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An antiinflammatory immunogen from yeast culture induces activation and alters chemokine receptor expression on human natural killer cells and B lymphocytes in vitro

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  • AIBMR Life Sciences Inc.

Abstract and Figures

The aim of this study was to evaluate the immunomodulating effects of a consumable yeast-based immunogen, EpiCor, on human leukocytes in vitro. The selection of antiinflammatory and lymphocyte activation assays was based on initial evidence for immunomodulating effects of EpiCor from an unusually low incidence of influenza among employees in a factory manufacturing EpiCor, along with a high oxygen radical absorbance capacity value. In the present study, EpiCor significantly reduced the production of reactive oxygen species by neutrophils (P < .005). EpiCor treatment of peripheral blood mononuclear cells (PBMCs) caused induction of the activation markers CD80 and CD86 on B lymphocytes, and CD69 and CD25 on CD3−CD56+ natural killer cells. This induction was also seen on enriched populations of natural killer and B lymphocytes, suggesting a direct effect not dependent on bystander cells. Coculturing of PBMC with EpiCor and phytohemagglutinin resulted in inhibition of phytohemagglutinin-induced T-cell proliferation and reduction of interferon gamma production. Fucoidan, a ligand for the homing molecule l-selectin (CD62L), is known to induce rapid up-regulation of several chemokine receptors on lymphocytes. EpiCor caused strong inhibition of Fucoidan-mediated expression of the chemokine receptors CXCR4 and CCR9 on PBMC. This suggested rapid altering of signal transduction pathways, or a direct competition for cell surface receptors, with an end result being an altered sensitivity to chemotactic signals from tissue. We conclude that EpiCor possesses significant antiinflammatory activity and induces direct activation and increased chemotactic awareness of lymphocyte subsets in vitro. This suggests further study of effects of EpiCor consumption on antiviral defense mechanisms and antibody production.
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An antiinflammatory immunogen from yeast culture induces
activation and alters chemokine receptor expression on human
natural killer cells and B lymphocytes in vitro
Gitte S. Jensen
a,
4, Aaron N. Hart
a
, Alexander G. Schauss
b
a
NIS Labs, Klamath Falls, OR 97601, USA
b
Natural amd Medicinal Products Research, AIBMR Life Sciences Inc, Puyallup, WA 98373, USA
Received 27 November 2006; revised 11 April 2007; accepted 13 April 2007
Abstract
The aim of this study was to evaluate the immunomodulating effects of a consumable yeast-based
immunogen, EpiCor, on human leukocytes in vitro. The selection of antiinflammatory and
lymphocyte activation assays was based on initial evidence for immunomodulating effects of
EpiCor from an unusually low incidence of influenza among employees in a factory manufacturing
EpiCor, along with a high oxygen radical absorbance capacity value. In the present study, EpiCor
significantly reduced the production of reactive oxygen species by neutrophils ( Pb.005). EpiCor
treatment of peripheral blood mononuclear cells (PBMCs) caused induction of the activation markers
CD80 and CD86 on B lymphocytes, and CD69 and CD25 on CD3
CD56
+
natural killer cells. This
induction was also seen on enriched populations of natural killer and B lymphocytes, suggesting a
direct effect not dependent on bystander cells. Coculturing of PBMC with EpiCor and
phytohemagglutinin resulted in inhibition of phytohemagglutinin-induced T-cell proliferation and
reduction of interferon gamma production. Fucoidan, a ligand for the homing molecule l-selectin
(CD62L), is known to induce rapid up-regulation of several chemokine receptors on lymphocytes.
EpiCor caused strong inhibition of Fucoidan-mediated expression of the chemokine receptors
CXCR4 and CCR9 on PBMC. This suggested rapid altering of signal transduction pathways, or a
direct competition for cell surface receptors, with an end result being an altered sensitivity to
chemotactic signals from tissue. We conclude that EpiCor possesses significant antiinflammatory
activity and induces direct activation and increased chemotactic awareness of lymphocyte subsets in
vitro. This suggests further study of effects of EpiCor consumption on antiviral defense mechanisms
and antibody production.
D2007 Elsevier Inc. All rights reserved.
Keywords: Saccharomyces cerevisiae; Human; Immune; NK cell; B cell; Chemokine receptor; Interferon; ROS;
Antiinflammatory
1. Introduction
EpiCor is an immunogen product based on anaerobic
fermentation of baker’s yeast (Saccharomyces cerevisiae)in
a proprietary medium. After fermentation, the whole liquid
is dried, resulting in a product that is high in yeast
metabolites, including vitamins, polyphenols, sterols, and
phospholipids. An unusually low incidence of influenza
infectivity among certain employees over several decades
working in a fermentation facility in Cedar Rapids, Iowa,
led to a theory that these employees’ exposure to a yeast-
based product produced at the facility and absorbed daily by
inhalation and/or ingestion might have an effect on their
0271-5317/$ – see front matter D2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2007.04.008
4Corresponding author. Tel.: +1 541 884 0112; fax: +1 541 884 0113.
