2006 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? Why does it matter? This project addresses objectives of the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." Identification of the beneficial and/or adverse biological effects of probiotics and their derivatives is the first step in understanding the potential impact they will have on health. It also addresses the Action Plan for Food Safety (animal and plant products - 108) related to objective to "Develop immunological based interventions, including vaccines and non-specific immune stimulants." The prevalence of allergic and inflammatory bowel diseases has increased in Western societies over the last 50 years. It has been proposed that the widespread use of antibiotics and processed foods have limited human exposure to bacterial antigens; however, appropriate animal models to test these claims have not been developed. The current proposal tests the hypothesis that probiotic microbes derived from humans can establish in pigs and induce beneficial effects on immunity to infection and control of inflammation and allergy. Increasing consumer awareness of the link between diet and health has promoted the introduction of probiotic bacteria into the diet of Americans; they have been commonly used in the diet of many European and Asian societies for decades. Probiotics are a class of microorganisms mainly from the Lactobacillus and Bifidobacterium species that have been originally isolated from the normal flora of Humans and animals. These microorganisms can establish and grow in a compartment of the host after consumption and may provide some positive health benefits including protection against pathogenic microorganisms and viruses, stimulation of the gut immune system and enhanced disease resistance, correction of some bowel diseases, reduced allergic disease, and protection against carcinogens. The basis of such claims, however, is often confounded by a lack of demonstrable growth and function of the probiotic strain in the gut. Scientific validation of many of the claims of probiotic activity is missing. Consumer concern about probiotic reliability, efficacy, and safety can be addressed by hypothesis-based testing of probiotic strains under controlled experimental conditions with adequate statistical testing of the data. Therefore, the overall objective of this research focuses on testing if dietary probiotics can safely enhance immune function and gut health by preventing the onset of allergenic responses and improving the immune response against infectious agents of the GI tract. Sound scientific evidence for probiotic efficacy will provide the food industry and regulatory agencies with relevant information for concerned consumers. Those at risk of chronic nutrition-related diseases will directly benefit. Development of an animal model to test the unique nature of various probiotics species and strains and their efficacy against particular disease conditions will benefit the functional food industry and health care providers that recommend dietary interventions to improve healthy outcomes. 2.List by year the currently approved milestones (indicators of research progress) Milestone 1 (1-3 years) - The research will define. 1)the colonization potential of probiotic bacteria in the porcine gut, and. 2)the activation of immune system in the pig after probiotic treatment to validate the pig as a large animal model for human-derived probiotics on immune development. Milestone 2 (1-4 years) - The research will characterize the role of dietary probiotics on the development and severity of allergic disease and test the hypothesis that the moderate activation of pro-inflammatory cytokines can regulate immediate-type hypersensitivity reactions. Milestone 3 (2-4 years) - This research will determine the impact of dietary probiotics in prevention of bacterial-induced pathologies of the GI tract. Milestone 4 (3-5 years) - This research will provide evidence to support the effect of dietary fiber (barley) on probiotic colonization of the pig intestine, and biological markers of host mucosal health and barrier function. 4a.List the single most significant research accomplishment during FY 2006. Probiotics activate local innate-immune-system: This accomplishment addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations". The immune response of probiotic treated pigs measured by changes in gene expression of immune mediators indicated that there is a local activation of the innate-immune-system at the intestinal mucosa. This response is enhanced in Bifidobacterium lactis-treated pigs from treated sows. The results suggested that administration of probiotics in the diet may be an alternative stimulant of the neonatal immune system improving responses to subsequent infectious agents. Other strains from the Lactobacillus species have been tested. The level of host innate-immune-system activation was down-regulated compared to vehicle-treated control pigs over several sites in the large and small intestinal mucosa. These results indicate that there are important differences among probiotic strains. Certain probiotic strains induce innate immune activation in intestinal sites where numbers of bacteria are high and can down-regulate genes in more distal sites in the small intestine mucosa. Other strains may have a more general pattern of down regulation of innate-immune gene expression throughout the intestine. This research promotes the concept that probiotics in the diet can differentially affect immune components that can enhance resolution of infectious and chronic disease. 