Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target Audiences include Veterinary/Animal Health Researchers, Veterinarians, Diagnosticians, and government agencies. Changes/Problems: No major problems were encountered, however over the 5-year course of the work prion diseases ceased to be of significant concern to stakeholders. This was largely due to the success of the implemented "feed ban", and failure to detect significant cattle prion disease in the United States. The focus of the newly approved followon project to the described work will focus on more acute measurement of acute immunity, including vaccine responsiveness. What opportunities for training and professional development has the project provided? A Phd Candidate, Dr. Maxim Lebedev, successfully defended his doctoral thesis in December 2014, based on work carried out under this project. In addition, at least one undergraduate Research Assistant participated in the project each year, including Olivia Kendall in the final year of the project. How have the results been disseminated to communities of interest? Results have been disseminated through publication of research results, as well as presentation of experimental data at regional and national meetings including the Autumn Immunology Conference (November 2013), Center of Excellence in Zoonotic and Animal Disease Annual Meeting (2014), and other regional biotechnology conferences. Two manuscripts based on the thesis work of Dr. Lebedev are currently in preparation. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Over the reporting period, the primary focus has been on publication of prior work completed on prion pathogenesis, and generation of new work on the immune response in domestic animals. Specifically, over the reporting period we published two distinct publications detailing our work on prion pathognesis. In the first report, we collaborated with Dr. Jason Bartz (former member of the TSE Multistate Research Committee) to examine mechanisms of prion replication in vitro. These data clearly demonstrated that prion clearance mechanisms could not be duplicated in vitro, although prion replication could be mimicked. In a second publication with ARS-USDA, we demonstrated the ability to detect infectoius prion protein associated with B cells in the blood of sheep. In a second series of experiments, we examined the development of specific bioassays of immune response to map vaccine effectiveness against a subunit Rift Valley Fever vaccine. In these studies, we used a peptide mapping approach to determine "marker" peptides that correlated with development of protective immunity against an experimental subunit vaccine against Rift Valley Fever. In these studies, one peptide in particular correlated well with development of immunity against the RVF virus. More importantly, we also developed assays of T cell immunity to the RVF vaccine, and while the anti-peptide responses were not predictive of neutralizing immunity, T cell responses did in fact correlate well with protection. These assays clearly illustrated the need for a diverse approach to predictive immunity against vaccine candidates, and illustrate new tools to develop antigen-specific assays of immunity in domestic animals. We currently have 2 publications in preparation describing this work.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Shikiya, R.A., Eckland, T.E., Young A.J., Bartz, J.C. (2014). Prion formation, but not clearance, is supported by protein misfolding cyclic amplification. Prion 8(6):415-420.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dassanayake, R.P., Truscott, T.C., Zhuang, D., Schneider, D.A., Madsen-Bouterse, S.A., Young, A.J., Standont, J.B., Davis, Wz.C., ORourke, K.I. (2014). Classical natural ovine scrapie prions are detected in practical volumes of blood by lamb and transgenic mouse bioassay. J. Vet. Sci. epub 2014/12/31.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Faburay, B., Lebedev, M., McVey, D.S., Wilson, W., Morozov, I., Young, A., Richt, J.A. (2014). A glycoprotein subunit vaccine elicits a strong Rift Valley Fever virus neutralizing antibody response in sheep. Vector Borne Zoonotic Dis. 14(10):746-756
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Rajput, M.K., Darweesh, M.F., Park, K., Braun, L.J., Mwangi, W., Young, A.J., Chase, C.C. (2014). The effect of bovine viral diarrhea virus (BVDV) strains on bovine monocyte-derived dendritic cells (Mo-DC) phenotype and capacity to produce BVDV. Virol. J. 11(1):44 doi 10.1186/1743-422X-11-44
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Wang, X., Zhang, H., Abel, A.M., Young, A.J., Xie, L., Xie, Z. (2014). Role of phosphatidylinositol 3-kinase (PI3K) and Akt1 kinase in porcine reproductive and respiratory syndrome virus (PRRSV) replication. Arch. Virol. DOI 10.1007/s00705-014-2016-4
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Progress 10/01/09 to 09/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project has ended, sumbitting final report to clear project out of REEport system.
