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The mycelium of the Trametes versicolor (Turkey tail) mushroom and its fermented substrate each show potent and complementary immune activating properties in vitro

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Background: The medicinal mushroom Trametes versicolor (Tv, Turkey Tail) is often prepared for consumption as a powder from the fungal mycelium and the fermented substrate on which it grew. The goal for this study was to evaluate the immune-modulating properties of the mycelium versus the fermented substrate, to document whether an important part of the immune-activating effects resides in the metabolically fermented substrate. Methods: Tv mycelium was cultured on rice flour. The mycelium and the fermented substrate were mechanically separated, dried, and milled. The initial substrate served as a control. Aqueous fractions were extracted and passed through 0.22-μm filters. The remaining solids were passed through homogenization spin columns without filtration. The aqueous and solid fractions of the initial substrate (IS), the fermented substrate (FS), and the Trametes versicolor mycelium (TvM) were tested for immune-activating and modulating activities on human peripheral blood mononuclear cell cultures, to examine expression of the CD69 activation marker on lymphocytes versus monocytes, and on the T, NKT, and NK lymphocyte subsets. Culture supernatants were tested for cytokines using Luminex arrays. Results: Both aqueous and solid fractions of TvM triggered robust induction of CD69 on lymphocytes and monocytes, whereas FS only triggered minor induction of CD69, and IS had no activating effect. The aqueous extract of TvM had stronger activating effects than the solid fraction. In contrast, the solid fraction of IS triggered a reduction in CD69, below levels on untreated cells. Both aqueous and solid fractions of FS triggered large and dose-dependent increases in immune-activating pro-inflammatory cytokines (IL-2, IL-6), anti-inflammatory cytokines Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 (IL-10), anti-viral cytokines interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-alpha (MIP-1α), as well as Granulocyte-Colony Stimulating Factor (G-CSF) and Interleukin-8 (IL-8). TvM triggered more modest cytokine increases. The aqueous extract of IS showed no effects, whereas the solid fraction showed modest effects on induction of cytokines and growth factors. Conclusion: The results demonstrated that the immune-activating bioactivity of a mycelial-based medicinal mushroom preparation is a combination of the mycelium itself (including insoluble beta-glucans, and also water-soluble components), and the highly bioactive, metabolically fermented substrate, not present in the initial substrate.
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R E S E A R C H A R T I C L E Open Access
The mycelium of the Trametes versicolor
(Turkey tail) mushroom and its fermented
substrate each show potent and
complementary immune activating
properties in vitro
Kathleen F. Benson
1
, Paul Stamets
2
, Renee Davis
2
, Regan Nally
2
, Alex Taylor
2
, Sonya Slater
2
and Gitte S. Jensen
1*
Abstract
Background: The medicinal mushroom Trametes versicolor (Tv, Turkey Tail) is often prepared for consumption as a
powder from the fungal mycelium and the fermented substrate on which it grew. The goal for this study was to
evaluate the immune-modulating properties of the mycelium versus the fermented substrate, to document
whether an important part of the immune-activating effects resides in the metabolically fermented substrate.
Methods: Tv mycelium was cultured on rice flour. The mycelium and the fermented substrate were mechanically
separated, dried, and milled. The initial substrate served as a control. Aqueous fractions were extracted and passed
through 0.22-μm filters. The remaining solids were passed through homogenization spin columns without filtration.
The aqueous and solid fractions of the initial substrate (IS), the fermented substrate (FS), and the Trametes versicolor
mycelium (TvM) were tested for immune-activating and modulating activities on human peripheral blood
mononuclear cell cultures, to examine expression of the CD69 activation marker on lymphocytes versus monocytes,
and on the T, NKT, and NK lymphocyte subsets. Culture supernatants were tested for cytokines using Luminex
arrays.
Results: Both aqueous and solid fractions of TvM triggered robust induction of CD69 on lymphocytes and
monocytes, whereas FS only triggered minor induction of CD69, and IS had no activating effect. The aqueous
extract of TvM had stronger activating effects than the solid fraction. In contrast, the solid fraction of IS triggered a
reduction in CD69, below levels on untreated cells.
Both aqueous and solid fractions of FS triggered large and dose-dependent increases in immune-activating pro-
inflammatory cytokines (IL-2, IL-6), anti-inflammatory cytokines Interleukin-1 receptor antagonist (IL-1ra) and
Interleukin-10 (IL-10), anti-viral cytokines interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-alpha
(MIP-1α), as well as Granulocyte-Colony Stimulating Factor (G-CSF) and Interleukin-8 (IL-8). TvM triggered more
modest cytokine increases. The aqueous extract of IS showed no effects, whereas the solid fraction showed modest
effects on induction of cytokines and growth factors.
Conclusion: The results demonstrated that the immune-activating bioactivity of a mycelial-based medicinal
mushroom preparation is a combination of the mycelium itself (including insoluble beta-glucans, and also water-
soluble components), and the highly bioactive, metabolically fermented substrate, not present in the initial
substrate.
Keywords: Anti-inflammatory, Immune modulation, CD69, Cytokines
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: gitte@nislabs.com
1
NIS Labs, 1437 Esplanade, Klamath Falls, Oregon 97601, USA
Full list of author information is available at the end of the article
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342
https://doi.org/10.1186/s12906-019-2681-7
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Medicinal mushrooms describe a category of edible mem-
bers of the kingdom Fungi, traditionally associated with
health-supporting properties. They have been used for cen-
turies to treat an array of ailments, particularly in trad-
itional Asian medicine and Eastern European traditions.
They are well regarded for supporting longevity, treating
infectious disease and cancer, and promoting overall well-
being [1,2]. Contemporary research has mainly focused on
the broad immune activity of mushrooms. Several preclin-
ical findings suggest that mushrooms may specifically sup-
port NK cell upregulation, [25] enhancement of T-cell
and NK cell cytotoxicity [6], and the induction of immune-
regulating cytokines such as TNF-α, IL-2, IFN-y, and IL-10
[710]. As a category, medicinal mushrooms stimulate
host defense and immunity due to the complex and vary-
ing polysaccharides; some well-studied examples include
(1,3;1,6)-β-glucans, proteoglycans, heteroglucans, that com-
prise the chitin-based fungal cell wall [1113]. Mushrooms
are also the source of other pharmacologically relevant
compounds, such as proteins like Ling zhi-8 in Ganoderma
lucidum [14] and lectins in several species [15], triterpenes
[16,17], phenols [18], and sterols [16]. While medicinal
mushrooms generally confer broad immune activity, indi-
vidual species often possess unique immunological proper-
ties. Trametes versicolor (Tv), commonly known as Turkey
tail and previously named Coriolus versicolor,isknownto
enhance innate and adaptive immune responses [19,20].
