OBJECTIVETo investigate the mechanism that glutamine (Gln) downregulates the cytokine expression in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PMBCs).

METHODSPMBCs were extracted from healthy volunteer by density gradient centrifugation, the cells were divided into two parts. The first part of PMBCs was pretreated with Gln of the concentration of 0, 8, 15 mmol/L for 0.5 h and 2.0 h respectively, then stimulated by LPS for 4.0 h. Cells and supernatants were collected. The second part of PBMCs was divided into group A, B and C. Group A and B were pretreated with HSP70 blocker (Quercetin) for 1.0 h, then were stimulated by LPS for 4.0 h. Cells and supernatants were also collected. The release of TNF-alpha and IL-10 was analyzed via enzyme-linked immunosorbent assay (ELISA) and HSP70 via Western Blot. In this experiment, the effect of Quercetin on TNF-alpha, IL-10 and HSP70 expression in human PBMCs was assessed.

RESULTSGln led to an increase in HSP70 expression, and decreased TNF-alpha, IL-10 release at 4.0 h after LPS stimulation when 8 mmol/L glutamine pretreated for 0.5 h and 2.0 h, 15 mmol/L glutamine pretreated for 0.5 h (P < 0.05). The expression level of HSP70 was significantly decreased, however, the expression of TNF-alpha and IL-10 was enhanced in Quercetin group (P < 0.05).

CONCLUSIONThe effect of glutamine attenuating cytokine release in PBMCs is related to the enhancement of HSP70 expression.

Cells, Cultured ; Cytokines ; metabolism ; Glutamine ; pharmacology ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Interleukin-10 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lipopolysaccharides ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the effects of rare earth exposure on human telomerase and apoptosis of human peripheral mononuclear cells (PBMNs).

METHODSRare earth mine lot in Xunwu county, the biggest ion absorptive rare earth mine lot of China, was selected as the study site. Another village of Xunwu county, with comparable geological structure and social environment was selected as the control site. Thirty healthy adults were randomly selected from the study site as exposure group and another 30 healthy adults randomly selected from the control site as control group. The blood content of 15 rare earth elements, including La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu and Y, were determined by inductive coupled plasma-source mass spectrometry (ICP-MS). The total contents of rare earth elements in the blood were calculated. The TRAP and FCM assays were carried out to analyse the telomerase and apoptosis of human PBMNCs respectively.

RESULTSIn the exposure group, the concentration of La, Ce, Dy and Y were significantly higher (P<0.001), and Pr, Nd, Sm, Gd and Yb were higher than those in the control group (P<0.05). The total content of rare earth in the blood of exposure group showed significant difference compared with control group (P<0.001). Telomerase activity in PBMNs of the exposure group was higher than that in the control group (P<0.05); there were 11 adults in the exposure group (30 adults) and 5 adults in control group (30 adults) showed positive telomerase activity. The average age of the exposure group was (38.69 +/- 8.02) years-old, while the control group was (40.45 +/- 9.02) years-old (P >0.05). It was found that there was a significant relationship between telomerase activity and the total content of rare earth elements (P <0.01). 3. The proportion of apoptosis was not different between the two groups (P >0.05), but the cells in the S-phase and G2-M phase were increased (P <0.01) in the exposed group.

CONCLUSIONThe telomerase activity of PBMNs in the rare earth elements exposed group was higher than that of the control group, and there is no effect on apoptotic rate of PBMNs, but may promote the diploid DNA replication, and increase the percentage of G2/M and S phase cells.

Apoptosis ; drug effects ; Environmental Exposure ; adverse effects ; Female ; Humans ; Leukocytes, Mononuclear ; cytology ; enzymology ; Male ; Metals, Rare Earth ; adverse effects ; analysis ; Telomerase ; metabolism

Adult ; Apoptosis ; drug effects ; Benzene ; poisoning ; Biomarkers ; analysis ; Bone Marrow Cells ; drug effects ; Female ; Flow Cytometry ; Humans ; Leukocytes, Mononuclear ; drug effects ; Lymphocytes ; drug effects ; metabolism ; Male ; Micronuclei, Chromosome-Defective ; drug effects ; metabolism ; Occupational Exposure ; analysis

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

OBJECTIVETo observe the effect of astragaloside on oxidative low-density lipoprotein (Ox-LDL) mediated oxidative damage of endothelial progenitor cells (EPCs).

METHODSPeripheral blood mononuclear cells(PBMCs) were isolated by Ficoll density gradient centrifugation, and EPCs were identified by flow cytometry. Adherent cells were collected after seven-day incubation and randomly divided into the normal control group, the Ox-LDL group (as the model group, at the dose of 100 microg/mL), the low, middle, and high astragaloside groups (with 100 microg/mL Ox-LDL plus 2, 10, and 50 microg/mL astragaloside). Twenty-four h later, the proliferation and adhesion capabilities of EPCs were observed using MTT colorimetry and the adhesion capability detection. Levels of superoxide dismutase (SOD) and malonaldehyde (MDA) in the cell supernate of each group were determined.

RESULTSAfter Ox-LDL damage, the proliferative and adhesive capacities of EPCs were significantly injured (53 +/- 8 vs 42 +/- 6, 0.49 +/- 0.12 vs 0.37 +/- 0.02, both P<0.05). The SOD content obviously decreased (21.95 +/- 1.43 vs 14.76 +/- 3.99, P<0.01), the MDA content obviously increased (3.72 +/- 0.30 vs 5.57 +/- 0.64, P<0.01). After intervened by astragaloside for 24 h, the proliferative and adhesive capacities of EPCs were significantly improved. The SOD contents of astragaloside intervention groups were obviously improved and the MDA content obviously lowered.

