OBJECTIVETo investigate effects of serum HDL(1) on the formation of foam cells from human peripheral blood monocyte-derived macrophages.

METHODSSectie density polyacrylamide gel electrophoresis (sd-PAGE) was applied for isolation and preparation of HDL(1) simultaneously. Monocytes were isolated from human peripheral blood by Ficoll-Hypaque density gradient centrifugation and plastic adsorptive process. The isolated monocytes were stimulated by phorbol 12-myristate 13-acetate (PMA) at a concentration of 50 nmol/L for 48 h and transferred to macrophages. The monocyte-derived macrophages were then coincubated with 80 mg/L ox-LDL and HDL(1) (0, 0.1, 1.0 and 10.0 mg/L) for 6, 12 and 24 h, respectively. The formation of foam cells was identified by transmission electron microscope (TEM), total cholesterol (TC), free cholesterol (FC) and protein (Pro) in cultured cells were quantitatively analyzed by high performance chromatography (HPLC) and modified lowry protein assay, respectively.

RESULTSHDL(1) isolated from human serum by sd-PAGE could significantly decrease TC/Pro ratio in foam cells in a concentration-dependent (0 mg/L: 36.9 +/- 1.1, 10.0 mg/L: 6.2 +/- 0.4, P < 0.01) and time-dependent (10.0 mg/L HDL(1) 6 h: 16.9 +/- 0.9, 24 h: 6.4 +/- 0.6, P < 0.01) manner.

CONCLUSIONHDL(1) is capable of inhibiting and attenuating the formation of foam cells by decreasing cellular TC, therefore, might play an important role in attenuating atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Cholesterol, LDL ; metabolism ; Foam Cells ; cytology ; metabolism ; Humans ; Lipoproteins, HDL ; blood ; Lipoproteins, LDL ; Monocytes ; cytology ; metabolism

The main aim of this study was to investigate the biological activities and immune modulation changes of chorionic villi mesenchymal stem cells (CV-MSC) after long term culture. The morphology of the CV-MSC of passage 3 and passage 9 were observed by microscopy, and their phenotypes were detected by flow cytometry. CV-MSC of passage 3 and 9 were co-cultured with PHA-stimulated PBMNC, and IFN-γ concentration in culture medium was detected by ELISA. The mRNA expression of COX-2, HGF and HLA-G in CV-MSC were detected by real-time PCR. The results showed that after long term culture, the CV-MSC kept the MSC morphology and most of the phenotypes including CD31, CD34, CD44, CD45, CD62L, CD73, CD90, CD105, CD117, CD151, CD235a, CD271 and HLA-DR, while the CD49d was significantly up-regulated. Immune modulation ability of CV-MSC was reduced and the mRNA expression of COX-2 and HGF was down regulated after long term culture, but the expression of HLA-G did not found to be obvious change. It is concluded that the long term in vitro expansion changes the expression of CD49d and reduces immune modulation of CV-MSC.
Cells, Cultured ; Chorionic Villi ; immunology ; Female ; Humans ; Integrin alpha4 ; metabolism ; Mesenchymal Stromal Cells ; cytology ; immunology ; Monocytes ; cytology ; Placenta ; cytology ; Pregnancy

OBJECTIVETo study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).

METHODSDCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.

RESULTSCompared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).

CONCLUSIONLong-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-12 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism

OBJECTIVETo investigate the expression changes of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) on Chlamydia pneumoniae (C.pn) induced foam cell formation.

METHODSHuman monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and were randomly allocated into four groups: negative control group (50 microg/ml LDL for 48 h); positive control group (50 microg/ml ox-LDL for 48 h); C.pn infection group (50 microg/ml LDL plus 1 x 10(5), 4 x 10(5), 5 x 10(5) and 1 x 10(6) IFU C.pn for 48 h or 1 x 10(6) IFU C.pn for 0, 24, 48 and 72 h); ACAT inhibitor 58-035 plus C.pn infection group (1, 5, 10 microg/ml ACAT inhibitor 58-035 pretreatment for 1 h, 50 microg/ml LDL and 1 x 10(6) IFU C.pn for 48 h). The mRNA and protein expressions of ACAT1 were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence.

