The purpose of this study was to explore the effect of NOD2 signalling pathway activated by muramyl dipeptide (MDP) on the immunomodulation effect of human monocyte-derived dendritic cells (DC) loaded with leukemia cell lysates. Peripheral blood mononuclear cells (PBMNC) were isolated by density gradient centrifugation, These cells were cultured with three cytokines for 7 days to induce their maturation. On the 5th day, cells were loaded with leukemia cell HL-60 lysates. NOD2 expression was detected by RT-PCR and Western blot. The phenotype of DC were analyzed by flow cytometry, and ELISA was used to assay levels of IL-12 (p40) . The results showed that MDP could trigger NOD2 mRNA and protein expression in different groups of DC, especially in sensitized DC+MDP group, which was significantly higher than that in the DC+MDP group and sensitized DC without MDP stimulation, the difference was statistically significant (P < 0.05). Besides, the expression of surface molecules (HLA-DR, CD80, CD83, CD86, CD40) in the group of DC loaded with leukemia cell lysate and stimulated by MDP (sensitized DC+MDP) reached the highest level, followed by the group of DC loaded with leukemia cell lysate without MDP and DC only stimulated by MDP, non-treated DC were the lowest (P < 0.05). Similarly, compared with untreated unstimulated DC, after loading with HL-60 lysates or only stimulating with MDP, the secretion of IL-12p40 increased, but IL-12p40 level (573.86 ± 32.09 pg/ml) in DC+MDP group was higher than that in group of sensitized DC (365.03 ± 28.86 pg/ml) (P < 0.05), and it in sensitized DC+MDP group reached the highest (898.30 ± 61.08) pg/ml, compared to other groups (P < 0.05). It is concluded that MDP can significantly enhance the NOD2 mRNA and protein expression in sensitized DC, promote the expression of HLA-DR, synergistic costimulatory molecules and adhesion molecules of DC, at the same time, MDP can increase secretion of inflammatory factors IL-12p40. This study will provide a new ideas for DC application in leukemia immunotherapy.
Acetylmuramyl-Alanyl-Isoglutamine ; pharmacology ; Cells, Cultured ; Dendritic Cells ; immunology ; metabolism ; HL-60 Cells ; Humans ; Interleukin-12 Subunit p40 ; secretion ; Leukemia ; immunology ; metabolism ; Leukocytes, Mononuclear ; metabolism ; Membrane Proteins ; metabolism ; Nod2 Signaling Adaptor Protein ; metabolism

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.
Antigens, Protozoan/*immunology ; Cell Line ; Humans ; Interleukin-12/*secretion ; Interleukin-23/*secretion ; Monocytes/*immunology/*parasitology ; Time Factors ; Toxoplasma/*immunology

OBJECTIVETo explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia (ITP) to further understand the effective mechanism of immunotherapy.

METHODSThe peripheral blood mononuclear cells (PBMCs) were isolated from remission patients with ITP before and after low-dose rituximab infusion, and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5% CO2 air at 37°C incubator. Then all of DCs were cultured with TNF-α for 48 hours. The morphology of DCs was monitored under inverted microscope daily, and the surface antigens of the DCs were analysed by flow cytometry, meanwhile the levels of IL-12p70 and TGF-β1 in supernatants were detected by ELISA, mix lymphocyte reaction was performed by MTT assay.

RESULTS(1) Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs. The former cells were small and most of nucleus were centric. (2) The expressions of HLA-DR, CD80, CD83 and CD86 on rituximab-treated-DCs \[56.37 ± 3.95)%, (36.41 ± 2.82)%, (30.45 ± 4.61)% and (41.98 ± 4.17)%, respectively\] were significantly lower than those untreated-DCs \[(73.71 ± 7.61)%, (55.14 ± 7.30)%, (80.91 ± 7.09)% and (59.03 ± 3.43)%, respectively\](all P < 0.05), the concentration of IL-12p70 was significantly lower, \[(66.87 ± 4.29)% vs (50.17 ± 14.52)%\], while that of TGF-β1 \[(9.70 ± 0.31)%\] higher than the untreated-DCs \[(2.70 ± 0.36)%\] (P < 0.05). (3) The abilities to activate T cells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs.

CONCLUSIONThe surface antigen of ITP-DCs and the concentration of IL-12p70 reduced after the low-dose rituximab infusion. The abilities to activate T cells proliferation reduced while the concentration of TGF-β1 increased. Rituximab may achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.

