Progress 08/01/99 to 01/17/04
Outputs
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, 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 naive 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. 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. More knowledge is needed to understand the effects of micronutrients on immune functions. Research conducted at the Western Human Nutrition Research Center, Davis, CA, in collaboration with 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 and their 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. What were significant activities that support special target populations? 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.
Impacts
(N/A)
Publications
- Drammeh BS, Marquis GS, Funkhouser E, Bates C, Eto I, Stephensen CB. A randomized, 4-month mango and fat supplementation trial improved vitamin A status among young Gambian children. Journal of Nutrition. 2002 Dec; 132(12):3693-9.
- Stephensen CB, Franchi LM, Hernandez H, Campos M, Colarossi A, Gilman RH, Alvarez JO. Assessment of vitamin A status with the relative-dose- response test in Peruvian children recovering from pneumonia. American Journal of Clinical Nutrition. 2002 Dec;76(6):1351-7.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? In the coming year (2004), we will continue these studies and plan to perform promoter analysis studies to determine if these genes are directly regulated via the RXR pathway. In the coming year and the following we will also determine if the genes regulated by RXR agonists play a role in Th2 development, and determine how T-cell function is altered by disruption of RXR-alpha. We will also pursue dietary studies to determine if vitamin A has the same effect on Th2 development as do synthetic RXR agonists during the next two years. In years two and three we will knock out expression of RXR-beta in T cells to determine how T cell function is altered. In years two and three we plan to determine if such genetic disruptions to RXR-alpha and -beta affect immune function and the pathogenesis of mouse model of asthma. 2. How serious is the problem? Why does it matter? Infectious and
immune-mediated chronic diseases are currently a major cause of morbidity in the U.S. population and worldwide. Diet can be a potent modulator of the immune response. Thus understanding the molecular role of micronutrients in modulating the immune response will help in making dietary recommendations for maintaining health and preventing disease. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The research evaluates the role of micronutrients in maintaining optimal immune function. It will thus help in setting dietary micronutrient requirements as well as defining the health-promoting properties of plant and animal foods. Both of these areas are key components of the ARS National Program 107 in Human Nutrition. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during the past year: Although oxidative stress contributes to the progression of chronic infectious
and inflammatory diseases (e.g., HIV infection and arthritis) by causing oxidative damage to cells and tissues, reliable measures of oxidative stress are not available for use in human studies. In a study of HIV-infected and uninfected adolescents done with collaborators from the University of Alabama at Birmingham and Iowa State University, investigators from the Western Human Nutrition Research Center examined the relation of blood levels of antioxidants and the antioxidant enzyme glutathione peroxidase to malondialdehyde, a product of oxidative damage to lipids. Analysis of these data revealed that glutathione peroxidase levels were elevated in HIV infection and were strongly associated with malondialdehyde concentrations, suggesting that elevated levels of glutathione peroxidase may be a useful marker of oxidative stress and that monitoring changes in this enzyme, or other antioxidant enzymes, may be a sensitive method for evaluating changes in oxidative stress for an
individual over time. B. Other significant accomplishments: Adolescents with HIV infection are at increased nutritional risk because of the demands of growth and disease as well as poor dietary habits. In a study of HIV-infected and uninfected adolescents done with collaborators from the University of Alabama at Birmingham and Iowa State University, investigators from the Western Human Nutrition Research Center collected dietary intake, anthropometric, biochemical, clinical, and sociodemographic data. HIV-infected participants with normal CD4+ T-cell counts had decreased iron intake and tended to have lower intakes of vitamins C and E compared to those with more advanced disease and HIV- uninfected youth. Given the increased micronutrient requirements during HIV infection, nutrition counseling with HIV-infected youth should focus on early increase of intake of foods rich in micronutrients to improve growth, slow disease progression, and increase survival. Maintaining adequate