E-mail address: gitte@holgernis.com (G.S. Jensen).
Nutrition Research 27 (2007) 327 – 335
www.elsevier.com/locate/nutres
immune defense. Bioactive components include the nutri-
ent/vitamin profile, cell wall components, and stress-
induced metabolites. Based on the known effects of yeast
cell walls on immune cell reactions in vitro, an overall
proinflammatory effect could be expected. However, the
product has an oxygen radical absorbance capacity (ORAC)
value within the range of 450 to 650 lmol Trolox
equivalents per gram [1] and a high reactive oxygen species
(ROS) scavenging activity [2]. The batch of EpiCor used in
this study had an ORAC value of 614 lmol of TE (trolox
equivalents) per gram. The overall combined effects of the
complex profile of pro- and antiinflammatory compounds
present in EpiCor have not previously been studied on
human immune cells in vitro. The decrease in incidence of
viral disease among the facility workers and the extremely
high antioxidant value of this food prompted this study to
assess for antiinflammatory potential and direct effects of
EpiCor on different lymphocyte subsets in vitro.
Among the known immunomodulatory compounds from
S. cerevisiae, various cell wall compounds have been
studied. The primary compound of the inner layer of cell
wall is b-1-3-glucan, interspersed with some b-1-6-glucan,
whereas the outer part of the wall is mostly composed of
mannans. Glucans are present in many plants and all fungi.
b-Glucans are known immunomodulators, and in vitro and
animal works indicate that b-glucan supports a shift toward
Th1 responsiveness [3]. Mice that received oral b-glucan
for 1 week before infection with anthrax showed dramat-
ically increased survival [4]. Orally administered b-glucans
have shown to protect against cancer in different experi-
mental models [5,6]. Of interest to our data is the previous
report on mouse spleen cells that particulate b-glucan from
S. cerevisiae, in the presence of a strong mitogenic
stimulus, results in reduced lymphocyte proliferation and
interferon gamma (IFN-c) production [7]. Mannans are
known as immunomodulators [8] and as potent activators
of natural killer (NK) cells [9]. Different mannans interact
with distinct toll-like receptors (TLRs) because mannan
from both S. cerevisiae and Candida albicans is recog-
nized by TLR-4, whereas phospholipomannan is recog-
nized by TLR-2 [10]. This allows for multifacetted
immunomodulating events in immune cells treated with a
complex product such as EpiCor. However, these cell wall
components are likely only minor contributing factors to
EpiCor’s overall effects because of the high amounts of
metabolites present in the product.
In this study, the effects of EpiCor were evaluated in a
broad panel of in vitro cell-based assays, using different
subsets of human leukocytes. Our hypothesis was that we
would observe NK-cell activation and antiinflammatory
effects in vitro. The antioxidant and antiinflammatory
properties were tested on human neutrophils and erythro-
cytes in an assay aimed at demonstrating inhibition of
formation of ROS. The immunomodulating properties were
evaluated by modulation of phytohemagglutinin (PHA)-
induced T-cell proliferation, demonstration of direct induc-
tion of calcium flux in lymphocyte subsets, and induction of
activation markers by EpiCor. The data indicate potent
antiinflammatory and antioxidant properties associated with
this food product, in combination with activation of cells
from the innate and adaptive immune response, particularly
NK-cell activation, which may help explain the reduction in
viral disease, such as influenza, seen in vivo.
2. Methods and materials
2.1. Study design
This study was conducted from February 2004 to June
2006 at NIS Labs, Klamath Falls, Ore. The study protocol
was designed in part based on data from employee health
records from a plant that produces EpiCor, showing
unusually low incidences of flu and other viral diseases
among employees with the highest daily exposure to EpiCor.
A follow-up study compared exposed and unexposed
employees, and showed increased ROS scavenging activity
and NK-cell activity and increased salivary IgA among
exposed employees [2]. Our in vitro study was therefore
designed to examine whether the specific mechanisms of
action of EpiCor included direct NK- and B-cell activation
and, at the same time, showed effects on ROS inhibition.
