietic progenitors, favoring reconstitution of the T- and B-lymphocyte, thrombocyte and red blood cell compartments.

  12. Studies on the effects of ionizing radiation and chemotherapeutic agents on hematopoiesis according to the stem-cell kinetics

    International Nuclear Information System (INIS)

    Hirashima, Kunitake

    1975-01-01

    The fundamental problem of the effects of ionizing radiation and antineoplastic drugs on hematopoiesis can be explained by the kinetic study on the hematopoietic stem-cell population. Quantitative comparison of a single x-irradiation and a single administration of several antineoplastic drugs on the stem-cell population was performed by the splenic colony-forming method. The repopulation pattern of stem-cells in mice after a single 150 rad irradiation was compared with that after the administration of corresponding dose of cyclophosphamide. It was demonstrated that the additional administration of cyclophosphamide immediately after the x-irradiation significantly accelerated repopulation of the stem-cell compartment. The mechanism of repopulation of the stem-cell compartment after partial irradiation was also studied according to the immigration theory of stem-cells. An in vitro colony-forming technique for the human bone marrow cells was introduced and compared with other assay methods for stem-cells. From the hematological observations of accidentally irradiated patients, it was determined that the thromboelastogram values were regarded as one of the most useful indicators for detecting the earliest recovery sign of the hematopoietic stem-cells. (Evans, J.)

  13. Mechanisms of growth inhibition of primary prostate epithelial cells following gamma irradiation or photodynamic therapy include senescence, necrosis, and autophagy, but not apoptosis

    International Nuclear Information System (INIS)

    Frame, Fiona M.; Savoie, Huguette; Bryden, Francesca; Giuntini, Francesca; Mann, Vincent M.; Simms, Matthew S.; Boyle, Ross W.; Maitland, Norman J.

    2015-01-01

    In comparison to more differentiated cells, prostate cancer stem-like cells are radioresistant, which could explain radio-recurrent prostate cancer. Improvement of radiotherapeutic efficacy may therefore require combination therapy. We have investigated the consequences of treating primary prostate epithelial cells with gamma irradiation and photodynamic therapy (PDT), both of which act through production of reactive oxygen species (ROS). Primary prostate epithelial cells were cultured from patient samples of benign prostatic hyperplasia and prostate cancer prior to treatment with PDT or gamma irradiation. Cell viability was measured using MTT and alamar blue assay, and cell recovery by colony-forming assays. Immunofluorescence of gamma-H2AX foci was used to quantify DNA damage, and autophagy and apoptosis were assessed using Western blots. Necrosis and senescence were measured by propidium iodide staining and beta-galactosidase staining, respectively. Both PDT and gamma irradiation reduced the colony-forming ability of primary prostate epithelial cells. PDT reduced the viability of all types of cells in the cultures, including stem-like cells and more differentiated cells. PDT induced necrosis and autophagy, whereas gamma irradiation induced senescence, but neither treatment induced apoptosis. PDT and gamma irradiation therefore inhibit cell growth by different mechanisms. We suggest these treatments would be suitable for use in combination as sequential treatments against prostate cancer

  14. Resistance of human and mouse myeloid leukemia cells to UV radiation

    International Nuclear Information System (INIS)

    Poljak-Blazi, M.; Osmak, M.; Hadzija, M.

    1989-01-01

    Sensitivity of mouse bone marrow and myeloid leukemia cells and sensitivity of human myeloid leukemia cells to UV light was tested. Criteria were the in vivo colony-forming ability of UV exposed cells and the inhibition of DNA synthesis during post-irradiation incubation for 24 h in vitro. Mouse bone marrow cells irradiated with a small dose of UV light (5 J/m 2 ) and injected into x-irradiated animals did not form hemopoietic colonies on recipient's spleens, and recipients died. However, mouse leukemia cells, after irradiation with higher doses of UV light, retained the ability to form colonies on the spleens, and all recipient mice died with typical symptoms of leukemia. In vitro, mouse bone marrow cells exhibited high sensitivity to UV light compared to mouse myeloid leukemia cells. Human leukemia cells were also resistant to UV light, but more sensitive than mouse leukemia cells. (author)

  15. Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam

    International Nuclear Information System (INIS)

    Courdi, A.; Mari, D.; Herault, J.; Chauvel, P.

    1995-01-01

    The aim of this study was to measure the biological efficiency of a low-energy argon beam (E=7.1 MeV/nucleon, LET=1590 keV/μm) on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colony-forming assay. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colony-forming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 μm 2 ; it was 39 μm 2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively, at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN. However, multiple (>2) MN in BNC were more frequently observed after low-dose Ar irradiation than after gamma-ray exposure (RBE > 1). Moreover, the frequency of multiple MN induction exceeded that expected from a Poisson distribution at all dose levels of Ar irradiation. (orig.)

