背景急性胰腺炎(acute pancreatitis,AP)是常见的急腹症,不同类型预后不同.AP的免疫应答和失衡免疫与其严重程度有关,炎症因子和相关免疫细胞在AP发病机制中至关重要,因而寻找炎症细胞和新炎症免疫因子对精准治疗AP具有重要意义.目的探...背景急性胰腺炎(acute pancreatitis,AP)是常见的急腹症,不同类型预后不同.AP的免疫应答和失衡免疫与其严重程度有关,炎症因子和相关免疫细胞在AP发病机制中至关重要,因而寻找炎症细胞和新炎症免疫因子对精准治疗AP具有重要意义.目的探讨AP患者外周血CD14^+CD16^+单核细胞表达B7-H2的临床意义.方法A P患者63例[轻度A P(m i l dA P,M A P)25例、中度AP(moderately severe AP,MSAP)20例、重度AP(severe AP,SAP)18例],对照组为健康体检者20例,采用流式细胞仪检测CD14^+CD16^+细胞亚群上B7-H2表达情况,评价其与胰腺炎严重程度关联性及临床意义.结果AP患者发病24hCD14^+CD16^+细胞B7-H2出现异常高表达,显著高于健康对照组(t=11.10,P<0.001);A P各组B 7-H 2在C D 14^+C D 16^+细胞膜上表达明显高于CD14^+C D16^-细胞膜上表达(P<0.01);SAP组CD14^+C D 16^+和CD14^+C D 16^-细胞B7-H2表达(373.30±89.72和78.62±13.05)最高,M S A P组(279.55±76.95/44.92±12.44)其次,均高于M A P组(181.15±35.75/23.32±4.28),各组两两比较差异有显著性(P<0.01);MAP组、MSAP组发病24 h、48 h、72hCD14^+CD16^+和CD14^+CD16^-单核细胞膜B7-H2动态表达差异无显著性(P>0.05),然而,SAP组无论C D14^+C D16^+还是CD14^+C D16^-细胞膜B7-H2表达24h、48h、72h均呈明显上升趋势,差异有显著性(P<0.05).结论CD14^+CD16^+和CD14^+CD16^-单核细胞膜B7-H2在AP患者体内高表达,与AP严重程度密切相关,且SAP呈动态升高变化;同时B7-H2在AP患者CD14^+CD16^+单核细胞膜表达较CD14^+CD16^-单核细胞明显升高,为进一步认识AP免疫应答和失衡提供了新的线索,为AP精准靶向治疗提供参考.展开更多
Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which c...Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.展开更多
Antigen-presentation via major histocompatibility complex(MHC)to T cells is the key event to initiate adaptive immune responses.In teleosts,as in mammals,the main types of professional antigen-presenting cells(APCs)ar...Antigen-presentation via major histocompatibility complex(MHC)to T cells is the key event to initiate adaptive immune responses.In teleosts,as in mammals,the main types of professional antigen-presenting cells(APCs)are dendritic cells(DCs),monocytes/macrophages,and B cells.In the current study,flow cytometry,immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon(Salmo salar L.)have antigen-presenting properties.The neutrophils were positive for MHC class II,CD83 and CD80/86,and upon in vitro bacterial exposure,gene expression analysis of purified neutrophils showed that IL-12p40,which is essential for proliferation of naïve T cells,was highly upregulated at both 6 and 24 h post bacterial exposure.Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation,we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs.This work makes an important basis for further exploring the potential of using neutrophils to develop new,targeted immunoprophylactic measures.展开更多
AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological character...AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.展开更多
