Stimulation of murine granulocyte macrophage-colony stimulating factor production by Pluronic F-68 and polyethylene glycol in transgenic Nicotiana tabacum cell culture

Pluronic F-68, PEG 8000, or PEG 20 000 added to cell suspension cultures of transgenic Nicotiana tabacum promoted cell growth and the production of the recombinant murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in a 5-l stirred tank bioreactor. The specific growth rates were enhanced from 0.27 d^–1 to 0.47 d^–1, 0.37 d^–1 and 0.4 d^–1 when Pluronic F-68, PEG 8000, or PEG 20 000 was added, respectively. The maximum cell density was also increased most to 13.6 g l^–1 when Pluronic F-68 was added (11.3 g l^–1 in the control culture). In terms of mGM-CSF production, PEG 8000 gave the greatest stimulation and with 2 g PEG 8000 l^–1, mGM-CSF increased from 1.6 to 6.6 ng ml^–1.     

A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli

Cytoplasmic expression is commonly used for production of recombinant human granulocyte macrophage-colony stimulating factor (rhGM-CSF) which most often comes with inclusion body formation. We expressed rhGM-CSF in periplasmic space of Escherichia coli and optimized its extraction by osmotic shock and purification by anion exchange chromatography. Our works show that MgCl2 at 2 mM in osmotic shock buffer improves extraction of the protein and reduces contamination with other proteins. To achieve a simplified purification procedure for rhGM-CSF, efforts were focused on the adjustment of pH of the buffers and application of proper concentration of salt. Following to measurement of the pI of 5.4 for rhGM-CSF by isoelectric focusing, the pH of dialysis buffer and buffers used in anion exchange chromatography were adjusted to 6.5 for optimal binding of the protein to the column and removal of proteins with higher pIs during washing of the column. In addition, it was found that appliance of NaCl at a concentration of 20 mM in dialysis and column washing buffers prior to elution with elution buffer containing 120 mM NaCl significantly improves purification of the protein. Starting with specific amount of total proteins obtained by osmotic shock, it was possible to recover 95% of which following to purification with a purification yield of 72% for rhGM-CSF along with appropriate biological activity.     

Identification and characterization of a nucleotide binding site on recombinant murine granulocyte/macrophage-colony stimulating factor.

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a regulatory cytokine important in the proliferative and functional activation of hematopoietic cells. It belongs to a family of 20 kDa or less acidic glycoprotein molecules found in a broad range of cellular sources. On the basis of the previously reported nucleotide-binding properties of interleukin-2 (IL-2), atrial natriuretic factor (ANF), and glucagon, the interaction of GM-CSF with nucleotides was investigated. Using radiolabeled 8-azidoadenosine-containing photoprobes of ATP ([gamma-32P]-8N3ATP) and Ap4A, the putative biological alarmone ([beta'-32P]-8N3Ap4A), we have identified a nucleotide binding site on recombinant murine GM-CSF (rmGM-CSF). Specificity of binding was demonstrated by saturation and competition experiments. Saturation of photoinsertion by [gamma-32P]-8N3ATP and [beta'-32P]-8N3Ap4A occurs with apparent Kd's of 10 and 0.7 microM, respectively. Using an immobilized Fe3+ affinity chromatography technique, developed specifically for the isolation of photolabeled peptides, a single radiolabeled peptide was isolated. It was identified as amino acids 5-14 near the N-terminus of GM-CSF. This peptide region has been shown in previous studies to be critical for biological activity. Also consistent with this observation is our finding that the photolabeled GM-CSF has lost most, if not all, of its biological activity, as determined by a cellular proliferation assay.     

