ROLE OF NUTRITION ON THE IMMUNE RESPONSE

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3210	1010	50%
302	3210	1090	50%

Progress 07/01/93 to 09/30/10

Outputs
OUTPUTS: The goals of this project are to find new ways to decrease the collateral cost of inflammatory processes and immune responses, to assure that useful products for controlling inflammation are safe and cost effective, and to find novel means of detecting and characterizing inflammatory events. During 2010, information on findings from this project were disseminated to target audiences through the use presentations at scientific meetings, presentation of materials to animal producers and companies that could ultimately use or produce the products, patent filings, and publication of peer reviewed journal articles. In some cases, invited lectures and seminars were present to groups of students and faculty on university campuses. Numerous one-on-one meetings were held with companies around the world that produce feed supplements for animal agriculture and the companion animal industries, and pharmaceuticals for animal agriculture and human health. Meetings with medical research doctors were also held on a number of occasions. PARTICIPANTS: Unless indicated, M.E. Cook served as the principal investigator of this project and studies were conducted at the University of Wisconsin. However, numerous other collaborators and principal investigators played significant roles on the projects reported. F. Assadi-Porter and D. Butz of the University of Wisconsin were responsible for the metabolome analysis. Assadi-Porter, Butz, and W. Porter conducted experiments on breath biomarkers. Elizabeth Bobeck was the graduate student that conducted the phosphate studies. M. Pariza was a co-writer of a paper on the flow chart for Food and Drug Approval of feed enzymes. V. Leone and S. Worzalla reported on the safety of conjugated linoleic acid, and D. Trott, and S. Huebner co-authored papers on the safety and uses of egg antibody. M. Yang and M. Etzel were experimenters on the heat stability of egg antibody. TARGET AUDIENCES: During 2010, the target audience focused mainly on users of new technology generated as a result of this project. The main target for the breath biomarker findings were medical research doctors who could begin clinical trials to determine its usefulness in monitoring the health of patients under intensive medical care. Findings involving novel egg antibody preparations were shared with numerous potential users or distributors of the products as well as pharmaceutical companies that may find tangent uses of the products. The target audience for the paper on the safety of feed enzymes was specifically designed for regulators in the Food and Drug Administration and producers of feed grade enzymes. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
With regards to development of useful products for decreasing the collateral cost associated with inflammation, one abstracts and two papers were published involving the use of egg antibody as a therapy for preventing immune-induced depression in body weight gain. An egg antibody to key gut peptides, such as secretory phospholipase A2, neuropeptide Y, and toll receptor 4 were found to improve growth and feed efficiency in animals. These antibodies may prove to be a useful adjunct or replacement of antibiotics as growth stimulants in animal agriculture. One abstract describes a model being developed to understand the role of phytate phosphorus on phosphate homeostasis in end stage renal failure. Patients with kidney disease often have trouble maintaining blood phosphate levels. As blood phosphate increases, patients have an increased risk of cardiovascular disease. Often, these patients are treated with 1, 25 dihydoxycholcalciferol (active D3). Studies showed that the use of active D3 increased the bioavailability of phytate phosphorus and increased the risk of hyperphosphatemia in patients with renal disease. In the published abstract, the effectiveness of pharmaceutical phosphate binders in preventing the increase in blood phosphate when treated with active D3 was demonstrated. Safety and cost effectiveness of known anti-inflammatory supplements were reported in two papers and one patent. A paper was developed for guiding animal feed regulators and enzyme producers for getting Federal approval of feed enzymes. A paper was published that demonstrates the development of chick embryos can be at risk if the maternal diet contains certain isomers of conjugated linoleic acid (CLA). A patent was filed on methods to heat stabilize egg antibodies so that they could be used in a cost effective manner in animal feeds that are processed with heat. In this patent, the use of select carbohydrates to form a glass around the antibody was found to be a cost effective means to improve the heat stability properties of antibodies. Two abstracts were published on the metabolome (collection of small molecular weight plasma molecules) of acute inflammatory disease. Changes in the concentration of these molecules provided insight into metabolic changes that occur during acute inflammatory disease. Patterns of change also allowed for the development of a suite of biomarkers as diagnostic tools for detection of infection. Findings using a biomarker involving the ratio of 13C/12C in exhale breath was found to be a early method of detecting peritonitis that leads to septic shock and death in a rodent model. This finding, as well as others, served as a preclinical study for approval of human studies that will determine the effectiveness of breath measurements as a early warning for inflammation that leads to sepsis in the intensive care patient. Our group has assisted medical groups in writing grants and getting institutional review board approval for these studies.

