Progress 11/01/05 to 10/31/10
Outputs
OUTPUTS: All chickens can be infected with MDV and MDV-neoplastically transformed cells occur in all chickens. However, some chickens are genetically resistant to MD lymphoma formation. Our objective is to identify genetic mechanisms responsible for the differences between MD susceptible and resistant chickens at the time of resistance to lymphoma development when MD lymphoma progressor and regressor chicken genotypes diverge which is ~21 days post infection (dpi) - in resistant chickens microscopic lymphoma lesions regress, whereas in susceptible chickens these lesions progress to gross lymphomas. We hypothesized that in resistant chickens the tissue or lymphoma environment is compatible with T cell immunity but in susceptible lines it is not. To test this hypothesis we used the B2 non-MHC-associated MD resistance/susceptibility (line [L]61/line [L]72) system and quantified the levels of key mRNAs. We measured gene expression in both whole tissues and, after tissue sectioning and laser capture micro-dissection, in the MD lesions themselves. Gene ontology-based modeling of our results suggested that overall environment in whole lymphomas as well as in microscopic lymphoma lesions in both L61 and L72 is pro T-regulatory cells (T-reg). But there are also pro T-helper (Th)-1 and anti Th-2 effects in L61 compatible with cell-mediated immunity. In contrast, L72 had anti Th1 and pro Th2 effects. The environment within the microscopic tumor lesion was pro T-reg, anti Th1 and pro Th2 in both L61 and L72. The predominance of pro T-reg phenotype in both L61 and L72 microscopic tumour lesions and the absence of pro Th1 phenotype suggest that the MD lesions and the transformation event is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity. In addition, Functional differences in expression of the host tumor antigen (CD30) and in the MDV "meq" oncogene promoter. Using PCR we have cloned 2.5 Kb 5' of the CD30 gene ATG (i.e. the gene promoter) from 5 MD-susceptible and 2 MD-resistant chicken lines. We have sequenced all of these and have identified polymorphisms many of which are in potential Meq binding sequences and phyogenetics shows that CD30 promoter polymorphisms exactly match chicken breeding history. We have completed functional assays to identify whether or not the polymorphisms affect the ability of MDV meq to transactivate the CD30 promoter. Transcription increased from the CD30 promoters of MD-susceptible, but decreased from MD-resistant, genotypes. To analyze functionality of the NF-κB binding site in the Meq promoter, we cloned the three main NF-κB isoforms (P65, P100, and P105) in an expression plasmid and the Meq promoter in reporter plasmid and did transcription assays as above. All NF-κBs stimulate transcription from Meq promoter not equally; the Meq oncoprotein itself further enhanced mRNA expression. We suggest that a positive feed forward loop exists between CD30 and Meq, similar to the LMP-1/CD30 system in Epstein Barr virus, and that perturbation of the CD30 system is highly evolutionarily conserved in virus induced lymphoma. PARTICIPANTS: SC Burgess. PD JJ Buza. Postdoctoral Researcher LA Shack, Research Associate. D Kunec, Research Associate. TARGET AUDIENCES: Vaccine companies, poultry breeding companies, biomedical researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work provides fundamental knowledge for maintaining the USA's poultry industry's competitiveness by as it aims to control Marek's Disease, one of the most economically-important diseases of poultry. This work is positively impacting our understanding of MD lymphomagenesis and for decreasing the expensive reliance on MDV vaccines to control MD tumors. We aim to identify potential novel measures for MD control by identifying genetic mechanisms to do so. Our work has also been important in developing novel proteomics and bioinformatics approaches and for annotating the chicken genome.
Publications
- D. Kunec S.C. Burgess. 2007. DNA sequence for predicting resistance to Marek's disease in chickens. USA Patent office, patent pending.