  16. Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoietic stem cells

    International Nuclear Information System (INIS)

    Bodine, D.M.; McDonagh, K.T.; Brandt, S.J.; Ney, P.A.; Agricola, B.; Byrne, E.; Nienhuis, A.W.

    1990-01-01

    Retroviral-mediated gene transfer into primitive hematopoietic cells has been difficult to achieve in large-animal models. The authors have developed an amphotropic producer clone that generates >10 10 recombinant retroviral particles (colony-forming units) per ml of culture medium. Autologous rhesus monkey bone marrow cells were cocultured with either high or low titer producer clones for 4-6 days and reinfused into sublethally irradiated animals. The proviral genome was detected in blood and bone-marrow cells from all three animals reconstituted with cells cocultured with the high-titer producer cells. In contrast, three animals reconstituted with bone marrow cocultured with the low-titer producer clone exhibited no evidence of gene transfer

  17. Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells.

    Science.gov (United States)

    Lin, Yang; Gil, Chang-Hyun; Yoder, Mervin C

    2017-11-01

    The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony-forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols. © 2017 American Heart Association, Inc.

  18. Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

    DEFF Research Database (Denmark)

    Bath, Chris; Yang, Sufang; Muttuvelu, Danson

    2013-01-01

    The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth......, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE......, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET)....

  19. Mesenchymal progenitor cells for the osteogenic lineage.

    Science.gov (United States)

    Ono, Noriaki; Kronenberg, Henry M

    2015-09-01

    Mesenchymal progenitors of the osteogenic lineage provide the flexibility for bone to grow, maintain its function and homeostasis. Traditionally, colony-forming-unit fibroblasts (CFU-Fs) have been regarded as surrogates for mesenchymal progenitors; however, this definition cannot address the function of these progenitors in their native setting. Transgenic murine models including lineage-tracing technologies based on the cre-lox system have proven to be useful in delineating mesenchymal progenitors in their native environment. Although heterogeneity of cell populations of interest marked by a promoter-based approach complicates overall interpretation, an emerging complexity of mesenchymal progenitors has been revealed. Current literatures suggest two distinct types of bone progenitor cells; growth-associated mesenchymal progenitors contribute to explosive growth of bone in early life, whereas bone marrow mesenchymal progenitors contribute to the much slower remodeling process and response to injury that occurs mainly in adulthood. More detailed relationships of these progenitors need to be studied through further experimentation.

  20. Accelerator Analysis of Tributyltin Adsorbed onto the Surface of a Tributyltin Resistant Marine Pseudoalteromonas sp. Cell

    OpenAIRE

    Mimura, Haruo; Sato, Ryusei; Sasaki, Yu; Furuyama, Yuichi; Taniike, Akira; Yoshida, Kazutoshi; Kitamura, Akira

    2008-01-01

    Tributyltin (TBT) released into seawater from ship hulls is a stable marine pollutant and obviously remains in marine environments. We isolated a TBT resistant marine Pseudoalteromonas sp. TBT1 from sediment of a ship’s ballast water. The isolate (109.3 ± 0.2 colony-forming units mL-1) adsorbed TBT in proportion to the concentrations of TBTCl externally added up to 3 mM, where the number of TBT adsorbed by a single cell was estimated to be 108.2. The value was reduced to about one-fif...

  1. Mesenchymal stem cells from cortical bone demonstrate increased clonal incidence, potency, and developmental capacity compared to their bone marrow–derived counterparts

    Directory of Open Access Journals (Sweden)

    Daniel Blashki

    2016-08-01

    Full Text Available In this study, we show that matrix dense cortical bone is the more potent compartment of bone than bone marrow as a stromal source for mesenchymal stem cells as isolated from adult rats. Lineage-depleted cortical bone-mesenchymal stem cells demonstrated >150-fold enrichment of colony forming unit–fibroblasts per cell incidence. compared to lineage-depleted bone marrow-mesenchymal stem cells, corresponding to a 70-fold increase in absolute recovered colony forming unit–fibroblasts. The composite phenotype Lin−/CD45−/CD31−/VLA-1+/Thy-1+ enriched for clonogenic mesenchymal stem cells solely from cortical bone–derived cells from which 70% of clones spontaneously differentiated into all lineages of bone, cartilage, and adipose. Both populations generated vascularized bone tissue within subcutaneous implanted collagen scaffolds; however, cortical bone–derived cells formed significantly more osteoid than bone marrow counterparts, quantified by histology. The data demonstrate that our isolation protocol identifies and validates mesenchymal stem cells with superior clonal, proliferative, and developmental potential from cortical bone compared to the bone marrow niche although marrow persists as the typical source for mesenchymal stem cells both in the literature and current pre-clinical therapies.

