The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and,...The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and, in some cases, prevent the development of the human hypernephroma in the cheek pouch of golden hamsters. The effect of enteropathgenic proteims may possibly consist in inducing the production of endogenous immune interferon which activates the host immune system and enhances the rejection of heterologous tumour cells.展开更多
In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the re...In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.展开更多
Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay ...Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.展开更多
AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in ...AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.展开更多
Predictive microbiology was utilized to model Staphylococcus aureus (S. aureus) growth and staphylococcal enterotoxin A (SEA) production in milk in this study. The modifed logistic model, modifed Gompertz model an...Predictive microbiology was utilized to model Staphylococcus aureus (S. aureus) growth and staphylococcal enterotoxin A (SEA) production in milk in this study. The modifed logistic model, modifed Gompertz model and Baranyi model were applied to model growth data of S. aureus between 15℃ and 37℃. Model comparisons indicated that Baranyi model described the growth data more accurately than two others with a mean square error of 0.0129. Growth rates generated from Baranyi model matched the observed ones with a bias factor of 0.999 and an accuracy factor of 1.01, and ft a square root model with respect to temperature; other two modifed models both overestimated the observed ones. SEA amount began to be detected when the cell number reached106.4 cfu ? mL-1, and showed the linear correlation with time. Besides, the rate of SEA production ftted an exponential relationship as a function of temperature. Predictions based on the study could be applied to indicate possible growth of S. aureus and prevent the occurrence of staphylococcal food poisoning.展开更多
Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins i...Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials;antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.展开更多
BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF...BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.展开更多
AIM To investigate the effects of Clostridium perfringens enterotoxin(CPE) on gastric cancer cells which highly expressed claudin-4(CL4) protein.METHODS In this study, we detected expression of CL4 protein in differen...AIM To investigate the effects of Clostridium perfringens enterotoxin(CPE) on gastric cancer cells which highly expressed claudin-4(CL4) protein.METHODS In this study, we detected expression of CL4 protein in different gastric cancer cell lines. Then, we investigated the effects of CPE on SGC7901 cells which highly expressed CL4 protein and the effects of CPE on sub-cutaneous tumor in nude mice models.RESULTS CL4 are highly expressed in SGC7901 cells. CPE expressedsignificant cytotoxicity in SGC7901 cells. Suppression of CL4 expression significantly decreased CPE-mediated cytotoxicity. CPE also inhibited tumor growth in subcutaneous tumor xenograft models.CONCLUSION CPE showed CL4 mediated cytotoxicity on gastric cancer cells SGC7901 and inhib-ited tumor growth in nude mice models.展开更多
This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork ...This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.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
[Objective] This study was conducted to investigate the pollution caused by staphylococcal enterotoxins(SE) in raw milk and the safety of dairy products in Jinan, and to provide a scientific basis for food safety risk...[Objective] This study was conducted to investigate the pollution caused by staphylococcal enterotoxins(SE) in raw milk and the safety of dairy products in Jinan, and to provide a scientific basis for food safety risk analysis. [Method] A total of 130 raw milk samples were collected from different regions of Jinan, and detected for Staphylococcus aureus by referring to GB4789.10-2010. Then, enzyme-linked immunosorbent assay was performed to detect whether the S.aureus strains produced enterotoxins, and the enterotoxin type was identified using colloidal gold-based immunochromatographic test strips. [Result] Fiftyseven of the raw milk samples were polluted by S.aureus, so the detection rate of S.aureus was 43.85%; and 11 of the strains produced enterotoxins. Among the 11 enterotoxin-producing strains, seven produced SEB, only one produced SEC, and the SE type of other three strains was not identified. [Conclusion] Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic test strips can be used in combination to rapidly detect staphylococcal enterotoxins and identify enterotoxin type, although there are some limitations. SEB is the main type of staphylococcal enterotoxin causing pollution in milk of Shandong Province.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
Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additional...Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additionally result in chest pain, dyspnoea, pulmonary oedema and respiratory failure. Severe exposure may be fatal and treatment relies on symptomatic support. At a cellular level, SEB up-regulates T-cell proliferation leading to a pathological inflammatory response. Deguelin, a rotenoid isolated from the African plant Mundulea sericea (Leguminosae), has been shown to reduce cellular proliferation by inhibiting the phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway. Using isolated murine splenocytes, we have demonstrated that treatment with deguelin reduces SEB inducing T cell proliferation by 60%. Deguelin treatment also decreased IL-2 and CCL2 secretion by splenocytes exposed to SEB. We demonstrate that targeting cellular proliferation can significantly reduce inflammation after SEB exposure and suggest that anti-proliferatives may have a role as potential generic medical counter measures if superantigens are used as biological weapons.展开更多
To study immune reactions and the mechanism of anergy induced by recombinant enterotoxin A (rSEA) of Staphylococcus aureus. The gene encoding SEA was cloned from standard strain of S. aureus and high efficiently expre...To study immune reactions and the mechanism of anergy induced by recombinant enterotoxin A (rSEA) of Staphylococcus aureus. The gene encoding SEA was cloned from standard strain of S. aureus and high efficiently expressed in E. coli. After immunization with purified rSEA, mice were examined for production of specific antibody, subtype of IgG, cytokine mRNA levels such as IFN-γ, IL-2 secretion and T-cell surface PD-1 expression. Results showed that high levels of specific antibodies were produced in two weeks of primary immunization shot. During this time, humoral immune reactions prevailed (IgG2a/ IgG1 【1). During the early phase, Th1 type cytokine mRNA is expressed at a higher level than Th2 type, indicating cellular immune reaction prevailed. Splen- ocyte IFN-γ secretion was significantly decreased after boosting immunization. The PD-1 expression was detected by a flow cytometry examination in the surface of T- lymphocytes which were induced by rSEA, and the expression of PD-1 molecules increased along with the number of boosting and the time after immunization.展开更多
The currently accepted model for superantigen (SAg) induced T cell activation suggests that SAg, without being processed, cross link both MHC class II, from Antigen Presenting Cells (APC), and V-β , from T-cell recep...The currently accepted model for superantigen (SAg) induced T cell activation suggests that SAg, without being processed, cross link both MHC class II, from Antigen Presenting Cells (APC), and V-β , from T-cell receptor (TCR), initi-ating nonspecific T-cell activation. This T-cell proliferation induces a massive cytokine release associated with several human diseases. It is thought that murine CD4+ T cells do not express MHC class-II molecules. However, we discov-ered that a subtype of mouse na?ve CD4+ T cells expresses MHC class II on their cell surface and that these CD4+ T cells can perform the role of both APC and T cells, able to present Staphy-lococcal enterotoxin A (SEA) to itself or neigh- boring CD4+ T cells via MHC class II, thus in-ducing massive CD4+ T cell proliferation. Treat- ment with neutralizing anti MHC class II anti-body inhibits this CD4+ T cell proliferation re-sponse. The fact that murine CD4+ T cells ex-press MHC class II offers new insight about SAg activity. Based on our findings, we propose re-vising and extending previous models for SAg induced T cell activation, altering previous models of MHC class II restriction of T cell re-sponses to SEA as well as the requirement for SAg processing.展开更多
Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines ...Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.展开更多
Objective : To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit (LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amp...Objective : To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit (LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L. Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Resuits: LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3. 36-10. 56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the preven- tion of mucosaroute evading pathogen.展开更多
文摘The effect of enterotoxins is to induce the production of endogenous IF. St. aureus enteropathogenic proteins (enterotoxins) possess an antitumour effect. After intraperitoneal inoculation, they decrease the size and, in some cases, prevent the development of the human hypernephroma in the cheek pouch of golden hamsters. The effect of enteropathgenic proteims may possibly consist in inducing the production of endogenous immune interferon which activates the host immune system and enhances the rejection of heterologous tumour cells.
