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Effect of medicinal extract from Agaricus blazei Murill on gene expression in a human monocyte cell line as examined by microarrays and immuno assays

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Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).
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Effect of a medicinal extract from Agaricus blazei Murill on gene
expression in a human monocyte cell line as examined by
microarrays and immuno assays
L.K. Ellertsen
a
, G. Hetland
c
, E. Johnson
d
, B. Grinde
b,
*
a
Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway
b
Division of Infectious Disease Control, Norwegian Institute of Public Health, Oslo, Norway
c
Department of Immunology and Transfusion Medicine, Ulleva˚l University Hospital, Oslo, Norway
d
Department of Gastroenterological Surgery, Ulleva˚ l University Hospital, Oslo, Norway
Received 21 June 2005; received in revised form 6 July 2005; accepted 18 July 2005
Abstract
Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against
cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order
to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line
(THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine
gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic
effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric
genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced
a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A,CXCL1,CXCL2 and CXCL3, as well as
PTGS2 (cyclooxygenase2).
D2005 Elsevier B.V. All rights reserved.
Keywords: Agaricus blazei Murill; Microarray; Cytokines; Lipopolysaccharide; Immune function
1567-5769/$ - see front matter D2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.intimp.2005.07.007
Abbreviations: AbM, Agaricus blazei Murill; BG, background;
FG, foreground; GO, gene ontology; HUGO, human genome orga-
nisation; LPS, lipopolysaccharide; MIAME, minimum information
about a microarray experiment; NAE, not appreciably expressed;
TLR, Toll-like receptor.
* Corresponding author. Fax: +47 22 042447.
E-mail address: bjorn.grinde@fhi.no (B. Grinde).
1. Introduction
The Agaricus blazei mushroom belongs to the
family Basidiomycetes and is native of Brazil. It
was brought to Japan due to alleged health effects,
and is today widely used both in the form of extracts,
and as an edible, medicinal mushroom. According to
tradition, it helps against a variety of diseases, includ-
ing cancer, diabetes, arteriosclerosis and hepatitis. In
International Immunopharmacology 6 (2006) 133 – 143
www.elsevier.com/locate/intimp
recent years, considerable research has been carried
out to investigate the putative effects.
An immunomodulating impact of extracts from
this mushroom seems to be well documented [1–6].
Moreover, several reports suggest a positive effect on
cancer [7–10], possibly mediated either via stimula-
tion of natural killer cells by fungal glucans and
subsequent apoptosis [1,3,7–9,11–14], or by an anti-
angiogenic effect mediated by ergosterol and pyroglu-
tamate [15,16]. As to antimicrobial activity, AbM has
been reported to inhibit western equine encephalitis
virus in cell culture [17]. Extracts may also be used as
an adjuvant to vaccines [18,19].
h-Glucans from other fungi have been shown to
have both antitumor and anti-infection effects [20–23].
The AbM extracts are rich in h-glucans that presum-
ably contribute to the observed activity [9,24], but
other substances are probably involved as well.
In the present study, a bacterial lipopolysaccharide
(LPS) preparation was used for comparison, as the
response to LPS has been well characterized [25–28].
LPS binds to Toll-like receptor 4 (TLR4), and thereby
activates mitogen-activated protein (MAP) kinases and
nuclear factor kappa B. The net result is to induce
proinflammatory cytokines such as IL8 and tumor
necrosis factor (TNF). The h-glucans are assumed to
exert their effect via Toll-like receptor 2 (TLR2), dec-
tin-1 (CLECSF12), and by binding to the lectin site of
complement receptor 3 (ITGAM) [29]. Myeloid differ-
entiation protein-88 (MYD88) appears to be involved in
signalling in the case of both LPS and h-glucans [30].
Previous reports have established that AbM can
induce certain cytokines, e.g., IL1B and IL6 in mouse
peritoneal macrophages upon oral administration [5],
and TNF and IL8 in rat bone marrow macrophages
upon in vitro administration [31]. A down-regulation of
IL2, IL4 and IFNG in human peripheral blood mono-
nuclear cells has also been documented [3]. Microar-
rays offer a powerful tool for investigating the global
gene expression effect of modulating agents. The
arrays used in the present study presumably cover the
vast majority of mRNAs made by human cells, as they
contain oligos reflecting 34580 known or putative
transcripts. Thus the present results offer a panorama
on how A. blazei preparations may act on human
monocytes.
As exemplified above, the HUGO gene symbols
will be used when discussing genes and gene pro-
ducts. Alternative names, e.g., those typically applied
to the corresponding proteins, will be included when
appropriate.
2. Materials and methods
2.1. Chemicals and cell culture
THP-1 (American Type Culture Collection, TIB-202)
cells were grown in plastic culture flasks (NunclonkSur-
face) in RPMI medium supplemented with 10% Foetal
Bovine Serum (Gibco, UK) and 1% Penicillin/Streptomycin
(Paa Laboratories GmbH, Austria) at 37 8C with 5% CO
2
.
The cells were seeded at a concentration of 7.510
5
cells/
ml (totally 10 10
6
THP-1 cells per flask), and stimulated
with either a 9% (v/v) solution of an aqueous extract of A.
blazei Murill (AbM) (Gold label, obtained from ACE Co.,
Ltd, Gifu-ken, Japan) containing 300 Ag/ml of h-glucans
according to the manufacturer, or 10 Ag/ml of E. coli
lipopolysaccharide (type L-2654 from Sigma, St.Louis,
MO, USA) for 24 h. Three independent experiments were
performed with each addition. The AbM extract was kept at
48C in dark glass bottles. Opened bottles were used for no
more than two weeks. The LPS content of the extract was
examined using the PyrogenekRecombinant Factor C
Endotoxin Detection System (BioWhittaker, Maine, USA)
and found to be negligible (i.e., 0.15 ng per ml).
2.2. RNA extraction
To stop the experiments, the cells were centrifuged
(800 g, 5 min), and the pellets immediately resuspended
in 600 Al of the lysis buffer supplied with the Agilent Total
RNA Isolation Mini Kit (Agilent Technologies, Palo Alto,
CA) used for RNA extraction. The lysed cells were kept at
70 8C until extraction according to manufacturer’s recom-
mendations. The RNA was eluted in 50 Al RNase-free water.
RNA concentration and quality were determined using UV
spectrophotometry as well as microcapillary electrophoresis
on a Bioanalyzer 2100 (Agilent Technologies, Palo Alto,
CA) according to manufacturer recommendations. The yield
was between 30 and 60 Ag of RNA per flask, with an UV
260 / 280 ratio between 2.016 and 2.050, and electrophoretic
profiles suggesting intact RNA.
