Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ethylene glycol) (PEG) is a water soluble polymer that when covalently linked to proteins, alters their properties in ways that extend their potential uses. PEG-modified conjugates are being exploited in many different fields. The improved pharmacological performance of PEG-proteins when compared with their unmodified counterparts prompted the development of this type of conjugate as a therapeutic agent. Enzyme deficiencies for which therapy with the native enzyme was inefficient (due to rapid clearance and/or immunological reactions) can now be treated with equivalent PEG-enzymes. PEG-adenosine deaminase has already obtained FDA approval. PEG-modified cytokines have been constructed and, interestingly, one of the conjugates, PEG-modified granulocyte-macrophage colony-stimulating factor, showed dissociation of two biological properties. This novel observation may open new horizons to the application of PEGylation technology. The biotechnology industry has also found PEG-proteins very useful because PEG-enzymes can act as catalysts in organic solvents, thereby opening the possibility of producing desired stereoisomers, as opposed to the racemic mixture usually obtained in classical organic synthesis. Covalent attachment of PEG to proteins requires activation of the hydroxyl terminal group of the polymer with a suitable leaving group that can be displaced by nucleophilic attack of the epsilon-amino terminal of lysine residues (other nucleophilic groups can also interact). Several chemical groups have been exploited to activate PEG, thereby giving rise to a variety of PEG-proteins. Some of these varieties retain part of the activating group as a coupling moiety between PEG and protein and others provide a direct linkage. For each particular application, different coupling methods provide distinct advantages.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The uses and properties of PEG-linked proteins. 145 45

For direct studies of growth control, a method was developed to purify viable human megakaryocytes to homogeneity from routine normal bone marrow aspirates. An initial separation of marrow over a 1.050 g/mL Percoll density cut was used to enrich megakaryocytes. After washing, the cells were specifically labeled with a fluoresceinated monoclonal antibody or F(ab')2 fragment to the platelet glycoprotein (GP) IIb/IIIa complex. Megakaryocytes were selectively sorted by using Becton Dickinson FACStar flow cytometer on the basis of a fluorescence intensity greater than 50-fold that of control cells. To increase resolution and purity the sorting rate was adjusted to one cell in 13 formed drops, and negative events that coincided with positive ones were aborted. Two thirds of the isolated cells were large, morphologically recognizable megakaryocytes with a forward light scatter fourfold that of the main cell population. Microscopic examination showed these cells to be greater than or equal to 98% megakaryocytes with a diameter of 20 to 46 microns and a ploidy range of 2N to 64N with a mode of 16N. The small highly fluorescent cells were 10 to 21 microns in diameter, and their ploidy range from 2N to 32N with main ploidy classes of 2N and 4N. The majority of these small cells also positively reacted with monoclonal antibody to platelet GPIb. The isolated cells were cultured in either Iscove's or leucine, lysine-deficient RPMI 1640 medium with 10% human plasma. The cells were maintained in culture more than three days and were capable of synthesis of both DNA and protein as assessed by radiolabeled thymidine and amino acid incorporation. Moreover, the isolated megakaryocytes were capable of responding to recombinant granulocyte-macrophage colony-stimulating factor. The data show that human megakaryocytes can be purified from routine marrow aspirates on the basis of a lineage marker and that they are capable of growth in vitro.
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PMID:Purification of human megakaryocytes by fluorescence-activated cell sorting. 331 39

High-affinity receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3, and interleukin 5 consist of ligand-specific alpha chains (low-affinity subunits) and a common beta chain (beta c) that converts each complex to a high-affinity form. Although beta c alone has no detectable cytokine-binding activity, amino acid substitutions for Glu-21 of human GM-CSF significantly reduce high-affinity but not low-affinity binding, implying that beta c interacts directly with GM-CSF during formation of the high-affinity receptor but only in the presence of the alpha chain. A potential GM-CSF-binding determinant was identified in the second hemopoietin domain of beta c, and the role of individual residues within this region was investigated by determining the ability of mutated beta c chains to confer high-affinity binding when coexpressed with the alpha subunit of the GM-CSF receptor in COS cells. Substitutions involving Met-363, Arg-364, Tyr-365, and Glu-366 did not affect high-affinity binding. However, substitution of His-367 by lysine or glutamine abolished high-affinity binding, suggesting that this residue may form an important part of the high-affinity GM-CSF-binding determinant. Consistent with the loss of high-affinity binding, higher concentrations of human GM-CSF were required to stimulate proliferation of CTLL-2 cell lines transfected with cDNAs for GM-CSF receptor alpha chain and His-367 beta c mutant than those expressing GM-CSF receptor alpha subunit and beta c wild type.
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PMID:Histidine-367 of the human common beta chain of the receptor is critical for high-affinity binding of human granulocyte-macrophage colony-stimulating factor. 827 75

