The potential role of staphylococcal enterotoxin B in the early intestinal injury in postburn Staphylococcus aureus sepsis

AIM: To investigate the role of staphylococcal enterotoxin B (SEB) in early intestinal injury in scald rats with Staphylococcus aureus sepsis. METHODS: 86 male Wistar rats were randomly divided into four groups as folows: normal controls (n=10), scald control group(n=10), postburn sepsis group (n=50) and SEB monoclonal antibody (MAb) treatment group (n=16). Plasma samples were collected to determine SEB, endotoxin, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). RESULTS: After scald injury followed by Staphylococcus aureus challenge, the levels of SEB, TNF-α and IFN-γ in plasma were significantly higher than those of normal controls, peaking at 2-6 h (P<0.05 or P<0.01), while the intestinal diamine oxidase (DAO) activity declined constantly (P<0.05). It was shown that plasma SEB levels were significant negatively correlated with intestinal DAO activity (r=-0.4398, P=0.0170), and SEB MAb pretreatment could ameliorate the intestinal injury to certain extent. Moreover, Staphylococcus aureus challenge could increase the endotoxin levels in plasma and various tissues, which were attenuated by SEB MAb pretreatment. CONCLUSION: In postburn sepsis, SEB might be involved in the development of intestinal barrier dysfunction, which in turn resulting in gut-derived endotoxin translocation and aggravating the pathophysiologic changes caused by Staphylococcus aureus challenge.     

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.     

Phenotype and immune activity of dendritic cells under interleukin-18 intervention

AIM: To investigate the phenotype and immune activity of dendritic cells using interleukin-18 as intervent.METHODS: Monocytes were isolated from human peripheral blood and induced into DCs with GM-CSF and IL-4. The cellular morphous was observed under inverted microscope. On the 5th day, 3 groups including IL-18 group, TNF-α group and IL-18+TNF-α group were set. IL-18, TNF-α or IL-18+TNF-α was used as intervents respectively to facilitate cell maturity. Supernatants were collected at 24 h, 48 h and 72 h. IL-12 in the supernatant, CD1a, HLA-DR, CD83 and CD86 were analyzed using flow cytometry. DCs of the 3 groups were co-cultured with T cells respectively on the ratio of 1∶〖KG-*2〗100, 1∶〖KG-*2〗50 and 1∶〖KG-*2〗10. T cell proliferation stimulated by DC was determined using MTT method. DCs were co-cultured with T cells on the ratio of 1∶〖KG-*2〗10, and the supernatant were collected at 24 h, 48 h and 72 h. IFN-γ in the supernatant was detected with ELISA method.RESULTS: Induced by GM-CSF and IL-4, then stimulated by IL-18, TNF-α or IL-18+TNF-α, monocytes showed typical morphous of DC. No morphological difference was observed among DCs of the 3 groups. No statistical difference showed in expression level of CD1a, HLA-DR, CD83 and CD86 between IL-18 group and TNF-α group (P>0.05). The positive rates of CD1a and CD83 in IL-18+TNF-α group were higher than those in other 2 groups. The positive rate of HLA-DR in IL-18+TNF-α group was higher than that in IL-18 group. No difference between IL-18 group and TNF-α group in the potency of stimulating T cell proliferation was found, whereas the stimulating potency in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group. IL-12 in IL-18+TNF-α group at 48 h and 72 h was higher than that in IL-18 group and TNF-α group (P<0.05). However, there was no difference between the latter 2 groups. There was also no difference between IL-18 group and TNF-α group in IFN-γ secretion. IFN-γ in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group (P<0.05).CONCLUSION: Using IL-18 as intervent, DC expresses high level of surface molecules, secretes high level of IL-12, stimulates T cell proliferating effectively and produces IFN-γ potently. The actions are stronger when used in combination with TNF-α. It suggests that IL-18 may serve as a promoting agent of DC maturity, or combination with TNF-α in DC induction will strengthen the immune activity of DC.     

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.     

SEB superantigen induces steroid resistance in PBMCs

AIM: To establish a model of staphylococcal enterotoxin B (SEB)-induced steroid resistance in human peripheral blood mononuclear cells (PBMCs), and to investigate the potential mechanism of SEB superantigen-induced steroid resistance in vitro. METHODS: PBMCs were isolated from normal children blood by Ficoll-Hypaque gradient centrifugation and stimulated with SEB at different concentrations. The proliferation rate of cells was measured by MTT assay. The subcellular localization of glucocorticoid receptor α (GRα) was examined by confocal microscopy. Protein phosphorylation was measured by means of Western blotting. RESULTS: SEB induced steroid resistance in a range of 10-500 μg/L and no significant difference among concentrations was observed. In SEB-stimulated PBMCs, the GRα did not translocate to the nuclear after dexamethasone treatment. ERK inhibitor U0126 significantly attenuated the inhibition of GRα nuclear translocation in SEB-stimulated PBMCs. SEB also induced more rapid and sustained phosphorylation of ERK1/2 in PBMCs. CONCLUSION: This study demonstrates that SEB may contribute to steroid resistance through ERK pathway and is associated with abrogation of GRα nuclear translocation.     