dietary quality and healthy weight status is important for maintaining optimal health among HIV-infected and uninfected adolescents. In a study of HIV-infected and uninfected adolescents done with collaborators from the University of Alabama at Birmingham (Craig Wilson) and Iowa State University investigators from the Western Human Nutrition Research Center collected dietary intake, anthropometric, biochemical, clinical, and sociodemographic data. About half the subjects (including both HIV-infected and uninfected), who were primarily African American and female, were overweight or obese and the overall diet quality was poor, particularly in those who were HIV- positive and sedentary. These data demonstrate that overweight and obesity are common nutrition problems for HIV-infected youth and suggest that nutrition education for HIV-infected youth should focus on helping youth improve their diet quality and increase physical activity to reduce health consequences associated with
both obesity and HIV infection. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project began in July, 1999 and has resulted in several significant accomplishments. We have found that active infection or inflammation - as indicated by elevated serum CRP concentrations - interfere with the assessment of vitamin A status using either serum retinol concentrations or the relative dose-response (RDR) test. With regard to the effect of vitamin A on immune function, we have found that stimulation of the retinoid X receptor pathway by vitamin A analogs enhances the development of T-helper type 2 cells, which protect against infection by many food- and waterborne pathogens, but can also be associated with chronic inflammatory diseases, such as asthma. We have also found that providing mango as a food-based intervention increases plasma beta-carotene and retinol levels in young children with low vitamin A intakes, indicating
that interventions using locally available foods can improve nutritional status. With regard to antioxidant micronutrients, we have also found that young, relatively healthy HIV-infected subjects show little evidence of oxidative damage but do have indicators of increased oxidative stress (elevated glutathione peroxidase levels), suggesting that monitoring antioxidant enzyme activity may be a useful method of monitoring oxidative stress in prospective nutrition intervention studies. 6. What do you expect to accomplish, year by year, over the next 3 years? We have continued to use DNA microarray analysis to identify genes that have increases mRNA levels following treatment with a synthetic RXR agonist (a vitamin A analog) during T-cell development. We have also made progress in determining which genes are directly regulated by the RXR agonist and to determine if these genes have a role in promoting Th2 development. In addition we have bred transgenic animals which should have a
disruption of the RXR-alpha receptor in T cells, to determine if how T-cell function is impaired by knocking out this receptor. In the coming year we will continue these studies and plan to perform promoter analysis studies to determine if these genes are directly regulated via the RXR pathway. In the year 2004 and the following we will also determine if the genes regulated by RXR agonists play a role in Th2 development, and determine how T-cell function is altered by disruption of RXR-alpha. We will also pursue dietary studies to determine if vitamin A has the same effect on Th2 development as do synthetic RXR agonists during the next two years. In years 2005 and 2006, we will knock out expression of RXR-beta in T cells to determine how T cell function is altered. We plan to determine if such genetic disruptions to RXR-alpha and -beta affect immune function and the pathogenesis of mouse model of asthma. 7. 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? Scientific information has been transferred directly to scientists via three scientific publication and two poster presentations (one at the American Association of Immunologists meeting in May, 2003 and one invited presentation at the FASEB Summer Conference on Nutritional Immunology in July, 2003) and an invited seminar given to the Nutrition Department at the University of Missouri (November, 2002). Information has also been transmitted more directly to consumers via a story featuring Dr. Stephensen's work in the ARS publication Agricultural Research in March, 2003 and in an upcoming article in Health Magazine. The findings in these presentations and publications (below) are intended for medical, nutrition and public health workers concerned with the impact of nutrition on
resistance to infectious and inflammatory disease, as well as for consumers interested in nutrition and health. The finding that antioxidant supplements did not benefit healthy younger men (publication #1), while such supplements do decrease oxidative damage in older subjects, suggests that young adults are more resistant to oxidative stress. Food-based supplementation programs using fresh fruit to improve vitamin A status have had beneficial effects and our publication (#2, below) indicated that dried mango is also beneficial in improving vitamin A status. We have also shown that clinical measures of vitamin A status must be interpreted carefully in subjects with ongoing infections, because the host response to infection can alter the test result (publication # 3). 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Vitamin
A Detectives Probe Puzzling Nutrient. Agricultural Research, March 2003, pp. 10-11. Quote in October 2003 issue of Health magazine regarding beneficial effects of dietary vitamin A (from pumpkin).