2.2. Preparation of EpiCor for in vitro work
The EpiCor product was supplied from Embria Health
Sciences, Cedar Rapids, Iowa, as dark brown flakes. A
solution was prepared in phosphate-buffered saline (PBS)
in preparation for in vitro cell-based assays by adding 0.5 g
of powder to 5 mL of PBS. This mixture was vortexed for
30 seconds and allowed to sit at room temperature for
1 hour. This allowed most of the powder to dissolve,
giving the PBS a dark brown color. The remaining solids
were removed by centrifugation at 2400 rpm for 10 minutes
and subsequent sterile filtration of the supernatant using a
0.22-lm syringe filter. A 10-fold dilution of the stock
solution was prepared in RPMI and incubated at 378C for 1
hour. This resulted in the formation of a white fluffy
mineral precipitate, which was removed by filtration before
adding the extract to cell cultures. The precipitate formed
in RPMI but not in PBS. Initial tests comparing bioactivity
of the extract before and after removal of the precipitate
showed no differences in terms of NK-cell activation. The
precipitate was removed in all subsequent experiments to
avoid interference with equipment readings, including
absorbance and flow cytometry.
2.3. Reagents and monoclonal antibodies
The following human lymphocyte-specific monoclonal
markers directly conjugated with fluorochromes were
purchased from Becton-Dickinson, San Diego, Calif:
CD3-PerCP, CD14-PE, CD25-FITC, CD45-FITC, CD56-
FITC, CD56-PE, CD69-FITC, CD80-PE, and CD86-PE.
Antibodies for CXCR4 and CCR9 were obtained from R&D
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335328
Systems, Inc, Minneapolis, Minn. The anti-CD19 B-cell
marker B4-FITC was purchased from Coulter, Hialeah, Fla.
Buffers including RPMI-1640, Histopaque 1119 and 1077,
and PBS without calcium or magnesium were from Sigma-
Aldrich, St Louis, Mo.
2.4. Purification of peripheral blood mononuclear cells and
polymorphonuclear cells
Peripheral venous blood samples were obtained, after
informed consent, from healthy human volunteers between
20 and 60 years old, as approved by the Merle West
Medical Center institutional review board. Samples were
drawn into sodium heparin and processed within 30 minutes
by gradient centrifugation [11,12]. In brief, whole blood
was layered onto a double gradient of Histopaque 1119 and
1077, and centrifuged for 25 minutes at 400g. The upper
peripheral blood mononuclear cell (PBMC)–rich interface
and the lower polymorphonuclear (PMN) cell–rich inter-
faces were harvested using sterile pipettes, transferred to
sterile vials, and washed twice in PBS at 2400 rpm for 10
minutes. After the second wash, the PMN cell pellet was
treated with 4 mL of nanopure water and gently mixed by
swirling for 30 seconds to lyse any remnant red blood cells.
Immediately, 4 mL of 1.8% saline was added to restore
physiologic isotonic strength. Four milliliters of PBS was
added, and the PMN cells were washed for 10 minutes
at 2400 rpm.
2.5. Assessment of ROS formation
The assessment of antiinflammatory capacity was
performed in a cell-based system [13] using freshly
purified PMN cells to examine whether treatment with a
test product led to reduced oxidative damage during an
oxidation challenge. The cells were pretreated with EpiCor
and loaded with the precursor dye DCF-DA (cat. no.
C6827; Molecular Probes, Eugene, Ore), which becomes
brightly green fluorescent after exposure to oxygen free
radicals. Subsequently, oxidative damage was triggered by
addition of H
2
O
2
. Parallel samples of freshly purified
human PMN cells were preincubated with EpiCor over a
range of dilutions from 10 to 0.01 lL/mL of the aqueous
extract, prepared as described hereinabove, at 378C and 5%
CO
2
for 90 minutes. A stock solution of DCF-DA was
prepared by adding 180 mL of DMSO and 20 lL of a 20%
solution of Pluronic F-127 in DMSO to a 50-lg aliquot of
DCF-DA, and vortexing 3 times for 15 seconds. A working
solution of DCF-DA was then prepared by adding a 10-lL
stock to 10 mL of PBS. The cells were washed twice in
PBS and then resuspended in the DCF-DA working
solution, and incubated for 1 hour at 378C. All samples,
except for the untreated control samples, were then
exposed to 167 mmol/L H
2
O
2
for a period of 45 minutes
to induce severe oxidative stress. Samples were washed
twice in PBS to remove the peroxide, transferred to cold
RPMI, and stored on ice in preparation for immediate
analysis by flow cytometry, using a FacsCalibur flow
cytometer (BD Biosciences, San Jose, Calif). Intracellular
levels of DCF-DA fluorescence intensity in untreated vs
challenged cells, in the presence vs absence of EpiCor,
were analyzed by flow cytometry. Positive control samples
were not pretreated with EpiCor to allow maximum ROS
formation. Negative control samples were treated with
neither EpiCor nor H
2
O
2
to establish baseline levels of
fluorescence in the samples. Two complete sets of positive
and negative controls were used in all experiments, where
flow cytometric analysis was performed on the first set of
controls at the beginning and the second set of controls at
the end of each set. This was to test for spontaneous
changes in oxidation in samples over the course of the flow
cytometry, ensuring that a reduced fluorescence seen with
EpiCor samples was not a result of changes in the samples.
