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TM The scientific information in this journal is educational and is not to be used as a substitute for a doctor's care or for proven therapy. PROVIDING SCIENTIFIC INFORMATION RELATED TO NUTRITIONAL SACCHARIDES AND OTHER DIETARY INGREDIENTS. AUGUST 3, 2001 VOL 2, NO 17 EXTERNAL EDITORIAL BOARD John Axford, BSc, MD, FRCP Consultant and Reader in Rheumatology and Clinical Immunology. St.George's Hospital Medical School University of London London, England Tom Gardiner, PhD Global Health Safety Environment and Regulatory Affairs Coordinator Shell Chemical Company (Retired) Houston, Texas Robert K. Murray, MD, PhD Professor (Emeritus), Biochemistry University of Toronto Toronto, Ontario, Canada Doris Lefkowitz, PhD Clinical Associate Professor of Microbiology University of South Florida College of Medicine Tampa, Florida MANNATECH INCORPORATED INTERNAL CONTRIBUTING AND CONSULTING EDITORS Stephen Boyd, MD, PhD, FRSM Bill McAnalley, PhD H. Reginald McDaniel, MD Stanley S. Lefkowitz, PhD Clinical Professor of Microbiology and Immunology University of South Florida College of Medicine Tampa, Florida James C. Garriott, PhD, D-ABFT Professor (Clinical Adjunct Faculty) University of Texas Health Science Center Consulting Toxicologist San Antonio, Texas TECHNICAL STAFF Gary Carter, BS Kia Gary, RN Barbara Kinsey Eric Moore, DChem Mary Wood GRAPHIC ARTIST Bruce Peschel MANAGING EDITOR Jane Ramberg, MS Alice Johnson-Zeiger, PhD Professor of Biochemistry (Retired) University of Texas Health Center Tyler, Texas EDITOR IN CHIEF Eileen Vennum, RAC Comparison of Natural Killer Cell Activation by Selected Nutritional Supplements: An In Vitro Study Stan Lefkowitz, PhD and Doris Lefkowitz, PhD ABSTRACT Protection from disease depends on the effective function of cells involved in the immune response. Macrophages, cells central to the immune response, have previously been shown to be powerfully stimulated by glyconutrients.1 Macrophages orchestrate the activity of other immune system cells, including natural killer (NK) cells. NK cells, white blood cells that circulate through the body and attack target cells (invaders), such as cancer cells, viruses, bacteria, and fungi, are crucial to normal immune system function. An extensive scientific literature has documented the effects of diet on proper immune function. This study was an in vitro comparison of the effect of two dietary supplements on human NK cell activity (a measure of the ability of NK cells to kill target cells). The first, a glyconutritional supplement (GN), is listed in the Physicians’ Desk Reference for Nonprescription Drugs and Dietary Supplements (PDR) as a supplement that can provide immune support.2 This effect has been documented in the scientific literature.1,3 GN is composed of various polysaccharides used by the body in glycoform synthesis and cell-to-cell communication. The TM second supplement tested, a bovine colostrum product with added constituents such as zinc, aloe vera and mushrooms (TFP), is not listed in the PDR (2001 edition). However, this product is promoted as an "immune system activator" on the web site of the distributing company. At no dose tested was TFP more effective than GN at stimulating NK cell activity. However, in amounts representing achievable levels of dietary intake, GN significantly enhanced natural killer cell activity 65% more than did TFP (p≤0.001.) GlycoScience & Nutrition (Official Publication of GlycoScience.com: The Nutrition Science Site). 2000;2(17):1-4. INTRODUCTION Natural killer (NK) cells, white blood cells from our "immunologic army", are constantly on the alert, circulating throughout the body looking for cancer cells or cells infected with viruses, bacteria, or fungi. Approximately 15% of the white blood cells in the peripheral blood are NK cells.4 In addition to residing in the peripheral blood, these cells are abundant in the spleen, an organ that filters circulating blood. The NK cell’s position in the peripheral circulation The Official Publication of www.usa.GlycoScience.com: The Nutrition Science Site Published by the Research and Development Department of Mannatech Incorporated, Coppell, Texas, USA. © 2000 All rights reserved. and the spleen enhances its chance of coming in contact with microorganisms that are trying to invade our bodies. NK cells are responsible for "immune surveillance." 