Publications
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Researchers in the field of Infectious Disease Veterinary Professionals Veterinary Vaccine Industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? A PhD student will be completing thesis work involving the vaccine adjuvant work in ruminants. An MS student successfully defended her thesis regarding equine immunology work in 2013, and papers are currently in preparation. How have the results been disseminated to communities of interest? A total of 3 publications relevant to this project were published in 2013, and a number of additional papers are currently under preparation. In addition, 2 abstracts relative to the project were presented in 2013. The PI on the project was an invited speaker at 4 meetings over the year where data relevant to the project was also presented. What do you plan to do during the next reporting period to accomplish the goals? We plan to further define the effect of adjuvant on the circulating immune system, in order to complete a total of 3 papers for the graduating PhD student. In addition, we will further pursue a novel flow-cytometry based assay to define immune responses to acute and chronic inflammation. Additional work will be carried out to define the utility of the FDC system to mirror germinal center function in domestic animals, to further enhance domestic-animal research and limit animal use in basic research.
Impacts
What was accomplished under these goals?
The immune system of domestic animals differs from the standard mouse and human paradigms, and as a result there is relatively limited information on the mechanisms used by these animals to combat and clear disease. This inhibits our ability to rapidly and effectively develop therapeutic and treatment regimens, particularly for emerging diseases. In 2012-2013, the deer industry (and to a less extent, cattle) were affected by an outbreak of Epizootic Hemorrhagic Disease. Simultaneously, Porcine Epidemic Diarrhea Virus broke in the United States, resulting in significant losses to the swine industry. As of January 2014, there are no effective fully licensed vaccines available to protect against these diseases, partially due to an incomplete understanding of the immune response to these agents. Furthermore, there are limited tools available to characterize the health and immune status of domestic animals relative to humans, presenting a challenge to producers and veterinary healthcare providers. The primary focus of this project is to further our knowledge of the response of these animal species’ to infectious diseases, particularly chronic diseases, and characterize specific similarities and differences in these responses relative to other known species. These data will then be directly applicable to a greater ability to measure and enhance the immune response of food animals and wildlife, increasing agricultural productivity and economic viability while furthering stakeholder protection from zoonotic diseases that affect both humans and animals. The results acquired over the past year largely focus on the immune response of ruminants to vaccine adjuvants, characterization of the role of a unique subset of immune cells found in sheep and cattle but not humans, and further analysis of the development and function of the equine immune system. This will not only directly benefit the formulation of new vaccines for veterinary use in the agricultural industry, but may also lead to development of new formulations for human use. The described data has been directly incorporated into vaccine formulations under development by federal and private partners. Our data on immune development of the horse, as well as it’s relationship to nutrition, may also lead to new therapeutics and production strategies for this agricultural species, and is already leading to new opportunities for assisting the equine nutrition community through joint projects. Our four primary objectives of this project are: (1) Examination of recirculating leukocyte populations as a measure of immune response to chronic diseases (2) Role of migratory Dendritic Cell subsets in the pathogenesis of Chronic Diseases (3) Role of FDCs in the pathogenesis of chronic diseases and support of local B cell responses and (4) Examination of novel, phenotypically-defined cell populations of domestic animals and cervids. Over the past year, we have continued to develop these objectives, resulting in 3 additional publications and presentation of 1 abstract at regional meetings. Information obtained as a result of this work has also been included in translational activities with regional biotechnology companies, as well as incorporated into course material presented in graduate courses offered through SDSU. More specifically, we have addressed goals 1 and 2 through an examination of the response of sheep to subunit protein vaccines emulsified in adjuvant in order to characterize the responding immune populations. To that end, sheep were immunized with subunit vaccines against Rift Valley Fever virus emulsified in a water-in-oil adjuvant, and the appearance of specific antibody and responding cells monitored in the bloodstream during development