Superantigen SEA antagonizes inhibitory effect of imatinib on T cell activation

AIM：To investigate the effects of staphylococcal enterotoxin A (SEA) on the inhibition of T lymphocyte activation induced by imatinib mesylate (IM). METHODS：Jurkat cells were stimulated with SEA (2 mg/L) and IM (5 nmol/L) for 24 h. The mRNA expression of CD3ε and ζ chains was measured by real-time fluorescence quantitative PCR. The protein levels of CD3ε and ζ chains were detected by Western blotting. RESULTS：The expression of CD3ε and ζ chains at mRNA and protein levels was down-regulated in the Jurkat cells stimulated by IM alone. These down-regulations of CD3ε and ζ chains were reversed by the stimulation of IM combined with SEA. The antagonistic effect of SEA on IM-mediated inhibition of CD3ε mRNA expression was significantly greater than that on CD3ζ mRNA. CONCLUSION:SEA antagonizes imatinib-mediated inhibitory effect on T cell activation.     

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.     

Dendritic cells transfected with tumor total RNA induce specific immune responses against hepatocellular carcinoma in vitro

AIM: To observe the ability of dendritic cells (DC) vaccine transfected with human hepatocellular carcinoma (HCC) total RNA induce specific cytotoxic T lymphocyte(CTL) response in vitro. METHODS: DCs generated from HCC patient’s peripheral blood mononuclear cells (PBMC) were incubated with recombinant human granulocyte macrophage colony-stimulationg factor (GM-CSF) and human interleukin (IL-4). Tumor total RNA was isolated from Hep G-2 cells and HCC cells. DCs transfected with tumor total RNA were used to induce specific CTL proliferation. Specific cytotoxicity was measured using MTT method. RESULTS: DC transfected with HepG-2 cell RNA and HCC RNA exhibited increased expression of CD83, CD86 and HLA-DR. The CTL from DCs transfected with HepG-2 cell RNA killed 5.84％， 14.26％， 25.19％, or 35.78％ of HepG-2 cells, and 5.26％， 11.67％， 14.68％, or 23.24％ of HCC cells, respectively, at an E/T ratio of 2.5, 5, 10, or 20. The CTL from DCs transfected with HCC cells RNA killed 4.65％， 12.23％， 15.61％, or 19.15％ of HepG-2 cells, and 7.20％， 12.83％， 27.21％, or 31.15％ of HCC cells,respectively, at an E/T ratio of 2.5, 5, 10, and 20. These CTL did not kill allogeneic malignant cells as human gastric carcinoma cells SGC-7901. CONCLUSION: DC transfected with tumor-derived total RNA could induce specific antitumor immune CTL response. These results suggest that CTL generation is applicable to adoptive immunotherapy of HCC.     

Antitumor effect of tumor necrosis factor-α in combination with interferon-γ on hepatocellular carcinoma

AIM: To investigate the antitumor effects of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on hepatocellular carcinoma (HCC). METHODS: Cytotoxicity of the combination of TNF-α and IFN-γ on HCC in vitro was measured by using a crystal violet (CV) staining method. Antitumor effects of the combination of TNF-α and IFN-γ on HCC in vivo were observed by intra-hepatic injection of TNF-α and IFN-γ to the tumor in a human HCC nude mice hepatic model. RESULTS: The growth of HCC cells was inhibited by TNF-α alone, which was dose-dependent. The cytotoxicity of TNF-α on HCC was enhanced by incubation with IFN-γ. TNF at 107 U/L, or IFN-γ at 106 U/L alone killed only 27.1% or 7.9 of HCC cells, respectively, when combined with IFN-γ, it killed 83.7% of HCC cells. A synergistic antitumor effect on HCC in vivo was observed in combination group, as tumor growth inhibition rate was 35.9% compared with 17.2% in TNF-α group and 5.6% in IFN-γ group. The survival period of mice bearing tumor was significantly prolonged and serum AFP was significantly decreased in combination group (P<0.05). Tumor necrosis was observed to be much severer in combination group. CONCLUSION: Our results suggest that the combination of TNF-α and IFN-γ by intratumor injection is a promising adjunctive modality to treat HCC.     

