The aim of this study was to explore the cytotoxicity of fresh cord blood(CB) NK cells and the influence of IL-12 and IL-15 on activity of the NK cells killing K562 and Jurkat cells lines. The NK cells were isolated from cord blood by depleting CD3(+) cells and then enriching CD56(+) cells using sorting with immunomagnetic beads. The experiment was divided into 3 groups: group A (fresh CB-NK cells without cytokines), group B (CB-NK cells cultured by IL-2) and group C (CB-NK cells cultured by IL-2 and IL-15). The purity of NK cells was determined by flow cytometry; the cytotoxity of fresh and different cytokine-treated CB-NK cells on K562 and Jurkat cell lines was detected by LDH release test. The results showed that the purity of NK cells before and after sorting was 14.88 ± 9.2% and 92.39 ± 0.8% respectively. After culture for 3 days, NK-forming colony amounts in group B and group C were 148.60 ± 13.0 and 831.80 ± 23.0 respectively, the comparison between group B and group C showed the significant difference (p < 0.05). The cytotoxicities of NK cells in group A, B and C on K562 and Jurkat cell lines were 27.76 ± 8.8%, 61.90 ± 9.1% and 87.62 ± 3.7%; 29.32 ± 2.5%, 69.43 ± 4.4% and 92.95 ± 3.2% respectively, the difference was significant (p < 0.05). It is concluded that the fresh isolated CB-NK cells show low cytotoxic activity. After stimulated with IL-2 or IL-2 plus IL15, cytotoxicity of CB-NK cells increases obviously, the effect of IL-2 plus IL-15 is much better than IL-2 alone for promoting the growth and enhancing the cytotoxicity of CB-NK cells.
Cytotoxicity, Immunologic ; drug effects ; immunology ; Fetal Blood ; drug effects ; immunology ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Jurkat Cells ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

OBJECTIVETo prepare an O/W ginseng saponins-based nanoemulsion and investigate its amplified immune response.

METHODThe formulation of ginseng saponins-based nanoemulsion was optimized via the range of nanoemulsion zone in phase diagrams and the solubility of ginseng saponins. Its physicochemical properties were investigated, including morphology, particle size distribution, pH, viscosity and stability. Ginseng saponins-based nanoemulsion as adjuvant was co-administrated with a model antigen ovalbumin (OVA) in mice. Two weeks after the boosting, the serum levels of OVA-specific antibody and its isotypes were determined.

RESULTThe optimized ginseng saponins-based nanoemulsion formulation consisted of ginseng saponins, IPM, Cremophor RH 40, glycerol and water (with the weight ratio of 2 : 4 : 17.8 : 17.8 : 58.4), which was a light yellow fluid. The shape of droplets was spherical under transmission electron microscopy with an average diameter of 72.20 nm and a polydispersity index of 0.052. The viscosity and pH value of it were 4.20 s and 6.02, respectively. And it showed good stability. When co-administered with OVA, no obvious side effects were observed in the mice immunized with ginseng saponin-based nanoemulsion. The serum levels of IgG, IgG1 and IgG2a antibody in the group of ginseng saponin-based nanoemulsion immunized mice was significantly increased compared to the groups of OVA and the saline solution of ginseng saponin. Compared with the adjuvant aluminium hydroxide, the serum levels of IgG and IgG1 antibodys in the groups of ginseng saponins-based nanoemulsion had no significant difference, but the level of IgG2a was obviously higher.

CONCLUSIONginseng saponin-based nanoemulsion could amplify the Th1 and Th2 immune responses, and can be used as the vaccine adjuvant.

