Effect of uric acid on the maturation and the biological function of BMDCs

AIM: To investigate the effect of uric acid on the maturation and the biological function of murine bone-marrow derived dendritic cells (BMDCs) in vitro.METHODS: BMDCs were cultured with GM-CSF, IL-4, LPS and uric acid. Cell phenotypes were analyzed by flow cytometry. MTT was used to detect the effect of uric acid on the function of BMDCs in stimulating the proliferation of T cells. IL-12 released by BMDCs was also detected. RESULTS: BMDCs were cultured and identified. Uric acid at concentrations of 200 mg/L and 400 mg/L increased the expression of the molecules (CD11c, CD83, CD86, IA/IE) on BMDCs surface and the IL-12 level in the culture supernatants (P<0.05), promoted the proliferation of T cells at the T: DC rate 5∶1, 10∶1, 20∶1 (P<0.05). However, uric acid at concentration of 70 mg/L had no effect on above molecule expression (P>0.05), no effect on T cell proliferation with BMDCs (P>0.05) was observed. CONCLUSION: Uric acid promotes the differentiation, maturation, the expression of co-stimulatory molecules, the IL-12 production in BMDCs and enhances the ability of BMDCs to stimulate the proliferation of T cell in a specical dose range.     

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.     

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.     

Effect of adriamycin combined with sensitized dendritic cells on cervical tumor-bearing mice

AIM：To explore the immunotherapeutic effect of adriamycin (ADM) combined with frozen-thawed antigen-sensitized dendritic cells (DCs) on cervical tumor-bearing mice. METHODS：The U14 cervical cancer model of Kunming mice was established by subcutaneous implantion of U14 cells in axillary fossa. DCs vaccine was prepared by U14 cervical cancer cell frozen-thawed antigen-sensitized mouse bone marrow-derived DCs. Mature phenotype of sensitized DCs was identified by flow cytometry. Tumor-bearing mice were randomly divided into 4 groups and treated for 3 cycles with PBS (control), DCs vaccine, ADM and ADM combined with DCs vaccine, respectively. The tumor volume was evaluated. The tumor weight and the levels of interleukin-2 (IL-2), IL-12 and interferon γ (IFN-γ) in the serum were determined by ELISA on the 21st day. RESULTS：Cancer cell frozen-thawed antigen-sensitized DCs had higher expression levels of CD11C, CD80 and CD86. The volume and weight of the tumor in ADM combined with DCs vaccine group were less than those in ADM group, DCs vaccine group and control group. The tumor inhibitory rate in combination group was higher than that in the other 3 groups. Compared with the other 3 groups, the serum levels of IL-2, IL-12 and IFN-γ in combination group significantly increased. CONCLUSION：ADM combined with tumor antigen-sensitized DCs vaccine can strengthen the animal antitumor immune response and effectively inhibit the growth of tumor in cervical tumor-bearing mice.     

Effects of glucocorticoid on miRNA-155 expression in CD4+T cells of asthmatic mice

AIM:To investigate the effects of glucocorticoid on the regulation of microRNA-155 (miRNA-155) expression in the CD4⁺ T cells of asthmatic mice. METHODS:The ovalbumin (OVA)-induced asthma mouse model was established and the mice were treated with glucocorticoid. The effects of glucocorticoid on the pulmpnary histopathological changes, the expression of miRNA-155 in the lung tissues and CD4⁺T cells, and the levels of cytokines in the bronchoal-veolar lavage fluid (BALF) were evaluated. RESULTS:The results of RT-qPCR showed that the expressions of miRNA-155 in the lung tissues and CD4⁺T cells from the spleen of asthmatic mice were significantly increased, and the level of miRNA-155 in the CD4⁺T cells was significantly increased with the increase in the allergen exposure time (P<0.01). HE and PAS staining showed that OVA significantly increased inflammatory cell infiltration as compared with control group, and the peribronchial and perivascular inflammation and mucus secretion of proliferative goblet cells were significantly reduced after glucocorticoid treatment. Glucocorticoid treatment inhibited the increase in the proportion of CD4⁺ CD8^- cells in the spleen and decreased the accumulation of CD4⁺ T cells in the lung tissues of asthmatic mice (P<0.01). After glucocorticoid treatment, the levels of interleukin-4 (IL-4), IL-5 and IL-13 in BALF were decreased, while the level of interferon-γ was increased significantly (P<0.01). CONCLUSION:Glucocorticoid reduces the accumulation of CD4⁺ T cells and inhibits the expression of miRNA-155 in the lung tissues and spleen CD4⁺ T cells of asthmatic mice.     