Publications

Assadi-Porter, F.M., Cook, M.E., Porter, W.P., and Butz, D.E.2010. Identification of disease characteristics using isotope ratios in breath. U. S. Patent Appl. 2010/0036274 A1.
Cook, M.E., Yang, M., and Etzel, M.R. 2010. Methods for heat-stabilizing proteins with specific binding activities. U. S. Patent 7,750,117 B2.
Butz, D.E., Cook, M.E., Porter, W.P., and Assadi-Porter, F. 2010. Early detection of the acute phase response in sepsis by identification of small molecule metabolism in breath and biofluids. FASEB J. 24:752.4.
Bobeck, E.A., and Cook, M.E. 2010. Phosphate-dependent physiological changes during short-term dietary phosphorus deficiency with or without dietary phosphorus binder. FASEB J. 24:917.22.
Cook, M.E., and Huebner, S.M. 2010. Host targeted antibody strategies for preventing growth depression due to microbial colonization. Anim. Sci J. 88:E-Suppl. 2:2.
Leone, V.A., Worzalla, S.P., and Cook, M.E. 2010. Evidence that maternal conjugated linoleic acid negatively impacts lipid uptake in late stage chick embryos resulting in increased embryonic mortality. Poultry Sci. 89:621-632.
Cook,, M.E., and Trott, D.L. 2010. IgY-immune components of eggs as a source of passive immunity for animals and humans. World Poultry Sci. 66:215-225.
Pariza, M.W., and Cook, M.E. 2010. Determining the safety of enzymes used in animal feeds. Reg. Toxicol. Pharma.56:332-342.
Cook, M.E. 2010 A review of science leading to host-targeted antibody strategies for preventing growth depression due to microbial colonization. J. Anim. Sci.PMID 21036928.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: During the course of study in 2009, eight papers, 2 abstracts, and 4 U.S. Patents/applications were published. Two student received their PhDs. Concepts that resulted from this research project were taught in lectures in Vaccinology, Biotechnology, and Pharmacology on UW's campus. Over 10 scientific presentations were given before scientist, industry groups and individual companies or animal agriculture producers. PARTICIPANTS: Dr. D. E. Butz (Scientist in Zoology and Animal Sciences, UW) was the lead investigator in the studies of breath isotope. Breath isotope work was also a collaboration that involved Professor W.P. Porter (Zoology, UW), Dr. F. Assadi-Porter (Scientist, Biochemistry, UW) and H.R. Eghbalnia (Professor, Univ Cinn.). All of the above plus, Dr. Michael Shortreed (Scientist, Chemistry, UW), Dr. L.D. Whigham (Scientist, OBGYN, Medicine, UW), Professor David Abbott (Primate Center, and Medicine, UW), Professor John Markley (Biochemistry, UW), Dr. Marco Tonelli (Scientist, Biochemistry, UW) and Professor L. Smith (Chemistry, UW) were involved in a metabolomic study of Polycystic Ovary Disease (PCOS). The PCOS project also resulted in the development of an inflammatory metabolome that also involved graduate students Shane Huebner, and Vanessa Leon, and undergraduates, James Campbell and Tyler Fulmer. At UW, students and employees involved in egg antibody investigations included, Dr. David Trott (recent PhD, Nutritional Sciences, UW), Elizabeth Bobeck (graduate student, Animal Sciences), D.E. Butz (above), Scientist/Professor at the Food Research Institute, UW (A.E. Larson, W.H. Trepp, and Professor E.A. Johnson). A side project involving one of the antibodies from our group (antibody to secretory phospholipase A2, aPLA2) resulted in a collaboration with Professor J.E. Gumperz and her laboratory (Medical Microbiology and Immunology, UW). This work led to a role of PLA2 in potential inflammatory responses involving natural killer T cells. A collaboration with Professor K.M. Koelkebeck and his research group (Animal Sciences, Univ Illinois) resulted in a finding of the value of spent laying hens for antibody production. Other projects lead by D.L. Trott (UW) resulted in new strategies to increase antibody production. Dr. Vanessa Leon (PhD, Animal Sciences, UW) in collaboration with Professor Mark Berres (Animal Sciences, UW), Professor R. Aydin (Animal Science, Kahramanmaras Sutcu Imam Univ, Turkey), and Dr.Assadi-Porter (above) advance the knowledge of CLA feeding of laying hens and subsequent embryo development. TARGET AUDIENCES: The development of biomarkers of inflammation are currently in early stages of development. Currently information related to the breath technology is being disseminated to the research medical community. Metabolomic analyses are primarily limited to a scientific audience. Advances in egg antibody technology are being shared with animal and aquaculture producers world wide. New egg antibody products and the methods to manufacture them have been disseminated to Federal agencies and other companies with specific needs. The CLA technology has global distribution. Knowledge involving laying hens is mainly intended for regulatory agencies, interested scientist, and poultry nutritionist. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
During this reporting year a number of papers, abstracts, and patents were published related to the study of nutrition and immune function. Investigations were centered around three major fields of study: 1) Investigations of markers of inflammation; 2) Investigations on the production and use of egg antibodies; 3) Understanding the mechanism by which an anti-inflammatory, conjugated linoleic acid (CLA), adversely effects chick embryonic development. We were able to generate both plasma and breath biomarkers of inflammation. The use of carbon isotopes in breath to detect early events of inflammation has found a potential use in new methods and devices for monitoring the health status of the intensive care patient. A plasma inflammatory metabolome was created and its usefulness in detecting inflammation in the human disease Polycystic Ovary Syndrome was investigated. We were able to develop methods and strategies to improve the production of egg antibody. We reported methods, adjuvant uses and the value of spent hens to improve the production of egg antibodies. We were also able to create antibodies to microbial toxins that have been viewed as bioterror agents. Our findings were reported both in publication and in a presentation at Fort Detrick, MD. We also found new anti-inflammatory uses for an antibody to secretory-phospholipase A2 (aPLA2). The study of aPLA2 and natural killer T cells has pointed to novel ways for regulating mucosal inflammation. We also reported on a new avian model to study methods to control increased blood phosphate in the end stage renal patient. CLA is a naturally occurring anti-inflammatory that has recently been approved for select animal and human foods. When CLA was fed to the laying hen, it had adverse effects on chick embryo development, a finding that could restrict its use. Considerable efforts were made to understand the mechanism by which CLA affects chick embryo development. Our data showed, that while CLA was not directly toxic to the chick embryo, it appeared to influence the transport of lipid across the yolk sac. The adverse effects of CLA on chick embryonic development could be prevented by the inclusion of select fats and oils in the hen's diet. Understanding CLA in the avian model was critical in gaining approval for the use of CLA in animal and human foods.