Recent clinical research involving consumption of Tv
mycelium on rice substrate by Standish and col-
leagues [21] suggests NK cell induction in women
with breast cancer. Other researchers cite antitumor
effects [4,22,23], but this is generally considered to
be a result of its underlying immunologic activity
[24]. Tv contains precursors to the proteoglycans
polysaccharide peptide (PSP) and polysaccharide-K
(Krestin, PSK), the latter of which is frequently pre-
scribed to gastric cancer patients in Japan [25]. The
constituents responsible for these immunological ef-
fects are believed to be the polysaccharides. However,
recent research suggests that the lipid fraction of PSK
isolated from Tv is instrumental to its TLR-2 induc-
tion activity [26].
The natural ecological role of mushrooms is to assist
the breakdown of dead plant matter, and therefore they
engage in a highly dynamic interaction with the environ-
ment in which they grow. The fungal organism contains
a vegetative state consisting of progressive extensions of
tissue into a substrate, as well as a reproductive state for
spore dispersal (Fig. 1). Mycelium is an aggregation of
multinucleate hyphae that typically appear as strands or
thin filaments. As a mycelium grows throughout its
environment, it secretes an array of compounds into its
substrate, altering the chemical nature of the substrate.
Fig. 1 Decomposition of a fallen tree log by Trametes versicolor (Tv). aFallen tree log, presenting fresh organic plant matter. bTv mycelium is
growing inside the log, decomposing the plant biomass by fermentation, in a highly dynamic exchange of solubilized nutrients from the tree log,
resulting from secreted fungal enzymes, combined with anti-microbial defense compounds to protect the mycelial territory. cFruitbodies serve
to spread the spores of the Tv mushrooms, and have a narrower chemical composition, focused on beta-glucans, spores, attractants to animals
that may eat and transport the spores, and protectants to protect the fruitbodies from bacteria and other fungi
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 2 of 14
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This enzyme-rich exudate helps catalyze the breakdown
of macromolecules for absorptionan example of which
are the extracellular lignin-modifying enzymes laccase,
lignin peroxidase, and manganese peroxidase [27]. Fungi
that engage in this type of enzymatic lignin biodegrad-
ation and decompose wood are known as white-rot
fungi, named for their white appearance. As a white rot
fungus with notable laccase production, Tv has the cap-
acity to enzymatically affect its environment, primarily
by degradation of its substrate [2830]. This enzymatic
activity is not limited to lignin-containing woody tissues;
rice bran can also function as an efficient substrate for
laccase production [31].
Of importance for the understanding of medicinal
mushrooms for human consumption, fungal hyphae also
secrete a wide variety of defense compounds to deter
predators and pathogens [32]. Secreted defense com-
pounds allow the fungi to maintain their territory and
evade invasion by bacteria and molds. These compounds
may be evolutionarily conserved and offer biological ef-
fects for other species such as humans. The medical sig-
nificance of this is apparent in the famous example of
the first antibioticpenicillin, isolated from the Penicil-
lium chrysogenum mold [33].
As structurally different as the mycelium is from the
fruitbody, so too are their biological functions. Whereas
the mycelium is the major biomass of a fungus and
serves to gather nutrients and interact with the substrate
during decomposition, the fruitbodies (the most com-
monly known form of edible mushrooms) are the instru-
ments of spore dispersal in higher fungi (Basidiomycota).
They commonly appear as a cap on top of a stem or
stalk, with either gills or pore structures underneath the
cap. Mycelium and fruiting bodies share similar cell wall
structures and contain the polysaccharide complexes
that enhance the innate and adaptive immune response
[12,34,35]. However, concentrations vary, and β-
glucans are considered to be present in higher concen-
trations in the fruiting body compared to the mycelium
[36], whereas the mycelial tissue may contain a broader
profile of bioactive compounds. Other metabolic vari-
ances may exist: recent proteomic research suggests that
40% more protein-coding genes in G. lucidum are
expressed in the mycelial state, compared to the fruiting
body [37].
The production of medicinal mushroom products uti-
lizes a wide spectrum of substrates, including sawdust to
mimic the natural habitat, as well as various grains. It is
well known that the biological properties of raw grain
are altered by fungal fermentation, likely due to the se-
creted enzymes. A simple fungal organism, namely yeast,
grown on red rice, is considered a dietary supplement,
and documented to reduce LDL in preclinical and
clinical settings [38], properties not associated with
consumption of plain unfermented rice. Another ex-
ample is the Saccharomyces yeast-based fermentate Epi-
Cor®, which is composed of the fungal cell walls as well
as secreted metabolites produced during the fermenta-
tion. The aqueous extract of the dried fermentate has
well-documented immune activating, anti-inflammatory,
and antioxidant properties [3941]. Consuming the
whole dried fermentate is associated with clinical bene-
fits including improved mucosal immune health [42],
and reduced incident and duration of colds [43] and
allergies [44]. Research using the SHIME model for di-
gestive health has shown beneficial effects on the gut
microbiome [45].
Many medicinal mushroom products are sold as a
crude powder consisting of mycelium and its fermented
substrate. While pre-clinical and clinical studies have
been performed on these products [19,21], the immuno-
logical contributions of the fermented substrate have not
been examined. The purpose of this study was to
characterize the immunological activity of each of the
components, namely the mycelia and the fermented sub-
strate, using the initial substrate as a control. Tv was se-
lected as a model organism for this effort because the
physical structure of the mycelium is well defined and
allows for harvest of both the mycelium and the fermen-
ted substrate (including secreted fungal metabolites)
when cultivated in specially designed solid substrate fer-
mentation (SSF) systems. The test model involved evalu-
ation of the early activation marker CD69 on different
subsets of immune cells and the induction of production
of cytokines and growth factors. The choice of CD69 is
based on its role in natural killer (NK) cell function,
where CD69 is rapidly induced in NK cells shortly after
activation [46] and has a direct role in NK cytotoxicity
(killing of target cells) [47].
