Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli.
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ABSTRACT:
SUBMITTER: Schrimsher JL
PROVIDER: S-EPMC1148387 | biostudies-other | 1987 Oct
REPOSITORIES: biostudies-other
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| S-EPMC10405220 | biostudies-literature
Project description:The formation of mature haemopoietic cells is controlled by hormones that specifically stimulate the progenitor cells of the granulocyte/macrophage, eosinophil, megakaryocyte and erythroid pathways. PWMSC medium (pokeweed-mitogen-stimulated spleen-cell-conditioned medium) is known to contain the biological activities that control the clonal proliferation of these four progenitor cells in vitro in semi-solid agar cultures. In this study the molecular properties of these biological activities were characterized, and all four colony-stimulating factors appear to be associated with glycoproteins. These factors were precipitated between 50 and 80%-satd. (NH(4))(2)SO(4) and could be concentrated by ultrafiltration over a 10000-mol.wt.-cut-off hollow-fibre membrane. Megakaryocyte- and erythroid-colony-stimulating factors were lost when the conditioned medium was dialysed at low ionic strength (<0.03m). Neither asialo- nor sialo-erythropoietin was detectable in concentrated PWMSC medium or in the fractions purified from it by gel filtration on Sephadex G-150. The factors bound to concanavalin A-Sepharose were eluted with alpha-methyl-d-glucopyranoside (0.10m). Analysis by gel filtration on Sephadex G-150 indicated that the apparent molecular-weight distributions of all colony-stimulating factors were identical (37000). Treatment with neuraminidase did not alter the biological activities of any of these factors, but when the molecular weights were analysed, after neuraminidase treatment, on Sepharose CL-6B in the presence of guanidine hydrochloride (6m) all were eluted with a mol.wt. of 24000. Although the apparent molecular weights of the different factors were identical, charge differences were detectable by isoelectric focusing on thin-layer granulated gels. There appeared to be considerable charge heterogeneity associated with each factor, as all were focused over 2-4 pH units. The maximum activity of the granulocyte/macrophage-colony-stimulating factor on isoelectric focusing was at pH4.8, whereas the maximum activity for the eosinophil-colony-stimulating factor was at pH5.8. The erythroid- and megakaryocyte-colony-stimulating activities were detected in the pH ranges 4.8-5.8 and 4.6-7.1 respectively. Chromatographic differences between the granulocyte/macrophage- and eosinophil-colony-stimulating factors were also detected by hydrophobic chromatography at low ionic strength (0.15m-NaCl) on Cibacron Blue-Sepharose and at high ionic strength [2m-(NH(4))(2)SO(4)] on phenyl-Sepharose. Eosinophil-colony-stimulating factor bound more strongly than the other factors to both matrices. The megakaryocyte- and erythroid-colony-stimulating activities were always associated with those for granulocytes/macrophages and eosinophils. Preparations highly enriched for eosinophil-colony-stimulating factor were also obtained by DEAE-cellulose chromatography. An overall purification of 100-fold for all of the factors was achieved with the present techniques, and, although differences were observed, only granulocyte/macrophage-stimulating factors and a small proportion of the eosinophil-stimulating factors could be completely separated from the others. Our results are consistent with the existence of separable factors for granulocyte/macrophage and eosinophil stimulation, but the megakaryocyte- and erythroid-stimulating activities were always associated with the granulocyte/macrophage- and eosinophil-stimulating activities. Thus there may be one molecule that is able to stimulate all four colony types or four very similar molecules that are difficult to separate.
| S-EPMC1161356 | biostudies-other
Project description:BackgroundCancer-associated pain is a major cause of poor quality of life in cancer patients and is frequently resistant to conventional therapy. Recent studies indicate that some hematopoietic growth factors, namely granulocyte macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), are abundantly released in the tumor microenvironment and play a key role in regulating tumor-nerve interactions and tumor-associated pain by activating receptors on dorsal root ganglion (DRG) neurons. Moreover, these hematopoietic factors have been highly implicated in postsurgical pain, inflammatory pain and osteoarthritic pain. However, the molecular mechanisms via which G-/GMCSF bring about nociceptive sensitization and elicit pain are not known.ResultsIn order to elucidate G-/GMCSF mediated transcriptional changes in the sensory neurons, we performed a comprehensive, genome-wide analysis of changes in the transcriptome of DRG neurons brought about by exposure to GMCSF or GCSF. We present complete information on regulated genes and validated profiling analyses and report novel regulatory networks and interaction maps revealed by detailed bioinformatics analyses. Amongst these, we validate calpain 2, matrix metalloproteinase 9 (MMP9) and a RhoGTPase Rac1 as well as Tumor necrosis factor alpha (TNF?) as transcriptional targets of G-/GMCSF and demonstrate the importance of MMP9 and Rac1 in GMCSF-induced nociceptor sensitization.ConclusionWith integrative approach of bioinformatics, in vivo pharmacology and behavioral analyses, our results not only indicate that transcriptional control by G-/GMCSF signaling regulates a variety of established pain modulators, but also uncover a large number of novel targets, paving the way for translational analyses in the context of pain disorders.
