Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain co...Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay(ELISA).Therefore,an anti-GP73 antibody with high specificity was highly demanded.In the present study,by hybridoma screening,we generated an anti-GP73 monoclonal antibody(mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression.The specificity of 6A2 was evaluated by Western blotting,immunohistochemistry and immunoprecipitation.The results showed that 6A2 recognized GP73 in both native and denatured forms.In addition,we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures,and measured the serum GP73 level of patients using this assay.Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls(P = 0.0036).Furthermore,for the first time,GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls(P = 0.0172).展开更多
Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 (HPCAL1), phos...Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 (HPCAL1), phosphatidylethanolamine binding protein 1 (PEBP1), lectin galactoside-binding soluble 7 (LGALS7), and serpin peptidase inhibitor clade E member 2 (SERPINE2) as biomarkers for pancreatic cancer detection in a single assay and to investigate antibodies' specificity and cross-reactivity. Capture antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2 proteins with different concentrations were incubated with the capture antibodies at different temperatures for different time periods. Biotinylated detection antibodies recognizing a different epitope on the captured proteins and a secondary detection molecule (Streptavidin-PE) were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture antibodies was at 500 μg/mL in PBST0.05 (PBS with 0.05% Tween-20), and the slides were incubated overnight at 4°C. The lowest protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding antigen in detecting a mixture of 4 proteins without significant cross-reactivity. The fluorescence and biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential biomarker detection for pancreatic cancer.展开更多
A large subset of corneal pathologies involves the formation of new vessels(neovascularization), leading to compromised visual acuity. This article aims to review the clinical causes and presentations of corneal neova...A large subset of corneal pathologies involves the formation of new vessels(neovascularization), leading to compromised visual acuity. This article aims to review the clinical causes and presentations of corneal neovascularization(CNV) by examining the mechanisms behind common CNV-related corneal pathologies, with a particular focus on herpes simplex stromal keratitis,contact lenses-induced keratitis and CNV secondary to keratoplasty. Moreover, we reviewed CNV in the context of different types of corneal transplantation and keratoprosthesis, and summarized the most relevant treatment available so far.展开更多
The cornea is maintained in an avascular state by maintaining an environment whereby anti-angiogenic factors take the upper hand over factors promoting angiogenesis. Many of the common pathologies affecting the cornea...The cornea is maintained in an avascular state by maintaining an environment whereby anti-angiogenic factors take the upper hand over factors promoting angiogenesis. Many of the common pathologies affecting the cornea involve the disruption of such equilibrium and the shift towards new vessel formation, leading to corneal opacity and eventually-vision loss. Therefore it is of paramount importance that the molecular underpinnings of corneal neovascularization(CNV) be clearly understood, in order to develop better targeted treatments. This article is a review of the literature on the recent discoveries regarding pro-angiogenic factors of the cornea(such as vascular endothelial growth factors,fibroblast growth factor and matrix metalloproteinases)and anti-angiogenic factors of the cornea(such as endostatins and neostatins). Further, we review the molecular underpinnings of lymphangiogenesis, a process now known to be almost separate from(yet related to) hemangiogenesis.展开更多
Antibodies are primary tools in several areas of biomedical sciences,including basic research,diagnostics,and molecular therapeutics.Antibodies are widely used in diagnostic applications for clinical medicine.Analysis...Antibodies are primary tools in several areas of biomedical sciences,including basic research,diagnostics,and molecular therapeutics.Antibodies are widely used in diagnostic applications for clinical medicine.Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections.Further,antibodies are making rapid inroads into medical therapeutics,driven by technological evolution from chimeric and humanized to fully human antibodies.The therapeutic antibody market has the potential to reach $30 billion by 2010.Our lab has developed a monoclonal antibody,named Met4 that was raised against the extracellular domain of Met specifically with the goal of measuring Met in FFPE tissues.The Met receptor kinase is expressed on the cell surface of a significant number and variety of human primary solid tumors and in their metastases.The characterization of the Met4 antibody suggests it should possess adequate performance for quantification of Met expression in clinical specimens.We have also generated a fully human Fab fragment against EGFR;conjugated it to taxol as an immuno-chemotherapy agent;and investigated its in vitro anti-tumor efficacy on EGFR positive A431 epidermoid carcinoma cells.The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner;furthermore,almost 100% of cells underwent apoptosis after treatment with Fab-Taxol.Our findings suggest that this Fab-Taxol conjugate could be a potential immuno-chemotherapeutic drug for clinical treatment of EGFR over-expressing tumors.展开更多