of a strong antibody response. In addition, a method previously developed was used to produce antigen-specific T cell lines specific for these proteins. Notably, we repeatedly saw an elevation in the frequency of a unique, previously undescribed subset of gamma-delta T cells expressing the cell-surface complement receptor CD11b (goal 4). More detailed analysis indicated that these cells also expressed a number of cell-surface receptors normally associated with innate immune responses, potentially indicating a role for them in antigen presentation and inflammatory responses. When adjuvant alone was injected into experimental animals, these cells were again elevated, indicating that it was the inflammatory response to injection with adjuvant rather than antigen-specific response that increased these cells in the bloodstream. In vitro cell culture further indicated that these cells were elevated when incubated with “stressed” cells, suggesting a potential role for pattern-recognition receptors in their differentiation. We are currently further analyzing the functional and phenotypic nature of these cells in animals, particularly as related to their recruitment during acute and chronic inflammation. A portion of this data was presented at a national meeting, and will be incorporated into a manuscript in the coming months. Related to this work, an invention disclosure was filed on our work involving Goal 3, and we anticipate patent application within the current year. In an attempt to address Goal 4, we further developed our knowledge of the developing equine immune system. In a second group of animals we significantly extended our map of the differentiation and phenotype of immune cell subsets in foals. The most notable observations were the rather unique interrelationship of the development of CD4+ T cells and B cells unique from that observed in any other species. In addition, we created qualitative ELISAs to monitor the development of Immunoglobulin subtypes in equine serum and colostrum, permitting us to map the relative development of these antibody subtypes in mares and foals. Two manuscripts are currently under preparation describing this data. The work we have described will have direct, and immediate impact on the public and private sector through the rational design of immunotherapeutics, and an enhanced ability to track and monitor immune responses in healthy and diseased domestic animals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Faburay, B., Wilson, W., McVey, D.S., Drolet, B.S., Weingartl, H.M., Madden, D., Ma, W., Young, A.J., Richt, J. (2013). Rift valley fever virus structural and non-structural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep. Vector Borne Zoonotic Dis. 13(9):619-629.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
2. Ran, Z., Chen, Y., Shen, H., Xiang, X., Liu, Q., Bawa, B., Qi, W., Zhu, L., Young, A., Richt, J., Ma, W., Li, F. (2013). In vitro and in vivo replication of influenza A H1N1 WSN33 viruses with different M1 Proteins. J. Gen Virol. 94(Pt 4):884-895.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Young, A.J., Richt, J.A. (2013) Food Safety Implications of Prion Disease in Foodborne Infections and Intoxications, 4th Ed. Academic Press. J. Glenn Morris Jr., Ed.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our four primary objectives of this project are: (1) Examination of recirculating leukocyte populations as a measure of immune response to chronic diseases (2) Role of migratory Dendritic Cell subsets in the pathogenesis of Chronic Diseases (3) Role of FDCs in the pathogenesis of chronic diseases and support of local B cell responses and (4) Examination of novel, phenotypically-defined cell populations of domestic animals and cervids. Over the past year, we have continued to develop these objectives, resulting in 3 additional publications and presentation of 6 abstracts at regional meetings. Information obtained as a result of this work has also been included in translational activities with regional biotechnology companies, as well as incorporated into course material presented in graduate courses offered through SDSU. Our focus continues to be examination of the recirculating immune system in domestic animals, it's development, and function in disease. To this end, we have collaborated with investigators to determine migratory characteristics of B cells in the skin, and the regulation of B cell function by chemokines. In collaboration with investigators at the USDA, we have continued our work to study the role of migratory B cells in the pathogenesis of sheep scrapie, as well as other diseases. In partnership with regional biotechnology industries, we are examining the use of a direct lymphatic cannulation model to define the efficacy of action of vaccine adjuvants and targets. This work has resulted in submission of a major proposal to the federal government, and further established partnerships to define novel approaches to animal health. In other work, we have examined the role of leukocyte subsets in response to vaccination with target antigens directed against pathogens of domestic animals including sheep, cattle, and deer. Experiments on vaccine targets