Impacts
(N/A)
Publications
- Drammeh BS, Marquis GS, Funkhouser E, Bates C, Eto I, Stephensen CB. A randomized, 4-month mango and fat supplementation trial improved vitamin A status among young Gambian children. Journal of Nutrition. 2002 Dec; 132(12):3693-9.
- Stephensen CB, Franchi LM, Hernandez H, Campos M, Colarossi A, Gilman RH, Alvarez JO. Assessment of vitamin A status with the relative-dose- response test in Peruvian children recovering from pneumonia. American Journal of Clinical Nutrition. 2002 Dec;76(6):1351-7.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 asthma. In this project we are examining the bioavailability and molecular role of nutrients, particularly vitamin A, in regulating the immune response in order to make better, more specific, recommendations about healthy dietary levels of these nutrients. 2. How serious is the problem? Why does it matter? Infectious and immune-mediated chronic diseases are currently a major cause of morbidity in the U.S. population and worldwide. Diet can be a potent modulator of the immune response. Thus understanding the molecular role of micronutrients in modulating the immune response will help in making dietary recommendations for maintaining health and preventing disease. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The research evaluates the role of
micronutrients in maintaining optimal immune function. It will thus help in setting dietary micronutrient requirements as well as defining the health-promoting properties of plant and animal foods. Both of these areas are key components of the ARS National Program in Human Nutrition NP107. 4. What was your most significant accomplishment this past year? A. Both vitamin A deficiency and excessive vitamin A intake can have adverse effects on susceptibility to or recovery from infectious diseases - presumably by modulating the immune response to these diseases - but the specific mechanisms involved have not been characterized. Working with collaborators from the University of Alabama at Birmingham (Dr. Pat Bucy and Dr. Casey Weaver) and Allergan, Inc. in Irvine, CA (Dr. Roshantha Chandraratna), we have examined the effects of synthetic vitamin A analogs on the development of T helper type 1 (Th1) cells, which regulate immune responses to viruses and other intracellular pathogens, and
Th2 cells, which regulate responses to many food- and water-borne bacterial and parasitic infections. In these experiments we found that vitamin A analogs that regulate gene expression via the retinoid X receptor (RXR) pathway (one of two pathways utilized by vitamin A to regulate gene expression) enhance Th2 development, while compounds that act via the retinoic acid receptor (RAR) pathway do not. This finding describes a mechanism that could explain how vitamin A deficiency impairs resistance to food- and water-borne pathogens (as has been previously described), and also suggests that excess vitamin A intake could exacerbate Th2-mediated inflammatory diseases, such as asthma. B. We have evaluated the utility of the relative dose-response (RDR) test (a test of vitamin A status) in children recovering from pneumonia to determine if infections interfere with the assessment of vitamin A status. In collaboration with clinical investigators at Universidad Peruana Cayetano Heredia
(including Drs. Herminio Hernandez, Miguel Franchi and Miguel Campos), we compared RDR test results in children who did or did not receive high-dose vitamin A supplements. We found that children with active infection (as indicated by elevated C-reactive protein - CRP - concentrations in the blood) were more likely to have a false positive RDR test result that were children without active infection. This finding indicates that active infections interfere with the assessment of vitamin A status. Vitamin A is lost in the urine during infections, thus diminishing vitamin A reserves, but the duration of this excretion is not known. With collaborators at the International Center for Diarrhoeal Disease Research in Bangladesh, we have evaluated the length of vitamin A excretion following an episode of diarrheal disease to determine if such excretion is transient or persists for several days. We have found that approximately two-thirds of children with high excretion on day 1 of