Chromosomes in human-mouse and human-(human-mouse)hybridomas wereanalysed by G-banding methods.It was found that most human chromosomes,exceptNo.13 and X,Y,were retained.The frequencies of chromosomes No.1,3,4,5,6,17,...Chromosomes in human-mouse and human-(human-mouse)hybridomas wereanalysed by G-banding methods.It was found that most human chromosomes,exceptNo.13 and X,Y,were retained.The frequencies of chromosomes No.1,3,4,5,6,17,19,21 and 22 were higher than those of other chromosomes in each hybridoma clone.The myeloma cell lines X63-Ag8.653 and SHM-D33 were also analysed.The morphologyof marker chromosomes was apparently different between hybridomas.There were 7 kindsof marker chromosomes in human-mouse hybridomas and 16 kinds of markerchromosomes in human-(human-mouse)hybridomas.Clones that retained humanchromosome No.1 were more stable and clones that did not retain human chromosomeNo.14 were still capable of secreting human immunoglobulin.Clones that retained humanchromosome No.2 did not secret human k light chain McAb while clones that retainedhuman chromosomes No.2 and No.22 only secreted λ light chain.展开更多
Although interferonα(IFNα)and anti-angiogenesis antibodies have shown appropriate clinical benefit in the treatment of malignant cancer,they are deficient in clinical applications.Previously,we described an anti-vas...Although interferonα(IFNα)and anti-angiogenesis antibodies have shown appropriate clinical benefit in the treatment of malignant cancer,they are deficient in clinical applications.Previously,we described an anti-vascular endothelial growth factor receptor 2(VEGFR2)-IFNαfusion protein named JZA01,which showed increased in vivo half-life and reduced side effects compared with IFNα,and it was more effective than the anti-VEGFR2 antibody against tumors.However,the affinity of the IFNv component of the fusion protein for its receptor-IFNAR1 was decreased.To address this problem,an IFNα-mutant fused with anti-VEGFR2 was designed to produce anti-VEGFR2-IFNamut,which was used to target VEGFR2 with enhanced anti-tumor and anti-metastasis efficacy.Anti-VEGFR2-IFNamut specifically inhibited proliferation of tumor cells and promoted apoptosis.In addition,antiVEGFR2-IFNαmut inhibited migration of colorectal cancer cells and invasion by regulating the PI3 K-AKT-GSK3β-snail signal pathway.Anti-VEGFR-2-IFNamut showed superior anti-tumor efficacy with improved tumor microenvironment(TME)by enhancing dendritic cell maturation,dendritic cell activity,and increasing tumor-infiltrating CD8+T cells.Thus,this study provides a novel approach for the treatment of metastatic colorectal cancer,and this design may become a new approach to cancer immunotherapy.展开更多
基金supported by the Science and Technology Foundation of Nanjing Medical University (No. 07NMUZ005).
文摘Recently,serum Golgi protein 73(GP73) levels have been found to be elevated in patients with hepatocellular carcinoma(HCC),and GP73 has been proposed as a novel marker for HCC.However,GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay(ELISA).Therefore,an anti-GP73 antibody with high specificity was highly demanded.In the present study,by hybridoma screening,we generated an anti-GP73 monoclonal antibody(mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression.The specificity of 6A2 was evaluated by Western blotting,immunohistochemistry and immunoprecipitation.The results showed that 6A2 recognized GP73 in both native and denatured forms.In addition,we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures,and measured the serum GP73 level of patients using this assay.Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls(P = 0.0036).Furthermore,for the first time,GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls(P = 0.0172).
基金supported by the Lustgarten Foundation for Pancreatic Cancer Research (No. RFA-08-003)
文摘Biomarkers play an important role in the detection at an early stage of pancreatic cancer. The aim of the present study was to optimize the conditions of antibody arrays for detecting Hippocalcin-like 1 (HPCAL1), phosphatidylethanolamine binding protein 1 (PEBP1), lectin galactoside-binding soluble 7 (LGALS7), and serpin peptidase inhibitor clade E member 2 (SERPINE2) as biomarkers for pancreatic cancer detection in a single assay and to investigate antibodies' specificity and cross-reactivity. Capture antibodies against HPCAL1, PEBP1, LGALS7 and SERPINE2 were printed on nitrocellulose coated glass slides. HPCAL1, PEBP1, LGALS7 and SERPINE2 proteins with different concentrations were incubated with the capture antibodies at different temperatures for different time periods. Biotinylated detection antibodies recognizing a different epitope on the captured proteins and a secondary detection molecule (Streptavidin-PE) were used to detect fluorescent signals. The arrays showed the strongest signals when the concentration of the capture antibodies was at 500 μg/mL in PBST0.05 (PBS with 0.05% Tween-20), and the slides were incubated overnight at 4°C. The lowest protein concentration for detection was 2 ng/mL. Each antibody demonstrated high specificity to the corresponding antigen in detecting a mixture of 4 proteins without significant cross-reactivity. The fluorescence and biomarker concentration displayed a linear correlation. The antibody microarray system could be a useful tool for potential biomarker detection for pancreatic cancer.
文摘A large subset of corneal pathologies involves the formation of new vessels(neovascularization), leading to compromised visual acuity. This article aims to review the clinical causes and presentations of corneal neovascularization(CNV) by examining the mechanisms behind common CNV-related corneal pathologies, with a particular focus on herpes simplex stromal keratitis,contact lenses-induced keratitis and CNV secondary to keratoplasty. Moreover, we reviewed CNV in the context of different types of corneal transplantation and keratoprosthesis, and summarized the most relevant treatment available so far.