Elevated levels of serum IL-6, ICAM-1 and P-selectin in stable survivors with liver transplantation

AIM: To study the profile of serum IL-6， ICAM-1 and P-selectin in stable survivors with clinical liver transplantation (LTx). METHODS: Flow cytometric analysis was used to determine the phenotype of T cell subsets in peripheral blood mononuclear cells （PBMCs） from stable survivors with liver transplantation (n=22), and healthy volunteers (n=12). Serum levels of the pro-inflammatory cytokines, tumor necrosis factor (TNF-α) and interleukin (IL-6), intercellular adhesion molecules (ICAM-1) and P-selectin in stable survivors with liver transplantation and healthy volunteers were assessed by enzyme-linked immunoabsordent assay (ELISA). Recently performed 6 cases of liver transplantation were also dynamically observed in this study. RESULTS: Percentage of CD4+ T cells， CD8+ T cells and CD3+ T cells, as well as ratio of CD4 to CD8 were no difference between two groups (P>0.05). However, a significant higher percentage of CD3+CD25+ T cells was found in stable liver transplantation group as compared to healthy group (P<0.05). Significantly increased concentrations of IL-6, ICAM-1 and P-selectin were found in stable liver transplantation group as compared to healthy group (P<0.05). A high TNF-α level was detected in stable liver transplantation group while no significant difference was found as compared to healthy volunteers group (P>0.05). There was not found no regular change of serum cytokines (IL-6, TNF-α) and adhesion molecules (ICAM-1, P-selectin) in 6 liver transplanted patients during post-operation from day 1 to day 30, indicating that was associated with the different status of patients before or after transplantation. CONCLUSIONS: Our data suggesting that increased levels of ICAM-1 and P-selectin, appears to participate in the processing of immunoregulation to transplanted livers, whereas elevated concentrations of IL-6 appear to be involved in the repair of the injury induced by TNF-α in allo-transplanted livers.     

Effect of monocyte/macrophages activated by CpG-oligodeoxynucleotides of bacteria on K562 cells

AIM: To study the effect of monocyte/macrophages treated with CpG-oligodeoxynucleotides on leukemic K562 cells. METHODS: The monocytes/macrophages from peripheral blood cells were isolated and induced. The expressions of CD14 and CD16 on monocytes/macrophages were detected by means of flow cytometry. After treated with synthetic CpG-oligodeoxynucleotides, and nonCpG-oligodeoxynucleotides for 24 hours respectively, the inhibiting effect of monocyte/macrophages on K562 cells were detected using MTT method. The secretions of TNF-α and IL-12 from monocytes/macrophages were determined using ELISA method. RESULTS: The monocytes/macrophages treated with CpG-oligodeoxynucleotides enhanced their antitumor effect on K562 cells and increased the secretion levels of TNF-α and IL-12. Whereas, there was no significant difference between antitumor effect and cytokine secretion of the monocytes/macrophages treated with nonCpG-oligodeoxynucleotide. CONCLUSION: CpG-oligodeoxynucleotides increases the cytotoxicity of macrophages on K562 cells in vitro, as well as facilitates the IL-12 and TNF-α secretion. It provides a new approach for immunologic treatment of leukemia.     

Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

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.     

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.     

Antitumor activity of Paecilomyces tenuipes polysaccharide and its mechanism in vitro

AIM:To investigate the antitumor activity and mechanism of Paecilomyces tenuipes polysaccharide(PTPS).METHODS:PTPS-I was obtained by water extraction and alcohol precipitation, and purified by DEAE-cellulose and Sephadex G-100 chromatography. Human erythroleukemia cell line K562, laryngocarcinoma cell line Hep2 and hepatic carcinoma cell line SMMC-7721were co-cultured with PTPS-I or the conditioned medium which prepared with PTPS-I-stimulated human mononuclear cells (PTPS-I-MNC-CM), and the proliferation of tumor cells was determined. The cell counting kit-8 (CCK-8) was used to determine the proliferation of MNCs. The FQ-RT-PCR was applied to investigate the expression of TNF-α and IL-6 mRNA in MNCs. RESULTS:PTPS-I-MNC-CM inhibited the proliferation of K562, Hep2 and SMMC-7721 cells in vitro (P<0.01). Cytotoxicity of PTPS-I against K562, Hep2 and SMMC-7721 cells was not observed (P<0.01). PTPS-I stimulated the proliferation of MNCs (P<0.01) and significantly enhanced the expression of TNF-α and IL-6 mRNA in MNCs (P<0.05, P<0.01). CONCLUSION:The results suggest that PTPS-I is an immunomodulator and its antitumoral activity is through the immunomodulatory mechanism rather than the direct cytotoxicity against tumor cells.     

