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

UNLABELLEDDendritic cells (DC) are very potent antigen-presenting cells (APC) with a unique ability to activate naive T cells to induce the differentiation of TH1/TH2. Monocytes can develop into DC in the presence of different cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. DCs are thought to play a key role in the initiation and maintenance of T cell immunity to inhaled antigens. While the density of DC within the bronchial mucosa is increased in asthma, there is little information currently available concerning the effects of DC in asthmatic children.

OBJECTIVETo investigate the role of dendritic cells in the pathogenesis of acute attack of asthma in children.

METHODSThomas' method was adopted. The adherent precursors of DC were isolated from peripheral blood of asthmatic children in acute attack stage and healthy controls. The adherent cells were induced with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha) to DC in vitro. The expression of the surface molecules CD80, CD86, HLA-DR etc. on the DC was examined by fluorescent activated cell sorter (FACS). And the ability to secret IL-10, IL-12 and their potentials to stimulate the proliferative reaction of DC inductive self T- lymphocyte were observed.

RESULTSThe results showed that in asthmatic children's acute attack stage, self T- lymphocyte proliferative reaction induced by DC was remarkably increased compared with normal control subjects (P < 0.01). At the same time, the asthmatic children in acute attack stage had remarkably decreased the ability to secret IL-10 compared with normal control subjects (P < 0.01), while the ability to secret IL-12 remarkably decreased compared with normal control subjects (P < 0.01); meanwhile, the HLA-DR and co-stimulating factor CD86(B(7-2)) expressed by DCs remarkably increased in the asthmatic children in acute attack stage compared with normal control subjects (P < 0.01).

CONCLUSIONDC possibly plays a vital role in the immunological mechanism of asthma by means of inducing the differentiation of TH1/TH2, that is DC may be the key factor in initiating the airway allergic reaction and the possible mechanism may involve interleukins (especially IL-10 and IL-12, etc.) secreted by DCs.

Adolescent ; Antigens, CD ; metabolism ; Asthma ; metabolism ; physiopathology ; Child ; Child, Preschool ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Female ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HLA-DR Antigens ; metabolism ; Humans ; Interleukin-10 ; secretion ; Interleukin-12 ; secretion ; Interleukin-4 ; pharmacology ; Male

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 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 study the effect of bifidobacterium genomic DNA on umbilical cord blood mononuclear cell (CBMC), and investigate the immunoregulation of bifidobacterium DNA and explore possible mechanisms by which bifidobacterium acts against allergic reaction.

METHODSBifidobacterium genomic DNA (bDNA) and human DNA (hDNA) were extracted with phenol/chloroform/isoamyl alcohol and stored at -20 degrees C for later use. Parts of bDNA were completely digested with DNaseI (d-bDNA) at 37 degrees C. CBMCs were isolated with Ficoll from umbilical cord blood and incubated at 37 degrees C in a 5% CO2 humidified incubator. These cells were divided into four groups, control group: without any stimulant; bDNA group: stimulated with 25 microg/ml bDNA; d-bDNA group: stimulated with 25 microg/ml d-bDNA; hDNA group: stimulated with 25 microg/ml hDNA. The cells were stimulated with different stimulants in vitro, at the end of incubation culture supernatant and cells were collected. IL-12 and IL-10 levels in the culture supernatant were measured by enzyme linked immuno sorbent assay (ELISA); cells secreting IL-4 and IFN-gamma were counted by enzyme linked immunospot (ELISPOT) assay; and total RNA was isolated from the cells to assay T-bet and GATA3 mRNA expression levels by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSSix hours after stimulation there was no significant difference in IL-12 level in supernatant among the four groups; 12 hours after stimulation, IL-12 level in supernatant of bDNA treated group was significantly higher than that of each of the other groups, so were the results obtained at 24 hours and 48 hours after stimulation (P < 0.05). No significant difference could be detected in IL-12 level in supernatant among the other 3 groups. On the other hand, 6 hours after stimulation there was no significant difference in IL-10 level in supernatant among the four groups. But 12 and 24 hours after stimulation IL-10 level in supernatant of bDNA treated group was lower than that of each of the other groups, but the difference was not statistically significant. The count of IFN-gamma secreting cells of bDNA treated group was higher than that of the other groups, while IL-4 secteting cells of bDNA treated group were lower than that of the other groups. After bDNA stimulation, nuclear factor T-box expressed in T cells (T-bet) mRNA expression was conspicuously enhanced as compared to the other three groups (P < 0.05). GATA3 mRNA transcription in CBMC had no significant change after bDNA stimulation.

CONCLUSIONbDNA could promote secretion of Th1 type cytokine IL-12, while Th2 type cytokine IL-10 level of cell supernatant was decreased. bDNA could stimulate secretion of IFN-gamma by CBMC and inhibit secretion of IL-4. T-bet mRNA expression was highly enhanced after bDNA stimulation. bDNA could upregulate Th1 type response, which may be one of important mechanisms by which bifidobacterium inhibit allergic response.