Adipose-derived stem cells mediate immunosuppression of Th17 in multiple sclerosis

AIM: To investigate how human adipose-derived stem cells (hASCs) regulates the differentiation of Th17 cells in multiple sclerosis. METHODS: hASCs were isolated from the adipose tissues. Magnetic-activated cell sorting (MACS) kit was used to isolate CD4⁺ T cells from peripheral blood mononuclear cells (PBMCs) which were isolated by density gradient centrifugation. The percentage of CD4⁺ T cells was detected by flow cytometry. The activated CD4⁺ T cells were co-cultured with hASCs for about 4 d at different ratios of hASCs to CD4⁺ T cells (1:4 and 1:10) in a Th17 polarised condition. Another group adding anti-leukemia inhibitory factor (LIF) antibody was set up. Th17 cell proportion of the CD4⁺ T cells was determined by flow cytometry. The level of LIF in the supernatant of co-cultured system was measured by ELISA. The mRNA expression of retinoid-related orphan receptor γt (RORγt), interleukin-6 receptor (IL-6R), interleukin-23 receptor (IL-23R), LIF and leukemia inhibitory factor receptor (LIFR) was detected by real-time PCR. RESULTS: The result of flow cytometry suggested there were mainly hASCs, and the percentage of CD4⁺ T cells in the PBMCs were above 90% after MACS. The Th17 cell proportion decreased in 1:4 and 1:10 co-cultured groups in a dose-dependent manner. The mRNA expression of IL-6R, IL-23R and RORγt was downregulated and the expression of LIFR and LIF was up-regulated. When the anti-LIF was added into the co-cultured system, the ratio of Th17 cells increased and reached to the control level. The protein level of LIF obviously increased after co-cultured. After anti-LIF added, the mRNA expression of RORγt and IL-6R was up-regulated. CONCLUSION: hASCs inhibits the differentiation of Th17 cells from multiple sclerosis patients through the competitive inhibition of LIF/IL-6 by secreting LIF.     

Effects of lipopolysaccharide and interleukin 1 receptor antagonist on proliferation and nitric oxide synthesis of mesangial cells

AIM: To investigate the effects of lipopolysaccharide (LPS) and interleukin-1 receptor antagonist (IL-1ra) on mesangial cells proliferation and nitric oxide synthesis. METHODS: Glomerular mesangial cells from SD rats were cultured. The first and second passages of cultured cells were used for the experiment. LPS and LPS plus IL-1ra were added in cell cultures, respectively. By using chemical method the nitrite in supernatants was measured, [³H]-TdR incorporation was determined to evaluate the GMC proliferation. Northern and slot hybridizations were performed to detect the expression of iNOS mRNA. RESULTS: There were expression of iNOS mRNA, more production of nitrite (0.64±0.25 vs 0.12±0.06 nmol/10⁴ cell) in supernatants and GMC proliferation (3735±1177.9 vs 1785±280.6) in LPS group compared to the control. While compared with LPS group, in LPS+IL-1ra GMC group, expression of iNOS mRNA decreased by 40%, nitrite increased (3.28±0.33nmol/10⁴ cell), proliferation of GMC decreased (818±77.27). CONCLUSION: LPS could activate the GMC to express iNOS mRNA and produce more nitrite. IL-1ra could partially inhibit the effects of LPS on the expression of iNOS mRNA in GMC, but not nitrite. There is no synchronous correlation between NO production and GMC proliferation.     

Immunization with mixed peptides derived from glucose-6-phosphate isomerase induces rheumatoid arthritis in DBA/1 mice

AIM: To establish an animal model of rheumatoid arthritis(RA) in DBA/1 mice induced by immunodominant mixed peptides derived from glucose-6-phosphate isomerase(GPI). METHODS: The DBA/1 mice were immunized with emulsified mixed peptide fragments of hGPI325-339+hGPI469-483 or single peptide hGPI325-339 in complete Freund's adjuvant by subcutaneous injection to induce the model of RA. Body weight, ankle joint symptom scores, the pathological change of the ankle joint, the levels of CD4⁺ T cells in the spleen and peripheral blood, the proportion of iNKT cells in the peripheral blood, and the levels of TNF-α and IL-6 in serum were detected to evaluate and analyze the model. RESULTS: The hind paw of the model mice appeared red swelling on the 8th day, and then aggravated gradually to the limbs. The red swelling reached peak on the 14th day, and then relieved gradually. Inflammation response dominated by lymphocytes and monocytes was observed in the ankle joint. The inflammatory effect of mixed peptides was more obvious than that of the single one(P<0.05). Compared with control group and the mice treated with single peptide, the weight gain was slow, the amount of CD4⁺ T cells in the peripheral blood and spleen were increased, the proportion of peripheral iNKT cells in the inflammatory peak was decreased(P<0.05), and the serum level of TNF-α was increased significantly(P<0.05) in the mice treated with mixed peptide fragments. CONCLUSION: The immunological characteristics of RA model induced by mixed GPI peptides in DBA/1 mice is closer to that in RA patients, especially in the immunopathology of iNKT cells. Therefore, this model can be used as a new tool for studying the mechanism and immunological intervention of RA.     