Antibodies, Monoclonal, Murine-Derived ; administration & dosage ; therapeutic use ; Cell Proliferation ; Cells, Cultured ; Dendritic Cells ; cytology ; metabolism ; secretion ; Female ; Humans ; Interleukin-12 ; metabolism ; Lymphocyte Activation ; Male ; Rituximab ; T-Lymphocytes ; immunology ; Thrombocytopenia ; drug therapy ; immunology ; metabolism ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo investigate immunomodulatory effect of Astragalus polysaccharides (APS) on IL-12-secreting dendritic cell (DC) subset CD11c(high)CD45RB(low) DC.

METHODSSpleen CD11c(high)CD45RB(low) DC and CD4(+)T lymphocytes in BALB/c mice were purified by magnetic beads sorting, and were treated with 0 (as control), 50, 100, 200 µg/mL APS. Immunofluorescence staining and flow cytometry were used to determine expressions of CD11c(high)CD45RB(low) DC surface molecules, including CD40, CD80, CD86, I-A/E, and Toll-like receptor (TLR) 4. IL-12 level in CD11c(high)CD45RB(low) DC culture supernatant was determined by ELISA. The CD4(+) T lymphocytes were divided into: normal control group, non-stimulation group (CD4(+)T lymphocytes cocultured with APS-unstimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC), high-dose APS stimulation+antibody 1 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody), high-dose APS stimulation+ antibody 2 group (CD4(+)T lymphocytes cocultured with 200 µg/mL APS-stimulated CD11c(high)CD45RB(low) DC and IL-12 antibody isotype). Proliferation ability of CD4(+) T lymphocytes was determined with MTT method. IL-4 level as well as IFN-γ level in CD4(+)T lymphocyte culture supernatant was determined by flow cytometry. Data were processed with one-way analysis of variance.

RESULTSCompared with those in control, the expressions of CD11c(high)CD45RB(low) DC surface molecules (except for CD86) on CD11c(high)CD45RB(low) DC surface, as well as IL-12-secreting level with dose-dependence were increased in cells stimulated with 50, 100, 200 µg/mL APS. Proliferation ability of CD4(+)T lymphocytes in high-dose APS stimulation group was higher as compared with that in non-stimulation group (F = 13.438, P < 0.05). IFN-γ level in high-dose APS stimulation group \[(2784 ± 137) pg/mL\] was higher than that in non-stimulation group \[(1952 ± 101) pg/mL, F = 12.177, P < 0.05\]. IL-4 level in high-dose APS stimulation group was (172 ± 20) pg/mL, which was lower than that in non-stimulation group \[(193 ± 19) pg/mL, F = 11.963, P < 0.05\]. Proliferation ability of CD4(+) T lymphocytes, IFN-γ level, and IL-4 level in high-dose APS stimulation + antibody 1 group were all ameliorated when compared with those in non-stimulation group; while levels of the 3 indexes in high-dose APS stimulation + antibody 2 group were similar to those in high-dose APS stimulation group.

CONCLUSIONSAPS can activate IL-12-producing CD11c(high)CD45RB(low) DC, and further induce the activation of immune function of T lymphocyte with shifting of Th2 to Th1 in vitro. APS can enhance the immune response via promoting the phenotypic and functional maturation of CD11c(high)CD45RB(low) DC.

Animals ; Astragalus Plant ; chemistry ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; secretion ; Interleukin-12 ; metabolism ; Mice ; Mice, Inbred BALB C ; Polysaccharides ; pharmacology ; Th1 Cells ; immunology

OBJECTIVETo prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.

METHODSA mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.

RESULTSMammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.

CONCLUSIONA vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.

Antigens, Neoplasm ; metabolism ; Cancer Vaccines ; immunology ; Carbonic Anhydrase IX ; Carbonic Anhydrases ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; genetics ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interferon-gamma ; secretion ; Interleukin-12 ; genetics ; metabolism ; Kidney Neoplasms ; metabolism ; pathology ; Plasmids ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection

This study was purposed to explore the changes in biological functions of human peripheral blood derived NK Cells after ex vivo expansion with different combinations of interleukin IL2 and/or IL12, IL15. According to different combination of cytokines, cultured NK cells were divided into 4 groups: group IL2, group IL2 + IL12, group IL2 + IL15 and group IL2 + IL15 + IL12. The group in which NK cells were cultured without cytokines was used as control. The cytotoxicity of cultured NK cells to target K562 cells was determined by using cell counting kit-8; the level of IFN-gamma in supernatants of NK cell culture was detected by ELISA; the perforin and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR. The results showed that the cytotoxicity of expanded NK cells in groups cultured with cytokines at different E:T ratio was significantly higher than that in group without cytokines (p < 0.01), although the cytotoxicity of NK cells in IL2 + IL15 + IL12 group seem to be slightly higher than that in IL2 + IL15 group, but there was no statistic difference (p > 0.05). The IFN-gamma levels in the supernatants of NK cell culture in the presence of cytokines significantly increased, and the IFN-gamma levels in IL2 + IL15 + IL12 group and IL2 + IL12 group were significantly higher than that in others (p < 0.01). The expressions of perforin and granzyme B mRNA of expanded NK cells in groups cultured with cytokines was significantly higher than that in control group (p < 0.01), and was consistent with cytotoxicity of NK cells. It is concluded that there are differences in the functions of NK cells cultured with different cytokines. IL2 and IL15 have synergistic effect on strengthening cytotoxicity of NK cells and promoting cell expansion. However, the main function of IL12 promotes NK cells to secrete IFN-gamma, which plays a role in immunoregulation.
Humans ; Interferon-gamma ; secretion ; Interleukin-12 ; administration & dosage ; pharmacology ; Interleukin-15 ; administration & dosage ; pharmacology ; Interleukin-2 ; administration & dosage ; pharmacology ; K562 Cells ; Killer Cells, Natural ; drug effects ; immunology

OBJECTIVETo observe the influence of traditional Chinese medicine (TCM) therapy for supplementing Pi () and nourishing Shen (, SPNS) on dendritic cell function in patients with chronic hepatitis B (CHB) treated by lamivudine.

METHODSSixty CHB patients with positive HBeAg were equally randomized by digital table into two groups: the observation group and the control group. Patients in the control group were treated with lamivudine only, while patients in the observation group were treated with lamivudine combined with SPNS fomula, for 12 weeks. The phenotype and function of dendritic cell, as well as its secretion factor interleukin 12 (IL-12) in all patients were determined after termination of therapy and the impacts on alanine transaminase (ALT) and HBVDNA were observed.

RESULTSThe phenotypes of dendritic cells such as CD1a, CD80, CD86, human leukocyte antigen (HLA-DR) and intercellular adhesion molecule-1, as well as the levels of stimulation index (SI) and IL-12 were higher in the observation group than those in the control group (P<0.05 or P<0.01). Meanwhile, signififi cant difference between the two groups was also shown in the normalizing rates of ALT and HBV-DNA (P<0.05).

CONCLUSIONTCM therapy for SPNS can signifificantly improve the function of dendritic cells in patients with CHB treated by lamivudine and enhance the early stage response of patients to the treatment.

Adult ; Alanine Transaminase ; metabolism ; DNA, Viral ; genetics ; Dendritic Cells ; drug effects ; pathology ; secretion ; Drug Therapy, Combination ; Female ; Hepatitis B, Chronic ; drug therapy ; enzymology ; physiopathology ; Humans ; Interleukin-12 ; secretion ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Phenotype ; Young Adult

The aim of this study is to investigate the effect of hyperin on the cccDNA of duck hepatitis B virus and its immunological regulation. Duck hepatitis B virus (DHBV) infection model and normal mouse spleen lymphocyte were used to evaluate the anti-HBV and immunoregulation effects. The DHBV-DNA of serum was detected at different time points by using serum DOT-BLOT hybridization. Polymerase chain reaction (PCR) was used for the determination of nuclear covalent closed circular DNA (cccDNA). Cytokine secretion was determined by ELISA method. DHBV-DNA were inhibited by hyperin (25 or 50 mg x kg(-1)), while cccDNA of liver could be eliminated efficiently by hyperin (25 or 50 mg x kg(-1), P < 0.05, P < 0.01). The T helper 1 effector cytokine was markedly enhanced by hyperin (25 or 50 microg x mL(-1), P < 0.01). In conclusion, hyperin has anti-HBV activity via multiple targets and pathways, and cccDNA may be one of the important targets.
Animals ; Antiviral Agents ; pharmacology ; DNA, Circular ; metabolism ; DNA, Viral ; metabolism ; Hepadnaviridae Infections ; virology ; Hepatitis B Virus, Duck ; genetics ; Hepatitis, Viral, Animal ; virology ; Interferon-gamma ; secretion ; Interleukin-12 ; secretion ; Liver ; virology ; Lymphocytes ; secretion ; Mice ; Quercetin ; analogs & derivatives ; pharmacology ; Spleen ; pathology ; virology

OBJECTIVETo examine whether the enhanced expression of CD40L cDNA on murine ovarian cancer (OVHM) cells could induce the secretion of Th1 cytokines from dendritic cells (DC).

