This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.
Cell Separation ; methods ; Humans ; Interleukin-2 ; analysis ; Lymphocytes ; cytology ; drug effects ; metabolism ; Phytohemagglutinins ; pharmacology

OBJECTIVETo study melanoma cell fusion and find a highly efficient fusion method for tumor cells.

METHODSMelanoma cells were labeled with green fluorescent protein and red fluorescent protein, respectively, and fused by a modified phytohaemagglutinin (PHA)-ECM830 fusion method. Melanoma fusion cells were selected by the fluorescence activated cell sorting. DNA content was determined by propidium iodide staining.

RESULTSMelanoma cells were labeled with green fluorescent protein and red fluorescent protein markers and successfully fused through PHA-ECM830 fusion method. The fusion efficiency (7.18%) was much higher compared with ECM830 electricfusion method (0.50%). Melanoma fusion cells were successfully obtained by the fluorescence activated cell sorting.DNA content was doubled in melanoma fusion cells compared to B16-F10 melanoma cells. The proliferation rate of melanoma fusion cells was significantly decreased in vitro and in vivo.

CONCLUSIONSWe successfully obtained the melanoma fusion cells by the improved PHA-ECM830 fusion method. The proliferation rate of melanoma fusion cells dramatically decreases.

Animals ; Cell Fusion ; methods ; Cell Line, Tumor ; Cell Proliferation ; Melanoma, Experimental ; pathology ; Mice ; Phytohemagglutinins ; pharmacology

The purpose of study was to investigate the in vitro proliferation ability of PHA-induced CIK cells and traditionally prepared CIK cells, the effector cell level and its influence on killing activity to K562 cells, and to analyze the difference between them. The peripheral blood mononuclear cells(PBMNC) of healthy persons were isolated and divided into A and B group. The CIK cells in A group were obtained by using traditional culture method, the CIK cells in B group were prepared by PHA induction. During the cultivation, the cell survival rate and cell absolute value in the cell culture system were counted every 3 days. On day 15 of culture, the cell immunophenotype of 2 groups were detected by flow cytometry, and the ratios of CD3(+)CD56(+), CD3(+)CD8(+) and CD3(+)CD4(+) cells in total cell amount of culture system were accounted. Meantime, the killing activity to K562 cells in different effector-target ratios was detected by using CCK-8 kit between the 2 groups. The results showed that the method of preparing CIK by PHA induction promoted the cell proliferation more than that of the traditional method (P < 0.05), moreover, both the survival rate of cells in 2 groups was more than 90%. The CD3(+)CD8(+), CD3(+)CD56(+) cell ratio in 2 groups obviously increased. As compared with traditional method, the CD3(+)CD8(+) cell level in B group was enhanced (P < 0.05); but there were no statistical differences in increase of CD3(+)CD56(+) cell level and decrease of CD3(+)CD4(+) cell level between 2 groups. while the effector-target ratio is 5:1, 10:1, 20:1 and 40:1, the killing activity of PHA-induced CIK cells to K562 cells was more stronger than traditionally-prepared CIK cells (P < 0.05), moreover, along with increase of effector-target ratio, the difference of killing activity to K562 cells in 2 groups significantly increased. It is concluded that compared with traditional method for preparing CIK cells, the new way by PHA induction can increase the proliferation of CIK cells obviously, enhance the ratio of CD3(+)CD8(+) cells and strengthen the killing activity to the K562 cells. This new way provides a new source of CIK cells and reliable evidence for cyto-immune therapy of leukemia and other tumors.
Cell Proliferation ; Cytokine-Induced Killer Cells ; cytology ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; Phytohemagglutinins ; pharmacology

OBJECTIVETo study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development.

METHODIn experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage.

RESULTLow concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death.