in sheep, deer, and horses has resulted in collaboration with USDA-ARS laboratories, several academic institutions, and internally with Drs. Christopher Chase, Michael Gonda, and Rebecca Bott. Our novel antigen-specific B cell assay developed last year has allowed us to examine simultaneously the development of antibody responses both in the serum, as well as the production of antigen-specific B cells in response to vaccination in deer and sheep. Finally, we have developed a growing research focus on the development and function of the equine immune system, resulting in submission of a proposal to NIFA this spring. Novel data acquired as a result of these studies was presented at a total of 3 international meetings over the past year, and is serving as background data for pursuit of further federal funding and at least 2 publications. In addition, collaborative work on the migration of B cells through skin resulted in a collaborative publication, detailing new information on the regulation of leukocyte responses to infection. PARTICIPANTS: Alan J. Young - Principal Investigator Maxim Lebedev - PhD Candidate. Developed all T cell assays to evaluate immune responses of domestic animals, assisted in all serological assays, assisted in all flow-cytometric based assay systems. Partner institutions provided support through collaboration, although no direct funding was received through this project. Partner Institutions included University of Pennsylvania, USDA-ARS Kerrville, Tx., USDA-ARS Manhattan KS., Kansas State University, USDA-ARS Pullman, Louisiana State University. Collaborators include Dr. Rebecca Bott (SDSU), Dr. Christopher Chase (SDSU), Dr. Michael Gonda (SDSU), Dr. Juergen Richt (KSU), Dr. Gudrun Debes (UPenn). TARGET AUDIENCES: Animal Health Researchers, Biomedical Researchers, Biotechnology and Animal Health Companies, Practising Veterinarians. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The majority of our work over the past year has focused on defining new tools for use in the measurement of the immune response of domestic animals. Using the sheep, we developed methodologies for the production of T cell lines specific for the target protein KLH, a "prototypic" protein antigen. Based on previous work, we adapted the technology to a platform protocol that can be used across species, and defined appropriate culture conditions for production of antigen-specific T cells in vitro. The most interesting observation we made during development was the apparent dependence of the production of CD4+ or CD8+ T cells respectively on the concentration of antigen used to generate the T cells. Further work is necessary to fully define the function of the T cell subsets, however our work suggests that by manipulation of local antigen concentration in vitro we can effectively manipulate the generation of antigen-specific CTLs or helper T cells. We will now use our established antigen-specific B and T-cell assays to define immunodominant epitopes for targeted vaccine design in agricultural animals. In a second project, we have partnered with researchers at the USDA to examine the immune response of deer to recombinant vaccine formulations. While ongoing, we were able to define the relative appearance of antigen-specific B cells in animals following vaccination, the development of specific antibody, and have partnered with regional industry to investigate the efficacy of these formulations for protecting both farmed and cultivated deer against zoonotic pathogens. A third focus of our work over the previous year has been to define basic immunological composition of the equine immune system. In preliminary studies, we defined a panel of monoclonal antibodies that were reactive against equine B, T, and other blood leukocyte subsets. These antibodies, once defined, were used to characterize the immune development of horse peripheral blood mononuclear cells from birth through 1 year of life. Intriguingly, B cells appeared to be significantly over-represented in the equine immune system relative to other domestic animal species. Although the frequency of B cells dropped following ingestion of colostrum, the overall frequency of these cells was significantly elevated in foals relative to young ruminants, suggesting significant differences in B cell development in horses as opposed to other hoofstock. Furthermore, we were able to define the contribution of maternal immunoglobulin to the blood of foals, and it's duration during the first few months of development. Together, this work has significantly expanded our understanding of comparative immunology, added significant tools to our ability to work with domestic animals, and provided new insight into vaccine design for animals of agricultural importance.
Publications
- Geherin, S.A., Rintushel, S.R., Lee, M.H., Patel, R.T., Alt, C., Young, A.J., Hay, J.B., Debes, G.F. (2012) The skin, a novel niche for recirculating B cells .J. Immunol. 188:6027-6035.
- Tamgueney, G., Richt, J.A., Hamir, A.N., Greenlee, J.J., Miller, M., Wolfe, L.L., Sirochman, T.M., Young, A.J., Glidden, D.V., Johnson, N.L., Giles, K., DeArmond, S.J., Prusiner, S.B. (2012). Salivary prions in sheep and deer. Prion 6(1):52-61.
