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Su.77. Immune Response to T-independent Antigen Type 2 in vitro
Abstracts
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genes involved, we used transcriptional profiling to identify differences between paired antigen-positive versus their isogenic antigen-negative melanoma cell lines. A common set of transcription factor genes were identified as differentially expressed in both tumor pairs. MITF-M was confirmed as a positive regulator of melanocytic gene expression, and several new candidate genes are currently undergoing further study. Candidate transcription factors identified in the paired tumors are being further validated in 6 additional tumor lines, and are being tested by transfection for differential effects on melanocytic gene expression. Expression changes are being monitored by antibody staining, RT-PCR, and melanocyte promoter luciferase reporter assays. Our aim is to identify transcription factors capable of enhancing antigen expression and thereby increase targeting of melanomas that would otherwise escape immune destruction. doi:10.1016/j.clim.2008.03.426
Laboratory Immunology Su.76. Investigation of the Mechanism Underlying Aristolochic Acid-induced Kidney Injury Jiun Liang Chen. Chang Gung Memorial Hospital, Taoyuan, Taiwan To investigate the mechanism underlying aristolochic acid-induced kidney injury, both cell model and animal model experiments were carried out to study the in vitro and in vivo effects of aristolochic acid. Renal tubular epithelial cells (NRK-52E) were cultured and the cells viability was measured by the MTT (3-[4,5-Dimethylthiazol2-yl]-2,5-dihenyltetrazolium bromide Thiazolyl blue) assay. Apoptotic cells were identified by propidium iodide (PI) staining, TUNEL(TdT-mediated dUTP nick end labelling) assay and Annexine-V FITC (Fluoreszeinthiocyanat) staining. Aristolochic acid in the concentration above 10μg/ml could significantly reduce the viability of NRK-52E rat kidney proximal renal tubule cell. TUNEL staining demonstrated the apoptosis of renal tubule cells induced by aristolochic acid. Annexine-V FITC (Fluoreszeinthiocyanat) staining further confirmed the apoptosis of renal tubule cells induced by aristolochic acid. Moreover, aristolochic acid in the concentration above 10μg/ml could increase the expression of Bax mRNA. Pretreatment of cyclosporin A (7.5μg/ml) could reduce the apoptosis of renal tubule cells induced by aristolochic acid. Radix salviae miltiorrhizae exerts dosedependent protective effect on the aristolochic acid-induced renal tubular epithelial cells injury. The FVB mice model of aristolochic acid nephropathy confirmed the cell apoptosis of kidney proximal renal tubule cells. We have set up the FVB mice model of Chinese herb-induced tubular interstitial nephritis which could help us to screen and study the Traditional Chinese medicines in the prevention or treatment of Chinese herb-induced tubular interstitial nephritis and the early renal fibrosis. doi:10.1016/j.clim.2008.03.427
Su.77. Immune Response to T-independent Antigen Type 2 In Vitro Ekaterina Sidorova, Marina Gavrilova, Irina Chernyshova. Mechnikof Institute of Vaccines and Sera of Russian Academy of Medical Sciences, Moscow, Russian Federation To study cellular mechanisms of B cell response to Tindependent antigens type 2 (TI-2 antigens) the original in vitro model was developed. Polyvinylpirrolidon (PVP) was used as TI-2 antigen. Immune response to PVP in the cultures of splenocytes, B-1 and B-2 cells of CBA mice was investigated. Cell viability in vitro depends on cell density. CBA/N mice do not respond to TI-2 antigens, therefore, their splenocytes may be used as filler cells to create optimal cell density and to study the role of cell interactions in the immune response of CBA B lymphocytes to TI-2 antigens. Thus, CBA cells were mixed with CBA/N splenocytes in different proportions and cultivated for 4 days in 96-well plates in RPMI 1640 medium with FCS and all necessary supplements with and without PVP. The numbers of antibody- and immunoglobulin-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The addition of 5% of CBA splenocytes to CBA/N cultures induced some increase in IFC number. At the 50% ratio of CBA and CBA/ N cells the number of IFC reached that in the cultures with 100% of CBA splenocytes. It means that splenocyte interactions per se induce B cell activation. The addition of PVP induced marked increase in AFC and IFC number, i.e. primary immune response in vitro. Similar results were obtained with 50% mixtures of CBA B-1 and B-2 cells with CBA/N splenocytes. Not only specific but polyclonal B cell activation as well was detected. The data obtained point out that polyclonal activation and immune response to TI-2 antigen in mixed cell cultures depend on CBA B cells. The model described will be used for study the role of different B cell subpopulations and their interactions with other cells in the immune response to TI-2. doi:10.1016/j.clim.2008.03.428
Su.78. An Immunoregulatory Role for Cyclooxygenase-2 in Human B Lymphocytes Stimulated with CpG Oligodeoxynucleotides: Implications for Vaccination Matthew Bernard,1 Richard Phipps. 2 1University of Rochester, Rochester, NY; 2University of Rochester, Rochester, NY Optimal immunization with antigens typically requires the use of adjuvants. Only aluminum based adjuvants are routinely used in the United States. Recently, synthetic CpG oligodeoxynucleotides (ODN), similar to DNA sequences found in certain microorganisms, have shown promise as adjuvants for humans by enhancing immune responses. Since antibody responses are key effectors and indicators of vaccination, it is important to understand how CpG ODN affects human B cell antibody production. Human B cells stimulated with synthetic CpG ODN sequences displayed increased steady-state Cox-2 mRNA levels and protein expression. Both naive and memory peripheral blood B cells were found to have elevated levels of Cox-2 following