文摘背景急性胰腺炎(acute pancreatitis,AP)是常见的急腹症,不同类型预后不同.AP的免疫应答和失衡免疫与其严重程度有关,炎症因子和相关免疫细胞在AP发病机制中至关重要,因而寻找炎症细胞和新炎症免疫因子对精准治疗AP具有重要意义.目的探讨AP患者外周血CD14^+CD16^+单核细胞表达B7-H2的临床意义.方法A P患者63例[轻度A P(m i l dA P,M A P)25例、中度AP(moderately severe AP,MSAP)20例、重度AP(severe AP,SAP)18例],对照组为健康体检者20例,采用流式细胞仪检测CD14^+CD16^+细胞亚群上B7-H2表达情况,评价其与胰腺炎严重程度关联性及临床意义.结果AP患者发病24hCD14^+CD16^+细胞B7-H2出现异常高表达,显著高于健康对照组(t=11.10,P<0.001);A P各组B 7-H 2在C D 14^+C D 16^+细胞膜上表达明显高于CD14^+C D16^-细胞膜上表达(P<0.01);SAP组CD14^+C D 16^+和CD14^+C D 16^-细胞B7-H2表达(373.30±89.72和78.62±13.05)最高,M S A P组(279.55±76.95/44.92±12.44)其次,均高于M A P组(181.15±35.75/23.32±4.28),各组两两比较差异有显著性(P<0.01);MAP组、MSAP组发病24 h、48 h、72hCD14^+CD16^+和CD14^+CD16^-单核细胞膜B7-H2动态表达差异无显著性(P>0.05),然而,SAP组无论C D14^+C D16^+还是CD14^+C D16^-细胞膜B7-H2表达24h、48h、72h均呈明显上升趋势,差异有显著性(P<0.05).结论CD14^+CD16^+和CD14^+CD16^-单核细胞膜B7-H2在AP患者体内高表达,与AP严重程度密切相关,且SAP呈动态升高变化;同时B7-H2在AP患者CD14^+CD16^+单核细胞膜表达较CD14^+CD16^-单核细胞明显升高,为进一步认识AP免疫应答和失衡提供了新的线索,为AP精准靶向治疗提供参考.
基金This work was supported by the National Natural Science Foundation of China(No. 39470293).
文摘Objective To express human B7. 2 extracellular domain with prokaryote expression system and to evaluate its biological activity in vitro. Methods PCR was used to amplify the extracellular region of human B7. 2 which contained both the IgV and IgC domains. The recombinant PGEX-4T-3/hB7. 2 (IgV+C) was obtained by cloning the PCR product into a prokaryote expression plasmid PGEX-4T-3 and was transformed into the host strain of DH5-a. Tke fusion protein consisted of GST and hB7. 2(IgV+C) was identified by SDS-PAGE and Western blotting. T cell activation was observed by exposing purified T lymphocytes to the fusion protein and [3H]-TdR incorporation with the presence of the first signal imitated hy anti-CD3 antibody. Results The fusion protein GST-hB7. 2 (IgV+ C) was produced and detected in inclusive body form from engineered bacterial cells. With the first signal existed,T lymphocytes proliferated when it was co-stimulated by the fusion protein. Conclusion These results indicated that the functional human B7. 2(IgV+C) fusion protein can be produced in bacterial cells and the fusion protein displays the co-stimulatory activity in T lymphocytes activation.
文摘Antigen-presentation via major histocompatibility complex(MHC)to T cells is the key event to initiate adaptive immune responses.In teleosts,as in mammals,the main types of professional antigen-presenting cells(APCs)are dendritic cells(DCs),monocytes/macrophages,and B cells.In the current study,flow cytometry,immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon(Salmo salar L.)have antigen-presenting properties.The neutrophils were positive for MHC class II,CD83 and CD80/86,and upon in vitro bacterial exposure,gene expression analysis of purified neutrophils showed that IL-12p40,which is essential for proliferation of naïve T cells,was highly upregulated at both 6 and 24 h post bacterial exposure.Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation,we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs.This work makes an important basis for further exploring the potential of using neutrophils to develop new,targeted immunoprophylactic measures.
基金Supported jby the Natural Science Foundation of Guangdong Province China,No.980180
文摘AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.
