AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial...AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.展开更多
AIM To investigate the pathogenic effect ofSEB and D-GalN on liver and the protection ofcyclosporin A, the relationship between hepaticapoptosis and necrosis and the possiblemechanism of acute hepatic necrosis.METHODS...AIM To investigate the pathogenic effect ofSEB and D-GalN on liver and the protection ofcyclosporin A, the relationship between hepaticapoptosis and necrosis and the possiblemechanism of acute hepatic necrosis.METHODS After staphylococcal enterotoxin B(SEB ) mixed with D--galactosamine (D-GaiN )were injected intraperitoneally into Balb/c miceand those previously treated with cyclosporin A,blood samples were collected and livers wereisolated at 2, 6, 12 and 24 h. Patterns othepatocellular death were studiedmorphologically and biochemically, circulatingcytokines (TNF-a, IFN--y ) and mice mortalitywithin 24h was assessed.RESU’LTS The SEB could induce the typicalapoptotic changes of hepatocytes, the D-GaiNcould induce hepatocytes apoptosis anddegeneration at the same time, and the micehaving received the SEB + D-GaiN injectionsdeveloped apoptosis at 2 and 6 h, but after 12 hhepatocytes were characterized by severein jury, whereas all the examinations in thecyclosporin A treated mice were normal.CONCLUSION Hepatic cell apoptosis might berelated to necrosis, and massive hepatocyteapoptosis is likely the initiating step of acutehepatic necrosis in mice. The effects induced bySEB and D--GaiN on hepatocytes might bemediated by T cells, and could be prevented bycyclosporin A.展开更多
Staphylococcal superantigen(SAg)toxins are the most notable virulence factors associated with Staphylococcus aureus,which is a pathogen associated with serious community and hospital acquired infections in humans and ...Staphylococcal superantigen(SAg)toxins are the most notable virulence factors associated with Staphylococcus aureus,which is a pathogen associated with serious community and hospital acquired infections in humans and various diseases in animals.Recently,SAg toxins have become a superfamily with 29 types,including staphylococcal enterotoxins(SEs)with emetic activity,SE-like toxins(SEIs)that do not induce emesis in primate models or have yet not been tested,and toxic shock syndrome toxin-1(TSST-1).SEs and SEIs can be subdivided into classical types(SEA to SEE)and novel types(SEG to SEIY,SE01,SE02,SEI26 and SEI27).The genes of SAg toxins are located in diverse accessory genetic elements and share certain structural and biological properties.SAg toxins are heat-stable proteins that exhibit pyrogenicity,superantigenicity and capacity to induce lethal hypersensitivity to endotoxin in humans and animals.They have multiple pathogenicities that can interfere with normal immune function of host,increase the chances of survival and transmission of pathogenic baaeria in host,consequently contribute to the occurrence and development of various infeaions,persistent infeaions or food poisoning.This review focuses on the following aspeas of SAg toxins:(1)superfamily members of classic and novelty discovered staphylococcal SAgs;⑵diversity of gene locations and molecular structural characteristics;(3)biological characteristics and activities;(4)multi-pathogenicity of SAgs in animal and human diseases,including bovine mastitis,swine sepsis,abscesses and skin edema in pig,arthritis and septicemia in poultry,and nosocomial infections and food-borne diseases in humans.展开更多
Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally w...Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally with SEB, D- GalN or both, blood samples were collected and livers were removed at 2,6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines(TNF, IFN-γ) were determined, and mice mortality within 24h was assessed. Results SEB could induce thetypical apoptotic changes of hepatocytes morphologically and biochemically. The mechanism is probably associatedwith the production and release of Cytokines (such as TNF, IFN- γ, etc).D - GalN could induce hepatocytesapoptosis and degeneration at the same time. Besides this, we confirmed hepatocytes of the mice which wereadministered SEB and D - GalN developing apoptosis at 2, 6h, but after 12h hepatocytes were characterized bysevere injury, the mice mortality within 24h is 50%. Conclusion SEB or D - GalN alone could induce the typicalapoptotic changes of hepatocytes. SEB+D-GalN developed hepatocytes apoptosis in the early stage and necrosisin the later. It suggests that there is some relationship between hepatic cell apoptosis and necrosis, and massivehepatocyte apoptosis is the probably initiating step of acute hepatic necrosis in mice.展开更多
