Methylglyoxal (MGO) is a vital signaling molecule that related to a variety of pathologies in both animals and plants. However, high levels of MGO are associated with several diseases. Therefore, developing a sensitiv...Methylglyoxal (MGO) is a vital signaling molecule that related to a variety of pathologies in both animals and plants. However, high levels of MGO are associated with several diseases. Therefore, developing a sensitive method for monitoring MGO levels in vivo and investigating its molecular mechanism is of great importance. Although most of the reported MGO fluorescence probes are designed for cells and animals, none have been used for study MGO levels in plants. Consequently, we herein report a fluorescent probe named CPDN, which is rational constructed utilizing coumarin derivatives and O-phenylenediamine as the fluorophore and the recognition group, respectively. In our study, CPDN have shown ability to selectively and sensitively detect MGO in solution and has been successfully exploited for imaging endogenous and exogenous MGO levels in living cells, zebrafish and Arabidopsis thaliana. Surprisingly, further investigation of CPDN has found that high MGO levels in Arabidopsis thaliana could inhibit the root growth. Moreover, it is demonstrated that the MGO levels in Arabidopsis thaliana increased when subjected to drought stress, which may be the main cause inhibiting root development and resulting in shorter root length. Therefore, the probe CPDN can be a powerful tool for studying the MGO levels under abiotic stress conditions and exploring its role in plant growth mechanisms. We believe that the application of CPDN in monitoring MGO levels in plants holds great values for deepening the understanding of plant growth mechanisms.展开更多
Shigellosis causes diarrheal disease in humans in both developed and developing countries, and multi-drug resistance in Shigella is an emerging problem. Understanding changing resistance patterns is important in deter...Shigellosis causes diarrheal disease in humans in both developed and developing countries, and multi-drug resistance in Shigella is an emerging problem. Understanding changing resistance patterns is important in determining appropriate antibiotic treatments. This meta-analysis systematically evaluated aminoglycoside resistance in Shigella. A systematic review was constructed based on MEDLINE and EMBASE databases. Randomeffect models or fixed-effect models were used based on P value considering the possibility of heterogeneity between studies for meta-analysis. Data manipulation and statistical analyses were performed using software STATA 11.0. By means of meta-analysis, we found a lower resistance to three kinds of aminoglycosides in the Europe-America areas during the 12 year study period than that of the Asia-Africa areas. Kanamycin resistance was observed to be the most common drug resistance among Shigella isolates with a prevalence of 6.88% （95%CI： 6.36%-7.43%）. Comparison of data from Europe-America and Asia-Africa areas revealed that Shigella flexneri resistance was greater than the resistance calculated for Shigella sonnei. Importantly, Shigella sonnei has played a significant role in aminoglycoside-resistance in recent years. Similarly, data showed that resistance to these drugs in children was higher than the corresponding data of adults. In conclusion, aminoglycoside-resistant Shigella is not an unusual phenomenon worldwide. Distribution in Shigella resistance differs sharply based on geographic areas, periods of time and subtypes. The results from the present study highlight the need for con- tinuous surveillance of resistance and control of antibiotic usage.展开更多
Apoptosis plays an important role in the pathogenesis of viral infections.In this study,we investigated the cell death processes during productive HHV-6A infection and the underlying mechanisms.Annexin V-PI staining a...Apoptosis plays an important role in the pathogenesis of viral infections.In this study,we investigated the cell death processes during productive HHV-6A infection and the underlying mechanisms.Annexin V-PI staining and electron microscopy indicated that HHV-6A is a strong inducer of apoptosis.HHV-6A infection decreased mitochondrial transmembrane potential and led to morphological changes of mitochondria.The cell death was associated with activation of caspase-3 and cleavage of DNA repair enzyme poly （ADP-ribose） polymerase,which is known to be an important substrate for activated caspase-3.Caspase-9 was activated significantly in HHV-6A-infected cells,whereas caspase-8 was not activated obviously.Moreover,HHV-6A infection upregulated Bax and downregulated Bcl-2.This is the first demonstration of mitochondrion-mediated,caspase-dependent apoptosis in HHV-6A-infected cells.展开更多
Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell t...Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.展开更多
Epstein-Barr virus infection is strongly associated with a number of malignancies.The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignanci...Epstein-Barr virus infection is strongly associated with a number of malignancies.The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies.In previous studies,the T cell epitopes of LMP2A have been identified systematically.However,the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly,which could be overcome by combination with appropriate adjuvants.Heat shock protein is a natural chaperon,which is able to activate the classical major histocompatibility complex class I antigen-processing pathway(cross-presentation).In this study,a minigene encoding LMP2A356-364(FLYALALLL)was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70.The epitope fusion protein was expressed and purified,and the cross-presentation of LMP2A_(356-364) by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated.Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation.Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A_(356-364) generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant(IFA).Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups.Our results suggested that MtHsp70-LMP2A_(356-364) fusion protein was more effective than the CD8^(+)T cell epitope alone on anti-tumor immunity.As a result,the MtHsp70-LMP2A_(356-364) fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.展开更多
Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an i...Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.展开更多
Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor de...Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.展开更多
基金support from the National Natural Science Foundation of China(No:21804102)the Open Project of Key Laboratory of Novel Biomass-Based Environmental and Energy Materials in Petroleum and Chemical Industry(No:BEEA0001)+1 种基金the Opening Project of Hubei Key Laboratory of Purification and Application of Plant Anti-cancer Active Ingredients(No:HLPAI2023011)the Graduate Innovative Fund of Wuhan Institute of Technology(No:CX2022429).
