Objective To detect the anti-colon cancer ability of whole cell lysates pulsed dendritic cell (DC) which acts as an adjuvant. Methods Whole cell lysates of LoVo cells were extracted with freeze thawing method, then the monocyte-derived DC were pulsed with this cellular antigen. Subsequently, the capability of antigen pulsed DC to promote T lymphocytes proliferation and the ability of T lymphocytes to kill LoVo cells were detected by 3H-TdR incorporation and lactate dehydrogenase release methods, respectively. Results The whole cell lysates of LoVo cells pulsed DC significantly stimulated T cells proliferation, and the cytotoxicity abilities of primed T cells to kill LoVo cells were also enhanced. Conclusion Tumor cell lysates which act as the cellular antigen to pulse DC can improve the efficacy of anti-cancer immune response， which provides us an experimental evidence for cancer immunotherapy.
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.
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 investigate the effects of FasL gene-modified dendritic cell (DC) on the airway inflammation in mice sensitized/challenged by house dust mite (HDM) allergen.Methods FasL gene-modified DC (FasL-DC) and control DC (nontransfection DC) were administrated into HDM sensitized and challenged mice by intratracheal injection respectively，then HDM sensitized and challenged mice were sacreificed two days later.Total and differentiation cell counts and levels of interleukin-4（IL-4），IL-5 and interferon-γ(IFN-) in bronchoalveolar lavage fluid (BALF) were detected and lung histological features were observed.Results After administration of FasL-DC，lung allergic inflammation was ameliorated while total cell counts，the percentage of eosinophil ,the levels of IL-4 and IL-5 in BALF decreased and the level of IFN- in BALF increased.Conclusion Administrating FasL-DC into HDM sensitized/challenged mice can inhibit Th2 cells activation and ameliorate airway allergic inflammation.
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 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.
Objective To investigate the feasibility of dendritic cells ( DCs ) as vector of immunotherapy through intratracheal injection. Methods The DCs obtained from the bone marrow of BALB/ c mice were cultured and isolated with CD11c-positive magnetic beads. Then DCs were overloaded with ovalbumin peptide 323-339 ( OVA 323-339) for 24 hours. The mice in the DC-OVA group were intratrachelly injected DCs overloaded with OVA 323-339 in dose of 2 ×106 cells per mouse. The mice in thenegative control group were intratracheally injected with DCs untreated by OVA 323-339. On the second day,all mice were challenged with 1% OVA in PBS lasting for five days. The asthma animal model established by classic method was used for the positive control. Pathologic changes in lung and cell numbers in bronchoalveolar lavage fluid ( BALF) were assayed 24 hours after challenged. Results Just like the lung tissues from the mice asthma models, the lung tissues from the mice instilled with DCs overloaded with allergen OVA 323-339 showed extensive inflammatory cells infiltration, most of which were eosinophils, neutrophils and lymphocytes. The lung tissues in the DC group showed no obvious inflammation. There were more cells in BALF in the DC-OVA group than that in the DC group. OVA-specific IgE in serum from the DC-OVA group was not significantly different from that in the mice asthma models [ ( 48. 22 ±4. 76) U/mL vs. ( 52. 75 ±4. 03) U/mL, P gt;0. 05] . Conclusion DCs overloaded antigen has the ability of transferring of antigen effectively and may be used as vectors of immunotherapy.
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.
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.