Publications

Aydin, R., ME Cook. 2009. Maternal dietary conjugated linoleic acid is not directly toxic for the developing chick embryo, but causes embryo mortality by altering fatty acid composition of the egg yolk in the chicken. J Sci Food Ag. 89:2687-2681.
Assadi-Porter, FM, ME. Cook, HR Eghbalnia, M. Tonelli, WP Porter, DE Butz. 2009 (April 23). Noninvasive measurement and identification of biomarkers in disease state. US Patent Appl. 20090104596 A1.
Cook ,M.E., D.L. Trott, M. Yang. 2009 (April 9). Immunogenic composition. US Patent appl. 200900926.39 A1.
Trott, D.L. 2009. Immunization of the laying hen: production and application of egg yolk antibodies. UW Dissertation
Leone, V. A. 2009. The effects of maternal dietary conjugated linoleic acid on embryonic and post-hatch development in an avian model. UW Dissertation.
Trott, DL, M Yang, PL Utterback, CW Utterback, KM Koelkeback, ME Cook. 2009. Utility of spent Single Comb White Leghorn hens for production of polyclonal egg yolk antibody. J. Appl. Poultry Res. 18:679-689.
Fox, LM, DG Cox, JL Lockridge, X Wang, X Chen, L. Scharf, DL Trott, RM Ndonye, N. Veerapen, GS Bersra, AR Howell, ME. Cook, EJ Adams, WH Hildebrand JE Gumperz. 2009. Recognition of lyso-phospholipids by human natural killer T lymphocytes. PLOS Biology 7: Oct 7 (10) e1000228.
Leone, VA, DL Stransky, R. Aydin, ME Cook. 2009. Evidence for conjugated linoleic acid-induced embryonic morality that is independent of egg storage conditions and changes in egg relative fatty acids. Poultry Sci. 88: 1858-1868.
Trott DL, M Yang, J Gonzalez, AE Larson, WH Trepp, EA Johnson, ME Cook. 2009. Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin. J Food Protection. 72:1005-1011.
Butz, DE, ME Cook, HR Eghbalnia, F Assadi-Porter, WP Porter. 2009. Changes in the natural abundance of (CO2)-C-13/(CO2)-C-12 in breath due to lipopolysacchride-induced acute phase response. Rapid Comm. Mass Spec. 23:3729-3735.
Leon VA, SP Worzalla, ME Cook. 2009. Body compositional changes and growth alteration in chicks from hens fed conjugated linoleic acid. Lipids 44:437-447.
Aydin, R., ME Cook. 2009. The effects of dietary conjugated linoleic acid alone or in combination with linoleic acid and oleic acid on fatty acid composition of egg yolk, embryo mortality and chick yolk sac content retention in chickens. Animal Feed Sci. Tech. 149:125-134.
Shortreed, M.R., F. Assadi-Porter, L.D. Whigham, M.E. Cook, D. Butz, W.P. Porter, D.H. Abbott, J.L. Markley, H. Eghbalnia, and L.M. Smith. 2009. Identification of key pathways in polycystic ovary syndrome by mass spectrometry, NMR and cavity ringdown spectroscopy. 57th Am Soc Mass Spec.
Bobeck, EA, K.M. Meyer, and ME Cook. 2009. Dietary sevelamer hydrochrloride prevents dietary 1 alpha-hydroxycholecalciferol (1 alpha-OH D3)-induced increases in plasma phosphorus. FASEB J 23:909.6.
Assadi-Porter, FM, ME Cook, WP Porter, DE Butz 2008. Identification of disease characteristic using isotope ratios in breath. US Patent 7465276B2.
Cook, ME, M Yang, K Roberson. 2009 Method for improving body weight uniformity and increasing carcass yield in animals. US Patent 7579002 B2.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: During the reporting period, results of reported products, processes and methods have been shared with commercial producers of the technology (e.g. Cognis Inc, Wisconsin Alumni Research Foundation, aOvatechnologies Inc., Isomark LLC, Cytochroma, Inc.), commercial users of the products developed and discovered (e.g. Tyson Foods Inc., The Wisconsin Egg and Poultry Producers Assoc., Midwest Foods Assoc.), academia and state extension agents (Animal Sciences Meeting, Poultry Science Meeting, County Extension Agent meetings), and U.S. Senate and Congressional Staff. When possible, information was disseminated as part of university teaching activity. The process by which the results were disseminated included scientific publications, popular press publications or video, presentations at scientific meetings, meeting with companies that are both users and producers of the products, transfer of key intellectual property to industry using the patent/licensing process, assuring the growth of start up companies, and informing students, producer organizations, and Senate and Congressional Offices of progress. Specific products reported during this period included: 1. Antibody that can be mass-produced using laying hens for the detection and neutralization of botulinium toxin: 2. Antibody that can prevent excessive fluid excretion into the gastrointestinal tract of Salmonella infected animals: 3. New techniques in vaccine development: 4. Dietary methods to alter the composition of the broiler chicken: 5. New information on the safety of feeding conjugated linoleic acid (CLA) to laying hens. PARTICIPANTS: At the University of Wisconsin, the following individuals were involved in some aspect of the project. The title of the person and their departmental affiliation is shown in parentheses. Personnel at UW included David Trott (graduate student with P.I., Nutritional Sciences), Shane Huebner (graduate student with the P.I., Nutritional Sciences), Vanessa Leone (graduate student with the P.I., Animal Sciences), Elizabeth Bobeck (graduate student with the P.I., Animal Sciences), Sean D. Ryan (undergraduate with the P.I., Medical Microbiology and Immunology), Sharon P. Worzalla (undergraduate with P.I., Animal Sciences), Amanda Murdoch (undergraduate with P.I., Biology), M. Yang (former graduate student and postdoc with P.I., Animal Sciences), D. E. Butz (former graduate student with PI, Scientist, Zoology), H, Eghbalnia (former scientist in the National NMR Laboratory, Biochemistry, currently Assistant Professor, University of Cincinnati), Rahim Aydin (former graduate student of P.I., Assistant Professor, Animal Science, Kahramanmaras Sutcu Imam University, Turkey), Jose Gonzales (summer minority NIHES trainee, Molecular and Environmental Toxicology Center), Ann Larson (Assistant Researcher, Bacteriology), William Tepp (Senior Research Specialist, Bacteriology), Eric Johnson (Professor, Bacteriology), Erica Hellestad (former research specialist with P.I., currently Veterinary Student, UW), Leah Whigham (former graduate student, currently Assistant Scientist, Medicine), David Abbot (Professor Medicine), Warren Porter (Professor, Zoology), Fariba Assadi-Porter (Assistant Scientist, Biochemistry), Marco Tonelli (Research Specialist, National NMR Laboratory), Michael Shortreed (Scientist, Chemistry). Collaboration with university personnel outside of Wisconsin included Colin Scanes (Dean of the Graduate School, Iowa State University, currently Dean of the Graduate School, University of Wisconsin-Milwaukee), R.W. Griffith (Professor, Veterinary Medicine, Iowa State University), S.A. Culter (Researcher, Iowa State University), P.L. Utterback (Research University of Illinois, Animal Sciences), Ken Koelkebeck (Professor, Animal Sciences, Univerity of Illinois). Collaborations with aOvatechnologies included Carrie Cook (Scientist), Briana Gelbach. TARGET AUDIENCES: The target audience for discoveries resulting from CLA research broadly impacts the general population since the product is available commercially in supplements and human foods. CLA used in animal agriculture currently would only impact the growers and processors of swine. The current egg antibody technology has direct effect on the UW spin off, aOvaTechnologies, and producers and processors of swine, poultry, fish, and cattle. Consumers are indirectly influenced by reduced food cost. Since the research project involves the training of undergraduates and graduate students, those involved in the laboratory research will be benefited. Our laboratory has participated over the years in several minority undergraduate mentoring programs associated with this project. PROJECT MODIFICATIONS: No major changes in the approach have occurred or envisioned. New tools, and models have been developed or used to better understand the role of inflammation on health. These have included the use of stable isotope mass spectrometry, NMR, and LC-mass spectrometry.

Impacts
Outcomes/impacts of the research on this project cannot be effectively measured within a one-year time frame except for the increase in knowledge through publications (shown in report). However, since this project has been on going for many years, the overall effects of the project on outcomes/impacts can be assessed. The result of this project, in collaboration with others, has resulted in the development of three start up companies. The first was sold to a large international company that now sells CLA worldwide. In 2008, CLA was approved for use in both animal and human foods by the Food and Drug Administration. Sales of the product in the U.S. food/feed market began in 2008. Sales of CLA as a dietary supplement worldwide, including the U.S. had resulted in over $10 million in royalty payments. The second company was a producer of egg antibody to phospholipase A2. In 2008, the company (7 employees) achieved its first sales in poultry, aquaculture, swine, and cattle species. Since this product is generated from eggs, new added value for egg products have been realized. Over 100 animal trials with the product have now been conducted, and global distribution may be realized in 2009. The third company is in its early stages and has yet to deliver a product to the market place.

