The effects of erythropoietin and recombinant cytokines (G-CSF, SCF, IL-3 and GM-CSF) on colony formation and self-renewal by erythroid burst-forming units (BFU-E) and granulocyte-macrophage progenitors (CFU-GM) from mobilized peripheral blood progenitor cells (PBPCs) of the normal donors and patients were investigated. To better understand how combinations of the cytokines may be appropriate for stem cell manipulation, a model involving replating of individual BFU-E and CFU-GM colonies has been used to study the effects of cytokines on colony formation and self-renewal. Based on granulocyte colony-stimulating factor (G-CSF) and erythropoietin(EPO) alone serve as a baseline with which to compare the effects of the combinations with stem cell factor (SCF), interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The results revealed that: 1. BFU-E derived from PBPCs produced a significantly great numbers of subcolonies in EPO plus IL-3 and EPO+SCF+IL-3, and a difference in EPO+SCF compared with EPO alone. 2. Compared patients to normal donors the self-renewal ability of BFU-E was not influenced after EPO plus SCF or IL-3. 3. There was a significantly increased frequency of CFU-GM in the presence of SCF, IL-3, and GM-CSF used in conjunction with G-CSF. Comparing the frequencies of CFU-GM in patients and donors, patients' PBPCs were more sensitive to G-CSF+SCF and GMix (G-CSF+SCF+IL-3+GM-CSF), especially to G-CSF alone than donors. 4. There was a significant increase in AUC (area under the curve of subcolony distribution) in the presence of G-CSF combined with other cytokines. GMix was identified as the optimal combination of cytokines for both the expansion of CFU-GM as well as the expansion of clonogenic progenitor cells. 5. Donors have a high AUC than patients, especially there was a significant increase AUC in G-CSF alone (P = 0.0067).
Cytokines ; pharmacology ; Erythroid Precursor Cells ; drug effects ; Erythropoietin ; pharmacology ; Humans ; Leukocytes, Mononuclear ; drug effects ; physiology ; Recombinant Proteins ; pharmacology ; Stem Cells ; drug effects

Intravenous immunoglobulin (IVIG) is being increasingly used to treat numerous immune-mediated diseases. However, there is a paucity of knowledge on the specific mode of action of IVIG in vivo. In this study, the in vitro effects of IVIG on peripheral blood mononuclear cell (PBMC) proliferation using phytohemagglutinin (PHA), anti-CD3 monoclonal antibody (MAb), phorbol myristate acetate (PMA), or purified protein derivatives (PPD) have been analyzed. The PBMCs were obtained from more than 10 individual donors. In all cases, IVIG almost completely inhibited PBMC proliferation at concentration above 20 mg/mL except when used in conjunction with PMA. PHA-induced proliferation of PBMCs at concentrations ranging from 1 to 15 mg/mL did not show significant differences. Anti-CD3 MAb-induced proliferation showed dose-dependent inhibition at concentrations ranging from 1 to 10 mg/mL. Interestingly, PMA-induced proliferation of PBMCs showed a dose-dependent increase at the same concentration range. PPD-induced proliferation of PBMC at concentrations ranging from 1 to 10 mg/mL did not show any statistically significant differences. These results suggest that high dose IVIG may be necessary to immune modulation in vivo and IVIG has various effects on PBMCs proliferation in limited concentration in vitro.
Cell Division/drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Human ; Immunoglobulins, Intravenous/*pharmacology ; Leukocytes, Mononuclear/*drug effects/physiology ; Tetradecanoylphorbol Acetate/pharmacology

Regulation of P2X7 receptor expression is of interest because activation of this receptor by extracellular ATP triggers a wide variety of cell functions in leukocytes. However, its expression and modulation in human peripheral blood mononuclear cells (PBMC) and monocytes remain unclear. RT-PCR was used to detect the constitutive level of P2X7 receptor and the levels upon stimulation with bacteria, bacterial product, mitogen and various cytokines in human PBMC and monocytes. P2X7 receptor mRNA was detected in PBMC and monocytes. P2X7 receptor expression in PBMC was up-regulated by interleukin-2, -4, -6 (IL-2, IL-4, IL-6) tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS) and heat-inactivated Staphylococcus aureus Cowan strain I (SAC). However, interferon-gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and phytohemagglutinin-M (PHA-M) had little effect on the expression of P2X7 receptor. Furthermore, LPS and M-CSF could up-regulate P2X7 receptor expression in monocytes, while IFN-gamma, TNF-alpha and GM-CSF had weak effects, but pretreatment with these inducers could not further enhance LPS-stimulated P2X7 receptor expression in monocytes. The results obtained demonstrate that inflammatory stimuli drive P2X7 expression, thus supporting the hypothesis that P2X7 receptor may play a role in the inflammatory responses against bacteria infection, which need further verification.
Humans ; Interleukin-2 ; physiology ; Interleukin-4 ; physiology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, Purinergic P2X7 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; physiology

