Objective To investigate the effects of nuclear factor kappa B decoy oligodeoxynucleotides ( NF-κB decoy ODN) transfection on biological characteristics of mature dendritic cells ( mDCs) in mice. Methods Immature DCs were harvested from Balb / c mice bone marrow, followed by the incubation with antigen OVA and LPS, and mature DCs were evaluated by the expressions of CD11c and MHC-Ⅱ detected by FACS. Mature DCs were transfected with NF-κB decoy ODN and the changes of NF-κB activity after the transfection were detected by EMSA. The expressions of the costimulatory molecules( CD40,CD80 and CD86) on DCs were detected by FACS and the proliferation of T cells was tested by mixed lymphocyte reaction( MLR) . Results The mature DCs were cultured successfully. The NF-κB activity of NF-κB decoy ODN transfected DCs was decreased significantly( P lt; 0. 05) . There was no difference in the expressions of CD40 and CD80, but the expression of CD86 was decreased significantly in NF-κB decoy ODN transfection group( P lt; 0. 05) . MLR test showed that the proliferation of T lymphocyte cells was inhibited by NF-κB decoy ODN transfected DCs, but was stimulated bly by the DCs of other groups. Conclusions Mature DCs transfected with NF-κB decoy ODN could inhibit the proliferation and activation of antigenspecical T cells, which was probably related to the down-regulation of CD86 on DCs. This modified DCs might be a promising vaccine for the treatment of asthma in the future.
Objective To observe the effect of transfer of immature mouse myeloid dendritic cells （DC） generated with low-dose granulocyte-macrophage colony-stimulating factor （GM-CSF） on cardiac allograft survival. Methods Mouse DC were generated with standard doses or low doses GM-CSF from bone marrow cells, the phenotype and functional properties of these DC were compared through fluorescence-activated cell sorting（FACS） analysis and mixed lymphocyte reaction（MLR）, 1. 0 × 106 DC generated with low doses GM-CSF were administered to the recipients 7 days before transplantation, and the cardiac allograft survival were observed. Results In contrast to DC generated with standard doses, DC generated with low doses were phenotypically immature DC （CD11c＋, CD80- , CD86- , MHCⅡlow）, and induced allogeneic T cell unresponsiveness, and administration of these DC to recipients prolonged cardiac allograft survival from 6.3±1.2 days to 14.3±1.9 days. Conclusions DC generated from mouse bone marrow progenitors in low doses of GM-CSF are phenotypically and functionally immature, and prolong cardiac allograft survival when they are administered 7 clays before transplantation.
Objective Respiratory syncytial virus ( RSV) is a primary cause of lower respiratory tract infections in children, and is also the cause for the development of asthma primarily in infants. However,the immunological mechanisms by which RSV enhances allergic sensitization and asthma remain unclear. The aimof this study was to examine the influence of RSV-infected airway epithelial cells on the activation and functions of rat myeloid dendritic cells ( mDCs) . Methods Rat airway epithelial cells ( RAECs) were infected by RSV. Then RSV-infected RAECs were co-cultured with rat mDCs, and the expression of cytokine and maturation markers on mDCs were examined by real time PCR and flow cytometry. To confirm this functional mDC maturation, allergenic mixed lymphocyte reaction ( MLR) were performed. Results Wefound that functional maturation of mDCs was induced by RSV-treated RAECs, as shown by their enhanced levels of OX40L and thymus- and activation-regulated chemokine ( TARC) mRNAs, which increased the expressions of major histocompatibility complex II ( MHCII) and CD86 costimulatorymolecules and promotedT-cell proliferation in mixed lymphocyte reactions. Conclusion Our results suggest that RSV-infected epithelial cells promote the maturation of mDCs that might support Th2 cell polarization and contribute to the pathogenesis of asthma.