4b.List other significant research accomplishment(s), if any. Pig model for the study of milk allergy: This accomplishment addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." A neonatal pig model has been developed to test the effect of dietary probiotics on the immune response to cow's milk allergy. Food allergy is a growing problem in children and can be expressed throughout life. Cow's milk was fed to sows and their piglets at one to three, and 5 to 7 days after birth, and expression of allergic disease was determined by clinical and molecular scoring at subsequent times after a challenge exposure to cow's milk. A partial evaluation of the data acquired has shown that glucose absorption in the intestine can be used as a marker of an allergic response and that reduced glucose absorption is corrected by use of a probiotic in the diet. This is part of a Trust Fund Agreement with Nestle USA, Inc., as part of 1235-52000-054-01T that addresses the issue of control over milk allergy in pediatric human populations through the uses of early probiotic feeding in the diet. 4c.List significant activities that support special target populations. None. 4d.Progress report. The focus of the project has been on measuring the presence of probiotic bacteria in intestine of treated animals and the effect that this colonization has on the immune markers (cytokines, receptors, or mediators of the immune response) of the host. Flow cytometric assays have been developed to determine changes in cellular phenotype of probiotic-treated animals versus controls. Certain probiotic bacterial strains induce activation of the immune system by up regulation of components of the innate immune response. Other probiotic strains induce a generalized down regulation of pro-inflammatory mediators. This effect would be particularly useful when inflammatory responses need to be reduced to minimize overt tissue damage that often accompanies infectious and chronic diseases. In addition, probiotics may have some beneficial effect in improving gut function. This experimental system provides opportunities for examining the role of diet and nutritional status from the fetus to mature adult in an animal species that has immune and physiological systems that function closely to that of humans. 5.Describe the major accomplishments to date and their predicted or actual impact. The feasibility of using the pig as a large animal model for human nutritional interventions (i.e., use of probiotics) has been tested in the current research project. Emerging new technologies such as quantitative gene expression using real-time PCR measurements and cDNA and protein microarray surveys have been used to assess the effect of nutritional status on pigs infected with parasitic worms as a surrogate for allergic responses at mucosal sites in the animal. These rapid and sensitive assays of immune status of healthy animals will be applied to studies of effective dietary interventions in humans and will provide robust identification of biomarkers of immune function that are responsive to changes in nutrition including probiotics in diet. These studies will provide quantitative data on the effect of probiotics and related functional foods on immune system development, and appropriate responses to allergic diseases and infectious agents that affect mucosal surfaces. The impact will be in the form of a large animal model to evaluate hypotheses on the effect of functional foods on healthy outcomes and the definition of definitive biomarkers. This research will provide identifiable dietary procedures that use probiotics to benefit the growing number of children that develop food allergy; it addresses the National Human Nutrition Action Plan (107) related to Performance Goal: "Health promoting intervention strategies for targeted populations." 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? The interest in this area of research is supported by a Trust Fund Cooperative Agreement with Nestle U.S.A., as part of project 1235-52000-054-01T, to study the effect of probiotics on immune function in neonatal pigs. This agreement has been expanded with additional funding to study the effect of a probiotic strain on the immune response against cow's milk allergy. A Trust Fund Cooperative Agreement with Yakult Central Institute for Microbiological Research, Japan, as part of project 1235-52000-054-02T is also in place to study the effect of Lactobacillus casei strain shirota on the development of immune function in neonatal pigs. 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). None. Review Publications Solano Aguilar, G., Dawson, H.D., Shea-Donohue, T., Madden, K., Jones, Y.L., Beshah, E., Restrepo, M., Schoene, N.W., Hare Jr, W.R., Urban Jr, J.F. 2006. Biofiobacterium lactis enhances toll-like receptor (tlr) pathway gene expression locally in the colon and enhances glucose uptake in the small intestine of pigs infected with parasitic nematode ascaris sum. American Society for Microbiology Proceedings. 106:45A.