AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell li...AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.展开更多
Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and t...Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.展开更多
Background: Anti-programmed death-1/programmed death-ligand 1(PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses...Background: Anti-programmed death-1/programmed death-ligand 1(PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra-and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy.This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules.Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+)T-cell density in primary tumors and PD-1 expression on CD8(+)T cells were detected with immunofluorescence. Univariate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival.Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors(45.4% vs. 38.7%, P = 0.005); the positive rate of PD-1 on CD8(+)T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes(both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression,PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis.Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stageT1-4 N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.展开更多
A 82-year-old man presented with an enlarged multiple superficial lymph nodes. The histological diagnosis of lymph node was peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), with an aberrant expression o...A 82-year-old man presented with an enlarged multiple superficial lymph nodes. The histological diagnosis of lymph node was peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), with an aberrant expression of CD20. Generally, PTCL lacks B cell antigen such as CD19 or CD20, however, rare cases have been reported in the literature that showed PTCL patients expressing the B cell antigens. It is considered that the prognosis of CD20 positive PTCL is poor, however, standard therapy has not been established. He was treated with eight cycles of CHOP regimen, but the enlargement of a part of lymph nodes still remained. Recently, it is reported that C-C Chemokine receptor type 4 (CCR4) is known to be expressed about 50% case of PTCL and CCR4 target therapy is effective. Our case was positive for CCR4 so mogamulizumab (anti-CCR4 antibody) was administered. Consequently, dramatic response was obtained and its combination of these therapy resulted in complete remission for 24 months. This is the first case of sustained remission by administration of mogamulizumab against CCR4/CD20 double positive PTCL. This strategy may be benefit to obtain the good prognosis.展开更多
Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often ...Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.展开更多
Objective To investigate the expression of Snail in bladder urothelial carcinoma and evaluate its relationship with E-cadherin and a subset of T cell groups. Methods Immunohistochemical method was used to detect the e...Objective To investigate the expression of Snail in bladder urothelial carcinoma and evaluate its relationship with E-cadherin and a subset of T cell groups. Methods Immunohistochemical method was used to detect the expression of Snail and E-cadherin proteins in tissue展开更多
AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</s...AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.展开更多
AIM: To study host gene expression and numberof immune cells in liver tissues from patients with fulminant hepatitis E(FH-E).METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_Bead Ch...AIM: To study host gene expression and numberof immune cells in liver tissues from patients with fulminant hepatitis E(FH-E).METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_Bead Chip arrays on post-mortem liver tissue from 5 patients with FH-E,and compared with similar tissue from 6 patients with fulminant hepatitis B(FH-B; disease controls) and normal liver tissue from 6 persons.Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E,than healthy liver tissue.For some genes that showed differential expression in FH-E,microarray data were validated using quantitative reverse transcription PCR.Differentially expressed gene lists were then subjected to "Gene Ontology" analysis for biological processes,and pathway analysis using Bio Carta database on the DAVID server.In addition,tissue sections were stained for CD4+,CD8+ and CD56+ cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups.RESULTS: Compared to normal livers,those from patients with FH-E and FH-B showed differential expression of 3377 entities(up-regulated 1703,downregulated 1674) and 2572 entities(up 1164,down 1408),respectively.This included 2142(up 896,down 1246) entities that were common between the two sets; most of these belonged to metabolic,hemostatic and complement pathways,which are active in normal livers.Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways,particularly those involving cytotoxic T cells.The fold-change values of m RNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis.At immunohistochemistry,CD8+ T cells showed an increase in liver biopsies from both FH-E [median 53.4 per arbitrary unit area(range 31.2-99.9)] and FH-B [median 49.3(19.3-51.0); P = 0.005] compared to control liver tissue [median 6.9(3.1-14.9)].CONCLUSION: FH-E patients show CD8+ T cell infiltration and increased gene expression of cytotoxic T cell pathways in liver,suggesting a possible pathogenetic role for these cells.展开更多
基金Supported by National Natural Science Foundation of China, No. 30771905
文摘AIM： To investigate the effect of hepatoma cells on up-regulation of programmed cell death-1 （PD-1）, and the function of PD-1 on T cells. METHODS： HepG2 or HepG2.2.1.5 cells were cocultured with a lymphoma cell line-Jurkat cells. PD-1 expression was detected by flow cytometry. IL：2, INF-γ and IL-10 in culture supernatant were detected by enzyme-linked immunosorbent assay （ELISA）. Cytotoxic action of T cells was determined by MIF reduction assay-direct mononuclear cell cytotoxicity assay. RESULTS： The PD-1 expression on Jurkat cells increased by 16.17% ± 2.5% and 17.43% ± 2.2% after HepG2 or HepG2.2.1.5 cells were co-cultured for 48 h. The levels of IL-2, INF-γ and IL-10 in the culture supernatant were 202.9 ＋ 53.0 pg/mL, 88.6 ± 4.6 pg/mL and 63.7± 13.4 pg/mL respectively, which were significantly higher than those （102.9 ± 53 pg/mL, 39.3 ± 4.2 pg/mL, and 34.6 =E13.7 pg/mL） in the control group （P 〈 0.05）. The OD value for MTT assay in the blocking group （0.29 ± 0.06） was significantly higher than that （0.19 ± 0.09） in the control group （P 〈 0.05）. CONCLUSION： PD-1 expression on Jurkat cells is upregulated by hepatoma cells, cytokines and cytotoxic action are elevated after PD-1/PD-L1 is blocked.