Publications

Scanes, C.G., S.A. Cutler, R.W. Griffith, M.Yang, and M.E. Cook. 2008. Effects of egg antibody to components of inflammatory activation (phospholipase A2 and Toll like receptor 4) on the response of young turkeys toSalmonella typhimurium challenge. Avian Biology 1: 167-175.
Trott, D.L., E.M. Hellestad, M. Yang and M.E. Cook. 2008. Additions of killed whole bacteria cell preparations to Freunds complete adjuvant alter laying hen antibody response to a soluble protein antigen. Poultry Sci. 87:912-917.
Trott, D.L. M. Yang, J. Gonzales, A.E. Larson, W.H. Tepp, E.A. Johnson, and M.E. Cook. 2009 Egg yolk antibodies for detection and neutralization of Clostridium botulinum type A neurotoxin. J. Food Protection In press.
Leone, V. A., S. P. Worzalla, and M.E. Cook. 2009. Body compositional changes and growth alterations in chicks from hens fed conjugated linoleic acid. Lipids. accepted
Aydin, R. and M.E. Cook. 2008. The effects of dietary conjugated linoleic acid alone or in combination with linoleic acid and oleic acid on fatty acid composition of egg yolk, embryo mortality and chick yolk sac content retention in chickens. Animal Feed Sci Tech. accepted.
Bobeck, E.A., C.L. Cook, B.E. Gelbach, M. Yang, and M.E. Cook. 2008. Thermoprotection of bioactive proteins added to animal feed. Proc. World Poultry Sci. Brisbane, Australia, Aug., 2008
Trott, D.L., S.D. Ryan, and M.E. Cook. 2008. Short-term administration of dietary corticosterone enhances hen antibody response to immunization. Proc. World Poultry Sci. Congress, Brisbane , Australia. Aug, 2008.
Leone, V. S. P. Worzalla, A.J. Murdoch, and M.E. Cook. 2008. Maternal dietary conjugated linoleic acid (CLA) in a low-fat laying hen diet delays embryonic lipid assimulation and growth during late incubation. Poultry Sci. 87:Suppl 1:120.
Cook, M.E. 2008. Strategies to minimize inflammatory taxation on animal performance. Proc. Joint ADSA-ASAS Meeting. Abstract # 26341.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: Previous experiments by our group showed that the feeding of mixed isomers of conjugated linoleic acid (CLA) were effective at reducing plasma levels of tumor necrosis factor (TNF) the first two hours after immune stimulation (preformed TNF). At the time of this study, the CLA isomer responsible for preventing a rise in TNF after acute endotoxemia (c9,t11- or t10,c12-CLA) could only be determined from in vitro cell culture studies (in vitro studies showed that the c9,t11-CLA isomer protected against increase endotoxin-induced TNF) due to inadequate quantities of pure isomers for feeding studies. In 2007, we reported that in animal feeding trial of acute endotoxin exposure, both isomers were effective in preventing endotoxin-induced increases in plasma TNF. The results did suggest that dietary c9,t11-CLA may be more effective in vivo in preventing the increase in plasma TNF after endotoxin exposure. Experiments were also conducted to determine the effectiveness of dietary egg antibodies to select targets to prevent decreases in growth response of animals with gastrointestinal inflammation (GII). A model of mild coccidiosis infection was used to induce GII. In this model, birds typically have a 10% reduction in growth rate when infected and compared to non-infected chicks. Feeding the chicks egg antibody produced against the coccidia prevented growth suppression caused by the infection. Antibody to an enzyme involved in inflammatory responses in the gastrointestinal tract called phospholipase A2 (PLA2) was tested in the coccidiosis model. Since a certain amount of "inflammation" is thought to be required to resist disease infection, would an antibody targeting an inflammatory process make the animal more susceptible to infection? Feeding antibody to PLA2 did not appear to exacerbate coccidiosis. The antibody to PLA2 increased animal growth whether they were infected or not infected with coccidiosis. The lack of interaction between feeding an antibody to PLA2 and coccidosis infection suggested that the PLA2 antibody improved growth independent of preventing the progression of the coccidosis. Over the last several years, our laboratory has been studying factors that regulate the quantity of antibodies deposited in the egg yolk. We recently reported that the inclusion of select microbial products in adjuvants for soluble protein antigens improved antibody responses (select gram positive whole bacteria products) as much as 1.5 fold, whereas the inclusion of other whole cell bacteria products (gram negative bacteria) adversely affect antibody production and levels in eggs. PARTICIPANTS: The work on the role of conjugated linoleic acid (CLA) and tumor necrosis factor was conducted in collaboration Y. Park (Department of Food Science, Univ. Mass.) and M. W. Pariza (Food Research Institute, Univ. WI-Madison). D.L Trott (Nutritional Sciences, Univ. WI-Madison) and E. Hellestad and J. Susko-Parrish (Research specialist, Animal Sciences, Univ. WI-Madison) played key roles in antibody studies (feeding studies and methods to enhance antibody production in eggs). Funding for these studies were supported by royalty income from Wisconsin Alumni Research Foundation (WARF) and the UW College of Agriculture and Life Sciences (CLA and antibody work), and Homeland Security (antibody titer improvement). TARGET AUDIENCES: The target audience for the CLA work has been mainly scientist conducting work on dietary mechanisms to control inflammation. Egg antibody work has been useful for both companies producing egg yolk antibodies, and animal producer groups testing alternative strategies to improve animal growth.

Impacts
Previously (Exp. Biol. Med 228:51-59, 2003) we showed in vivo that the CLA decreased serum tumor necrosis factor (TNF) following endotoxin challenge. Using in vitro macrophage cultures, we showed that the isomer responsible for this effect was the c9,t11-CLA isomer. The reduction in serum TNF after endotoxin exposure was observed as early as 1-2 hours after immune stimulation. These results strongly suggested that CLA's effects were being mediated independent of gene transcription/translation and probably was having an effect on the release of preformed TNF. At the time of this published report (2003), the quantity of pure CLA isomers for animal feeding trials was not sufficient. We now have demonstrated that mice fed pure c9,t11-CLA and to a lesser extent, t10,c12-CLA, have reduced plasma TNF-alpha levels 1 and 2 hours after endotoxin injection. Regulation of the release of preformed TNF plays a significant role in many inflammatory diseases. Antibodies directed toward TNF are now being used as standard pharmacological strategies to treat inflammatory diseases such as Crohn's disease and arthritis. CLA's ability to regulate plasma TNF may partly explain the mechanism by which CLA has been shown to be beneficial in several inflammatory diseases. Another method of regulating gastrointestinal inflammation (GI) involves the use of dietary antibodies. We reported that by targeting key inflammatory pathways during gut inflammation using egg yolk antibodies, animal growth and feed efficiency can be improved approximately 5%. The result of this research has already resulted in commercial antibody development. We were able to demonstrate that the targeting of phospholipase A2 (PLA2) with an antibody (aPLA2) would not negatively impact chick resistance to a coccidiosis challenge (a common GI protozoa). Also, we have shown that dietary egg antibodies to a mixture of coccidia were effective at preventing growth depression caused by a coccidiosis infection. Studies were also conducted to find new methods to enhance the level of antigen specific antibody in eggs. A commercial limitation on the commercial use of egg antibodies is the cost of egg. Any cost effective method to increase antibody production and deposition in eggs will increase the use of this technology commercially. We have successfully demonstrated a method to increase antibody levels 1.5 fold (or decreased the cost of antibody production 35%).

Publications

Cook, M.E., Trott, D.L., Hellestad, E.M. 2007. Immune components of eggs (IgY) as a source of passive immunity for animals and human. Proceedings of the IV International AMEVEA Conference. Lima, Peru. May 15-21.
Park, Y., Yang, M., Storkson, J.M., Albright, K.J., Liu, W., Cook, M.E., and Pariza, M.W. 2007. Effects of conjugated linoleic acid (CLA) on tumor necrosis factor-alpha concentrations in mice. J. Food Biochemistry 31:252-265.
Trott, D.L., Yang, M., Cook, M.E. 2007. Additions of killed whole bacteria cell preparations to Freund's complete adjuvant alters antibody response to soluble protein antigens. Poultry Sci. pending.
Trott, D.L., Hellestad, E.M., and Cook, M.E. 2007. Adjuvants containing diverse peptidoglycan structures modulate hen antibody response to immunization. Poultry Sci.86:Suppl. 1:51 #M149.
Hellestad, E., Susko-Parrish, J., and Cook, M.E. 2007. The effect of anti-coccidiosis antibody on growth performance in broiler chicks. Poultry Sci 86: Suppl. 1:249 (#T24).