Bifidobacterium ; cytology ; genetics ; Cell Culture Techniques ; DNA, Bacterial ; biosynthesis ; metabolism ; pharmacology ; Enzyme-Linked Immunosorbent Assay ; Fetal Blood ; cytology ; immunology ; GATA3 Transcription Factor ; genetics ; Humans ; Infant, Newborn ; Interferon-gamma ; immunology ; secretion ; Interleukin-10 ; immunology ; secretion ; Interleukin-12 ; immunology ; secretion ; Interleukin-4 ; immunology ; secretion ; Leukocytes, Mononuclear ; immunology ; secretion ; RNA, Messenger ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; T-Box Domain Proteins ; genetics ; Th1 Cells ; drug effects ; immunology ; secretion

OBJECTIVETo investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro.

METHODSSurvivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT.

RESULTSSurvivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P < 0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells.

CONCLUSIONSDCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Antigens, CD ; metabolism ; Dendritic Cells ; immunology ; Gastrointestinal Neoplasms ; therapy ; Humans ; Immunotherapy, Active ; In Vitro Techniques ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; secretion ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

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

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 the effects of IL-2 gene modification enhancement of the antigen-presenting function of the mouse bone marrow derived dendritic cells and on the activation of CTL induced by MHC class I molecule restricted antigen peptides as well as the related immunological mechanisms.

METHODSDCs were prepared from mouse bone marrow and modified by recombinant IL-2 adenovirus (DC-IL-2). The IL-12 and IFN-gamma levels in culture supernatant of DC and CTL were examined by ELISA, the expression of costimulatory molecules and fluorescent intensity of endocytosis of OVA-FITC in DC by FACS, the capacity of presenting 3LL cell tumor antigen by (3)H-TdR incorporation method, the MHC class I-restricted tumor-antigen-peptide Mut1 of 3LL cells pulsed DC-IL-2 to induce CTL cytotoxicity by (51)Cr 4-hr releasing assay.

RESULTSAfter IL-2 gene modification, DC-IL-2 could produce high level of IL-12 [(78.4 +/- 6.6) pg.(1 x 10(6) cells)(-1).ml(-1)]. The expression of costimulatory molecules on DC-IL-2 was increased, the fluorescent intensity of DC captured OVA-FITC was enhanced, and the proliferation of allo-T cells from 3LL bearing mouse pulsed with Mut1 was also enhanced. Mut1 antigen peptide pulsed DC-IL-2 could induce more potent antigen-specific CTL cytotoxicity and excrete high concentration of IFN-gamma [(1 168 +/- 58.4) pg/ml] in vivo.

CONCLUSIONIL-2 gene modification of DC can activate second signal for DC presenting antigen, and enhance the function for capturing and presenting tumor antigen. DC-IL-2 pulsed with MHC class I restricted tumor-antigen-peptide can induce specific anti-tumor immune response more effectively. Owing to IL-2 gene modification, the functions of IL-12 excretion and T cell activation of DC were promoted, so that the capacity of CTL excreting IFN-gamma was enhanced, which are relevant to the immune mechanism.

Adenoviridae ; genetics ; Animals ; Antigen Presentation ; immunology ; B7-1 Antigen ; genetics ; metabolism ; Dendritic Cells ; cytology ; immunology ; Female ; Interleukin-12 ; secretion ; Interleukin-2 ; genetics ; Lymphocyte Activation ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Recombination, Genetic ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; immunology

Cell mediated immune responses play a prominent role in syphilis, which is caused by Treponema pallidum. The role of dendritic cells (DC) in the syphilitic infection is not well understood in human. In the present study, we studied interaction of T. pallidum with DC, generated from human peripheral blood mononuclear cells with GM-CSF and IL-4. After adding T. pallidum for 16 hours to immature DC at culture day 7, the change of surface antigens on DC was monitored by flow cytometry, the amount of IL-12 in culture supernatant of DC was measured by ELISA and T cell stimulatory capacity of DC was checked in mixed lymphocyte reaction (MLR). We have observed an efficient phagocytosis of T. pallidum by electron microscopy as early as 2 hours after addition of T. pallidum to DC. Interaction of DC with T. pallidum resulted in increased surface expression of CD83 which was proportionally increased according to the number of T. pallidum. Expressions of CD80, CD86 and HLA-DR on DC were slightly increased. The amount of IL-12 in the culture supernatant of DC was increased (1, 099pg/ml) after the addition of T. pallidum. T. pallidum-infected DC also displayed enhanced T cell stimulatory capacity in MLR. As seen from the above, we observed phagocytosis of T. pallidum by DC as early as 2 hours after addition of T. pallidum to DC and found that T. pallidum can stimulate DC maturation which mean that DC modulate an protective immune response during T. pallidum infection.
Cells, Cultured ; Dendritic Cells/cytology/*immunology/*microbiology ; Human ; Interleukin-12/metabolism/secretion ; Lymphocyte Culture Test, Mixed ; Microscopy, Electron ; Phagocytosis/immunology ; Receptors, Cell Surface/immunology/metabolism ; Support, Non-U.S. Gov't ; Syphilis/*immunology ; T-Lymphocytes/immunology ; Treponema pallidum/*immunology/ultrastructure