基金The study was supported by National Basic Science Key Infrastructure Development Program (973 program), National Ministry of Science and Technology (Grant No: G19990559) and in part by E-institutes fund of Shanghai Municipal Education Commission. (Grant No:E03003)
文摘Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.
文摘Background: Anti-programmed death-1/programmed death-ligand 1(PD-1/PD-L1) immunotherapy has been proved to be effective on gastric cancer in ongoing clinical trials. However, the value of PD-L1 in predicting responses of patients with gastric cancer to anti-PD-1/PD-L1 immunotherapy is controversial. Some studies suggested that intra-and inter-tumoral heterogeneity of PD-L1 expression might explain the controversy.This study aimed to analyze the expression of PD-L1, PD-L2, and PD-1 as well as CD8(+) T-cell density in primary tumors and lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma to explore the heterogeneity of PD-1 signaling pathway molecules.Methods: In primary tumors and metastatic as well as non-metastatic lymph nodes from patients with stage T1-4 N+M0 gastric adenocarcinoma, we detected PD-L1 and PD-L2 expression with immunohistochemistry. CD8(+)T-cell density in primary tumors and PD-1 expression on CD8(+)T cells were detected with immunofluorescence. Univariate analysis was used to determine the prognostic values of them. Cox proportional hazard regression model was used to identify independent risk factors that affect patients' overall survival and disease-free survival.Results: Among 119 eligible patients who had undergone surgical resection, the positive rate of PD-L1 was higher in metastatic lymph nodes than in primary tumors(45.4% vs. 38.7%, P = 0.005); the positive rate of PD-1 on CD8(+)T cells was significantly higher in primary tumors and metastatic lymph nodes than in tumor-free lymph nodes(both P < 0.001). The intensity of PD-1 expression on CD8(+) T cells in primary tumors and in metastatic lymph nodes were stronger than that in tumor-free lymph nodes from the same patient. Beside, the positive rate of PD-L2 did not show any differences between primary tumors and metastatic lymph nodes. In multivariate analysis, PD-L1 expression,PD-L2 expression, a low density of CD8(+) T cells in primary tumors, and PD-1 expression on CD8(+) T cells in primary tumors were associated with poor prognosis.Conclusion: The expression of PD-L1 is heterogeneous in primary tumors and in metastatic lymph nodes from patients with stageT1-4 N+M0 gastric adenocarcinoma, which might explain the inconsistent results in assessing the prognostic value of PD-L1 expression in previous studies.
文摘A 82-year-old man presented with an enlarged multiple superficial lymph nodes. The histological diagnosis of lymph node was peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), with an aberrant expression of CD20. Generally, PTCL lacks B cell antigen such as CD19 or CD20, however, rare cases have been reported in the literature that showed PTCL patients expressing the B cell antigens. It is considered that the prognosis of CD20 positive PTCL is poor, however, standard therapy has not been established. He was treated with eight cycles of CHOP regimen, but the enlargement of a part of lymph nodes still remained. Recently, it is reported that C-C Chemokine receptor type 4 (CCR4) is known to be expressed about 50% case of PTCL and CCR4 target therapy is effective. Our case was positive for CCR4 so mogamulizumab (anti-CCR4 antibody) was administered. Consequently, dramatic response was obtained and its combination of these therapy resulted in complete remission for 24 months. This is the first case of sustained remission by administration of mogamulizumab against CCR4/CD20 double positive PTCL. This strategy may be benefit to obtain the good prognosis.
基金supported by the European Regional Development Fund(EFRE)and the German Ministry of Education and Research(BMBF 031B0831C).