  2. Petiveria alliacea L. extract protects mice against Listeria monocytogenes infection--effects on bone marrow progenitor cells.

    Science.gov (United States)

    Quadros, M R; Souza Brito, A R; Queiroz, M L

    1999-02-01

    In this study we have investigated the effects of Petiveria alliacea on the hematopoietic response of mice infected with Listeria monocytogenes. Our results demonstrate a protective effect of the crude extract of P. alliacea since the survival of the treated/infected was higher than that in the infected group. Moreover, the number of granulocyte/macrophage colonies (CFU-GM) and the serum colony stimulating activity levels were increased in the treated/infected mice in relation to the infected group. These results suggest an immunomodulation of Petiveria alliacea extract on hematopoiesis, which may be responsible, at least in part, for the increased resistance of mice to Listeria monocytogenes infection.

  3. Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

    Science.gov (United States)

    Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

    2011-10-01

    This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

  4. Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    International Nuclear Information System (INIS)

    Miyazaki, Nobuyoshi; Nagai, Kazuo; Tamura, Gakuzo

    1976-01-01

    A thermosensitive mutant ts 42, of Bacillus subtilis Marburg 168 thy trp2 which requires uracil, was examined as to the colony-forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in modified woese's medium. However, the cells retained the viability when sodium succinate or potassium chloride was added to the medium at that temperature, although uranil deficiency was unchanged. A little but significant incorporation of adenine-8- 14 C into RNA still continued even after the incorporation of N-acetyl- 3 H-D-glucosamine into the acid-insoluble fraction of the cells terminated in the modified Woese's medium at 48 0 C. Both incorporations as well as the increase of absorbance were slowed down in the presence of sodium succinate at 48 0 C. This mutant, ts42, was more sensitive to deoxycholate than the parent wild strain. The resoration of the colony-forming ability after the temperature shifted back from 48 0 to 37 0 C was suppressed by the addition of deoxycholate to the medium. However, the cells became resistant to deoxycholate when uracil had been added to the medium prior to the temperature shift. (Kobatake, H.)

  5. Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

    Science.gov (United States)

    MacLean, Glenn A.; Menne, Tobias F.; Guo, Guoji; Sanchez, Danielle J.; Park, In-Hyun; Daley, George Q.; Orkin, Stuart H.

    2012-01-01

    Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryionic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43+(Leukosialin)/CD235+(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. PMID:23045682

  6. An evaluation of the choice of feeder cell growth arrest for the production of cultured epidermis.

    Science.gov (United States)

    Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

    2015-12-01

    Growth arrested 3T3 cells have been used as feeder cells in human epidermal keratinocyte cultures to produce cultured epidermal autografts for the treatment of burns. The feeder cells were ideally growth-arrested by gamma-irradiation. Alternatively, growth arrest by mitomycin C treatment is a cost effective option. We compared the functional efficacy of these two approaches in keratinocyte cultures by colony forming efficiency, the net growth area of colonies, BrdU labeling and histological features of cultured epidermal sheets. The growth area estimation involved a semi-automated digital technique using the Adobe Photoshop and comprised of isolation and enumeration of red pixels in Rhodamine B-stained keratinocyte colonies. A further refinement of the technique led to the identification of critical steps to increasing the degree of accuracy and enabling its application as an extension of colony formation assay. The results on feeder cell functionality revealed that the gamma irradiated feeders influenced significantly higher colony forming efficiency and larger growth area than the mitomycin C treated feeders. The BrdU labeling study indicated significant stimulation of the overall keratinocyte proliferation by the gamma irradiated feeders. The cultured epidermal sheets produced by gamma feeders were relatively thicker than those produced by mitomycin C feeders. We discussed the clinical utility of mitomycin C feeders from the viewpoint of cost-effective burn care in developing countries. Copyright © 2015 Elsevier Ltd and ISBI. All rights reserved.

  7. Effect of salt on a thermosensitive mutant of Bacillus subtilis deficient in uracil and cell division

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, N; Nagai, K; Tamura, G

    1976-01-01

    A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp/sub 2/ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese's (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-/sup 14/C into RNA still continued even after the incorporation of N-acetyl-/sup 3/H-D-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48/sup 0/C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48/sup 0/C. This mutant, ts-42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back to 37/sup 0/C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.