Animals ; Drug Carriers ; chemistry ; Emulsions ; chemistry ; Female ; Immune System Phenomena ; drug effects ; Immunoglobulin G ; blood ; immunology ; Mice ; Mice, Inbred ICR ; Panax ; chemistry ; immunology ; Particle Size ; Random Allocation ; Saponins ; chemistry ; immunology ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; immunology

In order to investigate the effect of anti-TGF-beta1 monoclonal antibody on the expansion of cord blood CD34(+) cells, the purified cord blood CD34(+) cells were divided into three groups: blank control group: purified cord blood CD34(+) cells cultured on day 0; control group: cells cultured for 3 days in the culture system, containing SCF, IL-3, IL-6 and FLT3-L; test group: cells cultured for 3 days in the same culture system as control group, but with anti-TGF-beta1 monoclonal antibody. The mononuclear cell counting (MNC), the expression of CD34 and c-kit, and CFU-GEMM, BFU-E and CFU-GM counting were detected in all three groups. The result showed that the expansion of MNCs, CD34(+) cells and CD34(+)c-kit(+) cells in test group [(2.35 +/- 0.25) x 10(5), (1.16 +/- 0.29) x 10(5), (1.09 +/- 0.26) x 10(5)] was significantly higher than that in control group [(1.25 +/- 0.13) x 10(5), (0.55 +/- 0.19) x 10(5), (0.51 +/- 0.2) x 10(5)](p < 0.01). The expansion of more primitive CD34(+)c-kit(-) subpopulation in test group [(12.95 +/- 3.17) x 10(3)] was even significantly higher than in control group (1.71 +/- 0.83) x 10(3) (p < 0.01). Colony forming assay showed that the number of earlier progenitor colony CFU-GEMM, BFU-E in test group [(16.3 +/- 4.72) x 10(3), (60.5 +/- 20.96) x 10(3)] was higher than that in control group [(5.0 +/- 2.58) x 10(3), (16.25 +/- 7.93) x 10(3)] (p < 0.01). The number of relatively mature CFU-GM between test group and control group was not statistical significance [(6.33 +/- 2.85) x 10(3) vs (4.0 +/- 2.28) x 10(3)](p > 0.05), but both were higher than that in blank group (0.75 +/- 0.29) x 10(3). These results demonstrated that anti-TGF-beta1 monoclonal antibody promoted the expansion of MNC and CD34(+) cells, and even more marked expansion of the more primitive progenitor cells-CD34(+)c-kit(-) cells. Meanwhile, it enhanced the output of more immature colony CFU-GEMM and BFU-E, but had no evident influence on the mature myeloid colony CFU-GM. It is concluded that the anti-TGF-beta1 monoclonal antibody can synergize other cytokines to enhance the proliferation of cord blood CD34(+) progenitor cells effectively, and it is more important that can reserve more primitive progenitor cells.
Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD34 ; immunology ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; drug effects ; immunology ; Humans ; Transforming Growth Factor beta1 ; immunology

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

To study the effect of interleukin-15 (IL-15) on the proliferation, differentiation and apoptosis of MDS CD34(+) cells, CD34(+) cells of high enrichment were separated by MACS system, and cultured in liquid media with different concentration of IL-15 in treated group and without IL-15 in the control group. Apoptosis of hematopoietic precursors was assayed by propidium iodine staining and cell by FCM, and the other MDS CD34(+) cells were stained by cytochemical staining after culture. The results showed that after culture with IL-15 the proliferation and differentiation of MDS CD34(+) cells were obviously promoted. It was found the every lineage of mature cells developed, the expressions of cell surface antigens CD71, CD33 and CD19 all increased in the MDS CD34(+) cell treated with IL-15. It is suggested that IL-15 stimulates the proliferation and differentiation of MDS CD34(+) cells, and partly shows anti-apoptosis effects which may be applicable to the therapy MDS.
Antigens, CD ; immunology ; Antigens, CD19 ; immunology ; Antigens, CD34 ; immunology ; Antigens, Differentiation, Myelomonocytic ; immunology ; Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; immunology ; pathology ; Cell Cycle ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flow Cytometry ; Humans ; Interleukin-15 ; pharmacology ; Microscopy, Fluorescence ; Myelodysplastic Syndromes ; blood ; immunology ; pathology ; Receptors, Transferrin ; immunology ; Sialic Acid Binding Ig-like Lectin 3