文摘The cornea is maintained in an avascular state by maintaining an environment whereby anti-angiogenic factors take the upper hand over factors promoting angiogenesis. Many of the common pathologies affecting the cornea involve the disruption of such equilibrium and the shift towards new vessel formation, leading to corneal opacity and eventually-vision loss. Therefore it is of paramount importance that the molecular underpinnings of corneal neovascularization(CNV) be clearly understood, in order to develop better targeted treatments. This article is a review of the literature on the recent discoveries regarding pro-angiogenic factors of the cornea(such as vascular endothelial growth factors,fibroblast growth factor and matrix metalloproteinases)and anti-angiogenic factors of the cornea(such as endostatins and neostatins). Further, we review the molecular underpinnings of lymphangiogenesis, a process now known to be almost separate from(yet related to) hemangiogenesis.
文摘Antibodies are primary tools in several areas of biomedical sciences,including basic research,diagnostics,and molecular therapeutics.Antibodies are widely used in diagnostic applications for clinical medicine.Analysis of cells and tissues in pathology laboratories includes the use of antibodies on tissue sections.Further,antibodies are making rapid inroads into medical therapeutics,driven by technological evolution from chimeric and humanized to fully human antibodies.The therapeutic antibody market has the potential to reach $30 billion by 2010.Our lab has developed a monoclonal antibody,named Met4 that was raised against the extracellular domain of Met specifically with the goal of measuring Met in FFPE tissues.The Met receptor kinase is expressed on the cell surface of a significant number and variety of human primary solid tumors and in their metastases.The characterization of the Met4 antibody suggests it should possess adequate performance for quantification of Met expression in clinical specimens.We have also generated a fully human Fab fragment against EGFR;conjugated it to taxol as an immuno-chemotherapy agent;and investigated its in vitro anti-tumor efficacy on EGFR positive A431 epidermoid carcinoma cells.The Fab-Taxol conjugate inhibited A431 cell proliferation at low concentrations and in a dose-responsive manner;furthermore,almost 100% of cells underwent apoptosis after treatment with Fab-Taxol.Our findings suggest that this Fab-Taxol conjugate could be a potential immuno-chemotherapeutic drug for clinical treatment of EGFR over-expressing tumors.
文摘Chromosomes in human-mouse and human-(human-mouse)hybridomas wereanalysed by G-banding methods.It was found that most human chromosomes,exceptNo.13 and X,Y,were retained.The frequencies of chromosomes No.1,3,4,5,6,17,19,21 and 22 were higher than those of other chromosomes in each hybridoma clone.The myeloma cell lines X63-Ag8.653 and SHM-D33 were also analysed.The morphologyof marker chromosomes was apparently different between hybridomas.There were 7 kindsof marker chromosomes in human-mouse hybridomas and 16 kinds of markerchromosomes in human-(human-mouse)hybridomas.Clones that retained humanchromosome No.1 were more stable and clones that did not retain human chromosomeNo.14 were still capable of secreting human immunoglobulin.Clones that retained humanchromosome No.2 did not secret human k light chain McAb while clones that retainedhuman chromosomes No.2 and No.22 only secreted λ light chain.
基金supported by grants from“Double First-Class”University Project(CPU2018PZQ12 and CPU2018GY14,China)National Natural Science Foundation of China(Grant 81993223)+3 种基金National Natural Science Foundation of China(Grant 81773755)Natural Science Foundation of Jiangsu Province(BK20161459,China)Postgraduate Research&Practice Innovation Program of Jiangsu Province(SJCX19_0162,China)the National Students’Platform for Innovation and Entrepreneurship Training Program(201810316010Y,China)
文摘Although interferonα(IFNα)and anti-angiogenesis antibodies have shown appropriate clinical benefit in the treatment of malignant cancer,they are deficient in clinical applications.Previously,we described an anti-vascular endothelial growth factor receptor 2(VEGFR2)-IFNαfusion protein named JZA01,which showed increased in vivo half-life and reduced side effects compared with IFNα,and it was more effective than the anti-VEGFR2 antibody against tumors.However,the affinity of the IFNv component of the fusion protein for its receptor-IFNAR1 was decreased.To address this problem,an IFNα-mutant fused with anti-VEGFR2 was designed to produce anti-VEGFR2-IFNamut,which was used to target VEGFR2 with enhanced anti-tumor and anti-metastasis efficacy.Anti-VEGFR2-IFNamut specifically inhibited proliferation of tumor cells and promoted apoptosis.In addition,antiVEGFR2-IFNαmut inhibited migration of colorectal cancer cells and invasion by regulating the PI3 K-AKT-GSK3β-snail signal pathway.Anti-VEGFR-2-IFNamut showed superior anti-tumor efficacy with improved tumor microenvironment(TME)by enhancing dendritic cell maturation,dendritic cell activity,and increasing tumor-infiltrating CD8+T cells.Thus,this study provides a novel approach for the treatment of metastatic colorectal cancer,and this design may become a new approach to cancer immunotherapy.
