Suppression of tumor growth by mRNA DC vaccine in severe combined immune deficiency mouse

AIM: To establish HCC Hu-PBL-SCID(severe combined immune deficiency) chimeric model,and to observe the antitumor effect of mRNA-dendritic cell vaccine.METHODS: Hu-PBL-SCID chimeric model was established by intraperitoneal injection of human peripheral blood lymphocytes.Human IgG in mouse serum was detected by ELISA to identify the model.After the model was established,the mice were divided into four groups,and were inoculated with mRNA DC vaccine,anti CD4+,CD8++mRNA DC,naive DC,and PBS,respectively.Then the animals were injected subcutaneously with 2×106 HepG-2 cells.Tumorigenecity,latent period,and tumor volume were observed,and antitumor efficacy of CTL was measured.RESULTS: The concentration of human IgG in mouse serum in Hu-PBL- SCID model was detected,indicating that the Hu-PBL-SCID model was established successfully.No significant difference of tumorigenecity among the four groups was observed.However,tumors in mRNA DC vaccine group grew slowly,tumor volumes were significantly smaller than those in anti CD⁴⁺,CD⁸⁺+mRNA DC,PBS and naive DC groups.The spleen cells in mRNA DC vaccine group specifically killed the HepG-2 cells but not SGC-7901 cells.CONCLUSION: mRNA DC vaccine shows anti-tumor immune response in vivo by inducing CD⁴⁺,CD⁸⁺T lymphocyte immune responses.     

Immune function of dendritic cells in patients with hepatocellular carcinoma

AIM: To investigate the immune function of dendritic cells (DCs) in patients with hepatocellular carcinoma(HCC). METHODS: The DCs were cultured from human peripheral blood mononuclear cells (PBMC) by using GM-CSF and IL-4 for 7 days. Surface molecules CD86, HLA-DR of DCs were detected by flow cytometry. IL-12 production by DCs and IFN-γ production by T cells was measured with ELISA and ELISPOT, respectively. Allogenic mixed lymphocyte reaction was detected by MTT assay. RESULTS: CD86 expression in DCs in HCC patients were markedly lower than that in health control (91.7％ vs 83.5%, P<0.05). IL-12 production by DCs had no significant difference between the HCC patients and the health control ［(324.6±171.0)ng/L vs (436.5±142.7)ng/L, P>0.05］. However, after stimulated DCs with LPS, IL-12 production in HCC patients was significantly lower than that in health control ［(478.6±142.7)ng/L vs (630.0±151.9)ng/L, P<0.05］. IFN-γ production by T cells and T cell proliferation index (PI) in the HCC patients were all significantly lower than those in health control ［(IFN-γ: 133.4±51.2)103U/L vs (183.0±60.2)103U/L, P<0.05; PI: 2.3±0.7 vs 3.5±0.8, P<0.01］. CTL killing activity assay indicated that DC-induced CTL killing activity in HCC group was significantly lower than that in health group when the E/T ratio was 10∶1 and 20∶1, respectively. CONCLUSION: The immune function of DCs and T cells in the patients with HCC are significantly decreased. The CTL killing activity of HCC group is also markedly decreased.     

WT1 peptide-loaded DC triggers cytotoxic T lymphocytes and killing effects on K562 cells in vitro

AIM: To study the effects of WT1 peptide-loaded dendritic cells (DC) stimulating the cytotoxic T lymphocytes (CTL) on K562 cells in vitro. METHODS: DC were generated from normal human peripheral blood mononuclear cells (PBMC) in the presence of granulocyte-macrophage colony stimulating factor(GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-α) , DC were cultured with WT1 peptides , and then triggered T cells into specific CTL. RESULTS: Most suspended cells exhibited distinctive morphological features of DCs and they stimulated proliferation of allogenic lymphocytes. Under the effector : target ratio of 20∶1, CTLs derived from cultures with DC and WT1 peptides were showed 86.1%±26.8% cytotoxicity against K562 cells, cytotoxicity by CTLs derived from cultures with unloaded DC against K562 cells were 47.1%±20.8% and cytotoxicity by lymphocytes were 27.7%±15.3%. Cytotoxicity by CTLs derived from culture with WT1 peptides-loaded DC were the strongest among three groups （P＜0.05）. CONCLUSION: CTLs derived from cultures containing DC pulsed with WT1 peptides show the strongest cytolytic activities on K562 cells.     