- Furr, A., Young, A.J., Richt, J.A. (2012) The immune system in the pathogenesis and prevention of prion diseases. J. Bioterrorism and Biodefense. S1:012. doi:10.4172/2157- 2526.S1-012
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: We continue to focus on four objectives: (1) Examination of recirculating leukocyte populations as a measure of immune response to chronic diseases (2) Role of migratory Dendritic Cell subsets in the pathogenesis of Chronic Diseases (3) Role of FDCs in the pathogenesis of chronic diseases and support of local B cell responses and (4) Examination of novel, phenotypically-defined cell populations of domestic animals and cervids. Over the past year, we have focused on further definition of unique tools and assays to define immunity in domestic animals. Firstly, we have partnered with regional biotechnology industry to further develop unique diagnostic tools to define the health and disease status of domestic animals, and with the University of Illinois in a funded project to examine response to pox viruses by examination of draining lymph. Secondly, we have increased collaborative efforts among investigators through sharing of research resources and reagents specific to domestic animals, as reflected in one published and one pending manuscript. In order to further delineate the role of circulating B cells in the pathogenesis of chronic disease, we developed a new flow-cytometry based assay to enumerate antigen-specific B cells in domestic animals suitable for many antigens. Finally, our work to examine the role of migratory effector and regulatory T cells in the pathogenesis of disease has formed the basis of a collaboration with SDSU (Dr. Chris Chase, Bovine Viral Diarrhea Virus) and a funded project through the Kansas State DHS Center of Excellence in Zoonotic Disease. This work has resulted in 3 publications since the last report, and 3 pending publications submitted. A graduate student employed in the project is currently completing thesis work focused on the role of migratory leukocytes in prion diseases, which will further contribute publications in 2012. In addition, this work has formed the basis for additional translational research work in partnership with regional biotechnology industries to develop novel diagnostic tools for chronic veterinary disease. PARTICIPANTS: Katherine ORourke, Collaborator, USDA-ARS Christopher Chase, Collaborator, SDSU Maxim Lebedev, Student, SDSU. Juergen Richt, Collaborator, KSU Jason Bartz, Collaborator, Creighton U. TARGET AUDIENCES: Researchers in Domestic Animal Immunology; Producers; Biotechnology Industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In order to define subsets of migratory leukocytes potentially involved in the pathogenesis and progression of prion diseases, we continued to define unique B cell subsets found in normal and scrapie-infected sheep. We had previously demonstrated that the phenotype of circulating B cells was altered in scrapie-infected animals, such that there was a significant increase in the relative frequency of CD11c-positive B cells in infected animals associated with disease. These cells were virtually absent at birth, but gradually increased in normal animals throughout development to about 50% of the total B cell pool in adults. In scrapie-infected animals, these cells formed an even greater proportion, representing up to 70% of total B cells in affected animals. In an attempt to further define the function of these cells, we developed a flow-cytometry based assay to define antigen specific B cells in ruminants. In normal animals, antigen-specific B cells were approximately evenly distributed between CD11c positive and CD11c negative B cells. Following immunization, there was a marked increase in the frequency of antigen-specific CD11c positive but not CD11c negative B cells. These data are consistent with the conclusion that CD11c positive B cells represented memory cells in sheep. In other studies, CD11c positive B cells were found to express the memory B cell marker CD27. This confirmed the identity of CD11c positive B cells as memory cells. When sheep B cells from immunized animals were cultured on our Follicular Dendritic Cell cultures, we also saw an increase in the frequency of antigen-specific B cells. We are currently defining the ability of these culture systems to support B cell differentiation in vitro. In an attempt to develop tools for the study of domestic-animal immunology, we defined the reactivity of a panel of monoclonal antibodies across diverse mammalian species. Surprisingly, we found that when significant numbers of unique antibodies were used, there was a clear evolutionary linkage to the number of reactive antibodies for any related or unrelated species, although it was difficult to predict if any single antibody would cross-react. These data have been submitted for publication, and suggest that the conservation of tertiary antibody epitopes can be used for the study of evolutionary trees. More importantly, we have established a pool of reagents suitable for the study of immunological parameters in diverse species. In more defined studies, we further examined the development and differentiation of leukocyte subsets in cervids. Surprisingly, we found marked differences in the composition of T and B cell subsets in fetal, neonatal, and adult cervids. Specifically, there appeared to be significantly more B cells in fetal and neonatal cervids as compared to other ruminants. This may suggest unique pathways of B cell development and differentiation in deer as compared to sheep and cattle, and forms the basis of a pending research proposal. Together, this work has markedly expanded the immunological toolbox available to researchers of domestic livestock.
