Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles

Objectives

Granulocyte macrophage colony stimulating factor (GMCSF), an important therapeutic cytokine, was immobilized onto silica nanoparticles. Maintenance of structural integrity and biological performance in immobilized cytokine was assessed to augment its applicability in possible biomedical implications.

Results

Following its cloning and expression in E. coli, the recombinant human GMCSF (hGMCSF) was purified as a GST-tagged protein corresponding to a 42 kDa band on SDS-PAGE. The purified cytokine was immobilized onto biocompatible silica nanoparticles (~129.4 nm) by adsorption and the binding was confirmed by dynamic light scattering and infrared spectroscopy. Maximum binding of hGMCSF was at 6.4 µg mg^?1 silica nanoparticles. Efficient release of the cytokine from the nanoparticles with its structural integrity intact was deduced from circular dichroism spectroscopy. hGMCSF-immobilized silica nanoparticles efficiently increased the proliferation of RAW 264.7 macrophage cells with 50 % increase in proliferation at 600 ng hGMCSF µg^?1 silica nanoparticles.

Conclusions

Silica nanoparticles successfully immobilized hGMCSF maintaining its structural integrity. The release of the immobilized cytokine from silica nanoparticles resulted in the increased proliferation of macrophages indicating the potential of the system in future applications.

     

Expression of recombinant human mutant granulocyte colony stimulating factor (Nartograstim) in Escherichia coli

The human granulocyte colony stimulating factor (hG-CSF) plays an important role in hematopoietic cell proliferation/differentiation and has been widely used as a therapeutic agent for treating neutropenias. Nartograstim is a commercial G-CSF that presents amino acid changes in specific positions when compared to the wild-type form, which potentially increase its activity and stability. The aim of this work was to develop an expression system in Escherichia coli that leads to the production of large amounts of a recombinant hG-CSF (rhG-CSF) biosimilar to Nartograstim. The nucleotide sequence of hg-csf was codon-optimized for expression in E. coli. As a result, high yields of the recombinant protein were obtained with adequate purity, structural integrity and biological activity. This protein has also been successfully used for the production of specific polyclonal antibodies in mice, which could be used in the control of the expression and purification in an industrial production process of this recombinant protein. These results will allow the planning of large-scale production of this mutant version of hG-CSF (Nartograstim), as a potential new biosimilar in the market.     

Molecular cloning,expression in Escherichia coli and production of bioactive homogeneous recombinant human granulocyte and macrophage colony stimulating factor

Human granulocyte and macrophage colony stimulating factor (hGM-CSF) is a glycoprotein that activates and enhances the differentiation and survival of neutrophils, eosinophils and macrophages, which play a key role in the innate immune response. Here we describe the construction of the hGM-CSF encoding gene, cloning, expression in Escherichia coli, purification of recombinant hGM-CSF, N-terminal amino acid sequencing, and biological activity assay using TF-1 cells. The results presented show that the combination of experimental strategies employed to obtain recombinant hGM-CSF can yield biologically active protein, and may be useful to scaling-up production of biosimilar protein.     

The granulocyte macrophage colony stimulating factor (GM-CSF) regulates amyloid beta (Abeta) production

One of the hallmarks of Alzheimer’s disease (AD) is the accumulation of amyloid beta (Aβ) plaques in the brain parenchyma. An inflammatory component to AD has been suggested in association with increased cytokine release. We have previously shown that CD40L stimulation of microglia induces increases in pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-8 and GM-CSF. We have also shown that CD40L stimulation increases Aβ levels in HEK-293 cells over-expressing both the amyloid precursor protein (APP) and CD40 (HEK/APPsw/CD40). In this study, we show that GM-CSF neutralizing antibodies mitigate the CD40L-induced production of Aβ in HEK/APPsw/CD40 cells. In addition, we demonstrate that treatment of these cells with recombinant GM-CSF significantly increases Aβ levels. Furthermore, we show that shRNA silencing of the GM-CSF receptor gene significantly reduces Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Analysis of cell surface proteins revealed that silencing of the GM-CSF receptor also decreases APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). Taken together, our results suggest that GM-CSF operates downstream of CD40/CD40L interaction and that GM-CSF modulates Aβ production by influencing APP trafficking. GM-CSF signaling may be a suitable therapeutic target against Aβ production in AD.     

Expression of the fusion protein recombinant human granulocyte-macrophage colony stimulating factor and leukemia inhibitory factor in a baculovirus vector system

A fusion gene coding human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) cDNAs was inserted into the transfer vector pSXIVVI⁺ X3 with the control of Syn and XIV promoters. The Sf9 cells (Spodoptera frugiperda) were co-transfected with the recombinant plasmid and TnNPV DNA (Trichoplusia ni nuclear polyhedrosis virus DNA). The fusion protein recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and leukemia inhibitory factor (LIF) could be synthesized in cells infected with recombinant virus at a level of about 23% of their total cellular protein. Activity analysis of the fusion protein in infected cells revealed that it exhibited the dual activities of GM-CSF and LIF. Western blot analysis of the expressed fusion protein in infected larvae showed that the virus-mediated fusion protein, with a molecular weight of ∼35 kDa, is confirmed with immunoreactivity. Received 02 December 1998/ Accepted in revised form 07 May 1999     

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.     