Anticancer effect of mouse marrow-derived dendritic cells transfected by pcDNA3-hCEA

AIM: To prepare the vaccine of DCs(pcDNA3-hCEA) and observ the immunity effect of the DCs(pcDNA3-hCEA) inoculating on CT26(hCEA⁺) loaded in BALB/c mice. METHODS: DCs were generated from bone marrow in the presence of rmGM-CSF and rmIL-4. A new recombinant plasmid, pcDNA3-hCEA, reformed with inserting a 2.4 kb human CEA cDNA into pcDNA3. DC vaccine was prepared by transfection with pcDNA3-hCEA using lipofectamine. CEA mRNA expressed in DCs(pcDNA3-hCEA) was confirmed by RT-PCR, CEA expression level was detected with RIA method, and CEA specific CTL was induced in vitro. After vaccination of DCs(pcDNA3-hCEA), the survival time of the BALB/c mice challenged with critical loading CT26(hCEA⁺) was observed. RESULTS: G₄₁₈ test showed that about14% DCs were transfected with pcDNA3-hCEA. And CEA mRNA and protein could be detected respectively by RT-PCR and RIA in the genetically modified DCs. Furthermore, the DCs coud be targeted by specific CTL, the survival time of the mice challenged with CT26(hCEA⁺) was prolonged1-4 weeks. CONCLUSION: These results demonstrate that specific antitumor immune responses could be induced efficiently by vaccination of DCs(pcDNA3-hCEA), which is transfected eukaryotic expression vector encoding tumor antigen gene.     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

A dynamic observation on the levels of TNF-α、IL-6 and IFN-γ produced by human dendritic cells infected with Dengue virus

AIM: To study the dynamic levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and gamma interferon （IFN-γ） produced by human dendritic cells infected with Dengue virus. METHODS: Monocytes isolated from healthy human peripheral blood were incubated in medium with GM-CSF and IL-4 for more than 7 days. DCs were then collected and identified by scanning electron microscope, immunohistochemistry and lymphocytes stimulatory ability. Human dendritic cells （DC) were infected with Dengue-2 virus (DV-2) in vitro， culture supernatants were collected in different time postinfection (6 h, 12 h, 24 h, 48 h and 72 h)， DV antigen in human dendritic cells were demonstrated by an indirect immunofluorescent assay （IFA）, production of TNF-α, IL-6 and IFN-γ in the culture supernatants were evaluated by ELISA. RESULTS: After 7 days, typical dendritic cells could be obtained. Virus antigen were detected in infected DC by IFA. Dengue virus induces TNF-α and IL-6 secretion from DC and does not induce IFN secretion. CONCLUSION: Human dendritic cells are target of dengue virus infection. TNF-α， IL-6 production from DC are increased with DV infection. Dendritic cells may play an important role in DV pathogenicity and immunity.     

Enhanced effect of CD8+ T cells activated by tumor lysate-pulsed DCs on killing autologous tumor cells

AIM: To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell-mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by conglutination method. The immature dendritic cells were generated in the presence of interleukin-4(IL-4) and granulocyte/macrophage colony-stimulating factor(GM-CSF) from monocytes of healthy individuals. These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes (CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay against autologous human tumor cells.　RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor-pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay against target autologous tumor cells. The CD95(Fas) expression, IFN-γ and TNF-α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autologous tumor cells was significantly different(P<0.05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could induce the specific antitumor effect against autologous tumors . This in vitro model offers a new and simple approach to the development of DC+CTL-based immunotherapy.     

