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Objective: To investigate the effect of CD33-targeted bi-specific and tri-specific T-cell engagers on T-cell proliferation and explore their cytotoxicity on leukemia cells. Methods: The CD33-targeted bi-specific T-cell engager (CD33-BiTE) and tri-specific T-cell engager (CD33-TriTE) expression vectors were successfully constructed and expressed through a eukaryotic cell expression system. CD33-BiTE and CD33-TriTE were purified by affinity chromatography. The effects of CD33-BiTE and CD33-TriTE on T cells were analyzed through in vitro experiments. Results: ① CD33-BiTE and CD33-TriTE were successfully constructed and purified and could compete with flow cytometry antibodies for binding to the target cells. ② After 12 days of co-culture with CD33-BiTE and CD33-TriTE, the number of human T cells were expanded to 33.89±19.46 and 81.56±23.62 folds, respectively. CD33-TriTE induced a stronger proliferation of T cells than CD33-BiTE (P<0.05) . ③ Both CD33-BiTE and CD33-TriTE induced specific dose-dependent cytotoxicity on CD33(+) leukemia cells. ④ Compared to CD33-TriTE, leukemia cells were prone to express PD-L1 when co-cultured with T cells and CD33-BiTE. CD33-TriTE induced powerful cytotoxicity on leukemia cells with high PD-L1 expression. Conclusion: CD33-BiTE and CD33-TriTE expression vectors were constructed, and fusion proteins were expressed in eukaryotic cells. Our results support the proliferative and activating effects of BiTE and TriTE on T cells. Compared to that of CD33-BiTE, CD33-TriTE induced a stronger proliferative effect on T cells and a more powerful cytotoxicity on leukemia cells with high PD-L1 expression.
Subject(s)
Humans , B7-H1 Antigen/pharmacology , Leukemia, Myeloid, Acute/metabolism , Sialic Acid Binding Ig-like Lectin 3/pharmacology , T-LymphocytesABSTRACT
Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHC II and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.
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Objective: To predict B cell and T cell epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins. Methods: The sequences of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins which were derived from Orientia tsutsugamushi were analyzed by SOPMA, DNAstar, Bcepred, ABCpred, NetMHC, NetMHCⅡ and IEDB. The 58-kDa tertiary structure model was built by MODELLER9.17. Results: The 22-kDa B-cell epitopes were located at positions 194-200, 20-26 and 143-154, whereas the T-cell epitopes were located at positions 154-174, 95-107, 17-25 and 57-65. The 47-kDa protein B-cell epitopes were at positions 413-434, 150-161 and 283-322, whereas the T-cell epitopes were located at positions 129-147, 259-267, 412-420 and 80-88. The 56-kDa protein B-cell epitopes were at positions 167-173, 410-419 and 101-108, whereas the T-cell epitopes were located at positions 88-104, 429-439, 232-240 and 194-202. The 58-kDa protein B-cell epitopes were at positions 312-317, 540-548 and 35-55, whereas the T-cell epitopes were located at positions 415-434, 66-84 and 214-230. Conclusions: We identified candidate epitopes of 22-kDa, 47-kDa, 56-kDa and 58-kDa proteins from Orientia tsutsugamushi. In the case of 58-kDa, the dominant antigen is displayed on tertiary structure by homology modeling. Our findings will help target additional recombinant antigens with strong specificity, high sensitivity, and stable expression and will aid in their isolation and purification.
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Objective To study the expression of E6 and E7 mRNA in high-risk human papillomavirus (HPV) HPV-18 and the relationship between the expression of invasive gene and cervical carcinoma. Methods A total of 119 patients with cervical cancer, cervical erosion and cervical HPV infection who were diagnosed in our hospital were selected and randomly divided into two groups: cervical cancer group (n = 58) and non-cancerous group (n = 61). Another 60 patients with uterine leiomyoma were selected as normal control group. Detection of HPV18 E6, E7 mRNA expression and invasion, migration, proliferation inhibition genes, epithelial mesenchymal transition genes and proliferation related protein content. Results The relative expression of E6 and E7 HPV-18 in cervical cancer group was significant higher than that in non-cancerous group and control group (mRNA) (P < 0.05). The content of TRAF6 and c-FLIP in invasive cervical cancer group was significantly higher than that in non-cancerous group and control group (P < 0.05). The mRNA content of CD44v6 and MMP-9 in cervical cancer group was significantly higher than that in non-cancerous group and control group (P < 0.05). The content of DEC-1, IKK16, MBP-1 in cervical cancer group was significant lower than that in non-cancerous group and control group (P < 0.05). The mRNA content of beta -catenin and Vimentin in cervical cancer group was significantly lower than that in non cancerous group and control group (P < 0.05). The proliferation related protein E2F1 of cervical cancer group was significantly lower than that of non-cancerous group and control group, Bmi-1 content was significantly higher than non-cancerous group and control group (P < 0.05). Conclusions The expression of the detection of cervical cancer in high-risk human papilloma virus HPV-18 E6 and E7 mRNA, and the invasion, migration, proliferation inhibition gene, epithelial mesenchymal transition and proliferation related gene protein content, HPV expression rate of mRNA increased with the development of cervical cancer, the expression is also enhanced. The expression has a certain correlation between the level and development of cervical cancer. Through the above indicators, the development of cervical cancer monitoring and treatment to provide important clinical guidance.