Production of recombinant human granulocyte macrophage-colony stimulating factor in rice cell suspension culture with a human-like N-glycan structure

The rice α-amylase 3D promoter system, which is activated under sucrose-starved conditions, has emerged as a useful system for producing recombinant proteins. However, using rice as the production system for therapeutic proteins requires modifications of the N-glycosylation pattern because of the potential immunogenicity of plant-specific sugar residues. In this study, glyco-engineered rice were generated as a production host for therapeutic glycoproteins, using RNA interference (RNAi) technology to down-regulate the endogenous α-1,3-fucosyltransferase (α-1,3-FucT) and β-1,2-xylosyltransferase (β-1,2-XylT) genes. N-linked glycans from the RNAi lines were identified, and their structures were compared with those isolated from a wild-type cell suspension. The inverted-repeat chimeric RNA silencing construct of α-1,3-fucosyltransferase and β-1,2-xylosyltransferase (Δ3FT/XT)-9 glyco-engineered line with significantly reduced core α-1,3-fucosylated and/or β-1,2-xylosylated glycan structures was established. Moreover, levels of plant-specific α-1,3-fucose and/or β-1,2-xylose residues incorporated into recombinant human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced from the N44 + Δ3FT/XT-4 glyco-engineered line co-expressing ihpRNA of Δ3FT/XT and hGM-CSF were significantly decreased compared with those in the previously reported N44-08 transgenic line expressing hGM-CSF. None of the glyco-engineered lines differed from the wild type with respect to cell division, proliferation or ability to secrete proteins into the culture medium.     

Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM.

Spermatogenesis is initiated with the divisions of the type A spermatogonial stem cells; however, the regulation of this stem cell population remains unknown. In order to obtain a better understanding of the biology of these cells, type A spermatogonia were isolated from 80-day-old pig testes by sedimentation velocity at unit gravity. The cells were cultured for up to 120 h in Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F12) or a potassium-rich medium derived by the simplex optimization method (KSOM). At the end of the 120-h culture period, 30-50% of the spermatogonia were viable in KSOM, whereas in DMEM/F12 very few cells survived. Using KSOM as the culture medium, the effects of stem cell factor (SCF) and granulocyte macrophage-colony stimulating factor (GM-CSF) were studied. SCF significantly enhanced the percentage of cell survival at 100 ng/ml but not at lower concentrations. In comparison, GM-CSF promoted survival at relatively low concentrations (0.01, 0.1, and 1 ng/ml). At a higher dose (10 ng/ml), a significant reduction in percentage of cell survival was observed. The combination of SCF with GM-CSF had no significant effect on the percentage survival of type A spermatogonial cells. These data indicate that SCF and GM-CSF play a role in the regulation of survival and/or proliferation of type A spermatogonia.     

Marker rhythmometry with macrophage-colony stimulating factor (M-CSF).

In a patient with a debulked müllerian adenocarcinoma involving the ovary, an elevated serum concentration of macrophage-colony stimulating factor (M-CSF) (5.3 ng/ml) was lowered into the range of the age- and gender-matched controls by a 24-hour infusion of 135 mg/m2 of taxol, as was a Ca125 of 1480 U/ml by three such taxol courses given at 3-week intervals (to 14 U/ml). A downward trend of M-CSF in serum with an about-14-hour ultradian modulation during the first chemotherapy course resembles that of the concomitantly assessed Ca125. A decreasing trend modulated by an about-half-weekly component is found in M-CSF of fractionated urines collected at spontaneous voidings around the clock for 5 days. M-CSF may serve as a chronobiologic marker for optimizing, on an individualized basis, 1) the infradian scheduling of chemotherapy courses and 2) the ultradian-circadian within-course time patterns. Timing based on markers of the anticancer effect aims at teh as-yet unattained transfer from rodent to human of cancer cures that were not previously feasible without chronobiologic considerations. This goal can be pursued with M-CSF as well as Ca125 and UGP as possibly complementary chronobiologic markers in a chronotherapy trial with taxol in humans.     

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

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 "Aúladdering analysis"Aù, 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.     