|
Progress 11/01/08 to 10/31/09
Outputs
OUTPUTS: All chickens can be infected with MDV and MDV-neoplastically transformed cells occur in all chickens. However, some chickens are genetically resistant to MD lymphoma formation. Our objective is to identify genetic mechanisms responsible for the differences between MD susceptible and resistant chickens at the time of resistance to lymphoma development when MD lymphoma progressor and regressor chicken genotypes diverge which is ~21 days post infection (dpi) - in resistant chickens microscopic lymphoma lesions regress, whereas in susceptible chickens these lesions progress to gross lymphomas. We hypothesized that in resistant chickens the tissue or lymphoma environment is compatible with T cell immunity but in susceptible lines it is not. To test this hypothesis we used the B2 non-MHC-associated MD resistance/susceptibility (line [L]61/line [L]72) system and quantified the levels of key mRNAs. We measured gene expression in both whole tissues and, after tissue sectioning and laser capture micro-dissection, in the MD lesions themselves. Gene ontology-based modeling of our results suggested that overall environment in whole lymphomas as well as in microscopic lymphoma lesions in both L61 and L72 is pro T-regulatory cells (T-reg). But there are also pro T-helper (Th)-1 and anti Th-2 effects in L61 compatible with cell-mediated immunity. In contrast, L72 had anti Th1 and pro Th2 effects. The environment within the microscopic tumor lesion was pro T-reg, anti Th1 and pro Th2 in both L61 and L72. The predominance of pro T-reg phenotype in both L61 and L72 microscopic tumour lesions and the absence of pro Th1 phenotype suggest that the MD lesions and the transformation event is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity. In addition, Functional differences in expression of the host tumor antigen (CD30) and in the MDV "meq" oncogene promoter. Using PCR we have cloned 2.5 Kb 5' of the CD30 gene ATG (i.e. the gene promoter) from 5 MD-susceptible and 2 MD-resistant chicken lines. We have sequenced all of these and have identified polymorphisms many of which are in potential Meq binding sequences and phyogenetics shows that CD30 promoter polymorphisms exactly match chicken breeding history. We have completed functional assays to identify whether or not the polymorphisms affect the ability of MDV meq to transactivate the CD30 promoter. Transcription increased from the CD30 promoters of MD-susceptible, but decreased from MD-resistant, genotypes. To analyze functionality of the NF-κB binding site in the Meq promoter, we cloned the three main NF-κB isoforms (P65, P100, and P105) in an expression plasmid and the Meq promoter in reporter plasmid and did transcription assays as above. All NF-κBs stimulate transcription from Meq promoter not equally; the Meq oncoprotein itself further enhanced mRNA expression. We suggest that a positive feed forward loop exists between CD30 and Meq, similar to the LMP-1/CD30 system in Epstein Barr virus, and that perturbation of the CD30 system is highly evolutionarily conserved in virus induced lymphoma. PARTICIPANTS: SC Burgess. PD JJ Buza. Postdoctoral Researcher LA Shack, Research Associate. D Kunec, Research Associate. Kumar, S. PhD Student. TARGET AUDIENCES: Vaccine companies, poultry breeding companies, biomedical researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work provides fundamental knowledge for maintaining the USA's poultry industry's competitiveness by as it aims to control Marek's Disease, one of the most economically-important diseases of poultry. This work is positively impacting our understanding of MD lymphomagenesis and for decreasing the expensive reliance on MDV vaccines to control MD tumors. We aim to identify potential novel measures for MD control by identifying genetic mechanisms to do so. Our work has also been important in developing novel proteomics and bioinformatics approaches and for annotating the chicken genome.
Publications
- Parvizi, P., L. R. Read, M. F. Abdul-Careem, A. J. Sarson, C. Lusty, M. Lambourne, N. Thanthrige-Don, S. C. Burgess*, and S. Sharif*. 2009. Cytokine gene expression in splenic CD4+ and CD8+ T cell subsets of genetically resistant and susceptible chickens infected with Marek's disease virus. Vet Immunol Immunopathol 132:209-17.
- Kumar, S., J. J. Buza, and S. C. Burgess. 2009. Genotype-Dependent Tumor Regression in Marek's Disease Mediated at the Level of Tumor Immunity. Cancer Microenviron 2:23-31.