  8. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

    Science.gov (United States)

    Gerashchenko, O L; Zhuravel, E V; Skachkova, O V; Khranovska, N N; Filonenko, V V; Pogrebnoy, P V; Soldatkina, M A

    2013-06-01

    The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

  9. Gallic acid induced apoptotic events in HCT-15 colon cancer cells

    Science.gov (United States)

    Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

    2016-01-01

    AIM: To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. METHODS: The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2’,7’-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. RESULTS: MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells. PMID:27099438

  10. The Potential Role of Recombinant Hematopoietic Colony-Stimulating Factors in Preventing Infections in the Immunocompromised Host

    Directory of Open Access Journals (Sweden)

    James Rusthoven

    1991-01-01

    Full Text Available Hematopoietic colony-stimulating factors coordinate the proliferation and maturation of bone marrow and peripheral blood cells during normal hematopoiesis. Most of these factors are now available as recombinant human colony-stimulating factors, and preclinical and clinical testing is proceeding rapidly. Granulocyte and granulocyte/macrophage colony-stimulating factors have been the most extensively studied to date. In human clinical trials, granulocyte colony-stimulating factor improves neutrophil counts and function, reduces episodes of febrile neutropenia, improves neutrophil recovery after disease- or treatment-induced myelosuppression, and reduces the number of serious infections in several neutropenic disease states. Granulocyte/macrophage colony-stimulating factor has similar biological properties but may also improve eosinophil proliferation and function, and platelet cell recovery after myelotoxic bone marrow injury, Interleukin-1 boosts the effects of granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor, but also may promote the resolution of established infections in conjunction with antibiotics. The therapeutic realities and future therapeutic implications of these agents for the therapy of infections, cancer and hemopoietic disorders are discussed.

  11. Mobilization of hematopoietic stem cells with highest self-renewal by G-CSF precedes clonogenic cell mobilization peak.

    Science.gov (United States)

    Winkler, Ingrid G; Wiercinska, Eliza; Barbier, Valerie; Nowlan, Bianca; Bonig, Halvard; Levesque, Jean-Pierre

    2016-04-01

    Harvest of granulocyte colony-stimulating factor (G-CSF)-mobilized hematopoietic stem cells (HSCs) begins at day 5 of G-CSF administration, when most donors have achieved maximal mobilization. This is based on surrogate markers for HSC mobilization, such as CD34(+) cells and colony-forming activity in blood. However, CD34(+) cells or colony-forming units in culture (CFU-C) are heterogeneous cell populations with hugely divergent long-term repopulation potential on transplantation. HSC behavior is influenced by the vascular bed in the vicinity of which they reside. We hypothesized that G-CSF may mobilize sequentially cells proximal and more distal to bone marrow venous sinuses where HSCs enter the blood. We addressed this question with functional serial transplantation assays using blood and bone marrow after specific time points of G-CSF treatment in mice. We found that in mice, blood collected after only 48 hours of G-CSF administration was as enriched in serially reconstituting HSCs as blood collected at 5 days of G-CSF treatment. Similarly, mobilized Lin(-)CD34(+) cells were relatively enriched in more primitive Lin(-)CD34(+)CD38(-) cells at day 2 of G-CSF treatment compared with later points in half of human donors tested (n = 6). This suggests that in both humans and mice, hematopoietic progenitor and stem cells do not mobilize uniformly according to their maturation stage, with most potent HSCs mobilizing as early as day 2 of G-CSF. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  12. Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

    International Nuclear Information System (INIS)

    Pilgaard, L.; Lund, P.; Duroux, M.; Lockstone, H.; Taylor, J.; Emmersen, J.; Fink, T.; Ragoussis, J.; Zachar, V.

    2009-01-01

    Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

  13. Comparison between the radiosensitivity of human, mouse and chicken fibroblast-like cells using short-term endpoints

    International Nuclear Information System (INIS)

    Diatloff-Zito, C.; Loria, E.; Maciera-Coelho, A.; Deschavanne, P.J.; Malaise, E.P.

    1981-01-01

    A comparative study has been made of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells. (author)

  14. Relation between number of hemopoietic stem cells in newborn mice and their radiosensitivity

    International Nuclear Information System (INIS)

    Sutter, T.; Maes, J.; Gerber, G.B.; Leonard, A.

    1985-01-01

    Fractionation of a radiation exposure causes greater damage in newborn mice than a single application since it induces radioresistant foetal hemopoietic stem cells to differentiate prematurely to more radiosensitive adult ones. In the present investigation, it was studied whether other agents that give rise to extensive stem cell destruction also lead to such a change in radiosensitivity. Indeed, treatment with cytostatic drugs which reduces the number of spleen colony forming units (CFU-s) and total cells also diminished the D 0 value of the surviving cells 3 days later. Adriamycin was most effective in causing damage to hemopoietic stem cells and in inducing micronuclei in bone marrow; it also had the most marked action on the D 0 of the surviving stem cells. (orig.)