CONCLUSIONDepending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Animals ; Embryo, Mammalian ; drug effects ; Embryonic Development ; drug effects ; Female ; Male ; Mice ; Phaseolus ; chemistry ; Phytohemagglutinins ; pharmacology ; Pregnancy

To study and compare the immunomodulatory functions of human bone marrow mesenchymal stem cell (MSC) and cord blood mononuclear cell (CBMNC) in vitro, human bone marrow MSC were separated with Percoll (1.073 g/L) and cultured in low-glucose DMEM, and cord blood mononuclear cells were isolated with Ficoll (1.077 g/L). These two kinds of cells were added to mixed lymphocyte cultures and PHA induction transformation cultures with various concentrations. The proliferation of lymphocytes was measured by MTT method, effects of MSC and CBMNC on mixed lymphocyte response and PHA-induced lymphocyte transformation were investigated. The results showed that both 5 x 10(4), 1 x 10(4) MSC and 2 x 10(5) CBMNC could inhibit the mixed lymphocyte response (MLR) and PHA induced transformation. But with lower concentrations (MSC < or = 1 x 10(3), CBMNC < or = 1 x 10(5)), the inhibition effects of MSC and CBMNC were less consistent. 1 x 10(2) MSC and 1 x 10(4) CBMNC mainly increased the lymphocyte activation. In addition, the inhibition ratio of 5 x 10(4) MSC (MLC 65.3%, PHA 79.1%) was higher than that of 2 x 10(5) CBMNC (MLC 8.6%, PHA 37.3%). It is concluded that larger numbers of both MSC and CBMNC showed negative immunomodulatory functions and inhibited the mixed lymphocyte response and induction of transformation in vitro. Moreover, the inhibitory effect of MSC was much stronger than that of CBMNC.
Bone Marrow Cells ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; physiology ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Mesenchymal Stromal Cells ; physiology ; Phytohemagglutinins ; pharmacology

OBJECTIVETo observe the TH cell subset function in children with recurrent tonsillitis (RT) at the remission stage and to study the effects of astragalus membranacus (AM) on TH cell subset function.

METHODSThe peripheral blood mononuclear cells (PBMC) from 27 children with RT at the remission stage were stimulated with either phytohemagalutinin (PHA) (RT-PHA group) or PHA together with AM (RT-AM group) and were then cultured in vitro for 48 hrs. The samples from 21 healthy children stimulated with PHA were used as the Control group. The levels of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernatants of PBMC were detected using ELISA.

RESULTSThe IFN-gamma level and the ratio of IFN-gamma/IL-4 in the RT-PHA group were statistically lower than those in the Control group (P < 0.01). The level of IFN-gamma and the ratio of IFN-gamma/IL-4 in the RT-AM group were markedly higher than those in the RT-PHA group (P < 0.01), but were significantly lower than those in the Control group (P < 0.05). There were no differences in the IL-4 level among the three groups.

CONCLUSIONSTH1 cell subset dysfunction may exit in RT children at the remission stage, suggesting that TH1 cell subset dysfunction plays an important role in the pathogenesis of RT. AM can improve TH1 cell subset function and therefore shows an important significance in treating RT.

Adolescent ; Astragalus membranaceus ; Child ; Child, Preschool ; Female ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Male ; Phytohemagglutinins ; pharmacology ; Recurrence ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Tonsillitis ; etiology ; immunology

OBJECTIVEA growing body of evidence suggests that the pineal hormone, melatonin (MLT), has immunomodulatory properties; MLT can induce an increment of cell proliferation and an increase or a decrease of a number of cytokines in adults' peripheral blood mononuclear cells (PBMC). However, the influence of MLT on the modulation of neonatal cord blood mononuclear cells (CBMC) proliferation has not been reported. The present study aimed to investigate the possible regulatory effects of MLT on the proliferation of CBMC in vitro.