Human dendritic cells transfected with MUC1 mRNA induce lethal effect of cytotoxic T-lymphocytes on non-small cell lung cancer in vitro

AIM：To investigate the specific anti-tumor effects of mature dendritic cells (DCs) transfected with amplified mucin 1 (MUC1) mRNA in vitro. METHODS：DCs separated and purified from the peripheral blood mononuclear cells were induced in vitro and then identified by flow cytometry. pcDNA3.1（+）-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro. The MUC1 mRNA was transfected into DCs by electroporation. MUC1-transfected DCs were used to induce T cells to be cytotoxic T-lymphocytes. Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs. The proliferation of T cells was examined by MTT assay. The proportion of CD8+ cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay. The secretion of IFN-γ was detected by ELISA. RESULTS：The marker gene expression in the DCs transfected with MUC1 mRNA was significantly increased compared with control group, peaking at 24 h. The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10. The proportion of CD8+ cells in transfection group was higher than that in control group. The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group. The level of IFN-γ in the cell supernatant of transfection group was higher than that in control group. CONCLUSION：DCs plus MUC1 mRNA by electrical transfection induces specific anti-tumor effects, which provides an experiment evidence of using MUC1 as a target for immunotherapeutic strategy against non-small cell lung cancer.     

Anti-cancer effect of cytotoxic T lymphocytes activated by sensitized dendritic cells

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P<0.01). Injection of CTLs activated by HT-29 cell antigen sensitized DCs inhibited HT-29 transplanted tumor growth and prevented HT-29 tumor occurring in nude mice (P<0.05). PCNA expression level in tumor cells in CTL therapy group was dramatically lower than that in control (P<0.05). CONCLUSION: CTL activated by sensitized DCs kill tumor cells specifically, inhibit transplanted tumor growth and prevent tumor transplantation in nude mice.     

Expression and mechanisms of hsa_circ_087631 in patients with primary biliary cholangitis

AIM To explore the expression and mechanisms of circular RNA hsa_circ_087631 in the patients with primary biliary cholangitis （PBC）. METHODS RT-qPCR was used to detect the expression of hsa_circ_087631 in PBC patients and healthy controls. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was constructed and transfected into human acute T cell leukemia Jurkat cells， and then the expression levels of hsa_circ_087631， Bcl-6 mRNA and interleukin-21 （IL-21） mRNA were detected by RT-qPCR. Dual-luciferase reporter assay was performed to identify the interactions between hsa_circ_087631 and hsa-miR-346. RESULTS The expression of hsa_circ_087631 in the PBC patients was significantly increased compared with the healthy subjects. Hsa-miR-346-overexpressing lentiviral vector pLenti-EF1a-EGFP-F2A-Puro-CMV-MCS was successfully constructed. The expression of hsa-miR-346 was significantly increased after the hsa-miR-346-overexpressing lentiviral vector was transfected into the Jurkat cells， while the expression levels of hsa_circ_0087631， Bcl-6 mRNA and IL-21 mRNA were significantly decreased. After wild-type or mutant hsa_circ_087631 vector and hsa-miR-346 mimics were transfected into 293T cells， the results of dual-luciferase reporter assay showed that hsa-miR-346 significantly decreased the luciferase activity of wild-type hsa_circ_087631 （P<0.01）， but the regulation did not change significantly after mutation of the predicted binding site. CONCLUSION Peripheral blood hsa_circ_087631 level is elevated in the PBC patients. The hsa_circ_087631/hsa-miR-346/Bcl-6 signaling may take effect in human T cells. Hsa-miR-346 significantly reduces the expression of hsa_circ_087631， but it may not be regulated by predicted binding sites.     

Homoharringtonine and interferon α-2b promote maturation of leukemic dendritic cells derived from K562/A02 cells

