Effects of thymopeptide and 4 polysaccharides isolated from Chinese medicine on immune functions of spleen and tumor tissues in U14 cervical cancer-bearing mice

AIM: To identify the enhancement of relatively specific immune responses by thymopeptide and 4 polysaccharides isolated from traditional Chinese medicine in the mice bearing cervical cancer. METHODS: The model of cervical cancer-bearing mice was established by implantation of cervical cancer U14 cells into the armpit of the right forelimb of the mice. Beginning from the 2nd day of tumor implantation, 4 polysaccharides Lycium barbarum polysaccharide (LBP), Angelica sinensis polysaccharide (ASP), Ganodema lucidum polysaccharide (GLP) and Panax ginseng polysaccharide (PGP)], thymopeptide or saline solution were intragastric administered for 21 days according to the experimental design. The animals were then killed, the tumor inhibitory rate and spleen index in treatment groups were compared with those in control group. The splenic T-lymphocytes (TLC) and natural killer (NK) cells, and the content of IFN-γ and IL-10 in the tumor tissues were also determined by flow cytometry and immunohistochemical methods. RESULTS: The order of tumor inhibitory rates was: ASP < PGP < thymopeptide < LBP < GLP. The effects of GLP, LBP and thymopeptide were statistically different from that of control group. No difference of tumor inhibition between PGP/ASP groups and control group was observed. The order of spleen index was: ASP < tumor control group < thymopeptide < GLP < PGP < LBP, that of CD4/CD8 enhancement was: PGP < ASP < GLP < thymopeptide < LBP, and that of CD49b⁺ NK hyperplasia was: ASP < thymopeptide < LBP < GLP < PGP,with statistically significant differences among the groups. The changes of IFN-γ and IL-10 in the tumor tissues showed that Th1/Th2 in thymopeptide group, LBP group and GLP group shifted to Th1. No shift to Th1 in PGP group and ASP group was observed. CONCLUSION: LBP, GLP and thymopeptide show significant inhibitory effects on tumor growth in U14 cervical tumor-bearing mice and can enhance the immune functions in the animals.     

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.     

Suppressive effect of interferon γ on IL-13-induced fibrosis in fibroblasts

AIM：To investigate the suppressive effect of interferon γ (IFN-γ) on fibrosis induced by interleukin 13 (IL-13) in fibroblasts. METHODS：The fibroblasts were divided into IFN-γ (4×105U/L) group, IL-13 (100 μg/L) group, IFN-γ+IL-13 group and blank control group. At 24 h, 48 h and 72 h, the secreted collagen from fibroblasts was measured by hydroxyproline release assay. The mRNA expression of collagen type I α1 (Col1A1) in fibroblasts was examined by RT-PCR. The protein level of collagen type I synthesized in fibroblasts was analyzed by Western blotting. RESULTS：IFN-γ at 4×105U/ L significantly inhibited the proliferation of fibroblasts and down-regulated Col1A1 mRNA and cellular collagen. The mRNA expression of Col1A1 and the protein level of collagen type I in IFN-γ group were lower than those in blank control group at 48 h and 72 h. At 72 h, the mRNA expression of Col1A1 and the protein level of collagen type I in IL-13 group were substantially higher than those in blank control group, those in IFN-γ + IL-13 group were remarkable lower than those in blank control group, and those in IFN-γ group were also lower than those in blank control group. CONCLUSION：IFN-γ inhibits the fibrotic effect of IL-13 in fibroblasts.     