Progress 01/01/06 to 12/31/06

Outputs
In 2006. we showed that select isomers of conjugated linoleic acid (CLA) were inhibitors of cyclooxygenase-1 (COX-1) and effective in reducing platelet aggregation in a guinea pig blood model. The t10, c12 and t9, t11 isomer of CLA were the most effective inhibitors of platelet aggregation and inhibitors of macrophage release of thromboxane B2 (TXB2). The c9, t11 isomer of CLA had modest effects on platelet aggregation and TXB2 release, and c9, c12 linoleic acid or c9, c11 CLA had no effects on platelet aggregation or TXB2 release. The CLA cognate conjugated nonadecadienoic acid was found to also be effective at inhibiting platelet aggregation and TXB2 release. Previous work in our laboratory had suggested that the mechanism by which t10, c12 inhibited eicosanoid production was through the regulation of the NFkB signal transduction pathway. A mixture of c9, t11 and t10, c12-CLA was elongated one carbon to form a C19 fatty acid (c10, t12 and t11, c13 nonadecadienoic acid, or CNA). When applied to a previously used model of lipopolysaccharide (LPS)-induced COX-2 activity and transcriptional and translational up-regulation, it was found that CNA was effective in inhibiting COX-2 mRNA/protein expression, but unlike t10,c12-CLA the regulation appeared independent of NFkB. These results would suggest the anti-inflammatory and COX regulatory mechanism may be through a different signaling pathway than previously thought. Mechanism of anti-inflammatory effect of CLA gave insight into key pathways for regulating inflammatory disease that suppress growth and cause disease in farm animals. It became evident that an upstream step in inflammation mediated by eicosanoids was the enzyme phospholipase A2 (PLA2). Using a platform of antibody synthesis involving laying hens and egg yolk antibody, we previously showed that antibody made to PLA2 (aPLA2) would improve chick growth. We tested the growth enhancing effects of aPLA2 in a gut inflammation model we developed (coccidiosis). While aPLA2 was effective in improving growth in normal animals, growth suppression caused by inflammation (coccidosis or LPS-induced growth suppression) was not prevented using aPLA2. A number of companies around the world have begun to develop egg antibodies for use in animal agriculture and human applications. Numerous limitations remain in the commercial use of these products. Using the aPLA2 model we developed at UW, we began to explore economical ways to enhance the use of egg antibody for agriculture and medicinal uses. We were able to report on new discoveries involving the heat stability of egg antibody using trehalose. We also demonstrated the development of a new instrument to model steam conditioning of foods and feeds for studying methods to protect heat sensitive proteins. We demonstrate in collaboration with a faculty at University of Illinois (K. Koelkebeck) that force molting of laying hens has no adverse effect on egg antibody production, and that force molted hens were useful for antibody production. These results could prove to provide a new value to the "spent hen." COX inhibition was found to increase antibody production.

Impacts
Studies on dietary compounds that prevent inflammatory disease are the principal objective of this research project. Naturally occurring products that have anti-inflammatory action have previously been reported by the investigator's laboratory. Products that have been commercialized include conjugated linoleic acid (CLA) and egg antibody to key inflammatory products. More recent studies have investigated other biological activities of these products, the discovery of new products that have similar anti-inflammatory effects, and novel techniques and improved uses that will make these technologies of value for human and animal use. Additional studies have been conducted on successful anti-inflammatory products to better understand the basic mechanism by which they display such activity. Sales of conjugated linoleic acid exceed $US50 million per year. Estimates for egg antibody sales is not known at this time, but may prove to be a useful replacement for antibiotics as a growth promoting additive in animal feeds. Once this industry is established, sale could exceed $US100 million per year. Alternatives to antibiotics in the food animal industry could be potentially of value in reducing antibiotic resistant microorganisms in the environment. Use of CLA has already been proven to be effective in body fat reduction and reducing inflammation in humans.

Publications

Li, G., D.E. Butz, and M.E. Cook. 2006. Selective conjugated fatty acids inhibit guinea pig platelet aggregation. Eu. J. Pharmacology 545:93-99.
Li, G., B. Dong, D.E. Butz, Y Park, M.W. Paiza, and M.E. Cook. 2006 NF-kB independent inhibition of lipopolysaccharide-induced cyclooxygenase by a conjugated linoleic acid cognate, conjugated nonadecadienoic acid. Biochem. Biophy. Acta. 1761(9):969-972.
Bobeck, E.A., D.L. Trott, M.E. Cook, and M. Yang. 2006. Methods in heat stabilization of Gallus domesticus immunoglobulin Y (IgY). Poultry Sci. 85:Suppl 1:122.
Trott, D.L., M. Yang, P.L. Utterback, K.W. Koelkebeck, and M.E. Cook. 2006. Methods to improve the economics of egg antibody production. Poultry Sci.85:Suppl. 1:79.
Leone V.A. and M.E. Cook. 2006. Maternal dietary conjugated linoleic acid had no adverse effects on progeny development. Poultry Sci. 85:suppl1:12-13.
Schwartz, M., M. Yang, and M. Cook. 2006. Effects of egg antibody to phospholipase A2 (aPLA2) on preventing coccidian induced growth depression. Poultry Sci. 85:Suppl. 1: 38.
Trott, D.L., D.E. Butz, M.E. Cook. 2006. Effect of dietary cyclooxygenase inhibitors indomethacin and aspirin on egg yolk antibody synthesis. FASEB J 20:A1055.

Progress 01/01/05 to 12/31/05

Outputs
Conjugated linoleic acid (CLA) has now been recognized as a naturally occurring fatty acid that has anti-inflammatory activity. We have conducted an investigation into the mechanisms of CLA's anti-inflammatory action. We reported that the trans 10, cis 12 isomer of CLA decreased cyclooxygenase-2 (COX-2) activity, and protein and mRNA levels when induced in vivo and in vitro using lipopolysaccharide (LPS). We have proposed that the regulation of COX-2 is via the Nuclear Factor kappaB (NFkappaB) pathway. We have demonstrated that NFkappaB translocation to the nucleus during LPS challenage in vitro in Raw264.7 cells was inhibited by the t10, c12 CLA isomer. This inhibition appeared to be upstream of NFkappaB translocation since, phosphorylation of IkappaBalpha and its subsequent degradation were inhibited by treating the cells with the t10, c12 isomer. Currently it is not known how far upstream CLA regulates transcription of inflammatory genes. COX inhibition has been related to the prevention of immune- and cancer-induced body weight (muscle) wasting. Mice fed CLA were shown to have reduced gastrocnemius muscle wasting in tumor bearing mice. The mechanism appeared to be mediated by a down regulation of tumor necrosis factor receptor. Work also was conducted on another model of inflammatory disease, bone repair. While CLA has not been directly studied in the repair model, it was found that select events in the repair process, such as apoptosis and osteoclast recruitment could be targets for CLA effects in bone repair. Concern about CLA's effects on embryonic development lead to a study to determine if beef tallow from cows fed CLA would alter embryonic development when the tallow was fed to breeding hens. Tallow from CLA fed cows had no adverse effects on hen reproduction. To improve our understanding of the structure/function relationship between CLA and its ability to inhibit select enzymes involved in inflammation and prevent body fat accumulation, a 19 carbon derivative of CLA was made and named nonadecadienoic acid (CNA). CNA was more effective than CLA in decreasing markers associated with lipid accumulation in the adipocyte. CNA was also found to be a COX-1 and -2 inhibitor and was capable of inhibiting platelet aggregation. The data from the reports described above suggest that CLA is an effective dietary supplement or naturally occurring fatty acid that inhibits inflammatory events through an unknown process. The data also suggest that the addition of CLA to the diet of animals will not result in the production of a rendered fat that has a negative impact on fowl reproduction. Previous work from our group has shown that dietary CLA enhances bone mineral content. The effects of dietary CLA on the inflammatory events involving bone repair should be investigated using models such as the one described in this report.