2.3. Microarrays
RNA was converted to fluorescence-labelled cDNA with
the FairPlaykaminoallyl kit (Amersham International,
UK), according to the manufacturer’s recommendations,
using 20 Ag of total RNA per sample and Cy5 or Cy3 as
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143134
fluorescent dyes. The labelled cDNA was hybridized with
35 k human 70-mer oligonucleotide-based microarrays pro-
vided by the Norwegian Microarray Consortium (NMC,
http://www.mikromatrise.no). The microarrays were spotted
on Corning UltraGAPS glass-slides, using the Operon
Human Genome Oligo Set Version 3.0, with 34580 70-
mer probes representing 24,650 known or putative genes
and 37,123 gene transcripts (Operon Biotechnologies,
Huntsville, AL, USA). Controls in the form of Lucidea
Microarray ScoreCard v1.1 (Amersham Biosciences) were
included. Automated hybridization was performed for 12 h
at 45 8C with the ChipMap80 kit (including the Chi-
pHybe80 buffer) on the Ventana Discovery system (Ventana
Medical Systems, Tucson, AZ, USA). The arrays were
scanned on an Agilent fluorescence scanner (Axon Instru-
ments, Foster City, CA, USA) with a pixel size of 10 Am.
2.4. Microarray analyses
Quality control of the scanned images (tiff-files), and
conversion to numerical values were performed using the
GenePix Pro 4.1 (Axon Instruments). The gpr-files obtained
were further analysed using the freely available BASE BioAr-
ray Software Environment [32]; available at http://
alba.uio.no/base/. Analyses were based on mean FG–median
BG intensities. Flagged spots and controls were filtered away.
Next, the plug-in application Transformation Simsalabim (a
weak spot filter) was run with default settings, and then
normalization using Lowess. Subsequently, spots with both
channels mean FG–median BG b300 were removed, as well
as spots were log2 ratios were between 1 and 1. Only spots
where at least two of the three parallel experiments had passed
the previous criteria were retained, but for these spots data for
all three parallel experiments were included for further pro-
cessing. The files were transferred to Excel where the average
ratios for the three parallels were calculated. The following
filtering was performed: Reporters with average up- or down-
regulation above respectively 2or 4were selected, as
were reporters with average intensities above 1000. Table 1
shows the number of reporters retained as up- or down-
regulated at various stringency of filtering, as well as the
number of annotated genes these reporters corresponded to.
The gene ontology program eGOn (http://nova2.idi.ntnu.no/
egon/) and the Norwegian Microarray Consortium Dataware-
house (http://nova2.idi.ntnu.no/annotdb/) were used to exa-
mine gene function (based on gene annotations available as of
May 2004). The gene ontology analyses concentrated on
genes that were more than 4up- or down-regulated and
had mean intensities above 1000. Comparisons of up- or
down-regulated genes were performed with either the
bPaired-target testQ(for comparing AbM with LPS) or the
bTarget-Master testQ(for comparing either AbM or LPS up-
regulated genes with all expressed genes). Both tests are
available in the eGOn pack. MIAME compatible microarray
protocols and data are available in the ArrayExpress databank
http://www.ebi.ac.uk/arrayexpress/.
2.5. Determination of cytokine concentrations
The concentrations of IL-6, IL-8, IL-10 and IL-12p40 in
cell culture supernatants were measured using sandwich
enzyme immuno assays (ELISA), with antibodies from
R&D Systems (UK). Briefly, polystyrene plates (Costar
EIA/RIA Plate, Sigma-Aldrich Norway) were coated with
100 Al/well of anti-hIL-6 (1 Ag/ml) anti-hIL-8 (1 Ag/ml),
anti-hIL-10 (4 Ag/ml), or anti-hIL-12 p40 (4 Ag/ml) antibo-
dies in carbonate–bicarbonate buffer (50 mM, pH 9.6). Serial
two-fold dilutions (in 1% of BSA/Tris–Tween20) of recom-
binant cytokines were used as standards (R&D Systems).
The standards were distributed in triplicates (100 Al/well),
while culture supernatants were used undiluted and distrib-
uted in duplicates (100 Al/well). The incubation period was 2
h at room temperature. Between incubation steps the plates
were washed five times (Skan Washer 400, Model 12019,
Skatron, Norway) with 0.01 M of Tris/HCL-buffer (pH 7.4)
containing 0.05% Tween20. Biotinylated anti-human IL-6
Table 1
Number of up- or down-regulated transcripts at various stringencies of analysis
Treatment Transcripts
expressed–
annotated
a
Transcript
expressed N
1000
b
Transcripts
more than 2
up/down
c
Transcripts
more than 4
up/down
More than 4up/down and average
intensity N1000
Transcripts Annotated genes
AbM 5993–4195 1876 423/280 215/111 94/13 84/13
LPS 7261–5436 2360 768/539 371/233 177/13 155/12
a
The arrays contained 34,580 probes of which 24,897 reflected gene transcripts with annotations. Transcripts expressed denote reporters
where the intensity of at least one channel in one experiment had mean FG–median BG above 300 after normalization. The subset that had
annotations in the Datawarehouse (http://nova2.idi.ntnu.no/annotdb/) is indicated.
b
The number of transcripts where the average intensity (three experiments) of either red or green channel was above 1000.
c
The following columns indicate the number of up- or down-regulated transcripts based on increasing stringencies of analyses, as subsets of
the transcripts referred to in the first column.
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143 135
(50 ng/ml), IL-8 (0.04 Ag/ml), IL-10 (500 ng/ml), or IL-12
(0.3 Ag/ml) antibodies (R&D Systems) were added (100 Al/
well) as a third layer, and the plates were incubated for 1 h at
room temperature. StreptABComplex/AP (Dako A/S, Den-
mark) was added to the plates according to manufacturer’s
protocol. After 1 h at room temperature, the plates were
developed with 1 mg/ml p-nitrophenyl phosphate (Sigma
104R, St. Louise, MO, USA) in 10% diethanolamine buffer
(pH 9.8). Optical density (OD) was measured at 405 nm on a
MRX Microplate Reader connected to a PC using Revelation
software for instrument operation and calculations (Dynatech
laboratories, Chantilly, VA, USA).
Commercial ELISA kits were used to analyse IL-1h
(DuoSet, R&D Systems, UK) and TNF-a(TNF-a
CytoSetsk, Biosource International, USA). The assays
were performed as recommended by the manufacturers,
and OD measured as described above, but at 450 nm.
2.6. DNA and protein synthesis
Cells from two of the three parallel experiments
described above were used to examine total DNA and
protein synthesis by the incorporation of, respectively,
3
[H]thymidine and
3
[H]leucine. Cell suspensions (1 10
5
cells/well), either unstimulated or stimulated with AbM or
LPS, were added to 96-well plates in triplicates. In the
assay for thymidine incorporation, 1.25 ACi of
3
[H]thymi-
dine (Amersham Pharmacia Biotech, Buckinghamshire,
UK) was added to each well and the cells incubated for
21 h. A Skatron cell harvester (Skatron A/S, Lier, Norway)
transferred the cells onto filter papers (FilterMAT 11731)
that were subsequently washed and dried. In the assay for
3
[H]leucine incorporation, the cells were incubated for 21
h with 1 ACi/well (Amersham Pharmacia Biotech) in a
leucine free medium. The cells were subsequently centri-
fuged at 1000 rpm for 5 min and the pellets dissolved in
0.1 M NaOH. 100 Al of 20% trichloroacetic acid (TCA)
was added in order to precipitate the proteins, and the
precipitate transferred to the filterpaper using the Skatron
harvester (FilterMAT 11734). The filterpapers were mixed
with scintillation fluid, and the incorporated
3
[H]thymidine
or
3
[H]leucine measured in a scintillation counter (Tri-
Carb, Model 1500, Packard Instruments Co., Inc. Meridan,
CT, USA).