Influence of Cordyceps sinensis on cytokine profile of dendritic cells in chronic obstructive pulmonary rat model

AIM: To investigate the effect of Cordyceps sinensis (CS) on dendritic cells (DCs) in the rat model of chronic obstructive pulmonary disease (COPD). METHODS: Eighteen Sprague-Dawley male rats were randomly divided into 3 groups: control group, COPD group and CS group.The rats in the latter 2 groups were exposed to cigarette smoking for 8 weeks with (CS group) or without (COPD group) CS treatment. The rats in control group were maintained under normal condition. After 8 weeks,the histological changes of the right lung were observed under microscope. The DCs from the 3 groups were harvested and the supernatants of DCs were analyzed for the levels of TNF-α and IL-12 p70 by commercially available ELISA kit. The DCs were then washed and cocultured in vitro with autologous T cells purified by a nylon cotton column. The supernatants of DCs-T coculture were collected after 72 h incubation, and analyzed for the levels of interleukin-5 (IL-5) and interferon-γ (IFN-γ) by ELISA. RESULTS: Analysis of the rat lung parenchyma revealed a significant decrease in the mean alveolar number, an indicator of alveolar density, in COPD group (38±16) and CS group (48±9) in comparison with control group (62±8). The mean alveolar number tended to be increased in CS group than that in COPD group, although this difference did not achieve statistical significance (P>0.05). The concentrations of TNF-α and IL-12 p70 in the culture supernatants of DCs and IFN-γ in the supernatants of DCs-T cocluture were up-regulated in CS group as compared with those in COPD group and control group (P<0.05). The level of IL-5 in the DCs-T coculture supernatants of the 3 groups did not show differences with statistical significance (P>0.05). CONCLUSION: The therapeutic effects of CS on COPD rats may be related to modulation of Th1 and Th2 cell functions. This effect is probably mediated through IL-12 p70 produced by DCs and Th1 cytokine IFN-γ produced by autologous T cells.     

Modulatory function of high-dose hepatitis B surface antigen vaccine to cellular immune responses in mice

AIM:To observe the effects of high-dose hepatitis B surface antigen (HBsAg) vaccine on cellular immune response in BALB/C mice.METHODS:The mice were immunilized separately with low-dose and high-dose HBsAg vaccine by intramuscular injection two times. The specific proliferative activities of T lymphocytes were measured by -[H³]TdR incorporation assay. IL-2 as well as IFN-γ levels in the culture supernatant of T cells and anti-HBs IgG2a lever in sera were detected by enzyme-linked immunoabsorbent assay.RESULTS:After first vaccination with high-dose HBsAg, the proliferative activities of T cells in the experimental group were significantly stronger, both levels of IL-2 and IFN-γ were markedly higher than that in the control group and the percentage of mice to produce serum anti-HBs IgG2a was significantly higher compared to that of mice immunilized by low-dose HBsAg. All data in experimental groups were further increased after second dose of vaccine.CONCLUSION:Vaccination of mice with high-dose HBsAg can induce cellular immune responses tended to Th1(T helper 1 subset) response.     

The effect and mechanism of H. polygyrus infection in T-cell-mediated inflammatory bowel disease in mice

AIM: To investigate the effect and mechanism of Heligmosomoides polygyrus (H. polygyrus) infection in mouse inflammatory bowel disease (IBD) mediated by CD4⁺ helper T-cells. METHODS: Ovalbumin (OVA) -specific CD4⁺ helper T-cells were transferred into SCID (severe combined immunodeficiency) mice to establish an IBD model. The IBD mice were infected by H. polygyrus and sacrificed 14 days later. The histological changes of the colon were observed, and the expression of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in mesenteric lymph nodes was detected by ELISA and flow cytometry. Additionally, IL-4 monoclonal antibody was intraperitoneally injected into the H. polygyrus-infected IBD mice to block the secretion of IL-4. The IL-4-blocking IBD mice were sacrificed 9 days later and the above indexes were also determined.RESULTS: Compared with the non-infection group, the H. polygyrus-infected IBD mice had more severe colonic lesions, higher level of IL-4 and lower level of IFN-γ in mesenteric lymph nodes (all P<0.05). Compared with the non-blocking group, the H. polygyrus-infected IBD mice with IL-4 blockage had less colonic lesions, lower IL-4 level and higher IFN-γ level (all P<0.05).CONCLUSION: H. polygyrus infection in CD4⁺ T-cell-mediated IBD model promotes inflammation in the early stage probably by inducing the secretion of Th2 cytokine and inhibiting the secretion of Th1 cytokine. The finding suggests that using worms for treatment of IBD needs to be cautious.     