5,6 That is, they are always patrolling the body on a "search and destroy" mission. The NK cell is ready to eliminate any aberrant cells as they arise in the body. When an NK cell encounters an abnormal cell, it uses one of its many weapons to kill that cell. Unlike many other cells of our immunologic army, NK cells neither require being educated (sensitized) as to what the enemy is nor do they need any help to kill an abnormal cell. Thus, they can be regarded as acting like "hit men." When they see something they do not recognize, they kill the target without warning (or remorse!). These cells use various methods to kill their prey.4,6 They can secrete a substance called perforin that punches holes in the target cell membrane, causing it to burst. Or, they can punch a few holes in the target cell membrane and then insert tumor necrosis factor through the holes. This process causes the target cell to die slowly. Yet another mechanism is to secrete substances called granzymes. Granzymes are enzymes that cut up a cell’s DNA and thus also cause that cell to die. In addition to killing, NK cells have another major function. These cells help regulate the immune response.6,7 They do so by secreting various cell-cell communication signals called cytokines.4,7 Cytokines alert other cells of the immunologic army to the fact that there are abnormal cells present in the body, and then activate these cells. For example, NK cells secrete particular cytokines that cause macrophages (another white blood cell) to kill an invader.8 The invader may be a cancerous cell or a cell infected with bacteria, viruses, or fungi. Regardless of the methods chosen, NK cells are usually successful at accomplishing the goal of killing their prey. How do NK cells know which cells to kill and which cells to leave alone? Until recently, this was a mystery. It is now known that NK cells make contact with target cells. While adhering to the target cell, NK cells determine if the target cells have the correct glycoproteins (proteins with sugars attached) on their surface. These glycoproteins are collectively called major histocompatability complex (MHC). If a target cell does not have the correct MHC glycoproteins on its surface, it is marked for death (Figure 1).9,10 NK cells have two structures (glycoprotein receptors) with opposing functions on their surface that determine whether the NK cell will be activated or inactivated.6,11 Some of these receptors interact with carbohydrates on the target cell surface. Various sugars in the form of oligosaccharides on certain proteins, bind to receptors on the NK cell and activate it.12 Other NK cell receptors bind to carbohydrates on a target cell and the binding inactivates NK cell activity. This is an example of how carbohydrates modulate the immune system by either activating or inactivating NK cell activity (Figure 1). Another example of carbohydrate/cell interaction is the NK response to the influenza virus. After this virus infects a host cell, it inserts viral glycoproteins on the membrane of the host cell. NK cells bind to the carbohydrate part of these glycoproteins on the surface of virus-infected cells and subsequently kill the cell.13 Since the NK cell receptor for the GlycoScience Vol. 2, No. 17 viral carbohydrate is itself composed of carbohydrates, the end result is carbohydrate-carbohydrate interaction. As stated previously, NK cells are also intimately involved in regulation of the immune response. This is evident in a variety of situations, one of which is Chediak-Higashi Syndrome.6 In this condition, individuals have low NK cell counts, immune surveillance is impaired and there is an increased risk of cancer or infections.4 From studies involving this condition, a low NK cell count is known to be correlated with that person’s susceptibility to viral infections.4 Also, the number of functional NK cells that a patient with cancer undergoing chemotherapy has is a good prognosticator of his or her ability to eliminate the cancer.4 Conversely, an overabundance of NK cells has also been associated with autoimmune disease.7 In this situation the NK cell attacks the host’s cells, leaving a trail of destruction and inflammation that is ultimately harmful to the