  15. Capacity of ultraviolet-induced DNA repair in human glioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Hiroji

    1987-04-01

    A DNA repair abnormality is likely related to an increased incidence of neoplasms in several autosomal recessive diseases such as xeroderma pigmentosum, Fanconi's anemia, Bloom's syndrome and ataxia telangiectasia. In human glioma cells, however, there are only a few reports on DNA repair. In this study, an ultraviolet (UV)-induced DNA repair was examined systematically in many human glioma cells. Two human malignant glioma cell lines (MMG-851, U-251-MG) and 7 human glioma cell strains (4, benign; 3, malignant) of short term culture, in which glial fibrillary acidic protein (GFAP) staining were positive, were used. To investigate the capacity of DNA repair, UV sensitivity was determined by colony formation; excision repair by autoradiography and Cytosine Arabinoside (Ara-C) assay; and post-replication repair by the joining rate of newly synthesized DNA. As a result, the colony-forming abilities of malignant glioma cell lines were lower than those of normal human fibroblasts, but no difference was found between two malignant glioma cell lines. The excision repair of the malignant group (2 cell lines and 3 cell strains) was apparently lower than that of the benign group (4 cell strains). In two malignant glioma cell lines, the excision repair of MMG-851 was lower than that of U-251-MG, and the post-replication repair of MMG-851 was higher than that of U-251-MG. These results were considered to correspond well with colony-forming ability. The results indicate that there are some differences in each human malignant glioma cell in its UV-induced DNA repair mechanism, and that the excision repair of the malignant glioma cells is apparently lower than that of the benign glioma cells. These findings may be useful for diagnosis and treatment.

  16. Intrinsic radiosensitivity and PLD repair in osteosarcoma cell lines

    International Nuclear Information System (INIS)

    Sugimoto, M.; Toguchida, J.; Kotoura, Y.; Yamamuro, T.; Utsumi, H.

    1992-01-01

    The response to radiation of seven osteosarcoma cell lines was analysed by in vitro colony-forming assay and compared with that of eight human fibroblast strains. The values of D 0 , the surviving fraction after 2 Gy (S2Gy), and the mean inactivation dose (D-bar) of osteosarcoma cells in log-phase culture were significantly higher than those of fibroblast strains (p<0.01). PLD (potentially lethal damage) repair of osteosarcoma cells evaluated in the plateau phase of growth showed great variation for enhancement of survival, although all of the values were maximised within 12 h after irradiation. In the osteosarcoma, intrinsic radiosensitivity in vitro reflected the clinical response to radiation. However, the capacity for PLD repair might not be a good indicator for predicting the results of radiation therapy. (author)

  17. Radiosensitizing effect of epothilone B on human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Baumgart, T.; Kriesen, S.; Hildebrandt, G.; Manda, K. [Univ. of Rostock (Germany). Dept. of Radiotherapy and Radiation Oncology; Klautke, G.; Fietkau, R. [Friedrich-Alexander-Univ. Erlangen-Nuernberg, Erlangen (Germany). Dept. of Radiation Oncology; Kuznetsov, S.A.; Weiss, D.G. [Univ. of Rostock (Germany). Inst. of Biological Sciences, Cell Biology, and Biosystems Technology

    2012-02-15

    A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a {gamma}H2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC{sub 50} values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The {gamma}H2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel. (orig.)

  18. Action of caffeine on x-irradiated HeLa cells. VII. Evidence that caffeine enhances expression of potentially lethal radiation damage

    International Nuclear Information System (INIS)

    Beetham, K.L.; Tolmach, L.J.

    1984-01-01

    HeLa cells irradiated with 2 Gy of 220-kV X rays suffer a 60-70% loss of colony-forming ability which is increased to 90% by postirradiation treatment with 10 mM caffeine for 6 hr. The detailed postirradiation patterns of cell death and sister-cell fusion in such cultures and in cultures in which the colony-forming ability was brought to about the same level by treatment with a larger (4 Gy) X-ray dose alone or by longer (48 hr) treatment with 10 mM caffeine alone were recorded by time-lapse cinemicrography. Because the patterns of cell death and fusion differ radically in irradiated and in caffeine-treated cultures, the response of the additional cells killed by the combined treatment can be identified as X-ray induced rather than caffeine induced. The appearance of cultures after several days of incubation confirms the similarity of the post-treatment patterns of proliferation in cultures suffering enhanced killing to those occurring in cultures treated with larger doses of X rays alone. It is concluded that x rays do not sensitize cells to caffeine, but rather that caffeine enhanced the expression of potentially lethal radiation-induced damage

  19. Low/Negative Expression of PDGFR-α Identifies the Candidate Primary Mesenchymal Stromal Cells in Adult Human Bone Marrow

    DEFF Research Database (Denmark)

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra

    2014-01-01

    Human bone marrow (BM) contains a rare population of nonhematopoietic mesenchymal stromal cells (MSCs), which are of central importance for the hematopoietic microenvironment. However, the precise phenotypic definition of these cells in adult BM has not yet been reported. In this study, we show...... exhibited high levels of genes associated with mesenchymal lineages and HSC supportive function. Moreover, lin(-)/CD45(-)/CD271(+)/CD140a(low/-) cells effectively mediated the ex vivo expansion of transplantable CD34(+) hematopoietic stem cells. Taken together, these data indicate that CD140a is a key...... that low/negative expression of CD140a (PDGFR-α) on lin(-)/CD45(-)/CD271(+) BM cells identified a cell population with very high MSC activity, measured as fibroblastic colony-forming unit frequency and typical in vitro and in vivo stroma formation and differentiation capacities. Furthermore, these cells...

