2005 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? A fully competent immune system is necessary to control inflammatory diseases, such as heart disease and cancer, as well as infectious diseases. The cost for medical treatment and care exceeds $200 billion per year among those diseases linked strongly to diet. The staggering cost of nutrition-related diseases points to a need to develop effective strategies to lessen the cost of health care by prevention of disease. A determination of how dietary components contribute to the appropriate response of immune cells in the resolution and reduction in disease development is required in order to promote health. Immune activation of T helper 1 (Th1) and T helper 2 (Th2) cells is an important component of acquired immunity to infection. The Th1 cells typically respond to bacteria, viruses and protozoan infections that invade and reside inside of cells. This response, however, when unregulated or inappropriately induced can lead to tissue destruction as in the case of inflammatory bowel disease and arthritis. The Th2 response is appropriate to infectious agents, such as worm parasites, that live in the host, but outside of the cells of the body. When this response is inappropriately induced it can also lead to disease that is generally expressed as allergic disorders, such as food allergy and asthma. High Th1 responses tend to turn down Th2 responses and vice versa. These T cells can also be affected by nutritional components like certain vitamins and minerals. The role of dietary micronutrients in supporting cell signaling mechanisms that initiate an optimal immune response to disease agents will be studied in experimental animals fed diets where micronutrients are deficient or in excess of recommended levels and the outcome of the infection will be assessed. A large number of studies using experimental mice indicate that immune T cell differentiation and/or function is responsive to changes in nutritional status. This is particularly true for changes in vitamin A (VA) levels. Rapid and sensitive assays are needed to identify nutritionally relevant biomarkers of the immune status of healthy people as well as to evaluate the effects of dietary interventions on immune function. Assessment of VA status in the presence of infectious disease or inflammation is difficult because plasma VA is reduced by both of these stimuli. Current methods to assess VA status use plasma VA in combination with measurements of one or more markers of the acute phase responses to overcome this limitation. These combination assays have moderately improved the ability to determine VA status during infection or inflammation, but they still lack sensitivity and specificity. The recent development of new technologies such as laser capture micro-dissection and genomic and proteomic array analysis can isolate single cells or small populations of cells that can be identified and extracted for both Ribonucleic Acid (RNA) and protein material used in genomic and proteomic array analysis. Application of this technology to healthy human and animal subjects will provide a dynamic approach to the identification of biomarkers of immune status that are responsive to changes in nutrition. The use of swine to study food allergy provides a robust model more comparable to humans than found in rodent studies. Although rodents have been useful for screening genes for allergy susceptibility, they develop transient acute hypersensitivity and do not spontaneously exhibit allergy. Our analysis of 75 immunological variables in pig, human, and mouse shows that the pig immune system is more similar to humans than are mice for >80% of the variables compared while mouse is more similar to human than are pig <10%, and allergic disease is clinically similar to humans. Thus, data on food allergy derived from swine will be more relevant to humans than from mouse studies. This project addresses objectives of the National Human Nutrition Action Plan 107, related to “Research needed to identify biomarkers of nutrient intakes and status, nutritional adequacy and disease prediction (3.1.1.1),” since it will use both small and large animal models of infection with agents that represent similar infections in humans. It is important to demonstrate that dietary micronutrients can optimize control of infection without tissue destruction through an effective immune function, and enhance the health status of the U.S. population to prevent disease. 2.List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12 months)-Develop high throughput processing and analysis of porcine gene expression using real-time polymerase chain reaction (PCR) and microarrays. Milestone 2 (12–36 months)–Evaluate the primary immune responses to Ascaris suum or Trichuris suis in pigs given retinoic acid (ATRA). Milestone 3 (24–48 months)–Evaluate the primary or secondary immune responses to A. suum or T.suis in pigs with different vitamin A (VA) status. Milestone 4 (24–48 months)–Evaluate the effect of ATRA or VA status on the development of an allergic disease. Milestone 5 (40–60 months)–Technology transfer will be completed with recommendations for appropriate VA status to limit infectious and allergic disease. 4a.What was the single most significant accomplishment this past year? Real-time PCR arrays developed to evaluate pig immune response to infection and VA supplementation. It is necessary to reliably measure many genes simultaneously in order to fully understand the role of VA in the immune system. We have developed real-time PCR arrays for this purpose. There are several advantages of using real time PCR arrays over conventional microarrays in our experimental models since there are only 2 commercially available swine microarrays and swine microarrays that have been developed privately are biased toward genes involved in growth and reproduction. Most swine gene databases are not immunologically or nutritionally oriented or are poorly annotated for those research purposes. To address this problem, we initially applied our real time PCR technology to a protozoan disease model of infection in pigs with Toxoplasma gondii, and measured the expression of 380 genes in liver. The expression data was compared against human responses by extracting the information for the analogous human genes from a Pig Immunology and Nutrition (PIN) database that we developed. The data was mined using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) at National Institute of Allergy and Infectious Disease (NIAID), National Institute of Health (NIH). This analysis independently confirmed our internal annotation scheme and gene selection process and revealed a significant number of novel, interrelated, immune and metabolic pathways that we can reliably test with our assay library. This type of advanced analysis of gene expression in a model of infection is unparalleled, even compared to published human and rodent models. 