We studied the regulatory effects of various cytokines on the susceptibility to lymphocyte-mediated lysis of cell lines established from patients with acute T-lymphoblastic leukemia (T-ALL). None of the cytokines tested affected the sensitivity of these targets to natural killer activity. In contrast, specific cytokines, different for each cell line, enhanced the susceptibility to lymphokine-activated killer (LAK) cells, while interferon gamma (IFN)-gamma always induced resistance. The same cytokines that increased LAK susceptibility also induced proliferative responses. The TALL-101 cell line, which responded to granulocyte-macrophage colony-stimulating factor (GM-CSF) with increased susceptibility to lysis, and to IFN-gamma with resistance, was used as a model to analyze the mechanisms underlying these changes. Cold target inhibition and conjugate formation assays both indicated that the changes in LAK susceptibility were not at the level of effector-target (E/T) binding. Furthermore, no significant changes in surface expression of adhesion molecules involved in E/T binding were induced by either GM-CSF or IFN-gamma on TALL-101 cells. Finally, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl-esterase assays demonstrated no differences in the ability of these cytokines to trigger the secretion of cytolysins in the bound effectors compared to unstimulated cells. Taken together, these results suggest that the cytokine-modulated susceptibility to lysis of these T-ALL lines might occur at a post-binding stage with mechanisms involving an altered responsiveness to lytic factors.
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PMID:Cytokine modulation of the susceptibility of acute T-lymphoblastic leukemia cell lines to LAK activity. 844 46

The central role that tumor antigen-derived peptides play in induction of antitumor immunity makes them ideal candidates for peptide-based cancer vaccines. We have demonstrated that "transloading" is an efficient strategy for importing short peptide ligands into antigen-presenting cells in vitro. Postulating that the transloading procedure might effect peptide uptake by antigen-presenting cells in vivo as well, we tested this approach for the generation of peptide-based cancer vaccines. In the P815 mastocytoma system, we vaccinated mice by s.c. injection of a single, known natural peptide derived from JAK-1 kinase. Whereas vaccination with peptide alone or mixed with incomplete Freund's adjuvant was ineffective, application of the peptide in conjunction with the polycation poly-L-lysine protected a significant number of animals against tumor challenge. Dependent upon the type of poly-L-lysine applied, protection against tumor take was comparable to that achieved with irradiated whole-cell vaccines, genetically modified to secrete granulocyte-macrophage colony-stimulating factor. In the murine melanoma M-3, a combination of four putative tumor antigen-derived peptides was tested as a cancer vaccine. Administered in combination with polycations, these peptides evoked potent antitumor immunity that could not be obtained with the peptides alone or peptides emulsified in incomplete Freund's adjuvant. However, peptide-polycation vaccines applied to the M-3 model were not as efficient as cellular control vaccines, consisting of irradiated interleukin 2 or granulocyte-macrophage colony-stimulating factor-secreting tumor cells.
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PMID:Cell-free tumor antigen peptide-based cancer vaccines. 909 81

Increased numbers of eosinophils and mast cells in the bronchial mucosa are characteristic features in subjects with aspirin-sensitive asthma. Interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are involved in the activation, maturation, and perpetuation of survival of eosinophils. Immunohistochemical techniques were therefore used to study the expression of IL-5 and GM-CSF on frozen bronchial biopsies from 13 aspirin-sensitive asthmatic (ASA) and 8 non-ASA (NASA) subjects. Aspirin sensitivity was diagnosed by lysine-aspirin inhalation provocation. ASA airways demonstrated a significant 2-fold increase in the total number of submucosal inflammatory cells expressing IL-5 (p = 0.03) and approximate 4- and 2-fold increases in the numbers of mast cells expressing IL-5 and GM-CSF (p = 0.02 and p = 0.04, respectively). There was also a 4-fold increase in the number of eosinophils expressing IL-5 (p = 0.004). These results suggest a central role for the mast cell and eosinophil in regulation of the inflammatory cell infiltrate of ASA airways by secretion of the hemopoietic cytokines IL-5 and GM-CSF.
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PMID:Expression of interleukin-5 and granulocyte-macrophage colony-stimulating factor in aspirin-sensitive and non-aspirin-sensitive asthmatic airways. 937 49