Methods
Reagents
Roswell Park Memorial Institute 1640 medium, penicillin
streptomycin 100×, interleukin-2 (IL-2), phosphate-buffered
saline, and lipopolysaccharide (LPS) from Salmonella enter-
ica were purchased from Sigma-Aldrich Co. (St Louis, MO,
USA). CD69 fluorescein isothiocyanate, CD56 phycoeryth-
rin, CD3 peridinin chlorophyll protein, and heparin Vacutai-
ner tubes were purchased from Becton-Dickinson (Franklin
Lakes, NJ, USA). Customized Bio-Plex Prohuman cytokine
arrays were purchased from Bio-Rad Laboratories Inc.
(Hercules, CA, USA).
Trametes versicolor (Turkey tail) culture and separation of
mycelial and fermented substrate
The mycelial culture work and sample processing was
performed at Fungi Perfecti LLC, following a three-step
process of substrate preparation, mycelial culturing, and
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sample separation (Fig. 2). Certified organic rice flour
(Azure Farms, Dufur, Oregon, USA) mixed with water
to form a paste and sterilized by autoclaving at 1 bar for
60 min. This resulted in a solid biscuit-like disc of rice
grain media (0.40.45 g/g water content; aw 0.99). This
material constituted the initial substrate (IS). A Petri
dish containing 60 g (dry mass) of the milled and steril-
ized rice flour was inoculated with 50 mg of Trametes
versicolor agar media spawn. The resulting inoculated
media disc solid substrate fermentation microcosm was
stored at 20-24 °C for 42 days in a class 1000 clean room.
The Trametes versicolor mycelium spread radially over
the growth substrate, preferentially developing biomass
on the surface of the substrate where gas exchange was
highest. Mycelium was separated mechanically by re-
moving the surface mycelium from the underlying sub-
strate with a scalpel.
Preparation of mycelium and substrate for in vitro testing
The three powders were handled in the following man-
ner: 1) Liquid extraction using phosphate-buffered saline
(PBS) and referred to as the aqueous fraction; 2) Har-
vesting the non-aqueous, solid fractions left after
aqueous extractions were completed, and passing them
through homogenization spin columns (QIAshredder,
Qiagen, Hercules, CA). The aqueous fractions were
filtered through a 0.22-μm filter before adding to cell
cultures. The solid fractions were not filtered through a
0.22-μm filter. This provided two test products/
fractionsfrom each product, namely the aqueous frac-
tion and the solid fraction. From each fraction, serial
dilutions were made in phosphate-buffered saline.
Dry weight determinations of aqueous extracts
The data graphs show the biological activities per gram
starting material. However, in order to understand the
relative contributions from aqueous constituents, dry
weight assessments were performed for the aqueous
fractions. From each of the three powders, a 100 g/L sus-
pension was prepared in distilled H2O. The powder was
allowed to hydrate and water-soluble compounds were
extracted for 1 h under gentle agitation. Solids were pre-
cipitated by centrifugation in conical polypropylene vials
for 10 min at 400 g. The liquid fraction was harvested,
passed through a 0.22-μm cellulose acetate filter, and
dried at 100 °C. The weights of the filtrates were 45 mg/
g (4.5%w/w) for the initial substrate, 110 mg/g (11% w/
w) for the fermented substrate, and 120 mg/g (12% w/w)
for the mycelium.
Immune cell activation
Peripheral venous blood was drawn from three healthy
human donors upon written informed consent, as ap-
proval by the Sky Lakes Medical Center Institutional Re-
view Board, Federalwide Assurance 2603. The blood was
drawn into heparin vacutainer vials, and the peripheral
blood mononuclear cells (PBMC) isolated using Lym-
pholyte Poly (Cedarlane Labs, Burlington, Ontario, CA)
Fig. 2 Trametes versicolor (Tv) was used as an experimental model to isolate and compare the mycelium and its fermented substrate. aDiagram
showing the origin of the three test products compared: Initial substrate (rice flour), fermented substrate, and Tv mycelium. bPhoto of the three
powders: Initial substrate (left) is plain rice flour prior to use as a substrate for growing the Tv mycelium. The fermented substrate (center) is the
dried residual powder where the mycelium has been removed. The mycelium (right) is the collection of fungal hyphae, removed from the
fermented substrate on which it was grown
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 4 of 14
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by centrifugation for 35 min at 450 g. The PBMC were
washed twice in PBS, counted, and the density adjusted
to establish cultures with a cell density at 10
6
/mL, using
Roswell Park Memorial Institute 1640 medium contain-
ing penicillinstreptomycin and 10% heat-inactivated
fetal bovine serum (Gibco, Thermo Fisher Scientific,
Asheville, NC).
Serial dilutions of products or LPS were added to
cultures at a volume of 20 μL, and cultures were then incu-
bated at 37 °C, 5% CO2 for 24 h. The highly inflammatory
LPS from Salmonella enterica wasusedasapositivecon-
trol for immune-cell activation at a dose of 10 ng/mL. In
parallel, IL-2 was used as a positive control for natural killer
(NK)-cell activation, at a concentration of 100 IU/mL.
Untreated negative control cultures consisted of PBMC
exposed to phosphate-buffered saline in the absence of test
products. All treatments, including each dose of test prod-
uct and each positive and negative control, were tested in
triplicate. After 24 h, blood cells were isolated from each
culture well and stained for 10 min with fluorochrome-
labeled antibodies at the recommended concentration.
PBMC were then fixed using 0.5% formalin. The fluores-
cence intensities for CD3, CD56, and CD69 were measured
by flow cytometry, using an Attune acoustic-focusing flow
cytometer (Thermo Fisher Scientific).
During data analysis, gating on forward and side scat-
ter facilitated evaluation of the levels of CD69 expression
on lymphocyte and monocyte subsets. The lymphocyte
subpopulation was further analyzed for CD69 expression
on CD3+ T lymphocytes, CD3+ CD56+ NKT lympho-
cytes, and CD3- CD56+ NK cells.
Production of cytokines, chemokines, and growth factors
After 24 h of incubation, the supernatants were har-
vested from the PBMC cultures described above. Levels
of 10 cytokines and chemokines were quantified (IL-1ra,
IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, interferon gamma
(IFN-γ), tumor necrosis factor alpha (TNF-α), MIP-1α
(CCL3), and G-CSF) using Bio-Plex Promultiplex im-
munoassays (Bio-Rad Laboratories, Hercules, CA) and
utilizing xMAP technology (Luminex, Austin, TX, USA).
Statistical analysis
Average and standard deviation for each data set was
calculated using Microsoft Excel. Statistical analysis of
in vitro data was performed using the 2-tailed, independ-
ent t-test. Statistical significance was set at P< 0.05, and
a high level of significance at P< 0.01.
