The efficiency of dendritic cell-activated and cytokine-induced killer cell (DC-CIK) therapy on children with acute myeloid leukemia (AML) after chemotherapy was investigated. Mononuclear cells were collected from children achieving complete remission after chemotherapy, cultured in vitro and transfused back into the same patient. Interleukin-2 (IL-2) was injected subcutaneously every other day 10 times at the dose of 1 × 10(6) units. Peripheral blood lymphocyte subsets and minimal residual disease (MRD) were detected by flow cytometry. Function of bone marrow was monitored by methods of morphology, immunology, cytogenetics and molecular biology. The side effects were also observed during the treatment. The average follow-up period for all the 22 patients was 71 months and relapse occurred in two AML patients (9.1%). The percentage of CD3(+)/CD8(+) cells in peripheral blood of 15 patients at the 3rd month after DC-CIK treatment (36.73% ± 12.51%) was dramatically higher than that before treatment (29.20% ± 8.34%, P < 0.05). The MRD rate was >0.1% in 5 patients before the treatment, and became lower than 0.1% 3 months after the treatment. During the transfusion of DC-CIK, side effects including fever, chills and hives appeared in 7 out of 22 (31.82%) cases but disappeared quickly after symptomatic treatments. There were no changes in electrocardiography and liver-renal functions after the treatment. MRD in children with AML can be eliminated by DC-CIK therapy which is safe and has fewer side effects.
Adolescent ; Antineoplastic Agents ; therapeutic use ; Bone Marrow ; drug effects ; immunology ; pathology ; Child ; Child, Preschool ; Cytokine-Induced Killer Cells ; cytology ; immunology ; transplantation ; Dendritic Cells ; cytology ; immunology ; transplantation ; Female ; Humans ; Immunotherapy, Adoptive ; methods ; Injections, Subcutaneous ; Interleukin-2 ; therapeutic use ; Leukemia, Myeloid, Acute ; immunology ; pathology ; therapy ; Male ; Neoplasm, Residual ; Primary Cell Culture ; Recurrence ; Remission Induction ; Treatment Outcome

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects

OBJECTIVETo observe the anti-virus effects of andrographolide (AD) on the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) signaling pathway when immunological cells were infected with H1N1.

METHODSLeukomonocyte was obtained from umbilical cord blood by Ficoll density gradient centrifugation, and immunological cells were harvested after cytokines stimulation. Virus infected cell model was established by H1N1 co-cultured with normal human bronchial epithelial cell line (16HBE). The optimal concentration of AD was defined by methyl-thiazolyl-tetrazolium (MTT) assay. After the virus infected cell model was established, AD was added into the medium as a treatment intervention. After 24-h co-culture, cell supernatant was collected for interferon gamma (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunosorbent assay (ELISA) detection while immunological cells for real-time polymerase chain reaction (RT-PCR).

RESULTSThe optimal concentration of AD for anti-virus effect was 250 μg/mL. IL-4 and IFN-γ in the supernatant and mRNA levels in RLRs pathway increased when cells was infected by virus, RIG-I, IFN-β promoter stimulator-1 (IPS-1), interferon regulatory factor (IRF)-7, IRF-3 and nuclear transcription factor κB (NF-κB) mRNA levels increased significantly (P<0.05). When AD was added into co-culture medium, the levels of IL-4 and IFN-γ were lower than those in the non-interference groups and the mRNA expression levels decreased, RIG-I, IPS-1, IRF-7, IRF-3 and NF-κB decreased significantly in each group with significant statistic differences (P<0.05).

CONCLUSIONSThe RLRs mediated viral recognition provided a potential molecular target for acute viral infections and andrographolide could ameliorate H1N1 virus-induced cell mortality. And the antiviral effects might be related to its inhibition of viral-induced activation of the RLRs signaling pathway.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Coculture Techniques ; DEAD Box Protein 58 ; DEAD-box RNA Helicases ; genetics ; metabolism ; Dendritic Cells ; drug effects ; immunology ; virology ; Diterpenes ; pharmacology ; Fetal Blood ; cytology ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; immunology ; Influenza, Human ; drug therapy ; immunology ; virology ; Interferon-beta ; genetics ; metabolism ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Leukocytes, Mononuclear ; drug effects ; immunology ; virology ; Macrophages ; drug effects ; virology ; NF-kappa B ; genetics ; metabolism ; Promoter Regions, Genetic ; drug effects ; immunology ; RNA, Messenger ; metabolism ; Signal Transduction ; drug effects ; genetics ; immunology