  20. Sodium butyrate stimulates cellular recovery from UV damage in xeroderma pigmentosum cells belonging to complementation group F

    International Nuclear Information System (INIS)

    Nishigori, Chikako; Takebe, Hiraku

    1987-01-01

    Possible stimulation of the DNA repair capacity by sodium butyrate in normal and xeroderma pigmentosum (XP) cells was investigated. XP cells belonging to the complementation group F showed considerable stimulation of DNA repair by sodium butyrate in terms of both the amount of unscheduled DNA synthesis (UDS) and the colony-forming ability after UV irradiation. UDS in XP cells belonging to the complementation group A was not enhanced, while normal cells showed slight enhancement, but less than that of XP F cells. In XP A, XP C, and normal cells, sodium butyrate treatment enhanced the killing effect of UV irradiation. The residual repair capacity in XP F cells appeared to be stimulated by sodium butyrate. (author)

  1. Cancer stem cells in solid tumors: elusive or illusive?

    Directory of Open Access Journals (Sweden)

    Lehrach Hans R

    2010-05-01

    Full Text Available Abstract During the past years in vivo transplantation experiments and in vitro colony-forming assays indicated that tumors arise only from rare cells. These cells were shown to bear self-renewal capacities and the ability to recapitulate all cell types within an individual tumor. Due to their phenotypic resemblance to normal stem cells, the term "cancer stem cells" is used. However, some pieces of the puzzle are missing: (a a stringent definition of cancer stem cells in solid tumors (b specific markers that only target cells that meet the criteria for a cancer stem cell in a certain type of tumor. These missing parts started an ongoing debate about which is the best method to identify and characterize cancer stem cells, or even if their mere existence is just an artifact caused by the experimental procedures. Recent findings query the cancer stem cell hypothesis for solid tumors itself since it was shown in xenograft transplantation experiments that under appropriate conditions tumor-initiating cells are not rare. In this review we critically discuss the challenges and prospects of the currently used major methods to identify cancer stem cells. Further on, we reflect the present discussion about the existence of cancer stem cells in solid tumors as well as the amount and characteristics of tumor-initiating cells and finally provide new perspectives like the correlation of cancer stem cells and induced pluripotent cells.

  2. Closing the manufacturing process of dendritic cell vaccines transduced with adenovirus vectors.

    Science.gov (United States)

    Gulen, Dumrul; Abe, Fuminori; Maas, Sarah; Reed, Elizabeth; Cowan, Kenneth; Pirruccello, Samuel; Wisecarver, James; Warkentin, Phyllis; Northam, Matt; Turken, Orhan; Coskun, Ugur; Senesac, Joe; Talmadge, James E

    2008-12-20

    Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6+3.2% (mean+SEM), to an average frequency of 79.5+4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3+1.0x10(7) cells/ml, a viability of 93.8+2.2%, a purity of 88.9+3.3% and a total yield of 8.5+1.4x10(8) DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46+8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9x10(6) DCs/ml at an Ad5CMV-p53: DC ratio of 20

  3. Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells

    International Nuclear Information System (INIS)

    Boyle, J.M.

    1985-01-01

    Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [ 3 H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [ 14 C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, the authors have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities

  4. Long-term injury in B-lymphocyte precursor cells in repeatedly-irradiated mice

    International Nuclear Information System (INIS)

    Hendry, J.H.; Clarke, D.; Testa, N.; Kimber, J.

    1984-01-01

    Mice irradiated with 4 doses of 4,5 Gy X-rays at 3-week intervals, demonstrated long-term proliferative defects in B lymphocytes. There was a reduced mitogenic response to bacterial polysaccharide (30%), a lower concentration (35%) of B-lymphocyte colony-forming cells (BL-CFC) in agar with an increased proportion of clusters (x2), and a reduced concentration (30%) of plaque-forming cells. Grafts of thymocytes were able to restore the levels of BL-CFC in the short term, but in the long term large grafts of femoral marrow cells were much better in restoring the numbers of BL-CFC. The reduced mitogenesis (25%) of splenocytes by concanavalin A and the diminished number of plaque-forming cells, may suggest persistent injury in T-B cell cooperation

  5. Apoptosis induced by cold shock in vitro is dependent on cell growth phase.

    Science.gov (United States)

    Soloff, B L; Nagle, W A; Moss, A J; Henle, K J; Crawford, J T

    1987-06-15

    Chinese hamster V79 fibroblast cells were exposed to brief periods of cold but non-freezing temperatures at different points on the population growth curve. Upon rewarming, cells at the transition from logarithmic to stationary growth exhibited apoptosis (programmed cell death). Cells in other stages of growth, or after reentry into logarithmic growth by refeeding, did not exhibit apoptosis. Apoptosis was expressed by marked cytoplasmic blebbing, by a characteristic non-random fragmentation of DNA into nucleosomal-sized pieces, and by loss of colony-forming ability. The data suggest that cold shock served as a stimulus for susceptible cells to undergo apoptosis. Thus, the experiments describe a new in vitro system for studying the mechanisms of apoptosis.

