ObjectiveTo explore the antitumor effect of tumor vaccine fused from dendritic cells (DC) and Walker-256 cancer cells on implanted liver cancer in rats and the related mechanism of inhibition for tumor angiogenesis. MethodsWalker-256 cancer cells and mature DC were fused by 50% polyethylene glycol method for preparation of DC-Walker-256 fusion vaccines. Implanted liver cancer models were established through operations on healthy male SD rats at the age of 6－8 weeks. All the rats were divided into four groups, and rats in each group were injected subcutanely with fusion vaccine (group), mixed cultured cells (group), simple DC (group), and PBS (blank control group), respectively. On 28 d after making model, the rats were put to death, the tumor was observed and pathological essays were prepared. All rats’ spleens were collected and prepared into lymphocyte to detect antigenic specificity cytotoxic T lymphocyte (CTL) by enzymelinked immunosorbent spot (ELISPOT) method. The expressions of VEGF, ANG-1, ANG-2, and MVD were detected by immunohistochemistry. ResultsThe numbers of rats survived in the fusion vaccine group, mixed culture cells group, simple DC group, and blank control group was 8, 5, 6, and 3, respectively. The rats in the other three groups except for fusion vaccine group were manifested as inaction, anorexia, and gloomy fur in some degree as well as ascites. The tumorigenesis was found in all survival rats except for two in the fusion vaccine group. The weight of liver tumors of rats in the fusion vaccine group 〔(32.4±9.2) g〕 was significantly lighter than that in the mixed culture cells group 〔(67.3±5.1) g, P=0.031〕, simple DC group 〔(75.0±8.3) g, P=0.019〕, and blank control group 〔(86.6±10.5) g, P=0.008〕, respectively. The number of tumorspecific CTL of rats in the fusion vaccine group was also significantly higher than that in the other three groups (P=0.019, P=0.025, and P=0.001, respectively). The MVD of tumor tissue in the fusion vaccine group was (24.12±2.32) vessels/HP, which was significantly lower than that in the mixed culture cells group 〔(40.34±1.29) vessels/HP, P=0.025〕, simple DC group 〔(42.36±3.16) vessels/HP, P=0.035〕, and blank control group 〔(56.48±5.16) vessels/HP, P=0.006〕, respectively. The MVD of tumor tissue in the mixed cultured cells group and simple DC group was similar (P=0.165), however, which was significantly lower than that in the blank control group (P=0.040 and P=0.043). The positive rate of VEGFA protein expression was 23.2% in the fusion vaccine group, which was significantly lower than that in the mixed culture cells group (42.5%, P=0.031), simple DC group (61.3%, P=0.019), and blank control group (89.6%, P=0.003), respectively. The positive rate of VEGF-A protein expression in the mixed cultured cells and simple DC groups was similar (P=0.089), however, which was significantly lower than that in the blank control group (P=0.027 and P=0.038). The positive rate of ANG-1 protein expression in the fusion vaccine group (43.2%) was not different from that in the mixed culture cells group (46.3%, P=0.292), simple DC group (51.3%, P=0.183), or blank control group (49.6%, P=0.179), respectively, and the difference of pairwise comparison in latter three groups was not significant (P=0.242, P=0.347, and P=0.182). The positive rate of ANG2 protein expression was 19.2% in the fusion vaccine group, which was significantly lower than that in the mixed culture cells group (62.3%, P=0.007), simple DC group (67.3%, P=0.005), and blank control group (71.6%, P=0.004), respectively, however, the difference of pairwise comparison in latter three groups was not significant (P=0.634, P=0.483, and P=0.379). ConclusionFused vaccine can induce CD8+ CTL aiming at tumor cells and establish the effective antitumor immunity in vivo and also downregulate the level of VEGF and ANG-2 to suppress tumor angiogenesis and thereby achieve the purpose of curing tumor.
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.
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.
Objective To study the advances in the relationship between the number of infiltrating dendritic cells and the postoperative prognosis of digestive malignant tumor. MethodsThe literature in recent years on the relationship between the number of infiltrating dendritic cells and the postoperative prognosis of digestive malignant tumor was reviewed.ResultsThe number of infiltrating dendritic cells among esophageal cancer,and gastric carcinoma,colonic cancer and pancreatic cancer was associated with a better prognosis.Conclusion The population density of dendritic cells among the malignant tissue could be regarded as an independent indicator in estimating the postoperative prognosis of malignant tumor.
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.
Tumor-derived exosomes play a role in helping tumor cells with escape from immune surveillance, and it may also activate tumor-specific immune responses to eradicate tumor cells. Tumor cells release exosomes with major histocompatibility complex molecules and antigenic peptides on the surface membranes, which can induce dendritic cells (DC) and cytokine-induced killer (CIK) cells in vitro to produce the tumor antigen-specific T cells, and the obtained DC-CIK cells have a dual antitumor function with specificity and non specificity. This provides a new method for the treatment of cancers. This review briefly summarized the latest progress of adoptive immunotherapy with exosomes and DC-CIK.
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.
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.
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.
Abstract: Objective To investigate the phonetype and tolerogenic function of semimature dendritic cells (DC) transfected by myeloid differentiation marker 88（MyD88） small interfering ribonucleic acid（siRNA）. Methods Bone marrow of BALB/c mice was inoculated and cultured in vitro，and induced into DC by 10ng/ml recombinated granulocyte macrophage colony stimulating factor (rmGM-CSF) ，then DC was divided into three groups at the 8th day: blank control group: added nothing; lipopolysaccharide （LPS）group: added with 1μg/ml LPS; and experimental group: added with 1μg/ml LPS after transfected by MyD88 siRNA for 4 hours. The phonetype of three groups was analysed by fluorescenceactivated cell sorting (FACS). The concentration of interleukin 10（IL-10）and interleukin 12（IL-12） in culture supernatant was detected by enzyme linked immunosorbent assay（ELISA）.The function of stimulating alloreactive T cell roliferation were evaluated by primary and secondary mixed lymphocyte reaction （MLR）. The cardiac allograft survival time was compared after DC of three groups injected into recipient mice. Results The phonetype of blank control group DC was CD11c＋，CD25-，CD40low，CD80low，CD86low，MHC-Ⅱlow， which could be induced to mature DC by LPS. Experimental group DC was phonetypically semimature DC (CD11c＋，CD25－，CD40mid，CD80low，CD86low，MHC-IIid) and the IL-10/IL-12 ratio of semimature DC increases significantly. yporesponsiveness of alloactive T cells can be induced by experimental group DC and the survive time of heart allograft was prolonged. Conclusion Immature DC could become semimature DC after transfected by MyD88 siRNA and stimulation by LPS. These semimature DC are more tolerogenic than immature DC.