AIM: To study the maturational effect of homoharringtonine (HHT) and interferon α-2b on leukemia-derived dendritic cells(DCs). METHODS: Cytokines rhGM-CSF and rhIL-4 were added into the leukemia cells K562/A02. After 7 d induction, the cell-morphology was observed with the inverted microscope, the immunophenotype of cells was detected by flow cytometry and the cell function was evaluated by allogeneic mixed lymphocyte reactions, CTL responses and secretion of IL-12. Then homoharringtonine, interferon α-2b and homoharringtonine+interferon α-2b were added to these leukemia-derived DCs. Three days later, the DCs were redetected by the above-mentioned methods.RESULTS: After induced by homoharringtonine and interferon α-2b, the leukemia-derived DCs with typical dendritic morphology were increased. The expressions of CD83, HLA-DR and CD86 were (65.50±8.40)%, (32.00±4.32)% and (18.65±3.20)% respectively in 7 d leukemia-derived DCs, raised to (85.36±8.42)%, (39.58±7.68)% and (35.53±4.35)% respectively after exposing to homoharringtonine for 3 d, and increased to (87.15±7.59)%, (40.53±6.30)% and (38.45±6.42)% respectively after treated by interferon α-2b; and further increased to (94.38±6.59)%, (52.75±8.51)% and (42.98±9.87) % respectively after treated by homoharringtonine+interferon α-2b. These results were markedly different from unaffected cells. These DCs induced by HHT and interferon α-2b were upregulated significantly the capacity for stimulating allogeneic T cells. They also induced CTL to generate specific cytotoxic activity against K562/A02 cells and there was the strongest effect when the ratio of effector and targetor was 20∶1. The secretion of IL-12 was increased remarkably.CONCLUSION: Homoharringtonine and interferon α-2b induce the maturation of the leukemia-derived DCs and there is the strongest function when homoharringtonine cooperates with interferon α-2b.     

Effects of MAGEA3 inhibition by shRNA on apoptosis in human hepatocellular cancer cells

AIM:To investigate the inhibitory effect of vector-based RNA interference ( RNAi) on the expression of melanoma associated antigen A3 (MAGEA3) protein in hepatocellular carcinoma cells and on apotposis of hepatocellular carcinoma cells. METHODS:A vector for transcribing specific small hairpin RNA ( shRNA) targeting MAGEA3 gene was constructed ,introduced into hepatocellular carcinoma MEL-ED1 cells by Lipofectamine 2000. The MAGEA3 protein and mRNA expression levels of MEL-ED1 cells were detected by Western blotting and RT-PCR, respectively. The cell apoptosis was studied by DNA fragmentation, electron microscopy ，TUNEL assay, and annexin V/PI staining. RESULTS:The vector of RNA interference was successfully constructed and MAGEA3 expression was descreased significantly in MEL-ED1 cells. After the shRNA expression vector was transfected into the MEL-ED1 cells, the expression of MAGEA3 gene was inhibited significantly ( by 90% ). DNA fragmentation，electron microscopy and TUNEL assay showed classic apoptosis characters in the MEL-ED1 cells transfected with pSilencer-MAGEA3 plasmid with an apoptosis rate of 21.41% ±1.98%, significantly higher than those in the negative control group transfected with pSilencer-neo and in the non-transfected group (both P<0.01). CONCLUSION:The specific small hairpin RNA targeting MAGEA3 mRNA can inhibit the expression of MAGEA3 and cause apoptosis of hepatocellular carcinoma cells , which suggests inhibitory effect of MAGEA3 on apoptosis in cancer and provides an experimental basis for treating human tumors with RNAi.     

The in vitro immune effects of dendritoma formed by mouse hepatocellular carcinoma cells and lymphotactin gene modified dendritic cells

AIM: To investigate the in vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma cells and lymphotactin gene modified dendritic cells (DCs). METHODS: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol. RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein levels. The phenotypes and fusion efficiency were detected by FACS. The stimulatory capacity of DC to T cells was detected by mixed leukocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. RESULTS: Lymphotactin effectively expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells induced potent T cell proliferation effect and generated strong CTL reaction against allogenic H22 cells. CONCLUSION: Lymphotactin genetic modification enhanced the in vitro immune activity of dendritoma.     

