OBJECTIVES: To investigate the effect of granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) on expression of matrix metalloproteinase-2, 9 (MMP-2, 9) mRNA in mouse embryos. Materials and METHOD: From October 1997 to December 1998, morula stage mouse embryos were cultured for 48 hours with G-CSF and GM-CSF at concentrations of 0.1 pg/ml, 1 pg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml and 10 ng/ml, respectively. Embryos not treated with G-CSF or GM-CSF were served as control. Reverse transcription-polymerase chain reaction (RT-PCR) has been used to examine the expression of MMP-2, 9 mRNA in developed blastocysts. Following reverse transcription, strategically designed nested primers, optimized for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. Results were analyzed with Kolmogorov-Smirnov test and analysis of variance (ANOVA). The statistical significance was defined as p< 0.05. RESULTS: The relative quantities (relative volume x intensity) of MMP-2 mRNA expressed in embryos of all G-CSF treatment groups were significantly increased than in the control, especially in 10, 100 pg/ml and 1 ng/ml treatment groups. The relative quantities of MMP-2 mRNA in all GM-CSF treatment groups were also significantly increased than in the control, especially in 100 pg/ml treatment group. The relative quantities of MMP-9 mRNA of all GM-CSF treatment groups except 10 ng/ml group were significantly increased than in the control, especially 10, 100 pg/ml and 1 ng/ml treatment group. However, the relative quantity of MMP-9 mRNA was significantly increased in only 10 ng/ml G-CSF treatment group than in the control and other treatment groups. CONCLUSION: This study suggests that G-CSF and GM-CSF may increase the m-RNA expression of MMP-2 or 9 in mouse blastocysts with the concentration-specific manner.
Animals ; Blastocyst ; Colony-Stimulating Factors* ; Digestion ; DNA, Complementary ; Embryonic Structures* ; Granulocyte Colony-Stimulating Factor ; Granulocyte-Macrophage Colony-Stimulating Factor* ; Granulocytes* ; Matrix Metalloproteinase 2* ; Mice* ; Morula ; Reverse Transcription ; RNA, Messenger ; Sensitivity and Specificity ; Sequence Analysis

Administration of G-CSF may not always respond in rise of neutrophil counts in different patient population. In order to understand a possible inter-relationship between the G-CSF and GM-CSF induced leukocyte responses and expression levels of receptors for G-CSF (G-CSFr) and GM-CSF (GM-CSFr), the levels of each receptor and CSF were measured in patients with basophilia (8), eosinophilia (14) and bacterial infection showing neutrophilia (12) in comparison with normal healthy adults (12) and children (14). G-CSFr was expressed in neutrophils in the largest amount followed by monocytes, but GM-CSFr was expressed more in monocytes than neutrophils. Lymphocytes and basophils did not express G-CSFr or GM-CSFr. The amount of GM-CSFr in neutrophils was present less in patients with infection than normal control (P = 0.031). The neutrophils expressed more G-CSFr than GM-CSFr. The quantity of G-CSFr in eosinophil showed marked interval change, higher in acute stage. The plasma concentrations of G-CSF in patients with infection were much higher than normal adults or children (117.95 +/- 181.16 pg/ml, P < 0.05). Binding assay with excess amount of CSFs could discriminate the patient who did not show any response to G-CSF or GM-CSF administration. After incubation with excess CSFs, more receptors were blocked in children than in adults (G-CSF P = 0.024, GM-CSF P = 0.006). These results indicate that the amount of CSFr in leukocyte varies in different types of leukocyte, and changes according to the patients' condition even in the same type of leukocyte, and the CSFrs of children bind to CSFs more than those of adults.
Adult ; *Bacterial Infections ; Basophils/chemistry ; Breast Neoplasms ; Child ; Colony-Stimulating Factors/*blood ; Eosinophilia ; Human ; Leukemia, Myeloid, Chronic ; *Leukocyte Disorders ; Monocytes/chemistry ; *Neoplasms ; Neutrophils/chemistry ; Receptors, Colony-Stimulating Factor/*analysis ; Receptors, Granulocyte Colony-Stimulating Factor/analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/analysis

To explore the reasonable procedures and strategies of diagnosis and treatment of congenital neutropenia (CN), clinical data and laboratory examination results of a boy suspected of CN were collected; gene ELA2, GFI1, HAX1, and WASp of whom were sequenced, granulocyte colony-stimulating factor receptor (G-CSFR) expression on neutrophil was analyzed, and cytoplasmic domain of G-CSFR was sequenced. The results showed that the diagnosis of non-syndromic variants of CN (NSVCN) was made on this patient according to the criteria; sequencing results revealed no mutation occurred in ELA2, GFI1, HAX1 and WASp; a normal expression level of G-CSFR on neutrophil from this patient was detected and no truncated mutation was found in the intracellular domain of G-CSFR. It is concluded that reasonable procedure of diagnosis and treatment of CN is established, and a sporadic NSVCN with no recognized pathogenic mutation is confirmed in this patient.
Child ; DNA Mutational Analysis ; Humans ; Male ; Neutropenia ; congenital ; diagnosis ; genetics ; therapy ; Receptors, Granulocyte Colony-Stimulating Factor ; metabolism

OBJECTIVETo observe the short- term effects of hemogram in donors after peripheral blood stem cell（PBSC）collection and donors' tolerance.