Results
Induction of the CD69 activation marker on immune cell
subsets
The cell surface expression of the early activation marker
CD69 was measured on peripheral blood mononuclear
cells (PBMC), after 24 h incubation in the absence versus
presence of initial substrate (IS), fermented substrate (FS),
and Trametes versicolor mycelium (TvM). Representative
results from one blood donor are shown in Figure 3 and
4, and results from the 2 other blood donors are available
in Additional file 1. During flow cytometric data analysis,
the gating on the physical characteristics of the cell sub-
sets, allowed analysis on lymphocytes versus monocytes
(Fig. 3).
The aqueous fraction of IS showed no effect on CD69
expression (Fig. 3a). The solid fraction showed a very
minor increase in CD69 expression on lymphocytes, and
a highly significant suppression of CD69 expression on
monocytes (P< 0.001) (Fig. 3b).
The induction of CD69 on human lymphocytes by the
aqueous and solid fractions of FS showed higher CD69
induction by the solid fraction than the induction seen
by the aqueous fraction. The difference in CD69 induc-
tion by the aqueous and solid fractions of FS was statisti-
cally significant (P< 0.03).
The treatment of human lymphocytes with both
the aqueous and the solid fractions of TvM resulted
in a robust and statistically significant increase of
the CD69 marker, indicating immune cell activation
(Fig. 3a). The induction of CD69 on human lympho-
cytes by the TvM aqueous fraction was more robust
than the induction seen by the TvM solid fraction
(Fig. 3a),wherethedifferencebetweentheTvM
aqueous and solid fractions was statistically signifi-
cant (P<0.02).
In contrast, the induction of CD69 on human
monocytes by the TvM solid fraction was more ro-
bust than the induction seen by the TvM aqueous
fraction (Fig. 3b), where the difference between the
TvM aqueous and solid fractions was highly signifi-
cant (P<0.001).
The lymphocyte subset was further analyzed for ex-
pression of the CD69 activation marker of CD3+ T
cells, CD3+ CD56+ NKT lymphocytes, and CD3-
CD56+ Natural Killer (NK) cells (Fig. 4). It was found
that the aqueous extract from TvM triggered a very
potent activation of NKT cells (Fig. 4c), and a more
moderate activation of T cells and NK cells (Fig. 4a
and e). The aqueous extract of the fermented sub-
strate only induced minor increases in CD69 on all
three cell types, and the aqueous extract of the initial
substratedidnotinduceCD69onanyofthethree
cell types (Fig. 4a, c, e).
In contrast, the solid fractions of TvM and FS induced
comparable levels of cellular activation, as measured by
increased CD69 expression (Fig. 4b, d, f). The activation
was not as strong as what was seen for the aqueous ex-
tract of the mycelium but was stronger than the cell ac-
tivation by the aqueous extract of the fermented
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substrate. The solid fraction from the initial substrate
showed minor activation of NK cells and NKT cells
(Fig. 4d, f).
Increased production of pro-inflammatory, immune-
activating cytokines
The culture supernatants from the PBMC cultures
were tested for the levels of two cytokines involved in
immune cell activation, Interleukin-2 (IL-2) and
Interleukin-6 (IL-6). Representative results from one
blood donor are shown in Figure 5, and results from
the 2 other blood donors are available in Additional
file 1. Both the aqueous and the solid fractions of the
fermented substrate (FS) induced robust increases in
IL-2 and IL-6 levels (Fig. 5). The aqueous fraction of
Trametes versicolor mycelium (TvM) also induced IL-
2 and IL-6, but was more potent at doing so at lower
doses (Fig. 5a,c). The solid fraction of TvM had mild
effects on IL-2 and IL-6 production in the cultures,
and the induction was comparable to the solid frac-
tion of the initial substrate (Fig. 5b,d). The aqueous
fraction of the initial substrate did not have any effect
on IL-2 or IL-6 induction (Fig. 5a, c).
Increased anti-viral cytokine production
The treatment of human PBMC with the fungal ex-
tracts triggered increased production of two anti-viral
cytokines, namely Interferon-gamma (IFN-γ)and
MIP-1α(Fig. 6). Representative results from one
blood donor are shown in Figure 6, and results from
Fig. 3 Induction of the CD69 cellular activation marker on lymphocyte (a) and monocyte (b) subsets in human PBMC cultures. The PBMC cultures
were treated for 24 h in the presence of the aqueous versus solid fractions of initial substrate (IS), fermented substrate (FS), and Trametes
versicolor mycelium (TvM). Data are shown for the highest dose tested (2 mg/mL), where the dose represents the amount of starting material
used to produce a given fraction. Data are presented as mean ± standard deviation of the mean fluorescence intensities in triplicate cultures, and
represents one of three separate experiments using PBMC cells from three different healthy human donors. Positive controls included
lipopolysaccharide (LPS, 10 ng/mL) and Interleukin-2 (IL-2, 100 IU/mL). Statistical significance is indicated as * for P< 0.05 and **for P< 0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 6 of 14
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the 2 other blood donors are available in Additional
file 1. Both the aqueous and solid fractions from the
fermented substrate triggered robust increases in
these two cytokines, whereas treatment of cultures
with the Trametes versicolor mycelium (TvM) led to
modest increases in these cytokines. The aqueous
fraction of TvM showed more potent effect on IFN-γ
at lower doses than at higher doses (Fig. 6a).
Interestingly, treatment of human PBMC with the
solid fraction of the initial substrate showed a minor
increase in IFN-γand MIP-1αproduction. The induc-
tion of IFN-γexceeded that induced by the solid
Fig. 4 Induction of the CD69 cellular activation marker on immune cell subsets in human PBMC cultures. The PBMC cultures were treated for 24 h in
the presence of serial dilutions of Trametes versicolor mycelium (TvM), fermented substrate (FS), or initial substrate (IS). The percent change when
compared to untreated control cultures is shown for T lymphocytes (a-b), NKT cells (c-d), and NK cells (e-f). The effects of aqueous extracts are shown
in a,c,ande, and the effects of the solid fractions are shown in B, d,andf. Data are shown for three doses tested (0.08, 0.4, and 2 mg/mL), where the
doses represent the amount of starting material used to produce a given fraction. Data are presented as mean ± standard deviation of the percent
change seen in triplicate cultures, and represents one of three separate experiments using PBMC cells from three different healthy human donors.