ObjectivesThe aim of this meta-analysis was to evaluate the adjuvant efficacy of dendritic cell (DC) vaccines against advanced colorectal cancer.MethodsCNKI, CBM, WanFang Data, VIP, PubMed, Web of Science, The Cochrane Library and EMbase were searched to identify studies on dendritic cell vaccine for CRC up to August 13rd, 2017. After independently screening the literature and extracting data, two researchers evaluated the risk of bias in the studies, and used RevMan 5.3.5 software for meta-analysis.ResultsA total of 10 studies involving 2 050 patients were included. Meta-analysis showed that cellular immunotherapy based on DC vaccine treatment can improve the 2-year and 3-year overall survival rate of patients with advanced colorectal cancer (HR=0.33, 95%CI 0.17 to 0.27; 0.26, 95%CI 0.12 to 0.56, P<0.05), while there was no statistically significant difference in 1-year overall survival rate (HR=0.48, 95%CI 0.19 to 1.20, P=0.12); DC-CIK-based cellular immunotherapy could improve 2-year and 3-year overall survival rates (HR=0.27, 95%CI 0.10 to 0.75; HR=0.15, 95%CI 0.04 to 0.54, P<0.05), the difference of 1-year overall survival rate was not statistically significant (HR=0.39, 95%CI 0.13 to 1.13, P=0.08); DC combined with chemotherapy could improve 2-year and 3-year overall survival (HR=0.24, 95%CI 0.10 to 0.56; HR=0.22, 95%CI 0.04 to 0.54, P<0.05); the difference of 1-year overall survival rate was not statistically significant (HR=0.34, 95%CI 0.06 to 2.03, P=0.24); median overall survival in the DC vaccine group (MSR=1.25, 95%CI 1.16 to 1.34, P<0.05) and median progression-free survival (MSR=1.39, 95%CI 1.25 to 1.53, P<0.05) were superior to the control group. Fever was the most common adverse reaction and most patients could be relieved after treatment.ConclusionsDendritic cells vaccines-based immunotherapy can effectively improve the later overall survival rate and prolong median OS of patients with advanced colorectal cancer with mild adverse reactions, however the improvement of short term survival rate is not obvious.
Objective By using small interfering RNAs ( siRNAs) specific for spleen tyrosine kinase ( Syk) , to evaluate the role of Syk in maturation of bone marrow-derived dendritic cells. Methods The fragments of 21-23 bp siRNAs specific for mice Syk were chemo synthesized and transfected into the asthmatic murine bone marrow-derived dendritic cells ( BMDCs) by Lipofectamine 2000 transfection system for 48 hours. Then BMDCs were co-cultured with T cells from the normal mice spleen for 48 hours. The cytokines including IL-4, IL-13, IL-2 and INF-γin supernatant were detect by ELISA. The expression of Syk protein was measured by Western Blot to determine whether the Syk gene was silenced. Results The expression of Syk protein was obviously decreased in the siRNA-interference group. The secretions of IL-4 and IL-13 were significantly inhibited by siRNA interference ( P lt; 0. 05) , but the secretions of IL-2 and INF-γwere not interfered signficantly ( P gt;0. 05) . Conclusion Syk specific siRNA fragments can block the antigen presentation function of dendritic cells and block the activation and differentiation of T cells.
ObjectiveTo find out an effective method for amplification and purification of dendritic cells(DC) from peripheral blood of patients with pancreatic carcinoma. MethodsPeripheral blood mononuclear cells were purified from peripheral blood of health volunteers(control group,10 cases) and patients with pancreatic carcinoma (experimental group,12 cases) with incubation of granulocyte/macrophage colonystimulating factor(GMCSF) and interleukin4(IL4).The quality of DC were detected by immumofluorescence method and the expression levels of HLADR and B72 on DC were detected by flow cytometer after and before DC incubation with GMCSF and the IL4. ResultsThe expression level of HLADR and B72 of DC in experimental group were significantly less than those in control group(P＜0.01).DC in experimental group was significantly proliferated in the presence of GMCSF and IL4(P＜0.01).On day 7,the expression level of HLADR and B72 of DC in experimental group were significantly increased(P＜0.01) and there was no difference versus control group(Pgt;0.05).ConclusionIt is suggested that combination of GMCSF and IL4 can selectively and effectively enhance proliferation and immune function of DC from peripheral blood of patient with pancreatic carcinoma.
【Abstract】Objective To explore the effect against gastric cancer induced by Newcastle disease virus modified autologous tumor vaccine （NDV-ATV）pulsed dendritic cells(DCs). Methods The Newcastle disease virus infected the gastric cancer lines (MNK45) and was lost its activity. Peripheral blood mononuclear cell (PBMC) were cultured under condition of recombinant human granulocyte macrophage-colony stimulating factor (1 000 u/ml)+IL-4(1 000 u/ml) + TNF-α(100 ng/ml). The tumor antigen specific cytotoxic T lymphocytes (CTL) was generated from activated autologous T cell by the Newcastle disease virus infected the MNK45 pulsed DC. And Cyto Tox 96TM in vitro assayed the cytotoxicity of CTL to MNK45. Thawed gastric cancer cell antigen were used as control in these experiments. Results The killing rate of MNK45 by antigen specific CTL reached (90.15±9.82)%, which was nearly twice as high as that of control(60.57±5.74)%. The CTL had much higher cytotoxicity to different differentiated type of gastric cancer cells such as MGC803〔(52.23±6.45)% 〕 and SGC7901〔 (61.75±8.84)%〕, as compared with LOVO〔(9.11±3.42)%〕 and HepG2 〔 (8.30±3.12)%〕tumor cells(P＜0.05). Conclusion Efficient and specific of against gastric cancer immunoreaction can be induced in virtue of NDV-ATV pulsed DCs, NDV-ATV loaded DCs might provide a new kind of theraputic means for gastric cancer.
