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

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

OBJECTIVETo establish the method of cytological examination and the normal reference values for hypertonic saline solution-induced sputum of healthy children (age range from 5 to 15 years) with physical examination in Guangzhou.

METHODA total of 352 children, 5 to 15 years old, were enrolled from primary school and middle school in Guangzhou from January to December, 2010. All subjects completed a standardized questionnaire on the presence of respiratory, allergic symptoms and family history, the medical history and the physical examination was performed by doctors, lung function (forced expiratory volume at 1 s in predicted normal, FEV(1)%) was determined. There were 266 healthy children (137 males, 129 females) who were selected and undergone hypertonic saline solution induction of sputum, and cytological examination was performed. Hypertonic saline (5%) was nebulized and inhaled for 15 - 30 min. No expectoration within 30 min was defined as failure, and the procedure was terminated. The part of opaque and higher density sputum samples was detected by cytology. The proportion of neutrophils, lymphocytes, eosinophils, macrophages and monocytes was calculated. This study was approved by the institutional Ethics Review Committee of First Affiliated Hospital of Guangzhou Medical College. Informed consent was obtained from the legal guardians of all participants following a detailed description of the purpose and potential benefits of the study.

RESULTThere were 175 subjects' induced sputum specimens (175/266, 65.8%), non-qualified sputum samples were obtained from 16 of the subjects. The proportions of median (IQR) of lymphocytes were 0.012 (0.020), 95%CI were ranged from 0.015 to 0.022; neutrophils 0.207 (0.330), 95%CI 0.266 - 0.356 macrophages 0.761 (0.327), 95%CI 0.607 - 0.699; eosinophils 0.004 (0.019), 95%CI 0.013 - 0.022. There were no significant differences in proportions of cytological findings of female or male, different age groups and second-hand smoking or not (all P > 0.05). The incidence of adverse event was 4.40% (7/159).

CONCLUSIONThe method and the preliminary data may be used for research, diagnosis and treatment of patients with chronic cough and airway inflammation.

Adolescent ; Child ; Child, Preschool ; China ; Cough ; diagnosis ; physiopathology ; Eosinophils ; cytology ; Female ; Forced Expiratory Volume ; Humans ; Leukocyte Count ; Lymphocyte Count ; Lymphocytes ; cytology ; Male ; Monocytes ; cytology ; Neutrophils ; cytology ; Reference Values ; Saline Solution, Hypertonic ; chemistry ; Sputum ; cytology ; metabolism

PURPOSE: This study was done to evaluate the effects of psychosocial interventions on cortisol and immune response in adult patients with cancer. METHODS: MEDLINE via PubMed, Cochrane Library CENTRAL, EMBASE, CINAHL and domestic electronic databases were searched. Twenty controlled trials (11 randomized and 9 non-randomized trials) met the inclusion criteria with a total of 862 participants. Methodological quality was assessed using the Cochrane's Risk of Bias for randomized studies and the Risk of Bias Assessment tool for non randomized studies. Data were analyzed using the RevMan 5.2.11 program of Cochrane library. RESULTS: Overall, study quality was moderate to high. The weighted average effect size across studies was -0.32 (95% CI [-0.56, -0.07], p=.010, I2=45%) for cortisol concentration, -0.62 (95%CI [-0.96,-0.29], p<.001, I2=0%) for T lymphocyte (CD3) and -0.45 (95%CI [-0.74, -0.16], p=.003, I2=0%) for Th lymphocyte (CD4) numbers. Psychosocial interventions were not effective for Tc lymphocyte (CD4), NK cell, monocyte, and cytokine response. CONCLUSION: Although these results provide only small evidence of successful immune modulation, they support the conclusion that psychosocial interventions can assist cancer patients in reducing emotional distress and improving immune response.
CD4-Positive T-Lymphocytes/cytology/immunology ; Cytokines/metabolism ; Databases, Factual ; Humans ; Hydrocortisone/*analysis ; Killer Cells, Natural/cytology/immunology ; Monocytes/cytology/immunology ; Neoplasms/metabolism/pathology/*therapy ; Psychotherapy ; T-Lymphocytes/cytology/*immunology