The aim of this study was to compare cell proliferation and function of the T cells acquired under various culture conditions for establishing a simple, safe and efficient cell expansion protocol in vitro. The peripheral blood mononuclear cells (PBMNC) were isolated and stimulated with autologous dendritic cells (DC) and EBV-transformed B lymphoblastoid cell line (BLCL) weekly. The cell proliferation test, flow cytometry with PI and Annexin V double staining, Cr release test and ELISPOT test were used to detect the cell expansion level, frequency of IFN-γ producing T cells, killing activity of antigen-specific T cells, cell apoptotic status and cell differentiation potential, respectively. The results indicated that use of IL-2 combined with IL-7 and IL-15 resulted in the highest cell expansion comparing to the use of IL-2 alone and the use of CD3/28 Microbeads. Also the cells obtained under cultivating with IL-2, IL-7 and IL-15 together showed high frequency of IFN-γ producing cells, strong killing activity, high viability and high differentiation potential with large portion of CD3(+)CD8(+) population among the T cells. It is concluded that a protocol is established in which the use of IL-2 combined with IL-7 and IL-15 induces the biggest cell expansion, expanded cells show high viability, strong differentiation potential, high frequency of IFN-γ producing cells and strong killing activity.
Cell Line, Transformed ; Cell Proliferation ; Cell Separation ; Dendritic Cells ; cytology ; metabolism ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; T-Lymphocytes ; cytology ; drug effects ; metabolism

To explore the efficacy of the different combinations of stem cell factor (SCF), FLT3 ligand (FL) and interleukin (IL) 2, 7, 15 on the induction and expansion of cord blood (CB) derived CIK/ NK cells in vitro, according to the different combinations of cytokines, combinations of cytokines were divided into 3 groups: group A (SCF + IL2 + IL7 + IL15), group B (SCF + FL + IL2 + IL7 + IL15) and group C (IL2 + IL7 + IL15 as control group). At 21 days of culture, the proportion and the expansion rate of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells were measured. The results showed that at 21 days in culture, the percentages of CD3(+)CD56(+) CIK cells derived from CB-MNC in group A, B and C were (19.84 +/- 2.93)%, (26.20 +/- 4.05)%, (24.03 +/- 4.99%) and that of NK cells were (49.60 +/- 1.40)%, (51.16 +/- 6.45)% and (30.85 +/- 8.12)%, respectively. The proliferation of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells was the most effective in group B (SCF + FL + IL2 + IL7 + IL15). Expansion multiple of CIK cell number increased from 575.81 +/- 221.72 (control) to 796.09 +/- 278.47 with peak value of 1 313, and that of NK cells increased from 30.39 +/- 14.47 (control) to 65.85 +/- 30.83 with peak value of 121.06. In conclusion, a proper cytokines (SCF + FL + IL2 + IL7 + IL15) culture system can effectively induce and expand CB CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells simultaneously.
CD3 Complex ; analysis ; CD56 Antigen ; analysis ; Cytokines ; pharmacology ; Fetal Blood ; cytology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukin-7 ; pharmacology ; Killer Cells, Natural ; drug effects ; Membrane Proteins ; pharmacology ; Stem Cell Factor ; pharmacology

OBJECTIVETo investigate the effects of interleukin 7 (IL-7) on B7 molecules expression and immunogenicity of acute leukemia (AL) cells.

METHODSThe B7 molecules expression on fresh acute leukemia cells and on the IL-7 exposed leukemia cells was detected by FACScan cytometer. B7-1 and B7-2 mRNA in IL-7 treated HL-60 cells were detected by reverse transcription-PCR (RT-PCR). The stimulation of proliferation of allogeneic peripheral blood mononuclear cells (PBMNC) by IL-7 treated leukemia cells was detected by MTT method. The level of interferon-gamma (IFN-gamma) secreted by the stimulated PBMNC was determined using enzyme-linked immunosorbent assays (ELISA). The blocking experiments were performed using monoclonal antibodies against B7-1, B7-2 and W6/32.

RESULTSB7-1 was weakly expressed in three, whereas B7-2 did in only one of eleven AL patients. IL-7 significantly enhanced B7 molecules expression on AL cells in a time-dependent manner. Furthermore, IL-7 could induce higher expression of B7-1 and B7-2 mRNAs on HL-60 cells. IL-7 treated leukemia cells could stimulate PBMNC proliferation and promote their IFN-gamma production. Anti-B7-1 and anti W6/32 but not anti-B7-2 monoclonal antibodies significantly inhibited the stimulated PBMNC proliferation and IFN-gamma secretion.

