Dendritic cells （DCs） are bone marrow-derived professional antigen presenting cells （APCs）, they are crucial for initiation of both innate and adaptive immune responses. In this study, chicken bone marrow （chBM）...Dendritic cells （DCs） are bone marrow-derived professional antigen presenting cells （APCs）, they are crucial for initiation of both innate and adaptive immune responses. In this study, chicken bone marrow （chBM） cells were cultured in medium with recombinant chicken granulocyte-macrophage colony stimulating factor （rGM-CSF） and recombinant chicken interleukin-4 （rIL-4） for 7 days, displayed the typical morphology of DCs. These immature chicken bone marrow-derived DCs （chBM-DCs） showed signifcant up-regulation of the putative CD11c and of major histocompatibility complex class II （MHC II）, but CD40 and CD86 co-stimulatory molecules were almost no up-regulated. However, maturation with lipopolysaccharide （LPS）, surface expression of CD40, CD86 was greatly increased. The phagocytosis of chBM-DCs was assessed by neutral red, and the phagocytosis decreased after stimulation. In mixed lymphocyte responses （MLR）, stimulated chBM-DCs were more effective to T-cell stimulators than non-stimulated chBM-DCs. In addition, mRNA expression levels of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α, CXCLi1 and CXCLi2 were assessed by real-time qPCR （qRT-PCR）, and the results showed cultured chBM-DCs could be matured to a T helper cell type 1 （Th1）-promoting phenotype by LPS stimulation.展开更多
Little information was so far available about allergenic mechanism of the roasted peanut allergens during initial stages of allergy.The purpose of this study was to determine the influence of roasting(150℃,20 min)on ...Little information was so far available about allergenic mechanism of the roasted peanut allergens during initial stages of allergy.The purpose of this study was to determine the influence of roasting(150℃,20 min)on biochemical and biological properties of Ara h 3,a major peanut allergen.Allergenicity of roasted peanut emulsion to mice,differences in uptakes between Ara h 3 purified from raw peanuts(named as Ara h 3-Raw)and that purified from roasted peanuts(named as Ara h 3-Roasted)by bone marrow-derived dendritic cells(BMDCs)and the implication of cell surface receptors involving in uptake,and changes in glycosylation and structure of Ara h 3 after roasting were analyzed in this study.This study suggested that roasting increased allergenicity of peanut to BALB/c mice.Maillard reaction and structural changes of Ara h 3 induced by roasting significantly altered the uptake of Ara h 3-Roasted by BMDCs,and modified Ara h 3 fate in processes involved in immunogenicity and hyper allergenicity,indicating that food processing pattern can change food allergenicity.展开更多
OBJECTIVE This research was to induce dendritic cells （DCs） from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them. METHODS Embryonic stem ce...OBJECTIVE This research was to induce dendritic cells （DCs） from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them. METHODS Embryonic stem cells （ESCs） suspending cultured in petri dishes were induced to generate embryonic bodies （EBs）. Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM- CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined. RESULTS Growing mature through exposure to lipopolysaccharide （LPS）, both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-II and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction （MLR）. CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.展开更多
BACKGROUND： Bone marrow transplantation is an effective treatment for severe forms of various autoimmune disorders. Dendritic cell reconstitution is thought to be one factor contributing to host immune recovery and t...BACKGROUND： Bone marrow transplantation is an effective treatment for severe forms of various autoimmune disorders. Dendritic cell reconstitution is thought to be one factor contributing to host immune recovery and therapeutic efficacy. OBJECTIVE： To assess the effects of bone marrow transplantation on an animal model of experimental autoimmune encephalomyelitis （EAE）, and to investigate changes in dendritic cells and subgroups following bone marrow transplantation. DESIGN, TIME AND SETTING： This experimental, neuroimmunological study was performed in Sun Yat-sen University between August 2006 and May 2007. MATERIALS： A total of 30 female C57BL/6 mice, aged 6-8 weeks, served as recipients, and 20 female adult C57BL/6 served as donors. Myelin oligodendrocyte glycoprotein 35-55 amino acid peptide （MOG35-55） of mudne origin was synthesized by Bio-Scientific （Xi＇an, China, purity 〉 95%）. Complete Freund＇s adjuvant was purchased from Difco Laboratories, Detroit, MI; pertussis vaccine was purchased from Alexis, San Diego, CA; radiation device and flow cytometry for FACS analysis were purchased from Theratron 780-C, Canada and Coulter, Fullerton, CA, respectively. METHODS： The C57BL/6 mice, aged 6-8 weeks, were immunized by subcutaneous injection of MOG35-55 peptide emulsified in complete Freund＇s adjuvant, which contained 500 μg Mycobacterium tuberculosis H37RA. The mice were subsequently intravenously injected with pertussis vaccine to