Macrophage colony stimulating factor prevents NMDA-induced neuronal death in hippocampal organotypic cultures

Macrophage colony stimulating factor (M-CSF) and its receptor are up-regulated in the brain in Alzheimer's disease (AD), in transgenic mouse models for AD, and experimental models for traumatic and ischemic brain injury. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. We examined the role of M-CSF in excitotoxic neuronal cell death in organotypic hippocampal cultures. NMDA treatment induced neuronal apoptosis and caspase-3 activation in organotypic hippocampal cultures, whereas treatment with M-CSF protected hippocampal neurons from NMDA-induced apoptosis. Caspase-3 activation was inhibited by M-CSF treatment to the same degree as with the caspase inhibitor Z-VAD-FMK. These results suggest that M-CSF has neuroprotective properties through inhibition of caspase-3 that could promote neuronal survival after excitotoxic insult. The role of M-CSF in neurological disease should be reevaluated as a microglial activator with potentially neuroprotective effects.     

Cytotoxic T cell immunity against the non-immunogenic, murine, hepatocellular carcinoma Hepa1-6 is directed towards the novel alternative form of macrophage colony stimulating factor

Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells were transduced with the membrane form of macrophage colony stimulating factor (mM-CSF). When mM-CSF transduced Hepa1-6 cells were injected subcutaneously into mice, these cells did not form tumors. The spleens of these immunized mice contained cytotoxic CD8⁺ T lymphocytes (CTL) that killed the unmodified Hepa1-6 cells. We show that the alternative form of macrophage colony stimulating factor (altM-CSF) induced CTL-mediated immunity against Hepa1-6 cells. AltM-CSF is restricted to the H-2D^b allele. CTLs killed RMA-S cells loaded with exogenous altM-CSF peptide. Vaccination of mice with dendritic cells pulsed with the altM-CSF peptide stimulated anti-Hepa1-6 CTLs. Hyper-immunization of mice with mM-CSF Hepa1-6 cells showed inflammation of the liver and kidneys. Although altM-CSF was expressed within liver and kidney cells, its intensity was lower than Hepa1-6 cells. AltM-CSF was detected within the human HepG2 cell line. These studies suggest that altM-CSF may be a tumor antigen for HCC.     

重组人粒细胞-巨噬细胞集落刺激因子包涵体提取、复性和纯化的研究

由基因工程大肠杆菌表达的重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）以包涵体的形式存在于细胞中,通过破菌、洗涤获得包涵体,再经过溶解、凝胶过滤、复性、疏水和离子交换柱导析得到了均一的产品,经高压液相和ＳＤＳ－ＰＡＧＥ电泳测定纯度均大于９８％,ｒｈＧＭ－ＣＳＦ的比活为３．２×１０＾７ＩＵ／ｍｇ,纯化获得的ｒｈＧＭ－ＣＳＦ为一酸性蛋白,等电点约为５．２,ＮＨ２－末端有２０个氨基酸序列测定结果     

Pulsed ultrasound promotes melanoblast migration through upregulation of macrophage colony‐stimulating factor/focal adhesion kinase autocrine signaling and paracrine mechanisms

Repigmentation of vitiliginous lesions relies on the proliferation and migration of melanoblasts from hair follicles to the epidermis. Pulsed ultrasound has been demonstrated to have stimulatory effects on cell proliferation and migration and has been applied clinically to enhance tissue repair. To clarify the biologic effects and signaling mechanisms of pulsed ultrasound on melanoblast proliferation and migration, two melanoblast cell lines, the undifferentiated NCCmelb4 cells and the differentiated NCCmelan5 cells, were examined. We demonstrated that pulsed ultrasound increased cell migration in a dose‐dependent manner without altering cell proliferation. Pulsed ultrasound enhanced autocrine secretion of macrophage colony‐stimulating factor (M‐CSF), which subsequently activated the focal adhesion kinase (FAK) pathway to promote melanoblast migration. Furthermore, conditioned medium from mouse embryonic fibroblasts NIH 3T3 and primary human keratinocytes treated with pulsed ultrasound could stimulate melanoblast migration through a paracrine effect. Our results provide a novel mechanism to promote migration of melanoblasts by pulsed ultrasound stimulation.     