Impacts
The trans 10, cis 12 isomer of conjugated linoleic acid (CLA) is the anti-inflammatory isomer of CLA. Animals fed can be protected from a wide range of diseases associated with inflammation, including muscle wasting caused by select cancers. The one adverse environmental effect of this isomer in the food supply (fowl reproduction) can be prevented by a slight modification of the fatty acids in the diet; hence, there should be no environmental concerns with regard to feeding CLA on fowl reproduction when used by a knowledgeable nutritionist.

Publications

Aydin, R. and M. E. Cook. 2005. The influence of conjugated linoleic acid enriched tallow on egg hatchability and quality in chickens. Internl. J Poultry Sci. 4 : 538-542
Clark, W.D., E.L. Smith, K.A. Kim, J.R. Paul-Murphy, P. Muir, and M. E. Cook. 2005. Osteocyte apoptosis and osteoclast-like cell appearance in chicken radii following osteotomy. Calcified Tissue Internl. 77:327-336.
Graves, E., A. Hitt, M.W. Pariza, M.E. Cook, and DE. O. McCarthy. 2005. Conjugated linoleic acid preserves gastrocnemius muscle mass in mice bearing the colon-26 adenocarcinoma. Res. Nurs. Health 28:48-55.
Li, G. D. Butz, D. Barnes, D. Bjorling, and M. E. Cook. 2005. 10t, 12c-conjugated linoleic acid inhibits lipopolysaccharide-induced cyclooxygenase protein expression in vitro and in vivo. J. Lipids Res. 46:2134-2142.
Cook, M.E., M.W. Pariza, Y. Park, and G. Li. 2005. Conjugated nonadecadienoic acid compositions. US Patent 6,908,946 B6

Progress 01/01/04 to 12/31/04

Outputs
In 2004, we introduced the concept of using egg yolk antibodies to key processes of inflammation for animal health in a review article. In this article, which was presented at the Poultry Science Meeting in Madison, WI in 2003, we showed how antibodies to key gut peptides, receptors and enzymes could enhance animal growth. Egg antibody technologies are currently being developed by industry. Conjugated linoleic acid (CLA) is a fatty acid that is naturally occurring in select animal fats, and also appears to play a significant role in animal health. We have previously showed that it is a fatty acid that is important in preventing the collateral damage caused by an over active immune/inflammatory response. We have continued studies on the safety of CLA in animal foods. One adverse effect that CLA has been shown to have is in the reproducing fowl. Depending of the fatty acid content of the bird's diet, CLA can adversely affect hatchability. In studies involving Japanese quail, we were able to show that the maximum amount of CLA that could be fed to breeding fowl without adverse effects on hatchability was 0.25% of the diet. The fatty acid changes of yolk associated with CLA feeding were reported and were characterized by an increase in unsaturated fatty acids and a decrease in monounsaturated fatty acids (an indication of altered activity of stearoyl-CoA desaturase. While previous work from our laboratory has shown that the adverse effects of CLA on the hatch of eggs can be prevented, additional study of the mechanisms are forthcoming. However, considerable progress was made in determining the specific structure on CLA responsible for the prevention of fat accumulation in animals. Clear from this work is that the trans 10, cis 12 position and geometry of the diene in C18:2 is essential for preventing body fat accretion. This isomer also appears to play a predominate, but not sole, role in the decrease in hatchability, through the inhibition of stearoyl-CoA desaturase.

Impacts
Conjugated linoleic acid (CLA) and egg yolk antibodies (EA) are natural food substances that can prevent immune damage to an immune stress or immune diseased animal/human. CLA is currently sold world-wide as a supplement and is entering the human/animal food chain beginning in 2004. EA should be commercialized beginning in 2005. One of the commercial applications for EA is as a replacement for growth promoting antibiotics in animal feeds.

Publications

Cook, M.E. 2004. Antibodies:alternatives to antibiotics in improving growth and feed efficiency. J. Appl. Poultry Res. 13:106-119.
Aydin, R. and M. E. Cook. 2005. The effect of dietary conjugated linoleic acid on egg yolk fatty acids and hatchability in japanese quail. Poultry Sci. 83:2016-2022.
Park, Y., J.M. Storkson, W. Liu, K.J. Albright, M.E. Cook, and M.W. Pariza. 2004. Structure-activity relationship of conjugated linoleic acid and its cognates in inhibiting heparin-releasable lipoprotein lipase and glycerol release from fully differentiated #T#-L1 adipocytes. J Nutr. Biochem. 15:561-569.

Progress 01/01/03 to 12/31/03

Outputs
Dietary conjugated linoleic acid (CLA) was shown to prevent cachexia in a mouse lupus model. Using this mouse model, dietary CLA, post proteinuria, extended life 1.7 fold. Using BALB/c mice, it was found that the 9cis, 11 trans CLA isomer inhibited endotoxin-induced release of tumor necrosis factor, nitric oxide, and induced an increase in interleukin-2 and a decrease in interleukin-4. A proposed mechanism for the affects of dietary CLA is CLA's regulation of cyclooxygenase and lipoxygenase.

Impacts
Conjugated linoleic acid is a natural food substance that can prevent immune damage to an immune stress or immune diseased animal/human.

Publications

Cook, M. E., D. Butz, G. Li, M. W. Pariza, L. Whigham, and M. Yang. 2003. Conjugated linoleic acid enhances immune responses, but protects against the collateral damage of immune events. In: Advances in Conjugated Linoleic Acid Research,Volume 2, J. Sebedio, W. W. Christie, and R. Adlof, editors, AOCS Press, Champaign, IL, pp., 283-291.
Yang, M. and M. E. Cook. 2003. Dietary conjugated linoleic acid decreased cachexia, macrophage TNFa production and modifies splenocyte cytokines production. Exptl Biol. Med. 228:51-58.
Yang, M., and M. E. Cook. 2003. Dietary conjugated linoleic acid decreased weight loss and extended survival following the onset of kidney failure in NZB/W F1 mice. Lipids 38:21-24.

Progress 01/01/02 to 12/31/02

Outputs
Using a Lupus mouse model, we extended life 1.7 fold using conjugated linoleic acid (CLA). We defined the mechanism as possibly regulated by eicosaniod. CLA may be a selective cyclooygenase-2 inhibitor.

Impacts
We have come up with a diet that prevents the formation of cat hairballs. It is sold world wide. We published a LCMS/MS procedure for identifying 15 lipid mediators. We are actively involved in the creation of an antibody technology to eliminate antibiotics in animal food for improved growth and feed efficiency. This should maintain animal productivity at over $100 million/year.

Publications

Whigham, L. D., A. Higbee, D. E. Bjorling, Y. Park, M.W. Pariza, and M. E. Cook. 2002. Conjugated linoleic acid reduces anti-induced prostaglandin and leukotriene release from sensitized guinea pig lung trachea, and bladder as measured by HPLC-tandem mass spectrometry. Am. J. Physiol. Regulatory. Integrative Comp. Physiol. 282:R1104-1112.
Attie, A. D., Y Hamon, A. R. Brooks-Wilson, M.P. Gray-Keller, M. L. E. MacDonald, V. Rigot, A. Tebon, L. Zhang, J. D. Mulligan, R. R. Singaraja, J. J. Bitgood, M. E. Cook, J. J. P Kastelein, G. Chimini, and M. R. Hayden. 2002. Identification and functional analysis of a naturally occurring E89K mutation in the ABCA1 gene of the WHAM chicken. J Lipid Res. 43:1610-1617.
Cook, M. E. 2002. Method of using anti-phospholipase A2 antibodies to enhance growth or improve feed efficiency. US Patent 6,383,485.
Cook, M. E. , M. Yang, and M. W. Pariza. 2002. Method of increasing life longevity and preventing body weight wasting in autoimmune disease by using conjugated linoleic acid. US Patent 6,395,782.
Cook,M. E. 2002. Regulation of eicosanoid pathways: A pathway to health and development. Pakistan J. Nutr. 1:103-105.