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.     

The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus

Introduction

An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.

Methods

Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta^®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.

Results

B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4⁺ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.

Conclusion

The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.     

Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.     

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1)

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100-fold. Addition of CSF-1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF-1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF-1 reduce their protein synthetic rate by 50-60%. The protein synthetic rate commences to decrease at 2-3 h after CSF-1 removal. Readdition of CSF-1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF-1, BMM synthesize protein at a rate of approximately 8.7%/h and degrade it at a rate of approximately 0.9%/h. Removal of CSF-1 results in a decrease in the protein synthetic rate to approximately 3.4%/h and an increase in the rate of protein degradation to approximately 3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF-1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF-1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF-1 may be most significant for its survival inducing effect.     

Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation

Purified recombinant human granulocyte-macrophage (rhuGM)-CSF, rhuG-CSF, and rhuCSF-1 were evaluated for their capacity to influence the differentiation of U-937 cells and normal human monocytes. The human U-937 cell line represents an early stage of monocytic differentiation. It was found that rhuGM-CSF and rhuG-CSF, but not rhuCSF-1, induced phenotypic changes consistent with monocyte/macrophage differentiation in U-937 cells. After 3 days of culture in the presence of either rhuGM-CSF or rhuG-CSF, a small but significant proportion of U-937 cells were able to reduce nitroblue tetrazolium. Nitroblue tetrazolium reduction, however, was maximally induced when rhuGM-CSF and rhuG-CSF were added in combination. These changes were accompanied by increased alpha-naphthyl acetate esterase activity, acquisition of macrophage morphology, Mo-1 Ag expression, and decreased cell proliferation. rhuGM-CSF alone also induced expression of the c-fms proto-oncogene (CSF-1 receptor) in U-937 cells and this expression was enhanced by the combination of rhuGM-CSF and rhuG-CSF. In cultured normal human peripheral blood monocytes, representing a late stage of maturation, rhuGM-CSF and rhuCSF-1 differentially increased Mo-1 and My-4 Ag expression, respectively, whereas rhuG-CSF was without effect. Our results suggest that the interaction of GM-CSF, G-CSF, and CSF-1 may play a fundamental role in the early and late stages of the human monocyte/macrophage differentiation process.     

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.     

Efficacy of granulocyte colony stimulating factor in patients with severe alcoholic hepatitis with partial or null response to steroid (GRACIAH trial): study protocol for a randomized controlled trial

Background

Alcoholic hepatitis (AH) has the most severe presentation among alcohol-related liver diseases. Corticosteroids are the most widely recommended treatment for severe AH. However, more innovative, refined treatment measures are required because of its high mortality despite corticosteroid treatment. This study aims to determine whether granulocyte colony stimulating factor (G-CSF) treatment increases short-term survival in patients with severe AH refractory to corticosteroid treatment.

Methods/design

Patients with severe AH whose Maddrey’s discriminant function (MDF) score is ≥?32 and who will be treated with prednisolone (40?mg/day) for 1?week will be screened. Among them, 190 subjects with a partial response (PR) (Lille score 0.16–0.56), and 78 subjects with a null response (NR) (Lille score?≥?0.56) will be enrolled. Subjects with PR will be randomized to steroid plus placebo or steroid plus 12?G-CSF injections (5?μg/kg/day for 5?days followed by every 3?days) at a ratio of 1:1. Subjects with a NR will be randomized to the placebo or G-CSF group (1:1). Study subjects in the PR group will be treated with prednisolone for 28?days followed by dose tapering for an additional 2?weeks. The primary endpoint is the 2-month survival rate in the NR group and the 6-month survival rate in the PR group. Child–Turcotte–Pugh, model for end-stage liver disease score, and the change in the proportion of peripheral circulating CD34-positive cells will be analyzed as risk factors for mortality. Preliminary safety data for the initial 10 study subjects enrolled in the PR study will be assessed to determine whether the PR study would be continued, according to the G-CSF-mobilized, peripheral-blood stem cell donor assessment protocol of the National Marrow Donor Program.

Discussion

We hypothesized that G-CSF would prolong short-term survival of patients with severe AH refractory to corticosteroid treatment. This is a proof-of-concept trial designed to assess the efficacy of Lille-score-guided G-CSF treatment. This trial is also designed to identify a special subgroup in whom G-CSF rescue treatment would improve liver function and prolong survival.

Trial registration

ClinicalTrials.gov, NCT02442180. Prospectively registered on 13 May 2015.

     

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

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

Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross-reaction with a patient's endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX.     

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.