The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental grou...The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.展开更多
Microbial stimulation in early childhood may be necessary for proper maturation of the immune system. Infants colonized with Staphylococcus aureus have low risk of developing food allergy. Neonatal exposure to staphyl...Microbial stimulation in early childhood may be necessary for proper maturation of the immune system. Infants colonized with Staphylococcus aureus have low risk of developing food allergy. Neonatal exposure to staphylococcal superantigen improves oral tolerance and enhances protection in experimental allergy models. Here, we used three wild-type strains of S. aureus, naturally harboring genes for different superantigens (SElM/SElO alone, or in combination with SEA or TSST-1). We first investigated their in vitro stimulatory capacity of splenocytes from germ-free mice. Secondly, germ-free mice were colonized with the strains and their capacity to develop oral tolerance was tested in a food allergy model. In vitro, S. aureus with only SElM/SElO genes promoted the strongest B-cell stimulation. S. aureus carrying gene for SEA induced the highest proportion of CD4<sup>+</sup>FoxP3<sup>+</sup> T cells. The proportion of regulatory T cells was inversely correlated to B-cell proliferation, indicating suppressive ability of these cells. All strains were equally able to colonize the germ-free gut, initially achieving 10<sup>10</sup>CFU/g faeces, which decreased to 10<sup>5</sup> over a period of six weeks. Mice colonized with S. aureus carrying genes for SEA or TSST-1 had improved capacity to develop tolerance compared to germ-free mice. These results suggest that colonization by S. aureus producing superantigens may improve active tolerance to gut allergens.展开更多
Exposure of naive murine CD4 + T lymphocytes to superantigen such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell ...Exposure of naive murine CD4 + T lymphocytes to superantigen such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell population with SEB leads to the apoptotic events of activation-induced cell death (AICD). The signaling mechanism responsible for the AICD is a target of intensive investigation. However, the precise downstream signaling pathways of SEB-induced AICD remains unclear. Our results here show that the sequential activation of caspase-1/ICE-like and caspase-3/CPP32-like cysteine proteases probably plays a role in the signaling transduction of SEB-induced AICD, but caspase-3/CPP32-like proteases activation does not depend on caspase-1-like proteases activation. Herbimycin A, a specific inhibitor of protein tyrosine kinases, inhibit caspase-3/CPP32-like cysteine proteases activation. However, it does not prevent DNA fragmentation of CD4 + T cells apoptosis induced by SEB. These results indicate that protein tyrosine kinases pathway is probably involved in the signaling transduction of CD4 + T cells apoptosis induced by SEB and “crosstalks” with the pathway of caspase-3/CPP32-like proteases activation.展开更多