Data were collected in triplicate for controls and duplicate
for each sample concentration. Data analysis was per-
formed using the CellQuest Pro (BD Biosciences) and
FlowJo (TreeStar, Ashland, Ore) software packages. The
mean fluorescence intensity (MFI) of PMN cells was
compared among untreated, H
2
O
2
-treated, and EpiCor-
treated samples. A reduction in MFI in samples pretreated
with EpiCor before challenge with H
2
O
2
signified a
reduction in ROS production, as a result of antioxidant
and antiinflammatory effects.
2.6. Enrichment of NK and B lymphocytes using RosetteSep
To test whether the effect of EpiCor was the result of
direct activation of specific cell subsets, we took blood
samples and immediately treated it with RosetteSep
reagents from StemCell Technologies Inc, Vancouver,
BC, Canada, to enrich for certain cell types [14].For
B-cell purification, the B-cell enrichment cocktail was used
(cat no. 15024), and for NK cells, the NK enrichment
cocktail was used (cat no. 15025). The RosetteSep kits are
cocktails of antibodies directed at all the unwanted cell
subsets in the blood sample and assist with the cross-
linking of these cells to red blood cells. Upon treatment
with the antibodies, the blood was layered onto Histopaque
1077 and centrifuged as described above. The interface
consisted primarily of either NK (90% purity) or B cells
(95% purity). Cells were washed twice in PBS with 10%
autologous serum and used for culturing with EpiCor to
assess induction of activation markers.
2.7. Induction of cell surface markers and immunostaining
Freshly purified PBMCs were resuspended in culture
medium and exposed to serial dilutions over a range of
dilutions from 10 to 0.01 lL/mL of the aqueous extract of
EpiCor, for 18, 24, and 48 hours for NK- and T-cell
activation, and for 1, 3, and 7 days for B-cell activation.
Cells were harvested into V-bottom 96-well plates (Nunc,
Denmark) and washed in IF buffer (PBS containing 1%
bovine serum albumin and 0.02% sodium azide). Cells
were resuspended in 50 lL of IF (immunofluorescence)
buffer, and monoclonal antibodies were added in previous-
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335 329
ly established optimal amounts (CD45-FITC, CD14-PE,
CD3-PerPC, CD56-PE, CD69FITC, and CD25-FITC: 8 lL/
sample; CD19-FITC: 2 lL/sample; CD80 and CD86-PE:
10 lL/sample) and incubated in a dark at room temperature
for 10 minutes. An additional 110 lL of buffer was added
to each well, and the plates were centrifuged. The
supernatants were removed, and the cells were resuspended
in IF buffer and transferred to aliquots of 0.4 mL of 1%
formalin in PBS. Samples were stored dark and acquired
by flow cytometry within 24 hours using a FacsCalibur
cytometer (Becton-Dickinson, San Jose, Calif). Analysis
was performed using the software Flow Jo (Tree Star Inc,
Ashland OR). For evaluation of NK-activation markers,
electronic gating was performed on forward and side
scatter properties to include all lymphocytes while exclud-
ing nonviable cells as well as monocytes. Subsequently,
gating excluded CD3
+
cells and included CD56
+
cells for
final analysis of the 2 activation markers CD69 and CD25
on CD3
CD56
+
NK cells. For analysis of B-cell activation
markers, electronic gating on forward and side scatter
excluded nonviable cells and small lymphocytes, but
included all larger cells and lymphoblasts. Gating on
CD19
+
B cells within the population of large lymphoblasts
was performed, and then analysis of CD80 and CD86
expression on lymphoblasts, in proportion to all lympho-
cytes, was performed.
2.8. Cell proliferation assay
Freshly purified PBMCs were stained with a kit
containing a lipophilic membrane dye PKH26 (Sigma-
Aldrich), which is incorporated into the lipid bilayer of the
cell membrane in a highly stable manner and is distributed
between daughter cells at each mitotic division in culture
[15]. The staining was performed according to the
manufacturer’s guidelines. In brief, PBMCs were resus-
pended in diluent C supplied with the kit and rapidly added
to the PKH26 suspension. Cells were mixed by constant
gentle pipetting for 30 seconds and then gentle agitation of
the vial for an additional 4.5 minutes. The incorporation of
dye was stopped by addition of an equal volume of serum,
followed by dilution and repeated washing in RPMI
containing 10% fetal calf serum. The labeled cells were
resuspended at a concentration of 10
6
/mL in culture
medium and aliquoted into sterile round-bottom 96-well
cell culture plates (Nunc). Triplicate sets of samples were
prepared for each treatment, including various dilutions of
EpiCor, spanning a range of dilutions from 10 to
0.01 lL/mL of the aqueous extract, with and without
known stimuli in parallel sets of samples. As known
stimuli, we used the mitogen PHA (2 lg/mL) in parallel to
human recombinant IL-2 (50 IU/mL). Untreated samples
served as negative controls. The plates were incubated for
5 days at 378Cat5%CO
2
. Cells were transferred from the
culture plate to a V-bottom 96-well plate and washed twice
with PBS. The pellets were resuspended in 50-lL PBS and
transferred to 0.4 mL of 1% formalin in PBS. Data
acquisition was performed on a FacsCalibur flow cytometer
(Becton-Dickinson, San Jose, Calif). Analysis was per-
formed using the software FlowJo (TreeStar). Electronic
gating was performed on forward and side scatter
properties to exclude nonviable cells, and analysis deter-
mined the proportion of cells remaining in the parent
population as reflected by their fluorescence intensity.