文摘In this study,circular dichroism(CD)and molecular dynamics(MD)simulation were used to investigate the thermal unfolding pathway of staphylococcal enterotoxin B(SEB)at temperatures of 298–371 and 298–500 K,and the relationship between the experimental and simulation results were explored.Our computational findings on the secondary structure of SEB showed that at room temperature,the CD spectroscopic results were highly consistent with the MD results.Moreover,under heating conditions,the changing trends of helix,sheet and random coil obtained by CD spectral fitting were highly consistent with those obtained by MD.In order to gain a deeper understanding of the thermal stability mechanism of SEB,the MD trajectories were analyzed in terms of root mean square deviation(RMSD),secondary structure assignment(SSA),radius of gyration(R_(g)),free energy surfaces(FES),solvent-accessible surface area(SASA),hydrogen bonds and salt bridges.The results showed that at low heating temperature,domain Ⅰ without loops(omitting the mobile loop region)mainly relied on hydrophobic interaction to maintain its thermal stability,whereas the thermal stability of domain Ⅱ was mainly controlled by salt bridges and hydrogen bonds.Under high heating temperature conditions,the hydrophobic interactions in domain Ⅰ without loops were destroyed and the secondary structure was almost completely lost,while domain Ⅱ could still rely on salt bridges as molecular staples to barely maintain the stability of the secondary structure.These results help us to understand the thermodynamic and kinetic mechanisms that maintain the thermal stability of SEB at the molecular level,and provide a direction for establishing safer and more effective food sterilization processes.
基金This work was financially supported by Major Science and Technology Project of Yunnan Province(202302AE090022)Key Research and Development Program of Yunnan(202203AC100010)+4 种基金the National Natural Science Foundation of China(32160597,32160236,32371463)National Key Research and Development Program of China(2022YFC2601604)Cardiovascular Ultrasound Innovation Team of Yunnan Province(202305AS350021)Spring City Plan:the High-level Talent Promotion and Training Project of Kunming(2022SCP001)the second phase of“Double-First Class”Program Construction of Yunnan University.
文摘Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.
基金Supported by the National 863 Project of China,No.102-09-01-02National Natural Science Foundation of China,No.39770827
文摘AIM To prepare the conjugate of staphylococcal enterotoxin A (SEA) protein which is a bacterial SAg and the F(ab')2 fragment of mAb HAbl8 against human hepatocellular carcinoma (HCC), and identify its activity in order to use SAg in the targeting therapy of HCC.METHODS MAb HAbl8 was extracted from the abdominal dropsy of Balb/ c mice, and was purified through chromatography column SP-40HR with Fast protein liquid chromatography (FPLC) system. The F(ab')2 fragment of mAb HAb18 was prepared by papainic digestion method. The conjugate of mAb HAb18 F(ab')2fragment and SEA was prepared with chemical conjugating reagent N-succinimidyl-3-( 2-pyridyldithio) propionate (SPDP) and purified through chromatography column Superose 12with FPLC system. The molecular mass and purity of each collected peak were identified with SDS-PAGE assay. The protein content was assayed by Lowry's method. The antibody activity of HAb18 F (ab')2 against HCC in the conjugate was identified by indirect immunocytochemical ABC method, and the activity of SEA in the conjugate to activate peripheral blood mononuclear cells (PBMC) was identified with MTT assay.RESULTS The lgG mAb HAb18 was extracted,and purified successfully. Immunocytochemical staining demonstrated that it reacted with most of HHCC cells of human HCC cell line. There were two peaks in the process of purification of the prepared HAb18 F(ab)2-SEA conjugate. SDS-PAGE assay demonstrated that the molecular mass of the first peak was about 130 ku, and the second peak was the mixture of about 45 ku and a little 100 ku proteins. The immunocytochemical staining was similar in HAb18 F (ab ')2-SEAconjugate and HAb18 F (ab ')2, i.e., thecytoplasm and/or cell membranes of most HHCC cells were positively stained. The MTT assay showed that the optical absorbance (A) value at 490 nm of HAb18 F (ab')2-SEA conjugate was 0.182 ± 0.012, that of negative control was 0.033± 0.009, and there was significant difference between them ( P < 0.05).CONCLUSION SPDP is a good proteinconjugating reagent and can be used in preparing protein conjugate. The conjugate of mAb HAb18F(ab')2 fragment and SEA protein was preparedsuccessfully in present study and can be used in the experimental study of HCC targeting therapy with the conjugate of SAg and anti-HCC mAbs or their fragments.