CONCLUSIONSAstragaloside showed significant protection on Ox-LDL damaged EPCs. Its mechanism might be correlated with antioxidative damage.

Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Lipoproteins, LDL ; metabolism ; Malondialdehyde ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; drug effects ; Saponins ; pharmacology ; Stem Cells ; cytology ; drug effects ; metabolism ; Triterpenes ; pharmacology

OBJECTIVETo investigate the effect of Lang-chuang-ding Decoction (, LCD) on the expression of DNA methylation of CD70 gene promoter in peripheral blood mononuclear cells (PBMCs) of females with systemic lupus erythematosus (SLE).

METHODSPBMCs isolated from female patients with SLE or healthy donors were cultured and treated with LCD medicated serum or normal serum for 24 or 48 h. The mRNA expressions of CD70 gene in PBMCs were detected by reverse transcription polymerase chain reaction (PCR); the DNA methylation of the CD70 gene promoter region was detected by methylation-specific PCR.

RESULTSAfter treated with medicated serum for 48 h, the mRNA expression levels of CD70 in PBMCs of SLE patients were signifificantly higher than those of healthy donors (P<0.05); the DNA methylation levels of CD70 promoter region in PBMCs of SLE patients treated with medicated serum for 48 h were signifificantly higher than those treated with fetal bovine serum (P<0.01).

CONCLUSIONLCD could inhibit CD70 gene expression in PBMCs of SLE patients by promoting the DNA methylation of CD70 gene promoter.

Adult ; CD27 Ligand ; genetics ; metabolism ; DNA Methylation ; drug effects ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lupus Erythematosus, Systemic ; drug therapy ; genetics ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

In order to study how to activate T cells and their immunological characteristics, the anti-CD3 and anti-CD28 McAbs were used to stimulate PBMNC, then their related immunological changes, such as lymphocyte transformation function, the percentage of CD8(+)CD25(+) cells and TGF-beta expression were deleted by lymphocyte transformation assay, flow cytometry and RT-PCR respectively. The results showed that in costimulation with anti-CD28 antibody stimulation, the activity of anti-CD3 antibody was significantly enhanced, the ratio of CD8(+)CD25(+) cells of T cells was obviously increased, while TGF-beta expression was down-regulated. It was concluded that the anti-CD28 antibody costimulation could provide stimulatory signal II, which make T cells more active, while the expression of TGF-beta significantly down-regulated.
Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; immunology ; CD3 Complex ; immunology ; Down-Regulation ; Humans ; Leukocytes, Mononuclear ; immunology ; metabolism ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta ; biosynthesis

To investigate the role of anti-inflammatory cytokine in acute coronary syndrome (ACS), the effect of IL-10 on expression of tissue factor (TF) induced by IL-6 in peripheral blood mononuclear cells (PBMNC) were studied. PBMNC were allowed to culture with rhIL-10 before being stimulated by rhIL-6. One-step recalcification clotting time was used to evaluate procoagulant activity (PCA) of PBMNC. The expression and activity of TF protein were determined by ELISA and cell chromogenic substrate assay. The results showed that the expression of PCA, TF protein and its activity in PBMNC increased significantly after being stimulated by rhIL-6 (P < 0.01). In PBMNC, rhIL-6-induced PCA was regulated by rhIL-10 in different doses. This effect was associated with reduction of TF protein expression and activity by rhIL-10 (P < 0.01). In conclusion, IL-10 down-regulated expression PCA and TF in PBMNC, inhibitory effect of IL-10 on expression and activity of PBMNC TF may be important protective mechanism for ACS, regulation imbalance between inflammatory and anti-inflammatory cytokines may be important factor participating in coronary thrombosis.
Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-10 ; pharmacology ; Interleukin-6 ; genetics ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; metabolism ; Recombinant Proteins ; pharmacology ; Thromboplastin ; biosynthesis

OBJECTIVETo investigate the effect of prolactin on CD40 and CD154 expressions on the surface of peripheral blood mononuclear cells in patients with systemic lupus erythematosus (SLE) and explore the role of prolactin in the pathogenesis of SLE.

METHODSThe serum prolectin level was detected in 30 SLE patients and 20 healthy volunteers, from whom peripheral blood mononuclear cells (PBMCs) were also isolated to examine the expressions of CD40 and CD154 using flow cytometry.

RESULTSCD154 significantly increased on the PBMCs in SLE patients with high serum prolectin level in comparison with that in patients with normal prolactin level or the normal controls (P<0.05). When the PBMCs were incubated with recombinant human prolactin, CD154 expression was significantly increased in SLE patients with normal serum prolactin level (P<0.05), but not in the normal control group (P>0.05). Incubation of the PBMCs in the presence of bromocriptine did not result in significantly decreased CD154 expression in SLE patients irrespective of the prolactin level, nor was significant difference found in CD40 expression on the surface of PBMCs between SLE patients and the normal controls(P>0.05).

CONCLUSIONProlactin plays an important role in the pathogenesis of SLE by increasing CD154 expression on the PBMCs, and bromocriptine produces no significant inhibitory effect on either endogenous or exogenous prolectin.

Adult ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lupus Erythematosus, Systemic ; blood ; metabolism ; pathology ; Male ; Prolactin ; blood ; pharmacology