induce EAE. The mice were randomly assigned to transplantation and EAE model groups （n = 12 for each）. Bone marrow cells [（5-10） × 10^6] were transplanted into EAE mice via the tail vein 4-6 hours following total body irradiation, and the model group was not treated. MAIN OUTCOME MEASURES： Mouse behavioral changes following EAE were evaluated daily. Injured spinal cord tissue sections were stained with hematoxylin and eosin 20 days after the initial immunization to observe inflammatory infiltration using light microscopy. Flow cytometry was used to detect the ratio and absolute number of DC1 （CD6aCD11c＋） and DC2 （CD8a＋CD11c＋） in peripheral blood 36 days after bone marrow transplantation. RESULTS： Significant improvement in clinical signs was observed in EAE mice following bone marrow transplantation compared with the model group （P 〈 0.01 ）, as well as attenuated lymphocyte and macrophage infiltration. Compared with the model group, the absolute number of dendritic cells and DC1, as well as the DC1/DC2 ratio, was significantly greater following bone marrow transplantation （P 〈 0.05 or P 〈 0.01）. CONCLUSION： The increased proportion of dendritic cells and DC1 is proposed to contribute to EAE remission following bone marrow transplantation.展开更多
Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells （DCs） were electroporat...Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells （DCs） were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time ,pre-electroporation cell condition and serum concentration in electrical buffer, lnverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency （22.10%） could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer. Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.展开更多
Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their ...Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.展开更多
基金Supported by the National Technology and Research Project(2015BAD12B01-4)
文摘Dendritic cells （DCs） are bone marrow-derived professional antigen presenting cells （APCs）, they are crucial for initiation of both innate and adaptive immune responses. In this study, chicken bone marrow （chBM） cells were cultured in medium with recombinant chicken granulocyte-macrophage colony stimulating factor （rGM-CSF） and recombinant chicken interleukin-4 （rIL-4） for 7 days, displayed the typical morphology of DCs. These immature chicken bone marrow-derived DCs （chBM-DCs） showed signifcant up-regulation of the putative CD11c and of major histocompatibility complex class II （MHC II）, but CD40 and CD86 co-stimulatory molecules were almost no up-regulated. However, maturation with lipopolysaccharide （LPS）, surface expression of CD40, CD86 was greatly increased. The phagocytosis of chBM-DCs was assessed by neutral red, and the phagocytosis decreased after stimulation. In mixed lymphocyte responses （MLR）, stimulated chBM-DCs were more effective to T-cell stimulators than non-stimulated chBM-DCs. In addition, mRNA expression levels of IL-1β, IL-4, IL-6, IL-10, IL-12, IFN-γ, TNF-α, CXCLi1 and CXCLi2 were assessed by real-time qPCR （qRT-PCR）, and the results showed cultured chBM-DCs could be matured to a T helper cell type 1 （Th1）-promoting phenotype by LPS stimulation.
基金funded by the National Key Research and Development Program of China(2016YFD0501101)the project of Food Science Discipline Construction of Shanghai University and the National Natural Science Foundation of China(31201306)。
文摘Little information was so far available about allergenic mechanism of the roasted peanut allergens during initial stages of allergy.The purpose of this study was to determine the influence of roasting(150℃,20 min)on biochemical and biological properties of Ara h 3,a major peanut allergen.Allergenicity of roasted peanut emulsion to mice,differences in uptakes between Ara h 3 purified from raw peanuts(named as Ara h 3-Raw)and that purified from roasted peanuts(named as Ara h 3-Roasted)by bone marrow-derived dendritic cells(BMDCs)and the implication of cell surface receptors involving in uptake,and changes in glycosylation and structure of Ara h 3 after roasting were analyzed in this study.This study suggested that roasting increased allergenicity of peanut to BALB/c mice.Maillard reaction and structural changes of Ara h 3 induced by roasting significantly altered the uptake of Ara h 3-Roasted by BMDCs,and modified Ara h 3 fate in processes involved in immunogenicity and hyper allergenicity,indicating that food processing pattern can change food allergenicity.
基金supported by a grant from Program for New Century Excellent Talents in University,China(No.NECT-05-0820).
文摘OBJECTIVE This research was to induce dendritic cells （DCs） from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them. METHODS Embryonic stem cells （ESCs） suspending cultured in petri dishes were induced to generate embryonic bodies （EBs）. Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM- CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined. RESULTS Growing mature through exposure to lipopolysaccharide （LPS）, both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-II and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction （MLR）. CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.