文摘Chinese hamster ovary(CHO)cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications.However,toxic proteins and membrane proteins are often difficult-to-express in living cells.Alternatively,cell-free protein synthesis can be employed.This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both,cell-based and cell-free approaches.A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis.This resulted in elevated yields,while eliminating the necessity for exogenous additions during cell-free production,thereby substantially enhancing efficiency.Additionally,we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression.These findings provide promising advancements in bioproduction technologies,offering flexibility to switch between cell-free and cell-based protein production as needed.
文摘Objective To investigate the expression of Snail in bladder urothelial carcinoma and evaluate its relationship with E-cadherin and a subset of T cell groups. Methods Immunohistochemical method was used to detect the expression of Snail and E-cadherin proteins in tissue
文摘AIM To investigate the mechanisms ofsalvianolic acid A（SA-A）against liver fibrosisin vitro.METHODS NIH/3T3 fibroblasts were culturedroutinely,and incubated with 10<sup>-4</sup>mol/L-10<sup>-7</sup>mol/L SA-A for 22 h.The cell viability wasassayed by[<sup>3</sup>H]proline incorporation,cellproliferation by[<sup>3</sup>H]TdR incorporation,cellcollagen synthetic rate was measured with[<sup>3</sup>H]proline impulse and collagenase digestionmethod.The total RNA was prepared from thecontrol cells and the drug treated cellsrespectively,and α（1）I pro-collagen mRNAexpression was semi-quantitatively analyzedwith RT-PCR.RESULTS 10<sup>-4</sup>mol/L SA-A decreased cellviability and exerted some cytotoxiciy,while10<sup>-5</sup>mol/L-10<sup>-7</sup>mol/L SA-A did not affect cellviability,but inhibited cell proliferationsignificantly,and 10<sup>-6</sup>mol/L SA-A had the besteffect on cell viability among theseconcentrations of drugs.10<sup>-5</sup>mol/L-10<sup>-6</sup>mol/LSA-A inhibited intracellular collagen syntheticrate,but no significant influence on extracellularcollagen secretion.Both 10<sup>-5</sup>mol/L and10<sup>-6</sup>mol/L SA-A could decrease α（1）I pro-collagen mRNA expression remarkably.CONCLUSION SA-A had potent action againstliver fibrosis.It inhibited NIH/3T3 fibroblastproliferation,intracellular collagen syntheticrate and type I pro-collagen gene expression,which may be one of the main mechanisms of thedrug.
基金Supported by National Institutes of Health,Bethesda,United States,No.5R01AI076192
文摘AIM: To study host gene expression and numberof immune cells in liver tissues from patients with fulminant hepatitis E(FH-E).METHODS: Microarray-based expression profiling was done using Illumina Human WG-6_v3_Bead Chip arrays on post-mortem liver tissue from 5 patients with FH-E,and compared with similar tissue from 6 patients with fulminant hepatitis B(FH-B; disease controls) and normal liver tissue from 6 persons.Differential expression was defined as ≥ 2.0-fold change with Benjamini-Hochberg false discovery rate below 0.05 using t-test in liver tissue from FH-B and FH-E,than healthy liver tissue.For some genes that showed differential expression in FH-E,microarray data were validated using quantitative reverse transcription PCR.Differentially expressed gene lists were then subjected to "Gene Ontology" analysis for biological processes,and pathway analysis using Bio Carta database on the DAVID server.In addition,tissue sections were stained for CD4+,CD8+ and CD56+ cells using indirect immunohistochemistry; cells staining positive for each of these markers were counted and compared between groups.RESULTS: Compared to normal livers,those from patients with FH-E and FH-B showed differential expression of 3377 entities(up-regulated 1703,downregulated 1674) and 2572 entities(up 1164,down 1408),respectively.This included 2142(up 896,down 1246) entities that were common between the two sets; most of these belonged to metabolic,hemostatic and complement pathways,which are active in normal livers.Gene expression data from livers of patients with FH-E but not those of FH-B showed activation of several immune response pathways,particularly those involving cytotoxic T cells.The fold-change values of m RNA for selected genes in livers from FH-E than in normal liver tissue determined using quantitative reverse transcription PCR showed excellent concordance with microarray analysis.At immunohistochemistry,CD8+ T cells showed an increase in liver biopsies from both FH-E [median 53.4 per arbitrary unit area(range 31.2-99.9)] and FH-B [median 49.3(19.3-51.0); P = 0.005] compared to control liver tissue [median 6.9(3.1-14.9)].CONCLUSION: FH-E patients show CD8+ T cell infiltration and increased gene expression of cytotoxic T cell pathways in liver,suggesting a possible pathogenetic role for these cells.
