This study was aimed to investigate the effect of dexamethasone (Dex) on immunosuppressive ability of mesenchymal stem cells (MSC) during expansion and differentiation of MSC. MSC were cultured in 96-well flat-bottom plates. Proliferation assays were performed by using the BrdU colorimetric ELISA Kit. To explore the effect of Dex on MSC immunosuppressive ability, MSC were firstly cultured in complete culture medium for 14 d with Dex (10 nmol/L), and then, peripheral blood mononuclear cells (PBMNC) were co-cultured with MSC in 96-well flat-bottom plates for 3 d. Phytohemagglutinin A (PHA, 10 µg/ml) was used to stimulate activation of PBMNC. The concentrations of IFN-γ in culture supernatants was detected by ELISA. The results indicated that there was no obvious difference in representative phenotypes of MSC between experimental and control groups after MSC were treated with low concentration of Dex (10 nmol/L) for 14 d, but the suppression of Dex-treated MSC on lymphocyte activation in same concentration of cells was significantly reduced as compared with control group. After the Dex-treated MSC were co-cultured with IFN-γ for 12 h, the immunoregulatory ability of MSC was recovered in a certain degree. It is concluded that the Dex impairs the immunosuppressive ability of MSC, the IFN-γ can protect and reverse the immunosuppressive ability of MSC impaired by Dex, so that, when the immunoregulatory activity of MSC is investigated, it is necessary to avoid adding Dex in the culture medium.
Cells, Cultured ; Dexamethasone ; adverse effects ; Humans ; Immune Tolerance ; drug effects ; Interferon-gamma ; immunology ; Leukocytes, Mononuclear ; Lymphocyte Activation ; immunology ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology

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

OBJECTIVETo investigate the levels of T cell receptor rearrangement excision DNA circles (TRECs) within peripheral blood from workers exposed to lead, and thereby to evaluate the number of naive T cells and recent thymic output function.

METHODSQuantitative detection of TRECs in peripheral blood mononuclear cells (PBMNC) from 10 cases of workers exposed to lead was performed by real time PCR analysis. 11 workers without exposure to lead served as unexposed controls. In addition, the relationship between TRECs, age, length of service, blood lead, urea lead, blood ZPP and urea delta-ALA was investigated.

RESULTSThe mean value of TRECs in workers exposed to lead was (2.44 +/- 1.87)/1000 PBMC, significantly under (5.60 +/- 3.96)/1000 PBMC in unexposed controls. A significant negative correlation was found between the TRECs and urea-ALA. But there was no significant correlation between them after controlling for blood lead, urea lead.

CONCLUSIONLead exposure may damage thymic output naive T cells function. Furthermore, low-level exposure to lead may damage immune system and earlier than expected.

Adult ; DNA ; drug effects ; Gene Rearrangement, T-Lymphocyte ; drug effects ; Humans ; Lead ; toxicity ; Leukocytes, Mononuclear ; Male ; Occupational Exposure ; adverse effects ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes ; drug effects ; immunology ; Thymus Gland ; immunology

OBJECTIVETo observe the role of interleukin (IL) 21 alone, IL12 alone, and IL21 plus IL12 for inducing the antitumor activity of peripheral blood mononuclear cells (PBMCs) in patients with endometrial cancer.

METHODSPBMCs were isolated from peripheral blood in patients with endometrial cancer in vitro, and kept the culture with low-level IL2. IL2-stimulated PBMCs were cocultured under different conditions (with anti-IL21 antibody, IL21 alone, IL12 alone, or IL21 plus IL12) for 72 h. The cytotoxicity of PBMCs was then examined by lactate dehydrogenase(LDH) released assay. CD4(+) CD25(+) FOXP3(+) regulatory (Treg) cell and CD4(+) IL17A(+) T-helper (Th17) cell proportion were determined with flow cytometry. Cell proliferation and apoptosis were measured by cell counting kit-8(CCK-8)assay and flow cytometry, respectively.

