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United States Department of Agriculture

Agricultural Research Service

Research Project: Dietary Modulation of Immune Function and Oxidative Stress

Location: Immunity and Disease Prevention Research

2012 Annual Report

1a. Objectives (from AD-416):
Objective 1: Conduct a controlled, vitamin D supplementation trial in volunteers with vitamin D insufficiency (VDI) to determine if supplementation to achieve the proposed level of >75 nmol/L for maintenance of bone health is also appropriate for maintenance of immune function. Sub-objective 1A. Determine if supplements decrease the production of proinflammatory and increase the production of anti-inflammatory cytokines and chemokines by innate immune cells stimulated ex vivo. Objective 1B. Determine if supplements decrease serum markers of inflammation and autoimmune activity, and increase serum levels of defensive molecules. Objective 1C. Determine if supplements decrease blood levels of proinflammatory T-helper type 1 (Th1) and Th17 cells and increase levels of anti-inflammatory T-regulatory (Treg) and Th2 cells. Objective 2: Determine the impact of plant polyphenols and polyphenol-rich foods on immune cell function using cell culture systems, mouse models, and human volunteers. Examine anti-inflammatory and anti-cancer activities of polyphenols in animal models, as well as inflammation and oxidative damage in studies with human volunteers, including overweight/obese individuals. Objective 2A. Analyze the effects of polyphenol-rich foods and individual plant polyphenols on immune cell function in vivo and ex vivo. Ojective 2B. Examine anti-inflammatory activities of polyphenol-rich foods, individual plant polyphenols and vitamin A in mice and humans who are at risk for developing inflammatory disease, such as autoimmune mice and obese humans. Objective 2C. Evaluate the anti-cancer activity of polyphenol-rich foods and individual plant polyphenols. Objective 3: Examine the absorption of B-cryptoxanthin (CX) from supplements and foods, its contribution to vitamin A stores, and the impact of CX, other carotenoids and vitamin A on immune function. Objective 3A. Measure the absorption and metabolism of CX from Satsuma mandarin juice fed to healthy adult humans. Objective 3B. Estimate the impact of daily consumption of food sources of CX or B-carotene (BC) on plasma and breast milk concentrations of CX, BC and retinol in lactating women. Objective 3C. Determine the impact of CX on immune and bone marker status in the Mongolian gerbil. Objective 4: Determine if high-level vitamin A intake is associated with higher Th2 and Treg responses and lower Th1 and Th17 responses relative to adequate and deficient intake. Objective 4A. Using dietary and targeted gene disruption approaches in mice, determine if vitamin A enhances Th2 and Treg responses by acting directly on T cells. Objective 4B. Using subjects recruited in the vitamin D supplementation trial described under Objective 1, determine if vitamin A status is associated with higher blood levels of NK, NK-T, Th2 and Treg cells, and lower levels of Th1 and Th17 cells. Objective 5: Identify the role of dietary selenium and selenoproteins in regulating cellular responses to oxidative stress. Objective 5A. Identify the pro-inflammatory and anti-inflammatory proteins S-glutathionylated by selenoprotein W. Objective 5B. Determine the role of selenoprotein W in key inflammatory pathways.

1b. Approach (from AD-416):
The impact of selenium, vitamins A and D, and plant polyphenols, on immune function will be examined using cell culture systems, mouse models, and human intervention trials. The anti-cancer activities of polyphenols will be examined in animal models. Absorption of beta-cryptoxanthin will be examined in gerbils and humans. The effect of selenium on cell division and cell signaling will be examined in cell culture.

3. Progress Report:
Objective 1: A total of 18 subjects have now completed the full study. The study had been modified to examine the effect of vitamin D supplementation on vaccine-specific T-cell responses, in addition to the response of total T-cells. Thus subjects have been immunized with the tetanus toxoid vaccine as a booster immunization. Inclusion of this TT-specific endpoint has allowed reduction in the sample size to 24 subjects, based on data not available at the time the project was developed. Recruitment has stopped for the summer period because of improved vitamin D status at this time of year and to allow for laboratory analysis of samples that have been collected. Objective 2: (1) The U.S. High Bush Blueberry Council awarded a collaborative grant to study whether feeding humans blueberries will decrease the inflammatory response to a fatty meal. This study is in progress. (2) Feeding obese humans grapes decreased a type of LDL cholesterol linked to heart diseases, and increased the immune response against bacteria that is important in obese populations at high risk for developing infections. The mouse studies were not pursued due to insufficient resources. (3) Mouse studies completed in 2010 by us showed that feeding purified polyphenols at levels higher than typically found in foods did not kill leukemia cells. The diet studies planned for this year with polyphenol-rich foods will not be pursued as this approach is likely to be ineffective. Objective 3: We completed our food-based intervention study in Bangladesh, comparing the carotenoid and retinoid changes caused by feeding orange-fleshed sweet potatoes and mandarin oranges to lactating women with low vitamin A status. Beta-carotene concentrations in blood increased in the group fed orange-fleshed sweet potatoes and Beta-cryptoxanthin concentrations in blood increased in the group fed mandarin oranges. Beta-cryptoxanthin concentrations increased to a much greater extent than beta-carotene concentrations. This was similar to results we obtained in our in vitro digestion studies of beta-cryptoxanthin metabolism. We are now finishing analysis of the breast milk samples from the study. A second carotenoid intervention study using ‘biofortified’ cassava varieties with good amounts of beta-carotene found increased beta-carotene and vitamin A concentrations in women. Therefore, eating properly prepared ‘biofortified’ cassava has low risk, and can increase beta-carotene and vitamin A in people. Objective 5: We discovered a new mitogen-activated protein kinase pathway from MKK4 through p38-gamma, p38-delta, and JNK2 that controls phosphorylation of tumor suppressor protein p53 when selenoprotein W (SEPW1) is depleted. We showed SEPW1 is required for auto-phosphorylation and stabilization of the epidermal growth factor (EGF) receptor (EGFR). We demonstrated that activation of EGFR and 6 out of 6 other growth factor receptors present on breast cancer cells requires SEPW1.