| S-EPMC3852053 | biostudies-literature
Project description:The human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor used to prevent and treat neutropenia. G-CSF stimulates the bone marrow to produce infection-fighting granulocytes. Food and Drug Administration of the United States approved G-CSF in 1991 and its PEGylated version in 2002 as a prophylactic and therapeutic measure against neutropenia. Recombinant human G-CSF is produced in surrogate host Escherichia coli and is PEGylated at N-terminal. Besides neutropenia, G-CSF is also used in bone marrow transplantation for the mobilization and maturation of peripheral blood stem cells. Considering the requirement of producing G-CSF therapeutic in large quantities, construct designing for high expression is critical for the biopharmaceutical and industrial application. Earlier studies have employed approaches such as codon optimization, use of strong promoters, employment of protein tags, secretion signals, optimization of protein folding, etc., for increasing expression and yield of therapeutic proteins. In this study, it was observed that mRNA transcribed from the native human cDNA of G-CSF and the codon-optimized variant leads to low protein expression in E. coli. To understand the underlying reasons, the mRNA secondary structure of the 5' end of the G-CSF transcript was analyzed. This analysis revealed the presence of stable secondary structures at the 5' end of the G-CSF transcript, arising from the native human gene and even from the codon-optimized sequence. These secondary structures were disrupted through translationally silent mutations within the first 24 nucleotides of the transcript without affecting the protein sequence. Interestingly, through this approach, the G-CSF protein expression was increased 60 folds as compared to native G-CSF construct. We believe that these findings create a roadmap for optimization of G-CSF transcript for enhanced expression in E. coli and could be employed to increase the expression of other therapeutic proteins.
| S-EPMC7985328 | biostudies-literature
Project description:Human granulocyte colony-stimulating factor (G-CSF), and a mutant having a Ser for Cys substitution at residue 18 were produced in Escherichia coli strain W3110. About 60 mg of pure protein was obtained from 50 g of wet cells with a recovery of about 20%. The proteins were characterized physically and chemically, including determination of disulphide bonds, which were found to exist between residues 37-43 and 65-75. Cys-18 is not involved in disulphide bond formation and was substituted by Ser with no effects on gross protein conformation or biological activity. Both the wild-type and the mutant recombinant-derived proteins, although not glycosylated, possess colony-stimulating activities. In a bioassay using the murine myelomonocytic leukaemic cell line WEH1 3B D+, activities were obtained which were similar to those of natural G-CSF and of a glycosylated recombinant-derived human G-CSF produced in monkey cells.
| S-EPMC1135389 | biostudies-other
Project description:Concerns have been raised about potential toxic interactions when colony-stimulating factors (CSFs) and chemoradiation are concurrently performed. In 2006, the ASCO guidelines advised against their concomitant use. Nevertheless, with the development of modern radiotherapy techniques and supportive care, the therapeutic index of combined chemotherapy, radiotherapy, and CSFs is worth reassessing. Recent clinical trials testing chemoradiation in lung cancer let investigators free to decide the use of concomitant CSFs or not. No abnormal infield event was reported after the use of modern radiotherapy techniques and concomitant chemotherapy regimens. These elements call for further investigation to set new recommendations in favour of the association of chemoradiation and CSFs. Moreover, radiotherapy could induce anticancer systemic effects mediated by the immune system in vitro and in vivo. With combined CSFs, this effect was reinforced in preclinical and clinical trials introducing innovative radioimmunotherapy models. So far, the association of radiation with CSFs has not been combined with immunotherapy. However, it might play a major role in triggering an immune response against cancer cells, leading to abscopal effects. The present article reassesses the therapeutic index of the combination CSFs-chemoradiation through an updated review on its safety and efficacy. It also provides a special focus on radioimmunotherapy.
| S-EPMC7217575 | biostudies-literature
Project description:RationaleGranulocyte/macrophage colony-stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals.ObjectivesOur primary objective was to determine whether human GMAb would reproduce PAP in healthy primates. A secondary objective was to determine the concentration of GMAb resulting in loss of GM-CSF signaling in vivo (i.e., critical threshold).MethodsNonhuman primates (Macaca fascicularis) were injected with highly purified, PAP patient-derived GMAb in dose-ranging (2.2-50 mg) single and multiple administration studies, and after blocking antihuman immunoglobulin immune responses, in chronic administration studies maintaining serum levels greater than 40 microg/ml for up to 11 months.Measurements and main resultsGMAb blocked GM-CSF signaling causing (1) a milky-appearing bronchoalveolar lavage fluid containing increased surfactant lipids and proteins; (2) enlarged, foamy, surfactant-filled alveolar macrophages with reduced PU.1 and PPARgamma mRNA, and reduced tumor necrosis factor-alpha secretion; (3) pulmonary leukocytosis; (4) increased serum surfactant protein-D; and (5) impaired neutrophil functions. GM-CSF signaling varied inversely with GMAb concentration below a critical threshold of 5 microg/ml, which was similar in lungs and blood and to the value observed in patients with PAP.ConclusionsGMAb reproduced the molecular, cellular, and histopathologic features of PAP in healthy primates, demonstrating that GMAb directly cause PAP. These results have implications for therapy of PAP and help define the therapeutic window for potential use of GMAb to treat other disorders.