The aim was to study the roles that the bone marrow mesenchymal stem cells (MSC) and cytokines play in cord blood CD34(+) cell expansion ex vivo and the influence of culture ex vivo on expression of the adhesive molecule of CD44. CD34(+) cells sorted from cord blood cells had been cultured in each well of 24 well culture plates containing culture medium supplemented with mesenchymal stem cells layer or/and cytokines for a week, and then all kinds of indexes of different groups were compared. The results showed that as for cord blood cell expansion, there was no significant difference between the groups with cytokines SDF-1alpha + SCF + TPO + FL and SCF + TPO + FL no matter if MSC layer existed or not. The groups with MSC layer and cytokines were superior to the corresponding groups without MSC layer. In addition, the expression of the adhesion molecule CD44 had no distinct change after culture. It is concluded that SDF-1alpha has no distinct influence on the effect of cytokines SCF + TPO + FL on cord blood cell expansion ex vivo. MSC enhance the effect of cytokines on cord blood cell expansion ex vivo. Such expansion ex vivo may not influence the expression of the adhesive molecule CD44 on cord blood cells.
Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytokines ; pharmacology ; Female ; Fetal Blood ; cytology ; drug effects ; immunology ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; drug effects ; immunology ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; drug effects ; immunology ; Pregnancy

The aim of this study was to investigate the activation ability of CpG oligodeoxynucleotide (CpG ODN) 2216 on the peripheral blood mononuclear cells (PBMNCs) from leukemia patients in remission and the killing effect of activated PBMNCs on K562 cells. PBMNCs obtained from leukemia patients in remission were incubated with CpG ODN 2216. In control group, PBMNCs were incubated with normal saline (NS). The concentrations of cytokines (IFN-gamma, interleukin-12, interleukin-4, interleukin-10) in culture supernatant of PBMNCs from leukemia patients in remission were analyzed by using ELISA kits. The percentages of Th1, Tc1, Th2, Tc2 cells and killed K562 cells were detected by flow cytometry. The results showed that as compared with control group, CpG ODN 2216 induced higher concentrations production of IFN-gamma, IL-12 in supernatant (p < 0.01). There were no differences in IL-4, IL-10 in supernatant as compared with control group (p > 0.05). The percentages of Th1 and Tc1 cells increased significantly after culture with CpG ODN 2216 as compared with control group (p < 0.05). There was no difference between the percentages of Th2 and Tc2 cells in stimulated group and control group. The killing effect of PBMNCs on K562 cells was significantly different between the stimulated group and control group (p < 0.05). It is concluded that CpG ODNs 2216 can induce strong Th1-like immune activation, with the secretion of type-I cytokine and activation of strong CD8(+) T-cell responses. PBMNCs activated by CpG ODNs can more strongly kill k562 cells in vitro.
Adult ; Aged ; CD8-Positive T-Lymphocytes ; drug effects ; immunology ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; blood ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; K562 Cells ; Leukemia ; immunology ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Lymphocyte Activation ; Male ; Middle Aged ; Oligodeoxyribonucleotides ; pharmacology ; Th1 Cells ; drug effects ; immunology ; Th2 Cells ; drug effects ; immunology ; Young Adult

To investigate the effects of stromal cell-derived factor 1 (SDF-1) and platelet factor 4 (PF4) on the homing-related function of expanded ex vivo umbilical cord blood CD34(+) cells, purified cord blood CD34(+) cells were cultured in serum-free medium containing a HGF combination of FL + SCF + TPO (FST) with either 100 ng/ml SDF-1 alone, 100 ng/ml PF4 alone, or both of these 2 cytokines. The expansion rate of CD34(+) cells, colony formation, homing-related functions including expression of homing-related adhesion molecules of expanded CD34(+) cell, adhesion activity and chemotactic function of the re-selected expanded CD34(+) cells were evaluated at different time points. The results showed that expansion rate of CD34(+) cells and expansion multiple of CFU in SDF-1 groups were higher than those in control. The expression of CD49e on the expanded CD34(+) cells was remarkable up-regulated, in contrast, expression of CXCR-4 on the expanded CD34(+) cells was remarkable down-regulated in SDF-1 groups. The expression of CD49e, CD54 and CXCR-4 on the expanded CD34(+) cells were remarkably up-regulated in the PF4 groups. In all the SDF-1 group, PF4 group and SDF-1 plus PF4 group, the ability of expanded CD34(+) cells adhering to fibronectin layer were higher than those in the control on day 10. Spontaneous migration rate of expanded CD34(+) cells in SDF-1 groups were higher than those in control, while SDF-1-induced migration rate were lower than those in control on day 10. SDF-1-induced migration rate in PF4 groups were higher than those in control on day 10. Spontaneous and SDF-1-induced migration rate of expanded CD34(+) cells in the SDF-1 plus PF4 groups were higher than those in control on day 10. It is concluded that, SDF-1 and PF4 can up-regulate expression of adhesion molecules on expanded CD34(+) cells, and retain the adherent and migration ability of expanded CD34(+) cells, which is helpful for the homing of expanded CD34(+) cells. In short, SDF-1 and PF4 are helpful for the homing-related function of the expanded UCB HSPC.
Antigens, CD34 ; blood ; immunology ; Cell Adhesion ; drug effects ; Cells, Cultured ; Chemokine CXCL12 ; Chemokines, CXC ; pharmacology ; Chemotaxis ; immunology ; physiology ; Culture Media, Serum-Free ; Fetal Blood ; cytology ; immunology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Platelet Factor 4 ; pharmacology