hospitalization still have high excretion on day 3. These data suggest that a majority of children with significant vitamin A loss in the urine when initially seen at hospital are at risk of losing vitamin A in the urine for several days, and may thus be at risk of developing vitamin A deficiency. C. None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? This project began in July, 1999 and has resulted in several significant accomplishments. We have found that active infection or inflammation - as indicated by elevated serum CRP concentrations - interfere with the assessment of vitamin A status using either serum retinol concentrations or the relative dose-response (RDR) test. With regard to the effect of vitamin A on immune function, we have found that stimulation of the retinoid X receptor pathway by vitamin A analogs enhances the development of T-helper type 2 cells, which protect against infection by many food-
and waterborne pathogens, but can also be associated with chronic inflammatory diseases, such as asthma. We have also found that providing mango as a food-based intervention increases plasma beta-carotene and retinol levels in young children with low vitamin A intakes, indicating that interventions using locally available foods can improve nutritional status. 6. What do you expect to accomplish, year by year, over the next 3 years? This year we have used DNA microarray analysis to identify genes that have increases mRNA levels following treatment with a synthetic RXR agonist during T-cell development. In the coming year we will perform promoter analysis studies to determine if these genes are directly regulated via the RXR pathway. We will also begin to determine if the genes regulated by RXR agonists play a role in Th2 development. In years 2 and 3, we will pursue dietary studies to determine if vitamin A has the same effect on Th2 development as do synthetic RXR agonists. We
also plan to determine if such effects extend to affecting the pathogenesis of mouse model of asthma. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Scientific information on the effect of vitamin A on Th2 development has been transferred to scientists via one scientific publication and two poster presentations (one at the Keystone Nuclear Receptors Meeting in April, 2002; one at the FASEB Summer Retinoids Conference in July, 2002) and a seminar given to the Nutrition Department at the University of California, Davis (October, 2001). These results will help guide further experiments - in our lab and in other labs - to determine the effect of dietary vitamin A on immune function. Scientific information on the effect of infection on assessing vitamin A status using the RDR test have been
accepted for publication. This information will be available upon publication to nutrition professionals and will immediately affect the use of this test in assessing vitamin A status in subjects with infection. Scientific information on the duration and magnitude of urinary vitamin A excretion in children with watery diarrhea has been published in one article. This information is now available to clinicians and nutritionists responsible for the treatment of sick children at risk of vitamin A deficiency and should lead to increased monitoring of such children for the development of vitamin A deficiency.
Impacts
(N/A)
Publications
- Stephensen, C.B., Rasooly, R., Jiang, X., Ceddia,M.A., Weaver, C.T., Chandraratna, R.A.S., Bucy, R.P. Vitamin A Enhances in vitro Th2 Development via Retinoid X Receptor Pathway. Journal of Immunology. 2002. 168:4495-4503.
- Mitra, A.K., Wahed, M.A., Chowdhury, A.K., Stephensen, C.B. Urinary retinol excretion in children with acute watery diarrhoea. Journal of Health Population Nutrition. 2002. 20:12-17.
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Progress 10/01/00 to 09/30/01
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
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 asthma. In this project we are examining the bioavailability and molecular role of nutrients, particularly vitamin A, in regulating the immune response in order to make better, more specific, recommendations about healthy dietary levels of these nutrients.
2. How serious is the problem? Why does it matter?
Infectious and immune-mediated chronic diseases are currently a major cause of morbidity in the U.S. population and worldwide. Diet can be a potent modulator of the immune response. Thus understanding the molecular role of micronutrients in modulating the immune response will help in making dietary recommendations for maintaining health and preventing disease.
3. How does it relate to the National Program(s) and National Component(s)?
The research evaluates the role of micronutrients in maintaining optimal immune function. It will thus help in setting dietary micronutrient requirements as well as defining the health-promoting properties of plant and animal foods. Both of these areas are key components of the ARS National Program in Human Nutrition NP107.