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.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

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.     

“2012年园艺植物染色体倍性操作与遗传改良学术研讨会”预备通知

自2008年11月在中国农业科学院郑州果树研究所成功召开园艺植物染色体倍性操作与遗传改良研讨会以来,我国科研人员在该领域的研究取得了可喜的成绩。为进一步总结近几年来在该领域的研究进展,促进园艺植物倍性育种研究,同时为该领域的专家、学者和同仁们提供良好的交流平台。中国园艺学会定于2012年4月中旬在重庆召开园艺植物染色体倍性操作与遗传改良学术研讨会。欢迎从事该研究领域及相关研究工作的人员参加。     

Effects of iNKT cells from OVA-induced asthmatic mice combined with OVA on phenotype and function of bone marrow-derived dendritic cells in vitro

AIM:To investigate the effects of invariant natural killer T-cells (iNKT cells) from ovalbumin (OVA)-induced asthmatic mice combined with OVA on the phenotypic and functional characteristics of bone marrow-derived dendritic cells (BMDCs) in vitro. METHODS:The BMDCs from wild-type (WT) BLAB/c mice were co-cultured with purified iNKT cells from WT mice immunized and challenged with OVA in the presence of 100 mg/L OVA (iNKT cells plus OVA group) or PBS (iNKT cells plus PBS group) for 20 h, and were also cultured with 50 mg/L LPS (LPS group), 100 mg/L OVA (OVA group) or PBS (PBS group) for 20 h. The expression of MHC-Ⅱ, CD40, CD86, and CD80 on the BMDCs was measured by flow cytometric analysis, and the levels of interleukin-12 (IL-12) p70, IL-6, tumor necrosis factor-α (TNF-α) and IL-10 in the culture supernatant were measured by ELISA. Splenic CD4⁺ T cells from DO11.10 transgenic mice were co-cultured for 48 h with the above mentioned BMDCs, and then the concentrations of IL-4 and interferon-γ (IFN-γ) in culture supernatants were measured by ELISA. RESULTS:The expression of MHC-Ⅱ, CD80, CD86 and CD40, and releases of proinflammatory cytokines by BMDCs in iNKT cells plus OVA group were comparable to those in the LPS group (P>0.05), but significantly higher than those in iNKT cells plus PBS group, OVA group, and PBS group (P<0.05 or P<0.01). The concentration of IL-4 in culture supernatants from BMDCs in iNKT cells plus OVA group co-cultured with DO11.10 CD4⁺ T cells was similar to that in LPS group (P>0.05), but markedly higher than that in iNKT cells plus PBS group, OVA group, and PBS group (P<0.01). CONCLUSION:Bone marrow-derived dendritic cells undergo immunogenic maturation upon interaction with iNKT cells in the presence of OVA.     

Dihydroartemisinin enhances radiotherapy efficiency in H22 cell tumor-bearing mice by regulating PI3K/AKT signaling pathway

AIM To study the effect of dihydroartemisinin （DHA） on the radiotherapy efficiency in hepatocellular carcinoma H22 cell tumor-bearing mice and the role of phosphatidylinositol 3-kinase （PI3K）/protein kinase B （AKT） signaling pathway in this process. METHODS A model of H22 cell tumor-bearing mice was established. The mice was divided into model group, single radiotherapy group, 5-fluorouracil （5-FU） group, and low-, medium- and high-dose DHA groups. The body weight and tumor volume in each group were measured every other day. At the end of administration, blood was collected from the tail of the mice and the animals were killed by neck removal immediately. The synergistic effect of DHA on radiotherapy was determined, and tumor growth inhibitory rate was calculated. The degree of lymphocyte transformation and natural killer （NK） cell activity were measured by MTT, the serum levels of interleukin-2 （IL-2） and IL-4 were measured by ELISA, and the protein levels of PI3K, AKT and p-AKT were determined by Western blot. RESULTS The H22 cell tumor-bearing mouse model was successfully constructed. Compared with model group, the TGT₃ （tumor growth time to reach 3 times of volume） of single radiotherapy group was remarkably increased （P<0.05）, while tumor weight, lymphocyte transformation degree, NK cell activity, IL-2 and IL-4 levels, PI3K protein level and AKT phosphorylation level were remarkably decreased （P<0.05）. Compared with single radiotherapy group, TGT₃, EF （enhancement factor）, tumor inhibitory rate, lymphocyte transformation degree, NK cell activity, IL-2 level and IL-4 level were increased with the increase in DHA dose （P<0.05）, and the PI3K protein level and AKT phosphorylation level were decreased （P<0.05）. CONCLUSION DHA may enhance the immunity of tumor-bearing mice by inhibiting the activity of PI3K/AKT signaling pathway, thereby enhancing the efficacy of radiotherapy.     