Metastatic lung cancer continues to cause a high number of deaths due to high malignancy and poor prognosis,and the efficacy of typical chemotherapy or immunotherapy is less than ideal due to the low pulmonary accumul...Metastatic lung cancer continues to cause a high number of deaths due to high malignancy and poor prognosis,and the efficacy of typical chemotherapy or immunotherapy is less than ideal due to the low pulmonary accumulation and targeting of therapeutics.Here,a submicron-sized biomimetic liposome was formulated for the lung-targeted co-delivery of bacterial superantigen and paclitaxel.Recombinant staphylococcal enterotoxin C2(rSEC2),a bacterial superantigen,was expressed with the Escherichia coli system and showed potent immunostimulatory activities to mediate tumor cell death.The submicron-sized(w800 nm)biomimetic liposomes,namely 4T1 cell membrane-hybrid rSEC2 paclitaxel liposomes(TSPLs),exhibited high lung-accumulation efficiency and tumor homologous effect due to the suitable particle size and membrane hybridization of cancer cell membranes with phospholipids.Intravenous TSPLs remarkably inhibited metastatic lung cancer with limited systemic immune responses.TSPLs reversed the immunosuppressive state and increased the proportion of local CD4^(+) and CD8^(+) T cells in the lung;moreover,paclitaxel increased tumor cell apoptosis and reduced tumor burden.In summary,the high lung cancer targeting was achieved by particle size control and cell membrane hybridization,and the highly efficient anticancer effect was achieved by the co-delivery of superantigens and chemotherapeutic drugs.展开更多
Obejctive Superantigens are potent inflammatory stimuli which derive from pathogenic microbes such as bacteria, viruses and protozoa The aim of this study was to investigate the role of superantigens on the functio...Obejctive Superantigens are potent inflammatory stimuli which derive from pathogenic microbes such as bacteria, viruses and protozoa The aim of this study was to investigate the role of superantigens on the function of rabbit maxillary sinus epithelium Methods Twenty New Zealand white rabbits were divided into 4 groups Rabbit sinus mucosa was separated under a surgical microscope and mounted in Ussing chambers to record short circuit current, conductance and permeability to horseradish peroxidase (HRP) Group A was used as normal control Group B was stimulated with an injection of superantigen into the sinus for 4 hours The sinus mucosa of Group C was stimulated by the addition of tumor necrosis factor α (TNF α) into Ussing chambers Group D sinus mucosa was stimulated by superantigen after pretreatment with anti TNF α antibody Results Superantigen evoked increases in sinus epithelial cell baseline short circuit current, conductance and permeability to HRP stimulated by the addition of TNF α into Ussing chambers These were similar to results from superantigen stimulation in vivo The effect of superantigen on sinus epithelial cells could be blocked by pretreatment with anti TNF α antibody Conclusions Superantigen affected the function of sinus epithelial cells, including the capability of epithelial defensive barrier, which might be mediated by TNF α展开更多
基金Supported by the National Natural Science Foundation of China,No. 30271478
文摘AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.
文摘AIM To investigate the pathogenic effect ofSEB and D-GalN on liver and the protection ofcyclosporin A, the relationship between hepaticapoptosis and necrosis and the possiblemechanism of acute hepatic necrosis.METHODS After staphylococcal enterotoxin B(SEB ) mixed with D--galactosamine (D-GaiN )were injected intraperitoneally into Balb/c miceand those previously treated with cyclosporin A,blood samples were collected and livers wereisolated at 2, 6, 12 and 24 h. Patterns othepatocellular death were studiedmorphologically and biochemically, circulatingcytokines (TNF-a, IFN--y ) and mice mortalitywithin 24h was assessed.RESU’LTS The SEB could induce the typicalapoptotic changes of hepatocytes, the D-GaiNcould induce hepatocytes apoptosis anddegeneration at the same time, and the micehaving received the SEB + D-GaiN injectionsdeveloped apoptosis at 2 and 6 h, but after 12 hhepatocytes were characterized by severein jury, whereas all the examinations in thecyclosporin A treated mice were normal.CONCLUSION Hepatic cell apoptosis might berelated to necrosis, and massive hepatocyteapoptosis is likely the initiating step of acutehepatic necrosis in mice. The effects induced bySEB and D--GaiN on hepatocytes might bemediated by T cells, and could be prevented bycyclosporin A.
基金This study was supported in part by the JSPS KAKENHI Grant numbers 19590438(D.H.)21590475(D.H.)+1 种基金24590516(D.H.)16H05030(D.H.).