ObjectiveTo study the function of interleukin-10 (IL-10) in inhibiting the activation of dendritic cells (DC) in chronic severe hepatitis B patients. MethodsMonocytes were isolated from peripheral blood of 16 chronic severe hepatitis B patients between March and September 2012, by ficoll-hypaque density gradient centrifugation and then cultured with plastic-adherence method. Dendritic cells were induced and proliferated from the monocytes with granulocyte-macrophage colony stimulating factor and interleukin-4 for 8 days. Hepatitis B virus core antigen and IL-10 were used to the DC culture to treat DC. The expression of surface marker on dendritic cells was detected by fluorescence-activated cell sorter. The cytotoxic T lymphocyte activity, as well as the interferon (IFN)-γ, IL-12p70 secretion were observed. ResultsThe ratio of CD83, HLA-DR and CD86 positive cells, the concentration of IFN-γ and IL-12p70, as well as the cytotoxic T lymphocyte activity by dendritic cells were significantly increased in hepatitis B virus core antigen treated group and decreased in the IL-10 treated group compared with that in the control group. Meanwhile, the ratio of CD83, HLA-DR and CD86 positive cells, the concentration of IFN-γ and IL-12p70, as well as the cytotoxic T lymphocyte activity by dendritic cells were significantly decreased in IL-10 pretreated plus Hepatitis B virus core antigen treated group compared with that in the hepatitis B virus core antigen treated group. These results indicated that the hepatitis B virus core antigen could induce dendritic cells activation, and IL-10 could inhibit the activation of dendritic cells, even the Hepatitis B virus core antigen being added afterwards. ConclusionIL-10 can inhibit the activation of dendritic cells, and attenuate the cytotoxicity of autologous lymphocytes induced by DC.
Objective To study the method of obtaining a large number of dendritic cells （DC）. To study the specific cytotoxicity T lymphocyte (CTL) effect against tumor cells initiated by DC pulsed with peptide of cancer cell. Methods Development of cells with cytologic features of DC in bone marrow cultures supplemented with granulocyte macrophage-colony stimulus factor (GM-CSF) and IL-4. Determining the DC phenotype and the specific structure by electronic microscopy. The CTL effect against pancreatic carcinoma leading by the DC pulsed with tumor cells lysate in vitro was observed. Results A large number of typical DC was proliferated by supplementing with GM-CSF and IL-4 cytokines. DC had specific cell appearance and structure, and highly expressed various cell surface molecules. TNF-α had the ability of stimulating DC mature, the mature DC had the enhancing abilities of antigen presenting and IL-12 self-secreting, as well as, expressed higher levels of CD54， MHC-Ⅱ and CD86 molecules than control group (P＜0.05). T lymphoid cell stimulated by DC without tumor antigen could not recognize and kill the target cells, only if DC pulsed with peptide of cancer cell can lead a b immune response to special tumor cells. The inhibiting ratio of CTL was significantly higher than that in other groups (P＜0.01). Conclusion Bone marrow DC has b ability of inducing special CTL against determined cancer cells after they are pulsed with tumor cell lysate. DC vaccine is probably a new immunotherapeutic method against tumor in the near future.
Objective To explore the effect of dendritic cells (DCs) allergized by K-ras mutant peptide on expressions of chemokines CCL19, CCL22, and cytoskeletal protein fascin-1. Methods DCs were derived from peripheral blood in the presence of granuloceyte/macrophage-colony stimulating factor, interleukin (IL) -4 in vitro. The DCs were collected on day 7 after culture, and were divided into non-K-ras mutant peptide group (addition of RPMI 1604 culture solution 50 μg/ml) and K-ras mutant peptide group (addition of K-ras mutant peptide 50 μg/ml). Phenotype was identified by flow cytometry. The morphological structure was observed by scanning and transmission electron microscopies, respectively. The expressions of IL-12, CCL19, and CCL22 were tested continuously by enzyme-linked immunosorbent assay (ELISA). The expression of cytoskeletal protein fascin-1 was determined by Western blot. Results ①The expressions of CD1a, CD80, and CD86 after loading K-ras mutant peptide were higher than that before loading K-ras mutant peptide (Plt;0.01). ②The DCs with petal-like and branch-like profections after loading were observed under scanning electron microscopy; The DCs with irregular shapes, branch-like or burr-like were showed under transmission electron microscopy. ③The expressions of IL-12, CCL19, and CCL22 in the Kras mutant peptide group were higher than those in the non-K-ras mutant peptide group at different times (6, 12, 24, and 48 h) after loading Kras mutant peptide (Plt;0.01). ④The expression of fascin-1 in the K-ras mutant peptide group was also higher than that in the non-K-ras mutant peptide group (Plt;0.01). Conclusion K-ras mutant peptide can promote DC to mature and improve the expression of chemokines and cytoskeletal protein which will strengthen DC migration.