文摘Methylglyoxal (MGO) is a vital signaling molecule that related to a variety of pathologies in both animals and plants. However, high levels of MGO are associated with several diseases. Therefore, developing a sensitive method for monitoring MGO levels in vivo and investigating its molecular mechanism is of great importance. Although most of the reported MGO fluorescence probes are designed for cells and animals, none have been used for study MGO levels in plants. Consequently, we herein report a fluorescent probe named CPDN, which is rational constructed utilizing coumarin derivatives and O-phenylenediamine as the fluorophore and the recognition group, respectively. In our study, CPDN have shown ability to selectively and sensitively detect MGO in solution and has been successfully exploited for imaging endogenous and exogenous MGO levels in living cells, zebrafish and Arabidopsis thaliana. Surprisingly, further investigation of CPDN has found that high MGO levels in Arabidopsis thaliana could inhibit the root growth. Moreover, it is demonstrated that the MGO levels in Arabidopsis thaliana increased when subjected to drought stress, which may be the main cause inhibiting root development and resulting in shorter root length. Therefore, the probe CPDN can be a powerful tool for studying the MGO levels under abiotic stress conditions and exploring its role in plant growth mechanisms. We believe that the application of CPDN in monitoring MGO levels in plants holds great values for deepening the understanding of plant growth mechanisms.
基金funded by National Natural Science Foundation of China (No. 81000754)a grant from the Key Laboratory for Laboratory Medicine of Jiangsu Province of China (No. XK201114)a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions
文摘Shigellosis causes diarrheal disease in humans in both developed and developing countries, and multi-drug resistance in Shigella is an emerging problem. Understanding changing resistance patterns is important in determining appropriate antibiotic treatments. This meta-analysis systematically evaluated aminoglycoside resistance in Shigella. A systematic review was constructed based on MEDLINE and EMBASE databases. Randomeffect models or fixed-effect models were used based on P value considering the possibility of heterogeneity between studies for meta-analysis. Data manipulation and statistical analyses were performed using software STATA 11.0. By means of meta-analysis, we found a lower resistance to three kinds of aminoglycosides in the Europe-America areas during the 12 year study period than that of the Asia-Africa areas. Kanamycin resistance was observed to be the most common drug resistance among Shigella isolates with a prevalence of 6.88% （95%CI： 6.36%-7.43%）. Comparison of data from Europe-America and Asia-Africa areas revealed that Shigella flexneri resistance was greater than the resistance calculated for Shigella sonnei. Importantly, Shigella sonnei has played a significant role in aminoglycoside-resistance in recent years. Similarly, data showed that resistance to these drugs in children was higher than the corresponding data of adults. In conclusion, aminoglycoside-resistant Shigella is not an unusual phenomenon worldwide. Distribution in Shigella resistance differs sharply based on geographic areas, periods of time and subtypes. The results from the present study highlight the need for con- tinuous surveillance of resistance and control of antibiotic usage.
基金supported by the National Natural Science Foundationof China (No. 30771961 and No. 30901344)Science Development Foundation of Nanjing Medical University (No. 08NMUZ003)Jiangsu Province Laboratory of Pathogen Biology (No. 08bykf01)
文摘Apoptosis plays an important role in the pathogenesis of viral infections.In this study,we investigated the cell death processes during productive HHV-6A infection and the underlying mechanisms.Annexin V-PI staining and electron microscopy indicated that HHV-6A is a strong inducer of apoptosis.HHV-6A infection decreased mitochondrial transmembrane potential and led to morphological changes of mitochondria.The cell death was associated with activation of caspase-3 and cleavage of DNA repair enzyme poly （ADP-ribose） polymerase,which is known to be an important substrate for activated caspase-3.Caspase-9 was activated significantly in HHV-6A-infected cells,whereas caspase-8 was not activated obviously.Moreover,HHV-6A infection upregulated Bax and downregulated Bcl-2.This is the first demonstration of mitochondrion-mediated,caspase-dependent apoptosis in HHV-6A-infected cells.
基金supported by the National Nature Science Foundation of China (No.30571715)
文摘Epstein-Barr virus （EBV） associated nasopharyngeal carcinoma （NPC） is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A （LMP2A） is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three （LMP2A264.272, LMP2A426-434 and LMP2A3s6.364） of six peptides respectively showed that the numbers of spots forming cells （SFC） ranged from 55.7 to 80.6 SFC/5 x 104 CO8^＋ T cells and the responding index （RI） ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2- expressing target cells. As a result, LMP2A264.272 （QLSPLLGAV）, LMP2A426.434 （CLGGLLTMV） and LMP2A356.364 （FLYALALLL） were identified as LMP2A-specific CD8^＋ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC.