Progress 01/01/00 to 12/31/00

Outputs
The role of different derivatives of conjugated linoleic scid (CLA) on stearoyl-CoA desaturase activity was investigated. The t10, c12 isomer appeared to be the active isomer and the inhibition activity could not be enhanced by structural modification. Since this enzyme is thought to play a role in body fat accumulation, identifying the active isomer and ways to improve its inhibitory activity could be important in drug development. CLA was also shown to extend the life of mice prone to systemic lupus erythematosus (SLE) by 49% after first signs of proteinuria. Life extension of people suffering from this disease by a natural occurring fatty acid could improve human health. A human adenovirus has been found to markedly increase the adiposity of animals. A population of obese humans have been found to be seropositive for this virus. Demonstration of the role of a common human virus in obesity could prove value for developing a vaccine to prevent some forms of obesity. The role of antibiotics in human and animal health is well recognized. Currently there is increased concern that the use of antibiotics in animal feeds may result in antibiotic resistant organisms in the human population. The reasons antibiotics are used in animal feeds, the mechanisms by which they enhance growth, alternatives of antibiotics for growth promotion, and the potential harm banning antibiotics in animal feeds was reviewed. This information is already in the hands of congressional staff as they plan to deliberate the fate of antibiotics in animal feeds. Five patents issued in the year 2000. One of the patents shows a formulation of different isomer ratios of CLA that maximizes animal performance, but minimizes body fat. In another, a use patent issued showing that CLA increases certain muscle fiber types and improves meat quality. In a third patent, a method for enhancing CLA in eggs was disclosed. These 3 patents will be essential for protecting investments of companies as they generate an animal feed form of CLA. Two other patents present the immune enhancing effects of CLA and its mechanism through the regulation of cyclooxygenase-2, an enzyme involved in the synthesis of prostaglandins. These results provide the basis of understanding how CLA improves longevity of the SLE mouse and also explain the basis of CLA's anti-carcinogenic, anti-atherogenic, immune-modulating, and anti-cachectic effects.

Impacts
Conjugated linoleic acid has already had an impact on the development of new companies and divisions within companies. Products are commercially available world-wide. Scientific evidence of human health benefits is currently being reported.

Publications

Pariza, M.W., Y. Park and M.E. Cook, 2000. Mechanisms of action of conjugated linoleic acid. Evidence and speculation. Proc. Soc. Exptl. Biol. Med. 223: 8-13.
Cook, M.E., 2000. Skeletal deformities and their causes. Poultry Science 79:982-984.
Park, Y., J. Storkson, J. Ntambi, M.E. Cook, C.J. Sih and M.W. Pariza, 2000 Inhibition of hepatic stearoyl-CoA desaturase activity by trans 10, cis 12 conjugated linoleic acid and its derivatives. Biochim. Biophys. acta. 1486:285-292.
Yang, M., M.W. Pariza, M.E. Cook, 2000. Dietary linoleic acid protects against end stage disease of sytemic lupus erythematosus in the NZB/W F1 mouse. Immunopharma. Immunotox. 22:433-449.
Cook, M.E., 2000. The interplay between modern management practices and the chicken: how immune response and the physiological mechanism for growth and feed efficiency have adapted over time. Where do we go from here. In: Biotechnology in the Animal Feed Industry (eds., T.P. Lyons and K.A. Jacques). Nottingham University Press, pp 97-110.
Dhurandhar, N.V., B.A. Israel, J.M. Kolesar, G.F. Mayhew, M.E. Cook, and R.L. Atkinson, 2000. Increased adiposity in animals due to a human virus. Intl. J. Obesity 24:989-997.
Cook, M.E., D. Jerome, and M.W. Pariza, 2000. Method for selectively altering body fat levels, feed efficiency, or weight gain. U.S. Patent 6,020,378.
Cook, M.E., L.D. Whigham, and M.W. Pariza, 2000. Selective inhibition of cyclooxygenase-2. U.S. Patent 6,077,868.
Cook, M.E. and M.W. Pariza, 2000.Methods of treating animals to maintain or increase CD-4 and CD-8 cell populations. U.S. Patent 6,020376.
Cook, M.E., D.L. Jerome, M.W. Pariza, D.R. Buege, P. Mozdziak, 2000. Method of increasing fat firmness and improving meat quality in animals. U.S. Patent 6,060,087.
Cook, M.E., R. Aydin, and M.W. Pariza, 2000. Eggs enriched with conjugated linoleic acid and method of making same. U.S. Patent 6,113,973.
Aydin, R. 2000. The effects of dietary conjugated linoleic acid on avian lipid metabolism. Dissertation, University of Wisconsin, Madison, WI.
Whigham, L. D. 2000 Inhibition of lipid mediators in type 1 hypersensitivity immune reactions by conjugated linoleic acid. Dissertation, University of Wisconsin, Madison, WI

Progress 01/01/99 to 12/31/99

Outputs
Conjugated linoleic acid (CLA) is a naturally occurring fatty acid which has been shown to have anticarcinogenic and antiatherogenic activities. Previous research has also shown that it reduces body fat, increases lean mass, improves feed efficiency and enhances immune function while protecting nonlymphoidal cells against immune reactivity. We have found that CLA regulates both prostaglandin and leukotriene synthesis, that the trans 10, cis 12 isomer is responsible for fat reduction, that the cis 9, trans 11 isomer is a growth factor, that these isomers extend life 30-40% in lupus prone mice, that these isomers can inhibit avian embryonic survival in low fat diets which can be prevented with the addition of select dietary fatty acids, and that these isomers can attenuate the type I hypersensitive reaction. We have also found that CLA enhances the total number of natural killer cells which may be responsible for the anticarcinogenic activity of CLA.

Impacts
CLA is available world wide for human use. Anti-cholecystokinin is in commercial use in the poultry industry as a way to improve feed efficiency and replacement of antibiotics. We believe these alternative products will improve the effectiveness of antibiotics in human medicine.

Publications

Cook, M.E., 1999. Nutritional Effects on Vaccination. In: Veterinary Vaccines and Diagnostics (ed. R. D. Schultz). Advances in Veterinary Medicine Vol. 41: 53-59.
Cook, M.E., D. DeVoney, B. Drake, M.W. Pariza, L. Whigham, and M. Yang, 1999. Dietary control of immune-induced cachexia conjugated linoleic acid and immunity. In: Advances in Conjugated Linoleic Acid Research, Volume 1. (editors M.P. Yurawecz, M.M. Mossaba, J.K.G. Kramer, M.W. Pariza and G.J. Nelson) pp 226-237.
Park, Y., K. J. Albright, J. M. Storkson, W. Liu, M. E. Cook, and M. W. Pariza, 1999. Changes in body composition in mice during feeding and withdrawl of dietary conjugated linoleic acid. J. Lipids. Lipids 34: 243-248.
Pariza, Michael W., Yeonwha Park and Mark E. Cook, 1999. Conjugated Linoleic Acid and the Control of Cancer and Obesity. Toxicological Sciences. Toxicological Sciences. 52(Supplement): 107-110.
Cook, M.E., S. Kim, M.W. Pariza, D. DeVoney, 1999. Method of treating animals to enhance natural killer lymphocyte function. US Patent 5,914,346.
Cook, M.E., Y. Park, and M.W. Pariza, 1999. Method for controlling body fat and/or body weight in animals and pharmaceutical compositions for use therein. US Patent 5,855,917.
Cook, M.E., S. Kim, M.W. Pariza, D. DeVoney, 1999. Method of treating animals to enhance natural killer lymphocyte function. US Patent 5,914,346.
Cook, M.E., Y. Park, and M.W. Pariza, 1999. Method for controlling body fat and/or body weight in animals and pharmaceutical compositions for use therein. US Patent 5,855,917.