Renal tumor cell lysate antigen induces the activation of dendritic cells

AIM:To investigate the effects of renal tumor cell lysates and other cytokines on the activation of immature dendritic cells (iDCs), and to develop DCs vaccines for stimulation of renal tumour-specific immunity.METHODS:DCs induced from peripheral blood mononuclear cells of healthy volunteers were cultured and propagated in vitro using rhGM-CSF and rhIL-4, and then were cocultured with renal tumor cell lysates and different cytokines. A group: only with renal tumor cell lysates; B group: with renal tumor cell lysates and TNF-α; C group: with renal tumor cell lysates and IL-1β; D group: renal tumor cell lysates and TNF-α+IL-1β. RESULTS:iDCs were induced to mature in all four groups, and high level expressions of CD86, CD80 and HLA-DR were observed. Compared to other groups, DCs in D group expressed CD83 and CD54 at higher level (P<0.05), secreted higher quantity of IL-12 (P<0.01). Moreover, mDCs in D group induced multiplication of lymphopoiesis more effectively (P<0.05).CONCLUSION:Renal tumor cell lysates and TNF-α+IL-1β induce the mature phenotype and IL-12 production in DCs and synergistically promote the stimulatory effects of lymphopoiesis.     

Mechanisms underlying the induction of IL-2 secretion by PDB pl us ionomycin in CD4+CD25+ T cells from cord blood and adult peripheral blood

AIM:To confirm that CD4+CD25+ regulato ry T cells don't have an instinctive defection in IL-2 secretion,and to have an insight into the maturation state of CD4+CD25+ T cells in cord blood.METHODS:CD4+CD25+ and CD4+CD25- T cells were purified f rom cord blood of term infants (CB) and adult peripheral blood (PB) by autoMACS,and stimulated with PDB plus ionomycin.After 45 hours of culture,cells were d etected for expression of CD69 and CD25 by flow cytometry,and the supernatants were measured for 7 kinds of cytokines by Luminex.RESULTS:CD4+CD25+ T cells from both CB and PB proliferated comparably with CD4+CD25- T cells when stimulated with PDB plus ionomycin.A fter 45 hours of culture,however,the CD4+CD25+ T cells underwent a tendenc y of cell death.Expression of CD25 was further upregulated when CD25+ cells w ere activated.Under stimulation of PDB plus ionomycin,both CD4+CD25+ and C D4+CD25- T cells in PB secreted high levels of IFN-γ,IL-2 and TNF-α,with CD25+ cells secreted much higher level of IL-5,IL-4 and IL-10 than those in CD25- cells;CD4+CD25+ and CD4+CD25- T cells in CB also secreted high level of IL-2 and TNF-α but much lower level of IFN-γ than those in PB,and no secretion of IL-5,IL-4 and IL-10 was observed.CONCLUSION:CD4+CD25+ regulatory T cells don't have an i nstinctive defection in IL-2 secretion,otherwise there may be a different TCR signaling pattern in CD4+CD25+ T cells from traditional T cells.The CD4+C D25+ T cells in cord blood have not fully matured in function.     

Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

Effects of rapamycin-treated HSP60-pulsed dendritic cells on th e progression of the atherosclerotic plaque in mice

AIM:To evaluate whether tolerogenic dendri tic cells (DC) loaded with heat shock protein 60 (HSP60) inhibit the progression of aortic atherosclerotic plaque in hypercholesterolemic apolipoprotein E (Apo- E) -null mice.METHODS:Bone marrow derived DC of the mice were loaded with HSP 60 and co-cultured with rapamycin to generate tolerogenic DC.The tolerogenic DC ,DC loaded only HSP60 and PBS were injected into the ApoE-null mice at 8 weeks of age for three times at a one-week interval.8 weeks after the last injection,aorta were harvested for HE staining and anti-CD4+T cell immunostaining.Resp onses of pleenic cells to HSP60 were also evaluated.RESULTS:Compared with DC,DCHSP60 expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly,while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to p roliferation.After immunization with different injection,the numbers of CD4+ T cells in plaque were increased significantly in DCHSP60 group vs in PBS g roup (P<0.01).On the other hand,they were reduced significantly in rap-DC HSP60 group vs in PBS group (P<0.01).Plaque areas in the tolerog enic DC group were smaller than that in the PBS group (P<0.01).Stimulated by HSP60,pleenic cells in tolerogenic DC group secreted more IL-10,while in DC HSP60 group more IFN-γ secretion was observed.CONCLUSION:HSP60 specific tolerogenic DC immunization attenuate d the progression of plaque,indicating a new immune therapy for atherosclerosis.