RESULTSThe mRNA and protein expressions of ACAT1 were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C.pn infection in a time-dependent and concentration-dependent manner (all P < 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C.pn (at the concentrations of 5 x 10(5) and 1 x 10(6) IFU for 48 h) and C.pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could be significantly attenuated by ACAT inhibitor 58-035 (all P < 0.05).

CONCLUSIONChlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACAT1.

Cell Line, Tumor ; Chlamydophila pneumoniae ; Foam Cells ; cytology ; metabolism ; Humans ; Monocytes ; cytology ; Sterol O-Acyltransferase ; metabolism ; Up-Regulation

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Differentiation/*metabolism ; B-Lymphocytes/cytology/metabolism ; Basophils/cytology/metabolism ; Epitopes/genetics/metabolism ; *Flow Cytometry ; Gene Expression Regulation ; Granulocytes/cytology/metabolism ; Leukocytes/immunology/*metabolism ; Mice ; Monocytes/cytology/metabolism ; Rabbits ; T-Lymphocytes/cytology/metabolism

OBJECTIVETo analyze the effect of dexamethason (Dex) on blast composition in patients with myelodysplastic syndrome (MDS) and investigate its significance in diagnosis of MDS.

METHODSThe flow cytometry (FCM) was used to detect the blast rate and the expression of its antigens in 30 cases of MDS (10 cases were treated with Dex as DX group and 20 cases were treated without Dex as control group).

RESULTSThe difference of the CD34(+) cell number detected by FCM was not statistically significant between DX group and control group (P > 0.05); The rate of BM B cell precursors (BCP CD34(+)/CD19(+)/CD10(+) cells) increased in DX group significantly, and BM CD117(+) cells in CD34(+) cells was decreased significantly as compared with control group (P < 0.001). The expression of antigens between granulocyte and monocyte was not significantly different (P > 0.05).

CONCLUSIONThe dexamethasone can increase the rate of BCP significantly and decreased the rate of BM CD117(+) cells in CD34(+) cells significantly. There is significant influence on the blast composition in MDS patients after dexamethasone treatment and without significant influence on the other phenotypcs.

Antigens, CD34 ; metabolism ; Dexamethasone ; therapeutic use ; Flow Cytometry ; Granulocytes ; cytology ; Humans ; Monocytes ; cytology ; Myelodysplastic Syndromes ; drug therapy ; Precursor Cells, B-Lymphoid ; cytology ; Proto-Oncogene Proteins c-kit ; metabolism

OBJECTIVETo investigate the effect of simulated microgravity on the proliferation of human monocytic cells THP-1 and the expression of tissue factor (TF) mRNA.

METHODSTHP-1 cells were cultured under a simulated microgravity environment using the rotating cell culture system (RCCS). The changes in the cell proliferation after microgravity culture were assessed by cell counting and cell cycle analysis with flow cytometry. RT-PCR was used to detect the changes in the expression of TF mRNA in THP-1 cells.

RESULTSCulture under simulated microgravity resulted in a significant decrease in the cell number of THP-1 cells in comparison with that of the control cells (P<0.01). After a 24-h culture under microgravity, the G0-Gl phase cells increased from the control level of (46.57∓1.64)% to (67.64∓2.71)% (P<0.05). The cells in both groups showed a low level of TF mRNA expression in the absence of LPS stimulation. A 4-h stimulation with LPS caused up-regulated expression of TF mRNA in both cells, but the microgravity group showed a significantly smaller increase in the expression (2.301∓0.179) than the control group (9.210∓1.328) (P<0.05).

CONCLUSIONMicrogravity can inhibit the proliferation of THP-1 cells and suppress the cellular expression of TF mRNA.