Combination of R848 and Poly(I: C) promotes antigen presentation by DC and enhances killing effect of CTL on lung adenocarcinoma A549 cells

AIM: To investigate the effect of R848 (a Toll-like receptor 7/8 agonist) combined with poly-inosinic:polycytidylic acid[Poly(I:C), a Toll-like receptor 3 agonist] on dendritic cell (DC) maturation, and the killing effect of DC-induced cytotoxic T-lymphocytes (CTL) on human lung adenocarcinoma A549 cells. METHODS: Mononuclear cells were isolated from human peripheral blood and induced to differentiate into DC. The whole-cell lysate of A549 cells, namely tumor cell lysate (TCL), was used as antigen. R848 combined with Poly(I:C) was used as adjuvant to stimulate the DC. DC surface markers were analyzed by flow cytometry. The DC stimulated by antigen was co-cultured with T-lymphocytes for 7 d to induce CTL. The culture supernatant and CTL were collected. The levels of interleukin-12 (IL-12) p70, interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant were measured by ELISA. The CTL and A549 cells were co-cultured for 16 h, and the cytotoxicity was observed by LDH assay.RESULTS: The expression of CD83 and CD80 on the DC surface, and the secretion of IL-12 p70 in DC-R848+Poly(I:C) group were significantly increased compared with DC-TCL group (P<0.01). In addition, the cytotoxicity of CTL for A549 cells in DC-R848+Poly(I:C) group was significantly enhanced compared with DC-TCL group (P<0.01). The secretion levels of IFN-γ and TNF-α in DC-R848+Poly(I:C) group were significantly elevated compared with DC-TCL group (P<0.01). CONCLUSION: R848 combined with Poly(I:C) significantly promotes DC maturation and activation, and enhances the antigen-presenting effect of DC and the cytotoxicity of DC-induced CTL.     

Donor dendritic cells treated with B7-1, B7-2 antisense oligonucleotide prolonged mouse cardiac allograft survival

AIM:To investigate the effect of donor bone marrow derived dentritic cell (DC) treated with B7-1, B7-2 antisense oligonucleotide on mouse heart allografe survival time and its mechanism. METHODS: There were 7 groups of C57BL/10J (B10) mouse bone marrow DCs which were treated by 400 nM antisense oligonucleotide target to B7-1, B7-2 mRNA (AS B7-1/2), B7-1 mismatch oligo control, B7-2 mismatch control(mASB7-1/2), lipofectamine only and non-treatment, respectively. Each group of DC were named as ASB7-1 DC, ASB7-2 DC, mASB7-1DC, mAS B7-2DC, and Lipo DC, respectively.RESULTS: Flow cytometer results shown that AS B7-1/2 can inhibit B7-1(CD80)and B7-2 (CD86) molecule express on DC surface, while control groups have no effects. To observe their tolerogenicity in mouse cardiac allograft model, B10→C3H heterotopic heart transplantation were performed. Recepients were received 2×10⁶ of DC injection 7 days before transplantation. Results showed that both AS B7-1DC and AS B7-2 DC can prolong mouse cardiac allograft survival time to (18.6±0.89) days and (23.67±10.73) days, respectively, compared with IL-4 DC .Two mismatch control groups can slightly prolong while oligo DC has no effect. For understanding its mechanism, each group of DC was used as stimulator to stimulated C3H spleen T cell. Results suggested that AS B7-1DC and AS B7-2 DC had less allo-stimulate function, including MLR and generation CTL and IL-2 production than IL-4 DC but control groups have no effect.CONCLUSION: Donor bone marrow derived DC treated with AS B7-1 oligo and AS B7-2 oligo expressed lower level of CD80 and CD86, respectively. These cells can induce allogeneic T cells anergy in vitro and markedly prolong mouse heart allograft survival time in vivo.     

Effect of lung cancer cells transfected with interleukin 18 gene on the immunological activity of dendritic cells

AIM: To study the effect of interleukin 18 gene transfected lung cancer cells on the phenotype and immunological activity of dendritic cells (DC). METHODS: A secretive IL-18 expression vector containing IL-12 P40 signal sequence was constructed and transfected into NCI-H460 lung cancer cells. DC induced from human peripheral blood were divided into 4 groups (NT, PV, GT and PD). DC were stimulated by non-transfected NCI-H460 cells, pure vector transfected NCI-H460 cells and IL-18 transfected NCI-H460 cells respectively for group NT, PV, GT, and non-stimulated DC for group PD. CD54, CD80, CD83 and CD86 on DC in the 4 groups were detected with flow cytometry. T cell proliferation stimulated by DC in the 4 groups was assayed with MTT method. IL-12 release in cultured DC supernatant was measured by ELISA. RESULTS: Sequencing result of the secretive IL-18 was correct. The transfected cells expressed IL-18 fusion gene and 18 kD IL-18 protein. DC in GT group expressed more surface molecules than those in other 3 groups. T cell proliferation and IL-12 secretion in GT group were higher than those in other 3 groups. CONCLUSION: IL-18 gene transfected NCI-H460 cell increases surface molecule expressions on DC. It also enhances immunological activity and IL-12 secretion in DC.     

