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?
It is now well-accepted that maintaining good health requires eating a healthy diet. But what is a healthy diet? Healthy diets were once defined by their ability to prevent clinically-evident deficiency diseases. Now, with a more complete understanding of the physiological and molecular role of nutrients, definitions are changing. Vitamin A, for example, prevents blindness but has important functions beyond the eye which are not as obvious. Metabolites of vitamin A are now known to act as hormones and regulate gene expression in the immune system. The immune system also responds to oxidative stress that is produced during an infectious or inflammatory episode. Thus requirements for antioxidant micronutrients may increase and decrease depending on health status. Thus different dietary levels of micronutrients such as vitamin A, and the antioxidant micronutrients (including carotenoids, vitamin E and vitamin C) have the potential to affect resistance to infectious disease, response to vaccines, and the development of immune-mediated diseases, such as autoimmune diseases and asthma. In addition, many newly described, food-derived antioxidants have shown anti-cancer, anti-inflammatory, and anti-atherosclerotic activities. Few detailed studies of their beneficial or detrimental effects on the immune response have been performed.

This CRIS project is examining the effects of micronutrients (particularly vitamin A, but also antioxidants such as carotenoids and vitamin E) and other food-derived bioactive compounds (particularly plant-derived antioxidants) on immune function, particularly T lymphocyte-mediated immune responses. Some of these immune responses are beneficial (e.g., response to vaccination and infectious diseases) and others are not (e.g., autoimmune diseases and asthma). With regard to newly described, food-derived antioxidants, the specific goals of this project will be to understand how antioxidants found in foods may regulate lymphocyte activities and the immune response in general, and to define appropriate dietary intake levels of foods containing high concentrations of these antioxidants that would contribute to the production of a healthy immune system. The work of this CRIS project will allow us to make better, more specific, recommendations about healthy dietary levels of these compounds.

Public health will benefit from increased knowledge of the effects of vitamin A and antioxidants on immune function. The research will improve public understanding of appropriate dietary intake of vitamin A and food-derived antioxidants as whole foods and/or supplements that leads to a healthy immune system. Due to the anti-inflammatory nature of many food-derived antioxidants, further investigations will evaluate whether an increase in dietary intake of these substances may be useful for managing chronic inflammatory diseases, such as autoimmune disorders. Vitamin A may also have similar benefits, as indicated by work in animal models of human autoimmune and inflammatory diseases.

2.List the milestones (indicators of progress) from your Project Plan.
Vitamin A Studies

Objective 1: Use molecular techniques to identify vitamin A-responsive genes in naïve T-helper (Th0) cells and determine the role of these genes in the development of Th1 and Th2 memory cells, a process that is responsive to vitamin A. Year 1 (2004): (1) Complete microarray experiments. (2) Analyze microarray expression data (retinoid vs. control). (3) Identify candidate retinoid-responsive genes. Years 2-3: (1) Complete RT-PCR analyses of genes selected in year 1; (2) Determine if ligands for RXR partner receptors affect Th1/Th2 development. Year 4: Determine if candidate genes affect Th1/Th2 development

Objective 2: Determine if high-level dietary vitamin A, as well as vitamin A deficiency, affects development of T cell-mediated immunity and inflammation in vivo. Years 3-4: Conduct diet studies on Th1/Th2 development and asthma severity.

Objective 3: Use genetic techniques to produce transgenic mice in which expression of nuclear receptors for vitamin A are disrupted in T cells to determine how vitamin A affects development of T cell-mediated immunity and inflammation in vivo. Years 1-2: Breed knockout mice and cross onto appropriate genetic backgrounds. Years 3-5: Conduct RXR knockout studies on Th1/Th2 development and asthma severity.

Objective 4: Determine if immune function is impaired in men with marginal but "acceptable" vitamin A stores (as defined by U.S. dietary guidelines) have impaired T-cell mediated and innate immunity, as compared to men with high-level stores. Years 1-2: Conduct human studies on innate and adaptive immune response. Years 3-4: Analyze immune function and clinical data.

Plant-derived Antioxidant Studies Year 1 (FY 2004): Set up laboratory for Dr. Zunino and investigate the anti-inflammatory effects of vitamin A and food-derived antioxidants on autoimmune diabetes in mice using diets containing high levels of vitamin A and grape powder which contains multiple antioxidant compounds.

Year 2 (FY 2005): (1) Assess the inhibitory activity of plant-derived antioxidants on leukemia in mice. (2) Continue investigations on the mechanistic action of antioxidants on leukemic and normal lymphocytes. (3) Continue evaluation of the anti-inflammatory effects of vitamin A and food-derived antioxidants on chronic inflammatory diseases, such as autoimmune diabetes. (4) Evaluate the effects of liminoids from citrus fruits on immune cell function in a human dietary study.