Progress 01/01/98 to 12/31/98

Outputs
Immune stimulated animals systemically release cytokines (i.e. interleukin-1 (IL-1) and tumor necrosis factor) which in turn alter physiological and metabolic processes in the body. Their gross consequence associated with an immune effect is reduced feed intake, poor feed efficiency and weight loss or decreased rates of gain. For example, IL-1 stimulates prostaglandin E2 production in muscle which both induces muscle degradation and in turn can down-regulate immunity. IL-1 will also stimulate gut peptide release (such as cholesystekinin (CCK)) which induces anorexia. The publications below discuss methods to prevent immune induce changes in growth. Conjugated linoleic acid (CLA) decreases prostaglandin synthesis and protects from immune induced weight loss as well as enhancing immunity. Binding gut peptides with antibodies improves feed efficiency.

Impacts
Use of CLA has been licensed to 7 companies. Human grade supplements are marketed worldwide and feed grade products are now available. Gross value to the US swine industry is estimated to be over $800 mil/yr. Anti-CCK technology was licensed and is entering the industry as a growth stimulant and to improve feed efficiency. The value to the US broiler industry could exceed $100 mil/yr.

Publications

Cook, M.E., J.L. Pimentel and C.C. Miller. 1998. CCK antibodies used to improve feed efficiency. US Patent 5,827,517.
Cook, M.E. and D. L. Jerome. 1998. Method of improving the growth or the efficiency of feed conversion of an animal and compositions for use therein. US Patent 5,725,873.
Cook, M.E., M.W. Pariza, X. Yang and D. DeVoney. 1998. Methods of treating animals to maintain or increase CD-4 and CD-8 cell populations. US Patents 5,827,885.
Cook, M. E. and M. Pariza, 1998. The role of conjugated linoleic acid in health. Int. Dairy Journal. 8:459-462.

Progress 01/01/97 to 12/31/97

Outputs
Studies were conducted to investigate the role of conjugated linoleic acid (CLA) on immunological and physiological function. CLA was found to protect against the catabolic nature of the immune response and enhance immunological function. In addition, CLA caused a dramatic change in body composition with over a 50% reduction in body fat and up to a 14% increase in body lean. Changes in body composition were not associated with changes in body weight. CLA was found to be an inhibitor of adipocyte lipoprotein lipase activity and enhanced carnitine palmitoytransferase.

Impacts
(N/A)

Publications

Park, Y., K. J. Albright, W. Liu, J. M. Storkson, M. E. Cook, M. W. Pariza. 1997. Effect of conjugated linoleic acid on body composition in mice. Lipids 32:853-858.

Progress 01/01/96 to 12/30/96

Outputs
Mycotoxins produced by fusarium molds are known to cause immune suppression and other signs of toxicoses often the mechanism of immunotoxicosis is by way of altered nutrient metabolism. In studies investigation immunotoxicoses, field samples associated with altered human or animal health were studied. In the first study, feed and bedding from a Wisconsin dairy farm where cow were dying of hemorrhage and contain aseptic blister on their bucks and udders were tested to determine the role of mycotoxins as a cause. Moldy bedding was found to contain fusarium sporotrichioides. When bedding was placed on the backs of rats, similar blistering occurred. Analysis of the bedding reveal the presence of T-2 toxin which is well know to produce similar clinical sighs and immune suppression. A fusarium oxysporum was isolated from corn consumed by children in a remote region of China. The children had developed a skeletal defect known as Kasdrin Beck Syndrome. When baby chicks were fed corn cultures of f. Oxysporum the skeletal defect, tibial dysclrondroplasia, was induced. Plasma glycosaminoglycans were elevated in chickens as was previously reported in afflicted children, these data supported the hypothesis of a mycotoxin cause of the skeletal disease in these children. It was proposed that the broiler chicken could be a useful field model for screening corn for potential toxin.

Impacts
(N/A)

Publications

Chr, Q., W. Wu, M. E. Cook, and E. B. Smalley. 1996. Elevated glycosaminoglycunsin chickens with tibial dysdrondroplasia induced by a Fusarium Oxysporum Avian Dis 40:715-719.
Li, G., M. E. Cook, and W. Wu. 1996. Reduction of ferricyanide by thiamine or thiamine pyrophosphate. Biochem. Biophys. Res. Comm. 226:187-192.
Wu, W., M. E. Cook, F. S. Chu, T. Buttles, J. Hunger, and P. Sutherland. 1996. Case study of bovine dermatitis caused by oat straw infected with Fusarium Sporotrichioides. Vet Rec 140:399-400.
Cook, M. E. 1996. Diet-induced immunosuppression in: Poultry Immunology. Eds. T.S. Davison, T. R. Morris and L. N. Payne. Carfax Publishing Co. Oxfordshire, England. pp 317-328.

Progress 01/01/93 to 12/30/93

Outputs
Phellinus weirii is a fungal species that has been shown to cause butt rot in western red cedar and root rot in Douglas fir. Attempts have been made to separate the two forms of Phellinus weirii. Using laying hens, antibodies were produced against the two forms. Western blots were found to successfully separate the two forms based upon the presence of a 57 kDa band found in the fir form. ELISA also showed that the fir form was more closely related to Asian species P. sulphurascens. Again these data demonstrate the successful use of egg yolk antibodies in serological testing. The first report of the ability of conjugated linoleic acid (CLA) to prevent growth depression due to immune stimulation was published. Also reported were the immune-enhancing effects of CLA. Chicks, rats, mice, and rabbits fed CLA were found to be more efficient at converting food into body weight. These data suggest that CLA may be a valuable feed additive for animal agriculture. Previous reports have demonstrated that mammals are immunologically tolerant to food protein regularly consumed. Attempts were made to see if chickens could also be rendered immunologically tolerant to food antigens. While oral tolerance was produced in rats given casein or bovine serum albumen, chickens could not be made tolerant by orally-gavaged protein antigens. On the contrary, feeding chickens select protein antigens actually enhanced antibody responses following systemic exposure.

Impacts
(N/A)

Publications

BANIK, M.T., J.A. PAUL, H.H. BURDSALL, and M.E. COOK, 1993. Serological differentiation of two forms of Phellinus weirii. Mycologia: in-press.
COOK, M.E., C.C. MILLER, Y. PARK, and M. PARIZA. 1993. Immune modulation by altered nutrient metabolism. Nutritional control of immune-induced growth depression. Poultry Sci. 72:1301-1305.
MILLER, C. C. , and M. E. COOK. 1994. Evidence against the induction of immunological tolerance by feeding antigens to chickens. Poultry Sci. 73:(in press).
MILLER, C., Y. PARK, M.W. PARIZA, and M.E. COOK. 1994. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochim. Biophys. Res. Comm. 183:(in press).