2004 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? The role of antioxidant nutrients in the maintenance of mucosal immunity, gastrointestinal (GI) tract function, and resistance to orally-acquired enteric viral and bacterial infections is not well understood. Food-borne illnesses, ranging from mild to severe life-threatening syndromes, are caused by a variety of agents including viruses, bacteria, and parasites. The intestinal barrier is the first line of defense against orally acquired infections, but few studies have examined the role that nutrition plays in the maintenance of normal barrier function and mucosal immunity. Use of mouse models of orally acquired viral and bacterial infections is a practical approach to examining the role of nutrition, with an emphasis on antioxidant nutrients, in the maintenance of normal intestinal function and immunity to infectious agents. Seventy-six million cases of food-borne illness occur each year in the United States (U.S.) at a cost of $3-7 billion annually with approximately 31 million of these cases attributed to viruses. Of this number, 23 million represent infection with Norwalk-like viruses and roughly 4 million each to rota- and astro-virus. Salmonella and Campylobacter spp. induce the largest percentage of food-borne bacterial illnesses with Salmonella spp. accounting for about 26% of the hospitalizations and 31% of all deaths; making it one of the most important pathogens affecting the U.S. population today. Therefore, it is important to define the role that pro- and antioxidant nutrients have in maintenance of normal intestinal physiology and immunity. Any dietary considerations derived from this research could be used to define a health-promoting diet that would improve resistance or recovery from intestinal infections, have a broad impact on the large number of people affected by these illnesses, and could result in reduced health care costs. The proposed research falls under the Human Nutrition Program (107) and within Action Plan 3.1.1, Nutrition Requirements. The ARS Human Nutrition Program Rationale states that, "...A continuing need is to expand our understanding of the roles nutrients play in maintaining health and to identify those components of foods that are most beneficial." The proposed research clearly is aimed at understanding the role that dietary nutrients play in maintaining health and reducing the morbidity and mortality associated with acute viral and bacterial infections. This research promotes several goals of Action Plan 3.1.1 including: 1)the need "to establish healthful intakes of specific nutrients;" 2)"defining interventions for reducing disease risks;" and 3)"develop effective strategies, based on the results of sound human nutrition research, to lower the cost of health care by prevention of diseases."  2.List the milestones (indicators of progress) from your Project Plan. Milestone 1 (12-24 months)- Identify viral and bacterial models of food-borne pathogens suitable for use in nutritional studies by first identifying diet/pathogen combinations that result in enhanced oxidative stress and increased disease severity (pathogenicity). Milestone 2 (12-24 months)- Identify oxidative stress-induced innate and acquired immune deficits in response to viral and bacterial gastrointestinal (GI) infections as they are altered in response to nutritionally-induced oxidative stress. Milestone 3 (24-48 months)- The results from milestones 1 and 2 will be used to determine the parameters of innate and acquired mucosal immunity that relate to changes in GI physiological function including alterations in smooth muscle contractility, and epithelial cell absorption and secretion. Milestone 4 (12-48 months)- The redistribution of trace elements in response to GI infection and their possible role in developing local mucosal immune and physiological responses will be determined. Milestone 5 (48-60 months)- Completion of technology transfer on the interaction between immune and physiological responses to infections in the intestine will likely provide targets for control. The information on trace mineral redistribution will be a basis for examining mechanisms of action related to specific mineral requirements. 3.Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plan to do so. Milestone 1 (12-24 months)- A model to examine the effect of nutrition on orally-acquired bacterial infections has been established and initial studies examining the effect of selenium and/or vitamin E deficiencies are complete. Two virus models, reovirus T1L, and coxsackievirus B3 are currently being evaluated under these dietary conditions. Clinical, immunological and molecular changes in response to infection will now be analyzed. Milestone 2 (12-24 months)- The model developed in experiments described in Milestone 1 is being analyzed to determine the most critical diet and pathogen interactions that result in enhanced oxidative stress and increased disease severity. We