OK-432 enhances the effect of umbilical cord blood dendritic-cells treated with gastric cancer tumor vaccine

AIM: To evaluate the effect of replacing fetal cattle serum with umbilical cord plasma and application of OK-432 on dendritic cells from umbilical cord blood in vitro so as to offer a new technical way of preparation high-powered dendritic-cell-based vaccines for the cancer immunotherapy.METHODS: Human cord blood mononuclear cells (CBMCs) were cultured in RPMI-1640 containing 10% autologous plasma,GM-CSF and IL-4,some of which were supplemented with tumor lysates and/ or OK-432.MTT assay was applied to measure the antigen presenting ability of DCs in allo-MLR.Killing rates of autologous T lymphocytes induced by DCs on different target cells were measured by LDH method.RESULTS: Cells appeared typical morphology of DCs after culture and the allo-stimulate capacity of DCs and the CTL response in vitro were enhanced by treating with tumor lysates and OK-432.CONCLUSION: Mature DCs can be induced from human CBMCs by this means with fewer cytokines and less time.The tumor lysate antigens are captured by DCs treated with tumor lysates and OK-432,and presented to lymphocytes successfully,indicating a new way to develop dendritic-cell-based vaccines for clinical immunotherapy of gastric cancer and other tumors.     

Effect of glucocorticoid-treated dendritic cells on asthmatic Th2 response

AIM: To investigate the effect of dexamethasone-treated dendritic cells (DCs) on Th2 cytokine production from autologous T cells in asthmatic patients and explore the mechanisms by studying the effect of dexamethasone on differentiation, maturation and function of DCs from patients with asthma. METHODS: Human peripheral blood monocyte-derived DCs generated from asthmatic patients and healthy subjects were cultured in the absence or presence of dexamethasone. The phenotypic characterization of DCs was analyzed by flow cytometry. The mature DCs were harvested, washed, and then cocultured in vitro with autologous T cells purified by a nylon cotton column. The DC-T coculture supernatants were collected after 72 h incubation and analyzed for levels of IL-5 and IFN-γ by ELISA. RESULTS: The concentrations of IL-5 in the culture supernatants of DC-T coculture were significantly up-regulated in patients with asthma compared with that in healthy controls ［(145.13±89.76) ng/L vs (50.28±22.37) ng/L, P<0.01］. The level of IFN-γ in the DC-T coculture supernatants tended to be decreased in asthmatic patients than that in healthy controls, although this difference did not achieve statistical significance ［(197.58±76.32) ng/L vs (220.46±65.34) ng/L, P>0.05)］. There were significantly decreased levels of IL-5 by autologous T cells primed by dexamethasone-treated mature DCs from asthmatic patients ［(45.39±19.61) ng/L vs (145.13±89.76) ng/L, P<0.01］, alterations not observed from healthy controls (P>0.05). IFN-γ production was decreased by autologous T cells primed by dexamethasone-treated mature DCs from both asthmatic patients and healthy controls ［asthma group: (40.21±22.89) ng/L vs (197.58±76.32) ng/L, P<0.01; healthy controls: (56.78±20.37) ng/L vs (220.46±65.34) ng/L, P<0.01］. Dexamethasone-treated DCs exhibited decreased expression of CD83 (P<0.01) and increased expression of CD14 (P<0.01) in both asthmatic patients and healthy controls. CONCLUSION: DCs of asthmatic patients induce a Th2-skewed cytokine production from autologous T cells. Dexamethasone-treated DCs inhibit the Th2 reactions, and this effect is probably mediated through the pathway that dexamethasone inhibits DCs maturation and skews the macrophage/DC balance towards the macrophage side and thus directs the development more towards the macrophage lineage.