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Objective To study the regulatory effect and molecular mechanism of juglone on apoptosis of cervical cancer Hela cells. Methods Cervical cancer Hela cells were cultured and treated with different dosages of juglone (10, 20, and 40 μmol/L, respectively) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10, 20, and 40 μmol/L, respectively). Then cellular proliferative activity and the expression of JNK/c-Jun pathway molecule and apoptotic molecule in the cells were detected. Results After 6, 12, 18 and 24 h of treatment, the value for proliferative activity of cells treated with juglone was significantly lower than that of control group (P < 0.05), and the anti-proliferative effect was more significant as the treatment period and juglone dosage increased (P < 0.05). The protein expressions of Bax, CytC, Fas, FasL, Caspase-3, p-JNK and p-c-Jun in cells treated with juglone were significantly higher than those of control group (P < 0.05), and the protein expressions of Bax, CytC, Fas, FasL, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased (P < 0.05). In cells treated with 40 μmol/L juglone and SP600125, the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 were significantly lower than those of cells treated with 40 μmol/L juglone (P < 0.05), and the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 reduced more remarkably as the SP600125 dosage increased (P < 0.05). Conclusion Juglone can increase the expression of apoptotic molecules in mitochondrial pathway and death receptor pathway by activating JNK/c-Jun pathway, thus inducing apoptosis of cervical cancer cells.
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OBJECTIVE@#To study the expression of E6 and E7 mRNA in high-risk human papillomavirus (HPV) HPV-18 and the relationship between the expression of invasive gene and cervical carcinoma.@*METHODS@#A total of 119 patients with cervical cancer, cervical erosion and cervical HPV infection who were diagnosed in our hospital were selected and randomly divided into two groups: cervical cancer group (n = 58) and non-cancerous group (n = 61). Another 60 patients with uterine leiomyoma were selected as normal control group. Detection of HPV18 E6, E7 mRNA expression and invasion, migration, proliferation inhibition genes, epithelial mesenchymal transition genes and proliferation related protein content.@*RESULTS@#The relative expression of E6 and E7 HPV-18 in cervical cancer group was significant higher than that in non-cancerous group and control group (mRNA) (P < 0.05). The content of TRAF6 and c-FLIP in invasive cervical cancer group was significantly higher than that in non-cancerous group and control group (P < 0.05). The mRNA content of CD44v6 and MMP-9 in cervical cancer group was significantly higher than that in non-cancerous group and control group (P < 0.05). The content of DEC-1, IKK16, MBP-1 in cervical cancer group was significant lower than that in non-cancerous group and control group (P < 0.05). The mRNA content of beta -catenin and Vimentin in cervical cancer group was significantly lower than that in non cancerous group and control group (P < 0.05). The proliferation related protein E2F1 of cervical cancer group was significantly lower than that of non-cancerous group and control group, Bmi-1 content was significantly higher than non-cancerous group and control group (P < 0.05).@*CONCLUSIONS@#The expression of the detection of cervical cancer in high-risk human papilloma virus HPV-18 E6 and E7 mRNA, and the invasion, migration, proliferation inhibition gene, epithelial mesenchymal transition and proliferation related gene protein content, HPV expression rate of mRNA increased with the development of cervical cancer, the expression is also enhanced. The expression has a certain correlation between the level and development of cervical cancer. Through the above indicators, the development of cervical cancer monitoring and treatment to provide important clinical guidance.
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OBJECTIVE@#To study the regulatory effect and molecular mechanism of juglone on apoptosis of cervical cancer Hela cells.@*METHODS@#Cervical cancer Hela cells were cultured and treated with different dosages of juglone (10, 20, and 40 μmol/L, respectively) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10, 20, and 40 μmol/L, respectively). Then cellular proliferative activity and the expression of JNK/c-Jun pathway molecule and apoptotic molecule in the cells were detected.@*RESULTS@#After 6, 12, 18 and 24 h of treatment, the value for proliferative activity of cells treated with juglone was significantly lower than that of control group (P < 0.05), and the anti-proliferative effect was more significant as the treatment period and juglone dosage increased (P < 0.05). The protein expressions of Bax, CytC, Fas, FasL, Caspase-3, p-JNK and p-c-Jun in cells treated with juglone were significantly higher than those of control group (P < 0.05), and the protein expressions of Bax, CytC, Fas, FasL, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased (P < 0.05). In cells treated with 40 μmol/L juglone and SP600125, the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 were significantly lower than those of cells treated with 40 μmol/L juglone (P < 0.05), and the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 reduced more remarkably as the SP600125 dosage increased (P < 0.05).@*CONCLUSION@#Juglone can increase the expression of apoptotic molecules in mitochondrial pathway and death receptor pathway by activating JNK/c-Jun pathway, thus inducing apoptosis of cervical cancer cells.