4b.List other significant accomplishments, if any. Porcine Immunology and Nutrition (PIN) database is developed. We have completed development of our annotated pig gene expression database to “mine” data in conjunction with commercial Genespring or other analysis software (http://www.ars.usda.gov/Services/docs.htm?docid=6065). This database has been online since November 2004, and is scheduled for public release in August 2005. Included in the data base are originally described partial length sequences for porcine proteins T-box 21 (TBX21), Cell Differtiation (CD28), Forkhead Box P3 (FOXP3), Tyrosine Kenose (TXK), Interferon consensus sequence bending protein 1 (ICSBP1), Interleukin-22 (IL-22), Chemothine (C-C motif) Legand 11 (CCL11), and Myeloid differentiation primary response gene 88 (MYD88) that were cloned to expand the tools of immunological analysis to innate response genes and those involved in T cell-dependent regulation of acquired immune responses. This is a unique research tool for those interested in comparative gene expression between man and swine as a model for the study of infectious and metabolic disease. Retinoic acid augments the immune response to parasitic worm infection. Pathogenic organisms generally initiate infection through mucosal surfaces which are also the site of nutrient absorption in the intestine. The interaction between VA and common parasitic infections was tested in pigs to model the effect of dietary nutrient supplementation with VA on two important mucosal pathogens. Retinoic acid, an active metabolite of VA, affected development of predominantly Th1- and a Th2-immune response to A. suum. The data derived from these studies suggest that retinoic acid augmented the Th2 response to A. suum. This is important because Ascaris is the most common parasitic worm infection world wide and is a model for intestinal and pulmonary allergy. Inappropriate concentrations or timing of VA supplementation may negatively affect the host response. These disease outcomes can be used as an indicator of the biological activity of the retinoic acid supplementation because disease intensity was modified and cellular responses were altered. 4c.List any significant activities that support special target populations. None. 4d.Progress report. This project serves as a bridging vehicle for expanded analysis of the effects of micronutrients on immune responses at mucosal surfaces to infectious agents and immune based pathologies. A new project plan entitled, "Measuring Mucosal Derived Immunity in Swine with Different Vitamin A Status to Yield Biomarkers of Human Nutrient/disease Interactions" was approved in May 2005. The work is an extension of an initiative to evaluate the nutritional effects on immune function. There will be an expanded evaluation of type 2 immune cytokine response patterns that relate to protective immunity to parasitic worm infections, but also mimic the responses to allergens. The anti-inflammatory properties of the type 2 cytokines will also be evaluated under conditions of type 1-cytokine-induced pathology and inflammatory diseases at mucosal surfaces. Progress on the project has accrued rapidly because of the cooperative relationship between scientists in the Animal and Natural Resources Institute and the Beltsville Human Nutrition Research Center. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Measurement of changes in biomarkers within a defined large animal model of human infectious and allergic disease will be unique and may have potentially great impact to the study of the effect of diet on immunity. This will benefit agriculture and human health in the United States because these markers are predictive of appropriate and adequate immune status in commercially important livestock and humans, and provide molecular tools for hypothesis-based testing of the effect of nutrition on infectious and metabolic diseases. This component fits Strategic Plan Human Nutrition Requirements 3.1.1.1: To develop appropriate animal models that will be used to identify specific biomarkers that are associated with developmental effects or disease prevention. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Gene sequences were deposited in GenBank, making them available to the global research community free of charge. Technology transfer of real time PCR assays developed under this project have been transferred to other laboratories in ARS, and to numerous extramural collaborators within the U.S. and international collaborators in Scotland, Denmark, Japan, Spain, and Italy. The pig gene data base is currently on the "demonstration server" for the Beltsville Area and will become public in August 2005, to provide annotated use of the current information on pig gene expression and comparisons with similar genomic information in humans. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Recognition of the Porcine Immunology and Nutrition (PIN) database posted on The PigSite (August 30, 2005), http://www.thepigsite.com/LatestNews/ Default.asp?AREA=LatestNews&Display=9980>. Wisconsin Agconnection (August 29, 2005) http://www.wisconsinagconnection.com/story-national.cfm?Id=869 & yr=2005. Review Publications Dawson, H., Beshah, E., Nishii, S., Solano-Aguilar, G., Morimoto, M., Zhao, A. Madden, K., Ledbetter, T., Dubey, J.P., Shea-Donohue, T., Lunney, J.K., and Urban, J. F. Jr. 2005. Localized multi-gene expression patterns support an evolving Th1/Th2-like paradigm in response to infections with Toxoplasma gondii and Ascaris suum. Infection and Immunity. 73(2):1116-1128. Guthrie, H.D., Wall, R.J., Pursel, V.G., Foster Frey, J.A., Donovan, D.M., Dawson, H.D., Welch, G.R., Garrett, W.M. 2005. Follicular expression of a human B-cell leukemia/lymphoma-2 (Bcl-2)transgene does not decrease atresia or increase ovulation rate in swine. Journal Of Reproduction, Fertility And Development. 17(4):457-468. Schopf, L., Luccioli, S., Bundoc, V., Justice, P., Chan, C., Wetzel, B., Norris, H., Urban, J., Keane-Myers, A. 2005. Differential modulation of allergic eye disease by chronic and acute Ascaris infection. Investigative Ophthalmology and Visual Science. 46:2772-2780. Royaee, A.R., Husmann, R.J., Dawson, H.D., Calzada-Nova, G., Schnitzlein, W.M., Zuckermann, F., Lunney, J.K. 2004. Deciphering the involvement of innate immune factors in the development of the host responses to Porcine Respiratory and Reproductive Syndrome Virus (PRRSV) vaccination. Veterinary Immunology and Immunopathology. 102(3):199-216. Zarlenga, D.S., Dawson, H.D., Kringel, H., Solano Aguilar, G., Urban Jr, J.F. 2004. Molecular cloning of the swine IL-4 receptor and IL-13 receptor alpha 1 chains: effects of experimental toxoplasma gondii, ascaris suum and trichuris suis infections on tissue mrna levels. Veterinary Immunology and Immunopathology 101(3-4):223-234.