3. Results
3.1. Microarray analyses
Of the 34,580 transcript related reporters on the arrays,
5993 (corresponding to 4195 annotated transcripts) were
appreciably expressed (i.e., at least one channel in one
experiment had mean FG–median BG N300) in the experi-
ments with AbM, compared to 7261 (corresponding to 5436
annotated transcripts) in the experiments with LPS (Table
1). The difference between the AbM and the LPS experi-
ments was probably due partly to arbitrary variations in the
Fig. 1. Scatter plots showing spot intensities before normalization.
One representative experiment is shown for, respectively: A, AbM;
and B, LPS. The control cells were labelled green and the treated
cells red. Flagged spots and controls are not included. The regres-
sion lines F2SD are indicated. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web
version of this article.)
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143136
Table 2
Immune related genes up-regulated by AbM/LPS or otherwise of interest
HUGO Gene description AbM LPS
Ratio
(FSE)
Intensity
(average)
Ratio
(FSE)
Intensity
(average)
Selectively induced by AbM
CXCL3 Chemokine (CXC motif) ligand 3 60 (F18) 2230 NAE
PTGS2 Prostaglandin-endoperoxide synthase 2 (COX2) 41 (F3) 1756 15 (F2) 661
CXCL2 Chemokine (CXC motif) ligand 2 39 (F16) 1890 21 (F7) 831
CXCL1 Chemokine (CXC motif) ligand 1 31 (F6) 3470 12 (F4) 507
RGS1 Regulator of G-protein signalling 1 21 (F11) 1179 15 (F6) 930
IL1A Interleukin 1, alpha 13 (F4) 2201 4.0 (F1) 917
Induced by both AbM and LPS
IL8 Interleukin 8 972 (F416) 35,084 353 (F75) 19,018
IL1B Interleukin 1, beta 579 (F208) 36,846 407 (F43) 32,387
SCYA4 Small inducible cytokine A4 (=CCL4) 161 (F67) 6366 338 (F91) 27,989
CCL3 Chemokine ligand 3 (MIP-1a) 115 (F46) 6594 187 (F32) 13,675
IL23A Interleukin 23, asubunit p19 82 (F35) 3279 99 (F22) 8703
CCL3L1 Chemokine ligand 3-like 1 72 (F29) 6741 105 (F20) 16,831
SOD2 Superoxide dismutase 2, mitochondrial 67 (F17) 17,091 56 (F7) 27,435
SCYA20 Small inducible cytokine A20 (=CCL20) 48 (F22) 1963 37 (F9) 1742
NCF1 Neutrophil cytosolic factor 1 35 (F18) 2979 22 (F3) 9071
NFKBIA NFKB B-cells inhibitor, alpha 14 (F2) 4555 13 (F1) 9106
IFI30 Interferon, gamma-inducible protein 30 12 (F1) 1572 14 (F5) 3657
ALOX5AP Arachidonate 5-lipoxygenase-activating protein 11 (F6) 4560 6.1 (F0) 3113
FTH1 Ferritin, heavy polypeptide 1 11 (F4) 47,139 4.8 (F1) 47,140
NCF1 Neutrophil cytosolic factor 1 11 (F1) 2975 15 (F1) 10,000
IFNGR2 Interferon gamma receptor 2 11 (F3) 1317 6.6 (F1) 2141
CEBPB CCAAT/enhancer binding protein h11 (F2) 12,787 7.5 (F1) 20,342
PBEF Pre-B-cell colony-enhancing factor 10 (F3) 2996 7.4 (F0) 2724
G0S2 Putative lymphocyte G0/G1 switch gene 7.5 (F1) 2318 5.1 (F1) 1927
SCYA5 Small inducible cytokine A5 (=RANTES or CCL5) 7.0 (F1) 1525 4.6 (F1) 9876
NFKB1 NFK light polypeptide gene enhancer in B-cells 1 (p105) 6.1(F1) 1007 11 (F1) 2118
NFKBIE NFKB enhancer in B-cells inhibitor, epsilon 6.1 (F2) 1119 4.2 (F0) 1628
S100A9 S100 calcium binding protein A9 5.5 (F2) 4147 13 (F1) 11,606
CASP1 Caspase 1 (IL1B convertase) 4.4 (F0.1) 1075 4.8 (F0) 2232
Selectively induced by LPS
SCYA2 Small inducible cytokine A2 (=CCL2) 5.6 (F1) 384 211 (F43) 19,389
SCYB10 Small inducible cytokine B10 (=CXCL10) NAE 71 (F12) 3205
IFI27 Interferon, a-inducible protein 27 NAE 60 (F4) 7377
SERPINA1 Serine proteinase inhibitor A 1 8.4 (F1) 317 56 (F3) 2689
CYBB Cytochrome b-245, hpolypeptide 17 (F1) 783 49 (F6) 2773
TNFAIP6 TNF, a-induced protein 6 17 (F5) 744 48 (F9) 2128
ISG20 Interferon stimulated gene 20kDa NAE 40 (F7) 1600
EBI3 Epstein–Barr virus induced gene 3 13 (F2) 565 39 (F14) 3358
CD83 CD83 antigen 9.0 (F3) 367 37 (F7) 1449
IFIT1 Interferon-induced protein with tetratricopeptide repeats 1 NAE 27 (F2) 1503
TNF Tumor necrosis factor (=TNFA or TNFASF2) 10 (F6) 605 26 (F2) 5627
G1P2 Interferon, alpha-inducible protein NAE 21 (F2) 5838
IFI44L Interferon-induced protein 44 NAE 20 (F1) 2401
IL10RA Interleukin 10 receptor, alpha 8.0 (F0) 260 19 (F3) 1257
BCL6 B-cell CLL/lymphoma 6 11 (F0) 505 18 (F3) 1140
IL6 Interleukin 6 (interferon, beta 2) NAE 17 (F7) 1011
(continued on next page)
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143 137
intensity of the different arrays. However, as can be seen
from Table 1, LPS caused the induction of more genes (even
when correcting for differences in array intensities), an
effect that contributed to the bias in the number of genes
appreciably expressed.
Both AbM and LPS caused considerable changes in gene
expression (Table 1 and Fig. 1). As much as 12% (703/
5993) and 18% (1307/7261), respectively, of the expressed
genes were either up- or down-regulated at least 2. Even at
a high stringency of filtering, i.e., 4changes and average
intensities above 1000, respectively, 107 and 190 genes
were retained, here referred to as considerably up- or
down-regulated genes. As expected for substances that acti-
vate the cells, many more genes were up-regulated com-
pared to down-regulated.