originally proposed to look at the effect of selenium or vitamin E deficiencies on gut immunity to bacteria and viruses. However, initial studies indicate that deficiencies in these two nutrients may not significantly impact gut immunity to these microbial pathogens. Therefore, deficiencies in other nutrients important in antioxidant defense, including magnesium, are being evaluated for their impact on gut immunity and response to infection. Milestone 4 (12-48 months)- Mapping of redistribution of trace elements in response to GI infection and their possible role in developing immunity have been initiated. Data has been being acquired on the effect of GI parasitic infections on trace element distribution in both infected mice and pigs. We are currently acquiring additional tissue samples to increase the number of animals per groups to enhance statistical power of the analysis. B. List the milestones that you expect to address over the next 3 years(FY 2005, 2006, & 2007). What do you expect to accomplish year by year, over the next 3 years under each milestone. Milestone 2 (24-48 months)- We anticipate extending the model for studying the effects of nutrition on mucosal immunity to viral infections of the upper respiratory tract in FY 2005 as well. Milestone 3 (12-24 months)- We will continue to evaluate selenium and vitamin E as well as other nutrients including magnesium, copper, manganese, and iron for their effects on oxidative stress in the intestine and the impact on microbial infections. Once nutritional deficiencies are identified that increase the pathogenesis of bacterial and/or viral infections, those aspects of immune function that are altered will be identified. Genomic and proteomic methods will be employed to determine how nutrition is effecting the transcription and translation of genes important for mucosal immunity. We will employ real time PCR arrays to measure nutritionally induced changes in immune function-related gene expression including genes related to cytokine and chemokine levels. Proteomic analysis will follow to support the gene expression patterns observed including the use of ELISA and two-dimensional electrophoresis. Measures of oxidative stress will be assessed using biochemical approaches that quantify protein and lipid markers of oxidative damage. Immune cell changes will be evaluated using flow cytometric methods to identify alterations in immune cell populations as a result of nutritional deficiencies. Milestone 4 (12-48 months)- Research on the effect of GI nematode infections on trace element distribution in murine and porcine models will continue with anticipated identification of critical nutrients that affect immune function. We hypothesize that oxidative stress will alter immune function and that this is likely to occur at the genomic level via altered gene expression. Several important immune-related transcription activators will be studied to determine if their activity is altered under conditions of oxidative stress and the mechanism by which oxidative stress can alter their activity. NF-(B and AP-1 transcription factors will be studied along with the interferon regulatory factor family of transcription factors, ATF, NFAT, Elk-1, the STAT family of transcription factors, and Gata-3. 4.What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment During FY 2004: The demonstration that dietary deficiencies in selenium and/or vitamin E exacerbate the intensity and pathology of coxsackievirus B3 (CVB3) and influenza viral infections is a seminal observation. Since both selenium and vitamin E are important for the antioxidant defense of the host, it is believed that nutrient deficiency-induced oxidative stress is the driving force behind the rapid evolution of these viruses. We have conducted research to determine if deficiencies in selenium and/or vitamin E have more comprehensive affects on a variety of GI infections including parasitic worm infections which, in many ways, mimic allergic reactions to food allergens. The results clearly demonstrate that dietary deficiencies in selenium and/or vitamin E increased persistence (delayed worm expulsion) of worms. However, only vitamin E deficiency impaired the normal physiological response involved in worm expulsion indicating that selenium and vitamin E affected different mechanisms of action. The impact of these results is that specific physiological mechanisms in the intestine require better definition to evaluate selenium and vitamin E-dependent pathways. This suggests that a wider range of changes in the intestine may be susceptible to dietary deficiencies in these important micronutrients. Altered intestinal physiology leads to poor absorption as well as defects in appropriate mucosal immune response. B. Other Significant Accomplishment: The extent of selenium and vitamin E-dependent physiological changes in response to viral and bacterial infections of the GI tract would be important in the control of food borne infections. Initial studies do not indicate that resistance to experimental coxsackievirus B3 (CVB3) is impaired during an oral infection. This indicates that more specific interactions between a particular mucosal pathogen and the level of antioxidant stress in the gut need to be evaluated. However, changes in the level of magnesium can affect viral load indicating that more specific micronutrient and infection interactions need to be definitively evaluated. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress Report: This is a new CRIS approved in March 2004. Initial studies have been conducted to examine the effect of selenium and/or vitamin E deficiencies on orally-acquired reovirus or Salmonella typhimurium infections. These results indicate that clearance of these infections was not affected by deficiencies in selenium and/or vitamin E. Similar to deficiencies in selenium or vitamin E, a deficiency in magnesium also induces oxidative stress and the effect of a deficiency in magnesium on resistance to viral and bacterial infections are underway. Preliminary results indicate that magnesium deficiency may increase the viral load in coxsackievirus B3 infected mice. This result will be corroborated with additional studies using both viruses and bacterial pathogens. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Molecular analysis of the viruses isolated from the deficient mice indicated that specific mutations had occurred within the viral genomes that are associated with the increased virulence. Since both selenium and vitamin E are important for the antioxidant defense of the host, it is believed that nutrient deficiency-induced oxidative stress is the driving force behind the rapid evolution of these viruses. Based on the results of these original studies, nutritionally-induced oxidative stress was induced by dietary manipulation and the virulence and pathogenesis of other pathogens delivered orally was evaluated. Most important food borne and infectious agents that impact human and animal health enter through mucosal surfaces; however, little is known about nutrient-dependent immune mechanisms in the intestine. We anticipate that this research will increase our knowledge of the impact that oxidative stress has on immunity to and the pathogenesis of bacterial and viral infections. The results of this research will provide health-care professionals and the public with information they need to determine whether specific nutrients can improve disease resistance and GI function. The need for appropriate animal models to evaluate dietary effects on intestinal and mucosal immunity against viral and bacterial infections is limited. During the first 4 months of this CRIS we have characterized a mouse model for use in nutrition studies assessing the effect of antioxidant nutrients on mucosal immunity to bacterial and viral infections delivered orally. Initial studies have been conducted examining the effect of selenium and/or vitamin E deficiencies on orally-acquired reovirus or Salmonella typhimurium infections. These results indicate that clearance of these infections was not affected by deficiencies in selenium and/or vitamin E. However, similar to deficiencies in selenium or vitamin E, a deficiency in magnesium also induces oxidative stress and the effect of a deficiency in magnesium on resistance to viral and bacterial infections are underway. Preliminary results indicate that magnesium deficiency may increase the viral load in systemically infected coxsackievirus B3 infected mice. Magnesium deficiency is common among trauma patients that are treated in acute care facilities. These results would suggest that greater cautious should be exercised in the handling of trauma patients to prevent greater susceptibility to secondary microbial infections during acute injury. 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? A grant to the FDA intramural program on Women's Health Initiative was submitted in cooperation with scientists at the FDA Murkirk Road facility to address the issue of functional trace mineral and vitamin E deficiencies during pregnancy. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. None. Review Publications Smith, A.D., Madden, K., Zhao, A., Auyeung, K., Levander, O.A., Finkelman, F., Urban Jr, J.F., Shea Donohue, P.T. 2004. Selenium (se) and vitamin E (ve) deficiencies impair intestinal function and result in persistent infection with heligmosomoides polygyrus [abstract]. Federation of American Society of Experimental Biology Journal. 18(4):A9. Nelva, B., Levander, O.A., Hines, F., Smith, A.D. 2004. Study of the effect of copper deficiency on cvb3/0 and cvb3/20 infections: The light microscopic evaluation of the pancreata and hearts of c3h/hej mice [abstract]. Toxicologic Pathology. 32(1):146. Jun, W., Bhagwat, A.A., Gross, K.C., Smith, A.D., Angle, S.J. 2004. Membrane-derived oligosaccharides of Salmonella enterica serovar Typhimurium. American Society for Microbiology Annual Meeting. p. 107.