| S-EPMC2902758 | biostudies-literature
Project description:Background & aimsAnti-granulocyte macrophage-colony stimulating factor autoantibodies (aGMAbs) are detected in patients with ileal Crohn's disease (CD). Their induction and mode of action during or before disease are not well understood. We aimed to investigate the underlying mechanisms associated with aGMAb induction, from functional orientation to recognized epitopes, for their impact on intestinal immune homeostasis and use as a predictive biomarker for complicated CD.MethodsWe characterized using enzyme-linked immunosorbent assay naturally occurring aGMAbs in longitudinal serum samples from patients archived before the diagnosis of CD (n = 220) as well as from 400 healthy individuals (matched controls) as part of the US Defense Medical Surveillance System. We used biochemical, cellular, and transcriptional analysis to uncover a mechanism that governs the impaired immune balance in CD mucosa after diagnosis.ResultsNeutralizing aGMAbs were found to be specific for post-translational glycosylation on granulocyte macrophage-colony stimulating factor (GM-CSF), detectable years before diagnosis, and associated with complicated CD at presentation. Glycosylation of GM-CSF was altered in patients with CD, and aGMAb affected myeloid homeostasis and promoted group 1 innate lymphoid cells. Perturbations in immune homeostasis preceded the diagnosis in the serum of patients with CD presenting with aGMAb and were detectable in the noninflamed CD mucosa.ConclusionsAnti-GMAbs predict the diagnosis of complicated CD long before the diagnosis of disease, recognize uniquely glycosylated epitopes, and impair myeloid cell and innate lymphoid cell balance associated with altered intestinal immune homeostasis.
| S-EPMC10127946 | biostudies-literature
Project description:BACKGROUND:The protein hormone granulocyte colony-stimulating factor (GCSF) stimulates the production of white blood cells and plays an important role in medical treatment of cancer patients. METHODS:An efficient process was developed for heterologous expression of the human GCSF in E. coli BL21 (DE3). The feeding rate was adjusted to achieve the maximum attainable specific growth rate under critical value. In this method, specific growth rate was maintained at the maximum value of 0.55 h?¹ at the beginning of feeding to 0.4 h-1 at the induction time. Recombinant human GCSF (rh-GCSF) was produced as inclusion body. At first, inclusion bodies were released by cell disruption and then washed, solubilized and refolded. Finally, the rh-GCSF was purified by cation exchange chromatography. RESULTS:Obviouly, higher specific growth rate decreases process time and consequently increases productivity. The final concentration of biomass and GCSF was achieved 126 g DCW.l?¹ and 32.1 g.l?¹. Also, the final specific yield (YP/X) and total productivity of rh-GCSF were obtained 254 mg.g?¹ DCW and 1.83 g.l?¹.h?¹, respectively. According to the available data, this is one of the highest YP/X and productivity that has been reported for any human protein which is expressed in E. coli. Recovery yield of purification process was %40 and purity of recombinant protein was over than 99%. The circular dichroism spectra of purified rh-GCSF, Neupogen and PD-Grastim showed that all proteins have a similar secondary structure. CONCLUSION:Modified exponential feeding strategy for fed-batch cultivation of recombinant E. coli, results in minimum fed-batch duration and maximum productivity.
| S-EPMC4412921 | biostudies-literature
Project description:BackgroundMalignant tumours release factors, which attract myeloid cells and induce their polarisation to pro-invasive, immunosuppressive phenotypes. Brain-resident microglia and peripheral macrophages accumulate in the tumour microenvironment of glioblastoma (GBM) and induce immunosuppression fostering tumour progression. Macrophage colony stimulating factors (CSFs) control the recruitment of myeloid cells during peripheral cancer progression, but it is disputable, which CSFs drive their accumulation in gliomas.MethodsThe expression of CSF2 (encoding granulocyte-macrophage colony stimulating factor) was determined in TCGA datasets and five human glioma cell lines. Effects of stable CSF2 knockdown in glioma cells or neutralising CSF2 or receptor CSF2Rα antibodies on glioma invasion were tested in vitro and in vivo.ResultsCSF2 knockdown or blockade of its signalling reduced microglia-dependent glioma invasion in microglia-glioma co-cultures. CSF2-deficient human glioma cells encapsulated in cell-impermeable hollow fibres and transplanted to mouse brains, failed to attract microglia, but stimulated astrocyte recruitment. CSF2-depleted gliomas were smaller, attracted less microglia and macrophages, and provided survival benefit in tumour-bearing mice. Apoptotic microglia/macrophages were detected in CSF2-depleted tumours.ConclusionsCSF2 is overexpressed in a subset of mesenchymal GBMs in association with high immune gene expression. Tumour-derived CSF2 attracts, supports survival and induces pro-tumorigenic polarisation of microglia and macrophages.
| S-EPMC7403321 | biostudies-literature