Effects of taurine on the change of apoptosis induced by IL-1β, TNF-α and IFN-γ in rat pancreatic islet cells

AIM: To investigate the changes of apoptosis in isolated pancreatic islet cells, insulin secretion, expression of Bcl-xL and Bax induced by combination of IL-1β, TNF-α and IFN-γ, and effects of taurine on them.METHODS: Isolated pancreatic islet cells from Wistar rat were incubated in monolayer in vitro. NO^-₂/ NO^-₃ production, NOS activity, insulin secretion, the protein expression of Bcl-xL and Bax, percentage of islet cell apoptosis and DNA fragmentation in pancreatic islet cells incubated with combination of IL-1β, TNF-α and IFN-γ were measured, and the effects of taurine on the changes of them were further investigated. RESULTS: Combination of IL-1β, TNF-α and IFN-γ induced a significant increase in percentage of pancreatic islet cell apoptosis, NO^-₂/ NO^-₃ production and NOS activity, DNA ladder appearance, a decrease in insulin content, up-regulation in the protein expression of Bax and down-regulation in the protein expression of Bcl-xL (P<0.01), which were blocked by addition of taurine (P<0.01). These effects occurred in a dose dependent manner.CONCLUSION: Taurine attenuates β cell apoptosis induced by IL-1β, TNF-α and IFN-γ. The mechanism of which may be the inhibition of NOS activity and the decrease of NO production as well as the downregulation of Bax/Bcl-xL proportion.     

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.     

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.     

Progress on regulation of PD-L1 expression by major inflammatory cytokines in tumor microenvironment

Programmed cell death protein 1 ligand 1 （PD-L1） is an important immune checkpoint protein， and its high expression in tumors often affects the effect of immunotherapy and promotes the malignant progression of tumors. Besides the own factors of the cells， the expression of PD-L1 is regulated by a variety of factors. In recent years， many studies have shown that some inflammatory cytokines in the tumor microenvironment， such as interferon-γ （IFN-γ）， transforming growth factor-β （TGF-β）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6） and so on， regulate PD-L1 expression through various mechanisms， thus promoting the immune escape of tumor cells and affecting the prognosis of patients. In this article， we briefly review the regulatory effects of major inflammatory cytokines on tumor cell PD-L1 expression and the mechanisms in order to provide a reference for improving the clinical efficacy of anti-PD-L1 treatment and studying new tumor immunotherapy pathways.     

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.     

Effects of ganmao shuangjie heji on the inflammatory damage in the lungs of mice infected by IV FM1

AIM: To establish the influenza infected mouse model and study the anti-inflammatory effect of ganmao shuangjie heji. METHODS: IV FM1 infected mice were used as the animal model. The changes of pathology and the cytokine TNF-α, IFN-γ and IL-10 in the lung were observed by HE staining and ELISA (double antibody sandwich enzyme linked immunosorbent assay) after ganmao shuangjie heji treatment. RESULTS: After infected by influenza virus, severe interstitial pneumonia was induced in the model group. Mild interstitial pneumonia was observed in ganmao shuangjie heji treated group. The protein expressions of cytokine TNF-α, IFN-γ and IL-10 were higher in model group than those in the control group. The protein expressions of TNF-α and IFN-γ in ganmao shuangjie heji treated group decreased and IL-10 expression increased significantly compared with model group. CONCLUSION: Ganmao shuangjie heji decreases the expressions of TNF-α and IFN-γ, and increases the expression of IL-10, thus, alleviates inflammatory injury. The clinical application of this medicine can shorten the course of disease.     

Expansion capacity and cytokine releasing characteristics of TCRVα24+Vβ11+NKT cells in peripheral blood of patients with lymphoma