Proteomics screening of molecular targets of granulocyte colony stimulating factor in the mouse brain and PC12 cell line

Aims

Granulocyte colony stimulating factor (G-CSF), a new neuroprotective agent, binds to its specific receptors in the brain. In this study we hypothesized that at least a part of G-CSF's neuroprotective effect may be mediated through its interaction with other proteins in the brain.

Main methods

Using an immunoprecipitation (IP) kit, at first the antibody of G-CSF was covalently crosslinked to protein A/G agarose. Then the mouse brain or PC12 cell lysate mixed with G-CSF was added to the agarose beads plus antibody. After immunoaffinity isolation of target proteins, gel electrophoresis was performed and protein bands were identified using MALDI-TOF/TOF and MASCOT software.

Key findings

Our data show that G-CSF physically binds to cellular proteins like sodium/potassium-transporting ATPase, beta actin, aldehyde dehydrogenase, regucalcin and glutathione-s-transferase. These proteins are involved in membrane transportation, cell structure, signal transduction, enzymes involve in calcium related cell signaling and redox homeostasis.

Significance

Interaction of G-CSF with these proteins can explain some of its pharmacological effects in the CNS.     

hGM-CSF基因穿梭表达载体的构建及其在鱼腥藻7120中的克隆

人粒-巨噬细胞集落刺激因子(hGM-CSF)作为一种造血生长因子,能够刺激T细胞和巨噬细胞增殖、成熟和分化,具有极其重要的免疫调解功能.本研究运用PCR方法,从质粒pAG-MT-8中克隆该基因,并在其5′端添加有利于在蓝藻细胞中高效表达的SD序列,然后插入到表达载体(pRL-439)强启动子PpsbA的下游,进一步与穿梭表达载体pDC-08相连构建成穿梭表达载体pDC-GM.利用三亲接合转移方法将该穿梭表达载体(pDC-GM)转入丝状鱼腥藻7120,通过相应抗生素筛选后得到能稳定遗传的转基因藻.以该转基因藻的基因组DNA为模板进行PCR检测,结果表明hGM-CSF基因已转入鱼腥藻7120.这是首次尝试把蓝藻作为制备重组hGM-CSF的新宿主,具有潜在的经济价值和社会效益.     

Innate immune properties of the immortalized macrophage cell line I-9.5

Summary A colony stimulating factor-1-dependent macrophage cell line, I-9.5, originally derived from a BALB/c splenic macrophage colony, was maintained in culture and examined for the expression of certain properties key to its innate immune function. Chemotaxis, phagocytosis, and superoxide release were assessed in this cell line and compared to either freshly isolated elicited murine peritoneal or splenic macrophages from BALB/c mice. Three separate experiments indicated that I-9.5 displayed comparable phagocytosis of¹⁴C-radio-labeledStaphylococus aureus and similar levels of superoxide release in response to opsonized zymosan. I-9.5, however, demonstrated impaired chemotaxis toward the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, and displayed impaired random migration in response to a balanced salt solution. This observation suggests that I-9.5 may serve as an important model for elucidating the structural and molecular correlates of chemotaxis.     

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

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

Chemiluminescence sandwich enzyme immunoassay for determination of human granulocyte colony stimulating factor (G-CSF)

A chemiluminescence sandwich enzyme immunoassay, using a glucose oxidase (GO) label, was developed for detecting attomole amounts of human granulocyte colony stimulating factor (G-CSF). Purified goat F(ab′)₂ immobilized on a bead and purified goat Fab′ labelled with GO were selected in combination with a chemiluminescent detection system comprising luminol and ferricyanide. The detection limits for G-CSF were 4amol/assay (1 pg/mL) in buffer solution and 10 amol/assay (2.5 pg/mL) in human serum. Coefficients of variation within assay and between assay ranged from 5.5% to 7.8% and from 3.4% to 16.0%, respectively. The G-CSF content of serum from normal healthy individuals was measurable using this method. G-CSF in 24 normal human sera showed a mean value of 19.3 pg/mL and ranged from 3.6 to 83.0 pg/mL.     