Publications
- Dassanayake, R.P., Schneider, D.A., Truscott, T.C., Young, A.J., Zhuang, D., ORourke, K. (2011) Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma BMC Veterinary Research, 7:75-86.
- Furr, A., Knudsen, D., Hildreth, M., Young, A.J. (2011) Enhancement of immunohistochemical staining of scrapie proteins and immune cells within lymph nodes of early scrapie-infected sheep. J. Immunol. Methods. 371(1-2):1-7.
- Richt, J.A., Young, A.J. (2011) Chapter 14: Immunobiology of Prion Diseases in The Immune Response to Infection, ASM Press, Kaufmann, Rouse, Sacks Ed.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This project has 4 specific objectives. (1) Examination of recirculating leukocyte populations in lymph as a measure of immune response to chronic diseases (2) Role of migratory Dendritic Cell subsets in the pathogenesis of Chronic Diseases (3) Role of FDCs in the pathogenesis of chronic diseases and support of local B cell responses and (4) Examination of novel, phenotypically-define cell populations of domestic animals and cervids. Over the past year, we have made significant progress on each of these areas, reflected in several tangible outputs. Firstly, work derived through this project has been applied towards accomplishment of three externally funded collaborative grants with Industry to define new, immune-cell focused ultrasensivity diagnostic assays for prion disease. Secondly, work to define the role of migratory leukocytes in the pathogenesis of chronic disease was collaboratively applied to new grant applications in the study of Bovine Viral Diarrhea Virus and Prion Diseases, as well as reflected in a collaborative publication (listed below). Finally, our work to examine the role of migratory effector and regulatory T cells in the pathogenesis of disease formed the basis of a collaboration with the newly-funded Kansas State University Center of Excellence in Emerging Zoonoses and Animal Disease (funding pending). Portions of our work were presented at the Initial Meeting of CEEZAD, held at Kansas State University, and an additional three publications are currently under review for publication in 2011. In addition to these direct outputs, additional collaborations have been formed with Colorado State University, University of Wyoming, Pennsylvania State University, USDA-ARS, Kansas State University, and Loyola University to disseminate reagents prepared to specifically delineate the immune response in domestic animals, which is expected to lead to future publication opportunities focused on characterization of the immune response in domestic animals. PARTICIPANTS: Jingjing Bao - PhD Student Maxim Lebedev - PhD Student Ashley Peterson - Undergraduate student. TARGET AUDIENCES: Basic immunology researchers; Basic Prion researchers; Practicing veterinarians PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our first objective was to examine the composition of lymph in normal and diseased animals. Over the past 5 years, we have established a small cervid model to study the interaction of cervids with domestic animals in the disease process. The composition and flow of lymph in sheep has formed the basis of much of our knowledge regarding leukocyte recirculation in health and disease, and was directly compared to experimental values obtained in muntjac deer. Overall, the proportion of CD4 and CD8 T cells in efferent lymph was higher than that observed in other species, although the relative frequency of these two subsets was conserved. In contrast, there were significantly fewer B cells and gamma-delta T cells in muntjac lymph than is generally observed in sheep. This is, however, reflected in a lower proportion of B cells and gamma-delta T cells in muntjac blood, and therefore basic regulatory processes of leukocyte migration in cervids are likely similar to that observed in other species (Manuscript in preparation). In additional studies, we have examined the traffic of leukocytes through prion-infected sheep lymph nodes. In these studies, we repeatedly observed an overall increase in the release of CD21+/CD11c- B cells and gamma delta T cells, associated with a relative decrease in CD21-/CD11c- B cells (Manuscript under review). Given the recent publication that CD21- B cells in blood appear to harbor prion infectivity, it will be intriguing to investigate the mechanism behind this important observation. Under objective #2, we proposed to specifically examine mechanisms and subsets of migratory dendritic cells in the pathogenesis of chronic disease. Intriguingly, Dendritic Cell subsets were also increased in lymph concomitant with the detection of immunoreactive prion protein following acute exposure to infectious brain material from scrapie-infected sheep (Manuscript in preparation). Finally, we are attempting to identify novel cell subsets and immune cell functions in domestic animals and cervids. We are examining three distinct observations that appear to be specific to ruminants. Firstly, we continue to define the function and development of CD21-ve peripheral blood B cells, a population not found in mice or humans to the degree apparent in ruminants. While these B cells are a minor population in young sheep, they increase with age as a result of post-natal differentiation. Although they are present in deer, they are equally represented in both young and aged animals (Manuscript in preparation). WE continue to investigate the role of this subset in immune responses. We have also identified a subpopulation of CD8+ leukocytes in the peripheral blood of deer and sheep, based on production of an antibody apparently similar to human CD75s (Manuscript under review). Although this population represents over 20% of total PBMCs, it does not appear to show similarity to any known leukocyte population in mice or humans, and experiments are underway to define it's function in vitro.