AIM: To analyze the quantity of TCRVα24+Vβ11+ natural killer T (NKT) cells and cytokines production induced by α-galactosylceramide (α-Galcer) in vitro in lymphoma patients. METHODS: Flow cytometry was utilized to enumerate TCRVα24+Vβ11+NKT cells in peripheral blood mononuclear cells (PBMNCs) in 30 cases of lymphoma patients and 30 cases of age- and gender-matched healthy controls. NKT cells were activated with α-Galcer and interleukin-2 (IL-2) after expansion in vitro. The percentages of positive NKT cells which expressed intracellular interleukin-4 (IL-4), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were then determined by flow cytometry. RESULTS: The rates of NKT cells in PBMNCs were 0.17%±0.10% and 0.28%±0.18%(P<0.05) in lymphoma patients and controls, respectively. Seven days after expansion and activation with α-Galcer and IL-2, the fold of expansion of NKT cells in two groups was 101.37±44.61 and 129.66±56.31(P<0.05), respectively. The ratio of TCRVα24+NKT cells that secreted IFN-γ or TNF-α in lymphoma patients was significantly lower than that in controls (41.96%±15.06% vs 52.48%±18.85%, P<0.05; 46.30%±16.03% vs 71.37%±17.28%, P<0.05). While the ratio of TCRVα24+NKT cells that secreted IL-4 was not significantly different between the two groups (36.19%±11.74% vs 33.12%±12.95%, P>0.05). There was no significant difference in above parameters among different groups of lymphoma patients subdivided by pathology and clinical stage. CONCLUSION: The quantity of NKT cells in PBMNCs in lymphoma patients is lower than that in controls. The expansion capacity and the function of producing cytokines IFN-γ and TNF-α of NKT cells stimulated with α-Galcer are decreased. This decrease is independent of lymphoma pathology type or clinical stage.     

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.     

TCRVβ7.1 transfection activates signal protein ERK and insreases IFN-γ expression in PBMCs

AIM: To investigate the effect of tumor-specific T cell receptor (TCR) gene transfection on production of cytokine and signaling activation in T cells.METHODS: TCRVβ7.1 gene was transferred into peripheral blood mononuclear cells (PBMCs) obtained from healthy adults, and the expression of Vβ7.1 was detected by flow cytometry before and after transfection. The total quantities of protein and phosphorylation of ERK1/2 were detected by Western blotting. The expressions of IL-4 and IFN-γ were detected by ELISA.RESULTS: The results of flow cytometry showed that TCRVβ7.1 protein was efficiently expressed after transfection. The phosphorylation level of ERK increased significantly in TCRVβ7.1-modified PBMCs, and was related with the activation of T cells. The expression of IFN-γ was significantly higher in TCR-transfected cells than that in non-transfected cells. The expression of IL-4, however, has no distinct difference between groups.CONCLUSION: The transfection of TCRVβ7.1 induces phosphorylation of ERK1/2 and production of IFN-γ, and activates T lymphocytes.     

Establishment of T-lymphocytes that express CD20scFv-IgGFc-CD28-ζ and CD20scFv-IgGFc and their killing activity of B-lymphoma cells

AIM: To investigate the target killing effect of T lymphocytes with chimeric CD20scFv gene on Daudi cells and the activation of T lymphocytes. METHODS: Two kinds of plasmids were transfected into retrovirus-packed PA317 cell lines. The supernatant was collected from successfully transfected PA317 culture and was used to infect peripheral blood T lymphocytes. After one-week screening with G418, the cells were used to kill Daudi and K562 cells. The positive rates of AnnexinⅤ in Daudi cells were measured at different times points respectively by flow cytometry. Meanwhile, the level of IL-2 and IFN-γ were determined by ELISA. RESULTS: The Annexin V positive rate was significant higher in Daudi cells compared to control K562 cell lines at 24 h. No difference of AnnexinV in Daudi cells was observed in CD20 modification T lymphocyte groups. The secretions of IL-2 and IFN-γ in CD20scFv-CD80-IgGFc-CD28-ζ gene modified T cells co-cultured with Daudi cells were dramatically higher than that in CD20scFv-IgGFc group at 72 h. CONCLUSION: ① The two kinds of genetic modified specific T cells have no significant difference in inducing early apoptosis of Daudi cells. CD28-ζ cant affect Daudi cell early apoptosis at the CD20scFv target killing. ② The increase in the secretions of IL-2 and IFN-γ is more obvious in CD20scFv-IgGFc-CD28-ζ group, indicating that the self-activation takes place in CD3ζ and CD28 modified T cells without MHC restriction and then increases the activation and killing function of T cells.     

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.     