RESULTSIn comparison to control group, both IL21 and IL12 significantly enhanced the cytotoxicity of PBMCs. The IL21 plus IL12 group had superior effect to IL21 alone and IL12 alone. IL21 and IL12 significantly decreased the percentages of Treg cells and the rate of PBMCs apoptosis. IL21 or IL12 had no significant effect on the differentiation of Th17 cells and the proliferation of PBMCs.

CONCLUSIONSIL21 and IL12 can enhance the cytotoxicity of PBMCs in patients with endometrial cancer, which can be further strengthened with treatment of IL21 plus IL12. Such effects may be achieved by inhibiting the differentiation of Treg cells and the apoptosis of PBMCs, but not by the differentiation of Th17 cell.

Adult ; Aged ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endometrial Neoplasms ; immunology ; pathology ; Female ; Humans ; Interleukin-12 ; pharmacology ; Interleukins ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; immunology ; pathology ; Middle Aged ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVESTo explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV.

METHODSPBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR.

RESULTSWhen PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b.

CONCLUSIONIL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.

Adjuvants, Immunologic ; pharmacology ; Adult ; Female ; Hepatitis B Core Antigens ; pharmacology ; Hepatitis B virus ; drug effects ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-18 ; immunology ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; immunology ; Male ; Transfection

OBJECTIVETo explore an alternative method for easier and more effective establishment of allergen-specific T-cell clones (TCC) from peripheral blood monouclear cells (PBMCs) of allergic asthma patients with allogeneic feeding cells.

METHODSTo determine the optimal condition for T cell growth and effective dose and time of mitomycin-C (MMC) treatment of the feeding cells to prevent their proliferation, the PBMCs were treated with PHA, IL-2 or MMC at different concentrations, and the proliferation rate of the treated cells was analyzed by MTT assay. The effect of IL-4 on the growth and subset selection of TCC was also analyzed. Allergen-specific TCC was established by limiting dilution method with allogeneic PBMCs as the feeding cells, and the proliferation of the allergen-specific TCC was observed to evaluate the feasibility of the feeding cells.

RESULTSPHA at 25 microg/ml and IL-2 at 27 U/ml achieved optimal growth of the T cells, while MMC treatment at the dose of 60 microg/ml for 80 min effectively enriched the non-proliferative feeding cell from the PBMCs. IL-4 could not promote the survival of the TCC, but promoted the formation of CD(4)(+) TCC. Allergen-specific TCC obtained using allogeneic feeding cells required the presence of PHA, but the allergen reactivity of the TCC remained unpredictable.

CONCLUSIONIL-4 can promote the formation of CD(4)(+) TCC, but allogeneic feeding cells may fail to produce TCC with high allergen specificity.

Allergens ; immunology ; Asthma ; blood ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Cells, Cultured ; Clone Cells ; cytology ; drug effects ; immunology ; Humans ; Interleukin-2 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Mitomycin ; pharmacology ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; T-Lymphocytes ; cytology ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology

We have investigated in vitro proliferative responses of peripheral blood mononuclear cells and productions of interferon-gamma and soluble interleukin-2 receptors by these cells from 6 patients with chronic active hepatitis B immediately before and 24 hours after a single intravenous injection of 100 mg of polyadenylic.polyuridylic acid. Cell proliferations were assessed by the technique of tritiated-thymidine incorporation and productions of interferon-gamma and soluble interleukin-2 receptors were measured by enzyme-linked immunosorbent assay. The administration of polyadenylic.polyuridylic acid to the patients has resulted in significant increases of in vitro proliferations of their peripheral blood mononuclear cells as well as productions of interferon-gamma by these cells. However, in vitro productions of soluble interleukin-2 receptors were not changed significantly. These results suggest that the enhanced cellular responses by polyadenylic.polyuridylic acid might be due to the increased sensitivity rather than the increased expression of cellular interleukin-2 receptor.
Adult ; Hepatitis B/*immunology ; Hepatitis, Chronic/*immunology ; Human ; Immunity, Cellular/drug effects ; Interferon Type II/biosynthesis ; Leukocytes, Mononuclear/*drug effects/immunology ; Male ; Middle Age ; Poly A-U/*pharmacology ; Receptors, Interleukin-2/biosynthesis ; Solubility

OBJECTIVETo investigate the specific antitumor immunity induced by antigen peptide mixture prepared from different T lymphocytic leukaemia cells and the cross-reaction among the mixtures of different cell lines.