4. Accomplishments
1. Biofortified cassava may prevent vitamin A deficiency. Cassava is an important staple crop in many tropical countries but the calories it provides are considered “empty” because it has a low content of vitamins and minerals. The goal of “biofortification” is to select natural varieties of staple foods, such as cassava, which have improved nutritional quality. Introduction of biofortified varieties of cassava that are naturally rich in pro-vitamin A carotenoids such as beta-carotene could improve vitamin A intake in such settings, where vitamin A deficiency (which causes blindness and excess mortality) is often a significant public health problem. ARS scientists working at the Western Human Nutrition Research Center in Davis, CA, with a biofortified variety of cassava provided by the International Center for Tropical Agriculture in Cali, Colombia, evaluated the absorption of beta-carotene from this and standard cassava. The cassava varieties were fed to healthy women in a standard meal and the biofortified cassava produced a significantly greater increase in blood beta-carotene levels than the standard variety, indicating good absorption and thus a likely beneficial effect on vitamin A status. This study shows that biofortified cassava can be a good source of vitamin A and thus clears the way for larger studies to determine the effect of beta-carotene-rich cassava on preventing vitamin A deficiency in countries where cassava is a staple crop.

2. Dietary vitamin D2 competes with vitamin D3. Vitamin D deficiency causes loss of bone mineral density and osteoporosis in adults and is common in the US because few foods contain either vitamin D2 (found in some plants and mushrooms) or vitamin D3 (of animal origin). Vitamin D3 is also produced in human skin by sun exposure but this source is limited by skin pigmentation (thus African Americans are at high risk of deficiency) and lack of sun exposure. ARS scientists from the Western Human Nutrition Research Center in Davis, CA, tested the availability of vitamin D2 from mushrooms treated with UV light to increase their vitamin D2 content. The goal of the study was to determine if such mushrooms might be a useful source of vitamin D in the American diet. The study was done in healthy human volunteers during the summer and fall months and found that vitamin D2 was readily absorbed from mushrooms (and a supplement provided as a control) but that vitamin D3 levels decreased proportionally resulting in no net improvement of vitamin D status. These findings indicate that increased intake of vitamin D2 will not improve vitamin D status for individuals with significant vitamin D3 “exposure” from other foods or from sun exposure, suggesting that vitamin D3 is the preferred form of vitamin D for dietary intake.

3. Selenium and cellular proliferation. Selenium is an essential mineral that is incorporated into a specific class of proteins, termed selenoproteins, with diverse and poorly understood cellular functions. Understanding the biology of such selenoproteins is necessary to understand how selenium affects human health, including cancer risk, which may be decreased by the use of selenium supplements though data are equivocal on this point. ARS researchers working at the Western Human Nutrition Research Center in Davis, CA, discovered that selenoprotein W (SEPW1) is required for activation of a class of cellular proteins called receptor tyrosine kinases, or RTKs, which are responsible for triggering cellular proliferation. Controlled cellular proliferation is normal and healthy but uncontrolled cellular proliferation is a hallmark of cancer. Since RTK dysfunction is known to be involved in the development of most cancers these findings suggest that selenium may affect cellular proliferation, and perhaps cancer risk, via its effects on the RTK proteins. More work is needed to further understand SEPW1 function, how selenoproteins might be targeted in cancer therapy, and to evaluate the potential benefits and risks of selenium supplementation.