Year 3 (FY 2006): (1) Initiate human studies on the immune response to dietary intake of foods with high levels of antioxidants. (2) Continue the assessment of the inhibitory activity of plant-derived antioxidants on leukemia in mice. (3) Continue studies of mechanistic action of antioxidants at a cellular level on leukemic and normal lymphocytes. (4) Continue investigations of anti-inflammatory effects of food-derived antioxidants on chronic inflammatory diseases.

Year 4 (FY 2007): (1) Continue analysis on the immune response to dietary intake of foods with high levels of antioxidants using lymphocytes from healthy human volunteers. (2) Continue the mechanistic studies to determine activity of antioxidants at a cellular level in leukemic and normal lymphocytes. (3) Continue evaluation of anti-inflammatory effects of antioxidants from foods on chronic inflammatory diseases.

Year 5 (FY 2008). (1) Continue analysis on the immune response to dietary intake of foods with high levels of antioxidants using lymphocytes from healthy human volunteers. (2) Continue the mechanistic studies to determine activity of antioxidants at a cellular level in leukemic and normal lymphocytes. (3) Continue evaluation of anti-inflammatory effects of antioxidants from foods on chronic inflammatory diseases.

3.Milestones:
Vitamin A Studies

Objective 1: Year 1 (2004): (1) Complete microarray experiments. (2) Analyze microarray expression data (retinoid vs. control). (3) Identify candidate retinoid-responsive genes. All 3 milestones have been fully met.

Objective 3: Years 1-2: Breed knockout mice and cross onto appropriate genetic backgrounds. This milestone has been substantially met. Our RXR-alpha and RXR-beta mice have been produced and backcrossed onto C57Bl/6 mice to the extent that 104 out of 108 genomic markers are now derived from the C57Bl/6 strain (June, 2004). One to two more generations will be required to complete this backcross.

Objective 4: Years 1-2: Conduct human studies on innate and adaptive immune response. This goal has been substantially met as innate immune function has been examined in 32 subjects before and after adjustment of vitamin A stores and measurement with stable isotope dilution in Bangladesh.

Plant-derived Antioxidant Studies

Dr. Zunino has established her research laboratory at the WHNRC and studies examining the anti-inflammatory effects of vitamin A and food-derived antioxidants on autoimmune diabetes in mice using diets containing high levels of Vitamin A and grape powder which contains multiple antioxidant compounds.

3B. List the milestones (from the list in Question #2) 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?

Vitamin A Studies

Objective 1: Years 2-3 (FY2005-6): (1) Complete RT-PCR analyses of genes selected in year 1. (2) Determine if ligands for RXR partner receptors affect Th1/Th2 development. Year 4 (FY2007): Determine if candidate genes affect Th1/Th2 development.

Objective 2: Years 3-4 (FY2006-7): Conduct diet studies on Th1/Th2 development and asthma severity.

Objective 3: Year 2 (FY2005): Breed knockout mice and cross onto appropriate genetic backgrounds. Years 3-5 (FY2006-8): Conduct RXR knockout studies on Th1/Th2 development and asthma severity.

Objective 4: Year 2 (FY2005): Conduct human studies on innate and adaptive immune response. Years 3-4 (FY2006-7): Analyze immune function and clinical data.

Plant-derived Antioxidant Studies

Year 2 (FY 2005): (1) Assess the inhibitory activity of plant-derived antioxidants on leukemia in mice. By utilizing mice with leukemia as a model, we can evaluate whether certain plant-derived antioxidants can eliminate or reduce the presence of leukemia in a whole animal. We expect these substances to significantly reduce the presence of the leukemia in these mice. (2) Continue investigations on the mechanistic action of antioxidants on leukemic and normal lymphocytes. By comparing activities of antioxidants in leukemic lymphocytes and normal lymphocytes from human blood, we will determine both the anti-cancer and anti-inflammatory activities of these substances. (3) Continue evaluation of the anti-inflammatory effects of vitamin A and food-derived antioxidants (grape powder) on chronic inflammatory diseases, such as autoimmune diabetes. Vitamin A has been shown to inhibit development of TH1 subtype of lymphocytes involved in this autoimmune disease. The antioxidants found in grapes have been shown to have anti-inflammatory activity. We expect to observe inhibition of autoimmune diabetes by these substances. (4) Evaluate the effects of liminoids from citrus fruits on immune cell function in a human dietary study. Liminoids from citrus fruits have been shown to have anti-inflammatory activity. We will evaluate the immune responsiveness of lymphocytes isolated from human volunteers that have ingested liminoids over time to determine the effects of these substances on immune function. We expect to observe a decrease in inflammatory ability by liminoid ingestion which may be beneficial in humans with chronic inflammatory disease, including those with atherosclerosis.