The scatter plots obtained when analysing the effect of
AbM displayed a curious split, where approximately half the
reporters ended up above the regression line, and the other
HUGO Gene description AbM LPS
Ratio
(FSE)
Intensity
(average)
Ratio
(FSE)
Intensity
(average)
Selectively induced by LPS
LY6E Lymphocyte antigen 6 complex, locus E NAE 17 (F2) 2529
FOS V-fos FBJ murine osteosarcoma viral oncogene homolog NAE 15 (F5) 2453
MX1 Myxovirus resistance 1, interferon-inducible protein p78 NAE 14 (F2) 3562
SPP1 Secreted phosphoprotein 1 6.1 (F1) 717 12 (F3) 2063
SCYA8 Small inducible cytokine subfamily A8 (=CCL8) NAE 11 (F9) 1854
OAS2 2V-5V-oligoadenylate synthetase 2 1.2 (F0) 425 11 (F1) 5013
IL1RN Interleukin 1 receptor antagonist 2.6 (F0) 495 11 (F1) 2940
NFKB2 NFK light polypeptide gene enhancer in B-cells 2 5.9 (F0) 941 9.8 (F2) 1191
OAS3 2V-5V-oligoadenylate synthetase 3 3.3 (F0) 297 9.4 (F3) 1852
IFITM2 Interferon induced transmembrane protein 2 1.0 (F0) 1577 9.0 (F1) 24,115
OAS1 2V,5V-oligoadenylate synthetase 1 NAE 8.3 (F1) 1516
TNFAIP3 TNFa-induced protein 3 9.0 (F3) 778 8.1 (F3) 1136
CD53 CD53 antigen 5.7 (F2) 654 7.7 (F0) 1551
TAP1 Transporter 1, ATP-binding cassette, B 3.5 (F1) 501 6.7 (F0) 1727
IRF7 Interferon regulatory factor 7 1.8 (F0) 810 6.6 (F1) 3243
G1P3 Interferon, alpha-inducible protein 3.0 (F0) 3683 5.9 (F0) 47,559
S100A8 S100 calcium binding protein A8 3.4 (F2) 1526 5.8 (F1) 3890
IFI35 Interferon-induced protein 35 NAE 5.3 (F1) 1154
IFI16 Interferon, gamma-inducible protein 16 NAE 4.9 (F1) 1004
IFIT3 Interferon induced protein with tetratricopeptide repeats 1 NAE 4.7 (F0) 2054
Other immuno related genes of interest
TLR2 Toll-like receptor 2 3.6 (F0) 881 3.5 (F1) 996
TLR8 Toll-like receptor 8 NAE 3.7 (F1) 636
CLECSF12 C-type lectin, super-family 12 (Dectin-1) 1.4 (F0) 12,139 1.0 (F0) 7372
MYD88 Myeloid differentiation primary response gene 1.9 (F0) 240 0.9 (F0) 665
MAL T-cell differentiation protein 0.5 (F0) 478 0.5 (F0) 566
TRIF TIR domain containing adaptor inducing interferon-beta NAE 4.6 (F1) 321
TRAM1 Translocation associated membrane protein 1 1.1 (F0) 1179 1.3 (F0) 1694
CD14 Monocyte differentiation antigen NAE 23 (F3) 875
PTGS1 Prostaglandin-endoperoxide synthase 1 (COX1) 5.8 (F1) 374 4.1 (F1) 502
CCL13 Chemokine ligand 13 (SCYA13) NAE 11 (F2) 388
CCL22 Chemokine ligand 22 (SCYA22) NAE 2.0 (F0) 563
CXCL13 Small inducible cytokine B13 8.8 (F2) 276 22 (F3) 740
The average channel (red/ green) ratios, as well as the average red channel intensities, of three experiments are offered. The two data sets were
filtered for considerably up-regulated genes, i.e., reporters with ratios above 4 and intensities above 1000. The table is divided into genes
selectively induced by AbM, induced by both AbM and LPS, selectively induced by LPS, and other genes of interest. Data that do not satisfy the
filtering criteria are added in cursive. NAE stands for dnot appreciably expressedT, meaning that none of the channels were above 300 (mean
FG–median BG) in intensity.
Table 2 (continued)
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143138
half below (Fig. 1A). The effect was present in all three
parallels, but neither in those with LPS, nor in any other
arrays from our laboratory. Closer inspection of the arrays
gave no indications that the effect was due to artefacts.
Thus, the split apparently reflected an actual consequence
on gene expression of AbM treatment, presumably in the
form of either a large number of genes being somewhat
repressed, or a different sizeable subset of genes being
slightly activated.
3.2. Up- and down-regulated genes
Both treatments caused considerable down-regulation
of 13 transcripts, of which 5 (CAS2, CTSG, MS4A6A,
MYB and SCGF) were shared. The down-regulated genes
did not appear to converge on particular Gene Ontology
annotations.
In the case of up-regulated transcripts, however, genes
involved in immune processes were highly over-repre-
sented. In Table 2 is listed fold induction, as well as spot
intensities, of relevant immune related genes. Interestingly,
AbM proved to be a particularly potent activator of chemo-
kines, with a 30- to 60-fold increase in the mRNAs for
CXCL1, 2 and 3. Several other cytokines were considerably
up-regulated by both treatments, including IL8,IL1B ,
SCYA4,CCL3 (MIP1-a), IL23A,CCL3L1 and SCY15
(Rantes), while a number of cytokines were selectively
induced by LPS (such as SCYA2,SCYB10,IL6 and
SCYA8). Particularly in the case of LPS, there was also a
tendency towards up-regulation of genes involved in cell
death and apoptosis (data not shown).
The concentration of key cytokines was measured using
ELISA methods (Table 3). As can be seen, in the case of the
two most up-regulated transcripts, IL8 and IL1B, both
showed considerably increased levels of proteins. In the
case of TNF and IL12B, the protein levels appeared to
have increased more than the mRNA levels, in fact, there
was no appreciable expression of IL12B mRNA. IL6
appeared to be slightly induced, particularly in the case of
LPS, while IL10 was not appreciably detected either as
protein or mRNA.
3.3. Cytostatic effects
In order to evaluate the general cytostatic or cytotoxic
effect on the cells, protein and DNA synthesis were mea-
sured (incorporation of respectively [
3
H] leucine and [
3
H]
thymidine). AbM caused a 63% inhibition of DNA synth-
esis, compared to 69% for LPS, and a 30% inhibition of
protein synthesis, compared to 23% for LPS (all values are
mean of two experiments).
4. Discussion
Both LPS and AbM induced a Th1-related inflam-
matory response, as indicated by the increased expres-
sion of IL8,IL1B,IL12B, and TNF (Tables 2 and 3).