person. It should be readily apparent that NK cells are essential for a healthy immune system. Without their proper function, disease and severe problems follow. METHODS Experiments have been designed to compare the ability of a glyconutritional supplement (GN) and a colostrum supplement (TFP) to activate NK cells in vitro using a target-cell killing assay. In the laboratory, there are several ways to determine NK activity. The standard assay involves the use of target cells labeled with the radioactive isotope chromium 51Cr.14 NK cells are first exposed to the stimulus or inhibitor that is being investigated. Then, chromium labeled target cells are added to the NK cells. After a period of incubation, the cell growth fluid medium is collected and the amount of chromium measured. The more chromium released into the medium, the more target cells were killed by the NK cells. The amount of chromium is recorded using a gamma counter, which measures radiation released by chromium. The present study utilized NK-92 cells, a human NK cell line, and Raji cells, a human Burkitt’s lymphoma cell line, as the target cell. Both cell lines were obtained from the American Type Culture Collection. The NK cells were incubated overnight in dilutions of 10 and 100 mg/ml of the two August 3, 2001 2 nutritional products. Assuming 100% absorption and distribution of both products in an individual of average weight (75 kg or 165 pounds), 10 mg/ml in vitro is approximately equivalent to that attained in vivo using one-half teaspoon (750 mg) of GN or two capsules of TFP. A ten-fold increase (100 mg/ml) is approximately equivalent to one and twothirds tablespoons GN or 20 capsules TFP. Raji cells were incubated with radioactive chromium. These cells were then combined for 4 hours with ratios of NK-92 cells: Raji cells of 20:1, 10:1, 5:1, and 2.5:1. Culture fluids were then harvested and the amount of chromium (radioactivity) was measured as an indication of cell lysis. These data were converted into a comparison of the relative amount of target cell killing. The radioactivity counts from different ratios of NK cells to target cells were combined and applied to the formula published by Djeu.14 This calculation produced a number in Lytic Units that allows for quantitative comparison of data from each experiment. The experimental investigation was then repeated in its entirety. RESULTS The data in Figure 2 represent the relative amount of target cell killing by both supplements. Figure 2. Comparison of NK activity between GN and TFP. The cell line NK-92 was incubated overnight with the above nutrients then incubated with Raji cells for 4 hours. The lysis of Raji cells was measured by the release of 51Cr. The radioactivity counts from different rations of cells were combined and expressed as lytic units. Both treatments compared to the control were significantly different (p ≤ 0.001). It is apparent that both of the products tested stimulated NK cell activity. At the lower dose (10 mg/ml) GN and TFP induced approximately twice the cell lysis compared to NK cells only exposed to media (the control), and no difference between the effectiveness of GN and TFP was apparent. The higher amount of GN produced a significant increase of 65% in NK killing activity (p≤0.001). No increase was observed with TFP, even at 10 times the recommended serving size (Figure 2). Similar results were obtained when the experiment was repeated. DISCUSSION In summary, this experiment demonstrated that, at recommended serving sizes (represented by 10 mg/ml in vitro), GN and TFP produced approximately the same NK killing activity. However, at larger serving sizes, GN produced a 65% increase in NK killing activity. This experiment showed that GN can enhance natural killer cell activity significantly more than TFP (p≤0.001). Previous studies have suggested that GN show particular promise in immune system enhancement. In a recent in vitro study, enhancement of immune response by GN was demonstrated by enhanced killing of Candida albicans by macrophages.1 In a study of mice with sarcomas, animals treated with GN showed increased tumor regression and improved survival when compared with animals that did not receive supplementation.3 While the mechanism explaining this effect is as yet incompletely understood, it is known that