OBJECTIVETo investigate the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and GM-CSF/IL-3/IL-5 receptor common beta chain (beta c receptor) in an adult patient with idiopathic pulmonary alveolar proteinosis (PAP), so as to demonstrate the possible association of the GM-CSF and beta c receptor with the pathogenesis of human PAP.

METHODSThe GM-CSF levels were measured with a commercial ELISA kit (sensitivity 5 pg/ml) and the beta c receptor expression on the cell surface was detected by flow cytometry analysis. Reverse transcription polymerase chain reaction (RT-PCR) analysis was employed to detect the expression of the GM-CSF mRNA and the beta c receptor mRNA in peripheral blood mononuclear cells and alveolar macrophages. The entire coding regions of the GM-CSF cDNA and the beta c receptor cDNA were sequenced by the Sanger dideoxy-mediated chain termination method to detect possible mutations.

RESULTSThe patient with PAP failed to release the GM-CSF protein either from circulating mononuclear cells or from alveolar macrophages. The expression of the GM-CSF mRNA was normal after the stimulation of lipopolysaccharide, whereas a point mutation at position 382 of the GM-CSF cDNA from "T" to "C" was revealed by cDNA sequencing, which caused a change in amino acid 117 of the protein from isoleucine to threonine. The beta c receptor expression on the cell surface was normal, and the beta c receptor mRNA expression and the sequence of the entire coding region of the beta c receptor were also normal.

CONCLUSIONSThe decreased GM-CSF production is associated with the pathogenesis of human PAP. A point mutation of the GM-CSF cDNA may contribute to the decreased GM-CSF production in our adult PAP patient. The mutation of the beta c receptor in some of paediatric patients with PAP may not be a common problem in adult patients.

DNA, Complementary ; chemistry ; Granulocyte-Macrophage Colony-Stimulating Factor ; analysis ; biosynthesis ; Humans ; Male ; Middle Aged ; Pulmonary Alveolar Proteinosis ; etiology ; metabolism ; RNA, Messenger ; analysis ; Receptors, Cytokine ; biosynthesis ; genetics

Paeonia lactiflora root (baishao in Chinese) is a commonly used herb in traditional Chinese medicines (TCM). Two isomers, paeoniflorin (PF) and albiflorin (AF), are isolated from P. lactiflora. The present study aimed to investigate the protective effects of PF and AF on myelosuppression induced by chemotherapy in mice and to explore the underlying mechanisms. The mouse myelosuppression model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CP, 200 mg·kg(-1)). The blood cell counts were performed. The thymus index and spleen index were also determined and bone morrow histological examination was performed. The levels of tumor necrosis factor-α (TNF-α) in serum and colony-stimulating factor (G-CSF) in plasma were measured by Enzyme-Linked Immunosorbent Assays (ELISA) and the serum levels of interleukin-3 (IL-3), granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), and interleukin-6 (IL-6) were measured by radioimmunoassay (RIA). The levels of mRNA expression protein of IL-3, GM-CSF and G-CSF in spleen and bone marrow cells were determined respectively. PF and AF significantly increased the white blood cell (WBC) counts and reversed the atrophy of thymus. They also increased the serum levels of GM-CSF and IL-3 and the plasma level of G-CSF and reduced the level of TNF-α in serum. PF enhanced the mRNA level of IL-3 and AF enhanced the mRNA levels of GM-CSF and G-CSF in the spleen. PF and AF both increased the protein levels of GM-CSF and G-CSF in bone marrow cells. In conclusion, our results demonstrated that PF and AF promoted the recovery of bone marrow hemopoietic function in the mouse myelosuppression model.
Animals ; Antineoplastic Agents ; adverse effects ; Bridged-Ring Compounds ; administration & dosage ; Cyclophosphamide ; adverse effects ; Drugs, Chinese Herbal ; administration & dosage ; Glucosides ; administration & dosage ; Granulocyte Colony-Stimulating Factor ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Hematologic Diseases ; etiology ; genetics ; metabolism ; prevention & control ; Humans ; Interleukin-3 ; genetics ; metabolism ; Interleukin-6 ; metabolism ; Male ; Mice ; Monoterpenes ; administration & dosage ; Paeonia ; chemistry ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Animals ; Bone Marrow ; drug effects ; Catechin ; pharmacology ; Fabaceae ; chemistry ; Female ; Gene Expression Regulation ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; genetics ; Hematopoiesis ; drug effects ; Interleukin-6 ; blood ; genetics ; Male ; Mice ; Plants, Medicinal ; chemistry ; RNA, Messenger ; analysis

OBJECTIVETo construct mouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) expression vector and express, purify and refold mGM-CSF protein.