METHODSTen cord blood preparations from placenta vena umbilicalis of 10 healthy full-term normally delivered newborns and 10 healthy adult volunteers' peripheral blood preparations as controls were obtained. Cord/peripheral blood mononuclear cells suspension were prepared, 2 x 10(5) cells were added to 96-well plate and co-cultured with different stimulants (in cell cultures containing 5 microg phytohemagglutinin (PHA)/ml, 50 ng MLT/ml, 5 ng MLT/ml, 500 pg MLT/ml, 50 pg MLT/ml, 50 ng IL-2/ml, 5 ng MLT/ml + 5 microg PHA/ml or 5 ng MLT/ml + 50 ng IL-2/ml) for 72 h, while the cell suspension (with no stimulant) was used as controls, also cultured for 72 h. With the methods of microscopic examination and (3)H-TdR incorporation test, the influence of melatonin on CBMC morphology and proliferation were investigated, and the effects of MLT on the proliferation of CBMC and PBMC were compared with LSD-t T test and independent samples T test.

RESULTSIn CBMC, MLT (50 pg/ml - 50 ng/ml) increased the (3)H-TdR incorporation rates in a dose-dependent manner, the rate was the highest in the concentration of 5 ng/ml. After 72 h of cell culture, the number of cells in the MLT (5 ng/ml)-exposed group was higher than that recorded before incubation when observed under the high power microscope, including many big mononuclear cells. After adding different stimulants MLT (5 ng/ml), IL-2 (50 ng/ml), MLT plus PHA (5 microg/ml) or MLT plus IL-2 into mediums, the (3)H-TdR incorporation rates of CBMC (cpm) was 114 327 +/- 52 863, 16 087 +/- 9006, 118 360 +/- 59 207 and 17 682 +/- 7391 respectively. In comparison with the controls (14 133 +/- 8688), the incorporation rates of both MLT-exposed group and MLT + PHA-exposed group increased significantly (t = 5.9143, P < 0.001; t = 5.5078, P < 0.001); the rate of IL-2-treated group or MLT + IL-2-treated group demonstrated no significant changes (t = 0.4938, P > 0.05; t = 0.9839, P > 0.05); while the incorporation rates of MLT-exposed group or MLT + PHA-exposed group had no significant difference compared with that of PHA-exposed group (t = 0.1730, P > 0.05; t = 0.3286, P > 0.05). After adding different stimulants into the medium, the incorporation rates of CBMC were all higher than those of PBMC.

CONCLUSIONSMLT can promote not only the proliferation of PBMC, but also the proliferation of CBMC, and the effects of MLT on CBMC were stronger than those on PBMC. This suggests that MLT could be involved in the regulation of the newborn immune system and suggests a new immunotherapeutic strategy in the treatment of certain diseases of neonates.

Adult ; Blood Cells ; drug effects ; physiology ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; Cells ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; Humans ; Infant, Newborn ; Interleukin-2 ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; physiology ; Melatonin ; pharmacology ; Phytohemagglutinins ; pharmacology ; Pregnancy

OBJECTIVETo confirm that arsenic (As) induces oxidative DNA damage in phytohemagglutinin (PHA)-stimulated and unstimulated human lymphocytes.

METHODSThe alkaline comet assay combined with specific enzyme (Formamidopyrimidine-DNA glycosylase, FPG) digestion was used to measure As-induced base damage.

RESULTSThe enzyme-sensitive sites were readily detected with the alkaline comet assay after the cells were treated with 10 micromol As for 2 hours. The repair patterns observed for FPG-created DNA single strand breaks (SSBs) in As-treated cells were comparable to those in hydrogen peroxide (H(2)O(2))-treated cells. The enzyme-created SSBs, As-induced base damage, were more significantly revealed in PHA-stimulated lymphocytes. About 63% and 68% of SSBs induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes by 2-hour repair incubation, but about 34% and 43%, respectively, were repaired in unstimulated cells. About 40% and 49% of base damage induced by As and H(2)O(2), respectively, were repaired in PHA-stimulated lymphocytes, but about 19% and 21 %, respectively, were repaired in unstimulated cells.

CONCLUSIONSAs induces oxidative DNA damage in human lymphocytes within micromolar concentrations. Like the damage induced by H(2)O(2), As-induced DNA damage was more slowly repaired in unstimulated lymphocytes.