Effect of IFN-γ inhalation on the cellular immunity of the lungs

AIM:To study the effect of IFN-γ inhalation on the anti-infection ability of the lungs in the immunocompromised host. METHODS:The immunological factors in the immunocompromised rats and the immunocompromised rats administrated IFN-γ via aerosol were investigated after 1, 3, 7 days when they were injected Candida albicans via tracheal. The Canidda albicans count of the left lung was also determined after 7 days when injecting pathogen. RESULTS:The Canidda albicans count of the left lung in IFN-γ group was significantly less than that of control group. The phagocyting and bactericidal percentages, Ia antigen expression percentages, the levels of TNF-α, IL-1β and IL-6 in the culture supernatant of the AM, the activity of IFN-γ and TNF-α in BALF (except the TNF-α on 7 th day) in IFN-γ group were markedly higher than those in control group. The expression of IFN-γ and IL-1β pulmonary tissues in IFN-γ group was higher than that in control group. The expression of TNF-α in IFN-γ group was less than that in control group. The expression of IL-6 was no changes between two groups. The levels of IFN-γ, IL-1β and IL-6 in the blood (except IL-1β on 3 rd day), and the killing ability of the lymphocytes in blood had no difference between two groups. CONCLUSION:Administration of IFN-γ via aerosol obviously enhanced the anti-infection ability of the lungs in the immunocompromised host, but has no influence on the whole body cellular immunity.     

Farnesoid X receptor up-regulates thyrotropin embryonic factor and attenuates pathological injury of Con A-induced hepatitis

AIM：To observe how farnesoid X receptor (FXR) functioned in concanavalin A (Con A) -induced hepatitis (CIH) and the regulation of FXR-thyrotropin embryonic factor (TEF) pathway. METHODS：C57BL/6 mice were injected with Con A to induce hepatitis. The expression of FXR and TEF in the liver specimens was determined by qRT-PCR and Western blotting. The concentrations of serum ALT/AST and inflammatory cytokines IFN-γ, TNF-α, IL-4 and IL-2 in the blood samples were tested after Con A injection. RESULTS：FXR was down-regulated in CIH mice. TEF was up-regulated when FXR was activated by chenodeoxycholic acid (CDCA). Activation of FXR reduced the levels of aminotransferases and inflammatory cytokines IFN-γ, TNF-α, IL-4 and IL-2 in the CIH mice induced by Con A injection. CONCLUSION：FXR activation attenuates CIH mouse liver injury and reduces inflammatory cytokines. FXR activation results in TEF up-regulation. The FXR-TEF pathway may play a protective role in autoimmune hepatitis.     

Prostaglandin E2 receptors,EP2 and EP4, regulate expression of surface molecules and cytokines in B-cells of collagen-induced arthritic mice