METHODBased on previously constructed fusion protein hIL-2/mGM-CSF expression vector, pET-11c/mGM-CSF expression vector was constructed routinely and transformed into BL21 (DE3). The inclusion body protein was washed with our patented method and refolded with renaturation buffer containing low-concentration guanidinium chloride (Gu.Cl). The refolded protein was purified with affinity chromatography.

RESULTSpET-11c/mGM-CSF vector was constructed successfully. The host bacteria was cultured in TH broth and induced with 0.1 mmol/L IPTG at 32 degrees C, which resulted in the expression level of 60.6%. The best refolding effect was achieved with the renaturation media containing glutathione and 1.5 mol/L Gu.HCl. After purification with affinity chromatography, the purity of the target mGM-CSF protein reached 95% with activity of 5x10(6) U/mg.

CONCLUSIONEngineered bacteria BL21/pET- 11c/mGM-CSF with efficient mGM-CSF expression and laboratory scale renaturation and purification of mGM-CSF have been established, which facilitates further researches into the anti-tumor function of the dendritic cells and GM-CSF in vivo.

Animals ; Chromatography, Affinity ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; isolation & purification ; Mice ; Protein Folding ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; isolation & purification

OBJECTIVETo study the effect of total saponins of Panax ginseng (TSPG) on the expression of granulocytemacrophage colony-stimulating factor (GM-CSF) from human endothelial cells and monocytes and the relationship between TSPG and human granulocytopoiesis and monocytopoiesis modulation.

METHODSAdopting the hematopoietic progenitor cells culture in vitro, hematopoietic growth factor biological assay, immunocytochemistry and nucleic acid in situ hybridization, the GM-CSF expression in the endothelial cells and monocytes were detected.

RESULTSThe conditioned cultural media of endothelial and monocytes induced and prepared by TSPG, could significantly promote the proliferation and differentiation of human colony forming unit-granulocyte macrophage (CFU-GM), and enhance the protein and mRNA expression of GM-CSF in endothelial cells and monocytes.

CONCLUSIONTSPG could possibly through direct or indirect route, promote hematopoietic, induce endothelial cells and monocytes in the microenvironment to synthetize and secrete GM-CSF, so as to further promote the proliferation and differentiation of human CFU-GM.

Bone Marrow Cells ; cytology ; metabolism ; Cells, Cultured ; Culture Media, Conditioned ; Endothelial Cells ; metabolism ; Ginsenosides ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Hematopoiesis ; drug effects ; Humans ; Monocytes ; metabolism ; Panax ; chemistry ; Saponins ; pharmacology

OBJECTIVETo study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF).

METHODSSOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay.

RESULTSThe expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively.

CONCLUSIONThis research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.

Animals ; Arginine ; chemistry ; genetics ; metabolism ; Base Sequence ; Biological Assay ; Cell Proliferation ; Chromatography, Affinity ; Chromatography, Ion Exchange ; Cytokines ; metabolism ; Escherichia coli ; genetics ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; chemistry ; genetics ; isolation & purification ; metabolism ; Humans ; Interleukin-2 ; chemistry ; genetics ; isolation & purification ; metabolism ; Mice ; Molecular Sequence Data ; Protein Folding ; Protein Renaturation ; Recombinant Fusion Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

Cytokines have pleiotropic regulatory effects on hematopoietic cells and many other cell types that participate in host defence and repair processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages and regulates the biological functions expressed by mature cells of these lineages. Stem cell factor (SCF) is a multifunctional cytokine involved in hematopoiesis, melanogenesis and gametogenesis. In order to determine the complementary DNA (cDNA) of canine GM-CSF and canine SCF, cDNA clones were generated from lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMCs) and bone marrow cells by reverse transcription PCR amplification. The canine GM-CSF cDNA obtained in this study contains an open reading frame encoding 144 amino acid residues and has 53-75% homology with those of human, cat, sheep, pig, cow and mouse, Canine SCF cDNA consist of an open reading frame encoding 274 amino acid residues and shares 81-92% homology with those of human, cat, pig, cow and mouse.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; Cattle ; *Cloning, Molecular ; Codon ; DNA, Complementary/analysis/*genetics ; Dogs/blood/*genetics ; Gametogenesis ; Gene Amplification ; Granulocyte-Macrophage Colony-Stimulating Factor/chemistry/*genetics ; Hematopoiesis ; Humans ; Mice ; Molecular Sequence Data ; Open Reading Frames ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sheep ; Stem Cell Factor/chemistry/*genetics ; Swine

AIMTo study the effect of catechin, the active component of Spatholobus suberectus Dunn, on bone marrow cell cycle and the expression of IL-6 and GM-CSF mRNA in spleen cells of normal and marrow-depressed mice in order to clarify the mechanism of hematopoietic-supportive effect of catechin.