The molecular mechanism of tolegenic dendritic cells induced by IL-10 in mouse

AIM:To investigate the relation of tolerogenic dendritic cells (DC) induced by interleukin-10 (IL-10) and the paired immunoglobin-like receptor (PIR) A and B (PIR-A and PIR-B) in mouse. METHODS:The mouse dendritic cell line, DC2.4 cells were cultured with the IL-10 to develope the IL-10-DC and were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small inference RNA (siRNA) molecule of PIR-B was chemically synthesized and was transfected into IL-10-DC by Lipofectamine 2000 (Si-DC). The expression of PIR A and PIR B on DC2.4 cells were measured by semi-quantitative RT-PCR and flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using ［3H］-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. RESULTS:IL-10 up-regulated the PIR-B and down-regulated the PIR-A by semi-quantitative RT-PCR. On the contrary, LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. The expression of PIR, which is the common extra-membrane of PIR-A and PIR-B, was increased in both the IL-10-DC and the LPS-DC groups by FCM detection, but the higher expression was found in IL-10-DC group than that in LPS group. The IL-10 induced the higher PIR-B expression, inhibited allogenetic T cell proliferation and down-regulated the IFN-γ secretion. Special siRNA molecules of PIR-B in IL-10 group promoted the T cell proliferation and enhanced the IFN-γ secretion in MLR. CONCLUSION:IL-10 up-regulates the PIR-B expression and makes DC tolerance. Up-regulated PIR-B expression may be the molecular mechanism of tolerogenic dendritic cells induced by IL-10 in mouse.     

Gene expression of spleen lymphocytes induced by gp96-peptide complexes and its anti-tumor effect in vitro

AIM: To study the expressing variation of TNF-α and IFN-γ mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS: H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-α and IFN-γ mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy (LSCM) and transmission electron microscope (TEM).RESULTS: Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-α and IFN-γ mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups (P<0.05).The morphologic change of apoptosis of H22 tumor cells,which treated by the culture supernatant of experimental group was observed with LSCM and TEM.CONCLUSION: Heat shock protein gp96-peptide complexes increase the expression value of TNF-α and IFN-γ mRNA in spleen lymphocytes of mouse in vitro.Besides,apoptosis of H22 cells is induced by immunologic active material secreted by activated splenocytes.     

Allogeneic antigen-pulsed murine immature CD8α+ dendritic cells prevent T-cells proliferation in vitro

AIM: To explore the optimal condition of predominant immature CD8α⁺ dendritic cells(predo-iDCs) preventing T-cell proliferation pulsed by allogeneic antigen. METHODS: Predo-iDCs, induced with GM-CSF +IL-4 +SCF +Flt3L from SPF healthy C57BL/6 murine bone marrow cells, were pulsed by different doses (0, 2.5, 5, 10 and 20 mg/L)of allogeneic murine splenocyte antigen. Syngeneic T-cells were co-incubated with Ag-pulsed DCs (DCs/T=1∶ 1, 2∶ 1, 4∶ 1) and the T-cell proliferation was measured by MTT. The secretion of cytokines (IFN-γ and IL-10) in the co-incubated supernatants was detected by ELISA. The effect of prevention of T-cell proliferation generated by murine predo-iDCs pulsed by allogeneic antigen was detected. The control derived from mature dendritic cells(mDCs)induced by GM-CSF +IL-4 +TNF-α. RESULTS: The effect of Ag-pulsed predo-iDCs for stimulating T-cell proliferation was the slightest in predo-iDCs/T 1∶ 1 group, compared with that in predo-iDCs/T 2∶ 1 and 4∶ 1 group (P<0.05). The secretion of IFN-γ in mixed lymphocyte reaction(MLR) was significantly lower than the one in mDCs control group, while the secretion of IL-10 was higher than that in control group when low dose of antigen (<2.5 mg/L) was added into MLR. CONCLUSION: Predominant iDCs pulsed by low dose of allogeneic antigen (2.5 mg/L) mixed 1∶ 1 with T-cells is the optimal condition for the prevention of T-cells proliferation.     