cell surface carbohydrates play a crucial role in effective NK cell function. And, a body of scientific literature is rapidly growing that documents the fact that changes or abnormalities in glycoprotein formation significantly alter cellular function and, ultimately, the health of the individual.15,16,17,18,19 Further, some glyconutrients have been shown to be preferentially incorporated into glycoproteins.20,21 Additional research at the cellular level will be required to document the mechanism by which GN may enhance NK cell function and whether such enhancement can be demonstrated in vivo. ACKNOWLEDGMENTS Glyconutrients (Ambrotose® complex [Lot # 012104]), were obtained from Mannatech , Inc. Coppell TX and the thymic protein product (Transfer Factor Plus® [Lot # 20120]) was from 4Life- Research, Provo, UT. This study was funded by Mannatech. TM REFERENCE LIST 1. Lefkowitz SS, Lefkowitz DL. Macrophage candidicidal activity of a complete glyconutritional formulation versus aloe polymannose. Proc Fisher Inst Med Res. 1999;1(2):5-7. 2. Physicians' Desk Reference for Nonprescription Drugs and Dietary Supplements. 22nd Edition. Montvale, N.J.: Medical Economics, 2001. 3. Campbell BD, Busbee DL, McDaniel HR. Enhancement of immune function in rodents using a proprietary complex mixture of glyconutritionals. Proc Fisher Inst Med Res. 1997;1(1):34-37. 4. Kumar V, Bennett M. Natural Killer Cells. In: Frank MM, Austen KF, Claman HN, and Unanue ER, editor(s). Samter's Immunologic Diseases. Little, Brown and Company,1995: 311-319. 5. Whiteside TL, Herberman RB. The role of natural killer cells in immune surveillance of cancer. Curr Opin Immunol. 1995;7(5):704-710. REFERENCE LIST (continued next page) GlycoScience Vol. 2, No. 17 August 3, 2001 3 REFERENCE LIST (continued) 6. Kuby J. Cell-mediated effector responses. In: Goldsby RA, Kindt TJ, and Osborne BA, editor(s). Immunology. W.H. Freeman and Company,2000: 351-360. 7. Yokoyama WM. Natural killer cells. In: Paul WE, editor(s). Fundamental Immunology. Lippincott-Raven Publishers,1999: 575-603. 8. Abbas AK, Lichtman AH, and Pober JS. Innate Immunity. In: Abbas AK, Lichtman AH, and Pober JS, editor(s). Cellular and Molecular Biology. W.B. Saunders Company (A Harcourt Health Sciences Company),2000: 283-287. 9. Abbas AK, Lichtman AH, and Pober JS. Effector mechanisms of t cell-mediated immune reactions. In: Abbas AK, Lichtman AH, and Pober JS, editor(s). Cellular and Molecular Immunology. W.B. Saunders Company,1997: 293-295. 10. Carbone E, Terrazzano G, Melian A, et al. Inhibition of human NK cell-mediated killing by CD1 molecules. J Immunol. 2000;164(12):6130-6137. 11. Lanier LL. NK cell receptors. Annu Rev Immunol. 1998;16:359-393. 12. Bezouska K, Yuen CT, O'Brien J, et al. Oligosaccharide ligands for NKR-P1 protein activate NK cells and cytotoxicity. Nature. 1994;372(6502):150-157. 13. Mandelboim O, Lieberman N, Lev M, et al. Recognition of haemagglutinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature. 2001;409(6823):1055-1060. 14. Djeu JY. Natural Killer Activity. In: Burleson GR, Dean JH, and Munson AE, editor(s). Methods in Immunotoxicology . Wiley-Liss (A John Wiley & Sons, Inc., Publication),1995: 437-449. 15. Bond A, Alavi A, Axford JS. The relationship between exposed galactose and N-acetylglucosamine residues on IgG in rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and Sjögren’s syndrome (SS). Clin Exp Immunol. 1996;105 (1):99-103. 16. Malhotra R, Wormald MR, Rudd PM. Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein. Nat Med. 1995;1(3):237-243. 17. Freeze HH. Disorders in protein glycosylation and potential therapy: tip of an iceberg? J Pediatr. 1998;133(5):593-600. 18. Freeze HH. Human glycosylation disorders and sugar supplement therapy. Biochem Biophys Res Commun. 1999;255(2):189-193. 19. Freeze HH, Aebi M. Molecular basis of carbohydrate-deficient glycoprotein syndromes type I with normal phosphomannomutase activity. Biochim Biophys Acta. 1999;1455(2-3):167-178. 20. Alton G, Hasilik M, Niehues R. Direct utilization of mannose for mammalian glycoprotein biosynthesis. Glycobiology. 1998;8(3):285-295. 21. Berger V, Perier S, Pachiaudi C. Dietary specific sugars for serum protein enzymatic glycosylation in man. Metabolism. 1998;47(12):1499-1503. GlycoScience Vol. 2, No. 17 August 3, 2001 4