In vitro modulation of canine polymorphonuclear leukocyte function by granulocyte-macrophage colony stimulating factor

Granulocyte-macrophage colony stimulating factor (GMCSF) promotes the growth of granulocytes and macrophages from undifferentiated bone marrow cells and modulates the oxidative responses of polymorphonuclear leukocytes (PMN) to endogenous chemoattractants. We found that,in vitro, naturally occurring glycolsylated human GMCSF does not disturb the resting canine PMN membrane potential, may attentuate PMN oxidative responses to PMA, and is, to a small degree, chemotaxigenic. GMCSF, however, inhibits PMN chemotaxis to zymosanactivated plasma (ZAP). Compared to temperature controls, GMCSF (1-100 U/ml) produced up to 1.5-fold increases in H₂O₂ production after 15 minutes, while phorbol myristate acetate (PMA) treated cells increased H₂O₂ production 8–12-fold after 15 minutes. Preincubation of cells with GMCSF (1–100 U/ml) prior to PMA stimulation significantly reduced the H₂O₂ levels induced by PMA. H202 production was inhibited up to 15% after 15 minutes of GMCSF preincubation and up to 40% after 60 minutes of preincubation. As a chemotaxigenic agent, GMCSF (10–1000 U/ml) was able to elicit 49%–102% increases in quantitative cellular migration, compared to random migration. Total cellular chemotaxis to GMCSF was < 30% of the response to ZAP. Preincubation of PMNs with GMCSF for 15 minutes significantly inhibited ZAP-induced cellular migration. Human GMCSF does not appear to activate canine PMNin vitro and may actually down-regulate PMN inflammatory responses.Supported by the Armed Forces Radiobiology Research Institute, Defense Nuclear Agency, under work unit No. 00082. Views presented in this paper are those of the authors; no endorsement by the Defense Nuclear Agency has been given or should be inferred. Research was conducted according to the principles enunciated in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.     

Granulocyte-macrophage colony stimulating factor: Evaluation of biopharmaceutical formulations by stability-indicating RP-LC method and bioassay

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a cytokine that regulates the proliferation and differentiation of hematopoietic cells and activates granulocytes and macrophages. A reversed-phase liquid chromatography (RP-LC) method was validated for the assessing of the stability of non-glycosylated recombinant rhGM-CSF (Molgramostim) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C₄ column (250 mm × 4.6 mm i.d.), maintained at 45 °C. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA in acetonitrile, run at a flow rate of 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 29.2 min, and was linear over the concentration range of 2–300 μg/mL (r² = 0.9992). Specificity was established in degradation studies. Moreover, the in vitro cytotoxicity test of the degraded products showed significant differences (p < 0.05). The method was applied to the assessment of rhGM-CSF and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay. It is concluded that the employment of RP-LC in conjunction with current methods allows a great improvement in monitoring stability, quality control and thereby assures the therapeutic efficacy.     

Production of transgenic recloned piglets harboring the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene from porcine fetal fibroblasts by nuclear transfer

We used nuclear transfer (NT) to develop transgenic female pigs harboring goat beta-casein promoter/human granulocyte-macrophage colony stimulating factor (hGM-CSF). The expression of hGM-CSF was specific to the mammary gland, and the glycosylation-derived size heterogeneity corresponded to that of the native human protein. Although various cell types have been used to generate cloned animals, little is currently known about the potential use of fibroblasts derived from a cloned fetus as donor cells for nuclear transfer. The developmental potential of porcine cloned fetal fibroblasts transfected with hGM-CSF was evaluated in the present study. Cloned fetal fibroblasts were isolated from a recipient following the transplantation of NT embryos. The cells were transfected with both hGM-CSF and the neomycin resistance gene in order to be used as donor cells for NT. Reconstructed embryos were implanted into six sows during estrus; two of the recipient sows delivered seven healthy female piglets with the hGM-CSF gene (confirmed with PCR and fluorescent in situ hybridization) and microsatellite analysis confirmed that the clones were genetically identical to the donor cells. The expression of hGM-CSF was strong in the mammary glands of a transgenic pig that died a few days prior to parturition (110 d after AI). These results demonstrated that somatic cells derived from a cloned fetus can be used to produce recloned and transgenic pigs.     