OBJECTIVEA growing body of evidence suggests that the pineal hormone, melatonin (MLT), has immunomodulatory properties; MLT can induce an increment of cell proliferation and an increase or a decrease of a number of cytokines in adults' peripheral blood mononuclear cells (PBMC). However, the influence of MLT on the modulation of neonatal cord blood mononuclear cells (CBMC) proliferation has not been reported. The present study aimed to investigate the possible regulatory effects of MLT on the proliferation of CBMC in vitro.

METHODSTen cord blood preparations from placenta vena umbilicalis of 10 healthy full-term normally delivered newborns and 10 healthy adult volunteers' peripheral blood preparations as controls were obtained. Cord/peripheral blood mononuclear cells suspension were prepared, 2 x 10(5) cells were added to 96-well plate and co-cultured with different stimulants (in cell cultures containing 5 microg phytohemagglutinin (PHA)/ml, 50 ng MLT/ml, 5 ng MLT/ml, 500 pg MLT/ml, 50 pg MLT/ml, 50 ng IL-2/ml, 5 ng MLT/ml + 5 microg PHA/ml or 5 ng MLT/ml + 50 ng IL-2/ml) for 72 h, while the cell suspension (with no stimulant) was used as controls, also cultured for 72 h. With the methods of microscopic examination and (3)H-TdR incorporation test, the influence of melatonin on CBMC morphology and proliferation were investigated, and the effects of MLT on the proliferation of CBMC and PBMC were compared with LSD-t T test and independent samples T test.

RESULTSIn CBMC, MLT (50 pg/ml - 50 ng/ml) increased the (3)H-TdR incorporation rates in a dose-dependent manner, the rate was the highest in the concentration of 5 ng/ml. After 72 h of cell culture, the number of cells in the MLT (5 ng/ml)-exposed group was higher than that recorded before incubation when observed under the high power microscope, including many big mononuclear cells. After adding different stimulants MLT (5 ng/ml), IL-2 (50 ng/ml), MLT plus PHA (5 microg/ml) or MLT plus IL-2 into mediums, the (3)H-TdR incorporation rates of CBMC (cpm) was 114 327 +/- 52 863, 16 087 +/- 9006, 118 360 +/- 59 207 and 17 682 +/- 7391 respectively. In comparison with the controls (14 133 +/- 8688), the incorporation rates of both MLT-exposed group and MLT + PHA-exposed group increased significantly (t = 5.9143, P < 0.001; t = 5.5078, P < 0.001); the rate of IL-2-treated group or MLT + IL-2-treated group demonstrated no significant changes (t = 0.4938, P > 0.05; t = 0.9839, P > 0.05); while the incorporation rates of MLT-exposed group or MLT + PHA-exposed group had no significant difference compared with that of PHA-exposed group (t = 0.1730, P > 0.05; t = 0.3286, P > 0.05). After adding different stimulants into the medium, the incorporation rates of CBMC were all higher than those of PBMC.

CONCLUSIONSMLT can promote not only the proliferation of PBMC, but also the proliferation of CBMC, and the effects of MLT on CBMC were stronger than those on PBMC. This suggests that MLT could be involved in the regulation of the newborn immune system and suggests a new immunotherapeutic strategy in the treatment of certain diseases of neonates.

Adult ; Blood Cells ; drug effects ; physiology ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Cells ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; Humans ; Infant, Newborn ; Interleukin-2 ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; physiology ; Melatonin ; pharmacology ; Phytohemagglutinins ; pharmacology ; Pregnancy

OBJECTIVETo investigate the effects of Shexiang Baoxin Pill (SBP) on function of endothelial progenitor cells (EPCs) and its nitric oxide (NO) secretion.

METHODSTotal mononuclear cells were isolated from human peripheral blood by ficoll density gradient centrifugation and inoculated on the human fibro-ligandin encrusting plate. After 7 days of in vitro culture, adherent cells were collected and incubated with SBP for 24 h. The proliferation, migration, adhesive activity, vasculogenesis capacity and NO secretion of EPCs were assayed using MTT, Transwell chamber, adhesion determination, in vitro vasculogenesis kit and nitrate reductase method, respectively.