Year 3 (FY 2006): (1) Initiate human studies on the immune response to dietary intake of foods with high levels of antioxidants. It will be necessary to analyze the effects of antioxidants on immune responsiveness directly from humans that have added high levels of antioxidants to their diets. We can then determine bioavailability of antioxidants in the serum and analyze the effects of these substances on the health of the immune system. (2) Continue the assessment of the inhibitory activity of plant-derived antioxidants on leukemia in mice. We expect these substances to significantly reduce the presence of the leukemia in these mice. (3) Continue studies of mechanistic action of antioxidants at a cellular level on leukemic and normal lymphocytes. By comparing activities of antioxidants in leukemic lymphocytes and normal lymphocytes from human blood, we will determine both the anti-cancer and anti-inflammatory activities of these substances. These studies will require continued evaluation to elucidate the complex cellular mechanisms of action. (4) Continue investigations of anti-inflammatory effects of food-derived antioxidants on chronic inflammatory diseases. We expect to observe inhibition of autoimmune diabetes by these substances using diabetic mice. Continued analysis will reveal the mechanism of inhibition of disease progression.

Year 4 (FY 2007): (1) Continue analysis on the immune response to dietary intake of foods with high levels of antioxidants using lymphocytes from healthy human volunteers. It will be necessary to analyze the effects of antioxidants on immune responsiveness directly from humans that have added high levels of antioxidants to their diets. We can then determine bioavailability of antioxidants in the serum and analyze the effects of these substances on the health of the immune system. These studies will require continued evaluation to elucidate the effects of antioxidants on immune response. (2) Continue the mechanistic studies to determine activity of antioxidants at a cellular level in leukemic and normal lymphocytes. By comparing activities of antioxidants in leukemic lymphocytes and normal lymphocytes from human blood, we will determine both the anti-cancer and anti-inflammatory activities of these substances. These studies will require continued evaluation to elucidate the complex cellular mechanisms of action. (3) Continue evaluation of anti-inflammatory effects of antioxidants from foods on chronic inflammatory diseases. We expect to observe inhibition of autoimmune diabetes by these substances using diabetic mice. Continued analysis will reveal the mechanism of inhibition of disease progression.

4.What were the most significant accomplishments this past year?
A. The intake of micronutrients impacts immune function in humans. A study done among members of the Reaching for Excellence in Adolescent Health (REACH) cohort study with collaborators from the University of Alabama at Birmingham (Dr. Craig Wilson) and the Iowa State University (Dr. Grace Marquis and Lori Kruzich) found that African American adolescents and young adults in the early stage of HIV disease have low intakes of some micronutrients, particularly iron and vitamin E, important for optimal immune function. These low intakes may impair the immune response to infection and hasten the progression of HIV disease. This study will allow nutritionists to formulate more specific dietary recommendations for adolescents and young adults with HIV infection.

B. We succeeded in ablating the RXR-alpha gene in murine T lymphocytes using the cell-specific Cre-lox method for disruption of gene expression. This is important because it will allow us to specifically examine the role of this receptor, which binds vitamin A and lipids in order to regulate gene expression, in mediating T lymphocyte function. In collaboration with Dr. Kent Lloyd of UC Davis we produced a strain of mice in which the RXR-alpha gene is disrupted in T lymphocytes, but not other cells, including other cells of the immune system such as B lymphocytes. Use of these mice in future studies will allows us to determine if vitamin A and other nutrients that act via the RXR pathway modulate immune responses and autoimmune disease by acting on T lymphocytes, or if their activity involves other cells of the immune system.

C. Our analysis of data from the REACH population is providing information on the role of immune-modulating nutrients on progression of HIV disease in a cohort of subjects that are primarily young, African American women. A significant portion of subjects are also Hispanic.

D. None.

5.Describe the major accomplishments over the life of the project, including their predicted or actual impact.
This project is in its first year and accomplishments have been outlined above.

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?
Results of our research have been transmitted to the scientific community via publication in peer-reviewed journals.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work.
Stephensen, C.B. 2004. Vitamin A deficiency increases the development of IL-10 producing Th2 cells. In: Proceeding of Nutritional Immunology, April 17, 2004, Washington, D.C.

Review Publications
Stephensen, C.B. 2003. Commentary: a hypothesis concerning vitamin a supplementation, vaccines and childhood mortality. International Journal of Epidemiology. 32(5):828-829.