  6. Heparin concentration is critical for cell culture with human platelet lysate.

    Science.gov (United States)

    Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

    2013-09-01

    Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  7. Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

    Science.gov (United States)

    Yasui, T; Mabuchi, Y; Toriumi, H; Ebine, T; Niibe, K; Houlihan, D D; Morikawa, S; Onizawa, K; Kawana, H; Akazawa, C; Suzuki, N; Nakagawa, T; Okano, H; Matsuzaki, Y

    2016-02-01

    Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies. © International & American Associations for Dental Research 2015.

  8. Pattern changes in quantitative and qualitative markers of hematopoietic stem cells during acute and chronic exposure to Sr"9"0 isotope in cell culture

    International Nuclear Information System (INIS)

    Russu, Yi.Z.; Byil'ko, D.Yi.; Byil'ko, N.M.; Rodyionova, N.K.

    2015-01-01

    To study the condition of stem cells and their immediate progenitors we implemented cell culture methodology in vivo in gel diffusion capsules with subsequent analysis of the colonies and clusters. On the basis of experiments it was established that long-term effects of incorporated "9"0 Sr isotope leads to significant disturbances in the hematopoietic system and in particular, revealing changes in hematological parameters of irradiated animals such as the appearance of circulating progenitor cells in peripheral blood, reducing the colony-forming efficiency of the bone marrow derived progenitor cells, as well as quantitative and qualitative changes in the clones. Indices confirm the connection of the detected effects in individuals exposed to ionizing radiation described in the earlier publications and can serve as basis for developing criteria for the formation of risk groups among people exposed to "9"0 Sr

  9. Regeneration of hemopoietic precursor cells in spleen organ cultures from irradiated mice: influence of genotype of cells injected and of the spleen microenvironment

    International Nuclear Information System (INIS)

    von Melchner, H.; Lieschke, G.J.

    1981-01-01

    The regeneration of hemopoietic precursor cells (colony-forming cells, CFC) was monitored in spleen organ cultures from lethally irradiated mice injected with 10(7) normal syngeneic or allogeneic bone marrow cells. The important role of the microenvironment in supporting hemopoiesis was confirmed by the failure of mutant S1/S1d spleens to support CFC regeneration in organ cultures. However, the extent and quality of the CFC regeneration was clearly dependent on the genetic properties of the injected cells. Evidence for this was obtained from the regeneration patterns of various CFC types in organ cultured spleens derived from different mouse donor-recipient strain combinations (CBA/CBA, CBA/C57BL, CBA/BALB/c, C57BL/C57BL, C57BL/CBA, C57BL/BALB/c) that maintained the differences in the bone marrow frequency of various CFC types characteristic of the donor strain

  10. Sigma-2 ligands and PARP inhibitors synergistically trigger cell death in breast cancer cells

    International Nuclear Information System (INIS)

    McDonald, Elizabeth S.; Mankoff, Julia; Makvandi, Mehran; Chu, Wenhua; Chu, Yunxiang; Mach, Robert H.; Zeng, Chenbo

    2017-01-01

    The sigma-2 receptor is overexpressed in proliferating cells compared to quiescent cells and has been used as a target for imaging solid tumors by positron emission tomography. Recent work has suggested that the sigma-2 receptor may also be an effective therapeutic target for cancer therapy. Poly (ADP-ribose) polymerase (PARP) is a family of enzymes involved in DNA damage response. In this study, we looked for potential synergy of cytotoxicity between PARP inhibitors and sigma-2 receptor ligands in breast cancer cell lines. We showed that the PARP inhibitor, YUN3-6, sensitized mouse breast cancer cell line, EMT6, to sigma-2 receptor ligand (SV119, WC-26, and RHM-138) induced cell death determined by cell viability assay and colony forming assay. The PARP inhibitor, olaparib, sensitized tumor cells to a different sigma-2 receptor ligand SW43-induced apoptosis and cell death in human triple negative cell line, MDA-MB-231. Olaparib inhibited PARP activity and cell proliferation, and arrested cells in G2/M phase of the cell cycle in MDA-MB-231 cells. Subsequently cells became sensitized to SW43 induced cell death. In conclusion, the combination of sigma-2 receptor ligands and PARP inhibitors appears to hold promise for synergistically triggering cell death in certain types of breast cancer cells and merits further investigation. - Highlights: • PARPi, YUN3-6 and olaparib, and σ2 ligands, SV119 and SW43, were evaluated. • Mouse and human breast cancer cells, EMT6 and MDA-MB-231 respectively, were used. • YUN3-6 and SV119 synergistically triggered cell death in EMT6 cells. • Olaparib and SW43 additively triggered cell death in MDA-MB-231 cells. • Olaparib arrested cells in G2/M in MDA-MB-231 cells.