Structural comparison of recombinant human macrophage colony stimulating factor beta and a partially reduced derivative using hydrogen deuterium exchange and electrospray ionization mass spectrometry

Hydrogen deuterium exchange, monitored by electrospray ionization mass spectrometry, has been employed to characterize structural features of a derivative of recombinant human macrophage colony stimulating factor beta (rhm-CSFbeta) in which two of the nine disulfide bridges (Cys157/Cys159-Cys'157/Cys'159) were selectively reduced and alkylated. Removal of these two disulfide bridges did not affect the biological activity of the protein. Similarities between CD and fluorescence spectra for rhm-CSFbeta and its derivative indicate that removing the disulfide bonds did not strongly alter the overall three-dimensional structure of rhm-CSFbeta. However, differences between deuterium exchange data of the intact proteins indicate that more NHs underwent fast deuterium exchange in the derivative than in rhm-CSFbeta. Regions located near the disulfide bond removal site were shown to exhibit faster deuterium exchange behavior in the derivative than in rhm-CSFbeta.     

Generation of human innate immune responses towards membrane macrophage colony stimulating factor (mM-CSF) expressing U251 glioma cells within immunodeficient (NIH-nu/beige/xid) mice

The response of human peripheral blood mononuclear cells (PBMC) to cloned human HLA-A2+ U251 glioma cells (U251-2F11/TK) expressing membrane macrophage colony stimulating factor (mM-CSF) was investigated in vitro and in vivo. Enriched human monocytes derived from cancer patients produced a respiratory burst following 20min of interaction with mM-CSF expressing U251 glioma cells. This respiratory burst response was not observed in the enriched human monocytes following similar exposure to the viral vector control U251 (U251-VV) cells. Reactive oxygen species such as H(2)O(2) and HOCl produced death of the U251 cells. The U251-2F11/TK cells failed to grow in severely compromised combined immunodeficient (NIH-bg-nu-xidBR) mice that were depleted of murine monocyte/macrophages then reconstituted with human HLA-A2+ PBMC. Reactive oxygen species (ROS) were produced by PBMC, both in vitro and in vivo in response tomM-CSF expressing U251 cells. U251-2F11/TK cells failed to form subcutaneous tumors in macrophage depleted mice reconstituted with human PBMC; whereas, progressive growth of such tumors was observed with the U251-VV cells. U251-2F11/TK tumors formed if the initial inoculums of PBMC were depleted of monocytes. From this work it can be concluded that mM-CSF transduced U251-2F11/TK glioma cells can safely stimulate human innate immune responses in vivo.     

Oral administration of recombinant human granulocyte-macrophage colony stimulating factor expressed in rice endosperm can increase leukocytes in mice

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is used clinically to treat leucopenia typically caused by cancer chemotherapy or radiotherapy. This study used multiple strategies to obtain very high expression levels of OsrhGM-CSF (14 mug/seed) in rice endosperm. Electron micrographs of immunogold-labeled transgenic endosperm showed that rhGM-CSF was not only localized in protein bodies but was also distributed in the apoplast. A biological activity assay indicated that OsrhGM-CSF stimulated the growth of TF-1 cells in vitro. In addition, the transgene was used to effectively treat leucopenia by oral administration of the unprocessed transgenic grains. In cyclophosphamide-induced leucopenic mice, transgenic seeds produced a 27% (t = 0.021) gain in leukocytes after 14 days feeding. Even in non-leucopenic mice, leukocyte gain was 37% (t = 0.002) more than that of mice fed non-transgenic seeds. This study provides a novel approach to the use of oral unprocessed transgenic OsrhGM-CSF seeds to treat leucopenia.     