Cell Proliferation ; Cells, Cultured ; Humans ; Monocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Thromboplastin ; genetics ; metabolism ; Weightlessness ; Weightlessness Simulation

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Ligands ; Lipopolysaccharides ; adverse effects ; Mannose-Binding Lectin ; pharmacology ; Monocytes ; cytology ; metabolism ; Toll-Like Receptor 4 ; metabolism

In order to investigate the effect of stromal cell derived factor-1 (SDF-1)/CXCR4 on the proliferation of megakaryocytic line-HEL cells co-cultured with human umbilical cord blood-derived stromal cells (hUCBSCs) and to further elucidate the mechanism of SDF-1/CXCR4-mediated functions, the HEL cells were co-cultured with hUCBSCs or human bone marrow stromal cells (hBMSCs), the suspended HEL was used as control. The concentrations of SDF-1 in supernatant of hUCBSCs and hBMSCs were detected by ELISA assay. The expression of CXCR4 membrane-bound protein of HEL cells was detected by laser confocal scanning microscopy and flow cytometry, and the expression of CXCR4 mRNA was detected by RT-PCR. The result showed that the concentrations of SDF-1 in different groups were the same at the early stage of culturing. But at 6 days after seeding, the concentrations of SDF-1 increased significantly in the hUCBSCs group, even though the passage was done. By means of laser confocal microscopy, the expression of CXCR4 protein and also red dots of fluorescence could be detected in cytoplasm of HEL cells co-cultured with hUCBSCs. However, there was no significant differences of the CXCR4 mRNA level between different groups (p > 0.05). It is concluded that hUCBSCs may play important roles in secreting high level of SDF-1 and regulating megakaryocyte expression of CXCR4.
Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12 ; genetics ; metabolism ; Coculture Techniques ; Fetal Blood ; cytology ; metabolism ; Flow Cytometry ; Humans ; Megakaryocytes ; cytology ; metabolism ; Monocytes ; cytology ; RNA, Messenger ; genetics ; Receptors, CXCR4 ; genetics ; metabolism ; Stromal Cells ; cytology

In order to determine the precise mechanism of the interactions between different types of cells, which are common phenomena in tissues and organs, the importance of coculture techniques are becoming increasingly important. In the area of cardiology, artificial arteries have been developed, based on the understanding of physiological communication of the arterial smooth muscle cells (SMC), endothelial cells (EC), and the extracellular matrix (ECM). In the study of atherosclerosis, the modification of low-density lipoprotein (LDL), which result in the recruitment and accumulation of white blood cells, especially, monocytes/macrophages, and foam cell formation, are hypothesized. Although there are well known animal models, an in vitro model of atherogenesis with a precisely known atherogenesis mechanism has not yet been developed. In this paper, an arterial wall reconstruction model using rabbit primary cultivated aortic SMCs and ECs, was shown. In addition, human peripheral monocytes were used and the transmigration of monocytes was observed by scanning electron and laser confocal microscopy. Monocyte differentiation into macrophages was shown by immunohistochemistry and comprehensive gene expression analysis. With the modified form of LDL, the macrophages were observed to accumulate lipids with a foamy appearance and differentiate into the foam cells in the ECM between the ECs and SMCs in the area of our coculture model.
Animal ; Aorta/physiology* ; Aorta/cytology* ; Arteriosclerosis/etiology* ; Cell Differentiation/physiology ; Cell Movement ; Coculture ; Endothelium, Vascular/physiology ; Endothelium, Vascular/cytology ; Extracellular Matrix/metabolism ; Foam Cells/ultrastructure ; Foam Cells/cytology ; Macrophages/physiology* ; Macrophages/cytology ; Male ; Microscopy, Confocal ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Monocytes/ultrastructure ; Monocytes/physiology ; Muscle, Smooth, Vascular/physiology ; Muscle, Smooth, Vascular/cytology ; Myosin/metabolism ; Protein Isoforms/metabolism ; Rabbits