4. What were the most significant accomplishments this past year?
Vitamin A deficiency is a serious public health problem in many areas of the world which could be alleviated by better utilization of locally produced, vitamin A-rich foods, such as mango, which is rich in beta- carotene, the precursor to vitamin A. In a collaboration with Bakary Drammeh, a doctoral student from the University of Alabama at Birmingham, Dr. Grace Marquis, of Iowa State University, and investigators under the supervision of Dr. Andrew Prentice from the British Medical Research Council Laboratory in The Gambia, West Africa, we have examined the ability of dried mangoes to increase plasma beta-carotene and retinol (vitamin A) concentrations among rural Gambian children. We found that providing approximately 50% of the U.S. RDA for vitamin A per day as rehydrated mango (5 days per week) resulted in higher plasma concentrations of both beta-carotene and retinol after a four month intervention, as compared to a control group. This study demonstrates that locally produced
and processed (dried) mango is a good source of beta-carotene which can improve vitamin A status in young children. B. We are using a transgenic mouse model system to define the molecular role of vitamin A in regulating the development of T-helper cells, which play a central role in modulating the immune response to infectious and inflammatory diseases, as well as cancer. Working with collaborators from the University of Alabama at Birmingham (Dr. Pat Bucy and Dr. Casey Weaver) and Allergan, Inc. (Dr. Roshantha Chandraratna) we conducted in vitro experiments in our laboratory at the WHNRC to determine which of the two receptors utilized by vitamin A (retinoic acid receptor or retinoid X receptor) to regulate gene expression were active in T-cells during a primary immune response. Using synthetic analogs of vitamin A specific for each type of receptor, we found that stimulation of the retinoid X receptor (which modulates gene expression in response to the vitamin A metabolite 9-cis
retinoic acid) enhances development of type 2 T-helper cells (Th2 cells), which mediate the immune response to allergens, extracellular bacteria (e.g., enterotoxigenic E. coli) and some parasites that require serum (IgG and IgE) and secretory (IgA) antibody responses, and down-regulates the development of Th1 cells, which mediate the immune response to viruses and other intracellular pathogens that require cell-mediated immune responses. This finding suggests that dietary vitamin A enhances Th2-mediated responses and thus could improve resistance to enteric infections, such as foodborne bacteria and helminthic parasites, that require secretory IgA to clear primary infections and protect against reinfection. C. Because deficient intake of antioxidant micronutrients may exacerbate the progression of HIV disease, we are conducting a study to examine the relative antioxidant requirements of HIV-infected and uninfected adolescents (for maintaining blood levels of antioxidant nutrients),
and to determine if antioxidant intake is associated with the progression of HIV disease in this largely minority population (69% black, 22% Hispanic). In collaboration with Dr. Craig Wilson of the University of Alabama at Birmingham and Dr. Grace Marquis at the Iowa State University we have collected dietary intake data and blood samples at 15 clinical sites around the U.S. to measure micronutrient status and indicators of oxidative damage in HIV-infected and uninfected adolescents in an ongoing, NIH-funded project called the REACH study. Although previous studies in U.S. adolescents have found low intakes of vitamin C, we have found that dietary intake of vitamin C is at or above the RDA in 95% of subjects in this study. This result suggests that dietary intake of vitamin C is quite good in this diverse sample of adolescents, and suggests that deficient intake will not be a common risk for more rapid progression of HIV infection in this age group. D. During this year we have spent
a significant amount of effort on developing methods to evaluate gene expression in Th0 cells during their primary response to antigen, during which they begin to develop into Th1 and Th2 memory cell populations. This has included testing various methods for isolation of these Th0 cells after stimulation in the presence of a 10- to 25-fold excess of antigen presenting cells. These methods have included use of antibody coated beads and cell sorting by flow cytometry. The latter method has been more successful and achieves a purity of 98% or better. This requires use of the Optical Biology Core Facility at U.C. Davis, which has a high-speed cell sorter (which can sort cells from among 10,000 events - cells - per second). In addition, we have developed methods for examining the expression of retinoid receptor genes (RAR-alpha, -beta and -gamma as well as RXR- alpha, -beta and -gamma) and genes in the Th2 development pathway (including the IL-4 receptor, STAT6, GATA3 and c-maf). This