Dendritic cells (DCs) are crucial for the induction and maintenance of tumor-specific immune responses. Studies have shown that tumor-associated DCs are immunosuppressed in some human tumors. However, phenotype and function of DCs in retinoblastoma (RB) remain unclear. RB cell supernatant (RBcs) was used to treat DCs in vitro to explore the effect of RB cells on DCs. DCs were generated from peripheral blood mononuclear cells of healthy donors. On day 5 of culture, DCs were treated with RBcs for 24 h, and then purified using magnetic beads. The maturation of DCs was induced by TNF-α or LPS. After treatment with RBcs, expression of co-stimulatory molecules CD80 and CD86 was elevated in DCs, accompanied by increased production of IL-12p70, TNF-α, IL-6, IL-1β, and IL-8 but decreased production of IL-10. RBcs neither inhibited DC maturation nor promoted DC apoptosis. Moreover, RBcs-exposed DCs stimulated allogenetic T cell proliferation and T cell-derived cytokine production. These results indicate that RBcs can improve DCs' antigen presenting function and capability to activate T cells, suggesting that RB cells may have an immunostimulatory effect on DCs, and DC-based immunotherapy may be adopted in the treatment of RB.
B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Culture Media, Conditioned ; pharmacology ; Cytokines ; metabolism ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Humans ; Lipopolysaccharides ; toxicity ; Retinal Neoplasms ; metabolism ; pathology ; Retinoblastoma ; metabolism ; pathology ; T-Lymphocytes ; cytology ; immunology ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

BACKGROUNDSmoking causes frequent asthma attacks, leading to a rapid decline in lung function in patients with asthma, and it can also reduce the therapeutic effect of glucocorticoids in patients with asthma. Therefore, the present study aimed to investigate the effect of cigarette smoke on the expression of myeloid differentiation factor 88 (MyD88) in marrow dendritic cells (DCs) in asthmatic rats, and to explore the molecular mechanism of cigarette smoke exposure on asthma by DCs.

METHODSForty Wistar rats were randomly divided into the following groups: control, smoke exposure, asthma, and asthma combined with smoke exposure. The animal model was established, and then rat bone marrow-derived DCs were collected. Additionally, rat spleen lymphocytes and bone marrow-derived DCs were cultured together for mixed lymphocyte responses. Interferon (IFN)-gamma and interleukin (IL)-4, IL-10, and IL-12 expressions were determined by enzyme-linked immunosorbent assay (ELISA). MyD88 expression was determined by Western blotting. The proliferation of lymphocytes was examined with methyl thiazolyl tetrazolium (MTT) colorimetric assay.

RESULTSMyD88 expression was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01), and IL-10 and IL-12 expressions were decreased in the asthma combined with smoke exposure group compared to control group (P < 0.01). In addition, DCs stimulating activity on allogeneic lymphocytes were significantly decreased in the smoke exposure combined with asthma group compared to the control and asthma groups (P < 0.01). After allogeneic mixed lymphocyte responses, IL-4 expression was increased and IFN-gamma was decreased in the asthma group and the asthma combined with smoke exposure group compared to control group (P < 0.01). IL-4 expression was increased and IFN-gamma was decreased in the asthma combined with smoke exposure group compared to the asthma group (P < 0.01). The study also showed that MyD88 expression was positively correlated with IL-12 and IFN-gamma expressions and the activity of lymphocytes (P < 0.01), and negatively correlated with IL-4 expression (P < 0.01).

CONCLUSIONSSmoking aggravates asthma by weankening immunological mechanism. MyD88-dependent pathways may play a role in the immunological balance and activation of lymphocytes.

Animals ; Asthma ; immunology ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; Dendritic Cells ; drug effects ; metabolism ; Lymphocyte Activation ; drug effects ; Male ; Myeloid Differentiation Factor 88 ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Smoking ; adverse effects

OBJECTIVETo explore the mechanism of immunomodulatory activity of triptolide on primary immune thrombocytopenia (ITP)patients-derived plasmacytoid dendritic cells (pDCs).

METHODSpDCs in peripheral blood of ITP patients before therapy (group 1), ITP patients in complete response (ITP-CR, group 2) and healthy donors (group 3) were sorted by flow cytometry, then incubated with triptolide at 0, 5, 10 or 30 µg/L. After 24 hours, we collected the supernatants and then detected the concentrations of IFN-α, IL-6 and TNF-α using ELISA. After 5 days, the cultured cells were collected and CD11c, CD80 and CD86 expressions of myeloid dendritic cells (mDCs) were analyzed by flow cytometry, the morphology of mDC was observed by light microscope and electron microscope.

RESULTSAfter incubation with triptolide at 10 µg/L, the levels of IFN-α, IL-6 and TNF-α in group 1 \[(451.32 ± 85.77) ng/L, (105.68 ± 23.85) ng/L and (135.78 ± 30.62) ng/L\] and group 2 \[(391.71 ± 72.49) ng/L, (84.73 ± 17.77) ng/L and (108.16 ± 23.21) ng/L\] were significantly higher than those in group 3 \[(335.51 ± 67.54) ng/L, (73.62 ± 21.82) ng/L and (95.58 ± 32.85) ng/L\] (all P < 0.05); the levels of IFN-α, IL-6 and TNF-α in group 1 were significantly higher than those in group 2 (all P < 0.05) in a dose-dependent manner (P < 0.05). CD11c, CD80 and CD86 expressions of mDC in group1 and group 2 were significantly higher than those in group 3 (all P < 0.05); CD11c, CD80 and CD86 expressions of mDC in group 1 were significantly higher than those in group 2 (all P < 0.05) also in a dose-dependent manner (all P < 0.05). Triptolide could inhibit pDCs from differentiation into mDCs, the latter displayed more immature morphology than untreated-pDCs.