AIM: To observe the immunoregulatory effects of prostaglandin E₂ receptor (EP) subtypes EP2/EP4 on the B-cells of collagen-induced arthritic(CIA)mice. METHODS: DBA/1 mice were immunized with chicken type II collagen emulsified in Freunds complete adjuvant to induce arthritis. B-cells were isolated from the splenocyte suspension by positive selection using anti-CD19 monoclonal antibody immunomagnetic beads. The expression of MHC II, CD 80 and CD86 was examined by flow cytometry. The mRNA levels of EPs, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-6, IL-4, IL-10 and transforming growth factor-β (TGF-β) were detected by real-time RT-PCR. RESULTS: The rank of the mRNA levels of EPs was EP2>EP1>EP3>EP4 in B-cells and EP2/EP4 mRNA expression was obviously increased in CIA mice. EP2 antagonists inhibited the expression of MHC II, CD80 and CD86. EP4 antagonist had little effect on CD80. EP2/EP4 antagonists inhibited the mRNA expression of IFN-γ, TNF-α, and IL-6 (P<0.05 or P<0.01) and increased the expression of IL-10 (P<0.01 or P<0.05). Furthermore, the antagonists of EP2 and EP4 also increased the mRNA expression of IL-4 and TGF-β (P<0.01), respectively. CONCLUSION: PGE₂ modulate the pathogenesis of CIA via EP2/EP4 by regulating the expression of surface molecules and cytokines in B-cells. EP2/EP4 may be a new therapeutic target for treating rheumatoid arthritis.     

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.     

Effects of arsenic trioxide on T-bet/GATA3 signal pathway in MRL/lpr mice

AIM: To investigate the effect of arsenic trioxide (ATO) on T-bet/GATA3 signal pathway in MRL/lpr mice.METHODS: MRL/lpr mice and C57BL/6J mice at the age of 20 weeks were chosen and then divided in 2 different sub-groups, respectively. The mice in 2 sub-groups received ATO (0.4 mg·kg^-1·d^-1) and sodium chloride (NS, volume weight-determined) by intraperitoneal injection respectively for 2 months. Afterward, the spleens were isolated from the MRL/lpr and C57BL/6J mice under pathogen-free condition and the suspensions were prepared. The mRNA level of T-bet, GATA3, IFN-γ,IL-4 and the mRNA ratio of T-bet/GATA3 were detected by RT-qPCR. The protein expression of T-bet and GATA3 was determined by Western blot. The serum levels of IFN-γ and IL-4 were measured by ELISA.RESULTS: The mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 in NS group of MRL/lpr mice were higher than those in NS group of C57BL/6J mice (P<0.05). However, the GATA3 and IL-4 were lower in NS group of MRL/lpr mice in both mRNA and protein level (P<0.05). In MRL/lpr mice, the mRNA and protein levels of T-bet, IFN-γ and the mRNA ratio of T-bet/GATA3 were lower in ATO group compared with NS group (P<0.05), no difference was found in GATA3 and IL-4. No difference of the indexes mentioned above between ATO group and NS group in C57BL/6J mice was observed.CONCLUSION: ATO may affect the signaling pathway of T-bet/GATA3 to down-regulate the mRNA expression and the protein secretion of IFN-γ by decreasing the expression of T-bet in MRL/lpr mice.     

Effects of Foxp3 transfection on splenocyte functions in asthma mice

AIM: To study the expression and the effects of Foxp3 on the immunologic functions by transfecting the Foxp3 eukaryotic expression plasmid into the splenocytes of the asthma mice. METHODS: The mice were sensitized and challenged by ovalbumin to make asthma model. The splenocytes were harvested and cultured. The Foxp3 expression vector pcDNA3.1(-)-Foxp3 was transfected into the splenocytes with electroporation. The splenocytes transfected with empty vector and control splenocytes (non-transfected) were also set up. The expression of Foxp3 at mRNA and protein levels was detected by RT-PCR and Western blotting, respectively. The proportion of CD4⁺CD25⁺ Treg cells/CD4⁺ cells was measured by flow cytometry. Proliferation of the splenocytes was analyzed with MTT assay. ELISA was used to determine the levels of interleukin 4 (IL-4) and interferon γ (IFN-γ) in the supernatant of the splenocytes. RESULTS: The expression of Foxp3 at mRNA and protein levels in transfection group was significantly higher than that in empty vector group and control group. The proportion of CD4⁺CD25⁺Treg cells/CD4⁺ cells in transfection group was higher than that in empty vector group and control group. The proliferation of transfected cells was markedly inhibited compared with empty vector group and control group. The levels of IL-4 and IFN-γ were significantly lower in transfection group than those in empty vector group and control group. CONCLUSION: The transfected Foxp3 gene overexpresses in the splenocytes of asthma mice. Foxp3 increases the number of CD4⁺CD25⁺ T cells and inhibits the proliferation and production of Th1/Th2 cytokines in splenocytes.     

