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An Evaluation of the Immunological Activities of Commercially Available β β1, 3-Glucans

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9JANA Vol.10, No. 1, 2007
An Evaluation of the Immunological Activities
of Commercially Available Beta 1, 3 Glucans
Vaclav Vetvicka, PhD*, Jana Vetvickova, MS
University of Louisville, Department of Pathology, Louisville, KY
JANA ARTICLE
* Correspondence:
Vaclav Vetvicka, PhD.
ABSTRACT
Introduction
β1,3-Glucan’s role as a biologically active
immunomodulator has been well documented for over 40
years. Interest in the immunomodulatory properties of
polysaccharides was initially raised after experiments show-
ing that a crude yeast cell preparation stimulated
macrophages via activation of the complement system.1
Further work identified the immunomodulatory active com-
ponent as β1,3-glucan.2 Numerous studies (currently more
than 1,600 publications) have subsequently shown that
β1,3-glucans, either particulate or soluble, exhibit
immunostimulating properties, including antibacterial and
anti-tumor activities.3,4
Despite extensive investigations, no consensus on the
source, size and other biochemical or physicochemical
properties of β1,3-glucan has been achieved. In addition,
numerous concentrations and routes of administration have
been tested - including oral, intraperitoneal, subcutaneous
and intravenous applications.
This fact, together with the fact that there are probably
more than a hundred different samples on the U.S. market
alone, leads to confusion about the quality, biological
effects and overall efficiency of glucan. Therefore, we
decided to compare the basic immunological activities of a
group of glucans. The list of products chosen came from
those heavily advertised, commonly available and easily
obtained in the US, Europe, Southeast Asia and Japan. In
order to be certain that we are measuring the effects of glu-
can only, we picked the commercial samples with glucan
(either from one source or a mixture of different glucans) as
the only active ingredient.
The collection of tested biological reactions (phagocy-
tosis, surface markers on splenocytes, cytokine synthesis
and stimulation of antibody response) represents both the
humoral and cellular branches of the immune reaction, thus
offering insight as to the immunological activities of stud-
ied glucans.
MATERIAL AND METHODS
Animals
Female, 6 to 10 week old BALB/c mice were pur-
chased from the Jackson Laboratory (Bar Harbor, ME). All
animal work was done according to the University of
Louisville IACUC protocol. Animals were sacrificed by
CO2asphyxiation.
Materials
RPMI 1640 medium, sodium citrate, dextran, Ficoll-
Hypaque, antibiotics, sodium azide, bovine serum albumin
(BSA), Wright stain, Limulus lysate test E-TOXATE,
DRAFT
10JANA Vol.10, No. 1, 2007
Freund’s adjuvant and Concanavalin A were obtained from
Sigma Chemical Co. (St. Louis, MO). Fetal calf serum
(FCS) was from Hyclone Laboratories (Logan, UT).
ββ-1,3 glucans
The glucans used in this study were purchased from the
following companies: Now BETA glucan from Now Foods
(Bloomingdale, IL), IMMUTOL from Biotec (Tromso,
Norway), Immune Builder and Maitake Gold 404 from
Mushroom Science (Eugene, OR), Glucan #300 from
Transfer Point (Columbia, SC), Glucagel T from GraceLinc
(Christchurch, New Zealand), and Senseiro from Sundory
(Tokyo, Japan).
Antibodies
For fluorescence staining, the following antibodies
have been employed: anti-mouse CD4, CD8 and CD19,
conjugated with FITC were purchased from Biosource
(Camarillo, CA).
Flow cytometry
Cells were stained with monoclonal antibodies on ice
in 12 x 75-mm glass tubes using standard techniques.
Pellets of 5x105cells were incubated with 10 ?l of FITC-
labeled antibodies (1 to 20 ?g/ml in PBS) for 30 minutes on
ice. After washing with cold PBS, the cells were re-sus-
pended in PBS containing 1% BSA and 10 mM sodium
azide. Flow cytometry was performed with a FACScan
(Becton Dickinson, San Jose, CA) flow cytometer and the
data from over 10,000 cells/samples were analyzed.
Phagocytosis
The technique employing phagocytosis of synthetic
polymeric microspheres was described earlier.5,6 Briefly:
peritoneal cells were incubated with 0.05 ml of 2-hydrox-
yethyl methacrylate particles (HEMA; 5x108/ml). The test
tubes were incubated at 37°C for 60 min. with intermittent
shaking. Smears were stained with Wright stain. The cells
with three or more HEMA particles were considered positive.
The same smears were also used for evaluation of cell types.
Evaluation of IL-2 production
Purified spleen cells (2x106/ml in RPMI 1640 medium
with 5% FCS) were added into wells of a 24-well tissue cul-
ture plate. After addition of 1 mg of Concanavalin A into
positive-control wells, cells were incubated for 72 hrs. in a
humidified incubator (37°C, 5% CO2). At the endpoint of
incubation, supernatants were collected, filtered through
0.45 mm filters and tested for the presence of IL-2.7 Levels
of the IL-2 were measured using a Quantikine mouse IL-2
kit (R&D Systems, Minneapolis, MN).
RESULTS
The number of individual glucans is almost as great as
the number of sources used for their isolation. The rationale
for this combination of glucan samples was not only their
commercial availability and success, but most importantly,
we tried to include both soluble and insoluble glucans, and
also glucans from different sources, including yeast, mush-
rooms and cereals.
Glucagel barley β-glucan is a mixed link (13, 14)-ß-D-
glucose polymer, in which cellotriosyl and cellotetraosyl
residues occur in a ratio of ~3:1. The natural purification
process yields a reduced molecular weight ß-glucan (typi-
cally ~130 kDa) that is more readily hydrated than other
conventionally purified β-glucans. The typical carbohy-
drate content is 85-90%.
Senseiro is a soluble, high molecular weight glucan
isolated from Agaricus blazei, consisting of approximately
63% carbohydrate. Glucan #300 is a proprietary (13, 16)-
ß-D-glucan purified from Saccharomyces cerevisiae by
Biothera for Transfer Point and even when corresponding to
the glucan sold under WGP name, has much higher purity
(app. over 96%).
β-Glucans are generally considered to be potent stimu-
lators of cellular immunity, with macrophages and neu-
trophils being the most important targets. Not surprisingly,
we started our evaluation of glucan activities by phagocyto-
sis. We used the synthetic polymeric microspheres,
HEMA, since their use, dose and timing are already well
established in glucan studies.7-9 Results summarized in
Figure 1 show significant effects of glucan samples on
encapsulation of synthetic particles by peripheral blood
neutrophils. The significant stimulation of phagocytic activ-
ity was found with five glucans – Now Beta Glucan,
Maitake Gold, Immune Builder, IMMUTOL and Glucan
#300. The other samples, with the exception of Glucagel T,
also stimulated the phagocytosis, but at a much lower level
and the results were not significant.
Next, we compared the effects of tested glucans on the
expression of several membrane markers on splenocytes.
Twenty-four hours after an ip. injection of 100 ?g of indi-
vidual glucan, spleen cells were isolated and the surface
expression of CD4, CD8 and CD19 was evaluated by flow
cytometery. The results summarized in Figure 2 demon-
strated that only three glucans - Now Beta Glucan, Maitake
Gold, and Glucan #300 significantly increased the migra-
tion of CD4- and CD8-positive T lymphocytes; none of the
glucan had any significant effect on changes in presence of
CD19-positive B lymphocytes.
Evidence of the immunomodulating activity was also
demonstrated through effects on the production of IL-2 by
spleen cells (Figure 3). The production of IL-2 was mea-
sured after a 72 hr. in vitro incubation of spleen cells iso-
lated from control and glucan-treated mice. Again, treat-
ment of mice with Now Beta Glucan, Maitake Gold, and
Glucan #300 showed the highest stimulation of IL-2 pro-
duction. Immune Builder and IMMUTOL showed medium
11 JANA Vol.10, No. 1, 2007
Figure 1.
Figure 2.
Effect of an ip. administration of 100 ?g of different glucan samples on phagocytosis by peripheral blood granulocytes. Each value rep-
resents the mean ± SD. *Represents significant differences between control (PBS) and glucan samples at P 0.05 level.
Effect of ip. injection of 100 ?g of tested glucans on the expression of CD4, CD8 and CD19 markers by spleen cells. The cells from three
donors at each time interval were examined and the results given represent the means ± SD. *Represents significant differences between
control (PBS) and samples at P 0.05 level.
12JANA Vol.10, No. 1, 2007
level stimulation. As the secretion of IL-2 by non-stimulat-
ed splenocytes (PBS group) is almost zero, even low stimu-
lation by Glucagel T was significant. Another way to com-
pare the effect on IL-2 formation and/or secretion is to com-
pare it to the Con A stimulation.. In this case, only Glucan
#300 showed higher effects than Con A, whereas Now Beta
Glucan and Maitake Gold were comparable, and the rest of
the glucans show much smaller effects.
We then focused on the use of glucan as an adjuvant.
As an experimental model, we used immunization with
ovalbumin. Glucans were applied together with two
intraperitoneal doses of antigen; a commonly used Freund’s
adjuvant was used as additional positive control. The results
(Figure 4) showed that only Immune Builder and Senseiro
glucans were without any effects on antibody response. All
other glucans significantly supported the formation of spe-
cific antibodies. Glucans with the highest stimulation were
Glucagel T and Glucan #300. It must be noted, however,
that none of the glucans potentiated the humoral immunity
to the level of Freund’s adjuvant.
Table 2 summarizes the activities of individual glucans
in all tested functions. Clearly, the most active samples were
Glucan #300, followed by Now Beta Glucan and Maitake
Gold 404. Senseiro glucan was almost without any mea-
surable activity.
DISCUSSION
Despite the extensive amount of scientific reports
about glucans and their biological activities, most of the
studies are focused on the description of chemical and/or
biological properties of one particular glucan. Numerous
types of glucans have been isolated from almost every
species of yeast and fungi. For a long time, attention was
focused mainly on glucans isolated from yeast and mush-
rooms. Recently, the existence of a highly purified linear β-
1,3 glucan named Phycarine, and subsequent study showing
that Phycarine induced a broad range of defense reactions
in tobacco cells,10 brought new attention to seaweed-
derived glucans.11-13 More studies revealed that Phycarine
significantly stimulated phagocytosis, synthesis and release
of IL-1, IL-6 and TNF-a, and NK cell-mediated killing of
tumor cells both in vitro and in vivo.8 Similarly, recent clin-
ical trials demonstrated the high activity of glucan isolated
from barley.14 It is clear, therefore, that the biological activ-
ities of glucans might be related more to the purity and bio-
chemical/physiochemical characteristics than to the source.
Comprehensive reviews comparing several glucans are
rare. However, in one of those studies, Yadomae reviewed
how the structural properties of glucans affected biological
activities and found that branched or linear 1,4 glucans have
Figure 3.
Effects of glucans on Con A-stimulated secretion of IL-2 βy spleen cells. *Represents significant differences between control (PBS) and
samples at P 0.05 level.
Glucan used in this study
Name Source Manufacturer/Distributors Solubility
β-1,3/1,6-D-glucan Saccharomyces cerevisiae Now Foods No
Grifola frondosa
MaitakeGold 404 Grifola frondosa MushroomScience Yes
Immune Builder Agaricus blazei MushroomScience No
Cordyceps sinensis
Coriolus versicolor
Ganoderma lucidum
Lentinula edodes
Grifola frondosa
IMMUTOL Saccharomyces cerevisiae Biotec ASA No
Glucagel T Barley GraceLinc
Senseiro Agaricus blazei Sundory Yes
Glucan #300 Saccharomyces cerevisiae Transfer Point No
JANA Vol.10, No. 1, 200713
limited activity and β-glucans with a 1,3 configuration with
additional branching at the position 0-6 of the 1-3 linked D-
glucose residues have the highest immunostimulating activ-
ity.15 Readers seeking additional reviews might see Kogan16
or Vetvicka.17 However, it is important to keep in mind that
these reviews are oriented towards comparing results of
numerous publications and none of them offers a face-to-
face comparison of several glucans. At the same time, with
the high number of individual glucans and huge differences
in their biological activities, it is imperative to evaluate their
biological properties before any suggestions for use of a
particular glucan can be made.
In our paper, we compared seven commercially suc-
cessful glucans, differing both in source (mushroom, yeast
and barley) and solubility. At the same time, we used iden-
tical amounts of glucans from each sample. In the case of
complex mixtures (such as Immune Builder), the total
amount of used sample corresponded to the ratio of indi-
vidual glucans.
Table 1.
Table 2.
Comparison of individual glucans
Name Phagocytosis CD expression Il-2 production Antibody formation
Now Beta Glucan ++ +++ ++ ++
MaitakeGold 404 ++ +++ ++ ++
Immune Builder ++ + + -
IMMUTOL ++ + + +
Glucagel T - - +/- +++
Senseiro + + - -
Glucan #300 +++ +++ +++ +++
14JANA Vol.10, No. 1, 2007
As various glucans are well known to stimulate phago-
cytosis,18 one of the first tests of the immunological charac-
teristics of any glucan is phagocytosis. We used the 2-
hydroxyethyl methacrylate particles, which have only a
slight negative charge and thus do not nonspecifically
adhere to the cell surface. This guarantees that only phago-
cytosing cells will engulf these particles and significantly
lowers the chance of false negativity.19 Our investigation
showed that while most of the tested glucans stimulated
phagocytosis of synthetic microspheres (with the exception
of Glucagel T), the highest effects were obtained with
Glucan #300.
As some of the glucans are known to regulate the influx
of cells into the individual lymphatic organs,8 we compared
the effects of a single injection on expression of the basic
membrane markers present on splenocytes. Only three glu-
cans - Now Beta Glucan, Maitake Gold, and Glucan #300 –
changed the number of CD4- and CD8-positive lymphocytes.
No glucan significantly changed the percentage of B lym-
phocytes. The effects on CD4-positive cells corresponded to
the previously found effects of Phycarine8 or lentinan.20
In addition to the direct effect on various cells of the
immune system, the immunostimulating action of β-glu-
cans is caused by potentiation of a synthesis and release of
several cytokines such as TNF?, IFN?, IL-1 and IL-2. This
cytokine stimulating activity is dependent on the triple helix
conformation.21 The only glucan without a trace of pro-
inflammatory cytokine stimulation is PGG-glucan.22 We
focused on the stimulation of IL-2 production by spleen
cells in vitro and found that whereas all glucans (with the
exception of Senseiro) stimulated production of IL-2, only
two of the samples (Maitake Gold and Glucan #300)
showed stimulation comparable to the common stimulator
Concanavalin A. The activity of the most active glucan was
comparable to the previously published data.9,23
Glucans are usually considered stimulators or modula-
tors of the cellular branch of immune reaction and very lit-
tle attention has been focused on their potential effects on
antibody response. We decided to take advantage of the
recently published method of evaluating the use of glucan
as adjuvant.24 Our results rather surprisingly showed that
most of the tested glucans revealed some level of stimula-
tion of antibody response, the strongest being Glucagel T
and Glucan #300. In this case, however, the stimulation
was always significantly lower than in the case of combin-
ing antigen and Freund’s adjuvant.
Data presented in this study and summarized in Table
2 clearly demonstrated the differences in activities among
individual types of glucans. Also, it is clear that individual
glucans can be highly active in one particular part of
immune reactions (e.g., Glucagel T on antibody produc-
tion), and almost without any significant biological activity
in other parts of defense reaction. Glucan #300 showed not
only a broad range of action, but in all tested reactions (with
Effects of two ip. injections of tested glucans on formation of antibodies against ovalbumin. Mice were injected twice (two weeks apart)
and the serum was collected 7 days after last injection. Level of specific antibodies against ovalbumin was detected by ELISA. As positive
control, Freund’s adjuvant was used. *Represents significant differences between control (ovalbumin alone) and samples at P 0.05 level.
Figure 4.
15 JANA Vol.10, No. 1, 2007
the exception of the antibody formation where it was the
second most active sample) was the biologically most rele-
vant immunomodulator.
Several conclusions can be made: 1) Not all glucans are
created equal; 2) some of the commercial glucans have sur-
prisingly low activity; 3) most glucans differ in biological
effects based on tested characteristics; and 4) for good
results in immunomodulation, it is more imperative to find
a glucan from a solid vendor who is able to back the claims
with solid scientific data. Thinking about the biological
source of glucan is much less important.
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... Finally, the yeast cell wall can be submitted to further purification process in order to remove the mannan-protein outer darker layer to form a purified β-glucan (Figure 3). Various extraction methods for β-glucan exist, generating various products with distinct, but not always identical, immunomodulatory effects [13][14][15]. β-Glucans, the main part of the purified yeast cell wall,are macromolecules that present β conformation linkages in different degrees of polymerization, where the β-1,3 glucose linear chain core has abundant lateral branches initiated with β-1,6 linkages. The term glucan, in amore general meaning, represents a group of biologically active polysaccharides that can alsooriginate from other sources, including the cell wall of filamentous fungus, seaweed, oat, and barley [16]. ...
... The formation of antibodies was evaluated using ovalbumin as an antigen and ELISA assay [14]. Animals were subcutaneously injected twice (two weeks apart) with albumin (100μg per mice) and the serum was collected 7 days after the last injection. ...
... This studyadds more informationto the three previous studies, testing over 40 different glucans [14,22,39]. Our results suggest that the yeast derivatives have more limited immunological effects, maybe as a result of local intestinal immune reactions. ...
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... Although numerous routes for administration of Aloe vera and β-Glucan products existed, in the last decade oral application represents the most convenient route 19,21-23 . In addition, promoters offer a number of formulations that are widely available for consumption at various concentrations in liquid, powder, and tablet form 24,25,28,31,33,36 . It is demonstrated that immunomodulating and/or immunostimulating effects of these products are dependent on the activation of the innate immune cells (macrophages, neutrophils, lymphocytes and NK cells), synthesis and release of cytokines (TNF-α, IFN-α, IFN-γ, IL-1, IL-2, IL-6, IL-8), generation of enhanced cell-mediated responses, and induction of nitric oxide production 18-20,24,25,28,30-32,37 . ...
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