Positive controls included LPS and IL-2. The mean ± standard deviation percent change induced by LPS were 19 ± 2.1% for T lymphocytes, 54 ± 8.3%
for NKT cells, and 114 ± 10% for NK cells. The mean ± standard deviation percent change induced by IL-2 were 39 ± 0.9% for T lymphocytes, 150± 25%
for NKT cells, and 446± 60.0% for NK cells. Inserted tables: Statistical significance is indicated as * for P< 0.05 and ** for P<0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 7 of 14
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fraction of TvM (Fig. 6b). The MIP-1αinduced by
both aqueous and solid fractions of TvM and the
initial substrate were similar in magnitude (Fig. 6c, d).
Increased anti-inflammatory cytokine production
The treatment of human PBMC with the fungal extracts
triggered increased production of two anti-inflammatory
cytokines, namely Interleukin-1-Receptor Antagonist (IL-
1ra) and Interleukin-10 (IL-10) (Fig. 7). Representative re-
sults from one blood donor are shown in Figure 7, and re-
sults from the 2 other blood donors are available in
Additional file 1. Both the aqueous and solid fractions
from the fermented substrate triggered increases in both
these two cytokines, with the most robust induction being
associated with the solid fraction. The aqueous fraction of
Trametes versicolor mycelium (TvM) showed more potent
effect on both IL-1ra and IL-10 at lower doses than at
higher doses (Fig. 7a, c). Interestingly, treatment of human
PBMC with the solid fraction of the initial substrate
showed a moderate increase in IL-1ra production, exceed-
ing that induced by the solid fraction of TvM (Fig. 7b).
Increased production of markers involved in regenerative
processes
The treatment of human PBMC with the fungal extracts
triggered increased production of two biomarkers in-
volved in regenerative processes involving stem cells,
Granulocyte Colony-Stimulating Factor (G-CSF) and
Interleukin-8 (IL-8) (Fig. 8). Representative results from
one blood donor are shown in Figure 8, and results from
the 2 other blood donors are available in Additional file 1.
For the fermented substrate, both the aqueous and solid
fractions triggered increases in both these two markers.
The aqueous fraction of Trametes versicolor mycelium
(TvM) showed effects at a broad dose range (Fig. 8a, c),
whereas the effects of the solid fraction from the TvM
showed similar effects as the solid fraction from the initial
substrate (Fig. 8b, d).
Discussion
The principal finding of the work reported here was a
highly differentiated immune activating effect by the
Trametes versicolor mycelium (TvM) when compared to
Fig. 5 Changes in levels of the cytokines Interleukin-2 (IL-2) and Interleukin-6 (IL-6) in supernatants from human PBMC cultures. The PBMC were
cultured for 24 h in the presence of serial dilutions on Trametes versicolor mycelium (TvM), fermented substrate (FS), or initial substrate (IS). The
effects on IL-2 and IL-6, cytokines involved in immune activation, of aqueous extracts shown in aand c, and of the solid fractions are shown in band d.
Data are shown for three doses (0.08, 0.4, and 2 mg/mL), where the doses represent the amount of starting material used to produce a given fraction. Data
are presented as mean ± standard deviation of the percent change seen in triplicate cultures, and represents one of three separate experiments using
PBMC cells from three different healthy human donors. Inserted tables: Statistical significance is indicated as * for P < 0.05 and ** for P< 0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 8 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
its fermented substrate (FM). It was noteworthy that
both aqueous and solid fractions of both materials had
potent immune modulating activities.
TvM triggered robust increases in the CD69 activation
marker on lymphocytes and monocytes, unlike FM
which did not induce CD69 on immune cells. The cell
surface marker CD69 is rapidly upregulated on many
immune cell types after activation, and correlations have
been made between Natural Killer (NK) cell CD69 ex-
pression and NK cell-mediated tumor-killing activity in
the classical target cell-based assay, by a number of re-
search teams over the past 25 years. We have found the
induction of the CD69 activation marker a helpful tool
for natural products research, both in vitro [4854] and
in clinical studies [55,56]. When human NK cells are
co-cultured with K562 target cells, CD69 expression is
upregulated, and the increase significantly correlated
with NK cell activity, as measured by todays gold-
standard CD107 mobilization assay [57]. CD69 has the
capacity to activate the NK cytolytic machinery in the
absence of other NKtarget cell adhesion molecule in-
teractions [58]. A direct and highly significant cor-
relation between CD69 levels and NK cell activity was
demonstrated by Clausen et al 2003 [59], in a study in-
volving 14 breast cancer patients tested repeatedly dur-
ing chemotherapy.
NK cells do not function in a vacuum; they are regula-
tory cells engaged in crosstalk with other cell types [60].
Therefore, the work reported here focused not only on
NK cells, but also on NKT cells, T cells, and monocytes.
The TvM-mediated induction of CD69 expression on
lymphocytes and monocytes was triggered by both aque-
ous and solid fractions, however, the CD69 expression
on lymphocytes was more robust when cells were
treated with the aqueous fraction than the solid fraction,
with the aqueous fraction comparable to the induction
caused by LPS. Given that the aqueous fraction con-
tained almost 10 times less material than the solid
fraction, this further demonstrates the potency of the
aqueous compounds in the mycelium. For monocytes,
Fig. 6 Changes in levels of the cytokines Interferon-gamma (IFN-γ) and Macrophage Inflammatory Protein-1-alpha (MIP-1α) in supernatants from
human PBMC cultures. The PBMC were cultured for 24 h in the presence of serial dilutions of Trametes versicolor mycelium (TvM), fermented
substrate (FS), or initial substrate (IS). The effects on IFN-γand MIP-1α, cytokines involved in anti-viral immune defense activity, of aqueous
extracts shown in A and C, and of the solid fractions are shown in B and D. Data are shown for three doses (0.08, 0.4, and 2 mg/mL), where the
doses represent the amount of starting material used to produce a given fraction. Data are presented as mean± standard deviation of the
percent change seen in triplicate cultures, and represents one of three separate experiments using PBMC cells from three different healthy
human donors. Inserted tables: Statistical significance is indicated as * for P < 0.05 and ** for P < 0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 9 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
this was reversed, where the solid fraction triggered a
stronger CD69 expression than the aqueous fraction of
the TvM. This is expected, since monocytes are known
to be robustly activated by insoluble fungal beta-glucans
through Toll-Like receptors (TLR), specifically TLR-2
and TLR-4 [61].