RESULTSEPCs incubated with SBP showed the capacities higher than those of control in proliferation, migration, adhesion, in vitro vasculogenesis, and with a higher NO concentration in the culture supernatant.

CONCLUSIONSBP can improve the function of EPCs, which might be a novel mechanism of its effects in improving vascular endothelial function and promoting angiogenesis.

Cell Differentiation ; physiology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; physiology ; Humans ; Leukocytes, Mononuclear ; cytology ; Nitric Oxide ; biosynthesis ; Stem Cells ; cytology ; metabolism ; physiology

To explore the effect of retinoic acid (RA) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in murine bone marrow stromal cell (BMSC) and adhesive rate of human cord blood mononuclear cell (UCBMNC) to BMSC in vitro, the express ions of ICAM-1 and VCAM-1 of murine BMSC were detected by flow cytometry and the binding capacity of UCBMNC to BMSC was tested by MTT assay after co-culturing with 0.1, 1.0 and 10.0 micro mol/L RA, respectively. The results showed that 1.0 and 10.0 micro mol/L RA increased the expression of ICAM-1 and the adhesive rate of U CBMNC to BMSC, however, RA did not induced the increase of expression of VCAM-1. It was positive correlation between the increments of ICAM-1 expression and the adhesive rate (r = 0.7883, P < 0.05). It is concluded that RA up-regulated ICAM-1 expression of BMSC and increased the adhesion of UCBMNC to BMSC in vitro. These may clarify the correlation between adhesion molecules on BMSC and homing of hematopoietic stem cells, and provide the experimental basis for RA to promote the homing of umbilical cord blood stem cell.
Animals ; Bone Marrow Cells ; chemistry ; drug effects ; physiology ; Cell Adhesion ; drug effects ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Intercellular Adhesion Molecule-1 ; analysis ; Leukocytes, Mononuclear ; drug effects ; physiology ; Mice ; Stromal Cells ; chemistry ; drug effects ; physiology ; Tretinoin ; pharmacology ; Vascular Cell Adhesion Molecule-1 ; analysis

OBJECTIVEThis study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro.

METHODSPBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h.

RESULTSAfter six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity.

CONCLUSIONThis study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; physiology ; Phenotype ; Recombinant Proteins ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVEMany clinical evidences and epidemiologic data in the past suggested that Kawasaki disease (KD) is correlated with an acute immune dysfunction caused by infection. In our preliminary study, Toll-like receptor 4 signal pathway, which could activate nuclear transcription factor-kappaB and induce excessive product of proinflammatory cytokines, chemokines and co-stimulatory molecules, was observed to be significantly activated during acute phase of Kawasaki disease. But the causative factors and regulatory mechanism are still unknown. In this study, the authors further investigated the changes and significances of regulatory factors for signal pathway of Toll-like receptors (TLRs) in immunological pathogenesis of Kawasaki disease.

METHODSForty-eight children with KD, sixteen children with infectious disease (ID) and sixteen age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the expression levels of regulatory and effective factors in toll-like receptor 4 (TLR4) signal pathways and proinflammatory factors in peripheral blood monocyte/macrophage (MC). The expression of TLR4 protein in MC was analyzed by flow cytometry.

RESULTS(1) Expression levels of TLR4, MD-2, MyD88, IRAK-4, TRAF6, TAK1, TAB1 and TAB2 mRNA in KD group were elevated significantly during acute phase (P < 0.05). (2) Transcription levels of regulatory factors PRAT4B and STAP2 in patients with KD or ID were found to be higher than those in the healthy volunteers (P < 0.05), but no significant differences in these parameters were detected between KD patients and ID patients (P > 0.05). Transcription levels of regulatory factors such as FLN29, RP105 and MD-1 were up-regulated to some extents and expression level of DAP12 mRNA in KD patients were found to be lower than that in normal controls (P < 0.05), while all of the four regulatory factors were found to be lower than those in ID patients (P < 0.05). Expressions of proinflammatory cytokines such as L-1beta, IL-6 and TNF-alpha in KD patients were significantly higher than those in ID patients (P < 0.05). (3) Stimulation with lipopolysaccharide (LPS) elevated remarkably the expressions of regulatory factors PRAT4B and STAP2 in KD patients or healthy volunteers (P < 0.05). All of the four negative-regulatory factors were found to be significantly up-regulated after stimulation with LPS in controls (P < 0.05). No responses to LPS were observed in expression of FLN29, RP105 and MD-1 mRNA in KD patients (P > 0.05), except for increased transcription of DAP12. (4) The levels of PRAT4B and STAP2 mRNA in KD patients with coronary artery lesion (KD-CAL(+)) were detected to be higher than those in KD patients without coronary artery lesion (KD-CAL(-)) during acute phase (P < 0.05), while those of FLN29, RP105 and MD-1 in KD-CAL(+) group were lower than that in the latter (P < 0.05). No significant difference in DAP12 mRNA expression level was detected between the two groups (P > 0.05). Expressions of proinflammatory cytokines and TLR4 protein on surface of CD14-positive cells in KD-CAL(+) group were found to be higher than those in KD-CAL(-) group [(11.9 +/- 2.4)% vs. (6.5 +/- 1.7)%, P < 0.05].