None of the typical Th2 response cytokines (IL4, IL5,
IL9, and IL13) were expressed. The results contrast
with a previous report where LPS, but not a hemi-
cellulase-treated agaricus extract, was found to induce
proinflammatory cytokines in murine bone-marrow-
derived dendritic cells [2]. Both the nature of the
extract, and the type of cell, may help explain the
discrepancy. Yet, in order to rule out the possibility
that the present effect of AbM could be due to con-
tamination with LPS, the LPS concentration was
assayed. It was found to be miniscule, i.e., 0.15 ng/
ml of LPS equivalents. In contrast, the LPS prepara-
tion used contained 10 Ag/ml. Moreover, our findings,
as to the capacity of agaricus extracts to induce key
Table 3
Comparison of microarray data with data on expressed proteins
HUGO Gene description AbM LPS
Cytokine vs. control
(pg/ml)
mRNA
(times induced)
Cytokine vs. control
(pg/ml)
mRNA
(times induced)
IL8 Interleukin 8 39,467 (F664) vs. b1500 972 (F416) 24,900 (F2558) vs. b1500 353 (F75)
IL1B Interleukin 1h2154 (F96) vs. 18 (F2) 579 (F208) 1676 (F315) vs. 13 (F4) 407 (F43)
TNF Tumor necrosis factor a7234 (F1580) vs. 54 (F16) 10 (F6) 46,852 (F32466) vs. 46 (F8.8) 26 (F2)
IL12B Interleukin 12 subunit p40 1254 (F582,2) vs. b125 NAE 4525 (F1053) vs. b125 NAE
IL6 Interleukin 6 (interferon, h2) 72 (F20) vs. b32 NAE 178 (F40) vs. b32 17 (F7)
IL10 Interleukin 10 V490 vs. b490 NAE b490 vs. b490 NAE
Both protein and mRNA data are based on changes in expression compared to control (non-treated cells), and offered as the average of three
independent experiments. NAE stands for dnot appreciably expressedT. The symbol bimplies that the result was below the (indicated) detection
limit of the assay.
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143 139
Th1 cytokines, are consistent with those of other
reports [5,31,33]. We therefore assume that the pre-
sent effect of AgM is due to other components than
LPS, of which h-glucans (300 Ag/ml) may be of
particular importance.
While LPS binds primarily to TLR4 (which presum-
ably was present on the cells, although not appreciably
expressed as mRNA), glucans bind to TLR2, dectin-1
(CLECSF12)[30,34,35] and CD11b (ITGAM) [29].
TLR2 was appreciably induced by both AbM (3.3)
and LPS (3.2). CLECSF12 was highly expressed in
these cells, and induced 1.5by AbM while remaining
unchanged in the case of LPS. ITGAM was not appre-
ciably expressed or induced. The reason why TLR4 did
not appear on the arrays may be that the receptor has a
low turnover and therefore does not require continual
gene expression.
The microarray approach presumably allowed for
an evaluation of possible changes in most of the genes
expressed by the cells. A reasonably large fraction of
the mRNAs changed considerably upon treatment
with AbM, and even more genes displayed altered
expression upon the addition of LPS (Table 1). The
more drastic effect of the latter is in line with the
general understanding that TLR4 ligands activate two
or more pathways, including the MYD88 signalling
pathway used by TLR2; and that macrophages
respond more strongly to TLR4 ligands compared to
TLR2 ligands [28].
As may be expected from substances that cause
cellular activation, many more genes were up-regu-
lated compared to down-regulated. Genes belonging
to both groups were analysed as to molecular func-
tion, biological process and cellular component using
the gene ontology based program eGOn (http://
nova2.idi.ntnu.no/egon/). While the down-regulated
genes did not appear to cluster, both AbM and LPS
up-regulated genes clustered highly significantly ( p-
values from 10
5
to 10
29
) to immune related pro-
cesses such as immune response, inflammatory
response, response to pathogen, and response to exter-
nal biotic stimulus. These results substantiate the
validity of the use of microarrays to discern the effect
of immune modulators on gene expression, at least
when looking at genes with a certain level of tran-
script concentration. The main genes involved in the
microbial product based activation of the cells will be
briefly discussed.
MYD88 is assumed to be essential for the signal-
ling of all TLRs with the exception of TLR3 and TLR4
[25].MYD88 was expressed in the cells (Table 2), but
the mRNA concentration was neither high nor appre-
ciably altered. Several MAP kinases typically
involved in the downstream events were also detected
on the arrays, but at low levels and with no obvious
induction. Genes such as these may reflect a limitation
of the microarray approach. That is, genes that need
only to be expressed at low levels even when induced,
e.g., due to a signalling cascade type of effect, may
fall below the mRNA concentration range where
microarrays perform best. Moreover, genes that are
induced only temporarily and have short-lived
mRNAs, are also easily missed. The point is reflected
in the data on the cytokines IL12B and IL6, which
displayed a considerable increase in protein level in
the absence of detectable mRNA expression (Table 3).
As to the NF-kappa-B transcription regulator,
both NFKB1 and NFKB2 were induced by AbM
and LPS, as were the related inhibitors NFKBIA
and NFKBIE (Table 2). The induction of both the
regulator and its inhibitors suggest a fine-tuned reg-
ulation of NFKB-mediated transcriptional control.
Other negative regulators of the TLR-response,
such as IRAKM, SOCS1, NOD2 and TOLLIP
[36], were not appreciably expressed or induced.
However, as will be discussed below, apoptosis
related genes were induced, particularly in the case
of LPS.
IL8 and IL1B were the dominant cytokines to
come out of the activation with either AbM or LPS.
These genes were up-regulated several hundred
folds to high expression levels, and the correspond-
ing proteins were found at high concentrations in
the cell supernatants. Both AbM and LPS also
caused considerable increase in three interesting
chemokine receptor ligand mRNAs: CCL3 (MIP-
1a), CCL3L1, and SCYA5 (Rantes). These ligands
may inhibit HIV replication, possibly by binding to
the HIV uptake receptor CCR5 [37,38], suggesting a
potential role for AbM in the treatment of HIV
patients. It is also interesting to note that AbM
was particularly potent in inducing CXC chemo-
kines, while both treatments induced several CC-
type chemokines. The C-C and CXC chemokines
are well known stimulators of, respectively, mono-
cytes and neutrophil granulocytes.
L.K. Ellertsen et al. / International Immunopharmacology 6 (2006) 133–143140
Although several additional immune related genes
were induced by both preparations (Table 2), there were
notable differences in the effect of the two preparations.
CD14, which is involved in the TLR4 response [39],
was induced 23-fold by LPS, but, as expected, not by
AbM. Other cytokines (e.g., SCYA2,SCYA8,SCYB10,
and IL6) and interferon related genes (e.g., IFI27,
ISG20,IFIT1,IFI44L,IFITM2,IFI35 ,IFI16, and
IFIT3) were also selectively induced by LPS. Notably,
the interferons themselves were not detectably induced.
TRIF, another gene involved in TLR4 signalling [28],
appeared, as expected, to be induced by LPS and not
AbM, but again the level of expression was very low.
Interestingly, AbM was more potent as to the induction
of the key prostaglandin synthesis enzyme cox2
(PTGS2). PTGS1 increased upon both treatments, but
was expressed at low levels.
AbM, but to a less extent LPS, induced RGS1,a
regulator of G-protein signalling. RGS1 is crucial for
the activity of the G-protein-coupled rhodopsin-like
chemokine/cytokine receptor super-family. This family
includes the chemotaxis related receptors for IL8
(CD128), complement C5a anaphylatoxin (CD88),
bacteria-derived formyl peptides (fMLP) and leuko-
trienes (LTB4) [40–43]. Combined with the drastic
up-regulation of IL8, this may suggest that AbM
is involved in eliciting the directional migration of
leukocytes.