基金Sun Yat-sen University Clinical Research 5010 Program,No. 2007027Science and Technology Project of Guangdong Province,No. 2006B36004003the Natural Science Foundation of Guangdong Province,No. 8151008901000104
文摘BACKGROUND： Bone marrow transplantation is an effective treatment for severe forms of various autoimmune disorders. Dendritic cell reconstitution is thought to be one factor contributing to host immune recovery and therapeutic efficacy. OBJECTIVE： To assess the effects of bone marrow transplantation on an animal model of experimental autoimmune encephalomyelitis （EAE）, and to investigate changes in dendritic cells and subgroups following bone marrow transplantation. DESIGN, TIME AND SETTING： This experimental, neuroimmunological study was performed in Sun Yat-sen University between August 2006 and May 2007. MATERIALS： A total of 30 female C57BL/6 mice, aged 6-8 weeks, served as recipients, and 20 female adult C57BL/6 served as donors. Myelin oligodendrocyte glycoprotein 35-55 amino acid peptide （MOG35-55） of mudne origin was synthesized by Bio-Scientific （Xi＇an, China, purity 〉 95%）. Complete Freund＇s adjuvant was purchased from Difco Laboratories, Detroit, MI; pertussis vaccine was purchased from Alexis, San Diego, CA; radiation device and flow cytometry for FACS analysis were purchased from Theratron 780-C, Canada and Coulter, Fullerton, CA, respectively. METHODS： The C57BL/6 mice, aged 6-8 weeks, were immunized by subcutaneous injection of MOG35-55 peptide emulsified in complete Freund＇s adjuvant, which contained 500 μg Mycobacterium tuberculosis H37RA. The mice were subsequently intravenously injected with pertussis vaccine to induce EAE. The mice were randomly assigned to transplantation and EAE model groups （n = 12 for each）. Bone marrow cells [（5-10） × 10^6] were transplanted into EAE mice via the tail vein 4-6 hours following total body irradiation, and the model group was not treated. MAIN OUTCOME MEASURES： Mouse behavioral changes following EAE were evaluated daily. Injured spinal cord tissue sections were stained with hematoxylin and eosin 20 days after the initial immunization to observe inflammatory infiltration using light microscopy. Flow cytometry was used to detect the ratio and absolute number of DC1 （CD6aCD11c＋） and DC2 （CD8a＋CD11c＋） in peripheral blood 36 days after bone marrow transplantation. RESULTS： Significant improvement in clinical signs was observed in EAE mice following bone marrow transplantation compared with the model group （P 〈 0.01 ）, as well as attenuated lymphocyte and macrophage infiltration. Compared with the model group, the absolute number of dendritic cells and DC1, as well as the DC1/DC2 ratio, was significantly greater following bone marrow transplantation （P 〈 0.05 or P 〈 0.01）. CONCLUSION： The increased proportion of dendritic cells and DC1 is proposed to contribute to EAE remission following bone marrow transplantation.
基金Supported by National Natural Science Foundation of China (30570828,30471691, and30170915)
文摘Objective To explore the optimal electroporation parameters for transfection of plasmid DNA into murine bone marrow-derived dendritic cells. Methods Murine bone marrow-derived dendritic cells （DCs） were electroporated with plasmid DNA in varied conditions, such as electrical voltage, pulse time ,pre-electroporation cell condition and serum concentration in electrical buffer, lnverted fluorescence microscope and flow cytometer were used to determine the transfection efficiency. Some of the DCs genetically modified under different conditions were stained with trypan-blue and its viability was observed microscopically 48h after electroporation. Results Highest transfection efficiency （22.10%） could be reached when electrical voltage was 250V and pulse time was 20ms. Refreshing the culture medium pre-electroporation may help the cells recover more easily from gene transfer. Besides, electrical buffer containing serum may benefit the viability of DC after electroporation and temperature may has little influence on transfection efficiency. Conclusion Our observations demonstrated plasmid DNA could be efficiently transferred into murine bone marrow-derived DCs by electroporation. These data may helpful for cancer research related to murine DC transfection.
基金supported by a grant from the State Scholarship Fund under the China Scholarship Council (No.2003850064)the Chongqing Education Commission (KJ080319)
文摘Objective To explore the immune stimulation effect of recombinant E.coli LLO/OVA on mice bone marrow-derived dendritic cells （BMDCs） and T lymphocytes in vitro.Methods After BMDCs stimulated by E.coli LLO/OVA,their Toll-like receptor （TLR） and nucleotide-binding oligomerization domain （NOD） receptor signalling pathway were examined by superarray hybridization;and the priming effect of the vaccine activated BMDCs on CD4＋T and CD8＋T was determined by [3H]thymidine uptake and ELISA,the tumor cytotoxic effect of activated CD8＋T cells was determined by cytotoxic assay.Results After BMDCs were activated by E.coli LLO/OVA via TLR4,NOD1 receptor and NF-κB signalling pathway,the expression of their surface molecules including MHC class Ⅰ,MHC class Ⅱ,CD40,CD80 and CD86 significantly up-regulated;the secretion of IL-12 and IFN-？ increased also.The mature BMDCs stimulated the allergic CD4＋T and CD8＋T cells proliferation and their IL-2 and IFN-γ secretion,and the activated CD8＋T cells effectively killed B16-OVA melanoma cells and RMA-S/OVA lymphoma cells in vitro.Conclusion E.coli LLO/OVA is effective in inducing BMDCs maturation via activating TLR4 and NOD1 receptor signalling pathway and promoting specific anti-tumor T cell immunity in vitro.