Anti-Sonic hedgehog blocking antibody enhances killing effect of PBMCs on cervical carcinoma HeLa cells

AIM: To investigate the effect of anti-Sonic hedgehog(Shh) blocking antibody on the killing effect of peripheral blood mononuclear cells(PBMCs) on cervical carcinoma HeLa cells. METHODS: The expression levels of Shh and Shh signaling molecules in HeLa cells were detected by immunocytochemistry and RT-PCR. PBMCs from health peoples were isolated by the method of Ficoll density gradient centrifugation, and then co-cultured with HeLa cells in vitro. The expression of CD3, CD69 and CD71 was assayed by flew cytometry. The concentrations of IFN-γ, IL-10 and IL-4 in culture supernatants were detected by ELISA. The killing effect of PBMCs on HeLa cells was observed under microscope. RESULTS: Shh and Shh signaling molecules were expressed in HeLa cells. The level of Shh expression didn't change significantly in the 6th passage of HeLa cells. CD3⁺ cells were increased in the co-culture system. The expression of CD69 and CD 71, and the secretion of IFN-γ were increased, while the secretion of IL-10 was decreased in the co-culture system treated with anti-Shh blocking antibody. Anti-Shh blocking antibody has no effect on the secretion of IL-4. The killing effect of PBMCs on HeLa cells was strengthened by anti-Shh blocking antibody. CONCLUSION: Anti-Shh blocking antibody promotes the activation of PBMCs and enhances the killing effect of PBMCs on cervical carcinoma HeLa cells.     

Effects of chronic Clonorchis sinensis infestation on inflammatory reaction of macrophages

AIM：To evaluate the immune state in rats with chronic Clonorchis sinesis (Cs) infestation by investigating the effects of Cs on macrophage polarization and inflammatory reactions. METHODS：Sprague-Dawley rats were used in the study. Chronic Cs infestation model was reproduced by intragastric perfusion with Cs eggs. Twenty rats were randomly divided into normal group (n=10) and Cs infestation group (n=10). The serum levels of interleukin (IL-4) and IL-10, tumor necrosis factor α(TNF-α) and interferon γ (IFN-γ) were detected by ELISA. The macrophages were harvested by peritoneal lavage. The differentiation proportion of M1 and M2 macrophages were detected by flow cytometry. The macrophages were divided into control group, normal group and chronic Cs infestation group according to the sources of macrophages. The levels of TNF-α and IL-10 in the culture supernatants were detected by ELISA at 0, 2, 12 and 24 h after lipopolysaccharide (LPS, 10 μg/L) stimulation in vitro. RESULTS：Compared with normal group, chronic Cs infestation increased the serum levels of TNF-α, IFN-γ, IL-4 and IL-10. The differentiation proportion of M1 detected by flow cytometry was 92.1% in normal group and that of M2 macrophages was 93.8% in Cs infestation group. The levels TNF-α and IL-10 in culture supernatants were increased at 2~24 h after LPS stimulation both in normal group and Cs infestation group, but the levels of TNF-α were lower in chronic Cs infestation group than that in normal group at 2 h,12 h and 24 h after LPS stimulation. The level of anti-inflammatory cytokine IL-10 was higher in Cs infestation group than that in normal group at 2 h, 12 h and 24 h after LPS stimulation. CONCLUSION：Chronic Cs infestation increases the serum levels of both pro-inflammatory cytokines and anti-inflammatory cytokines, thus inducing the polarization of M2 macrophages. The macrophages derived from chronic Cs-infected rats produce tolerance in the inflammatory process against LPS in vitro.