  11. Mammalian-enabled (MENA) protein enhances oncogenic potential and cancer stem cell-like phenotype in hepatocellular carcinoma cells.

    Science.gov (United States)

    Hu, Kunpeng; Huang, Pinzhu; Luo, Hui; Yao, Zhicheng; Wang, Qingliang; Xiong, Zhiyong; Lin, Jizong; Huang, He; Xu, Shilei; Zhang, Peng; Liu, Bo

    2017-08-01

    Mammalian-enabled (MENA) protein is an actin-regulatory protein that influences cell motility and adhesion. It is known to play a role in tumorigenicity of hepatocellular carcinoma (HCC) but the underlying molecular mechanism remains unknown. This study aimed to investigate the oncogenic potential of MENA and its capacity to regulate cancer stem cell (CSC)-like phenotypes in HCC cells. Real-time-PCR and western blot were used to assess mRNA and protein levels of target genes in human HCC tissue specimens and HCC cell lines, respectively. Stable MENA-overexpressing HCC cells were generated from HCC cell lines. Transwell cell migration and colony formation assays were employed to evaluate tumorigenicity. Ectopic expression of MENA significantly enhanced cell migration and colony-forming ability in HCC cells. Overexpression of MENA upregulated several hepatic progenitor/stem cell markers in HCC cells. A high MENA protein level was associated with high mRNA levels of MENA, CD133, cytokeratin 19 (CK19), and epithelial cell adhesion molecule (EpCAM) in human HCC tissues. Overexpression of MENA enhanced epithelial-to-mesenchymal transition (EMT) markers, extracellular signal-regulated kinases (ERK) phosphorylation, and the level of β-catenin in HCC cells. This study demonstrated that overexpression of MENA in HCC cells promoted stem cell markers, EMT markers, and tumorigenicity. These effects may involve, at least partially, the ERK and β-catenin signaling pathways.

  12. Radiation-induced changes in nucleoid halo diameteres of aerobic and hypoxic SF-126 human brain tumor cells

    International Nuclear Information System (INIS)

    Wang, J.; Basu, H.S.; Hu, L.; Feuerstein, B.G.; Deen, D.F.

    1995-01-01

    Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propodium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similiar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors. 22 refs., 5 figs

  13. Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

    DEFF Research Database (Denmark)

    Jantzen, Kim; Roursgaard, Martin; Madsen, Claus Desler

    2012-01-01

    Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stress-related damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung...... treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine...... relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells....

  14. Knock-down of NDRG2 sensitizes cervical cancer Hela cells to cisplatin through suppressing Bcl-2 expression

    International Nuclear Information System (INIS)

    Liu, Junye; Guo, Guozhen; Yang, Le; Zhang, Jian; Zhang, Jing; Chen, Yongbin; Li, Kangchu; Li, Yurong; Li, Yan; Yao, Libo

    2012-01-01

    NDRG2, a member of N-Myc downstream regulated gene family, plays some roles in cellular stress, cell differentiation and tumor suppression. We have found that NDRG2 expression in cervical cancer Hela cells increases significantly upon stimulation with cisplatin, the most popular chemotherapeutic agent currently used for the treatment of advanced cervical cancer. This interesting phenomenon drove us to evaluate the role of NDRG2 in chemosensitivity of Hela cells. In the present study, RNA interference was employed to down-regulate NDRG2 expression in Hela cells. RT-PCR and Western blot were used to detect expression of NDRG2, Bcl-2 and Bax in cancer cells. Real-time PCR was applied to detect miR-15b and miR-16 expression levels. Drug sensitivity was determined with MTT assay. Cell cloning efficiency was evaluated by Colony-forming assay. Apoptotic cells were detected with annexin V staining and flow cytometry. In vitro drug sensitivity assay revealed that suppression of NDRG2 could sensitize Hela cells to cisplatin. Down-regulation of NDRG2 didn’t influence the colony-forming ability but promoted cisplatin-induced apoptosis of Hela cells. Inhibition of NDRG2 in Hela cells was accompanied by decreased Bcl-2 protein level. However, Bcl-2 mRNA level was not changed in Hela