|
Progress 11/01/07 to 10/31/08
Outputs
OUTPUTS: All chickens can be infected with MDV and MDV-neoplastically transformed cells occur in all chickens. However, some chickens are genetically resistant to MD lymphoma formation. Our objective is to identify genetic mechanisms responsible for the differences between MD susceptible and resistant chickens at the time of resistance to lymphoma development when MD lymphoma progressor and regressor chicken genotypes diverge which is ~21 days post infection (dpi) - in resistant chickens microscopic lymphoma lesions regress, whereas in susceptible chickens these lesions progress to gross lymphomas. We hypothesized that in resistant chickens the tissue or lymphoma environment is compatible with T cell immunity but in susceptible lines it is not. To test this hypothesis we used the B2 non-MHC-associated MD resistance/susceptibility (line [L]61/line [L]72) system and quantified the levels of key mRNAs. We measured gene expression in both whole tissues and, after tissue sectioning and laser capture micro-dissection, in the MD lesions themselves. Gene ontology-based modeling of our results suggested that overall environment in whole lymphomas as well as in microscopic lymphoma lesions in both L61 and L72 is pro T-regulatory cells (T-reg). But there are also pro T-helper (Th)-1 and anti Th-2 effects in L61 compatible with cell-mediated immunity. In contrast, L72 had anti Th1 and pro Th2 effects. The environment within the microscopic tumor lesion was pro T-reg, anti Th1 and pro Th2 in both L61 and L72. The predominance of pro T-reg phenotype in both L61 and L72 microscopic tumour lesions and the absence of pro Th1 phenotype suggest that the MD lesions and the transformation event is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity. In addition, Functional differences in expression of the host tumor antigen (CD30) and in the MDV "meq" oncogene promoter. Using PCR we have cloned 2.5 Kb 5' of the CD30 gene ATG (i.e. the gene promoter) from 5 MD-susceptible and 2 MD-resistant chicken lines. We have sequenced all of these and have identified polymorphisms many of which are in potential Meq binding sequences and phyogenetics shows that CD30 promoter polymorphisms exactly match chicken breeding history. We have completed functional assays to identify whether or not the polymorphisms affect the ability of MDV meq to transactivate the CD30 promoter. Transcription increased from the CD30 promoters of MD-susceptible, but decreased from MD-resistant, genotypes. To analyze functionality of the NF-κB binding site in the Meq promoter, we cloned the three main NF-κB isoforms (P65, P100, and P105) in an expression plasmid and the Meq promoter in reporter plasmid and did transcription assays as above. All NF-κBs stimulate transcription from Meq promoter not equally; the Meq oncoprotein itself further enhanced mRNA expression. We suggest that a positive feed forward loop exists between CD30 and Meq, similar to the LMP-1/CD30 system in Epstein Barr virus, and that perturbation of the CD30 system is highly evolutionarily conserved in virus induced lymphoma. PARTICIPANTS: SC Burgess. PD JJ Buza. Postdoctoral Researcher LA Shack, Research Associate. D Kunec, Research Associate. TARGET AUDIENCES: Vaccine companies, poultry breeding companies, biomedical researchers PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This work provides fundamental knowledge for maintaining the USA's poultry industry's competitiveness by as it aims to control Marek's Disease, one of the most economically-important diseases of poultry. This work is positively impacting our understanding of MD lymphomagenesis and for decreasing the expensive reliance on MDV vaccines to control MD tumors. We aim to identify potential novel measures for MD control by identifying genetic mechanisms to do so. Our work has also been important in developing novel proteomics and bioinformatics approaches and for annotating the chicken genome.
Publications
- D. Kunec S.C. Burgess. 2007. DNA sequence for predicting resistance to Marek's disease in chickens. USA Patent office, patent pending.
- Shyamesh Kumar, Joram J. Buza and Shane C. Burgess**. 2009. Genotype dependent tumor regression in Mareks Disease is mediated at the level of tumor immunity. Cancer Microenvironment. In press.
|
Progress 11/01/06 to 10/31/07
Outputs
OUTPUTS: All chickens can be infected with MDV and MDV-neoplastically transformed cells occur in all chickens. However, some chickens are genetically resistant to MD lymphoma formation. Our objective is to identify genetic mechanisms responsible for the differences between MD susceptible and resistant chickens at the time of resistance to lymphoma development when MD lymphoma progressor and regressor chicken genotypes diverge which is ~21 days post infection (dpi) - in resistant chickens microscopic lymphoma lesions regress, whereas in susceptible chickens these lesions progress to gross lymphomas. We hypothesized that in resistant chickens the tissue or lymphoma environment is compatible with T cell immunity but in susceptible lines it is not. To test this hypothesis we used the B2 non-MHC-associated MD resistance/susceptibility (line [L]61/line [L]72) system and quantified the levels of key mRNAs. We measured gene expression in both whole tissues and, after tissue sectioning and laser capture micro-dissection, in the MD lesions themselves. Gene ontology-based modeling of our results suggested that overall environment