CONCLUSIONFresh AL cells express low level of B7-1 and B7-2 molecules. IL-7 enhances the B7 molecules expression on AL cells. The IL-7-treated leukemia cells can significantly stimulate the proliferation of allogeneic PBMNC and induce their IFN gamma secretion.

B7-1 Antigen ; genetics ; metabolism ; B7-2 Antigen ; genetics ; metabolism ; Humans ; Interleukin-7 ; pharmacology ; Leukemia ; immunology ; metabolism ; RNA, Messenger ; genetics ; Tumor Cells, Cultured ; Up-Regulation ; drug effects

AIMTo investigate the expression of interleukin-2 receptors (IL-2Rs) on MCF-7 cells, estradiol's regulation of IL-2Rs expression and the influence of IL-2 on the proliferation of MCF-7 cells.

METHODSImmunocytochemistry and flow cytometric analysis were used to investigate the expression of interleukin-2 receptors (IL-2Rs) by using of specific IL-2R polyclonal antibody; MTT method and 3H-TdR incorporation method were used to examine the changes of proliferation of MCF-7 cells.

RESULTSIL-2Ralpha, beta, gamma like immunoreactive substances can be found on MCF-7 cells and the IL-2Rgamma immunostaining was more strong than the other two. Estradiol of 10(-6) mol/L can increase the percentage of immunoreactive cells of IL-2Ralpha, beta and the expression of IL-2Rgamma. Exogenous addition of recombinant IL-2 of 100 U/ml to 1 000 U/ml can significantly increase the proliferation of MCF-7 cells.

CONCLUSIONMCF-7 cell can express IL-2R and estradiol can regulate their expression, IL-2 can influence the proliferation of MCF-7 cells.

Breast Neoplasms ; genetics ; metabolism ; Cell Division ; Estradiol ; pharmacology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; Receptors, Interleukin-2 ; metabolism

OBJECTIVETo investigate the effects of different concentrations of lipopolysaccharide (LPS), tumor necrosis factor α (TNFα), interleukin-6 (IL-6), dexamethasone (Dex), and insulin on the mRNA and protein expressions of GPR54 in the MCF7 cell line in vitro.

METHODSMCF7 breasr cancer cells were cultured and treated with different concentrations of LPS (10 and 20 µg/ml), TNFα (20 and 100 ng/ml), IL-6 (10 and 20 ng/ml), Dex (10(-6) and 10(-7) mol/L), and insulin (0.01 and 0.1 IU/L). Those treated with culture fluid only served as controls. The mRNA and protein expressions of GPR54 were measured by real-time PCR and Western blot, respectively, after 6, 24, 48, and 72 hours of treatment.

RESULTSCompared with the blank con- trol, LPS (10 and 20 µg/ml), TNFα (20 and 100 ng/ml), IL-6 (10 and 20 ng/ml), Dex (10(-6) and 10(-7) mol/L), and insulin (0.01 and 0.1 IU/L) significantly increased the expressions of GPR54 mRNA (P < 0.05) and protein (P < 0.05).

CONCLUSIONLPS, TNFα, IL-6, Dex, and insulin evidently increase the expression of GPR54 in the MCF7 cell line, indicating their influence on the function of gonads by regulating the GPR54 level.

Blotting, Western ; Dexamethasone ; administration & dosage ; pharmacology ; Glucocorticoids ; administration & dosage ; pharmacology ; Gonads ; drug effects ; metabolism ; Humans ; Hypoglycemic Agents ; administration & dosage ; pharmacology ; Insulin ; administration & dosage ; pharmacology ; Interleukin-6 ; administration & dosage ; pharmacology ; Lipopolysaccharides ; administration & dosage ; pharmacology ; MCF-7 Cells ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, G-Protein-Coupled ; drug effects ; genetics ; metabolism ; Receptors, Kisspeptin-1 ; Time Factors ; Tumor Necrosis Factor-alpha ; administration & dosage ; pharmacology

Interleukin-7 (IL-7) is known as a growth factor for pre B-cell and mature T-cells in human. But in leukemic cells, IL-7 effect is variously reported. To investigate the effect of IL-7 on the cells of childhood acute leukemia we used 3H-Thymidine assay. Twelve Acute lymphoblastic leukemia (ALL), seven T-ALL and three Acute myelogenous leukemia (AML) were involved in this study. Two out of twelve ALL and three out of seven T-ALL bone marrow (BM) cells were stimulated by IL-7 in 3H-Thymidine incorporation. In normal and AML BM cells, IL-7 had no stimulatory activity as in various leukemic cell lines. Two normal peripheral blood T-cells responded to IL-7 dose dependently. We have seen the effect of IL-7 to stimulate T-lineage cells but, for precise conclusion, further study using more purified samples will be needed.
Adolescent ; Child ; Child, Preschool ; Dose-Response Relationship, Drug ; Female ; Humans ; Infant ; Interleukin-4/pharmacology ; Interleukin-7/*pharmacology ; Leukemia, Myeloid, Acute/*immunology ; Leukemia-Lymphoma, Adult T-Cell/*immunology ; Lymphocytes/drug effects/*immunology ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*immunology ; Tumor Cells, Cultured

OBJECTIVETo explore the roles of NF-κB in factor VIIa-induced proliferation and migration of a colon cancer cell line (SW620) in vitro and its possible mechanism.