The peculiar scatter plots observed in the case of
cells treated with AbM (Fig. 1) testify further to the
different nature of the two treatments. The split in the
scatter plots suggests that most genes were either
(slightly) up- or down-regulated. Alternatively (if one
of the two legs reflects status quo), that half the genes
were either up- or down-regulated. If the split in the
scatter plots was caused by a broad down-regulation of
genes, one might speculate that the effect reflected an
induction of apoptosis or other forms of cell death.
In order to assess a possible cytostatic or cytotoxic
effect, protein synthesis ([
3
H]leucine incorporation)
and DNA synthesis ([
3
H]thymidine incorporation)
were measured. Both AbM and LPS caused an
approximately 25% inhibition of protein synthesis
and 65% inhibition of DNA synthesis. Moreover,
LPS, but to a less extent AbM, significantly induced
genes involved in programmed cell death and cell
growth regulation (as indicated by gene ontology
analyses); and LPS, but not AbM, caused an appreci-
able increase in the number of cells being stained with
the vital dye trypan blue (data not shown). Thus,
cytotoxic or cytostatic effects do not appear to explain
the peculiar microarray scatter plots.
An alternative explanation might be that AbM
caused the cells to differentiate, in which case one
might expect a massif change in gene expression.
However, markers associated with monocyte differ-
entiation, i.e., macrophage receptors such as CD23,
CD25 and CD69, were not induced.
In addition to the annotated genes, several tran-
scripts with no suggested functions were induced to
high levels either by one or by both treatments (for
details, consult data submitted to ArrayExpress). The
elucidation of the function of these genes would be
interesting.
The Th1 response includes both proinflammatory
and anti-inflammatory cytokines, and plays a role in
fighting off infections caused by viruses, bacteria and
fungi, as well as combating cancer. The Th2 response
is presumably designed for parasites, but its activation
is also associated with allergy. The Th1/Th2 paradigm
postulates that stimulation of the Th1 response should
down-regulate the Th2 response [44]. The recent iden-
tification of regulatory cells as a third arm of the
immune response has modified the paradigm [45],
but the Th1/Th2 balance is still of interest. Conse-
quently, AbM may theoretically be used not only to
fight infections and cancer, but also to reduce allergy.
This option is currently being investigated.
Acknowledgements
The present study was supported by The National
Programme for Research in Functional Genomics in
Norway (FUGE) under The Research Council of Nor-
way, and by the Norwegian Asthma and Allergy
Foundation. We would like to thank Marc Gayorfar,
Jan Inge Helseth and A
˚se Eikeset for excellent tech-
nical assistance.
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... For example, significant induction of IL12p40 expression, which is important for driving T-helper cell (Th) 1 differentiation, was detected following treatment with CWC from strains B, C, and D at 24 h. Other studies have also shown that β-glucans stimulate production of macrophage IL-12p40 (Gantner et al., 2003;Rogers et al., 2005;Ellertsen et al., 2006), which promotes cellmediated immunity against Mycobacterium spp. For example, IL-12p40 deficient mice were more susceptible to chronic mycobacterial infection, and a higher rate of mortality was observed after pulmonary infection with M. tuberculosis (Hölscher et al., 2001). ...
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The yeast Saccharomyces cerevisiae and its components are used for the prevention and treatment of enteric disease in different species; therefore, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium ssp. paratuberculosis (MAP). The objective of this study was to identify potential immunomodulatory S. cerevisiae components using a bovine macrophage cell line (BOMAC). The BOMAC phagocytic activity, reactive oxygen species production, and immune-related gene (IL6, IL10, IL12p40, IL13, IL23), transforming growth factor β, ARG1, CASP1, and inducible nitric oxide synthase expression were investigated when BOMAC were cocultured with cell wall components from 4 different strains (A, B, C, and D) and 2 forms of dead yeast from strain A. The BOMAC phagocytosis of mCherry-labeled MAP was concentration-dependently attenuated when BOMAC were cocultured with yeast components for 6 h. Each yeast derivative also induced a concentration-dependent increase in BOMAC reactive oxygen species production after a 6-h exposure. In addition, BOMAC mRNA expression of the immune-related genes was investigated after 6 and 24 h of exposure to yeast components. All yeast components were found to regulate the immunomodulatory genes of BOMAC; however, the response varied among components and over time. The in vitro bioassessment studies reported here suggest that dead yeast and its cell wall components may be useful for modulating macrophage function before or during MAP infection.
... This and other immuno-modulating effects such as increased cytokine production [43,44], upregulation of adhesion molecules on leukocytes [45] and dendritic cell activation [46], are brought about by AbM-and probably also He and Gf-stimulation of monocytes, granulocytes and NK cells via TLR2, and probably dectin-1 and CD11b/18 [47,48]. In vitro Andosan™ stimulation of monocytic cells has shown increased expression of genes related to immune function [49], including the gene for IL-23 in the IL-12 family. This is in line with the current finding of an Andosan™-induced increase in the Th1 cytokine IL-12p70, which we have observed previously ex vivo in spleen cells harvested from Andosan™ treated Balb/c mice in the allergy model [8]. ...
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Background The novel A/J Min/+ mouse, which is a model for human Familial Adenomatous Polyposis (FAP), develops spontaneously multiple adenocarcinomas in the colon as well as in the small intestine. Agaricus blazei Murill (AbM) is an edible Basidiomycetes mushroom that has been used in traditional medicine against cancer and other diseases. The mushroom contains immunomodulating β-glucans and is shown to have antitumor effects in murine cancer models. Andosan™ is a water extract based on AbM (82%), but it also contains the medicinal Basidiomycetes mushrooms Hericeum erinaceus and Grifola frondosa. Methods and findings Tap water with 10% Andosan™ was provided as the only drinking water for 15 or 22 weeks to A/J Min/+ mice and A/J wild-type mice (one single-nucleotide polymorphism (SNP) difference), which then were exsanguinated and their intestines preserved in formaldehyde and the serum frozen. The intestines were examined blindly by microscopy and also stained for the tumor-associated protease, legumain. Serum cytokines (pro- and anti-inflammatory, Th1-, Th2 -and Th17 type) were measured by Luminex multiplex analysis. Andosan™ treated A/J Min/+ mice had a significantly lower number of adenocarcinomas in the intestines, as well as a 60% significantly reduced intestinal tumor load (number of tumors x size) compared to control. There was also reduced legumain expression in intestines from Andosan™ treated animals. Moreover, Andosan™ had a significant cytotoxic effect correlating with apoptosis on the human cancer colon cell line, Caco-2, in vitro. When examining serum from both A/J Min/+ and wild type mice, there was a significant increase in anti-tumor Th1 type and pro-inflammatory cytokines in the Andosan™ treated mice. Conclusions The results from this mouse model for colorectal cancer shows significant protection of orally administered Andosan™ against development of intestinal cancer. This is supported by the finding of less legumain in intestines of Andosan™ treated mice and increased systemic Th1 cytokine response. The mechanism is probably both immuno-modulatory and growth inhibition of tumor cells by induction of apoptosis.
