Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for can... more Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for cancer vaccine development. Here, we report that an adenovirus vector carrying hepatocellular carcinoma (HCC) antigen HCA661 and infected into DCs generates T-cell immunity against hepatoma cells. HCA661 is a novel cancer/testis (CT) antigen screened by SEREX from sera of an HCC patient. We constructed a recombinant adenovirus expressing the full-length cDNA of HCA661 gene and then transduced immature DCs, which had been generated with GM-CSF and IL-4 from peripheral blood mononuclear cell of HLA-A2(+) healthy donors. The resulting adenovirus-transduced DCs differentiated in the presence of monocyte-conditioned medium and poly [I] : poly [C], expressing the surface markers of mature DCs, including CD83, CD80, CD86 and HLA-DR. After maturation, the transduced DCs transcribed HCA661 mRNA and were able to prime the naïve T cells to become cytotoxic T lymphocytes (CTLs). Intracellular flow cytometry and enzyme-linked immunospot assay showed that these CTLs were able to target a hepatoma cell line, HepG2, which is HLA-A2 and HCA661 positive. In summary, we found that this recombinant adenovirus can help to induce DC maturation and these mature DCs can activate T cells to target hepatoma cells. Therefore, this recombinant adenovirus may have potential for use in liver cancer immunotherapy.
ABSTRACT The military's need to reduce both fuel and battery resupply is a real time requ... more ABSTRACT The military's need to reduce both fuel and battery resupply is a real time requirement for increasing combat effectiveness and decreasing vulnerability. Mobile photovoltaics (PV) are a technology that can address these needs by leveraging emerging, flexible space photovoltaic technology. In this ongoing project, the development and production of a semi-rigid, lightweight, efficient solar blanket with the ability to mount on, or stow in, a backpack and recharge a warfighter's battery was undertaken. The blanket consists of a 10 × 3 solar array of 20 cm2 epitaxial lift-off (ELO) solar cells. In the first two phases of the project, single-junction cells with an efficiency of ~21% under AM1.5G illumination were used. Several of these systems were outfitted during Limited Objective Experiments (LOE) in February 2012 and August 2012. In the third and most current phase of this project, the panels will be made from IMM triple-junction cells with an efficiency of 28-30% under AM1.5G illumination. The results of laboratory tests of the new prototypes, as well as a test plan and expected outcome for a field experiment are presented here.
The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program asses... more The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.
To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem ce... more To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.
Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although t... more Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although they express MHC class I molecules and ICAM-1. HCC's poor immunogenicity may therefore be due to lack of CD80 molecules. This study first investigated whether CD80 molecules could provide minimal co-stimulatory signal for establishing an efficient anti-tumor immunity in HCC and second, whether the transfection of CD80 into the BEL-7402 cell line could induce T cell activation for targeting other HCC cell lines expressing shared common antigens. The transfection of cDNA encoding CD80 into ICAM-1+ HCC BEL-7402 cells was confirmed by flow cytometrical analysis. The CD80-transfected cells could enhance the immunogenicity of BEL-7402 cells as detected by T cell proliferation assay, and also activated the T cells at a higher proliferation rate comparing with the BEL-7402 cells transfected with vector only. The CD80-transfected cell line was also found able to activate T cells which subsequently induced cell lysis of SMMC-7721, QGY-7703 and parent BEL-7402 cell lines as detected by cytotoxicity assay. It can be concluded that the cytotoxicity was due to MHC class I restricted CD8+ cytotoxic T lymphocytes, but not natural killer (NK) cells, since this cytotoxic effect could be blocked by anti-MHC class I antibody and the cytotoxicity was shown very low in NK-cell-sensitive K562 cell line. Electroporation of CD80 cDNA into human HCC cells could increase the expression of the functional CD80 molecules and enhance the immunogenicity of the genetically-modified HCC cells to activate T cells for targeting 3 HCC cell lines in an HLA-restricted manner.
Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 an... more Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 and ICAM-I molecules, all of which are essential for activation of naïve T cells. In this study, dendritomas were formed by fusion of hepatocellular carcinoma (HCC) SMMC-7721 cells with autologous DCs in vitro. DCs were obtained from adherent monocytes cultured in the presence of GM-CSF and IL-4 and were matured in monocyte-conditioned media. Expression of MHC class II and HCC-specific antigen by these dendritomas were determined using a specific murine anti-HCC monoclonal antibody (mAb) specific for HCC cell line SMMC-7721, and a murine anti-human HLA-DR mAb, and was also confirmed using bi-dimensional flow cytometry and immuno-histostaining. Dendritomas were co-cultured with autologous T cells, resulting in activation of T cell proliferation and priming of naïve T cells to induce MHC class I restricted lysis of HCC SMMC-7721 cells. The results imply that these dendritomas may have potential for use in HCC immunotherapy.
HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HC... more HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HCC). To search for immunogenic peptides of HCA661, bioinformatics analysis and CD8(+) T cell IFN-gamma ELISPOT assay were employed, and two HLA-A *0201 restricted peptides, H110 and H246, were identified. These two HCA661 peptides are naturally processed in dendritic cells (DCs) and when used for DCs loading, they are sufficient to prime autologous CD8(+) T cells to elicit cytotoxic response against HCA661(+) human cancer cells. The HCA661 peptides, H110 and H246, are hence attractive candidates for human cancer immunotherapy.
Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for can... more Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for cancer vaccine development. Here, we report that an adenovirus vector carrying hepatocellular carcinoma (HCC) antigen HCA661 and infected into DCs generates T-cell immunity against hepatoma cells. HCA661 is a novel cancer/testis (CT) antigen screened by SEREX from sera of an HCC patient. We constructed a recombinant adenovirus expressing the full-length cDNA of HCA661 gene and then transduced immature DCs, which had been generated with GM-CSF and IL-4 from peripheral blood mononuclear cell of HLA-A2(+) healthy donors. The resulting adenovirus-transduced DCs differentiated in the presence of monocyte-conditioned medium and poly [I] : poly [C], expressing the surface markers of mature DCs, including CD83, CD80, CD86 and HLA-DR. After maturation, the transduced DCs transcribed HCA661 mRNA and were able to prime the naïve T cells to become cytotoxic T lymphocytes (CTLs). Intracellular flow cytometry and enzyme-linked immunospot assay showed that these CTLs were able to target a hepatoma cell line, HepG2, which is HLA-A2 and HCA661 positive. In summary, we found that this recombinant adenovirus can help to induce DC maturation and these mature DCs can activate T cells to target hepatoma cells. Therefore, this recombinant adenovirus may have potential for use in liver cancer immunotherapy.
ABSTRACT The military's need to reduce both fuel and battery resupply is a real time requ... more ABSTRACT The military's need to reduce both fuel and battery resupply is a real time requirement for increasing combat effectiveness and decreasing vulnerability. Mobile photovoltaics (PV) are a technology that can address these needs by leveraging emerging, flexible space photovoltaic technology. In this ongoing project, the development and production of a semi-rigid, lightweight, efficient solar blanket with the ability to mount on, or stow in, a backpack and recharge a warfighter's battery was undertaken. The blanket consists of a 10 × 3 solar array of 20 cm2 epitaxial lift-off (ELO) solar cells. In the first two phases of the project, single-junction cells with an efficiency of ~21% under AM1.5G illumination were used. Several of these systems were outfitted during Limited Objective Experiments (LOE) in February 2012 and August 2012. In the third and most current phase of this project, the panels will be made from IMM triple-junction cells with an efficiency of 28-30% under AM1.5G illumination. The results of laboratory tests of the new prototypes, as well as a test plan and expected outcome for a field experiment are presented here.
The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program asses... more The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.
To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem ce... more To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.
Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although t... more Human HCC cell lines (BEL-7402, SMMC-7721 and QGY-7703) do not express CD80 molecules, although they express MHC class I molecules and ICAM-1. HCC's poor immunogenicity may therefore be due to lack of CD80 molecules. This study first investigated whether CD80 molecules could provide minimal co-stimulatory signal for establishing an efficient anti-tumor immunity in HCC and second, whether the transfection of CD80 into the BEL-7402 cell line could induce T cell activation for targeting other HCC cell lines expressing shared common antigens. The transfection of cDNA encoding CD80 into ICAM-1+ HCC BEL-7402 cells was confirmed by flow cytometrical analysis. The CD80-transfected cells could enhance the immunogenicity of BEL-7402 cells as detected by T cell proliferation assay, and also activated the T cells at a higher proliferation rate comparing with the BEL-7402 cells transfected with vector only. The CD80-transfected cell line was also found able to activate T cells which subsequently induced cell lysis of SMMC-7721, QGY-7703 and parent BEL-7402 cell lines as detected by cytotoxicity assay. It can be concluded that the cytotoxicity was due to MHC class I restricted CD8+ cytotoxic T lymphocytes, but not natural killer (NK) cells, since this cytotoxic effect could be blocked by anti-MHC class I antibody and the cytotoxicity was shown very low in NK-cell-sensitive K562 cell line. Electroporation of CD80 cDNA into human HCC cells could increase the expression of the functional CD80 molecules and enhance the immunogenicity of the genetically-modified HCC cells to activate T cells for targeting 3 HCC cell lines in an HLA-restricted manner.
Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 an... more Mature dendritic cells (DCs) have highly expressed CD1a, MHC class I, MHC class II, B7-1, B7-2 and ICAM-I molecules, all of which are essential for activation of naïve T cells. In this study, dendritomas were formed by fusion of hepatocellular carcinoma (HCC) SMMC-7721 cells with autologous DCs in vitro. DCs were obtained from adherent monocytes cultured in the presence of GM-CSF and IL-4 and were matured in monocyte-conditioned media. Expression of MHC class II and HCC-specific antigen by these dendritomas were determined using a specific murine anti-HCC monoclonal antibody (mAb) specific for HCC cell line SMMC-7721, and a murine anti-human HLA-DR mAb, and was also confirmed using bi-dimensional flow cytometry and immuno-histostaining. Dendritomas were co-cultured with autologous T cells, resulting in activation of T cell proliferation and priming of naïve T cells to induce MHC class I restricted lysis of HCC SMMC-7721 cells. The results imply that these dendritomas may have potential for use in HCC immunotherapy.
HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HC... more HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HCC). To search for immunogenic peptides of HCA661, bioinformatics analysis and CD8(+) T cell IFN-gamma ELISPOT assay were employed, and two HLA-A *0201 restricted peptides, H110 and H246, were identified. These two HCA661 peptides are naturally processed in dendritic cells (DCs) and when used for DCs loading, they are sufficient to prime autologous CD8(+) T cells to elicit cytotoxic response against HCA661(+) human cancer cells. The HCA661 peptides, H110 and H246, are hence attractive candidates for human cancer immunotherapy.
Uploads
Papers by Ray Chan