Publications
- Mathiason, C.K., Hayes-Klug, J., Hays, S.A., Powers, J., Osborn, D.A., Dahmes, S.J., Miller, K.V., Warren, R.J., Mason, G.L., Telling, G.C., Young, A.J., Hoover, E.A. 2010. B cells and platelets harbor prion infectivity in the blood of deer infected with chronic wasting disease. J. Virol. 84(10:5097-5107.
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Progress 10/01/09 to 12/31/09
Outputs
OUTPUTS: This project commenced September 2009. It is the focus of this work to specifically define the lymphoid pathogenesis of prion agents and bovine viral diarrhea virus. To accomplish this goal, we have the following specific aims: 1. To define the role(s) of regulatory T cells in the acute and chronic immune response to persistent diseases. 2. To define the role(s) of migratory dendritic cells in the pathogenesis of persistent diseases 3. To map the mechanisms of Follicular Dendritic Cells in the maintenance of chronic diseases 4. To map the function of unique leukocyte subsets in domestic animals To that end, we have assembled appropriate reagents and collaborations to specifically identify T cell subsets in sheep. Through collaborative efforts, we have succeeded in identification of regulatory T cell subsets in sheep. PARTICIPANTS: Ruth Burroughs - MS Student; Stacy Dreis - Research Technician TARGET AUDIENCES: Basic researchers, practitioners, Biotechnology Industry professionals specializing in domestic animal therapeutics and vaccine development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our initial work has been focussed on defining previously undescribed leukocyte subsets in the blood of sheep and deer that may be involved in the pathogenesis of chronic diseases. To that end, we have collaborated to obtain antibodies specific for ovine FoxP3, a marker of regulatory T cells. This antibody was used in conjunction with flow cytometry to stain sheep peripheral blood leukocytes from animals of various ages. To date, we have succeeded in optimizing this protocol for use in sheep. In addition to these known subsets, we have been focussing on defining two novel leukocyte subsets defined by new monoclonal antibodies derived in our laboratory. In sheep, an antibody which appears to selectively recognize ovine CD75s, a sialic acid dependent epitope, has been found to react with a subset of ovine CD8+ T cells. Based on work in the mouse, this subset may be analagous to CD8+ regulatory T cells, and therefore are candidates for an important role in the maintenance of chronic diseases of domestic animals. Initial work has been devoted to further defining the phenotype of this unique subset. To date, it has been difficult to define any significant differences between CD75s/CD8+ T cells and other subsets, however additional reagents including CD25 have been obtained for further detailed analysis. Morphologically, these cells appear similar to all other T cells. In deer, we have also isolated a unique subset of myeloid cells in the peripheral blood. Specifically, we have identified a significant subset of myeloid lineage cells in the peripheral blood which are morphologically similar to monocytes, but fail to express CD14. To date, we have identified no significant characteristics of these cells to define their nature, however based on their morphology and cell-surface expression of unique proteins may be a novel myeloid dendritic cell. Further work is necessary to define this subset fullly.
Publications
- No publications reported this period
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