Unfractionated heparin ameliorates lipopolysaccharide-induced expression of interleukin-8 in human endothelial cells through Toll-like receptor 4 signaling

AIM:To determine the effect of unfractionated heparin(UFH) on lipopolysaccharide(LPS)-induced expression of interleukin-8(IL-8) and the role of Toll-like receptor 4(TLR4) signaling. METHODS:Human pulmonary microvascular endothelial cells(HPMECs) were either exposed to LPS alone(10 mg/L) or in combination with 100 U/L or 10³ U/L UFH. UFH was added to the cells 15 min prior to stimulation with LPS. Those samples not receiving LPS or UFH received an equal volume of phosphate-buffered saline. The concentrations of IL-8 in the cell culture supernatants were detected by ELISA at 2, 6 and 12 h. The mRNA expression of IL-8, CD14 and TLR4 in HPMECs was detected by real-time fluorescence quantitative polymerase chain reaction at 2, 6 and 12 h. RESULTS:Compared with normal control group, the mRNA expression of IL-8 in LPS group was increased, and reached the peak at 6 h. The protein level of IL-8 reached the peak at 12 h. The mRNA expression of TLR4 in LPS group reached the peak at 6 h. They were down-regulated in UFH group. The mRNA expression of CD14 was not detected. CONCLUSION:The expression of IL-8 is obviously increased in LPS-treated HPMECs. UFH might exert its therapeutic effect through TLR4 signaling.     

Expression of IFN-γ, IL-4 and IL-17A in asthmatic mice vaccinated with BCG and HepB in neonatal period

AIM: To investigate the expression of IFN-γ, IL-4 and IL-17A in asthmatic mice vaccinated with bacillus Calmette-Guérin (BCG) and hepatitis B (HepB) in the neonatal period. METHODS: BALB/c mice were randomly divided into BGG+HepB+ovalbumin (OVA) group (B/H/O group), B/O group, H/O group, B/H group, OVA group, BCG group, HepB group and normal saline (NS) group (n=6). The mice in B/H/O group and B/H group at 0, 7 and 14 d received subcutaneous injection of 1×10⁵ CFU BCG for 3 times, while at 0 and 28 d received intramuscular injection of 1.5 μg HepB on the hindlimb twice. The mice in other groups were individually vaccinated with BCG or HepB. OVA sensitization and aerosol inhalation were performed to establish the asthma model. The lung tissues were collected for HE staining. Bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) were collected, and the number of eosinophils (EOS) in BALF was counted. The serum levels of IFN-γ and IL-4, and the level of IL-17A in lung tissue homogenate were measured by ELISA. RESULTS: The pathological changes of the lung in OVA group, B/O group, B/H/O group and H/O group were observed. There were extensive inflammatory cell infiltration around the bronchus, and epithe-lial cell hypertrophy. Those in B/H/O group and H/O group were worse than those in OVA group, while those in B/O group was better than those in OVA group. Total BALF cell counts in B/H/O group, B/O group and H/O group were decreased (P<0.05) as compared with OVA group. The BALF EOS count in B/H/O group was higher than that in B/H group, that in B/O group was higher than that in BCG group, and that in H/O group was higher than that in HepB groups (P<0.05). Compared with H/O group, OVA group and NS group, the serum IFN-γ/IL-4 ratio in HepB group was increased (P<0.05), and compared with B/H/O group, B/O group, OVA group and NS group, that in B/H group was also increased (P<0.05). Compared with OVA group, the level of IL-17A in the lung tissues of B/H/O group and B/O group was decreased (P<0.05), and compared with B/O group, that in B/H/O group was further decreased (P<0.05). CONCLUSION: Combined vaccination of BCG and HepB reduces the inflammotory responses in the lung tissues of asthmatic mice. The mechanism may be related with the decrease in the release of IL-4, the increase in IFN-γ/IL-4, and the inhibition of IL-17A expression.     