in whole lymphomas as well as in microscopic lymphoma lesions in both L61 and L72 is pro T-regulatory cells (T-reg). But there are also pro T-helper (Th)-1 and anti Th-2 effects in L61 compatible with cell-mediated immunity. In contrast, L72 had anti Th1 and pro Th2 effects. The environment within the microscopic tumor lesion was pro T-reg, anti Th1 and pro Th2 in both L61 and L72. The predominance of pro T-reg phenotype in both L61 and L72 microscopic tumour lesions and the absence of pro Th1 phenotype suggest that the MD lesions and the trsnformation event is essentially the same in both L61 and L72 and that resistance/susceptibility is mediated at the level of tumor immunity. In addition, Functional differences in expression of the host tumor antigen (CD30) and in the MDV "meq" oncogene promoter. Using PCR we have cloned 2.5 Kb 5' of the CD30 gene ATG (i.e. the gene promoter) from 5 MD-susceptible and 2 MD-resistant chicken lines. We have sequenced all of these and have identified polymorphisms many of which are in potential Meq binding sequences and phyogenetics shows that CD30 promoter polymorphisms exactly match chicken breeding history. We have completed functional assays to identify whether or not the polymorphisms affect the ability of MDV meq to transactivate the CD30 promoter. Transcription increased from the CD30 promoters of MD-susceptible, but decreased from MD-resistant, genotypes. To analyze functionality of the NF-κB binding site in the Meq promoter, we cloned the three main NF-κB isoforms (P65, P100, and P105) in an expression plasmid and the Meq promoter in reporter plasmid and did transcription assays as above. All NF-κBs stimulate transcription from Meq promoter not equally; the Meq oncoprotein itself further enhanced mRNA expression. We suggest that a positive feed forward loop exists between CD30 and Meq, similar to the LMP-1/CD30 system in Epstein Barr virus, and that perturbation of the CD30 system is highly evolutionarily conserved in virus induced lymphoma. PARTICIPANTS: SC Burgess. PD JJ Buza. Postdoctoral Researcher LA Shack, Research Associate D Kunec, Research Associate TARGET AUDIENCES: Vaccine companies, poultry breeding companies, biomedical researchers PROJECT MODIFICATIONS: NIL
Impacts
This work provides fundamental knowledge for maintaining the USA's poultry industry's competitiveness by as it aims to control Marek's Disease, one of the most economically-important diseases of poultry. This work is positively impacting our understanding of MD lymphomagenesis and for decreasing the expensive reliance on MDV vaccines to control MD tumors. We aim to identify potential novel measures for MD control by identifying genetic mechanisms to do so. Our work has also been important in developing novel proteomics and bioinformatics approaches and for annotating the chicken genome.
Publications
- D. Kunec S.C. Burgess. 2007. DNA sequence for predicting resistance to Marek's disease in chickens. USA Patent office, patent pending.
- Shack*, L. A., J. Buza*, J., and S. C. Burgess. 2008. The neoplastically-transformed (CD30hi) Marek's Disease lymphoma cell phenotype most closely resembles T-regulatory cells. Cancer Immunology and Immunotherapy. In press. DOI: 10.1007/s00262-008-0460-2
|
Progress 11/01/05 to 11/01/06
Outputs
All chickens can be infected with MDV and MDV-neoplastically transformed cells occur in all chickens. However, some chickens are genetically resistant to MD lymphoma formation. Our hypothesis is that specific mechanisms intrinsic to normal lymphocyte biology are disregulated to a greater or lesser degree in MD and it is the degree of disregulation from normal that determines whether or not gross lymphomas will form after MDV infection. Our objective is to identify genetic mechanisms responsible for the differences between MD susceptible and resistant chickens at the time of resistance to lymphoma development when MD lymphoma progressor and regressor chicken genotypes diverge which is ~21 days post infection (dpi). Functional differences in expression of the host tumor antigen (CD30) and in the MDV meq oncogene promoter. Using PCR we have cloned 2.5 Kb 5' of the CD30 gene ATG (i.e. the gene promoter) from 5 MD-susceptible and 2 MD-resistant chicken lines. We have
sequenced all of these and have identified polymorphisms. We are now starting functional assays to identify whether or not the polymorphisms affect the ability of MDV meq to transactivate the CD30 promoter. We are also testing the hypothesis that a self-sustaining cycle between CD30 and meq exists. Meq contains putative NFkB transcription factor binding sites and we know meq transactivates CD30. We have now cloned the three main isoforms of NFkB in expression plasmids and the MEQ promoter in a reporter plasmid. We are now doing the functional transcription assays.
Impacts
This work provides fundamental knowledge for maintaining the USA's poultry industry's competitiveness by as it aims to control Marek's Disease, one of the most economically-important diseases of poultry. This work is positively impacting our understanding of MD lymphomagenesis and for decreasing the expensive reliance on MDV vaccines to control MD tumors. We aim to identify potential novel measures for MD control by identifying genetic mechanisms to do so. Our work has also been important in developing novel proteomics and bioinformatics approaches and for annotating the chicken genome.
Publications
- No publications reported this period
|
|