METHODSFlow cytometry was adopted to investigate the influence of catechin on bone marrow cell cycle in mice and the expression of IL-6 and GM-CSF mRNA induced by catechin in spleen cells was detected by RT-PCR technique simultaneously.

RESULTSThe cell proportion of normal and marrow-depressed mice in G0/G1 phase was reduced significantly, while that in S + G2/M phase increased significantly. Being induced by catechin, IL-6 mRNA and GM-CSF mRNA in spleen cells were markedly up-regulated.

CONCLUSIONCatechin (2 g x L(-1), intraperitoneally injected to mice daily immediately after irradiation for 7 consecutive days) was shown to promote the expression of IL-6 mRNA and GM-CSF mRNA in spleen cells of mice, through which it can accelerate bone marrow cells of normal mice into cell cycle and help those of marrow-depressed mice to get out of "G1-phase-block", enter into cell cycle and radically accelerate the proliferation and differentiation of hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC).

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Catechin ; isolation & purification ; pharmacology ; Cell Cycle ; drug effects ; Fabaceae ; chemistry ; Female ; Gene Expression Regulation ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; Hematopoietic Stem Cells ; cytology ; Interleukin-6 ; biosynthesis ; genetics ; Male ; Mice ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Spleen ; cytology ; metabolism

OBJECTIVETo observe the effects of active ingredients from Chinese drugs for activating blood circulation and detoxicating, including notoginseng saponins (drug 1), Coptis chinensis (drug 2), giant knotweed rhizome (drug 3) and rhubarb (drug 4), on blood lipids and inflammatory reaction of aortic atherosclerotic plaques in ApoE knockout mice.

METHODSApoE knockout mice were fed with high-fat diet for 26 weeks, then they were randomized into 6 groups, the untreated model group and the test groups treated with various test drugs respectively. After ending the 13 weeks of treatment, all the mice were sacrificed with their blood lipids detected, and their heart and aorta were taken out to make slices with paraffin embedding. Four sections from aortic root of each mouse were chosen to measure and calculate the percentage of lipid core (LC) in the total area of plaque (TP) and the lipid/collagen ratio (L/C) in the plaque by HE and Movat staining respectively, and the mean value of the four sections was taken for analysis. The expressions of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) in mice's aorta root were determined by immunohistochemical staining as well.

RESULTSAfter being treated for 13 weeks, either the percentage of LC in TP and the L/C ratio was significantly lower in all the test drug treated groups than those in the model group, respectively (P < 0.01), especially prominent in the group treated with drug 3. Although lowering of the two indexes presented in all the 3 groups treated by drug 1, 2 and 3, significant difference still presented between drug 3 treated group vs drug 1 and 2 treated group (P < 0.05). As for the expressions of GM-CSF and TNF-alpha, in comparing with the untreated model group, significant decreasing of the TNF-alpha showed only in the drug 4 treated group, while that of GM-CSF could be found in all the test drug treated groups (P < 0.05).

CONCLUSIONAll the 4 drugs tested in the recommended dosage can stabilize the vulnerable plaques in ApoE knockout mice by improving the constitution of plaque, among them, drug 3 and 4, the drugs possess both the actions of activating blood circulation and detoxicating, show more significant effect, and their mechanisms may be related to their actions in regulating lipid metabolism and inhibiting inflammatory reaction.

Animals ; Aorta ; drug effects ; metabolism ; pathology ; Apolipoproteins E ; genetics ; Atherosclerosis ; blood ; pathology ; prevention & control ; Blood Circulation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Granulocyte-Macrophage Colony-Stimulating Factor ; analysis ; Immunohistochemistry ; Lipids ; analysis ; blood ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Random Allocation ; Saponins ; pharmacology ; Tumor Necrosis Factor-alpha ; analysis