Association of IFN-γ and IL-4 gene polymorphisms with childhood bronchial asthmatic susceptibility and the levels of plasma IFN-γ,IL-4 and IgE

AIM: To investigate the gene polymorphisms of interferon-γ(IFN-γ) and interleukin-4(IL-4) and the association with asthmatic susceptibility and the levels of plasma IFN-γ, IL-4 and IgE of asthmatic children. METHODS: 100 asthmatic children and 122 control children were enrolled the study. The genotypes of IFN-γ gene-179G/T polymorphism, IL-4 gene-33C/T and-589C/T polymorphisms were tested by PCR-RFLP.The genotype of IFN-γ gene +874A/T polymorphism was tested by AS-PCR.The CA repeat polymorphism of IFN-γ gene was detected by capillary electrophoresis technique.The levels of serum IFN-γ, IL-4 and IgE were measured by ELISA. RESULTS: 100 asthmatic children and 122 control children were all GG homozygotes at -179 locus of IFN-γ gene.-179 locus of IFN-γ gene has no mutation. The genotypes and allele frequency of IFN-γ gene +874A/T and CA repeat polymorphisms showed no significant difference between asthmatic children and the control(P>0.05). An association was revealed between IFN-γ gene +874A/T polymorphism and the level of plasma IFN-γ.The level of IFN-γ was lower in AA genotype than in AT genotype(P<0.05). The genotypes and allele frequency of IL-4 gene -33C/T and -589C/T polymorphisms showed significant difference between asthmatic children and the control(P<0.05).The levels of plasma IL-4 and IgE were higher in TT genotype at -33 locus and -589 locus than those in CT genotype, but only -33C/T polymorphism was associated with the level of plasma IL-4(P<0.05). CONCLUSION: The IFN-γ gene +874A/T and CA repeat polymorphisms were not correlated with asthmatic susceptibility, but there is significant correlation between the level of IFN-γ and +874A/T polymorphism. TT genotype of IL-4 gene -33 locus and -589 locus maybe the susceptible genotype of asthma in children, and the -33 locus polymorphism is associated with the level of IL-4.     

Effects of dendritic cells induced by superantigen staphylococcal enterotoxin B or staphylococcal enterotoxin C on T lymphocytes

AIM: To evaluate the effect of staphylococcal enterotoxin B (SEB) or staphylococcal enterotoxin C (SEC) combining with dendritic cells (DC) on T cell functions and in vitro anti-HcaF tumor cytotoxicity of activated T cells. METHODS: S-100 protein expression in DC was detected by immune histochemistry staining. The expressions of I-Eκ and CD80 molecules on DC, the expression of CD69 molecule on T cells and the production of IL-2 and TNF-α by T cells were determined with flow cytometry. The proliferation of T cells and its cytotoxicity to HcaF tumor cells were detected by MTT assay. RESULTS: In vitro experiments showed that isolated DC expressed high level of S-100 protein. SEB or SEC-induced DC highly expressed I-Eκ and CD80 molecules and that SEB or SEC-induced DC promoted the activation and proliferation of T cells. 100 μg/L of SEB or SEC was the most effective concentrations to induce T cells to secret IL-2 and TNF-α. The T cells activated by SEB or SEC combined with DC showed significant cytotoxicity to HcaF cells, appearing a stronger role than tumor antigen combined with DC. There was no difference in the role for T lymphocytes between both SEB and SEC. CONCLUSIONS: The results indicate that SEB or SEC combined with DC is an effective way to enhance T cell functions, producing stronger cytotoxicity to HcaF tumor cells than tumor antigen-loaded DC used at present, which offers a forceful evidence for the possibility of superantigen SEB or SEC combining with DC to be applied to clinical tumor immunotherapy.     

Effects of antioxidant and NF-κB on the induction of IL-8 in human colon cancer cell line HT-29 cells in vitro

AIM: To investigate the role of nuclear factor κB (NF-κB) in the induction of IL-8 gene by TNF-α in colon cancer cells and the effect of antioxidant on the induction of IL-8. METHODS: ELISA was used to detect the concentrations of IL-8. IL-8 mRNA was analyzed by using RT-PCR. NF-κB in the cell nuclei was detected with electrophoretic mobility shift assay. RESULTS: (1) IL-8 production and IL-8 mRNA expression induced by TNF-α was blocked by pyrrolidine dithiocarbamate (PDTC). (2) TNF-α triggered the activation and translocation of NF-κB and PDTC inhibited the activation of NF-κB induced by TNF-α. CONCLUSION: The induction of IL-8 gene and protein by TNF-α is dependent on the activation of NF-κB. Antioxidants may inhibit the induction of IL-8 gene and protein through inhibiting NF-κB activation.     

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.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.