METHODSThe expression levels of NF-κB (p65), inhibitory protein of NF-κB (IκB-α), caspase-7, interleukin 8 (IL-8) and tissue factor (TF) in SW620 cells treated with factor VIIa, PDTC (an inhibitor of NF-κB) and other factors were measured by Western-blotting and real-time PCR. Proliferation and migration of the cells were analyzed by flow cytometry and Transwell assay, respectively.

RESULTSFactor VIIa down-regulated the IκB-α level in SW620 cells and increased the intranuclear level of NF-κB. Those effects of factor VIIa were blocked by anti-TF or anti-PAR2 antibodies. The effects of factor VIIa on proliferation and migration of SW620 cells, expression of IL-8, TF as well as caspase-7, were interfered by PDTC (the inhibitor of NF-κB).

CONCLUSIONSTF/VIIa complex activates NF-κB pathway via PAR2, thereby up-regulates IL-8 and down-regulates caspase-7 expression in SW620 cells, finally promotes proliferation and migration of colon cancer cells. In addition, TF/VIIa/PAR2/NF-κB pathway also upregulates TF expression, thus to create a positive feedback loop of TF/VIIa/PAR2/NF-κB/TF.

Antineoplastic Agents ; pharmacology ; Caspase 7 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-8 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; metabolism ; Thiocarbamates ; pharmacology ; Thromboplastin ; genetics ; metabolism ; Transcription Factor RelA ; antagonists & inhibitors ; metabolism

BACKGROUNDBone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs.

METHODS2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted.

RESULTSThe XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF.

CONCLUSIONSThe treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Colony-Stimulating Factor ; genetics ; Humans ; Interleukin-11 ; genetics ; Interleukin-6 ; genetics ; Interleukin-7 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Stem Cell Factor ; genetics ; Transforming Growth Factor beta ; pharmacology

BACKGROUNDRapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells.

METHODSPeripheral CD4(+)CD25(-) naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4(+)CD25(-) T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4(+)CD25(+)CD127(-) subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4(+)CD25(+) T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1 x 10(5) carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4(+)CD25(-) T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25 x 10(5)/well) in 96-well round-bottom plates for 6 days. Then 1 x 10(5) or 0.25 x 10(5) converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4(+)CD25(-) T cells using flow cytometry. Data were analyzed using CellQuest software.

RESULTSWe found that RAPA can convert peripheral CD4(+)CD25(-) naive T Cells to CD4(+)Foxp3(+) Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity.

CONCLUSIONOur findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigen-Presenting Cells ; drug effects ; immunology ; metabolism ; B-Lymphocytes ; drug effects ; immunology ; metabolism ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; metabolism ; Cell Proliferation ; drug effects ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Forkhead Transcription Factors ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-7 Receptor alpha Subunit ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mitomycin ; pharmacology ; Polymerase Chain Reaction ; Sirolimus ; pharmacology ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism

OBJECTIVETo investigate the mechanisms that coagulation factor VIIa promotes proliferation and migration of a colon cancer cell line (SW620 cells) in vitro.

METHODSThe expression of interleukin 8 (IL-8), tissue factor (TF), caspase-7 and p-p38 MAPK in SW620 cells treated with factor VIIa or protease activated receptor 2 agonist (PAR2-AP) was measured by ELISA, Western-blotting and QT-PCR.

RESULTSFactor VIIa and PAR2-AP induced IL-8 expression at both mRNA and protein levels, upregulated TF mRNA expression and TF activity, but down-regulated caspase-7 mRNA and p-p38 MAPK levels in SW620 cells. The effects of factor VIIa were not only blocked by anti-TF but also by anti-PAR2 antibodies.

CONCLUSIONFactor VIIa binds to TF on cell surface, forming a complex which activates PAR2, then provoking IL-8 and TF expression, and suppresses caspase-7 expression, thus promotes the tumor cell proliferation and migration. p38 MAPK may negatively regulate this process.

Caspase 7 ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; Interleukin-8 ; biosynthesis ; genetics ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; agonists ; Thromboplastin ; biosynthesis ; genetics ; p38 Mitogen-Activated Protein Kinases ; metabolism