METHODSOVHM cells were transfected with the full-length mouse CD40L cDNA by lipofectamine 2000 and then G418 resistant cells as positive cells were selected. They were examined for their expression of CD40L with flow cytometry. Bone marrow cells were firstly depleted of erythrocytes, macrophages, T and B cells with PE-conjugated magnetic beads, and then cultured in 10% FCS RPMI 1640 medium supplemented with recombinant mouse GM-CSF and IL-4 for 10 days. PKH67-labeled tumor cells were cultured with DC, and then the stained cells were analyzed for the expression of MHC-I, MHC-II, CD80, CD86, CCR7 in DC with flow cytometry. The expression of p40, p19, p35, p28, EBI3 subunits, IL-18, IFN-gamma, Mig gene in cocultured DC-tumor cells were detected by RT-PCR.

RESULTSThe CD40L cDNA was successfully transfected into OVHM cells. Bone marrow-derived DCs, when cultured with CD40L/OVHM, formed clusters with the tumors and showed an upregulated expression of MHC- I, MHC-II, CD80, CD86, CCR7. Expression of the IL-12, IL-23, IL-27, IL-18, interferon-gamma (IFN-gamma) and Mig (monokine induced by IFN-gamma) genes was induced in the DCs that were cultured with CD40L/OVHM but not with OVHM cells.

CONCLUSIONThese data directly showed that the expression of CD40L on ovarian cancer cells facilitates the interaction between DCs and tumors, enhances the maturation of DCs, induces secretion of Th1 cytokines, especially for IL-12, IL-23 and IL-27, which maybe one of the possible antitumor mechanism for CD40L-transfected ovarian cancer cell line.

Animals ; CD40 Ligand ; genetics ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Cytokines ; secretion ; DNA, Complementary ; genetics ; Dendritic Cells ; cytology ; metabolism ; Female ; Interleukin-12 ; secretion ; Interleukin-23 ; secretion ; Interleukins ; secretion ; Mice ; Ovarian Neoplasms ; metabolism ; pathology ; Th1 Cells ; secretion ; Transfection

OBJECTIVETo investigate the transduction efficiency of serotype 1, 2, 5, 6, 7, 8, 9, 10 recombinant adeno-associated viruses (rAAV) in human lung cancer cell line A549 cells and compare the transduction efficiency of conventional AAV vectors with that of self-complementary AAV (scAAV) vectors. Furthermore, the capacity of A549 cells expressing transgenic CD40L to stimulate dendritic cells (DCs) was evaluated.

METHODSLung cancer A549 cells were infected with 1 x 10(4) particules per cell of AAV encoding the green fluorescent protein (GFP) or human CD40L driven by CMV promotor, and transgene expression was analyzed by flow cytometry and fluorescence microscopy. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin-12 with immunoassay.

RESULTSSerotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. The transduction efficiency of scAAV2/5 was significantly higher than that of conventional AAV2/5. Furthermore, pre-treatment with carboplatin substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L was used to generate CD40L. A549 cells transduce by these vectors were co-cultured with immature human DCs. As a consequence, interleukin-12 was released and measured in the culture supernatant. Specificity of immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L.

CONCLUSIONscAAV2/5 transduce lung adenocarcinoma A549 cell efficiently, and co-administration of chemotherapeutic agent carboplatin further enhances its transduction efficiency. It is confirmed that lung cancer cells infected with a CD40L-encoding scAAV2/5 construct can activate human DCs to secrete interleukin-12. Our findings provided a basis for future immunotherapeutic approaches including intratumoral transfer of stimulating factors.

Antineoplastic Agents ; pharmacology ; Blotting, Western ; CD40 Ligand ; genetics ; metabolism ; physiology ; Carboplatin ; pharmacology ; Cell Line ; Cell Line, Tumor ; Coculture Techniques ; Dendritic Cells ; cytology ; secretion ; Dependovirus ; classification ; genetics ; Flow Cytometry ; Gene Expression ; drug effects ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunoassay ; methods ; Interleukin-12 ; secretion ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Serotyping ; Transfection