2.9. Interferon gamma production
Interferon gamma is a proinflammatory cytokine often
associated with NK-cell activation. The effect of EpiCor on
IFN-cproduction in conjunction with 2 known stimuli (PHA
and IL-2) was examined. Freshly purified PBMCs were
resuspended at 10
6
/mL in culture medium and aliquoted into
sterile round-bottom 96-well plates (Nunc). Serial dilutions
of EpiCor, with and without addition of the mitogen PHA vs
interleukin 2, were added to the wells, with each treatment
being performed in triplicate. Doses of EpiCor, PHA, and
IL-2 were added as described above. The plates were
cultured for 5 days at 378Cat5%CO
2
. The supernatants
were collected and assessed for content of IFN-cusing the
DuoSet ELISA kit from R&D Systems. The absorbance was
read at 450 nm, blanking on 570 nm, using a PowerWave X
microplate reader (BioTek Instruments, Winooski, Vt). Data
were exported to Excel for statistical analysis.
2.10. Induction of chemokine receptor expression by
Fucoidan
Fucoidan is an l-selectin ligand that triggers a rapid
expression of the chemokine receptor CXCR4 on human
lymphocytes [16]. The relative expression of different
chemokine receptors dictates the potential for a chemotactic
response involved in lymphocyte recruitment to tissue. To
evaluate whether EpiCor would alter the chemokine
Fig. 1. The antiinflammatory properties of EpiCor were quantified by
measuring the inhibition of formation of intracellular ROS in human PMN
cells. The PMN cells were pretreated with EpiCor and loaded with the ROS
reporter dye DCF-DA before triggering formation of ROS by H
2
O
2
.
Reactive oxygen species formation within the PMN cells was measured as
MFI of the dye, which becomes green fluorescent upon exposure to ROS.
The data shown are mean FSD of triplicate tests and are representative of
4 similar experiments. A dose-dependent inhibition of ROS formation by
EpiCor was seen. This ROS inhibition was highly significant at the highest
dose of EpiCor at 1 lL/mL ( Pb.005).
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335330
receptor profile, we resuspended freshly purified PBMCs in
RPMI 1640 and aliquoted it into microwell plates at a
concentration of 2 10
5
cells/well, and then exposed it to
Fucoidan (0.1 mg/mL). In parallel, PBMCs were exposed to
either EpiCor alone (10 lL/mL) or a premixed cocktail of
Fucoidan with EpiCor. The samples were incubated for 5 to
60 minutes, washed with IF buffer, and stained using PE-
conjugated monoclonal antibodies specific for the CXCR4
and CCR9 chemokine receptors (R&D Systems) at 8 lL/
sample. Untreated cells were used for assessment of
baseline expression of CXCR4 and CCR9.
2.11. Statistical analysis
Statistical analysis was performed using Microsoft Office
Excel 2003 (Microsoft Corp, Redmond, Wash). The
averages of identical control vs test samples were calculated,
and the statistical significance was tested using the
independent sample ttest, also known as Student ttest
[17,18]. A probability ( P) value of less than .05 indicated
that the averages between 2 groups of data were signifi-
cantly different.
3. Results
3.1. Reduction of ROS in PMN cells by EpiCor
Human PMN cells were used for testing of the combined
antioxidant and antiinflammatory effect of EpiCor. The
PMN cells were pretreated for 90 minutes with EpiCor
before loading with the oxidation-sensitive dye DCF-DA
and the induction of reactive oxygen burst, which generates
large amounts of intracellular free oxygen radicals. Untreat-
ed cells were assayed to establish the baseline level of
oxidation of the indicator dye DCF-DA in the absence of an
oxidative challenge. EpiCor pretreatment resulted in signif-
icant reduction of ROS formation when an oxidative burst
was triggered in PMN cells challenged with hydrogen
peroxide (Fig. 1). The effect was dose dependent, and at the
highest dose of EpiCor, the effect was highly significant
(Pb.005). This assay does not distinguish between
compounds with a direct antioxidant effect vs compounds
mediating an antiinflammatory effect, which, by binding to
cell surface receptors and altering signaling, leads to the
reactive oxygen burst. However, the assay showed that,
overall, EpiCor had a highly significant antiinflammatory
effect in this cell-based system.
3.2. Modulation of PHA-induced lymphocyte proliferation
by EpiCor
The proliferative response to the T-cell mitogen PHA was
used to compare untreated PBMC to PBMC exposed to
EpiCor for 5 minutes before exposure to PHA. No
proliferative response was induced in response to EpiCor,
indicating that EpiCor was not in itself mitogenic for human
Fig. 2. EpiCor modulated the proliferative response of human PBMC to the
known T-cell mitogen PHA. The costimulation of PBMC with PHA and
EpiCor resulted in a significant increase of nonproliferating cells compared
with PHA alone ( Pb.000 01), indicating that compounds within EpiCor
altered the cell signaling by PHA. EpiCor showed no direct mitogenic effect
on human PBMC. Data are shown on triplicate samples from 1 experiment
(mean FSD, n = 3), representative of 3 experiments with cells from
different individual donors.
Fig. 3. Flow cytometry analysis showed that EpiCor caused expression of
the NK activation marker CD69. (A) Induction of the CD69 activation
marker on NK cells from PBMC cultures after 18 hours of incubation in the
presence vs absence of EpiCor (10 lL/mL) was evaluated by multicolor
immunofluorescence, where electronic gating allowed analysis of CD69 on
CD3
CD56
+
NK cells. The data shown are based on samples from 1 of 3
representative experiments. The data show that CD69 was induced by
EpiCor on the CD56
medium
cells, but not on the CD56
bright
cells. (B)
EpiCor-mediated induction of the CD69 activation marker on human NK
cells was seen both on NK cells from PBMC cultures and on enriched NK
cells. The induction of CD69 was statistically significant both on NK cells
from PBMC cultures ( Pb.03) (white bars) and from enriched NK-cell
cultures ( Pb.02) (black bars). Data are shown on triplicate samples from
one experiment (mean FSD, n = 3). The induction of CD69 was lower on
purified NK cells than on mixed PBMC, indicating that other cells within
the PBMC may further enhance the NK-activating effect of EpiCor.
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335 331
lymphocytes (Fig. 2). However, proliferation was signifi-
cantly reduced in the presence of EpiCor ( Pb.000 01)
compared with PHA alone, indicating that EpiCor was
engaging signaling components, so subsequent signaling by
PHA was modulated on human T lymphocytes.
3.3. Induction of the CD69 activation marker on NK cells by
EpiCor
Evaluation of activation of NK cells was examined using
18-hour culturing followed by immunostaining for NK- and
T-cell lineage markers in combination with the activation
markers CD69 and CD25 (the IL-2 receptor). EpiCor
treatment resulted in the induction of CD69 on CD3
CD56
+
NK cells ( Pb.03) (Fig. 3). The induction of CD69 on NK
cells was strictly limited to those NK cells with a moderate
expression of CD56 and was not observed on the small
subset of CD56
bright
NK cells (Fig. 3A). The increase in
CD69 expression was of similar proportions when examin-
ing the NK cells in PBMC cultures by electronic gating on
CD3
CD56
+
cells and when NK cells were physically
purified by the RosetteSep method (Fig. 3B). These highly
enriched NK cells also displayed significant up-regulation
of the CD69 marker when exposed to EpiCor ( Pb.02),
meaning that the NK-activating effect of EpiCor was at least
in part caused by a direct activation of NK cells and not
dependent on bystander cells.
3.4. Inhibition of PHA-induced and IL-2–induced IFN-c
production by EpiCor
Interferon gamma is a proinflammatory cytokine made
by certain cell types including activated NK cells.
Peripheral blood mononuclear cell culture supernatants
from 5-day cultures were tested for IFN-cproduction in
the presence vs absence of EpiCor. Two known stim-
ulators of IFN-cproduction, PHA and IL-2, were used
alone and in combination with EpiCor. EpiCor treatment
of PBMC reduced the levels of PHA-i nduced ( Pb.02)
as well as the IL-2–induced ( Pb.09) IFN-cproduction
(Fig. 4). EpiCor alone did not induce IFN-cproduction.
Fig. 4. Inhibition of IFN-cproduction EpiCor. Human PBMCs were
incubated for 5 days in the absence vs presence of EpiCor (10 lL/mL)
with and without PHA or IL-2. Culture supernatants were harvested and
assayed for IFN-cby enzyme-linked immunosorbent assay. The data
shown are mean FSD (n = 3). When comparing the level of IFN-c
production as a result of PHA alone vs PHA + EpiCor treatment, a
statistically significant inhibition was seen ( Pb.02). When comparing
the level of IFN-cproduction as a result of IL-2 alone vs IL-2 + EpiCor
treatment, an inhibition was seen but did not meet the criterion for
significance ( Pb.09).
Fig. 5. EpiCor-mediated activation of B cells. (A) The expression of CD80
and CD86 on large B lymphoblasts was evaluated by immunostaining and
flow cytometry. The induction of these 2 B-cell activation markers was
greater in cultures treated with EpiCor (10 lL/mL) than in cultures where
B cells were activated with Staphylococcus aureus protein A (protein A,
10 lg/ml). The differences in activation marker expression between
untreated and EpiCor-treated cells were statistically significant for both
CD80 ( Pb.0001) and CD86 ( Pb.02). Data are shown on triplicate
samples from 1 experiment (mean FSD, n = 3) and are representative of
3 experiments with cells from different individual donors with similar
results. (B) The EpiCor-mediated B-cell activation was a result of direct
effect of EpiCor on B cells. Data are shown on triplicate samples from one
experiment (mean FSD, n = 3). White bars indicate the CD86 expression
on B cells electronically gated from PBMC cultures based on their
expression of the B-cell marker CD19. Black bars show the CD86
expression on enriched B cells. The induction of CD86 on enriched B
cells was highly significant ( Pb.000 01).
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335332
These data showed that even though EpiCor activated NK
cells, it did not lead to production of the proinflammatory
cytokine IFN-c, and it reduced IFN-cproduction as a
result from other stimuli.
3.5. Induction of activation markers on human B cells by
EpiCor
Based on previous data showing that in vivo effects of
EpiCor included increased salivary IgA production, it was
speculated that a direct effect of EpiCor on B-cell activation
was a key part of its mechanism of action. We showed that
EpiCor induced a significant expression of the 2 B-cell
activation markers, CD80 and CD86, in vitro, with a
maximal expression of CD80 ( Pb.0001) as well as
CD86 ( Pb.02) on day 3 of culture (Fig. 5A). As a positive
control for B-cell activation, protein A was used to induce
B-cell activation. The coculturing of PBMC with EpiCor
and protein A resulted in an enhanced B-cell activation
when compared with either substance alone (Fig. 5A). To
evaluate whether the effect of EpiCor on human B cells was
a result of a direct activation or collaborative events
requiring the presence of several cell types, the testing
was repeated with highly enriched B cells. The B cells were
enriched from PBMC using a RosetteSep antibody cocktail
and gradient centrifugation, and cultured with EpiCor. A
highly significant expression of CD86 ( Pb.000 01) was
observed in these cultures (Fig. 5B), indicating that at least
in part the effect of EpiCor on B cells was direct.
3.6. EpiCor modulation of
l
-selectin–mediated expression
of chemokine receptors on NK cells
The expression of adhesion molecules and chemokine
receptors on lymphocytes determines their ability to
respond to recruitment signals from tissue and is thus an
important factor in immune surveillance. Peripheral blood
mononuclear cell treated for 24 hours with EpiCor showed
an almost complete loss of staining with the monoclonal
antihuman l-selectin antibody TQ1, which is specific
toward the ligand-binding area of human l-selectin
(Fig. 6A). This significantly reduced binding of TQ1 to
PBMC ( Pb.0001 at 24 hours) could indicate that
l-selectin was lost from the cell surface as a result of
EpiCor treatment, possibly as a result of shedding of the
extracellular portion of l-selectin from the cell surface via
protein kinase C–mediated cell activation. Alternatively, the
reduced binding of TQ1 to the l-selectin could be a result
of a direct competition for binding between the TQ1
antibody and compounds in the EpiCor extract. Because the
binding of ligands to l-selectin is known to modulate the
profile of chemokine receptors, thus, altering chemotactic
potential during leukocyte trafficking, further work on
chemokine receptors was undertaken. Fucoidan, a known
ligand for l-selectin, was used to provoke externalization of
intracellular reservoirs of the chemokine receptors CXCR4
and CCR9. EpiCor interfered with the Fucoidan-mediated
Fig. 6. EpiCor modulation of l-selectin–mediated expression of chemokine
receptors on NK cells. (A) Binding of the monoclonal antibody TQ1,
directed toward the ligand-binding area of the homing molecule l-selectin,
was significantly reduced after exposure of lymphocytes to EpiCor
(10 lL/mL) (24 hours, Pb.0001). Data are representative of 3 similar
experiments. (B) EpiCor served as an agonist for l-selectin–mediated
chemokine expression on NK cells. When PBMCs were incubated with
Fucoidan, a known l-selectin ligand, the cells rapidly (within minutes) and
transiently externalized the chemokine receptors CXCR4 and CCR9 many-
fold above background expression level. Brief 10-minute preincubation of
PBMC with EpiCor drastically reduced the Fucoidan-induced CXCR4
(Pb.005) and CCR9 ( Pb.0007) expression on NK cells. Data are
expressed as mean FSD, n = 3, and are representative of 3 similar
experiments performed on lymphocytes from different donors.
G.S. Jensen et al. / Nutrition Research 27 (2007) 327– 335 333
expression in a significant manner for both CXCR4 ( Pb
.005) and CCR9 ( Pb.0007) (Fig. 6B). This supports the
data indicating a possible role of l-selectin in the EpiCor-
mediated modulation of cellular responses to chemotactic
signals during lymphocyte trafficking and migration.
4. Discussion
Chronic inflammation has grown to endemic propor-
tions in industrialized countries, in correlation with a fast-
paced lifestyle and diet [19-22]. An unbalanced immune
activation, in response to pathogens from the gut lumen,
has been implicated as a risk factor for many chronic
inflammatory conditions, including irritable bowel disease,
ulcerative colitis, and Crohn disease, and has been a
contributing factor in obesity, heart disease, Alzheimer
disease, and autoimmune diseases such as rheumatoid
arthritis and psoriasis. Furthermore, chronic inflammation
facilitates carcinogenesis and tumor progression [23,24].
There is a growing interest in unique foods and supple-
ments that combine vitamins, antioxidants, and immuno-
modulating compounds to use nutritional strategies to
reduce inflammation and support various aspects of
immune function [25,26].
EpiCor is a new S. cerevisiae–based immunogen product
with a high content of metabolites, including antioxidant
compounds resulting in a reported high ORAC value [1] and
high ROS scavenging activity [2]. Data presented here show
that EpiCor possesses significant antioxidant and antiin-
flammatory activity over a wide range of dilutions in a cell-
based assay. EpiCor inhibited ROS formation, as induced by
H
2
O
2
, in human PMN cells. As several ROS species play
roles in cell signaling, it is likely that EpiCor is capable of
reducing the background noise of chronic inflammation,
thereby increasing the capacity for maintaining balanced
immune responses.
Furthermore, our data on EpiCor strongly indicate that it
contains compounds that directly provide signals for
human lymphocyte subsets. The direct activation of NK
cells and B cells is significant. In addition, the data points
to modulation of cell signaling are reflected by changes in
proliferative activity, cytokine production, and chemokine
receptor expression. This suggests a change in lymphocyte
responsiveness and may indicate a shift from cellular (Th1)
toward humoral (Th2) immune response in the presence of
EpiCor. Of particular interest is the strong inhibition of IL-
2–induced IFN-cproduction. Interferon gamma is unique
among the interferons and is the principal molecule for
activation of macrophages. Because IFN-cis produced by
activated T-lymphocytes as part of an immune response,
this supports the notion that EpiCor may favor Th2
responsiveness while simultaneously producing a strong
activation of B cells and NK cells. It may also play a role
in the physiology of chronic inflammatory conditions by
reducing inflammation while optimizing the humoral
response to pathogens.
Realistically, in a given person at a given time, Th1-
type and Th2-type reactions are happening simultaneously
in different microanatomical locations throughout the
body. The spatial and temporal distance allows for fine-
tuning of the overall immune defense. However, under
chronic inflammatory conditions, the failure to balance
Th1 and Th2 responses, possibly due in part to increased
levels of inflammatory mediators, may contribute to a
reduced capacity for optimal immune response to
various pathogens.
Given the strong inhibition of chemokine receptor
expression seen on human lymphocyte subsets after
exposure to EpiCor in vitro, and based on EpiCor as a
consumable product, a major antiinflammatory impact
might be seen in the gut. Effects could hypothetically
include modulation of trafficking of specific immune cell
types and/or subsets. Chemokine antagonists are intensively
researched as potent and highly selective pharmaceutical
antiviral and antiinflammatory compounds [27-29]. Partic-
ularly, CCR9 plays a key role in homing to the gut a subset
of CD4
+
T cells [30] and plasma cells [31]. Also, the
homing of T cells to the liver in chronic liver disease is
mediated by CCR9 [32].
Based on these in vitro studies, EpiCor has the potential
to act as an immune modulator while at the same time
helping to reduce gastrointestinal and, possibly, systemic
inflammation. The data presented here on EpiCor can be
seen as support for the benefit of further clinical work to
determine the possible effect of EpiCor consumption on
chronic inflammatory disorders, as well as viral diseases.
Acknowledgment
The authors thank Lue Ann Zaske, Kelly M. Patterson,
Anna Gawlicka, John R. Endres, and Donna Kachinskas for
their excellent technical assistance. The study was spon-
sored by Embria Health Sciences, Cedar Rapids, Iowa.
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