基金Supported by"Academic Backbone"Project of Northeast Agricultural University(15XG26)the National High-level Talents Special Support Program of China
文摘Predictive microbiology was utilized to model Staphylococcus aureus (S. aureus) growth and staphylococcal enterotoxin A (SEA) production in milk in this study. The modifed logistic model, modifed Gompertz model and Baranyi model were applied to model growth data of S. aureus between 15℃ and 37℃. Model comparisons indicated that Baranyi model described the growth data more accurately than two others with a mean square error of 0.0129. Growth rates generated from Baranyi model matched the observed ones with a bias factor of 0.999 and an accuracy factor of 1.01, and ft a square root model with respect to temperature; other two modifed models both overestimated the observed ones. SEA amount began to be detected when the cell number reached106.4 cfu ? mL-1, and showed the linear correlation with time. Besides, the rate of SEA production ftted an exponential relationship as a function of temperature. Predictions based on the study could be applied to indicate possible growth of S. aureus and prevent the occurrence of staphylococcal food poisoning.
文摘Staphylococcus aureus represents a public health challenge all over the world. Therefore, this study aims to analyze the prevalence of five genes (sea, seb, sec, see and seg) encoding the staphylococcal enterotoxins in S. aureus isolated from different sources and to evaluate the association of these toxins in comparison to susceptibility towards 12 antimicrobials;antimicrobial susceptibility was conducted by disc diffusion method. Detection of staphylococcal enterotoxins was performed by PCR and the ability to express these genes was assessed among isolates by RT-PCR. The most common enterotoxin gene was sea gene (66%), followed by seb, sec, see and seg (38%, 23%, 19% and 5%) respectively. Expression of sea, seb and seg genes was variable. However, sec and see genes were not expressed by any of the tested isolates. No statistically significant association exists between (seb, sec and see) and isolation sources, while the sea was significantly associated with clinical isolates. High significant correlation was found between elevated sea expression and multidrug-resistance. Our findings indicate that the pathogenic potential of S. aureus may be greater than previously thought. This emphasizes the utmost need to implement proactive measures and more emphasis will be placed on the application of hygiene practices in hospitals to control S. aureus infection and enterotoxins production.
基金Supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education,Science and Technology,South Korea,No.NRF-2018R1D1A1B07043350
文摘BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.
基金Supported by The National Natural Science Foundation of China,No.81300268
文摘AIM To investigate the effects of Clostridium perfringens enterotoxin(CPE) on gastric cancer cells which highly expressed claudin-4(CL4) protein.METHODS In this study, we detected expression of CL4 protein in different gastric cancer cell lines. Then, we investigated the effects of CPE on SGC7901 cells which highly expressed CL4 protein and the effects of CPE on sub-cutaneous tumor in nude mice models.RESULTS CL4 are highly expressed in SGC7901 cells. CPE expressedsignificant cytotoxicity in SGC7901 cells. Suppression of CL4 expression significantly decreased CPE-mediated cytotoxicity. CPE also inhibited tumor growth in subcutaneous tumor xenograft models.CONCLUSION CPE showed CL4 mediated cytotoxicity on gastric cancer cells SGC7901 and inhib-ited tumor growth in nude mice models.
基金Development of Application Technology Project(No:2015-114)issued by Science and Technology Committee of Shanghai Municipal GovernmentNational Key Scientific Instruments Project(No:2013YQ150557)issued by Ministry of Science and Technology of the P.R.China.