Stimulation of human neutrophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor-alpha (TNF) results in increased superoxide (O2-) release and adherence. O2- release and adherence are dependent on activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Possible participation of serine proteases in GM-CSF- or TNF-induced activation of human neutrophils was explored with various serine protease inhibitors, including phenylmethylsulfonyl fluoride, L-1-tosylamido-2-phenylethyl-chloromethyl ketone and N-alpha-p-tosyl-L-lysine-chloromethyl ketone. GM-CSF- or TNF-induced O2- release and adherence were inhibited in parallel by pretreatment of neutrophils with these inhibitors. On the other hand, GM-CSF- or TNF-induced phosphorylation of ERK and p38 MAPK was unaffected by these inhibitors at the concentrations effective for the inhibition of O2- release and adherence. These findings suggest that serine proteases are involved in GM-CSF- and TNF-induced O2- release and adherence in human neutrophils and that serine proteases function downstream or independently of the activation of ERK and p38 MAPK.
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PMID:Serine protease inhibitors inhibit superoxide release and adherence in human neutrophils stimulated by granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha. 1273 68

Smoking is associated with lung inflammation and a protease-antiprotease imbalance. We previously reported that cigarette smoke extract (CSE) stimulates human lung fibroblasts to release chemotactic cytokines. We hypothesized that serine protease inhibitors might modulate lung fibroblast release of chemotactic cytokines in response to CSE. To test this hypothesis, serine protease inhibitors (FK706, alpha1-antitrypsin, methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone, or Nalpha-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) from human fetal lung fibroblasts by the blind-well chemotactic chamber. Metalloproteinases and cysteine proteinases were not examined in this study. Similarly, the release and gene expression of chemokines and nuclear factor-kappaB (NF-kappaB) activation were measured by means of enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Release of NCA, MCA, chemotactic chemokines including interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor, and the expression of interleukin-8 and monocyte chemoattractant protein-1 mRNA were attenuated by FK706. Furthermore, FK706 suppressed NF-kappaB activation. These data suggest that serine protease inhibitors attenuate the CSE-induced release of NCA and MCA from human fetal lung fibroblasts and that the inhibitory action of antiproteases might depend on NF-kappaB signaling pathway.
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PMID:Serine protease inhibitors modulate smoke-induced chemokine release from human lung fibroblasts. 1273 88

We previously described the requirement of tumour necrosis factor-alpha (TNF-alpha) and the role of beta2 integrins in the Fc-gamma receptor IIa (FcgammaRIIa)-mediated mechanism of neutrophil activation by antiproteinase-3 (anti-PR3) or anti-myeloperoxidase (anti-MPO) antibodies. In the present study, we assessed the involvement of FcgammaRIIIb by studying the respiratory burst activation of completely FcgammaRIIIb-deficient neutrophils primed by TNF-alpha and exposed to anti-PR3 or anti-MPO. Activation of the NADPH oxidase occurred normally in these neutrophils, which indicates that engagement of FcgammaRIIIb is not essential in our model. Experiments performed with neutrophils from severe leucocyte adhesion deficiency (LAD) patients confirmed that beta2 integrins play a pivotal role in this activation. We next studied whether adhesion per se, beta2-integrin-mediated adhesion, or beta2-integrin ligation without adhesion is necessary or sufficient for this activation. Anti-PR3 or anti-MPO induced an FcgammaRIIa-dependent burst in TNF-primed neutrophils incubated in wells coated with poly-L-lysine, known to induce beta2-integrin-independent adhesion, but this reaction was still inhibited by blocking CD18 antibodies. In a system with granulocyte-macrophage colony-stimulating factor (GM-CSF)-primed neutrophils, which did not enhance adhesion, we measured a similar activation by anti-PR3 or anti-MPO and inhibition by CD18. We also noticed that treatment with the beta2-integrin-activating CD18 MoAb KIM185 per se is insufficient for neutrophil activation by anti-PR3 or anti-MPO. We therefore conclude that ligation of beta2 integrins rather than adherence per se is essential for this activation, and that TNF-alpha or GM-CSF is needed for priming but not for adherence.
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PMID:Involvement of Fcgamma receptors and beta2 integrins in neutrophil activation by anti-proteinase-3 or anti-myeloperoxidase antibodies. 1461 97

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn's disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut(S) strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2-3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.
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PMID:PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization. 1621 50


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