Cloning and expression of recombinant macrophage-colony stimulating factor - A progressive strategy for economical production

Macrophage-colony stimulating factor (M-CSF) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, recombinant M-CSF (rM-CSF) became widely used as a biological research reagent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. rM-CSF is a glycoprotein that activates and enhances the differentiation and survival of macrophages, which play a key role in the osteoclastogenetic response. Here, we describe the construction of the gene encoding rM-CSF, its cloning, and expression in Escherichia coli, as well as the purification of rM-CSF protein, and its activity in a biological assay in mouse bone marrow cells. Our results show that the combination of experimental strategies employed to obtain recombinant rM-CSF can yield a biologically active protein, and may be useful when scaling-up production of other biologically similar proteins.     

Clinical applications of recombinant macrophage-colony stimulating factor (rhM-CSF).

rhM-CSF has diverse in vitro and in vivo hematologic and metabolic effects. Clinical trials with this new biopharmaceutical are now in progress and early results have confirmed preclinical findings. The major demonstrated effects of rhM-CSF include an increase in number and activation of monocytes and macrophages, the ability to enhance antibody-dependent cellular cytotoxicity, enhancement of macrophage microbial phagocytic and killing activity, cholesterol lowering, and platelet lowering. The range of potential indications for the use of rhM-CSF makes the further clinical development of this molecule both a challenge and an opportunity. Investigation of combination therapies with other cytokines or monoclonal antibodies will be an important aspect of future investigations.     

Production of biologically active human granulocyte colony stimulating factor in the milk of transgenic goat

We have developed a transgenic female goat harboring goat beta-casein promoter/human granulocyte colony stimulating factor (G-CSF) fusion gene by microinjection into fertilized one-cell goat zygotes. Human G-CSF was produced at levels of up to 50g/ml in transgenic goat milk. Its biological activity was equivalent to recombinant human G-CSF expressed from Chinese hamster ovary (CHO) cell when assayed using in vitro HL-60 cell proliferation. Human G-CSF from transgenic goat milk increased the total number of white blood cells in C57BL/6N mice with leucopenia induced by cyclophosphamide (CPA). The secreted human G-CSF was glycosylated although the degree of O-glycosylation was lower compared to CHO cell-derived human G-CSF.     

Mutagenesis of human granulocyte colony stimulating factor

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.     

The beta common chain (beta c) of the granulocyte macrophage-colony stimulating factor, interleukin-3 and interleukin-5 receptors

The hematopoietic cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), Interleukin (IL)-5 and IL-3 utilise a common receptor signalling molecule, the beta common chain (beta c). This shared receptor component explains, in part the overlapping actions of these cytokines. Mice lacking beta c have a low circulating eosinophil level, have impaired eosinophilic responses to parasitic infection and develop lung disease analogous to human pulmonary alveolar proteinosis (PAP). Surprisingly however, mature hematopoietic cell function is relatively intact, although all GM-CSF-mediated mature cell responses, including glucose transport are absent. Intriguing observations suggesting altered susceptibility to some infectious agents and amelioration of responses to inflammatory stimuli, require further clarification.     

绵羊粒细胞集落刺激因子原核表达与提纯及用于颗粒细胞培养的效果

文中旨在研究粒细胞集落刺激因子(Granule cell stimulating factor,GCSF)对绵羊颗粒细胞体外培养过程中细胞增殖和凋亡的影响,明确GCSF对绵羊颗粒细胞生存的调节作用,为今后该蛋白用于羊繁育方面的研究奠定基础。原核克隆表达纯化羊GCSF蛋白,纯化的蛋白用M-NSF60细胞检测生物学活性,将纯化的GCSF添加到颗粒细胞培养基中作为试验组,以培养基中未添加GCSF的细胞为对照,利用Alarmarblue检测细胞增殖情况,流式细胞仪检测细胞周期和凋亡的变化。结果表明,羊GCSF可以原核表达并纯化,并且具有生物学活性。在24 h和48 h时,在0.06–600 ng/mL的范围内随着加入GCSF的终浓度增加,细胞活力升高。试验组颗粒细胞体外培养24 h后,与阴性对照相比,细胞周期的分布显著改变,S期细胞比例显著减少(P<0.05),G2/M期细胞比例显著增多(P<0.05)。试验组凋亡率和对照组相比,48 h检测时凋亡率显著降低(P<0.05)。综上表明,GCSF在体外培养的绵羊颗粒细胞中,可调控绵羊颗粒细胞周期,促进细胞增殖,抑制细胞凋亡。     

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50°C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50°C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.     