4. Grapes are heart-healthy and boost immunity. Many studies show that adequate intake of fruits and vegetables are associated with low risk of heart disease and improvements in other measures of good health such as a healthy immune system. However, data on the actual benefits of specific fruits and vegetables is quite limited. To address this deficit, ARS researchers working at the Western Human Nutrition Research Center in Davis, CA, investigated the effect of consuming table grapes (provided in a freeze-dried form for the sake of uniformity throughout the study period) on risk factors for heart disease, such as blood cholesterol, and markers of a healthy immune system, including the response of white blood cells to stimulation with a product of bacteria. The study was conducted in obese adults and showed that eating grapes decreased blood levels of a class of cholesterol associated with heart disease, suggesting that consuming grapes may be heart-healthy. Consuming grapes also increased the response of white blood cells to a microbial product, a finding consistent with improved protection against infections. These results suggest a beneficial effect of consuming grapes in the obese population known to have a higher risk of both infections and heart disease.

Review Publications
Hawkes, W.C., Alkan, Z. 2012. Selenoprotein W depletion induces a p53- and p21-dependent delay in cell cycle progression in RWPE-1 prostate epithelial cells. Journal of Cellular Biochemistry. 113:61-69.

Parelman, M.A., Storms, D.H., Kirschke, C.P., Huang, L., Zunino, S.J. 2012. Dietary strawberry powder reduces blood glucose concentrations in obese and lean C57BL/6 mice and selectively lowers plasma C-reactive protein in lean mice. British Journal of Nutrition. 1-11. DOI: 10.1017/S0007114512000037.

Zunino, S.J., Parelman, M., Freytag, T.L., Stephensen, C.B., Kelley, D.S., Mackey, B.E., Woodhouse, L.R., Bonnel, E. 2011. Effects of dietary strawberry powder on blood lipids and inflammatory markers in obese human subjects. British Journal of Nutrition. 1-10. DOI: 10.1017/S0007114511006027.

Turner, T., Burri, B.J. 2012. Rapid isocratic HPLC method and sample extraction procedures for measuring carotenoid, retinoid, and tocopherol concentrations in human blood and breast milk for intervention studies. Chromatographia. 75:241-252. DOI 10.1007/s10337-012-2193-9.

Havens, P.L., Stephensen, C.B., Hazra, R., Flynn, P.M., Wilson, C.M., Rutledge, B., Bethel, J., Pan, C.G., Woodhouse, L.R., Van Loan, M.D. 2012. Vitamin D3 decreases parathyroid hormone in HIV-infected youth being treated with tenofovir: a randomized, placebo-controlled trial. Clinical Infectious Diseases. 54(7):1013-1025. DOI: 10.1093/cid/cir968.

Turner, T., Burri, B.J., La Frano, M.R. 2012. Beta-cryptoxanthin: A vitamin A-forming carotenoid. In: Yamaguchi, M., editor. Carotenoids: Properties, Effects and Diseases. Hauppauge, NY: Nova Publishers. p. 331-354.

Zunino, S.J., Storms, D.H., Newman, J.W., Pedersen, T.L., Keen, C.L., Ducore, J.M. 2012. Resveratrol given intraperitoneally does not inhibit growth of high-risk t(4;11) acute lymphoblastic leukemia cells in NOD/SCID mouse model. International Journal of Oncology. 40(4):1277-84. DOI:10.3892/IJO.2011.1316.

Burri, B.J. 2012. Red palm oil as an intervention food to prevent vitamin A deficiency.. Comprehensive Reviews in Food Science and Food Safety. 11:221-233. DOI: 10.1111/j.1541-4337.2011.00181.x.

Stephensen, C.B., Zerofsky, M., Burnett, D., Lin, Y., Hammock, B.D., Hall, L.M., Mchugh, T.H. 2012. Ergocalciferol from mushrooms or supplements consumed with a standard meal increases 25-hydroxyergocalciferol but decreases 25-hydroxycholecalciferol in the serum of healthy adults. Journal of Nutrition. 142(7):1246-1252. DOI: 10.3945/jn.112.159764.

Hawkes, W.C., Alkan, Z. 2012. Delayed cell cycle progression in selenoprotein W depleted cells is regulated by a mitogen-activated protein kinase kinase 4–p38–p53 pathway. Journal of Biological Chemistry. DOI: 10.1074/jbc.M112.346593.

Armstrong, P., Kelley, D.S., Newman, J.W., Staggers, F., Hartiala, J., Allayee, H., Stephensen, C.B. 2012. Arachidonate 5-lipoxygenase gene variants affect response to fish oil supplementation by healthy African Americans. Journal of Nutrition. 142(8):1417-1428. DOI: 10.3945/jn.112.159814.

Stephensen, C.B. 2012. Vitamin A and immune function. In: Preedy, V.R., editor. Food and Nutritional Components in Focus No. 1: Vitamin A and Carotenoids: Chemistry, Analysis, Function and Effects. Cambridge, UK: Royal Society of Chemistry. p. 501-515. DOI:10.1039/ISBN

Last Modified: 06/26/2017
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