Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE...Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.展开更多
Background & Aims: Colon cancers with defective DNA mismatch repair (MMR) have peculiar molecular, pathologic, and clinical features, including high-level microsatellite instability, conspicuous lymphocytic infilt...Background & Aims: Colon cancers with defective DNA mismatch repair (MMR) have peculiar molecular, pathologic, and clinical features, including high-level microsatellite instability, conspicuous lymphocytic infiltration, preferential location in the proximal colon, and better prognosis. Our aim was to characterize the transcriptional profile of this colon cancer subset. Methods: An oligonucleotide microarray containing 12,625 probes was used to evaluate gene expression in 25 proximal colon cancers, 10 samples of normal colon mucosa, and 14 colon cancer cell lines. Transcriptional profiles of MMR-deficient cancers and cell lines were compared with those of their MMR-proficient counterparts. Results: Unsupervised anal-ysis of microarray data showed that MMR status exerts a predominant influence on the gene expression profile of proximal colon cancers. Hierarchical clustering divided the cancers into 2 groups corresponding almost perfectly with their MMR status. Supervised analysis identified numerous gene expression changes that represent a genetic signature of MMR-deficient colon cancers. Changes in genes involved in apoptosis and the immune response were consistent with the better prognosis of MMR-deficient cancers. In MMR-deficient cancers and cell lines, 4-1BBL, a crucial gene in the anti-tumor immune response, was, respectively, 2.4 and 6.0 times more expressed than in their MMR-proficient counterparts. This difference was con- firmed by quantitative reverse-transcription polymerase chain reaction and flow cytometric assessment of 4-1BBL protein expression in colon cancer cell lines. Our analysis also showed novel possible gene targets of microsatellite instability. Conclusions: MMR inactivation produces distinct changes in the cellular messenger RNA pool, which is consistent with a unique tumorigenesis pathway.展开更多
Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes （Lm） infection model, ...Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes （Lm） infection model, we demonstrate that dendritic cells （DCs） require the support of macrophages to elicit protective immunity against Lm infection. DCs themselves were inefficient at taking up Lm but capable of taking up microparticles （MPs） released by Lm-infected macrophages. These MPs transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. MP-mediated Lm antigen transfer required M HC class I participation, since M HC class I deficiency in macrophages resulted in a significant reduction of T-cell activation. Moreover, the vaccination of mice with MPs from Lm-infected macrophages produced strong protective immunity against Lm infection. We here identify an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasize that macrophages also play an essential role in DC-elicited Lm-specific T-cell responses.展开更多
文摘Objective：To express the melanoma associated gene MAGE-A9 recombinant protein, obtain the anti-MAGE-A9 monoclonal antibody and to examine the expression of MAGE-A9 in hapatocellular carcinoma specimens. Methods：MAGE-A9 cDNA was cloned from human hepatocellular carcinoma tissue by using RT-PCR, and then subcloned into the plasmid pMD18-T. After sequencing, the MAGE-A9 was cloned into the prokaryotic expression vector pBAD/gⅢ to construct the recombinant expression vector pBAD/gⅢ - MAGE-A9, and was transformed into E. coli TOP10. The recombinant MAGE-A9 protein was expressed under induction of L-Arabinose, and was purified through Hitrap column. The anti-MAGE-A9 monoclonal antibody was generated. The expression of MAGE-A9 in hepatocellular carcinoma specimens was examined through ABC assay. Results：The cDNA sequence of the cloned MAGE-A9 gene was consistent with the reported sequence. By affinity column and SDS-PAGE, the purified MAGE-A9 fusion protein displayed a band of Mr 35,000, and subsequently the anti-MAGE-A9 monoclonal antibody was obtained. We found that MAGE-A9 expressed in the cytoplast of positive cells and MAGE-A9 antigen was detected in 8 cases out of 39 （21%） hepatocellular carcinoma specimens. Conclusion：MAGE-A9 antigen was expressed in a fair proportion of hepatocellular carcinoma specimens, these patients might be suitable candidates for immune involving antigen, encoded by the MAGE-A9 gene.
文摘Background & Aims: Colon cancers with defective DNA mismatch repair (MMR) have peculiar molecular, pathologic, and clinical features, including high-level microsatellite instability, conspicuous lymphocytic infiltration, preferential location in the proximal colon, and better prognosis. Our aim was to characterize the transcriptional profile of this colon cancer subset. Methods: An oligonucleotide microarray containing 12,625 probes was used to evaluate gene expression in 25 proximal colon cancers, 10 samples of normal colon mucosa, and 14 colon cancer cell lines. Transcriptional profiles of MMR-deficient cancers and cell lines were compared with those of their MMR-proficient counterparts. Results: Unsupervised anal-ysis of microarray data showed that MMR status exerts a predominant influence on the gene expression profile of proximal colon cancers. Hierarchical clustering divided the cancers into 2 groups corresponding almost perfectly with their MMR status. Supervised analysis identified numerous gene expression changes that represent a genetic signature of MMR-deficient colon cancers. Changes in genes involved in apoptosis and the immune response were consistent with the better prognosis of MMR-deficient cancers. In MMR-deficient cancers and cell lines, 4-1BBL, a crucial gene in the anti-tumor immune response, was, respectively, 2.4 and 6.0 times more expressed than in their MMR-proficient counterparts. This difference was con- firmed by quantitative reverse-transcription polymerase chain reaction and flow cytometric assessment of 4-1BBL protein expression in colon cancer cell lines. Our analysis also showed novel possible gene targets of microsatellite instability. Conclusions: MMR inactivation produces distinct changes in the cellular messenger RNA pool, which is consistent with a unique tumorigenesis pathway.
基金This work was supported by the National Basic Research Program of China （2012CB932500）, Funds for International Cooperation and Exchange of the National Natural Science Foundation of China （30911120482）, the Program for New Century Excellent Talents in University （NCET-08-0219） and the Fundamental Research Funds for the Central Universities （HUST-2010JC024, HUST-2011TS027）.
文摘Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes （Lm） infection model, we demonstrate that dendritic cells （DCs） require the support of macrophages to elicit protective immunity against Lm infection. DCs themselves were inefficient at taking up Lm but capable of taking up microparticles （MPs） released by Lm-infected macrophages. These MPs transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. MP-mediated Lm antigen transfer required M HC class I participation, since M HC class I deficiency in macrophages resulted in a significant reduction of T-cell activation. Moreover, the vaccination of mice with MPs from Lm-infected macrophages produced strong protective immunity against Lm infection. We here identify an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasize that macrophages also play an essential role in DC-elicited Lm-specific T-cell responses.