method involves isolation of RNA from sorted cells, reverse transcription to make cDNA, and amplification of cDNA by PCR using the LightCycler instrument (Roche) to quantify number of copies of each gene. In addition, we clone each specific PCR product to prepare a standard curve for this assay. the identity of each clone is confirmed by DNA sequence analysis at the UC Davis Sequence Analysis Core facility. Furthermore, we have been preparing for in vivo studies to confirm our in vitro observations on the effect of retinoids on Th1/Th2 development. This work has taken two tacks. First, we have confirmed that we can administer retinoic acid and synthetic analogs to mice by gastric gavage and achieve appropriate blood levels of these compounds (confirmed by HPLC). This approach has taken precedence over dietary studies, as we wish to confirm if receptor-selective compounds are active in vivo, as they are in vitro. Following these studies, we will perform dietary interventions to
determine if vitamin A has the same effect on Th2 development in vivo. In preparation for these in vivo studies, we have also confirmed that we can adoptively transfer our DO11.10 transgenic T- cells into recipient mice, administer retinoids during primary immunization, and identify (by flow cytometry) the adoptively transferred cells a month later in order to characterize the development of Th1 and Th2 memory populations. Performing the latter analysis required development of intracellular cytokine staining protocols to identify murine Th1 and Th2 cells, a process that we are currently completing. This Th1/Th2 analysis will be pursued in advance of other studies examining the effect of retinoid treatments and dietary vitamin A interventions on antibody responses mediated by the DO11.10 transgenic T- cells. This year we have also tested methods for these (secretory IgA) antibody studies. Another component of our analysis of Th1/Th2 development is the collaborative study that is
being carried out with Dr. Nelly Zavaleta of the Instituto de Investigacion Nutricional in Lima, Peru. We are currently following infants in a community study providing vitamin A-, zinc- and folate-fortified weaning food. In preparation for analysis of the effect of these supplements on Th1/Th2 development, we have established intracellular cytokine staining protocols to examine Th1/th2 development in human T-cells. As part of this process we have characterized the Th1/Th2 memory cell population of 10 healthy adult volunteers in Davis. Preliminary analysis of lymphocytes from the Peruvian subjects is now in progress. With regard to our studies of antioxidant nutrients in HIV-positive and HIV-negative adolescents, we completed several analyses this year(protein carbonyls as a measure of protein oxidation, plasma CRP and cerruloplasmin levels) and are currently analyzing samples for plasma lipid peroxides by HPLC in collaboration with Dr. Robert Jacob's laboratory.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
This project began in July, 1999 and the accomplishments described above are the major accomplishments over the life of the project.
6. What do you expect to accomplish, year by year, over the next 3 years?
In the coming year we plan to continue studies to examine two mechanisms by which 9-cis retinoic acid promotes Th2 development: (1) We will continue to identify genes which are regulated by 9-cis retinoic acid and synthetic RXR agonists during Th2 development. Current candidates include genes in the Th2 development pathway. The role of these genes in mediating the effects of 9-cis retinoic acid on Th2 development will be examined in the coming two years. (2) We will continue experiments to define which retinoid receptors (RAR-alpha, -beta or gamma; RXR-alpha, -beta or -gamma) mediate the Th2-promoting activity of 9-cis retinoic acid. These studies will continue through year 3. In years 2 and 3, we will pursue dietary studies to determine if vitamin A has the same effect on Th2 development as does 9-cis retinoic acid. We also plan to determine if such effects extend to affecting the pathogenesis of mouse model of asthma.
7. 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 & durability of the technology product?
Scientific information on the effect of 9-cis retinoic acid and receptor selective retinoid agonists on Th2 development has been transferred to scientists via four oral and one poster presentation at Experimental Biology 2001 (April, 2001), a poster presentation at the Midwinter Immunology Conference (January, 2001), and seminars given to the Nutrition Department at the University of California, Davis (October, 2000) and the Department of Nutrition and Food Science at the University of Florida (February, 2001). Data concerning Th1/Th2 development from FY2001 were published in abstract form and a manuscript was submitted for publication. A related review article was published in 20001. It is too early to determine how implementation of these results regarding the impact of retinoic acid on T-cell development will affect health in the U.S. population.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
None.