文摘Staphylococcal superantigen(SAg)toxins are the most notable virulence factors associated with Staphylococcus aureus,which is a pathogen associated with serious community and hospital acquired infections in humans and various diseases in animals.Recently,SAg toxins have become a superfamily with 29 types,including staphylococcal enterotoxins(SEs)with emetic activity,SE-like toxins(SEIs)that do not induce emesis in primate models or have yet not been tested,and toxic shock syndrome toxin-1(TSST-1).SEs and SEIs can be subdivided into classical types(SEA to SEE)and novel types(SEG to SEIY,SE01,SE02,SEI26 and SEI27).The genes of SAg toxins are located in diverse accessory genetic elements and share certain structural and biological properties.SAg toxins are heat-stable proteins that exhibit pyrogenicity,superantigenicity and capacity to induce lethal hypersensitivity to endotoxin in humans and animals.They have multiple pathogenicities that can interfere with normal immune function of host,increase the chances of survival and transmission of pathogenic baaeria in host,consequently contribute to the occurrence and development of various infeaions,persistent infeaions or food poisoning.This review focuses on the following aspeas of SAg toxins:(1)superfamily members of classic and novelty discovered staphylococcal SAgs;⑵diversity of gene locations and molecular structural characteristics;(3)biological characteristics and activities;(4)multi-pathogenicity of SAgs in animal and human diseases,including bovine mastitis,swine sepsis,abscesses and skin edema in pig,arthritis and septicemia in poultry,and nosocomial infections and food-borne diseases in humans.
文摘Objective To observe the role of superantigen staphylococcal enterotoxin B(SEB) andD - galactosamine (D - GalN) on Balb/c mouse hepatocytes and its mechanism. Methods After Balb/c mice wereinjected intraperitoneally with SEB, D- GalN or both, blood samples were collected and livers were removed at 2,6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines(TNF, IFN-γ) were determined, and mice mortality within 24h was assessed. Results SEB could induce thetypical apoptotic changes of hepatocytes morphologically and biochemically. The mechanism is probably associatedwith the production and release of Cytokines (such as TNF, IFN- γ, etc).D - GalN could induce hepatocytesapoptosis and degeneration at the same time. Besides this, we confirmed hepatocytes of the mice which wereadministered SEB and D - GalN developing apoptosis at 2, 6h, but after 12h hepatocytes were characterized bysevere injury, the mice mortality within 24h is 50%. Conclusion SEB or D - GalN alone could induce the typicalapoptotic changes of hepatocytes. SEB+D-GalN developed hepatocytes apoptosis in the early stage and necrosisin the later. It suggests that there is some relationship between hepatic cell apoptosis and necrosis, and massivehepatocyte apoptosis is the probably initiating step of acute hepatic necrosis in mice.
文摘The anti-tumor effect and mechanism of the staphylococcal enterotoxin A (SEA) were studied. The mouse gastric tumor model was produced by subcutaneously inoculating gastric tumor ceils (MGC80-3). The experimental group was treated with SEA, and the control group was treated with normal saline. The percentage of tumor generation and tumor mass was measured. The results showed that the percentage of the tumor generation in the SEA-treated mice was lower than in the control group, but there was no significant difference (P>0. 05). However, the tumor mass in the experimental group was significantly lighter than in the control group, with the difference being very significant (P<0. 001). There were more CD4+ T cells and CD8+ T cells in the tumor of the mice treated with SEA than those of the control group. SEA has an obvious anti-tumor effect on mice gastric tumor. The mechanism might be that SEA induces the effect of superantigen-dependent cell mediated cytotoxicity to the tumor cells.
文摘Microbial stimulation in early childhood may be necessary for proper maturation of the immune system. Infants colonized with Staphylococcus aureus have low risk of developing food allergy. Neonatal exposure to staphylococcal superantigen improves oral tolerance and enhances protection in experimental allergy models. Here, we used three wild-type strains of S. aureus, naturally harboring genes for different superantigens (SElM/SElO alone, or in combination with SEA or TSST-1). We first investigated their in vitro stimulatory capacity of splenocytes from germ-free mice. Secondly, germ-free mice were colonized with the strains and their capacity to develop oral tolerance was tested in a food allergy model. In vitro, S. aureus with only SElM/SElO genes promoted the strongest B-cell stimulation. S. aureus carrying gene for SEA induced the highest proportion of CD4<sup>+</sup>FoxP3<sup>+</sup> T cells. The proportion of regulatory T cells was inversely correlated to B-cell proliferation, indicating suppressive ability of these cells. All strains were equally able to colonize the germ-free gut, initially achieving 10<sup>10</sup>CFU/g faeces, which decreased to 10<sup>5</sup> over a period of six weeks. Mice colonized with S. aureus carrying genes for SEA or TSST-1 had improved capacity to develop tolerance compared to germ-free mice. These results suggest that colonization by S. aureus producing superantigens may improve active tolerance to gut allergens.