CONCLUSIONTriptolide could decrease the immune function of pDCs from ITP, inhibit pDCs from differentiation and maturation.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Female ; Humans ; Male ; Middle Aged ; Phenanthrenes ; pharmacology ; Thrombocytopenia ; etiology ; immunology ; Young Adult

This study was aimed to investigate the effect of 1,25(OH)(2) vitamin D(3) [1,25(OH)(2) Vit D(3)] on the differentiation, maturation and function of human dendritic cells (DC) in vitro and its mechanism. Human peripheral blood mononuclear cells were induced to differentiate to DC in vitro. The DC in test group were cultured with 1,25(OH)(2) Vit D(3) 1 nmol/L for 9 d, while the DC in control group were cultured with the equivalent of absolute alcohol. The expression of co-stimulatory molecules on DC were analyzed by flow cytometry. T cell proliferation induced by DC was assessed by MTT method. The expression of indoleamine 2, 3-dioxygenase (IDO) protein was determined by Western blot. The results showed that compared with the control group, the expression of CD80, CD83 and CD86 on DC in test group was significantly down-regulated (P < 0.05), while the CD1a was up-regulated (P < 0.05). The expression rate of CD80, CD83, CD86, CD1a in test group were (40.43 ± 9.83)%, (20.04 ± 4.73)%, (14.45 ± 5.38)%, (58.48 ± 10.72)% respectively, while in control group were (29.36 ± 13.34)%, (35.91 ± 10.19)%, (27.15 ± 11.64)%, (72.20 ± 12.79)% respectively. Compared with the control group, 1,25(OH)(2) Vit D(3)-treated DC exhibited a markedly reduced ability to stimulate allogenic T cell proliferation and up-regulated IDO protein expression.It is concluded that 1,25(OH)(2) Vit D(3) efficiently inhibits the maturation of DC and DC-mediated T cell proliferation, which may be related to the up-regulation of IDO protein expression.
Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cholecalciferol ; pharmacology ; Dendritic Cells ; cytology ; immunology ; Flow Cytometry ; Humans ; Immune Tolerance ; drug effects ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism

OBJECTIVETo examine the effect of ginsenoside Rb1 (GRb1) on the immune maturation of monocyte-derived dendritic cells (DCs) induced by oxidized low-density lipoprotein (OX-LDL).

METHODSHuman monocytes purified by CD14+ immuno-magnetic beads were differentiated and induced into immature DCs, which were randomly divided into 6 groups, Group A treated with PBS, Group B treated with OX-LDL, Group C and D treated respectively with GRb1 and ciglitazone, Group E and F were pretreated with the two testing drugs respectively followed by OX-LDL. The immuno-phenotypic expression (CD40, CD1a, and HLA-DR) and endocytosis function of DCs were examined using flow cytometry, the concentration of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) in the culture supernatants were measured with ELISA.

RESULTSCompared with Group B, Group E showed significantly lowered immuno-phenotypic expression of DCs in terms of CD40 (67.4 +/- 1.62 vs. 145.69 +/- 14.86), CD1a (79.64 +/- 3.04 vs. 159.89 +/- 6.09), and HLA-DR (46.43 +/- 2.85 vs. 99.33 +/- 17.11), as well as higher endocytosis level (88.13% +/- 1.06% vs. 25.90% +/- 5.77%, all P < 0.01). Meantime, the serum levels of IL-12 (88.65 +/- 5.59 ng/L vs. 716.69 +/- 36.35 ng/L) and TNF-alpha (133.27 +/- 11.98 ng/L vs. 968.10 +/- 36.42 ng/L) obviously decreased (P < 0.01). The surface molecular expression of DCs and the secretion of inflammatory factors in Group F also obviously decreased, showing insignificant difference from Group E (P > 0.05).

CONCLUSIONGRb1 could obviously inhibit the OX-LDL-induced maturation of DCs, showing similar effects to ciglitazone.

Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Ginsenosides ; pharmacology ; Humans ; Lipoproteins, LDL ; Monocytes ; cytology ; drug effects ; immunology

We investigated the immunostimulatory effects of a novel beta-glucan purified from Paenibacillus (P.) polymyxa JB115 on bone marrow-derived dendritic cells (DCs), a type of potent antigen-presenting cells. beta-glucan isolated from P. polymyxa JB115 enhanced the viability and induced the maturation of DCs. beta-glucan markedly increased the cytokine production of DCs and surface expression of DC markers. In addition, DCs treated with beta-glucan showed a higher capacity to stimulate allogeneic spleen cell proliferation compared to those treated with medium alone. These results demonstrate the effect of beta-glucan on DC maturation and may increase the use of beta-glucan.
Animals ; Bone Marrow Cells/cytology/*drug effects/*immunology ; Cell Survival/drug effects/*immunology ; Dendritic Cells/cytology/*drug effects/*immunology ; Flow Cytometry ; Immunophenotyping/methods ; Interleukin-12/analysis/immunology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/analysis/immunology ; Paenibacillus/*chemistry ; Tumor Necrosis Factor-alpha/analysis/immunology ; beta-Glucans/isolation & purification/*pharmacology