Impacts
(N/A)
Publications
- Kemper, C., Leung, M., Stephensen, C.B., Pinkert, C.A., Liszewski, M.K., Cattaneo, R., Atkinson, J.P. Membrane cofactor protein (MCP; CD46) expression in transgenic mice. Clinical and Experimental Immunology 124:180-89, 2001.
- Stephensen, C.B. Vitamin A, Infection and Immune Function. Annual Review of Nutrition 21:167-92, 2001.
- Stephensen, C.B., Rasooly, R., Jiang, X., Ceddia, M.A., Weaver, C.T., Chandraratna, R.A.S., Bucy, R.P. Retinoic Acid Modulates in vitro Th1/Th2 Phenotype Development of Naive T-cells from DO11.10 T-cell Receptor Transgenic Mice. FASEB J 15:A1093, 2001.
- Stephensen, C.B., Jacob, R., Aiello, G., Marquis, G., Kruzich, L. Wilson, C.M. for the REACH Project. Vitamin C intake and plasma ascorbic acid in HIV+ and HIV- U.S. adolescents and young adults in the REACH study. FASEB J 15:A739, 2001.
- Mitra A.K., Wahed, M., Chowdhury, A., Stephensen, C.B. factors associated with urinary loss of vitamin A in children with watery diarrhea. FASEB J 15:A256, 2001.
- Kruzich, L.A., Marquis, G.S. Stephensen, C.B., Wilson, C.M. for the REACH Study. Reported micronutrient intakes among HIV-positive and HIV-negative adolescents and young adults in the REACH study. FASEB J 15:A624, 2001
- Drammeh, B., Marquis, G.S., Stephensen, C.B., Eto, I. A randomized fruit-based intervention study to improve vitamin A status or rural Gambian children. FASEB J 15:A732, 2001.
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Progress 10/01/99 to 09/30/00
Outputs
1. What major problem or issue is being resolved and how are you resolving it?
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 asthma.
2. How serious is the problem? Why does it matter?
Infectious and immune-mediated chronic diseases are currently a major cause of morbidity in the U.S. population and worldwide. Diet can be a potent modulator of the immune response. Thus understanding the molecular role of micronutrients in modulating the immune response will help in making dietary recommendations for maintaining health and preventing disease.
3. How does it relate to the National Program(s) and National Component(s)?
The research evaluates the role of micronutrients in maintaining optimal immune function. It will thus help in setting dietary micronutrient requirements as well as defining the health-promoting properties of plant and animal foods. Both of these areas are key components of the ARS National Program 107 Human Nutrition.
4. What were the most significant accomplishments this past year?
A. Infectious and inflammatory diseases induce the acute phase response, which has protective functions but also affects serum concentrations of several micronutrients, thus potentially interfering with the assessment of nutritional status. In collaboration with Ginny Gildengorin at the WHNRC, we examined the relationship of an active acute phase response (as indicated by elevated serum concentrations of C-reactive protein, or CRP) to serum retinol concentration using data from the Third National Health and Nutrition Examination Survey (NHANES III). We found that (1) elevated CRP correlated strongly with lower serum retinol concentrations in all age groups, (2) the effect was more pronounced in men than women, and (3) serum CRP was elevated in subjects with common infectious and chronic diseases. These data indicate that an active acute phase response causes mis-classification of vitamin A status in U.S. population samples. B. We examined the role of vitamin A metabolites in
regulating the development of T-helper type 1 and type 2 (Th1 and Th2) cells to understand how vitamin A affects the development of an immune response to stimuli such as infectious organisms, vaccines, allergens or cancer cells. In collaboration with Drs. Pat Bucy and Casey Weaver from the University of Alabama at Birmingham we examined the effect of all-trans and 9-cis retinoic acid on the development of Th1 and Th2 memory cells (which develop from antigen-naive Th0 cells) in vitro using cells from DO11.10 T-cell receptor transgenic mice. We found that 9-cis retinoic acid enhanced the development of Th2 cells (which produce interleukins 4, 5 and 10, promote responses against extracellular pathogens, such as bacteria and parasites, and mediate the symptoms of asthma) and diminishes the development of Th1 cells (which produce interferon-gamma and promote responses against intracellular pathogens, such as viruses and tuberculosis). This work will help us understand how vitamin A