To investigate whether the dendritic cells (DC) could grow up in cultural system with umbilical cord serum (UCS), the UCS was used in the culture instead of fetal calf serum. The phenotype of dendritic cells was detected by flow cytometry and the antigen presenting ability of DC in allo-MLR was measured by MTT assay. The results showed that DC grown in UCS (UCS-DC) had higher expression rate of CD86, CD83 and HLA-DR than that in grown in FCS (FCS-DC). (P < 0.05), and their expression of CD1a was lower than that of FCS-DC. The ability to induce T cell proliferation had no difference between UCS-DC and FCS-DC. It is suggested that dendritic cells with more mature phenotype had been produced in the medium containing UCS than those in the medium containing FCS, and UCS-DC possessed potent ability to stimulate proliferation of allogeneic T cells.
Animals ; Antigens, CD ; immunology ; B7-2 Antigen ; Cattle ; Cell Culture Techniques ; methods ; Cell Division ; drug effects ; Culture Media ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; immunology ; Fetal Blood ; physiology ; HLA-DR Antigens ; immunology ; Humans ; Immunoglobulins ; immunology ; Membrane Glycoproteins ; immunology

OBJECTIVE: To investigate the protective effect of Zengye Decoction (, ZYD) on the submandibular glands (SMGs) in nonobese diabetic (NOD) mice. METHODS: Twenty-seven female NOD mice were randomly equally divided into 3 groups: the model group, the hydroxychloroquine (HCQ) group, and the ZYD group. Nine C57/B6 mice served as the normal group. After 1-week acclimation, the HCQ and ZYD groups were intragastrically administered with HCQ and ZYD, respectively, and the normal and model groups were administered with normal saline. Changes in the salivary flow rate were observed. Mice from all 4 groups were sacrificed at the age of 20 weeks. The serum and SMGs were collected. Serum cytokines gamma-interferon (IFN-γ), interleukin-10 (IL-10) were detected by enzyme-linked immunosorbent assay. Histological changes in the submandibular glands were examined by hematoxylin and eosin staining. The mRNA expression of IFN-γ, IL-10 and vasoactive intestinal peptide (VIP) in the submandibular glands were measured by real-time polymerase chain reaction. RESULTS: Compared with the model group, the salivary flow of the ZYD group significantly increased (P<0.05), the extent of the histological changes was ameliorated (P<0.05), and the Th1/Th2 cytokine imbalance was remedied (P<0.05). In the ZYD-treated mice, the VIP mRNA was up-regulated (P<0.05). CONCLUSIONS ZYD is beneficial in protecting structure and function of SMGs in NOD mice. The mechanism may be associated with the correction of the Th1/Th2 cytokine imbalance, and with the prevention of a progressive decline of the VIP level.
Animals ; Cytokines ; blood ; Drugs, Chinese Herbal ; pharmacology ; Female ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Salivation ; drug effects ; Sjogren's Syndrome ; drug therapy ; immunology ; Submandibular Gland ; drug effects ; pathology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vasoactive Intestinal Peptide ; genetics