METHODSA total of 166 related allogeneic donors were selected from The First Affiliated Hospital of Medical School of Zhejiang University between January 2013 and December 2014, including 86 male and 80 female. All donors accepted granulocytecolony- stimulating factor（G-CSF）5-10 μg·kg⁻¹·d⁻¹ until collection finished and were measured by blood cells count before and after PBSC collection.

RESULTSAfter PBSC collection, the hemoglobin level decreased from 145（94-181）g/L to 138（93-167）g/L, and the platelet counts decreased in all donors from 231（105- 490）× 10⁹/L to 95（39- 210）× 10⁹/L. The amount of hemoglobin contamination in collection products was weak correlated with the decreased hemoglobin in peripheral blood（r=0.297, P=0.017）, and the platelet contamination was high correlated with that decreased in peripheral blood（r=0.719, P<0.001）. The decline of hemoglobin level after twice PBSC collection was of no significant difference between four groups in different ages（P≥0.05）. The decline of platelet counts was out of a significant difference（P> 0.05）. In addition, the decline of hemoglobin level after once and twice PBSC collection was of a significant difference between four groups in different body mass index（BMI）（P=0.003 and P<0.001）, especially in thinner group with obvious decrease. But the decline of platelet counts was out of a significant difference （P>0.05）.

CONCLUSIONThe hemoglobin level decreased mildly in healthy allogeneic hematopoietic stem cell donors after PBSC collection and it is better to adjust parameters every time to ensure their safety for thinner donors. However, it will increase the risk of platelet decline, which is unrelated with ages and BMI and can be tolerated.

Blood Donors ; Blood Platelets ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cells ; cytology ; Hemoglobins ; analysis ; Humans ; Male ; Platelet Count

The ability to generate dendritic cells (DCs) in sizeable numbers has enormous implications for the development of clinically-effective antigen presentation procedures for cancer immunotherapy. We evaluated the generation of immunostimulatory DCs from peripheral blood CD34+ cells collected from healthy donors. CD34+ cells purified from leukapheresis product were seeded at 1 x 10(4) cells/mL in complete medium supplemented with GM-CSF, TNF alpha, IL-4, c-kit ligand, and flt3 ligand (FL). By day 14 of culture in the presence of GM-CSF + TNF alpha, the total cell number increased by 23.4 +/- 5.4-fold compared to the starting number of CD34+ cells. When the c-kit and FL were added to GM-CSF and TNF alpha, the cell number increased by 109.8 +/- 11.2-fold without affecting the immunophenotype of recovered cells. Flow cytometric analysis indicated that cells with the markers of mature dendritic cells, i.e., CD1a +CD14 -HLA-DR+, and CD80+CD86+HLA-DR+, constituted 49.0% +/- 7.5%, and 38.9% +/- 6.5%, respectively. This pattern of expression of surface antigen was unchanged whether the c-kit ligand and/or FL was added. The irradiated CD1a+HLA-DR+ cells recovered from in vitro cultures elicit a vigorous proliferation of allogeneic peripheral blood T-cells, irrespective of cytokine combinations. These findings provide advantageous tools for the large-scale generation of DCs that are potentially usable for clinical protocols of immunotherapy or vaccination in patients undergoing cancer treatment.
Antigens, CD34/analysis* ; Dendritic Cells/physiology* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology ; HLA-DR Antigens/analysis ; Hematopoietic Stem Cells/physiology* ; Human ; Interleukin-4/pharmacology ; Tumor Necrosis Factor/pharmacology

