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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
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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
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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
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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.
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Economics, 2001.
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