Bone marrow-derived mesenchymal stem cell-conditioned medium atte-nuates LPS-induced acute lung injury in mice

AIM:To explore the effects of bone marrow-derived mesenchymal stem cells-conditioned medium (MSCs CdM) on lipopolysaccharide (LPS)-induced acute lung injury. METHODS:Lung injury was induced in mice by intraperitoneal injection of LPS. The mice were given a tail vein injection of MSCs CdM or normal saline 1 h after LPS administration. The mice were killed by an intraperitoneal injection of pentobarbital 6 h after LPS injection for either bronchoalveolar lavage fluid (BALF) and serum collection or lung histological analysis. RESULTS:Compared with control group, the BALF levels of protein, interleukin-10 (IL-10) and keratinocyte growth factor (KGF), the serum levels of tumor necrosis factor α (TNF-α) and IL-6, and the myeloperoxidase (MOP) activity in the lung tissues were significantly higher in LPS group, and severe pathological damages in the lung tissues were also observed. Treatment with MSCs CdM significantly reduced the BALF prtein level, the seum TNF-α and IL-6　levels and the lung MPO activity, and attenuated the lung pathological damages, but further increased the levels of IL-10 and KGF in the BALF. CONCLUSION:Treatment with MSCs CdM attenuates the lung injuries induced by LPS, which may be via regulating the expression of TNF-α, IL-6, IL-10 and KGF.     

Protective effect of MUC2 in mice with colitis and the expression of anti-CBir1 antibody in serum

AIM: To investigate the protective effect of mucin 2 (MUC2) on intestinal mucosa of colitis model mice, and to explore the correlation between the expression of anti-CBir1 flagellin antibody and MUC2. METHODS: The mice were randomly divided into normal control group, 2,4,6-trinitrobenzenesulfonic acid (TNBS) group, lipopolysaccharide (LPS)+ovalalbumin (OVA)+TNBS group and ketotifen+TNBS group. The expression of MUC2 in colon tissue was determined by PAS staining and immunohistochemistry, and the anti-CBir1 antibody level in the serum of mice in each group was measured by ELISA. RESULTS: The scores of disease activity index and histological index in TNBS group were higher than those in normal control group (P<0.05). The scores in LPS+OVA+TNBS group were much higher than those in TNBS group (P<0.05). However, the values in ketotifen+TNBS group were lower than those in TNBS group (P<0.05). PAS staining showed a decrease in goblet cells in TNBS group. Compared with TNBS group, the colonic mucosa integrity in LPS+OVA+TNBS group was destroyed, and the number of goblet cells in ketotifen+TNBS group increased significantly. Immunohistochemical staining showed that the expression of MUC2 in the intestinal tract of each mo-del group was basically consistent with the results of PAS staining. The serum anti-CBir1 antibody level in TNBS group was higher than that in normal control group (P<0.05), and that in LPS+OVA+TNBS group was significantly higher than that in TNBS group (P<0.05), whereas that in ketotifen+TNBS group was decreased slightly (P<0.05). CONCLUSION: MUC2 plays a protective role in the pathogenesis of colitis in mice, and there is a negative correlation between the expression of MUC2 and the bacterial flagellin in the intestinal mucosa of mice with colitis.     

The expression of CD14 in rat Kupffer cells

AIM: To explore the expression of CD14 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS or the mediators from KCs induced by LPS, the changes of CD14 expression were measured by RT-PCR and immunohistochemistry.The expressions of TNFα mRNA、IL-6 mRNA or the concentrations of TNFα、IL-6 were estimated by in situ hybridization and radioimmunoassay, respectively. RESULTS: LPS increased the expression of CD14 in KCs in a dose-dependent fashion (LPS, 1 μg/L-10 mg/L) and in a time-dependent fashion(0.5 h-24 h, peaked at 3-6 hours). While the expression of CD14 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 hour were obviously increased. CONCLUSIONS: There was a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14. It is implied that the increase in CD14 expression may be induced by LPS and the cytokines produced by KCs, it also reveals that there is a auto-regulated loop in CD14 expression.     

中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛征文通知

"中国园艺学会2012年学术年会暨庆祝《园艺学报》创刊50周年—园艺学进展论坛"将于2012年10月在西安举行,即日起征集:①研究论文摘要,②有关园艺学进展的综述文章。经审查合格的摘要和综述将分别收入"中国园艺学会2012年学术年会论文摘要集"和"庆祝《园艺学报》创刊50周年—园艺学进展论坛专辑",并于会前以     

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.     

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.     