文摘This study was conducted to analyze the effects of sodium nitrite,nisin,potassium sorbate,and sodium lactate against Staphylococcus aureus(S.aureus)growth and staphylococcal enterotoxins(SEs)production in cooked pork sausage by inoculating sausage samples containing preservative with an S.aureus strain producing staphylococcal enterotoxin A(SEA)and then storing them at 37℃ for 36 h.Samples were analyzed every 3 h to count the S.aureus colonies and to detect SEA.The modified Gompertz model was used to describe S.aureus growth in the samples under various conditions,and the preservatives with a significant antimicrobial effect were selected.In addition,the antimicrobial effects of the selected preservatives under various concentrations were tested.Results showed that sodium nitrite,nisin,and potassium sorbate had a weak effect against S.aureus growth and had no effect against SEA production,whereas sodium lactate could significantly inhibit S.aureus growth and SEA production.Moreover,the antimicrobial effect of sodium lactate was concentration-dependent,wherein sodium lactate concentration<12 g/kg showed no inhibitory effect,but when the concentration was increased to 24 g/kg,sodium lactate could effectively inhibit S.aureus growth and SEA production,and at 48 g/kg,sodium lactate had a significant inhibitory effect.
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[Objective] This study was conducted to investigate the pollution caused by staphylococcal enterotoxins(SE) in raw milk and the safety of dairy products in Jinan, and to provide a scientific basis for food safety risk analysis. [Method] A total of 130 raw milk samples were collected from different regions of Jinan, and detected for Staphylococcus aureus by referring to GB4789.10-2010. Then, enzyme-linked immunosorbent assay was performed to detect whether the S.aureus strains produced enterotoxins, and the enterotoxin type was identified using colloidal gold-based immunochromatographic test strips. [Result] Fiftyseven of the raw milk samples were polluted by S.aureus, so the detection rate of S.aureus was 43.85%; and 11 of the strains produced enterotoxins. Among the 11 enterotoxin-producing strains, seven produced SEB, only one produced SEC, and the SE type of other three strains was not identified. [Conclusion] Enzyme-linked immunosorbent assay and colloidal gold-based immunochromatographic test strips can be used in combination to rapidly detect staphylococcal enterotoxins and identify enterotoxin type, although there are some limitations. SEB is the main type of staphylococcal enterotoxin causing pollution in milk of Shandong Province.
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Staphylococcal Enterotoxin B (SEB) is considered a potential biological weapon. It is toxic by both inhalation and ingestion. Effects of ingestion include fever, vomiting and diarrhoea, while inhalation may additionally result in chest pain, dyspnoea, pulmonary oedema and respiratory failure. Severe exposure may be fatal and treatment relies on symptomatic support. At a cellular level, SEB up-regulates T-cell proliferation leading to a pathological inflammatory response. Deguelin, a rotenoid isolated from the African plant Mundulea sericea (Leguminosae), has been shown to reduce cellular proliferation by inhibiting the phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathway. Using isolated murine splenocytes, we have demonstrated that treatment with deguelin reduces SEB inducing T cell proliferation by 60%. Deguelin treatment also decreased IL-2 and CCL2 secretion by splenocytes exposed to SEB. We demonstrate that targeting cellular proliferation can significantly reduce inflammation after SEB exposure and suggest that anti-proliferatives may have a role as potential generic medical counter measures if superantigens are used as biological weapons.
文摘To study immune reactions and the mechanism of anergy induced by recombinant enterotoxin A (rSEA) of Staphylococcus aureus. The gene encoding SEA was cloned from standard strain of S. aureus and high efficiently expressed in E. coli. After immunization with purified rSEA, mice were examined for production of specific antibody, subtype of IgG, cytokine mRNA levels such as IFN-γ, IL-2 secretion and T-cell surface PD-1 expression. Results showed that high levels of specific antibodies were produced in two weeks of primary immunization shot. During this time, humoral immune reactions prevailed (IgG2a/ IgG1 【1). During the early phase, Th1 type cytokine mRNA is expressed at a higher level than Th2 type, indicating cellular immune reaction prevailed. Splen- ocyte IFN-γ secretion was significantly decreased after boosting immunization. The PD-1 expression was detected by a flow cytometry examination in the surface of T- lymphocytes which were induced by rSEA, and the expression of PD-1 molecules increased along with the number of boosting and the time after immunization.