... These effects are probably in part mediated by binding of glucans in the extract to Toll-like receptor 2 [17], the dectin-1 receptor [18], the lectin-binding site of CD11b/18 [19] and possibly CR4 CD11c/18 [20]. As, however, AndoSan TM is an extract of the mushrooms 0 mycelium rather than their fruit bodies, it contains less ß-glucan than anticipated from the published data of ß-glucan content in the fruit bodies [21]. Therefore, action also of other yetnot-identified immunomodulating substances in the extract must be considered to explain the observed effects. ...
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Ingestion of the Agaricus blazei Murill-based mushroom extract AndoSan(™) has been shown in randomized placebo-controlled studies to improve symptoms in Crohn's disease (CD) and ulcerative colitis (UC) and also fatigue and quality of life in the latter patients. The aim was to examine whether this clinical impact of AndoSan(™) intake could be explained by influence on foremost pro-inflammatory cytokines in the patients. Fifty patients with symptomatic UC and CD were randomized and blinded for oral daily intake of AndoSan(™) or placebo. Blood samples taken before (visit 1) and after 21 days' (visit 3) consumption were analyzed for cytokines IL-1ß, IL-2, IL-4-8, IL-10, IL-12-13, IL-17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1ß and TNF-α. Baseline cytokine levels were similar in CD and UC. In CD cytokine levels at visit 1 versus visit 3 were unaltered within the AndoSan(™) - and the placebo groups. Only IL-2 was significantly reduced at visit 3 in the Andosan(™) - compared with the placebo group. However, when combining IL-1ß, IL-6 and G-CSF in the CD patients, there was a significant reduction in the Andosan(™) group from visit 1 to visit 3. In UC levels of IL-2, IL-5 and MIP-1ß were reduced within the AndoSan(™) group. IL-5 was also reduced at visit 3 compared with placebo. Generally, the effect on reduction in systemic cytokine levels by consumption of AndoSan(™) was limited, and supported only marginally anti-inflammatory effects in these patients. Therefore, other explanations behind the clinical anti-inflammatory effects than the contribution of cytokines seem more pertinent, including anti-allergic and anti-oxidant activities. This article is protected by copyright. All rights reserved.
... The experimental setup allowed us to examine changes in gene expression in leucocytes from the patients prior to and after treatment [30]. As might be expected, the changes were less pronounced compared to previous studies looking at similar effects on monocytes treated in vitro [32]. Moreover, the cytokine genes most strongly induced in vitro were not induced in vivo. ...
... Production of inflammatory cytokines and mediators enhances the ability to counteract viral and bacterial or fungal challenge in innate immunity [10]. Furthermore, antiinflammatory activities [16], ability in activating naïve T lymphocytes [17] and even anticancer properties [18] of ␤-glucans have been also concluded from in vitro studies. ...
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The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterization of traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway-based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields.
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Background: The mushroom Agaricus blazei has evoked considerable scientific and practical interest in several fields, especially those linked to its medicinal properties. This review aims to summarize and evaluate the past decade findings related to nutritional and therapeutic uses of A. blazei, with especial emphasis on the most recent discoveries regarding its chemical composition and clinical investigations. Methods: The specialized literature was searched for basic and clinical studies. The main isolated and identified compounds or fractions are described and confronted with their corresponding bioactivities. Results: Basic research of high quality using ex vivo and in vivo conditions are quite abundant in the specialized literature, but ony 17 clinical studies and two case reports were found. A great number of active molecules have been identified, and they can be divided into three categories, (1) hydrophilic small molecules (e.g., phenolics), (2) lipophilic or partially lipophilic small molecules (e.g., agarol) (3) and macromolecules (e.g., β-glucans). At least the following bioactivities can be considered as being supported by experimental evidence: antioxidant activity (in aging or disease), immunomodulation and cell signaling, anti-inflammatory activity, antiparasitic actions, antimicrobial activity, anticancer effects and tumor growth inhibiting effects, antimutagenic activity, hepatoprotection against chemical or viral infection and antidiabetic activity. Conclusion: The amount and quality of the evidence that has been accumulating during the last decade strongly speaks in favor of the health benefits of the ingestion of A.blazei or derived products. However, there are many uncertainties and limitations when attempts are made to extrapolate or to demonstrate their biological effects in the human organism in health or disease. Clearly, more clinical trials, using reliable statistical methods and standardized preparations are needed to establish the efficacy of A. blazei as a therapeutic agent.
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Background Ingestion of AndoSan™, based on the mushroom Agaricus blazei Murill, has previously been shown to exhibit anti-inflammatory effects because of reduction of pro-inflammatory cytokines in healthy individuals and patients with ulcerative colitis. In this randomized single-blinded placebo controlled study we examined whether intake of AndoSan™ also resulted in clinical effects. Methods and Findings 50 patients with symptomatic ulcerative colitis were block-randomized and blinded for oral daily intake of AndoSan™ or placebo for the 21 days’ experimental period. The patients reported scores for symptoms, fatigue and health related quality of life (HRQoL) at days 0, 14 and 21. Fecal calprotectin and general blood parameters were also analyzed. In the AndoSan™ group (n = 24) symptoms improved from baseline (day 0) to days 14 and 21, with respective mean scores (95% CI) of 5.88 (4.92–6.83), 4.71 (3.90–5.52) (p = 0.002) and 4.50 (3.70–5.30) (p = 0.001). Corresponding improved mean scores (±SD) for total fatigue were 16.6 (5.59), 14.1 (4.50) (p = 0.001) and 15.1 (4.09) (p = 0.023). These scores in the placebo group (n = 26) were not improved. When comparing the two study groups using mixed model statistics, we found significant better scores for the AndoSan™-patients. HRQoL for dimensions bodily pain, vitality, social functioning and mental health improved in the AndoSan™ group. There were no alterations in general blood samples and fecal calprotectin. Conclusions Beneficiary effects on symptoms, fatigue and HRQoL from AndoSan™ consumption were demonstrated in this per-protocol study, supporting its use as a supplement to conventional medication for patients with mild to moderate symptoms from ulcerative colitis. The patients did not report any harms or unintended effects of AndoSan™ in this study. Trial Registration ClinicalTrials.gov NCT01496053
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The effects of F III-2-b (Agaricus blazei Murill polysaccharide) with or without 5-fluorouracil (5-FU) on immune responses were investigated in Meth A tumor-bearing and normal mice. The i.p. administration of F III-2-b (10 mg/kg/day × 30) moderately inhibited the growth of Meth A tumor cells implanted s.c. in mice. Development of implanted tumors was strongly inhibited by the combination of F III-2-b and 5-FU. The picryl chloride-induced delayed type hypersensitivity (PC-DTH) response in mice was depressed after the implantation of tumor and treatment with 5-FU. F III-2-b restored the suppression of PC-DTH by 5-FU, but did not increase the PC-DTH of normal mice. F III-2-b not only enhanced the degree of spleen cell-mediated sheep red blood cells (SRBC) hemolysis (quantitative hemolysis of SRBC), the indexes of the spleen and thymus, and the number of spleen cells but also restored the suppressive effect of 5-FU. In the group receiving F III-2-b, the percentages of splenic Thy1.2-, L3T4- and asialo GM1-positive cells were significantly increased as compared with the tumor-bearing mice treated with saline. Furthermore, the L3T4+/Lyt2+ ratio showed a tendency to increase, and the Lyt2+/Thy1.2+ ratio was markedly decreased. These results suggest that the antitumor effect of F III-2-b may be correlated with the changing pattern of the Thyl.2-, L3T4- and asialo GM1-positive cells.
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Subset analysis of splenic lymphocytes using flow cytometry showed that the percentages of Thy1.2-(pan T-cells), L3T4-(CD4, helper T-cells), and Lyt2-(CD8, cytotoxic T-cells) positive cell populations were significantly increased in mice orally administered a hot water-soluble fraction from Agaricus blazei as compared with mice treated only with saline. 13C-NMR data indicates that the main component in the active polysaccharide is the complex of alpha-1,6- and alpha-1,4-glucan, which had already been shown to have anti-tumor activity against Sarcoma 180. It seems that the polysaccharide from Agaricus blazei may be an effective prophylactic, protecting humans against cancer by stimulating lymphocytes such as cytotoxic T-cells.
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The antimicrobial effect of soluble β-1,3-d-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3–48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.
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Some polysaccharide-containing materials were successively extracted from the fruiting bodies of Agaricus blazei with aqueous ammonium oxalate and sodium hydroxide, fractionated, and assayed for antitumor activity. From chemical analyses and n.m.r. data, it was concluded that the most active fraction, FIII-2-b, was comprised of protein and a (1----6)-beta-D-glucan.
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The antitumor activity of a highly branched (1----3)-beta-D-glucan, SSG, purified from the liquid culture filtrate of Sclerotinia sclerotiorum IFO 9395 and its several derivatives were treated in ICR mice bearing Sarcoma 180 cells. SSG was effective by both systemic (intraperitoneal and intravenous) and local (intratumoral) administrations on the solid form of Sarcoma 180 in ICR mice and the mice acquired resistance to subsequent inoculation of Sarcoma 180. However, SSG was not effective on the ascites form Sarcoma 180. The pretreatment of ICR mice with carrageenan suppressed the antitumor activity, suggesting the involvement of macrophages on the antitumor activity. Derivatives prepared from SSG by periodate oxidation/borohydride reduction showed antitumor activity, but those obtained after acetylation, carboxymethylation and hydroxyethylation were less active. From these results, it is suggested that SSG is a useful antitumor glucan which modifies biological responses and can be used as a source for some antitumor derivatives.
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Lentinan, a β-(1-3)-glucan with some β-(1-6)-gluco-pyranoside branchings, has been extracted and purified from Lentinus edodes a most popular edible mushroom in Japan. This substance has been shown to act as an immunostimulating agent through host defense mechanisms as reported by Chihara et al (1, 2). Lentinan exerts it’s antitumor activity on both syngeneic and spontaneous tumors. The cellular mechanisms of antitumor activity have been clarified by Hamuro et al (3), in that lentinan appears to stimulate host defense mechanisms to induce cytotoxic T cells, natural cytotoxicity and/or augmented macrophages against tumor cells. This suggests that lentinan may be effective for patients with malignant diseases. Based on the results of Phase I and II clinical trials conducted by Taguchi et al (4), the administration conditions for lentinan have been determined to be intravenous administration at doses of 0.5 to 1.0 mg/person/day once or twice a week in combination with chemotherapeutic agents for patients with advanced or recurrent cancer. In order to clarify the clinical efficacy of lentinan administration a Phase III randomized control trial has been conducted on patients with advanced or recurrent gastric or colorectal cancer.
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We conducted studies with mice, rats, and monkeys which demonstrated the ability of glucan to induce either nonspecific or specific enhancement of host resistance to infectious diseases. Intravenous pretreatment of mice with glucan significantly enhanced the survival of mice challenged with either Venezuelan equine encephalomyelitis (VEE) virus or Rift Valley fever virus. Pretreatment was beneficial when initiated 3 days before challenge with VEE virus and 7 days before challenge with Rift Valley fever virus. Treatment of mice after VEE challenge did not increase their survival compared with controls. Glucan pretreatment of rats provided increased resistance to both intraperitoneal and low-dose aerosol challenges with virulent Francisella tularensis when the glucan was given intravenously, but not when it was administered intranasally. In contrast, intranasal glucan pretreatment enhanced the survival of mice when they were challenged by aerosol with Pseudomonas pseudomallei, whereas intravenous glucan pretreatment did not increase survival. mice given glucan combined with a marginally immunogenic dose of VEE vaccine were more resistant to homologous virus challenge than were mice given either Freund complete adjuvant plus vaccine or vaccine alone. Similarly, both primary and secondary VEE antibody titers in cynomolgus monkeys given glucan with VEE vaccine were significantly greater than titers in vaccine controls.
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The effects of F III-2-b (Agaricus blazei Murill polysaccharide) with or without 5-fluorouracil (5-FU) on immune responses were investigated in Meth A tumor-bearing and normal mice. The i.p. administration of F III-2-b (10 mg/kg/day x 30) moderately inhibited the growth of Meth A tumor cells implanted s.c. in mice. Development of implanted tumors was strongly inhibited by the combination of F III-2-b and 5-FU. The picryl chloride-induced delayed type hypersensitivity (PC-DTH) response in mice was depressed after the implantation of tumor and treatment with 5-FU. F III-2-b restored the suppression of PC-DTH by 5-FU, but did not increase the PC-DTH of normal mice. F III-2-b not only enhanced the degree of spleen cell-mediated sheep red blood cells (SRBC) hemolysis (quantitative hemolysis of SRBC), the indexes of the spleen and thymus, and the number of spleen cells but also restored the suppressive effect of 5-FU. In the group receiving F III-2-b, the percentages of splenic Thy1.2-, L3T4- and asialo GM1-positive cells were significantly increased as compared with the tumor-bearing mice treated with saline. Furthermore, the L3T4+/Lyt2+ ratio showed a tendency to increase, and the Lyt2+ ratio was markedly decreased. These results suggest that the antitumor effect of F III-2-b may be correlated with the changing pattern of the Thy1.2-, L3T4- and asialo GM1-positive cells.
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The molecular cloning of the C5a receptor places this molecule in the superfamily of G-protein coupled receptors. This superfamily is characterized by the presence of signature motifs including seven hydrophobic domains which span the cell membrane and impart a predicted serpentine topology to the receptor proteins. The identification of other members of this family, including receptors for the chemokines IL-8 and Mip-1/Rantes, thrombin, formyl peptide, and platelet activating factor, provide new tools for understanding structure-function relationships relevant to the inflammatory process. This review focuses on the recent biological studies concerning the ligand C5a and its cellular receptor, the structure/activity relationships so far discerned, signal transduction mechanisms, progress toward identification of receptor antagonists, and some likely directions for future studies. Where appropriate, relevant work on related seven transmembrane segment receptors is discussed.