The cytokine induction by aqueous and insoluble com-
pounds in the fermented substrate was seen in the ab-
sence of CD69 up-regulation, suggesting activation via
alternate pathways, such as has been demonstrated for
NK cell activation, which may involve CD69 expression
or as an alternative mode of activation involve upregula-
tion of the Interleukin-2 receptor CD25. This was dem-
onstrated by Clausens team to be associated with
distinct functional differences, where CD69 expression is
associated with cytotoxicity as described above, whereas
the CD25 expression is associated with increased cell
proliferation [59].
Interestingly, the initial un-fermented substrate was
not very bioactive, devoid of aqueous bioactive com-
pounds, and only minor effects on cytokine induction by
the solid fraction. This further helps demonstrate the
uniqueness of the fermented substrate, in terms of fer-
mentation of the rice along with fungal exudates. This is
important for the many consumable products that are
produced from mycelia along with their fermented
substrates.
The aqueous extracts were produced by cold-water
extraction, and not using heat or pressure. This is in
contrast to other types of fungal extracts and teas, where
heat and sometimes pressure is applied to produce the
extract. Examples include the use of batch reactors and
subcritical water extraction, with heat up to 300 °C, to
produce extracts from golden oyster mushrooms
(Pleurotus citrinopileatus)[62] and Chaga mushrooms
(Inonotus obliquus)[63].
The results clearly demonstrate that most of the
bioactivity for cytokine induction lies in the fermented
substrate. Based on the known enzyme secretions by my-
celium during active growth, the fermented substrate
likely represents a broad array of fungal products, in
conjunction with breakdown products from the substrate.
Fig. 7 Changes in levels of the cytokines Interleukin-1 receptor antagonist (IL-1ra) and Interleukin-10 in supernatants from human PBMC cultures.
The PBMC were cultured for 24 h in the presence of serial dilutions of Trametes versicolor mycelium (TvM), fermented substrate (FS), or initial
substrate (IS). The effects on IL-1ra and IL-10, both involved in anti-inflammatory processes as part of the resolution of inflammatory processes, of
aqueous extracts are shown in aand c, and of the solid fractions are shown in band d. Data are shown for three doses (0.08, 0.4, and 2 mg/mL),
where the doses represent the amount of starting material used to produce a given fraction. Data are presented as mean ± standard deviation of
the percent change seen in triplicate cultures, and represents one of three separate experiments using PBMC cells from three different healthy
human donors. Inserted tables: Statistical significance is indicated as * for P< 0.05 and ** for P< 0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 10 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Tv has proved to be an effective model for demon-
strating the bioactivities of mycelium versus its fermen-
ted substrate. This model will be useful for further
evaluation of Tv and other medicinal mushrooms, and
this model can help mycology research in the isolation
and identification of bioactive compounds. There may
be potentially pharmaceutical uses of isolated novel
compounds form both mycelium and fermentate, how-
ever, one of the more unique and interesting challenges
will be to understand their synergistic relationships in
traditional medicinal use of the whole crude fermentate.
Further work from our team is ongoing and includes this
evaluation of synergy, by pretreating isolated NK cells
and monocytes with TvM, FM, and a blend thereof,
followed by the classical NK activity assay in co-culture
with tumor cells, and by co-cultures of Tv-primed
monocytes with lymphocytes to evaluate the role of NK
cells and monocytes in the overall immunological cross-
talk between cell types. In addition, a clinical trial will
compare the TvM, FM, and the blend thereof. This will
help further document the importance of the multi-
faceted and complex actions of the natural mixture of
TvM and its fermented substrate, traditionally used for
immune support in the integrative medicine setting.
Conclusions
The work reported here has helped demonstrate that the
mycelial fermentation of its substrate dramatically alters
the biological effects of the fermented substrate. Further-
more, the mushroom mycelium has distinctly different
biological and immune-modulating properties than its
fermented substrate. The mycelium was very potent in
terms of triggering immune cell activation, whereas the
fermented substrate was very active in terms of cytokine
induction. Complex immune-activating bioactivity of
mycelial-based medicinal mushrooms go beyond effects
of insoluble beta-glucans, as potent effects were also
seen in the aqueous fraction. The results suggest that
overall medicinal effects are associated both with the
mycelium itself (including insoluble beta-glucans, but
Fig. 8 Changes in levels of the growth factor Granulocyte-Colony Stimulating Factor (G-CSF) and the cytokine Interleukin-8 in supernatants from
human PBMC cultures. The PBMC were cultured for 24 h in the presence of serial dilutions of Trametes versicolor mycelium (TvM), fermented
substrate (FS), or initial substrate (IS). The effects on the stem cell mobilizing growth factor G-CSF and Interleukin-8 (IL-8) of aqueous extracts are
shown in aand c, and of the solid fractions are shown in band d. Data are shown for three doses (0.08, 0.4, and 2 mg/mL), where the doses
represent the amount of starting material used to produce a given fraction. Data are presented as mean ± standard deviation of the percent
change seen in triplicate cultures and represents one of three separate experiments using PBMC cells from three different healthy human donors.
Inserted tables: Statistical significance is indicated as * for P< 0.05 and ** for P< 0.01
Benson et al. BMC Complementary and Alternative Medicine (2019) 19:342 Page 11 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
also water-soluble components), and the highly bioactive
fermented substrate. Novel applications for animal and
human immune health may be identified in the future
for components isolated from fermented substrates,
independent of mushroom mycelium.
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12906-019-2681-7.
Additional file 1. The graphs in the paper shows representative data on
immune effects on cells from one of three healthy donors. The full sets
of data from all 3 blood donors are shown in the supplementary
information.
Abbreviations
FS: Fermented substrate; G-CSF: Granulocyte-Colony Stimulating Factor; IFN-
γ: Interferon-gamma; IL-10: Interleukin-10; IL-1ra: Interleukin-1 Receptor
Antagonist; IL-2: Interleukin-2; IL-6: Interleukin-6; IL-8: Interleukin-8; IS: Initial
substrate; LPS: lipopolysaccharide; MIP-1α: Macrophage Inflammatory Protein
1-alpha; PBMC: Peripheral Blood Mononuclear Cells; TNF-α: Tumor Necrosis
Factor-alpha; Tv: Trametes versicolor (Turkey Tail mushroom); TvM: Trametes
versicolor mycelium
Authorscontributions
PS and AT conceived of the questions to be tested. GSJ and KFB wrote the
research protocol and designed the study. KFB, SS, and AT conducted the
work presented here. KFB and GSJ performed the data analysis. GSJ, RD, RN,
and KFB wrote the manuscript. All co-authors participated in the writing and
final edit of the manuscript.