Mutation of macrophage colony stimulating factor (Csf1) causes osteopetrosis in the tl rat

Osteopetrosis results from a heterogeneous group of congenital bone diseases that display inadequate osteoclastic bone resorption. We recently mapped tl (toothless), a mutation that causes osteopetrosis in rats, to a genetic region predicted to include the rat Csf1 gene. In this study, we sequenced the coding sequence of the rat Csf1 gene to determine if a mutation in Csf1 could be responsible for the tl phenotype. Sequencing revealed a 10-base insertion in the coding sequence of mutant animals that produces a frameshift and generates a stop codon early in the mutant Csf1 coding sequence. The 41 amino acid polypeptide predicted to be produced from the Csf1 promoter would have only the first nine amino acids of the wild-type rat protein. These data suggest that osteopetrosis develops in tl/tl rats because they cannot produce functional mCsf, a growth factor required for osteoclast differentiation and activation.     

Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea

Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 μM Cd and/or 15.3 μM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.     

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

Intravenously injected granulocyte macrophage colony‐stimulating factor (GM‐CSF) has shown efficacy in Alzheimer's Disease (AD) and Parkinson's Disease (PD) animal studies and is undergoing clinical evaluation. The likely need for dosing of GM‐CSF to patients over months or years motivates pursuit of avenues for delivering GM‐CSF to circulation via oral administration. Flow cytometric screening of 37 yeast‐displayed GM‐CSF saturation mutant libraries revealed residues P12, H15, R23, R24, and K72 as key determinants of GM‐CSF's CD116 and CD131 GM‐CSF receptor (GM‐CSFR) subunit binding affinity. Screening combinatorial GM‐CSF libraries mutated at positions P12, H15, and R23 yielded variants with increased affinities toward both CD116 and CD131. Genetic fusion of GM‐CSF to human transferrin (Trf), a strategy that enables oral delivery of other biopharmaceuticals in animals, yielded bioactive wild type and variant cytokines upon secretion from cultured Human Embryonic Kidney cells. Surface plasmon resonance (SPR) measurements showed that all evaluated variants possess decreases in CD116 and CD131 binding K_D values of up to 2.5‐fold relative to wild type. Improved affinity led to increased in vitro bioactivity; the most bioactive variant, P12D/H15L/R23L, had a leukocyte proliferation assay EC₅₀ value 3.5‐fold lower than the wild type GM‐CSF/Trf fusion. These outcomes are important first steps toward our goal of developing GM‐CSF/Trf fusions as orally available AD and PD therapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:668–677, 2015     

粒细胞集落刺激因子对失代偿期肝硬化患者骨髓干细胞的动员效果及安全性观察

目的观察失代偿期肝硬化患者行自体骨髓干细胞移植前粒细胞集落刺激因子（G-CSF）对骨髓干细胞的动员效果及安全性。方法在51例失代偿期肝硬化患者行自体骨髓干细胞经肝动脉移植术前,连续2 d给予G-CSF 4μg/（kg·d）动员骨髓干细胞。抽取骨髓的当日化验血常规、肝肾功等指标;从患者髂后上棘抽取骨髓150-200 ml,分离收集骨髓单个核细胞并计数,应用流式细胞仪检测CD34＋细胞并计数,观察应用G-CSF期间不良反应的类型和发生率。患者治疗前后比较采用配对t检验进行统计学分析。结果G-CSF皮下注射后,外周血白细胞由术前（3.31±0.96）×10^9/L升至（11.35±1.92）×10^9/L（P〈0.01）,骨髓单个核细胞数（1.91±0.83）×10^9/kg,CD34＋细胞为（2.02±1.29）×10^7/kg;患者皮下注射后,发热率17.6﹪,体温最高38℃,停药后降至正常;腹部胀痛3例,四肢皮肤散发皮疹2例,均未给予特殊处理,2-3 d后恢复正常。结论给予G-CSF皮下注射后提取骨髓干细胞移植治疗失代偿期肝硬化的是一种临床确切有效的、安全的干细胞动员方法。