基金This work was funded by the National Natural Science Foundation of China(No.30170880 and No.30571715)Science Committee Foundation of Jiangsu Province(No.BJ98100)Project of Key Laboratory of Laboratory Diagnosis of Jiangsu Province(No.XK200731).
文摘Epstein-Barr virus infection is strongly associated with a number of malignancies.The EBV latent membrane protein 2A has been implicated as one of the most attractive candidates for immunotherapy of related malignancies.In previous studies,the T cell epitopes of LMP2A have been identified systematically.However,the epitope-based vaccine generally meets inefficient immunogenicity when used in vivo directly,which could be overcome by combination with appropriate adjuvants.Heat shock protein is a natural chaperon,which is able to activate the classical major histocompatibility complex class I antigen-processing pathway(cross-presentation).In this study,a minigene encoding LMP2A356-364(FLYALALLL)was genetically fused to the carboxy-terminal of mycobacterial heat shock protein 70.The epitope fusion protein was expressed and purified,and the cross-presentation of LMP2A_(356-364) by monocyte-derived dendritic cells pulsed with the epitope fusion protein was evaluated.Results showed that the epitope fusion protein-pulsed mDCs were much more efficient than the single peptide-pulsed mDCs on CTL activation.Immunization of HLA-A2.1 transgenic mice with MtHsp70-LMP2A_(356-364) generated peptide specific CTL more effectively than a single peptide plus incomplete Freund's adjuvant(IFA).Growth of LMP2A expressing B16 melanoma tumor cells was suppressed in the vaccinated groups.Our results suggested that MtHsp70-LMP2A_(356-364) fusion protein was more effective than the CD8^(+)T cell epitope alone on anti-tumor immunity.As a result,the MtHsp70-LMP2A_(356-364) fusion protein is considered to be a promising candidate vaccine for EBV related malignancies.
基金Acknowledgements We thank Professor Xueguang Zhang （Soochow University, Suzhou, China） for kindly providing plasmid pGEZ, pHIT456, pHIT60 and 293T cell lines. This work was supported by the National Natural Science Foundation of China （No. 30772003 and No. 30170880）, Jiangsu Province＇s Outstanding Medical Academic Leader programme （RC2007057） and Science Development Foundation of Nanjing Medical University （NMUZ009）.
文摘Type Ⅱ Epstein-Barr virus （EBV） associated malignancies such as nasopharyngeal carcinoma and non-Hodgkin＇s lymphomas consistently express latent membrane 2A （LMP2A） proteins, which have been suggested to be an ideal target for immunotherapy. In previous studies we have demonstrated that using LMP2A protein loaded dendritic cells, the most powerful antigen processing cells in the body can elicit specific and robust anti-tumor cellular immune response in vitro. In this paper, we further investigated the T cell profile of the anti-tumor immune response. We found that LMP2A specific CD4＋ and CD8＋ T cells could be stimulated by LMP2A protein loaded dendritic cells （DCs）. The Thl type immune response is dominant in the immune response mediated by LMP2A specific CD4^＋ T cells. The CD8^＋ cytotoxic T cells can lyse LMP2A bearing cells effectively and specifically. The CD8^＋ cytotoxic T cells can also secrete high level of intracellular IFN-γ, which indicates these cells are EBV-LMP2A specific cytotoxic T cells. Altogether, our studies proved that LMP2A protein loaded DCs can elicit anti-tumor cellular immune responses efficiently. This study provides a rationale for the DC-based immunotherapy against EBV-LMP2A expressing malignancies.
基金the National Nature Science Foundation of China (No. 30170880 and No. 30571715)Science Committee Foundation of Jiangsu Province (No. BJ98100)Health Department Foundation of Jiangsu Province (No. H200336)
文摘Epstein-Barr virus （EBV）, a potential oncogenic herpesvirus, has been found to be associated with several malignancies. It＇s critical to elicit cellular immunity of the body to fight against EBV-associated tumor development. Using dendritic cells （DCs） loaded with latent membrane protein 2A （LMP2A） to elicit T cell response against tumor may be one of the most direct and safest immunotherapy approaches. The present study aimed to develop DCs-based cancer vaccine （DC loaded with LMP2A protein） and study its biological characteristics and immune functions. Purified LMP2A protein was extracted from a cell line L929/LMP2A stably expressing LMP2A. LMP2A could be loaded on DCs with no significant changes of the DC surface markers and cytomorphology. The percentage of DCs loaded with LMP2A was above 80%. LMP2A-loaded DCs markedly enhanced the proliferation of antigen-specific CD8^＋ T and CD4^＋ T cells by 3H-TdR incorporation assay. Besides, the specific cytotoxicity of the CTLs against LMP2A target cells was also significantly increased. These results indicated that DC-based vaccine loaded with virus antigen could elicit potent CTL response and provide a foundation for further study on the DC-based immunotherapy for nasopharygeal carcinoma and other EBV associated tumors. Cellular ＆ Molecular Immunology.