基金This study was supported by a grant from the Natural Science Foundation of China (No.30070703)
文摘Exposure of naive murine CD4 + T lymphocytes to superantigen such as staphylococcal enterotoxin B (SEB) induces a strong proliferative response. Prolonged exposure or subsequent restimulation of the responding T cell population with SEB leads to the apoptotic events of activation-induced cell death (AICD). The signaling mechanism responsible for the AICD is a target of intensive investigation. However, the precise downstream signaling pathways of SEB-induced AICD remains unclear. Our results here show that the sequential activation of caspase-1/ICE-like and caspase-3/CPP32-like cysteine proteases probably plays a role in the signaling transduction of SEB-induced AICD, but caspase-3/CPP32-like proteases activation does not depend on caspase-1-like proteases activation. Herbimycin A, a specific inhibitor of protein tyrosine kinases, inhibit caspase-3/CPP32-like cysteine proteases activation. However, it does not prevent DNA fragmentation of CD4 + T cells apoptosis induced by SEB. These results indicate that protein tyrosine kinases pathway is probably involved in the signaling transduction of CD4 + T cells apoptosis induced by SEB and “crosstalks” with the pathway of caspase-3/CPP32-like proteases activation.
基金partially supported by the Medical Innovation Research Project(23SWAQ12)National Natural Science Foundation of China(82404486)Beijing Natural Science Foundation(L232085,China).
文摘Metastatic lung cancer continues to cause a high number of deaths due to high malignancy and poor prognosis,and the efficacy of typical chemotherapy or immunotherapy is less than ideal due to the low pulmonary accumulation and targeting of therapeutics.Here,a submicron-sized biomimetic liposome was formulated for the lung-targeted co-delivery of bacterial superantigen and paclitaxel.Recombinant staphylococcal enterotoxin C2(rSEC2),a bacterial superantigen,was expressed with the Escherichia coli system and showed potent immunostimulatory activities to mediate tumor cell death.The submicron-sized(w800 nm)biomimetic liposomes,namely 4T1 cell membrane-hybrid rSEC2 paclitaxel liposomes(TSPLs),exhibited high lung-accumulation efficiency and tumor homologous effect due to the suitable particle size and membrane hybridization of cancer cell membranes with phospholipids.Intravenous TSPLs remarkably inhibited metastatic lung cancer with limited systemic immune responses.TSPLs reversed the immunosuppressive state and increased the proportion of local CD4^(+) and CD8^(+) T cells in the lung;moreover,paclitaxel increased tumor cell apoptosis and reduced tumor burden.In summary,the high lung cancer targeting was achieved by particle size control and cell membrane hybridization,and the highly efficient anticancer effect was achieved by the co-delivery of superantigens and chemotherapeutic drugs.
文摘Obejctive Superantigens are potent inflammatory stimuli which derive from pathogenic microbes such as bacteria, viruses and protozoa The aim of this study was to investigate the role of superantigens on the function of rabbit maxillary sinus epithelium Methods Twenty New Zealand white rabbits were divided into 4 groups Rabbit sinus mucosa was separated under a surgical microscope and mounted in Ussing chambers to record short circuit current, conductance and permeability to horseradish peroxidase (HRP) Group A was used as normal control Group B was stimulated with an injection of superantigen into the sinus for 4 hours The sinus mucosa of Group C was stimulated by the addition of tumor necrosis factor α (TNF α) into Ussing chambers Group D sinus mucosa was stimulated by superantigen after pretreatment with anti TNF α antibody Results Superantigen evoked increases in sinus epithelial cell baseline short circuit current, conductance and permeability to HRP stimulated by the addition of TNF α into Ussing chambers These were similar to results from superantigen stimulation in vivo The effect of superantigen on sinus epithelial cells could be blocked by pretreatment with anti TNF α antibody Conclusions Superantigen affected the function of sinus epithelial cells, including the capability of epithelial defensive barrier, which might be mediated by TNF α