Nutrigenomics refers to research that investigates the interaction between nutrition and the human genome. Caffeine in tea and coffee is widely and routinely consumed by people. This study was performed to confirm the effect of caffeine treatment on the gene expression and cytokine profiling in 3T3-L1 adipocyte cells using microarray and protein array methodology. Treatment of caffeine in 3T3-L1 adipocyte cells increased expression of several genes related with obesity including adipocyte C1Q and collagen domain containing (ACDC), Adipsin (ADN), uncoupling protein 3 (UCP3), while glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is known as lipid storage enzyme, was decreased by caffeine treatment. Furthermore, cytokines, such as interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-13 (IL-13), granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF), were decreased in caffeine treated 3T3-L1 adipocyte cells. These results provided interesting information about the genes related with caffeine and cytokine expression profiling in obesity.
Adipocytes* ; Caffeine* ; Coffee ; Collagen ; Colony-Stimulating Factors ; Complement Factor D ; Cytokines ; Gene Expression ; Genome, Human ; Granulocyte-Macrophage Colony-Stimulating Factor ; Granulocytes ; Humans ; Interleukin-12 ; Interleukin-13 ; Interleukin-3 ; Nutrigenomics ; Obesity ; Oxidoreductases ; Protein Array Analysis ; Tea ; Vascular Endothelial Growth Factor A

OBJECTIVETo explore the roles of granulocyte colony-stimulating factor in the pathogenesis of moyamoya disease.

METHODSSerum G-CSF concentrations were measured using enzyme linked immunosorbent assay (ELISA) in 20 children with moyamoya disease and 20 healthy children.

RESULTSSerum G-CSF concentration (35.7+/-10.3 pg/mL) in children with moyamoya disease was significantly higher than that in healthy controls (23.5+/-3.8 pg/mL) (p<0.01).

CONCLUSIONSThe elevated serum G-CSF concentration in children with moyamoya disease suggests that G-CSF may play an important role in the pathogenesis of moyamoya disease.

Child ; Child, Preschool ; Female ; Granulocyte Colony-Stimulating Factor ; blood ; physiology ; Humans ; Male ; Moyamoya Disease ; blood ; etiology ; Vascular Endothelial Growth Factor A ; analysis ; physiology

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

OBJECTIVE: Granulocyte-macrophage colony-stimulating factor (GM-CSF) facilitates mammalian embryonic development and implantation. However, its biological function after implantation is not elucidated. The aim of this study is to assess the changes of gene expression by GM-CSF in human trophoblast obtained in early pregnancy. METHODS: Human trophoblast obtained in early pregnancy was cultured with or without GM-CSF. The difference of gene expression was evaluated with microarray and selected genes were reevaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Microarray analysis revealed that the expressions of 468 genes were increased while those of 40 genes were decreased by GM-CSF. These genes were evaluated according to the known biologic pathways. The regulation of actin cytoskeleton and focal adhesion pathways were mostly influenced by GM-CSF. Annexin A2, thymosin-like 3, vimentin, myogenin, ACK1, and tensin1 genes were selected for real-time RT-PCR. The increased expressions of of vimentin and ACK1, and decreased expressions of tensin1 were confirmed by real-time RT-PCR. CONCLUSION: GM-CSF activates focal adhesion pathway in human trophoblast by increasing the expression of vimentin and ACK1, and decreasing the expression of tensin1.
Actin Cytoskeleton ; Annexin A2 ; Embryonic Development ; Female ; Focal Adhesions ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; Humans ; Microarray Analysis ; Myogenin ; Pregnancy ; Trophoblasts ; Vimentin

The objective was to observe the effect of G-CSF as a mobilizer of hematopoitic stem cells on the absolute counts of T-cell subsets in peripheral blood and their relevance with the mobilized CD34(+) cells. The examples of peripheral blood from 26 patients performed of autologous stem cell transplantation were taken before and after mobilization by G-CSF. Flow cytometry was used for detecting CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(+)CD4(+)CD8(+) and CD3(+)CD4(-)CD8(-) cells. Concurrently, their correlations with mobilized CD34(+) cells in peripheral blood were compared. The results showed that after the mobilization by G-CSF, the amounts of CD3(+), CD3(+)CD4(+), CD3(+)CD4(+)CD8(+) and CD3(+)CD4(-)CD8(-) cells in peripheral blood increased by 2.23, 2.62, 2.99 and 10.96 fold respectively, but that of CD3(+)CD4(-)CD8(+) cells was nearly no changed (P = 0.243). The correlation coefficient of CD3(+)CD4(-)CD8(-) cells and mobilized CD34(+) cells was 0.796, (P = 0.000) and no correlation with other T-cell subsets. It was concluded that when CD34(+) cells were mobilized by G-CSF from bone marrow to peripheral blood, the absolute counts of the peripheral T-cell subsets got changed. The increase of CD3(+)CD4(-)CD8(-) cells had correlated with mobilization effect of CD34(+) cells into peripheral blood.
Adolescent ; Adult ; Antigens, CD34 ; analysis ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoietic Stem Cell Mobilization ; Humans ; Male ; Middle Aged ; T-Lymphocyte Subsets ; drug effects