Role of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis of bone marrow derived stem cells

Macrophage colony stimulating factor (M-CSF) and osteoclast differentiation factor (ODF) regulate osteoclastogenesis in vivo. Regulation of osteoclast development in vitro by these cytokines has been reported in the present study. Simultaneous addition of ODF and M-CSF during initiation of bone marrow culture inhibited osteoclastogenesis. However, delayed addition of ODF (three days after initiation of the culture) resulted in dramatic increase in phenotypically and functionally mature osteoclast cells. Delayed addition of ODF beyond day three decreased osteoclastogenesis. Further, removal of M-CSF as early as day three inhibited ODF-induced osteoclastogenesis. These studies provided evidence for the importance of co-ordinated regulation of osteoclastogenesis by M-CSF and ODF.     

The effect of granulocyte macrophage-colony stimulating factor on the prostaglandin E2-like activity of the small intestine of rat during total body irradiation

Radiation-induced gastrointestinal toxicity is important for subjects receiving radiation to the pelvis. Eicosanoids and free radicals may be involved in the mechanism. rHuGM-CSF is a subcutaneously administered drug which may reduce some side effects of radiation. This experimental study was undertaken to determine the effectiveness of rHuGM-CSF on PGE₂-like activity of the small intestine in rats. Thirty-two adult male Wistar-Albino rats entered the study to be randomized to one of the four groups: Group I. Control; II. Drug administered; III. Irradiated; IV. Irradiated and drug administered. Radiation was by total body irradiation, 800 rads with Cobalt 60. On the 9th day the animals were killed and biopsies were taken from the terminal ileum. PGE₂-like activity was evaluated. Animals were weighed on the day of irradiation and end of the experiment. A statistically significant difference was found according to pre- and post-treatment weights in the irradiated and nonirradiated drug administered groups (Groups II and IV) (P=0.035 and 0.018, respectively). PGE₂-like activity in the intestinal tissue was statistically significant higher in the drug-treated animals, both in non-irradiated and irradiated groups. Surprisingly, irradiation was found to decrease the PGE₂-like activity in the intestinal tissue (P=0.008). rHuGM-CSF was found to increase PGE₂-like activity in the intestinal tissue. The cellular mechanisms underlying this must be clearly determined and weighed carefully in considering the drug for clinical usage.     

The effect of granulocyte macrophage-colony stimulating factor on the prostaglandin E(2)-like activity of the small intestine of rat during total body irradiation

Radiation-induced gastrointestinal toxicity is important for subjects receiving radiation to the pelvis. Eicosanoids and free radicals may be involved in the mechanism. rHuGM-CSF is a subcutaneously administered drug which may reduce some side effects of radiation. This experimental study was undertaken to determine the effectiveness of rHuGM-CSF on PGE(2)-like activity of the small intestine in rats. Thirty-two adult male Wistar-Albino rats entered the study to be randomized to one of the four groups: Group I. Control; II. Drug administered; III. Irradiated; IV. Irradiated and drug administered. Radiation was by total body irradiation, 800 rads with Cobalt 60. On the 9th day the animals were killed and biopsies were taken from the terminal ileum. PGE(2)-like activity was evaluated. Animals were weighed on the day of irradiation and end of the experiment. A statistically significant difference was found according to pre- and post-treatment weights in the irradiated and nonirradiated drug administered groups (Groups II and IV) (P=0.035 and 0.018, respectively). PGE(2)-like activity in the intestinal tissue was statistically significant higher in the drug-treated animals, both in non-irradiated and irradiated groups. Surprisingly, irradiation was found to decrease the PGE(2)-like activity in the intestinal tissue (P=0.008). rHuGM-CSF was found to increase PGE(2)-like activity in the intestinal tissue. The cellular mechanisms underlying this must be clearly determined and weighed carefully in considering the drug for clinical usage.