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genes involved, we used transcriptional profiling to identify differences between paired antigen-positive versus their isogenic antigen-negative melanoma cell lines. A common set of transcription factor genes were identified as differentially expressed in both tumor pairs. MITF-M was confirmed as a positive regulator of melanocytic gene expression, and several new candidate genes are currently undergoing further study. Candidate transcription factors identified in the paired tumors are being further validated in 6 additional tumor lines, and are being tested by transfection for differential effects on melanocytic gene expression. Expression changes are being monitored by antibody staining, RT-PCR, and melanocyte promoter luciferase reporter assays. Our aim is to identify transcription factors capable of enhancing antigen expression and thereby increase targeting of melanomas that would otherwise escape immune destruction. doi:10.1016/j.clim.2008.03.426
Laboratory Immunology Su.76. Investigation of the Mechanism Underlying Aristolochic Acid-induced Kidney Injury Jiun Liang Chen. Chang Gung Memorial Hospital, Taoyuan, Taiwan To investigate the mechanism underlying aristolochic acid-induced kidney injury, both cell model and animal model experiments were carried out to study the in vitro and in vivo effects of aristolochic acid. Renal tubular epithelial cells (NRK-52E) were cultured and the cells viability was measured by the MTT (3-[4,5-Dimethylthiazol2-yl]-2,5-dihenyltetrazolium bromide Thiazolyl blue) assay. Apoptotic cells were identified by propidium iodide (PI) staining, TUNEL(TdT-mediated dUTP nick end labelling) assay and Annexine-V FITC (Fluoreszeinthiocyanat) staining. Aristolochic acid in the concentration above 10μg/ml could significantly reduce the viability of NRK-52E rat kidney proximal renal tubule cell. TUNEL staining demonstrated the apoptosis of renal tubule cells induced by aristolochic acid. Annexine-V FITC (Fluoreszeinthiocyanat) staining further confirmed the apoptosis of renal tubule cells induced by aristolochic acid. Moreover, aristolochic acid in the concentration above 10μg/ml could increase the expression of Bax mRNA. Pretreatment of cyclosporin A (7.5μg/ml) could reduce the apoptosis of renal tubule cells induced by aristolochic acid. Radix salviae miltiorrhizae exerts dosedependent protective effect on the aristolochic acid-induced renal tubular epithelial cells injury. The FVB mice model of aristolochic acid nephropathy confirmed the cell apoptosis of kidney proximal renal tubule cells. We have set up the FVB mice model of Chinese herb-induced tubular interstitial nephritis which could help us to screen and study the Traditional Chinese medicines in the prevention or treatment of Chinese herb-induced tubular interstitial nephritis and the early renal fibrosis. doi:10.1016/j.clim.2008.03.427
Su.77. Immune Response to T-independent Antigen Type 2 In Vitro Ekaterina Sidorova, Marina Gavrilova, Irina Chernyshova. Mechnikof Institute of Vaccines and Sera of Russian Academy of Medical Sciences, Moscow, Russian Federation To study cellular mechanisms of B cell response to Tindependent antigens type 2 (TI-2 antigens) the original in vitro model was developed. Polyvinylpirrolidon (PVP) was used as TI-2 antigen. Immune response to PVP in the cultures of splenocytes, B-1 and B-2 cells of CBA mice was investigated. Cell viability in vitro depends on cell density. CBA/N mice do not respond to TI-2 antigens, therefore, their splenocytes may be used as filler cells to create optimal cell density and to study the role of cell interactions in the immune response of CBA B lymphocytes to TI-2 antigens. Thus, CBA cells were mixed with CBA/N splenocytes in different proportions and cultivated for 4 days in 96-well plates in RPMI 1640 medium with FCS and all necessary supplements with and without PVP. The numbers of antibody- and immunoglobulin-forming cells (AFC and IFC, respectively) were determined by ELISPOT method. The addition of 5% of CBA splenocytes to CBA/N cultures induced some increase in IFC number. At the 50% ratio of CBA and CBA/ N cells the number of IFC reached that in the cultures with 100% of CBA splenocytes. It means that splenocyte interactions per se induce B cell activation. The addition of PVP induced marked increase in AFC and IFC number, i.e. primary immune response in vitro. Similar results were obtained with 50% mixtures of CBA B-1 and B-2 cells with CBA/N splenocytes. Not only specific but polyclonal B cell activation as well was detected. The data obtained point out that polyclonal activation and immune response to TI-2 antigen in mixed cell cultures depend on CBA B cells. The model described will be used for study the role of different B cell subpopulations and their interactions with other cells in the immune response to TI-2. doi:10.1016/j.clim.2008.03.428
Su.78. An Immunoregulatory Role for Cyclooxygenase-2 in Human B Lymphocytes Stimulated with CpG Oligodeoxynucleotides: Implications for Vaccination Matthew Bernard,1 Richard Phipps. 2 1University of Rochester, Rochester, NY; 2University of Rochester, Rochester, NY Optimal immunization with antigens typically requires the use of adjuvants. Only aluminum based adjuvants are routinely used in the United States. Recently, synthetic CpG oligodeoxynucleotides (ODN), similar to DNA sequences found in certain microorganisms, have shown promise as adjuvants for humans by enhancing immune responses. Since antibody responses are key effectors and indicators of vaccination, it is important to understand how CpG ODN affects human B cell antibody production. Human B cells stimulated with synthetic CpG ODN sequences displayed increased steady-state Cox-2 mRNA levels and protein expression. Both naive and memory peripheral blood B cells were found to have elevated levels of Cox-2 following