Authorsinformation
KFB is a molecular geneticist with a specific interest in the human
microbiome, including the normal and diseased microbial composition in
gut, skin, and blood.
PS is a mycologist with a broad spectrum of interests, and active as advisor
on NIH panels for complementary and alternative medicine. PS is also deeply
engaged in ecological research, particularly in successful anti-viral protection
of endangered honey bees by mushroom extracts. PS served as a consultant
to Star Trek Discovery, pertaining to contemporary and futuristic visions for
astrobiology/astromycology.
RD is a writer, herbalist, and researcher with an interest in botanical and
mycological medicine to improve clinical treatment strategies and health
outcomes. She has been involved in health policy research and consultations
in indigenous tribal communities, and research in botanical medicine around
womens health and herb-drug interactions.
RN is an organic chemist whose academic research focused on the synthesis
of supramolecular systems capable of altering their physical properties based
upon environmental stimuli. She has a profound interest applying scientific
tools to the development of the natural products industry.
AT is a biological systems engineer specializing in medicinal mushroom
cultivation and fungal biotechnology. He has 8 yrs of academic and industry
experience spanning diverse topics in fungal biotechnology including
development of quantitative methods for assessing biomass production,
evaluation of field-scale fungal deployment for environmental restoration,
and development of mushroom-derived antiviral formulations. His research
interests include microbial growth dynamics, biofiltration, biological biomass
conversion, mathematical modeling and industrial fermentation.
SS is a research scientist with over 18 yrs of experience working in molecular
biology and immunology in Seattle area biotech companies and the
University of Washington with a focus on B cell directed drug therapies:
Cloning of recombinant DNA molecules, subsequent protein expression in
mammalian cell lines, protein purification and characterization,
immunological assays to test the efficacy of the protein against B cell tumors
and in autoimmune diseases.
GSJ has an academic background in cancer research, with a special focus on
immune surveillance and systems biology. This is combined with a strong
interest in natural products, nutrition, herbal and complementary medicine.
She has applied her research to serving traditional integrative medicine as
well as novel developments in the natural products industry with
mainstream research tools.
Funding
The study was sponsored by Fungi Perfecti, LLC, a grower and producer of
commercially available mushroom mycelia and finished consumable
products. Authors RD, RN, PS, AT, and SS are scientists from the funding
body and their roles in the design, execution, and analysis is described
below.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Ethics approval and consent to participate
Written informed consent, as approved by the Sky Lakes Medical Center
Institutional Review Board (Federalwide Assurance 2603), was obtained from
the healthy blood donors from which venous blood samples were obtained
for this work.
Consent for publication
Not applicable.
Competing interests
GSJ and KFB declare that they have no competing interests. RD, RN, AT, and
SS are employed by the sponsor of the study. PS holds several patents on
topics related to the presented work, and is the founder and owner of the
sponsoring company.
Author details
1
NIS Labs, 1437 Esplanade, Klamath Falls, Oregon 97601, USA.
2
Fungi Perfecti,
Postal Box 7634, Olympia, Washington 98507, USA.
Received: 25 November 2017 Accepted: 11 September 2019
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... The chemical analysis results revealed significant improvement in the nutritive value of MS compared with untreated sorghum. This confirmed the report by Benson et al. [26], who noted that the biological properties of raw grain were altered by fungal fermentation, and was also consistent with previous studies [10,14] when Pleurotus mycelium enhanced the feeding value of corn stover. The increase in DM content of MS implies that MS would potentially offer more nutrients when fed to the animals than untreated sorghum, which might have resulted from the release of bound nutrients during the solid-state fermentation [14]. ...
... According to Duodu et al. [28], low protein digestibility of sorghum is linked to the tough inner protein wall surrounding the seed, the protein (karifin proteins) crosslinking, as well as the interaction of sorghum proteins within themselves and with non-protein components which impede amino acids to proteolysis; however [6], this is attributed the low protein digestibility to the presence of anti-nutritional factors. Meanwhile, solid-state fermentation has been reported to increase the protein and amino acid concentrations of substrates or products [26,29]. In agreement, Kim et al. [30] reported higher CP contents in spent Agaricus bisporus substrates. ...
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... The fermented substrate-induced increase in the immune-activating proinflammatory cytokines. The mycelium itself along with fermented substrate accounted mostly for the immune-activating bioactivity of a mycelial-based medicinal mushroom preparation (Benson et al., 2019). ...
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... Used in cancer treatments to boost efficacy, these mushrooms also aid in treating muscle weakness and fatigue. Additionally, they contain bioactive compounds like fatty acids (linoleic, oleic, stearic, linolenic), polyphenols (vanillic, phenolic acids, homogentisic, p-hydroxybenzoic, protocatechuic), and polysaccharides (Benson et al. 2019). ...
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... This result suggests that the choice of substrate significantly impacts the crude protein content of T. versicolor mushrooms (Angelova et al. 2022). Moreover, substrates such as S5 and S1 demonstrate potential for enhancing the protein content of the mushrooms, highlighting the importance of substrate selection for enhanced nutritional quality and potentially influencing the therapeutic properties of the mushrooms (Benson et al. 2019). Conversely, the lower protein content observed in S7 indicates the need for careful substrate formulation to achieve desirable nutritional outcomes in mushroom cultivation. ...
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... Medicinal mushrooms enhance the host immune system through their bioactive compounds, such as (1,3;1,6)-b-glucans, heteroglucans, proteoglycans, lectins, phenols, sterols, triterpenes and pharmacologically relevant proteins like Ling zhi-8 (Kino et al., 1989). Several preclinical studies suggest that mushrooms can induce cytokinins like IL-2, IL-10, TNF-a, and IFN-y, enhancethe cytotoxicity by NK cells and T-cells, and upregulation of NK cells (Benson et al., 2019). Turkey tail, one of the most researched mushrooms,has been used for centuries in Chinese and Asian medicine for its medicinal health benefits. ...