CONCLUSIONDisturbance of negative-regulatory factors may be one of the factors causing aberrant immunological function in KD.

Child ; Coronary Vessels ; drug effects ; physiology ; Cytokines ; metabolism ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lipopolysaccharides ; toxicity ; Macrophages ; drug effects ; pathology ; Mucocutaneous Lymph Node Syndrome ; immunology ; metabolism ; physiopathology ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; physiology ; Toll-Like Receptors ; immunology ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation

BACKGROUNDO(6)-methylguanine-DNA-methyltransferase (MGMT) is a specific DNA revising enzyme transferring alkylated groups from DNA to its cysteine residue to avoid the abnormal twisting of DNA. Therefore, it is one of the drug resistant genes targeted in the treatment of cancer. This study explored the protective effect of MGMT gene transferred into mammalian cells.

METHODSMammalian expression vector containing the MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells (PBMCs) via liposome, then assayed for gene expression at RNA and protein levels. MTT assay was used to check the drug resistance of cells transfected with MGMT gene.

RESULTSMGMT gene was successfully cloned. Real-time PCR showed that the mRNA expression in gene transfected groups in K562 cell line and PBMC were 13.4 and 4.0 times that of the empty vector transfected groups respectively.

RESULTSof Western blotting showed distinct higher expression of MGMT in gene transfected group than in other two groups. The IC(50) values increased to 7 and 2 times that of the original values respectively in stable transfected K562 cells and transient transfected PBMC.

CONCLUSIONThe alkylating resistance of eukaryotic cells is enhanced after being transfected with MGMT gene which protein product performs the protective function, and may provide the reference for the protective model of peripheral blood cells in cancer chemotherapy.

Blotting, Western ; Cell Survival ; drug effects ; genetics ; physiology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Enzymologic ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Microscopy, Fluorescence ; Nitrogen Mustard Compounds ; pharmacology ; O(6)-Methylguanine-DNA Methyltransferase ; genetics ; metabolism ; physiology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Astragalus membranaceus may be a potential therapy for childhood asthma but its driving mechanism remains elusive. The main components of A. membranaceus were identified by HPLC. The children with asthma remission were divided into two combination group (control group, the combination of budesonide and terbutaline) and A. membranaceus group (treatment group, the combination of budesonide, terbutaline and A. membranaceus). The therapeutic results were compared between two groups after 3-month therapy. Porcine peripheral blood mononuclear cells (PBMCs) were isolated from venous blood by using density gradient centrifugation on percoll. The levels of FoxP3, EGF-β, IL-17 and IL-23 from PBMCs and serum IgE were measured. The relative percentage of Treg/Th17 cells was determined using flow cytometry. The main components of A. membranaceus were calycosin-7-O-glucoside, isoquercitrin, ononin, calycosin, quercetin, genistein, kaempferol, isorhamnetin and formononetin, all of which may contribute to asthma therapy. Lung function was significantly improved in the treatment group when compared with a control group (P < 0.05). The efficacy in preventing the occurrence of childhood asthma was higher in the treatment group than the control group (P < 0.05). The levels of IgE, IL-17 and IL-23 were reduced significantly in the treatment group when compared with the control group, while the levels of FoxP3 and TGF-β were increased in the treatment group when compared with the control group (P < 0.05). A. membranaceus increased the percentage of Treg cells and reduced the percentage of Th17 cells. A. membranaceus is potential natural product for improving the therapeutic efficacy of combination therapy of budesonide and terbutaline for the children with asthma remission by modulating the balance of Treg/Th17 cells.
Animals ; Asthma ; drug therapy ; immunology ; Astragalus propinquus ; chemistry ; Budesonide ; administration & dosage ; Cells, Cultured ; Child ; Child, Preschool ; Cytokines ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Humans ; Immunologic Factors ; administration & dosage ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lung ; drug effects ; physiology ; Male ; Swine ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Terbutaline ; administration & dosage ; Th17 Cells ; cytology ; drug effects ; Treatment Outcome