Adult ; Arsenic ; pharmacology ; DNA Damage ; DNA Repair ; DNA, Single-Stranded ; drug effects ; DNA-Formamidopyrimidine Glycosylase ; Electrophoresis ; methods ; Humans ; Hydrogen Peroxide ; pharmacology ; Lymphocytes ; drug effects ; N-Glycosyl Hydrolases ; Oxidation-Reduction ; Phytohemagglutinins ; pharmacology

Familial hypercholesterolemia(FH) is a disease based on defects of low-density lipoprotein receptors(LDL-R). To interrupt and control the natural course of this disease, early identification of these patients is important. The routine lipid profile tests for hypercholesterolemia can not differentiate objectively FH from secondary hypercholesterolemia. The exact diagnosis of FH heterozygotes is especially essential because it is easier to develop premature coronary heart diseases compared with secondary hyper-cholesterolemia. A simplified rapid and precise method for the mass screening of FH patients and the differentiation between FH heterozygote and secondary hyperlipidemia was needed. For the test, lymphocytes were used as target cells in LDL-R assay. After a 5 day culture with anti-CD3 Ab as a mitogen, indirect immunofluorescence stain and flow cytometric analysis were applied. The results were as follows; 74 +/- 9% of the stimulated lymphoblasts from normal controls expressed LDL-R activity. Cultured, but unstimulated, lymphocytes of normal controls showed 27 +/- 8% positivity and total cultured lymphocytes showed positivity of 46 +/- 11% positivity. Lymphoblasts, unstimulated lymphocytes, and total cultured lymphocytes from hyper-cholesterolemia without FH showed 74 +/- 10%, 25 +/- 10% and 50 +/- 17%, respectively, which showed no significant differences from normal control groups. FH Heterozygotes showed LDL-R positivity, 21 +/- 11% in lymphoblasts, 11 +/- 6% in unstimulated lymphocytes and 18 +/- 7% in total cultured lymphocytes. These data imply that adequately stimulated lymphocytes might be used for detecting defects in LDL-R and used to differentiate FH from secondary hypercholesterolemia.
Adult ; Antibodies/*pharmacology ; Antigens, CD3/*immunology ; Female ; Human ; Hypercholesterolemia, Familial/*blood ; Lipids/blood ; Lymphocyte Activation/drug effects ; Lymphocytes/drug effects/*ultrastructure ; Male ; Middle Age ; Phytohemagglutinins/pharmacology ; Receptors, LDL/*analysis ; Support, Non-U.S. Gov't

OBJECTIVETo explore the effect of mouse bone marrow mesenchymal stem cells (MSCs) on the expression of chemokine receptors in T lymphocytes in vitro.

METHODSMouse bone marrow MSCs were separated with Percoll, cultured and expanded in low glucose DMEM. C57BL/6 mouse spleenocytes were cultured in the 24-hole flasks by the density of 1 x10(6)/hole. Phytohemagglutinin (PHA) was then added to the holes and cultured for 72 hrs. This study consisted of three groups. Groups A and B were co-cultured by adding MSCs as the ratio of 0.1 and 0.01 to spleenocytes respectively. The control group was cultured without MSCs. Three days later the suspended spleenocytes were harvested for detecting the expression of three chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes by the flow cytometry.

RESULTSThe expression of CD3(+)CCR5(+) and CD3(+)CCR7(+) were statistically different among the three groups. Group A had the strongest expression, followed by group B and the control group. The expression of CD3(+)CXCR3(+) in group A was statistically higher than that in group B and the control group.

CONCLUSIONSMSCs could up-regulate the expression of chemokine receptors CXCR3, CCR5 and CCR7 in T lymphocytes stimulated by PHA.

Animals ; Bone Marrow Cells ; physiology ; Cells, Cultured ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; physiology ; Mice ; Mice, Inbred C57BL ; Phytohemagglutinins ; pharmacology ; Receptors, CCR5 ; analysis ; Receptors, CCR7 ; analysis ; Receptors, CXCR3 ; analysis ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology