Scientific Research Articles on MG Beta Glucan Preparation and Immune Potentiation
The following articles published in
recent years, including 2005, are
authored by the research team at the University of Nevada School of Medicine,
School of Microbiology, involved in extensive on-going beta glucan research
since 1998 (Mode of Action of B-Glucan Immunopotentiators), continuing in 2014. The article reviews a portion of the research project
sponsored by the State of Nevada and Nutritional Scientific Corporation.
An additional current peer-reviewed article in Immunology Letters published in
May 2004 and authored by Kenneth W. Hunter, Jr., Sally duPre and Doug Redelman
is summarized in an abstract available by clicking "2004
MG Beta Glucan Research Abstract."
Additional research papers are available on this website by clicking on "Additional
MG Glucan Research."
The nonaggregated microparticulate beta glucan with superior
immunopotentiating capabilities described is the MG glucan now processed in the
NSC Laboratories by a U.S. patent
protected process and patents pending. Nutritional Scientific Corporation
expresses our appreciation and thanks to Dr. K. W. Hunter, Jr., Dr. Ruth Gault,
M.D. Berner and Sally duPre of the Department of Microbiology and Immunology,
University of Nevada School of Medicine, Reno, NV; Doug Redelman of the
Department of Biology, University of Nevada, Reno, NV; and the many who have contributed to the successful research to date
and who continue to make new beneficial discoveries almost daily.
upregulates the expression of B7.1, B7.2, B7-H1, but not B7-DC on cultured
murine peritoneal macrophages
Kenneth W. Hunter, Jr.
a, Sally duPre’a
and Doug Redelmanb
a Department of Microbiology and Immunology, University of Nevada
School of Medicine, Reno, NV 89557, USA
b Department of Biology, University of Nevada, Reno, NV 89557, USA
Immunology Letters – February 2005
from a variety of biological sources has been shown to enhance both humoral and
cellular immune responses to a variety of antigens, infectious agents, and
tumors. Nevertheless, its mode of action has not been fully defined. We sought
to determine whether a 1–2 m
diameter microparticulate form of -glucan
(MG) from the yeast Saccharomyces cerevisiae could regulate expression of
B7 family glycoproteins on resident peritoneal macrophages from BALB/c mice. We
discovered that MG unregulated B7.2 mRNA expression and enhanced the surface
membrane expression of B7.2 glycoprotein. Although B7.1 mRNA was not upregulated
above constitutive levels, MG treatment enhanced B7.1 glycoprotein expression on
the macrophages, albeit to a lesser extent than B7.2. At the same time, the gene
and cell surface expression of B7-H1, a putative negative regulator of T cell
activity, was also upregulated by MG.
The expression of B7-DC, another B7
family molecule with negative regulatory activity, was not affected by
incubation with MG. This study has demonstrated that a microparticulate form of -glucan
can enhance B7 co-stimulatory molecule expression on macrophages, thereby
enabling these antigen-presenting cells to deliver the second signal to
T-lymphocytes that express CD28. In addition, because MG also induces the
expression of B7-H1, it may enable macrophages to provide a concomitant
downregulatory signal to T-lymphocytes expressing PD-1 or related receptors.
Author Keywords: -Glucan;
B7.1; B7.2; B7-H1; B7-DC; Macrophages; Co-stimulation
As part of an Abstract related to Microparticulate Glucan
as a Vaccine Adjuvant (Jan 2003), Kenneth W. Hunter, Jr. ScD from the University
of Reno School of Medicine, Dept of Microbiology reported,
"Nutritional Supply Corporation has
recently developed a novel microparticulate form of beta-1,3-(D)-glucan (MG)
from Saccharomyces cerevisiae...The uniform 1-2 micron diameter MG is rapidly
enocytosed by APC's (macrophages and dendritic cells), and most importantly,
upregulates the expression of B7 family co-stimulatory molecules in the APC's.
Without co-stimulation, APC's not only fail to activate T lymphocites, but they
may actually induce an unresponsive state called tolerance."
APC's are antigen presenting cells
which are essential to a proper immune response wherein T Cells are properly
activated. "Enocytosis" is the process of cellular ingestion in which the plasma
membrane folds inward to bring substances into the cell.
|Letters in Applied
Volume 35 Issue 4 Page 267 - October 2002
of microparticulate B-glucan
from Saccharomyces cerevisiae for use in immune potentiation
Hunter Jr, R.A. Gault and M.D. Berner
To develop a method for the preparation of an immunologically active,
homogeneous, nonaggregated, microparticulate
material from the budding yeast Saccharomyces cerevisiae.
Methods and Results: Using a combination of sonication and
spray-drying, a homogeneous preparation of 1-2-µ diameter
particles was made from alkali- and acid-insoluble yeast cell wall material.
remained in suspension longer and, following oral administration at 0·1 mg
for 14 d, enhanced phagocytosis of mouse peritoneal macrophages
significantly better than did aggregated
Conclusions: A new sonication and spray-drying method can be
employed to overcome the problem of aggregation of
microparticles in aqueous media.
Significance and Impact of the Study: A microparticulate form of
that remains in suspension longer for pharmaceutical applications and has
superior immune potentiation characteristics has been developed.
are polymers of
[with or without
side chains] found in the cell walls of many bacteria, plants and yeasts.
There is an extensive literature describing the immunomodulating effects of
both water soluble and insoluble
with macrophages as the principal target cells (Reynolds
et al. 1980;
Cleary et al. 1999). While various soluble and particulate
have been used in pharmaceutical applications (Williams
et al. 1992;
Babineau et al. 1994) particulate
preparations derived from the yeast Saccharomyces cerevisiae are
widely used as over-the-counter nutritional supplements. Examination of
several commercially available products consistently revealed a predominant
'globular' morphology consisting of aggregated
particles ranging in size from 5 to 100-µ diameter, with some unaggregated
single particles in the 1-2-µ range.
preparations have immune potentiating activity, it was thought that a
homogeneous preparation of smaller particles would be more efficient at
activating macrophages, as well as more suitable for incorporation into
pharmaceutical and cosmetic formulations. As 1-2-µ diameter particles are
optimally phagocytized by macrophages (Tabata
and Ikada 1988), our goal was to increase the number of
microparticles in this size range in the
However, even after extensive grinding and sieving of
extracted from yeast cell walls, it was discovered that the
particles formed aggregates when suspended in aqueous media. Therefore,
we devised a sonication and spray drying method that yielded a consistent
1-2-µ diameter particle that remained dispersed upon hydration. Although
both aggregated and microparticulate glucans enhanced peritoneal macrophage
activation when administered orally to mice, the microparticulate glucan was
significantly better than the aggregated form.
Materials and Methods
Processing of yeast glucan
The starting S. cerevisiae
material was obtained from Nutritional Supply Corporation (Carson City, NV,
USA). This material was processed from common baker's yeast using the
following procedure. Active dry yeast was added to 0·1 mol l
of NaOH and stirred for 30 min at 60 °C. The material was then heated to 115
°C at 8·5 psi for 45 min and then allowed to settle for 72 h. The sediment
was resuspended and washed in distilled H2O by centrifugation
(350 g for 20 min). The alkali insoluble solids were combined with 0·1 mol l
acetic acid and heated to 85 °C for 1 h, then allowed to settle at 38 °C.
The acid insoluble solids were drawn off and centrifuged as above. The
compacted solid material was mixed with 3% H2O2 and
refrigerated for 3 h with periodic mixing. The material was then centrifuged
and the pellet washed twice with distilled H2O followed by two
washes in 100% acetone. The harvested solid material was dispersed on drying
trays and dried under vacuum at 38 °C for 2 h in the presence of Ca2SO4,
and then further dried overnight under vacuum at room temperature. This
procedure yielded a white powder with less than 5% protein, lipid and
nucleic acid. Carbohydrate analysis revealed 85% hexoses (using the anthrone
method) with 4·5% chitin (measured as N-acetylglucosamine).
Preparation of microparticulate
Examination of the
powder dispersed in saline revealed aggregates ranging from 5 to 100 µ in
diameter (20 µ on average). To make a uniform 1-2-µ diameter particulate
preparation, the aggregated
material was first hydrated in distilled H2O overnight at 4 °C. A
1·5% suspension of the hydrated material was subjected to sonic energy via a
19-mm probe utilizing a 300-V/T Sonic Dismembrator (BioLogics, Gainesville,
VA, USA). Using an ultrasonic output frequency of 20 kilohertz per s at 192
watts, the glucan suspensions were sonicated on ice for 12 min (12 48-s
cycles of sonication with a 12-s pause between cycles). The sonicated
suspension was spray-dried using a Buchi 190 Mini-Spray Dryer (Buchi,
Germany) with an inlet air temperature of 110-170 °C, an outlet air
temperature of 90-120 °C and an atomizer pressure of 30-100 psi. Using flow
cytometric analysis with an EPICS XL-MCL Flow Cytometer (Coulter
Electronics, Hialeah, FL, USA), 1 mg of sonicated glucan consisted of 1·81
Morphology and sedimentation of the
preparations were suspended in normal saline and viewed with a Nikon Eclipse
E400 microscope under bright-field illumination. Photomicrographs were taken
with a Kodak DC digital camera. Dried
samples were placed on an s.e.m.
specimen holder and sputter-coated with gold to an approximately 200-Å
thickness. Prepared samples were viewed on a JEOL TSM T300 Scanning Electron
Suspensions of aggregated
were prepared in distilled H2O (1·5% w/v). Each suspension was
vortexed for 10 s and allowed to settle in one gravity in a 15-ml test tube
for 0, 2, 5, 10, 20, 30 or 60 min. Photographs were taken of the
sedimentation using a Kodak digital camera.
Phagocytosis of bioparticles by peritoneal
macrophages from mice treated orally with
Six- to eight-week old female BALB/c mice were obtained from Jackson
Laboratories (Bar Harbor, ME, USA). The mice were housed and cared for under
an approved protocol in accordance with NIH/USDA guidelines. They were given
daily oral doses of 0·1 mg kg
adsorbed on a food pellet (vehicle control was food pellet only) for 2
weeks. On day 14 the mice were killed and their resident peritoneal
macrophages removed by lavage with cold RPMI-1640 medium without serum.
Because Gram-negative bacterial endotoxin or lipopolysaccharide is a potent
macrophage activator and inducer of proinflammatory mediators, all
biochemical reagents and culture media were obtained as endotoxin-free or
screened for LPS by the Limulus test (BioWhittaker, Walkersville, MD, USA).
also triggers clotting in the standard Limulus test, we employed the
LPS-specific Glucospecy test to screen for LPS in our
preparations (Shigegaku, Tokyo, Japan).
All reagents and
preparations had < 10 pg ml
LPS. After 1 h of incubation at 37 °C in a 5% CO2 in air
atmosphere, nonadherent cells were removed by washing in warm culture medium
and the adherent cells (> 85% macrophages by morphology) were cultured for
24 h in RPMI-1640 medium containing 10% fetal bovine serum. The macrophages
were tested for their ability to ingest fluorescein isothiocyanate-labelled
bioparticles using the Vybrant
Phagocytosis Assay Kit (Molecular Probes, Eugene, OR, USA). The percentage
of cells ingesting fluorescent bioparticles, and the number of fluorescent
bioparticles per cell were determined using a Nikon Eclipse E400 fluorescent
microscope. At least 100 cells in each of 10 replicate wells per treatment
were counted after the fluorescence of noninternalized bioparticles was
quenched with trypan blue. Differences between the means of the treatment
groups were evaluated using Student's t-test for paired samples.
Results and Discussion
material resulting from the chemical extraction process detailed in the
Methods section was examined by light microscopy after hydration in
distilled H2O or saline.
material was determined to be a heterogeneous mixture of individual
microparticles (1-2 µ in diameter) and glucan particle aggregates ranging
from 5 to 100-µ diameters (Fig.
1a). As there was evidence that macrophages, key target cells for the
immunopharmacological activity of
preferentially ingest particles in the 1-2-µ diameter size range (Tabata
and Ikada 1988), we wanted to develop a method for making
microparticulate glucan. However, initial attempts to disrupt the aggregates
by vigorous vortexing, heating (100 °C), or treatment with strong acid (2 N
HCl) or strong base (2 N NaOH) failed (data not shown). Because ultrasonic
energy has been used to prepare microparticles in other systems (Hata
et al. 2000), we investigated sonication as a method of
disrupting the glucan aggregates.
Although disaggregation was accomplished by sonication using the
optimized conditions outlined in the Methods section (Fig.
1b), when the sonicated material was air-dried (either directly or after
addition of various organic solvents such as acetone) the resulting dry
material had the consistency of cardboard. This material could be ground
into a fine powder with a mortar and pestle, but upon hydration in distilled
H2O or saline it demonstrated significant aggregation (Fig.
1c). To overcome this re-aggregation problem, we employed a spray-drying
technique. The fine powder resulting from this spray-drying process when
hydrated in distilled H2O or saline resulted in a homogeneous
suspension of 1-2-µ diameter particles with very few small aggregates (Fig.
Interestingly, the addition of an excipient like maltodextrin did not
significantly improve the process. A similar ultrasonic approach was used by
Levis and Deasy (2001) to achieve particle size reduction of
microcrystalline cellulose. These authors discovered that re-aggregation in
aqueous media was substantially reduced by spray-drying, with or without the
addition of a surfactant. Just how sonication and spray-drying alters the
chemical or physical attributes of particles to mitigate against
re-aggregation remains to be determined.
and the microparticles obtained following the sonication and spray-drying
procedure were gold shadowed and examined with a s.e.m..
Figure 1(e) shows the morphology of a typical aggregate with a diameter
of approximately 35 µ. Note that this aggregate appears to be composed of
subunits in the 1-2-µ diameter size range ().
Sonication and spray drying results in separate and discrete microparticles
in the 1-2-µ diameter size range (Fig.
1f). This analysis indicates that the aggregated
is composed of discrete subcomponents that can be disrupted into 1-2-µ
diameter microparticles by a combination of sonication and spray-drying. The
chemical composition (-glucan
and chitin) and size of the
microparticles suggest that they may be yeast bud scars (Bacon
et al. 1969;
Manners et al. 1973). We are presently investigating this notion.
demonstrate that the microparticulate
preparation had a lower sedimentation rate, we performed the experiment
Fig. 2. As can be seen from this figure, after 1 h of sedimentation at 1
g the microparticulate
demonstrated very little settling, where as the aggregated
preparation had nearly sedimented fully. Indeed, some settling of the
was observed at even the earliest time point. Because the microparticulate
remains in aqueous suspension longer, it can be more easily formulated into
gels and creams for dermatological applications.
bind to glucan receptors on phagocytic cells (Goldman
Czop and Kay 1991;
Brown and Gordon 2001) and cause these cells to become 'activated'
1983). Earlier studies by
Suzuki et al. (1990) in mice showed that oral administration of a
derived from the fungus Sclerotinia sclerotiorum enhanced the
phagocytic activity of peritoneal macrophages. Therefore, we compared the
ability of orally administered microparticulate and aggregated
preparations given at 0·1 mg kg
daily for 14 d to enhance peritoneal macrophage phagocytosis. Note that this
dosage is equivalent to a 10-mg capsule of
given orally to a 75-kg human. As shown in
Table 1, cultured peritoneal macrophages taken from mice treated with
increased the percentage of peritoneal macrophages ingesting bioparticles
over the vehicle control (P< 0·05).
In addition, the microparticulate
was more stimulatory than the aggregated
(P=0·06). Also, the number of bioparticles ingested/cell was
increased over controls by both aggregated
(P< 0·05), and macrophages from microparticulate
mice ingested more bioparticles/cells than macrophages from mice treated
(P=0·06). These data imply that both microparticulate and aggregated
can survive transit through the gastrointestinal tract in forms capable of
being absorbed and ultimately of interacting with
receptors on the surfaces of resident peritoneal macrophages. It appears
that the same dose of microparticulate
is better able to enhance macrophage phagocytosis than aggregated
In conclusion, we have developed a new method for preparing homogeneous,
nonaggregated, 1-2-µ diameter
particles from yeast cell walls. Compared with the aggregated form of
microparticles remain in suspension longer for pharmaceutical applications
and are more effective at enhancing phagocytosis by peritoneal macrophages
following oral administration.
We thank Brandon Carter for technical assistance.
This work was supported by an Applied Research Initiative Matching Grant
from the State of Nevada and Nutritional Supply Corporation, Carson City,
Nevada, NV, USA.
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