regulates Th1/Th2 development, and thus how dietary vitamin A modulates the immune response to infectious diseases and the pathogenesis of immune-mediated chronic diseases, such as asthma. Because deficient intake of antioxidant micronutrients may exacerbate the progression of HIV disease, we have begun a study to examine the relative antioxidant requirements of HIV-infected and uninfected adolescents (for maintaining blood levels of antioxidant nutrients), and to determine if antioxidant intake is associated with the progression of HIV disease. In collaboration with Dr. Craig Wilson of the University of Alabama at Birmingham and Dr. Grace Marquis at the Iowa State University we have begun collecting dietary intake data and blood samples at 15 clinical sites around the U.S. to measure micronutrient status and indicators of oxidative damage in HIV-infected and uninfected adolescents in an ongoing, NIH-funded project called the REACH study. Work is ongoing but we have found evidence
that blood levels of some antioxidants are lower than normal cut-offs in HIV-infected subjects. Eventually, the results of this study should be useful in making recommendations for antioxidant micronutrient intakes in subjects with HIV infection. C. None.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
This project began in July, 1999 and the accomplishments described above are the major accomplishments over the life of the project.
6. What do you expect to accomplish, year by year, over the next 3 years?
In the coming year we plan to begin studies to examine two mechanisms by which 9-cis retinoic acid promotes Th2 development: (1) We will identify genes which are regulated by 9-cis retinoic acid during Th2 development. The role of these genes in mediating the effects of 9-cis retinoic acid on Th2 development will be examined in years 2 and 3. (2) We will plan and initiate experiments to define which retinoid receptors (RAR-alpha, beta or gamma; RXR-alpha, -beta or -gamma) mediate the Th2-promoting activity of 9-cis retinoic acid. These studies will continue through year 3. In the coming year, and in years 2 and 3, we will pursue dietary studies to determine if vitamin A has the same effect on Th2 development as does 9-cis retinoic acid. We also plan to determine if such effects extend to affecting the pathogenesis of mouse model of asthma. Next year we will also initiate studies to determine if vitamin A fortification of the diets of Peruvian infants affects the development of
Th1/Th2 responses, and characterize the relationship between micronutrient status and development of tuberculosis. In addition, we plan to complete analysis of blood samples from the HIV/antioxidant study in the coming year, and statistical analysis in the following year.
7. 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 & durability of the technology product?
Scientific information on the effect of 9-cis retinoic acid on Th2 development has been transferred to scientists via an oral presentation at Experimental Biology 2000 (April, 2000), a poster presentation at the FASEB Summer Retinoids Symposium (June, 2000), and seminars given to the Nutrition Department at the University of California, Berkeley (May, 2000), and the Retinoids Research Group, Allergan (August, 2000; a pharmaceutical company and collaborator that is developing synthetic retinoids). Retinoic acid results from FY2000 were published in abstract form in 2000 and are likely to be published as an article in 2001. Scientific information on the effect of the acute phase response on assessment of vitamin A status are being published in the American Journal of Clinical Nutrition in December, 2000. It is too early to determine how implementation of these results regarding the impact of retinoic acid on T-cell development will affect health in the U.S. population. Nutrition
researchers are currently examining ways of using indicators of the acute phase response to identify likely misclassification of micronutrient status, and the work on the relationship of serum CRP to serum retinol will directly aid these efforts.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Local Lab Seeks to Solve Food-Health Mysteries. Sacramento Bee. June 2000. p. A1.
Impacts
(N/A)
Publications
- Stephensen, C.B., Ceddia, M.A., Weaver, C.T., Bucy, R.P. Retinoic Acid (RA) Treatment of T-helper (Th) Cell Cultures Modulates Th1/Th2 Phenotype Development. FASEB Journal. 2000.v. 14. p. A557.
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