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Medicinal mushroom are known for their therapeutic capabilities and one such notable example is the Turkey tail mushroom (Trametes versicolor). This fascinating medicinal fungus, belonging to the genusTrametes that has been in used for centuries in traditional medicine systems, particularly in Asia. Turkey tail mushroom look-alikes due to the existence of diversity and variation in species.It is crucial to observe key morphological features for proper identification of fruiting bodies. The presence of phenols, polysaccharides, terpenoids, and other compounds in Turkey tail enrich its biological activities as a potent anticancer, antioxidant, antimicrobial, antiaging, hepatoprotective, and anti-diabeticpropeties. Recent studies have explored the various metabolic activities of T. versicolor, highlighting important polysaccharides such as Polysaccharopeptides (PSP) and Polysaccharopeptide Krestin (PSK), which have been thoroughly researched for their effects on cancer cells. This review attempts to disclose the importance of Turkey tail mushrooms and their untapped medicinal potential to further benefit immunology studies.
... 11,16,17 Including fungal mycelia in the fermented substrate makes it possible to exploit the range of functional metabolites and components, namely those in the mycelia, those released into the substrate, as well as those in the fermented transformed substrate. Benson et al. 18 ...
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Mushrooms are widely cited for their medicinal qualities, yet very few human intervention studies have been done using contemporary guidelines. The aim of this study was to determine whether consumption of whole, dried Lentinula edodes (shiitake) mushrooms could improve human immune function. Primary objectives were to ascertain whether L. edodes consumption would improve γδ-T cell proliferation and activation responses, quantify a dose response, and elicit cytokine secretion patterns. Secondary objectives included determining changes in natural killer T (NK-T) cell proliferation and activation, secretory immunoglobulin A (sIgA) in saliva, and C-reactive protein (CRP) in serum. Fifty-two healthy males and females, aged 21-41 years, participated in a 4-week parallel group study, consuming either 5 or 10 g of mushrooms daily. Each subject had blood drawn before and after 4 weeks of daily L. edodes consumption. Saliva and serum were also collected. Peripheral blood mononuclear cells were cultured in autologous serum for 24 hours or 6 days, stained, and examined by flow cytometry. Eating L. edodes for 4 weeks resulted in increased ex vivo proliferation of γδ-T (60% more, p < 0.0001) and NK-T (2-fold more, p < 0.0001) cells. Both cell types also demonstrated a greater ability to express activation receptors, suggesting that consuming mushrooms improved cell effector function. The increase in sIgA implied improved gut immunity. The reduction in CRP suggested lower inflammation. The pattern of cytokines secreted before and after mushroom consumption was significantly different; consumption resulted in increased interleukin (IL)-4, IL-10, tumor necrosis factor (TNF)-α, and IL-1α levels, a decreased macrophage inflammatory protein-1α/chemokine C-C ligand 3 (MIP-1α/CCL3) level, and no change to IL-6, IL-1β, MIP-1β, IL-17 and interferon (IFN)-γ levels. Regular L. edodes consumption resulted in improved immunity, as seen by improved cell proliferation and activation and increased sIgA production. The changes observed in cytokine and serum CRP levels suggest that these improvements occurred under conditions that were less inflammatory than those that existed before consumption.
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Background/aim: We investigated the relationship between the expression of natural killer group 2, member D ligands (NKG2DLs) and the antitumor effects of protein-bound polysaccharide-K (PSK). Materials and methods: PSK was administered to evaluate its effectiveness against tumor growth. The expression of Rae-1 and H60 were analyzed in multiple cell lines. Results: PSK showed the highest antitumor effects in mice implanted with cells expressing neither Rae-1 nor H60. PSK had little antitumor effect in mice implanted with cells expressing both Rae-1 and H60. A correlation between the expression of NKG2DLs and the antitumor effect of PSK was observed. After PSK administration, INF-γ production in CD8(+) T cells increased in mice with cells expressing neither Rae-1 nor H60, but did not change in mice implanted with cells expressing both Rae-1 and H60. Conclusion: We demonstrated that the expression of NKG2DLs affects tumor immunity and the efficacy of immuno therapy in tumor-bearing mouse model.
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Mushrooms are popular folk medicines that have attracted considerable attention because of their efficient antitumor activities. This review covers existing research achievements on the mechanisms of isolated mushroom polysaccharides, particularly (1→3)-β-D-glucans. Our review also describes the function in modulating the immune system and potential tumor-inhibitory effects of polysaccharides. The antitumor mechanisms of mushroom polysaccharides are mediated by stimulated T cells or other immune cells. These polysaccharides are able to trigger various cellular responses, such as the expression of cytokines and nitric oxide. Most whose polysaccharides could bind other conjugate molecules like polypeptides and proteins, which conjugation always possess strong antitumor activities. The purpose of this review is to summarize available information, and to reflect the present situation of polysaccharide research filed with a view for future direction.
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In the US market, there is a variety of mushroom preparations available, even within the same species of mushroom. Nonetheless, little is known about whether species or the various extraction methods affect biological activity and potency of the immune modulatory activity of mushroom extracts. After discovering that protein-bound polysaccharide-K, a hot water extract from Trametes versicolor, was a potent Toll-like receptor (TLR)-2 agonist that stimulates both innate and adaptive immunity, this study was initiated to evaluate whether other medicinal mushroom products also have TLR2 agonist activity and immune-enhancing potential as measured by the induction of tumor necrosis factor (TNF)-α in J774.A1 murine macrophage cells. Furthermore, the products were divided by extraction method and species to determine whether these factors affect their immunomodulatory activity. The results showed that the majority (75%) of mushroom products tested had TLR2 agonist activity and that there was a significant correlation between TLR2 agonist activity and TNF-α induction potential in the mushroom products analyzed. In addition, the data demonstrated that hot water mushroom extracts are more potent than ground mushroom products in activating TLR2 and inducing TNF-α. These data provide evidence that extraction methods may affect the biological activity of mushroom products; thus, further studies are warranted to investigate the structural differences between various mushroom products.
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Mechanisms of fungal antagonism and defense often include the production of biologically active metabolites by one species that exert effects on potential competitors and (or) predators. Studies carried out in our laboratory and others clearly indicate that such ecological phenomena can serve as valuable leads to the discovery of novel and potentially useful bioactive fungal metabolites. There is evidence that some of these compounds may render advantages to the producing organism, although careful and definitive ecological studies are required to determine this. Nevertheless, the results summarized here demonstrate the broad array of possible benefits that can arise from interdisciplinary studies in this area. This paper focuses primarily on our own investigations of the chemistry involved in fungal antagonism and defense using coprophilous and sclerotial fungi as model systems. These results have potential implications in many areas of study, including fungal ecology, secondary metabolism, chemotaxonomy, organic chemistry, structure determination, antifungal chemotherapy, and insect control.