文摘The currently accepted model for superantigen (SAg) induced T cell activation suggests that SAg, without being processed, cross link both MHC class II, from Antigen Presenting Cells (APC), and V-β , from T-cell receptor (TCR), initi-ating nonspecific T-cell activation. This T-cell proliferation induces a massive cytokine release associated with several human diseases. It is thought that murine CD4+ T cells do not express MHC class-II molecules. However, we discov-ered that a subtype of mouse na?ve CD4+ T cells expresses MHC class II on their cell surface and that these CD4+ T cells can perform the role of both APC and T cells, able to present Staphy-lococcal enterotoxin A (SEA) to itself or neigh- boring CD4+ T cells via MHC class II, thus in-ducing massive CD4+ T cell proliferation. Treat- ment with neutralizing anti MHC class II anti-body inhibits this CD4+ T cell proliferation re-sponse. The fact that murine CD4+ T cells ex-press MHC class II offers new insight about SAg activity. Based on our findings, we propose re-vising and extending previous models for SAg induced T cell activation, altering previous models of MHC class II restriction of T cell re-sponses to SEA as well as the requirement for SAg processing.
文摘Mucosal vaccination has been getting more and more recognition because of its compliance and low risk of spreading infectious disease by contaminated syringes used in subcutaneous immunization. However, most vaccines are unable to induce immune responses when given mucosally, and require the use of strong adjuvant for effective delivery systems. Heat-labile enterotoxin (LT) and Cholera toxin(CT) are powerful mucosal adjuvants when co-administered with soluble antigens. But high toxicity hampers their use in humans. Thanks to the fine knowledge of the structure-function relationship of LT and CT, many nontoxic or low toxic mutants have been generated, part of them retain high adjuvanticity of mucosal immunization. Among these mutants, LTS63K, LTA72R, LTR192G and CTE29H, CTE112K have been widely investigated. LTS63K and CTE112K are fully non toxic, whereas LTA72R and CTE29H are low toxic, and LTR192G is nontoxic in vitro(it remains the same toxicity as wild type LT in vivo). These mutants are extremely active as mucosal adjuvants when co-administrated with a variety of antigens in different animal models. They will be investigated more widely and deeply in the future. Some of them will be tested soon in human bodies.
基金Supported by the National Natural Science Foundation ofChina (No. 30070848)
文摘Objective : To construct plant transformation vector containing Escherichia coli heat-labile enterotoxin B subunit (LT-B) gene and generate LT-B transgenic tobacco plants. Methods: The LT-B coding sequence was amplified from pMMB68 by PCR, subcloned into middle vector pUCmT and binary vector pBI121 to obtain plant expression vector pBI-LTB, in which LT-B expression was controlled under the Cauliflower mosaic virus (CaMV) 35S promoter. The tobacco plants (Nicotiana tobacum L. Cuttivar Xanthi) were transformed by co-cultivating leaf discs method via Agrobacterium tumefaciens LBA4404 harboring the plant expression vector. The regenerated transgenic tobacco plants were selected by kanamycin and confirmed by PCR, Southern blot, Western blot and ELISA. Resuits: LT-B gene integrated in the tobacco genomic DNA and were expressed in 9 strains of transgenic tobacco plants. The yield was varied from 3. 36-10. 56 ng/mg total soluble tobacco leaf protein. Conclusion: The plant binary expression vector pBI-LTB was constructed successfully, and transgenic LT-B tobacco plants was generated, and confirmed by Southern blot. The protein LT-B expressed by engineered plants was identified by Western blot analysis and had the expected molecular weight of LT-B pentamer protein. This result is an important step close to developing an edible vaccine and supplying a mucasal immunoajuvant, which will contribute to the preven- tion of mucosaroute evading pathogen.