Effect of mesenchymal stem cells on secreting function of T lymphocytes from patients with idiopathic thrombocytopenic purpura in vitro

AIM: To analyze the effect of mesenchymal stem cells (MSCs) on secreting cytokines by T lymphocytes from patients with idiopathic thrombocytopenic purpura (ITP) in vitro.METHODS: Human bone marrow-derived MSCs were isolated by Ficoll Hypaque and cultured for proliferating to passage cells. Allogeneic T lymphocytes of ITP were isolated from peripheral blood by Ficoll Hypaque and nylon cotton column. Then the stromal feeder layers of different numbers (2×103, 1×104, 5×104 per well) of MSCs treated with mitomycin were co-cultured with above-mentioned T lymphocytes. The supernatant were respectively collected on day 2, 4 and 6 after co-culture, then the levels of IL-2, IFN-γ, IL-4, IL-10 secreted by T lymphocytes were measured by enzyme linked immunosorbent assay (ELISA) dynamically.RESULTS: The levels of IL-2 and IFN-γ secreted by T cells from ITP were higher than those from normal control (P<0.05, respectively). Inversely, IL-4 and IL-10 were lower than those in normal control (P<0.05, respectively). After co-cultured with T lymphocytes, MSCs significantly inhibited the cytokine levels of IL-2 and IFN-γ secreted by T lymphocytes from ITP or health adults (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect was more obvious when co-cultured for 4 days or 6 days than that for 2 days (P<0.05, respectively). However, MSCs significantly promoted the releases of IL-4 and IL-10 by T lymphocytes from ITP patients (P<0.05, respectively) in a dose dependent manner (P<0.05, respectively), and the effect on IL-10 was in a time dependent way (P<0.05), while the effect on IL-4 had no obvious difference among 2 d, 4 d and 6 d(P>0.05). As for health control group, when cell numbers exceeded above 1×104, MSCs obviously promoted IL-4 and IL-10 levels secreted by T lymphocytes (P<0.05) in a dose dependent manner (P<0.05), and both of the effects were more noticeable when co-cultured for 4 d or 6 d than that for 2 d(P<0.05, respectively).CONCLUSION: MSCs regulate the balance between Th1 and Th2 reaction and partly correct ITP Th1 polarization in vitro.     

Roles of dendritic cells treated with 17β-estradiol in immune tolerance induction in skin allograft

AIM: To study the roles of bone marrow-derived dendritic cells from donor mouse treated with 17β-estradiol (E₂) in immune tolerance induction in skin allograft. METHODS: Bone marrow-derived dendritic cells from C57 mouse as donor were cultured respectively treated with E₂ (E₂ group). BALB/c mouse as recipient received respectively one injection of dendritic cells of E₂ group, mature dendritic cell group and immature dendritic cell group intravenously. Skin transplantation was performed in the absence of immunosupression after 7 d. Mice that received PBS were served as control. The time of skin survival was observed after transplantation. Flow cytometry was used to analyze the percentage of CD4+CD25+ T cells in peripheral blood respectively before and after transplantation. RESULTS: Compared with immature dendritic cells and control group, the time of skin survival in E₂ group was significantly longer (P<0.01), especially, the time of skin survival still prolonged 10.6 d after skin rejection in immature dendritic group. The percentage of CD4+CD25+ regulatory T cells in E₂ group was significantly higher than that in immature dendritic cell group and control group (P<0.01). CONCLUSION: In skin allograft model, dendritic cells treated with E₂ prolong the allograft survival time.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Effect of mesalazine on Th1, Th17 and Treg cells in mice with DSS-induced ulcerative colitis

AIM: To investigate the effect of mesalazine treatment on regulation of Th1, Th17 and Treg cells in mice with dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). METHODS: The expression of IL-17, IFN-gamma and Foxp3 in the peripheral blood mononuclear cells (PBMC) and intestinal mucosa lamina propria mononuclear cells (LPMC) of DSS-induced UC mice was detected by flow cytometry analysis. The effect of mesalazine treatment on regulaiton of Th1, Th17 and Treg cells in the mice with DSS-induced ulcerative colitis was examined.RESULTS: The expression of IL-17, IFN-γ and Foxp3 on CD4+T cells were significantly higher in the PBMC of DSS-induced mice than those in control group. CD4+ IFN-γ+T cells and CD4+ Foxp3+T cells were higher in LPMC than those in control group, except CD4+IL-17+T cells. Moreover, the Th1, Th17 and Treg cells were higher in DSS group than those in control group in LPMC. However, only Tregs was higher in PBMC. Pre-treatment with mesalazine significantly decreased the number of Th17, Th1 and Treg cells of UC model mice both in PBMC and LPMC.CONCLUSION: The Th1, Th17 and Tregs cells in DSS-induced mice were significantly higher than those in control mice, suggesting that CD4+T cell subsets play an important role in the pathogenesis of UC. Mesalazine may play a role in the treatment of UC by regulating the Th1, Th17 and Tregs cells.