METHODSAntigen peptide mixtures were prepared from different leukaemia cell lines and then bound with Hsp70 in vitro. The activation and proliferation of peripheral blood mononuclear cell (PBMC) were observed after the stimulation by different Hsp70-peptide complexes. The cytotoxicity of such activated PBMCs to different target cells was assayed.

RESULTSThe antigen peptides from different leukaemia cell lines were mixed ones, which could activate PBMC effectively with Hsp70 and stimulate the activated PBMC to proliferate. The proliferative PBMC had specific cytotoxicity to the corresponding leukaemia cells. To Hut-78 cell, Molt-4 cell and Jurkat cell, the cytotoxicity of PBMC activated by either Hut78-peptides or Molt-4-peptides was significantly stronger than that of PBMC activated by HL-60-peptides (P < 0.05). The cytotoxicity to Jurkat cell of PBMC activated by Hut78/Molt-4-peptides was significantly stronger than that of PBMC activated by Hut78-peptides or Molt-4-peptides alone (P < 0.05).

CONCLUSIONAntigen peptide mixture from T lymphocytic leukaemia cells is able to induce specific antitumor immunity. There is a cross-reactivity among antigen peptide mixtures from different T lymphocytic leukaemia cell lines, with the more crossed antigen peptides obtained from the mixtures of different antigen peptides from different T lymphocytic leukaemia cell lines, which suggests that the antigen peptide mixture with broad antigenic spectrum could possibly be prepared by using multiple leukaemia cell lines.

Antigens, Neoplasm ; metabolism ; pharmacology ; Cross Reactions ; HL-60 Cells ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Leukemia, T-Cell ; immunology ; Leukocytes, Mononuclear ; cytology ; drug effects ; Peptides ; pharmacology ; T-Lymphocytes, Cytotoxic ; drug effects ; immunology ; Tumor Cells, Cultured ; chemistry ; immunology

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects

Purpose of this study was to establish an effective method in vitro to proliferate natural killer T (NKT) cells from umbilical cord blood (UCB) and peripheral blood (PB), and to study their different phenotype. Mononuclear cells (MNC) from UCB and PB were cultured in the presence of IL-2 (100 U/ml), with or without alpha-Galcer. TCR Valpha24 Vbeta11 double positive natural killer T-cells (NKT cells) and their other phenotypes were determined by flow cytometry. The results showed that after expansion for 7 days, TCRValphabeta(+) NKT cells from UCB-MNCs increased by (8.74 +/- 4.37) x 10(2) times as much, but most of them did not express NK1.1 and its TCR Vbeta11(+) was higher than TCR Valpha24(+). After expansion for 14 days, TCR Valphabeta(+) NKT cells from PB-MNCs increased by (3.72 +/- 2.01) x 10(2) times, the expression of NK1.1 was high and its TCR Vbeta11(+) was almost equal to TCR Valpha24(+). It is concluded that human TCR Valpha24 Vbeta11 double positive NKT cells can expand by addition of alpha-Galcer. The proliferation efficiency in UCB-MNCs is greater than that in PB-MNCs. Most of the UCB-NKT is NK1.1(-), while the PB-NKT is NK1.1(+), a new subset of NKT cells.
Cell Proliferation ; Cells, Cultured ; Fetal Blood ; cytology ; Galactosylceramides ; pharmacology ; Humans ; Interleukin-2 ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Leukocytes, Mononuclear ; cytology ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology