Clinical Effects and Cytokine Responses from Ingestion of AndoSanTM

in Patients with Ulcerative Colitis and Crohn´s Disease

A Randomized Placebo Controlled Study

Stig Palm Therkelsen

Department of Gastrointestinal and Pediatric Surgery Faculty of Medicine

Oslo University Hospital, Ullevål, Norway University of Oslo

© Stig Palm Therkelsen, 2018

Series of dissertations submitted to the

Faculty of Medicine, University of Oslo

ISBN 978-82-8377-189-3

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Print production: Reprosentralen, University of Oslo.
Table of Contents

Preface and acknowledgements ……….. 3

List of papers ……….. 5
Abbreviations ……….. 6
General introduction ……….. 7
TM
Composition of AbM and the mushroom extract AndoSan ……….. 10
Toxicology and safety of AbM and AndoSanTM ……….. 14
Immunological and clinical effects of AbM and AndoSanTM ……….. 16
The immune response ……….. 19
Immune stimulation by mushrooms ……….. 20
CLRs ……….. 23
TLRs ……….. 24
NLRs ……….. 25
Non-dectin-1 β-glucan receptors (other than TLR2/TLR6) ……….. 25
T cell responses to fungi ……….. 25
Intestinal absorption of β-glucans ……….. 26
Cytokines ……….. 27
Inflammatory bowel disease ……….. 34
Immunopathogenesis in IBD ……….. 35
The gut microbiota and epithelial barrier ……….. 35
Genetics ……….. 36
Innate immunity ……….. 37
Adaptive immunity ……….. 41
Cytokines in IBD ……….. 42
Other factors contributing to the pathogenesis of IBD ……….. 43
Methodological considerations ……….. 45
Aims of the study ……….. 50
General summary ……….. 51
General discussion ……….. 53
Conclusion ……….. 58
Future perspectives ……….. 59
References ……….. 59

2
Preface and acknowledgments
The present work was performed between 2012 and 2017 at the Departments of
Gastrointestinal and Pediatric Surgery, Medicine, and Medical Biochemistry, Oslo
University Hospital, Ullevål. During this period I was a research fellow from 2012–14
at Institute of Clinical Medicine, University of Oslo, followed by a position as registrar
from 2014–17 at the Department of Gastrointestinal and Pediatric Surgery, Oslo
University Hospital, Ullevål, where my work was equally divided between research and
patient care.
This thesis builds upon the work of many others. Dr. Bernardshaw (2005) and Dr.
Førland (2011) did their thesis on the same medicinal mushroom Agaricus blazei Murill
and the mushroom extract AndoSanTM, and their thorough research forms the basis for
my work.
Professor Egil Johnson has been my supervisor. I am forever grateful for his advice,
corrections, and constructive feedback throughout the research process. Without his
support and patience, this project would not have been completed. It has been a
pleasure having him as a mentor with all his academic and surgical experience, together
with his enthusiasm and good mood.
My co-supervisor Professor Geir Hetland has given me great support and has been a
key player in all the aspects of the study, from the very beginning until the end. His
contributions are many, where especially great knowledge in immunology and
constructive feedback in writing the manuscripts has been mostly valuable.
I am grateful for the contribution in this project by chief investigator Dr. Torstein
Lyberg, and the staff, especially Lisbeth Sætre, at the Department of Medical
Biochemistry for technical support and guidance, and to Hans Christian Dalsbotten
Aass for the excellent analysis of cytokines.
An important contribution was all the help with timely recruitment of patients by Dr.
Idar Lygren at the Department of Medicine. A special thank to nurse Gunnhild Seim
who took care of much of the practical work in meeting with the patients.
Professor Leif Sandvik at Oslo Center for Biostatistics and Epidemiology made the
hurdles in the statistical analyses passable and was of great help in the interpretation of
my results. Nihal Pereira made an important contribution in making the database for my
study.

3
 I would also like to thank Dr. Bjørn Atle Bjørnbeth, the previous head of the
Department of Gastrointestinal and Pediatric Surgery, and Dr. Tom Glomsaker the
present head of our Department, for all the support in my academic and surgical career.

And most importantly, special thanks to my dear family - Jannicke, Ida, Silje and
Kaja, for continuously reminding me of the most important things in life.
Without your love and support, I could not have pulled this through.

Oslo, April 2017

Stig Palm Therkelsen

4
List of papers

I Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016).

Effect of a Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSanTM,
on Symptoms, Fatigue and Quality of Life in Patients with Ulcerative Colitis in a
Randomized Single-Blinded Placebo Controlled Study.
PLoS One 11: e0150191. doi: 10.1371/journal.pone.0150191 PMID: 26933886.

II Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016).

Effect of the Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSanTM,
on Symptoms, Fatigue and Quality of Life in Patients with Crohn's Disease in a
Randomized Single-Blinded Placebo Controlled Study.
PLoS One 11: e0159288. doi: 10.1371/journal.pone.0159288 PMID: 27415795.

III Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016).

Cytokine levels after consumption of a Medicinal Agaricus blazei Murill-based
Mushroom Extract, AndoSanTM, in Patients with Crohn's disease and Ulcerative
Colitis in a Randomized Single-Blinded Placebo Controlled Study.
Scand J Immunol. doi: 10.1111/sji.12476 PMID: 27588816.

5
Abbreviations

AMP: antimicrobial peptide

APC: antigen-presenting cell
CLR: C-type lectin receptor
CR: complement receptor
DAMP: damage-associated molecular patterns
DC: dendritic cell
Dectin-1: dendritic-cell-associated C-type lectin-1
EWAS: epigenome-wide association studies
G-CSF: granulocyte colony-stimulating factor
GALT: gut-associated lymphoid tissue
GM-CSF granulocyte-monocyte colony-stimulating factor
GWAS: genome-wide association studies
IEC: intestinal epithelial cell
IFNγ: interferon γ
IL: interleukin
ILC: innate lymphoid cell
M cell: microfold cells
MAMP: microbe-associated molecular patterns
MCP-1: monocyte chemotactic protein-1
MHC: major histocompatibility complex
MIP-1ß: macrophage inflammatory protein - 1ß
MMP: matrix metalloproteinase
NLR: NOD-like receptor
NOD: nucleotide-binding oligomerization domain
NF-ĸB: nuclear transcription factor – kappa B
PAMP: pathogen-associated molecular patterns
PRR: pattern recognition receptor
ROS: reactive oxygen species
TCR: T cell receptor
TLR: Toll-like receptor
TNFα: tumor necrosis factor α
UPR: unfolded protein response

6
General introduction

Mushrooms are macrofungi with a distinctive fruiting body and are large enough to be
seen with the naked eye. Most of the macrofungi belong to the class Basidiomycetes,
but there are also others from the class Ascomycetes. The number of existing mushroom
species in nature is estimated at approximately 10,000, from 550 genera and 80
families, of which about 10% are likely to be edible, and perhaps only 10% of the
named species are known to science [1-4]. Out of these, approximately 700 species
have been found to be medicinally useful [5, 6].
Humans have used mushrooms in their food since ancient times, with the oldest
archaeological record dating from 3500 BC. For many centuries mushrooms were used
as nutrients in the human diet, as agents of fermentation in the production of food and
drink, and finally as medicine [5]. Mushrooms also have a nutritional value as a
potential source of carbohydrates, proteins, amino acids, and minerals.
Medicinal mushrooms and fungi are thought to possess approximately 130 medicinal
functions, including immunomodulation, antioxidant, radical scavenging,
cardiovascular, antihypercholesterolemic, antiviral, antibacterial, antiparasitic,
antifungal, detoxification, hepatoprotective, and antidiabetic effects [7]. Many, if not
all, higher Basidiomycetes mushrooms contain biologically active compounds in
fruiting bodies, cultured mycelium, and cultured broth. Special attention has been paid
to the mushrooms´ bioactive polysaccharides and polysaccharide-protein complexes
described to enhance innate and cell-mediated immune responses in animals and
humans [7]. Modern clinical practice in Japan, China, Korea, Russia, and several other
countries rely on mushroom-derived preparations in the treatment of patients [8-10]. In
Japan, Agaricus blazei Murill (AbM) is used by an estimated 500,000 people and is the
most popular complementary and alternative medicine taken by cancer patients [11].
Nowadays, medicinal mushrooms are used as dietary food and as dietary supplement
products. The world mushroom production was 30 million metric tons in 2012 [12, 13],
and the market of medicinal mushrooms as dietary supplement products is quickly
growing. It has a value of more than 18 billion US dollars per year [14], including use
as “mushroom pharmaceuticals”, natural bio-control agents in plant protection, and in
cosmeceutical industry. Mushrooms are currently evaluated for their nutritional value
and acceptability, as well as for their pharmacological properties. In particular, and

7
most importantly for modern medicine, medicinal mushrooms represent an unlimited
source of polysaccharides (especially β-glucans) and polysaccharide-protein complexes
with immunomodulating properties [10, 15]. Furthermore, higher Basidiomycetes
mushrooms also contain biological high- and low-molecular-weight compounds
(triterpenes, lactones, alkaloids, and other compounds) in fruiting bodies, cultured
mycelia, and cultured broth [10, 15, 16].
Historically, it is a fact that substances for medicinal use have emerged from
components extracted from mushrooms. Prominent examples are the
immunosuppressive drug, cyclosporine A, isolated from the fungi Tolypocladium
inflatum and penicillin isolated from Penicillium notatum [17, 18]. One of the natural
compounds with immunomodulating properties that have attracted considerable interest
are β-glucans, a group of branched glucose polymers [19].
AndoSanTM is an extract prepared from edible, medicinal Basidiomycetes mushrooms,
mainly Agaricus blazei Murill, but it also contains Hericeum erinaceus and Grifola
frondosa, all of which have immunomodulating properties. The commercial dietary
supplement AndoSanTM that we used in this study is a sterilized mixture from the
mycelia of these three mushrooms as described in the following.
Hericium erinaceus (He) (Lion´s Mane Mushroom, Bearded Tooth Mushroom,
Hedgehog Mushroom, Satyr´s Beard, Bearded Hedgehog Mushroom, pom pom
mushroom, Bearded Tooth Fungus, and Yamabushitake (jp.)) has a long history of use
in traditional Chinese medicine, an edible delicacy that is one of the famous four dishes
in China. Recent studies demonstrates antibiotic properties (against MRSA [20],
Salmonella typhimurium [21], Helicobacter pylori [22]), antioxidant [23],
immunoregulatory [24, 25] and anticancer effects [26]. He contains a number of
polysaccharides, such as β-glucan, heteroglucans, heteroxylans, as well as several
cyanthane derivate triterpens known as hericenone and erinacine [27]. He is a good
source of exogenous antioxidants with promising results on oxidative stress-related
neurological diseases, such as Alzheimer´s disease. Erinacines and hericenones
stimulate the release of nerve growth factor in rat brains and cultured nerve brain tissue
[28]. He has free-radical-scavenging activity, including reducing power ability,
chelating effects on ferrous ions, 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) free-radical-
scavenging activity, β-carotene bleaching, and inhibition of lipid peroxidation [29, 30].
Grifola frondosa (Gf) (Hen of the Woods, Ram´s Head, Sheep’s Head, signorina, and
Maitake (jp.)) is one of the most popular mushrooms in traditional Chinese medicine,

8
where it among others has been used as a remedy for pain and inflammation [31].
Extracts from the fruiting body or liquid-cultured mycelium of Gf has been shown to
exert antitumor, antimutagenic, antihypertensive, antidiabetic, hypolipidemic effects, as
well as increased synthesis of collagen in mice [32-35]. The fruiting body of this
mushroom is rich in β-glucans. In a recent in vivo rat model of inflammatory bowel
disease (IBD), oral administration of a water extract of Gf (GFW) for five days (1g/kg
per day) demonstrated suppressed production of TNFα as well as its signaling through
NF-κB leading to the expression of inflammatory chemokines, MCP-1 and IL-8. The
results from this study also indicated that GFW contains strong antioxidant components
that inhibit production of reactive oxygen species (ROS) by TNFα, and thus, ultimately
suppress the TNFα-induced recruitment of leukocytes to epithelial cells, thereby
suggesting GFW as an alternative medicine for IBD [36].
Agaricus blazei Murill (AbM) (fig. 1) is an edible mushroom naturally growing in the
coastal Piedade rain forest area near the city of São Paulo, Brazil, and has for centuries
been utilized as a food ingredient in the normal diet by the local population who were
found to have less prevalent diseases such as atherosclerosis, hepatitis, hyperlipidemia,
diabetes, viral infections and cancer than did neighbouring populations, presumably
owing to constant consumption of AbM in their normal diet [6]. AbM was found to be
particularly rich in different forms of β-glucans, such as β-(1→3)-, β-(1→4)-, and β-
(1→6)-glucans [37, 38]. These glucans, which are an integral part of the cell wall of
mushrooms, exhibit immunomodulatory effects on monocytes, macrophages, and
natural killer (NK) cells [39-41]. In addition to β-glucans, the mushroom´s effect on the
immune system is believed to be due to other biologically active substances like α-
glucans [42], proteoglucans [37], lectins [43], ergosterol (provitamin D2) [44],
riboglucan [45], glucomannan [45], sodium pyroglutamate [46], blazein [47], agaritine
[48], isoflavonoids [49], antioxidant substances [50], anti-inflammatory substances
such as isolated alkaline and aqueous extracts [51], and the steroid 4-hydroxy-17-
methylincisterol (4-HM) [52]. Cellular and animal research has shown that AbM may
stimulate the production of cytokines, such as interferons and interleukins [41]. AbM is
known to have antiviral properties in cell culture [53, 54]. However, except for a brief
pilot study in patients with chronic hepatitis C virus (HCV) infection where AbM
(AndoSanTM) had a small but non-significant reduction in serum HCV levels, the ability
to inhibit viruses in the human body has not been studied [55]. Additional research
suggests that the mushroom has a beneficial effect on cholesterol [56], hyperglycemia

9
and improvement of insulin resistance [56-58], and as an adjuvant for vaccines [54, 59-
61] as well as inhibition of tumor growth and angiogenesis [46, 62].
Taxonomy and origin of AbM have for many years raised controversies among
researchers (Wasser et al. 2002 [63] and 2014 [7] (Agaricus brasiliensis), Kerrigan
2005 [1] and Wisitrassameewong et al. 2012 [45] (Agaricus subrufescens)). Hence, this
species is found under different names but is most frequently referred to as Agaricus
blazei Murill (sensu Heinemann). Taxonomically this mushroom is classified to the
kingdom of Fungi, division Basidiomycetes, order Agaricales, family Agaricaceae,
genus Agaricus. AbM is also known as Royal Sun Agaricus, Himematsutake (jp.),
Kawariharatake, Agaricus rufotgulis, Songrong, Cogmelo de Dos, and almond
mushroom. Agaricus blazei Murill was introduced to the health food market in Japan in
the 1960s and effects of AbM and other Basidiomycetes mushrooms such as He [64]
and Gf [65] have received an increasing research effort [38].

Inflammatory bowel diseases like ulcerative colitis (UC) and Crohn´s disease (CD)
are bothersome conditions of unknown etiology. We have shown in a previous pilot
study anti-inflammatory effects by ingestion of AndoSanTM in IBD patients as
measured by reduction in pro-inflammatory cytokines and also of fecal calprotectin
[66]. On this background, the main task of this study was to determine whether the
effect of AndoSanTM could be reproduced clinically and by measurement of cytokines
in a prospective and randomized study.

Composition of AbM and the mushroom extract AndoSan™

The mushroom extract AndoSanTM was provided by the company Immunopharma AS
(organization no. 994924273), Oslo, Norway, and produced by the company ACE CO. LTD.,
Gifu-ken, Japan. The commercial manufacturing processes of this mushroom extract are GMP
(Good Manufacturing Practices) certified, with established and identified optimal growth
conditions (e.g., substrate, temperature, fermentation, and timing) and extraction processes. Its
complex and time-consuming production process guarantees a formula that is safe and
consistent. It was stored at 4 °C in metal cans and used under sterile conditions ex vivo and
kept sterile until taken by volunteers for in vivo experiments.
The AbM mixed powder contains per 100 g the following constituents (specified by the
manufacturer): moisture 5.8 g, protein 2.6 g, fat 0.3 g, carbohydrates 89.4 g, of which ß-

10
glucan constitutes 2.8 g, and ash 1.9 g. AndoSanTM is a mushroom extract from the mycelia of
three different Basidiomycetes and contains 82.4% from AbM 14.7% from He and 2.9% from
Gf, and its final concentration was 340 g ⁄ l. The amount per liter of the extract was for sodium
11 mg, phosphorus 254 mg, calcium 35 mg, potassium 483 mg, magnesium 99 mg and zinc
60 mg. The LPS content of AndoSanTM was found, using the Limulus amebocyte lysate test
(COAMATIC Chromo-LAL; Chromogenix, Falmouth, MA, USA) with detection limit 0.005
EU ⁄ ml (1 EU = 0.1 ng ⁄ ml), to be a minuscule concentration of <0.5 pg ⁄ ml. The
concentrations of heavy metals were conformable with strict Japanese regulations for health
foods. AndoSanTM had been heat-sterilized (124 °C for 1 h) by the producer and quality
controlled by an independent company, Meiji Co, Japan.
The fruiting body of AbM (fig. 2) is particularly rich in proteoglucans and different forms of
the ß-glucans [37, 38]. The main structure of ß-glucans in AbM is a β- (1→3)-backbone with
β- (1→6)-side branches. Differences in biological activity of β-D-glucans could be correlated
to solubility in water, the size of the molecules, branching rate and form, and the β- (1→6)
bounding system in the β- (1→3) major chain [67]. Kept together by hydrogen bounding, the
three β- (1→3)-D-polymers with β- (1→6)-D-branches form a triple helical structure that
links covalently to chitin in the cell wall, forming an insoluble complex in an alkaline milieu
(fig. 3) [68]. A reduction in pH alters this triple confirmation into a single helical and random
coil structure [69]. The host’s immune responses to biological response modifiers (BRM),
such as β-glucans, are related to their structural composition.

11
Figure 1. The medicinal mushroom, Agaricus blazei Murill

Figure 2. The basic structure of β-glucans of AbM

β-Glucans comprise a major component of many fungal cell walls and occur mainly in linear (β-(1→3)) or
branched (β-(1→6)) forms. This figure is a modified version of two figures (used with permission) from Dag T
Førlands thesis “Studies on a medicinal Agaricus blazei Murill based mushroom extract” (2011).

12
Figure 3. Structure of the outer wall of fungi including β-glucans and receptors

The figure shows the principal layers from the cell wall of mushrooms with key receptor-ligand interactions, also
including receptors with unknown ligands. CR3, complement receptor 3; DC-SIGN, DC-specific ICAM-3-
grabbing nonintegrin; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN, interferon; IL,
interleukin; MR, mannose receptor; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; TNF,
tumor necrosis factor. Reprinted from the International Journal of Medicinal Mushrooms with permission from
Begell House, Inc. [70].

An antiallergic effect of AndoSanTM has been demonstrated in a previous study as measured

by reduction of IgE levels in mice sensitized with the allergen ovalbumin (OVA). When
AndoSanTM prior to oral ingestion was dialyzed against a membrane with a cut-off of 12.5
kDa, the observed reduction in specific IgE antibodies in serum was rendered not statistically
significant [71]. Hence, small molecular substance(s) in AndoSanTM contribute(s) to its anti-
allergy effect(s) [71]. Such substances are most probably not β-glucans, both because they are
usually bigger molecules and because it has previously been shown that β-glucan from yeast
rather had a positive adjuvant effect on OVA sensitization in the very same allergy model in
mice [71]. Therefore, these low-molecular-weight substances (yet not identified) may also
contribute to the immunomodulatory effects of AndoSanTM, e.g. by lowering the anti-

13
inflammatory response [71]. After more specific analyses the carbohydrate content was found
to be 2% of the 4.5 mg/ml dry material [72] after lyophilization. Further, the glucan content in
AndoSanTM was less than stated by the manufacturer, with β-glucan 0.1% vs. 2.8% [72]. This
is probably so because the mycelial extract of the three Basidiomycetes contains less
carbohydrate than their respective fruiting bodies. In addition, the carbohydrate profile of the
extract was analyzed with the findings of 26% xylose, 23% glucose, 11% arabinose, and 10%
mannose [72]. Together with researchers at Norwegian University for Life Sciences at Aas,
protein profiles and identification of peptides were done on the digested fractions of
AndoSanTM, with findings of protein concentration of 13 mg/ml [73]. All these proteins,
mainly containing actin, histone H4 and endo-xylanase with molecular weights (MW) of 97,
30 and 14 kDa, respectively, were degraded into peptides and amino acids when exposed to
human gastrointestinal enzymes in vitro [73].
Although several sophisticated methods are available for isolation of AbM, the laboratory
procedures have certain established isolation steps in common [6, 69]. Initially, the dried
mushroom is denaturated using different solvents (e.g. NaOH, EtOH, MeOH, Hexane,
Chloroform) before boiling. Then there is a new round with the use of solvents and freeze-
drying to obtain a precipitate containing polysaccharides that are isolated (e.g. by
chromatography) and tested for biological effect. It is crucial to be aware of that differently
available AbM extracts exhibit different effects, as demonstrated in a sepsis study [41] where
most were ineffective because they are prepared by using different mushroom strains and
subspecies, grown on different substrates and by different protocols. Previous reports show
that there are various compositions of β-glucans in AbM extracts [74] and the concentration
of active ingredients in each component depends on the methods of extraction [49, 75] and on
the substrate (rotting woods) they are grown on.

Toxicology and safety of AbM and AndoSanTM

The Department of Cancer Research and Molecular Medicine, Norwegian University of
Science and Technology, Trondheim, Norway, has previously investigated AndoSanTM for in
vitro inhibitory potential on P-gp-mediated transport of digoxin in the Caco-2 intestinal cell
line [76]. They found inhibition of P-gp in vitro by AndoSanTM in a similar concentration as
for green tea without affecting the viability of the cells [76]. Thus, AndoSanTM may interact
with P-gp substrates such as vinblastine anticancer agent, digoxin cardiac agent and
cyclosporine immunosuppressive agent [77] and loperamide antidiarrhea agent – hence, it

14
should not be given to individuals using such drugs. AndoSanTM and AbM should also not be
given together with other P-gp inhibitors such as verapamil and quinidine. AndoSanTM was
also tested for in vitro inhibition of cytochrome P-450 (CYP3A4 isoform) metabolism and
found to have an inhibitory effect, but 20 times less than did green tea [78]. The P-450
enzyme is involved in the metabolism of 50% of drugs [79] and is therefore also of interest
for drug-herb interactions. Researchers Engdal and Nilsen concluded, “although Agaricus
inhibited CYP3A4 metabolism in vitro, clinical relevant systemic or intestinal interactions
with CYP3A4 were considered unlikely” [78].
Depending on the manufacturer, accumulation of unwanted harmful chemicals in the dried
mass from fruiting body and mycelium vary substantially. Cultivated mushrooms may
generate toxic compounds from non-toxic substrates, like the hydrazine-derived substance
agaritine, which makes up approximately 1% of the dried mass of the fruiting bodies [80, 81].
AbM has been studied and assessed for possible side effects of agaritine and its derivates [82,
83] as these are suspected to be genotoxic and possibly carcinogenic or tumorigenic agents.
This molecule is thought to be capable of binding to the DNA of organs after administration
to mice models [84]. The genotoxicity of agaritine is, however, very limited. To date, such
effects of AbM have not been demonstrated [84].
Several tests from Japan Food Research Laboratories (authorized by the Japanese
Government) were done in March 2012, December 2013, October 2014, April 2015 and
February 2016. The tests were for pH, arsenic, lead, cadmium, tin, aerobic plate count,
coliform bacteria (MPN), viable molds count, viable yeasts count, mesophilic aerobic spores,
refractometric brix degree and specific gravity (15°C) – and all of the results were within the
recommended quantitation limits. AndoSanTM also passed the water quality test (acceptable
levels of bacteria, as well as ions, pH, taste, colour and odor). An accelerated aging test
(within four months) was performed with an almost unchanged character of the mushroom
drink. Since the Fukushima accident in 2011 in Japan, AndoSanTM has also been tested for
radioactivity, with no detection of Cesium-137, Cesium-134 and Iodine-131 (Meiji Co,
Japan). In addition, the Norwegian Food Safety Authorities found no radioactivity in
AndoSanTM (2013, data not shown).
Several human studies [55, 66, 85-90] have demonstrated the safety of AndoSanTM and
AbM when taken orally. In these studies, there were no subjective side effects or adverse
effects on hematological parameters, electrolyte balance, liver, pancreatic and renal function
[78]. Regarding safety, AndoSanTM has also been tested at HLF Sports Science, an Olympic
committee-approved antidoping facility in Oxford, UK, and found by Liquid Chromatography

15
and Mass Spectrometry to be free of any of the 130 illegal substances on the international
antidoping drug list (WADA). It was also found by gas chromatography and mass
spectrometry to be free of steroids, and thus it was cleared for the use by competing athletes
[91].

Immunological and clinical effects of AbM and AndoSanTM

AbM per se and the AbM-based extract, AndoSanTM, have been shown to exhibit multiple
biological effects including antitumor, antiallergic and both pro-inflammatory and anti-
inflammatory effects as reviewed [38, 92]. At the Norwegian Inst. of Public Health, Hetland
et al. [41] compared, in 2003, five AbM-based extracts from main Japanese health food
producers in a lethal Gram-positive pneumococcal infection model in mice. The only AbM
extract with statistically significant decrease in bacteremia and resulting in significant
increase of survival rate was Agaricus Gold Label, later named AndoSanTM, produced by
ACE Co. Ltd., Gifu-ken. [93]. Accordingly, this particular mushroom extract was chosen for
use in further animal and clinical studies.
Several studies have previously demonstrated immunomodulatory effects of glucans,
especially β-glucans, on monocytes, macrophages, and natural killer (NK) cells [39-41]. All
of these cells originate from a common precursor cell found in bone marrow. The influx of
new cells from bone marrow is steady but limited. β-glucans are shown in vivo to stimulate
the production of precursor cells in bone marrow, resulting in a more rapid flow of new cells
into the blood stream and all lymphoid organs [94].
In vitro, AndoSanTM stimulates human monocytes and human vein endothelial cells
(HUVEC) to secrete the pro-inflammatory cytokines IL-1ß, IL-6, IL-8 and TNFα [93], and in
addition, also the chemokine MIP-1β [95] in monocyte-derived dendritic cells. One
mechanism behind these effects is probably mediated by binding of glucans in the extract to
Toll-like receptor 2 [96, 97] as well as to the dectin-1 receptor [98], the lectin-binding site of
CR3 CD11b/18 [99] and possibly CR4 CD11c/18 [100]. Lactosylceramide (LacCer), a
glycosphingolipid receptor in the plasma membrane of many cells, is found to be stimulated
by β-glucans [101]. These receptors also stimulate the cells to release nitric oxide and
hydrogen peroxide in order to kill intruding microbes [102, 103]. The results on cytokine
synthesis in HUVEC were supported by the fact that AndoSanTM-stimulated promonocytic
THP-1 tumor cells [104] demonstrated upregulation of genes for IL-1ß, IL-8, TLR-2 and the
co-operative molecule MyD88, but not for TLR-4. However, in another in vivo study, daily

16
consumption of 60 ml of AndoSanTM for a week in patients with chronic HCV infection had
no effect on expression of these genes in blood cells [55]. Rather, foremost genes associated
with antitumor properties were upregulated. Further, AndoSanTM was found to activate innate
immune cells by inducing NF-κB activation via stimulation of their TLR-2, and also to inhibit
TLR-4-induced NF-κB activation [97]. In this experiment, also the pure mushroom
ingredients contained in this mixed extract were tested in the NF-κB activation assay to
examine which mushroom was most responsible for the all-over stimulatory effect of
AndoSanTM on TLR-2. The observed contributions of He and Gf to NF-κB activation were
equally negligible, although a potential synergetic effect could not be ruled out [97]. Another
commercial mycelial AbM-based extract, obtained from Chang Gung Biotechnology, Taipei,
Taiwan, demonstrated activation of the NLRP3 inflammasome in vitro in a monocytic
leukemia THP-1 cell line, causing caspase-1-dependent IL-1β secretion [105]. The increased
levels of this pro-inflammatory cytokine is important in stimulation of the innate immune
response with the recruitment of phagocytic cells in the defense against tumors, infections and
inflammation [105].
Since AndoSanTM, which is an extract of the mushrooms´ mycelium and not their fruiting
bodies, has recently been found to contain less ß-glucan than anticipated [72], action of other
yet not identified immunomodulating substances in the extract is believed to part-take to
render the observed effects [106]. An example is an isolated polar high-molecular-weight
fraction of AndoSanTM that was found to inhibit the activity of macrophages in vitro of the
tumor-associated and pro-inflammatory protease, legumain (asparaginyl endopeptidase)
[106]. Legumain probably activates pro matrix metalloproteinases and processing of
cathepsins, leading to pro-inflammatory activity [106].
Antitumor effects of AbM have also been reported in mice (e.g. fibrosarcoma, myeloma,
ovarian-, lung-, and prostate cancer), in humans (gynecological cancer and leukemia) and in
vitro in cancer cell cultures [44, 86, 90, 92, 107, 108]. Interestingly, AndoSanTM has recently
been found to inhibit intestinal tumorigenesis in mice, in which also IL-1ß and IL-12 were
elevated [109]. In addition to β-glucan in AbM and AndoSanTM, ergosterol and agaritine also
exhibit antitumor activity, respectively, by oral administration in sarcoma 180 bearing mice
[44] and by induction of apoptosis in leukemic cells [48]. Moreover, isoflavonoids, another
isolated subcomponent of AbM, demonstrated a reduction in blood glucose levels in diabetic
rats [49]. There was also an antiallergic effect in mice sensitized to ovalbumin (OVA), as
demonstrated by the reduction of specific anti-OVA IgE antibodies, both when AndoSanTM
was given before or after the OVA immunization [110]. Additionally, in this allergy model,

17
there was an increase in TH1 relative to TH2 cytokines in spleen cell cultures ex vivo obtained
from the animals treated with AndoSanTM. This finding is in line with the reduced specific
IgE levels in these animals, supporting an antiallergic effect of AndoSanTM, through
engagement of the adaptive immune response. This observation is compatible with the
improvement of the Th1/Th2 imbalance in tumor-bearing and asthma-induced mice, after
ingestion of Agaricus blazei extracts [111]. Extracts of AbM have also been used successfully
as adjuvants in DNA vaccines to improve their efficacy against hepatitis B virus infection and
foot-and-mouth disease [59, 60]. Antiviral activities have also been reported about the AbM’s
mycelia in vitro, but not their fruiting bodies, with inhibition of the toxic effect of Western
equine encephalitis virus on VERO cells in culture [54].
In a human pilot study, with oral intake of AndoSanTM (60 ml/day) over 12 days in eight
healthy volunteers, there was a reduction in intracellular levels of ROS (mainly superoxide
ion) in granulocytes and monocytes in vivo, also supporting an anti-inflammatory effect [112].
In a randomized placebo-controlled clinical study in 57 elderly females, there were no
difference in levels of the chosen cytokines IL-6, TNFα, and IFNγ after daily consumption of
the AbM dry extract (900 mg) or placebo (600 mg) for 60 days [113]. Thus, AbM had no
modulating effect on the levels of these classical pro-inflammatory cytokines. However, in a
placebo-controlled study in 100 patients with gynecological cancer, treatment with AbM
(AbM Kyowa) in addition to chemotherapy was reported to increase NK cell activity in blood
and improved quality of life [86]. In another study with this AbM-based mushroom extract, ex
vivo stimulation of whole blood resulted in a pronounced release of many cytokines being
pro-inflammatory (IL-1ß, IL-6, TNFα), anti-inflammatory (IL-10), chemokines (IL-8, MIP-
1ß, MCP-1, leukocyte growth factors (G-CSG, GM-CSF), pleiotropic (IL-7, IL-17) as well as
of the TH1-type (IFNγ, IL-2, IL-12) and TH2-type (IL-4, IL-5, IL-13) cytokines [85]. In
addition, when eigth healthy volunteers were given AndoSan (60 ml per day) for a 12 day
periode, there was a significant reduction in cytokine levels in plasma of IL-1ß, TNFα, IL-6,
IL-2 and IL-17, whilst levels of the remaining 12 cytokines in the analysis were unaltered,
pointing to an anti-inflammatory effect in vivo, when given by the oral route [85]. Similarly,
as for healthy individuals, 11 patients with CD and 10 patients with UC who likewise
consumed the mushroom extract, cytokine levels were reduced in both untreated and in LPS-
stimulated blood ex vivo [66]. For CD respective reductions in cytokine levels were for IL-2,
IL-8, IL-17 and IL-1ß, MIP-1ß, MCP-1, IL-8, IL-17, G-CSF and GM-CSF, while MCP-1 and
MIP-1ß, IL-6, IL-1ß, IL-8, G-CSF, MCP-1, GM-CSF were reduced in UC. For the UC
patients, the level of fecal calprotectin was also reduced [66].

18
 The divergent results of levels of cytokines ex vivo and in vivo in human pilot studies, also
pointed to a potential anti-inflammatory effect of AndoSanTM when given the oral route, that
deserved further studies.

The immune response

The immune system is divided into the innate and adaptive immune system that protects the
host against a wide range of pathogens. The innate immune response acts in a rapid, non-
specific and conserved manner, and is the dominant system of host defense in most
organisms. The host cells express pattern recognition receptors (PRRs), particularly
represented by macrophages, DCs and NK cells, that sense pathogen-associated molecular
patterns (PAMPs), which makes it possible to discriminate “self” from “non-self” cells.
Importantly, the immune system also senses endogenous alarm or danger signals from
infected or damaged host tissue, many of which signal through the same receptors as do
PAMPs. Different PRRs initiate downstream intracellular events that promote the activation
of the immune system, with the specific immune response generated depending on the cell
type involved. The innate immune system is usually sufficient to fight off most pathogens on
its own but also has the ability to alarm and activate the adaptive immune system when
needed.
The adaptive immune system allows for a stronger immune response as well as
immunological memory, where each pathogen is “remembered” by a signature antigen (Ag).
The leukocytes of the adaptive immune system are the lymphocytes that are divided into B-
and T-cells. The adaptive immune response is Ag-specific and requires the recognition of
specific “non-self” Ag when DCs, mononuclear phagocytes and B cells present them to T
cells. For the activation of the adaptive immune system, Ags must be internalized and
processed by Ag-presenting cells (APCs; dendritic cells (DCs), macrophages and B cells). In
lymph nodes, the Ag is presented to T cells together with a peptide of the major
histocompatibility complex (MHC) class I or II. Then, clonal expansion of activated
lymphocytes occurs, with the generation of antibody producing plasma cells and helper- and
cytotoxic T cells. In contrast to the innate immune system, the Ag-specific adaptive immune
system needs time to get operational. B cells are involved in the humoral immune response,
whereas T cells are involved in the cell-mediated immune response. There are two major
subtypes of T cells: the cytotoxic T cell and the helper T cell (TH). In addition, there are
regulatory T cells that have a role in modulating the immune response. Killer T cells only

19
recognize Ag coupled to class I MHC molecules, while TH and regulatory T cells (Treg) only
recognize antigens coupled to class II MHC molecules. These two mechanisms of Ag
presentation reflect the different roles of the two subtypes of T cells.
The reference for this chapter, meant as a brief presentation of the immune system, is the textbook “Janeway´s
Immunobiology. 9th ed.” [160].

Immune stimulation by mushrooms

The complex fungal cell wall is the main source of PAMPs that are recognized by PRRs on
mammalian cells [114]. From inner- to outmost, the three layers of the fungal wall are i)
chitin, which is a polymer of N-acetylglucosamine, ii) β-glucans and iii) mannans, which are
chains of mannose molecules coupled to fungal proteins by N- or O-linkages (Fig. 3) [70].
The edible and harmless medicinal mushrooms like AbM, He, and Gf, share PAMPs with
other highly poisonous species, resulting in an effective and rapid engagement of the innate
immune system by pathogen recognition receptor (PRRs) [70]. Such mushrooms and fungi
are usually a health threat due to their action of toxins. β-glucan, which is a major cell wall
component of fungi and has no host-cell analogs, acts as a major PAMP and serves as a key
foreign molecule with immunostimulating and antitumor activities [70]. Representative PRRs
of the innate immune system include Toll-like receptors (TLRs), nucleotide oligomerization
domain (NOD)-like receptors (NLRs), and C-type lectin receptors (CLRs) that detect
invading bacteria, fungi, and viruses, and initiate downstream intracellular events that
promote generation of a specific immune response, depending on the cell type involved.
Mushroom β-glucans activate PRRs expressed on the innate immune cells to protect the host
from invading pathogens [70]. Mammalian non-immune and immune cells, including NK
cells, DCs, monocytes, and macrophages, as well as B- and T-cells, all express TLRs, and
thereby play critical roles in the early innate immune response and also induce adaptive
immunity [115,116]. In addition, fungal β-glucans bind to the dectin-1 receptor in
combination with the dimers of TLR2/TLR4, TLR2/TLR6, or TLR4/TLR6 of macrophages or
DCs, thereby activating adaptive immunity, such as targeted cell lysis and humoral- and cell-
mediated responses [115, 116].
In addition to PAMPs, mammalian PRRs also recognize damage-associated molecular
patterns (DAMPs), mainly being damaged host cell components exemplified by nucleic acids
and alarmins [117]. The PAMP- and DAMP-induced immune responses are coordinated by
the alarmin S100B via the signals from TLRs and the receptor for advanced glycation end-
products (RAGE) [118]. The cross-talk between RAGE and TLRs represents a regulatory

20
circuit in infection, whereby an endogenous danger signal protects the host against pathogen-
induced inflammation and a nucleic acid-sensing mechanism terminates the inflammation
induced by the endogenous danger signal.
In summary, TLRs and CLRs activate multiple intracellular pathways upon binding to
specific fungal PAMPs, including β-glucans, chitin, mannans, β- (1→2)-linked
oligomannosides and fungal nucleic acids. These signals activate NF-κB and the NLRP3
inflammasome, and this culminates in the production of defensins, chemokines, cytokines,
and reactive oxygen species (ROS) [119]. The major signaling pathways, which further will
be described, are elegantly detailed in figure 4.

21
Figure 4. Signaling pathways in innate recognition of fungi

Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) that are
present during fungal infections are recognized by pattern recognition receptors (PRRs). The major PRRs are
Toll-like receptors (TLRs); C-type lectin receptors (CLRs; such as dectin-1 and -2, DC-SIGN, mincle and the
mannose receptor; galectin family proteins (such as galectin 3) and receptor for advanced glycation end-products
(RAGE). TLRs and CLRs activate multiple intracellular pathways upon binding to specific fungal PAMPs,
including β-glucans, chitin, mannans linked to proteins through N- or O-linkages, β-(1,2)-linked
oligomannosides and fungal nucleic acids. These signals activate canonical or non-canonical NF-κB and the
NLRP3 inflammasome, and this culminate in the production of defensins, chemokines, cytokines, ROS and IDO.
Complement receptor 3 and members of the scavenger receptor family (such as CD36) mediate recognition of β-
glucans and the fungal adhesin BAD1 (Blastomyces adhesion 1). After TLR activation, protease-activated
receptors (PARs) sense proteolytic virulence factors and tissue injury and contribute to fungal recognition. In
addition, the alarmin S100B, through the spatio-temporal integration of signals from TLRs and RAGE, allows
the immune system to discriminate between pathogen-derived and endogenous danger signals. By forming

22
complexes with various TLR2 ligands, S100B, and this accounts for its anti-inflammatory activity. However, the
ability of S100B to bind nucleic acids results in the activation of intracellular TLRs that signal through TIR
domain-containing adaptor protein inducing IFNβ (TRIF) and this eventually resolves damage-associated
inflammation through transcriptional downregulation of S100B gene expression. ASC; apoptosis-associated
speck-like protein containing a CARD; BCL-10, B cell lymphoma 10; CARD9, caspase recruitment domain-
containing protein 9; ERK, extracellular signal-regulated kinase; FcRγ, Fc receptor γ-chain; IL, interleukin;
IRF3, IFN-regulatory factor 3; MALT1, mucosa-associated lymphoid tissue lymphoma translocation protein 1;
MYD88, myeloid differentiation primary response protein 88; SYK, spleen tyrosine kinase.
Reprinted with permission from © Nature Publishing group [119].

CLRs
C-type lectin receptors (CLRs) are a large family of proteins that have one or more
carbohydrate-recognition domains. CLRs exist as transmembrane and soluble proteins, and
include type 1; decalektin and mannose receptor (MR), and type 2; dectin-1, dectin-2,
macrophage-inducible C-type lectin (mincle), DC-specific ICAM-3-grabbing nonintegrin
(DC-SIGN), and DC, NK lectin group receptor-1 (DNGR-1) [120]. To protect the host from
fungal infection, various receptors are involved in cytokine secretion and phagocytosis
through N-linked mannan recognition. This includes the MR on macrophages and DCs, DC-
SIGN of DCs [121], the β-galactoside receptor galectin-3 of macrophages [122], the lectin
receptor mincle of macrophages [123], and FcRγ-coupled dectin-2 of macrophages and DCs
[124]. Mincle is not only an essential component of the innate response that protects against
infection by pathogenic fungi but also is a receptor for endogenous DAMPs of damaged
necrotic cells [125]. DC-SIGN is a CLR expressed on the surface of DCs and plays important
roles in orchestrating the adaptive immunity of DCs with T lymphocytes against the bacterial,
fungal, and viral pathogens [126].
The immune system detects infection and tissue damage through PRRs. DNGR-1 is one of
the immune receptors expressed by a small subset of DCs that interacts with damaged cell
components. DNGR-1 is responsible for the recognition of intracellular DAMPs derived from
damaged body components, engulfs necrotic cells and digests them in endosomes [127]. It is
not known whether fungal β-glucans regulate the expression of DNGR-1 on DCs.
Dendritic-cell-associated C-type lectin-1 (dectin-1) exists on many cell types including
DCs, macrophages, monocytes, polymorphonuclear granulocytes and a subset of T cells [128,
129]. Dectin-1 is found abundantly at the portals of pathogen entry (lung and intestine) [130],
and its expression is influenced by various cytokines, chemokines and microbial stimuli
[131]. The expression of dectin-1 is markedly increased by Th2 response cytokines, especially
IL-4 and IL-13, whereas IL-10 and LPS down-regulate this expression [131]. Dectin-1 is the
main PRR that recognizes β-glucans and, following ligand binding, it induces the production

23
of pro- and anti-inflammatory cytokines and chemokines [132], and also plays a dual role in
the internalization and cellular immune responses to β-glucan in macrophages and DCs. The
immune response is achieved through the activation of two distinct signaling pathways
downstream of dectin-1, the SYK/CARD9 (spleen tyrosine kinase - caspase recruitment
domain-containing protein 9) pathway and the MAPK (mitogen-activated protein kinase)
pathway. These pathways mutually interact to activate nuclear factor-κB (NF-κB) for
modulation of cytokine gene expression [133]. The SYK-CARD9 pathway also activates the
NLRP3 (NOD-, LRR- and pyrin domain-containing 3) inflammasome, which causes
proteolytic activation of the cytokines IL-1β and IL-18 by caspase 1 [119]. Furthermore,
recent studies indicate that dectin-1 and TLR2/TLR6 cooperate to induce NF-κB and
inflammatory cytokines such as IL-12 and TNFα in the presence of β-glucan [134, 135].
When TLRs are activated, dectin-1 activation stimulates macrophages and DCs to produce
ROS [136].

TLRs
Toll-like receptors (TLRs) are a family of membrane-spanning proteins that recognize
PAMPs as well as DAMPs at the surface of cells. There are 11 different TLRs found in
humans, TLR1-11. They are the most important sensors of the innate immune system and
signal to the host that a microbial pathogen is present or that tissue damage has occurred. The
TLRs are characterized by an ectodomain with varying numbers of leucine-rich repeat motifs
and a cytoplasmic Toll/IL-1 receptor (TIR) domain that recruits adaptors, such as the myeloid
differentiation primary response protein 88 (MyD88) and TIR-domain-containing adaptor
protein including IFNβ (TRIF) [119]. TLRs -2, -4 and -9 are involved in detecting fungal
components like zymosan, phospholipomannan, O-linked mannans and fungal DNA [137]. A
study in mice has shown that the lack of MyD88 is highly susceptible to infections with
various fungi [138]. This finding support that TLRs are involved in the defense against fungal
infections, although their relative contribution in this battle is still unclear. Presentation of
fungal Ags by DCs are promoted by TLRs, which is a prerequisite for adequate T cell
responses and further processing of both self and non-self components [139].
In addition, during inflammation, host- and fungal-derived proteases induce the presence of
protease-activated receptors (PARs), which belongs to the family of G protein-coupled
receptors (GPCRs) [140]. The stimulation of TLRs by fungi unmasks the divergent roles of
PAR1 and PAR2 in downstream signaling and inflammation. After fungal recognition by
TLRs, PARs become activated to sense proteolytic virulence factors and tissue injury, to

24
mediate pro-inflammatory (PAR1) or anti-inflammatory (PAR2) responses and to modulate
the activity of TLRs. Thus, TLRs regulate PAR signaling and vice versa [140].

NLRs
NLRs are a family of cytosolic proteins that recognize PAMPs and endogenous ligands. The
recognitions of ligands induce a signaling cascade leading to activation of NF-κB, or the
inflammasome, to produce pro-inflammatory cytokines. NLRs are also involved in signaling
for cell death. Although cytoplasmic receptors for fungi have yet to be described, the NLRs
are implicated in sensing fungi and, once activated, these receptors induce production of IL-
1β and IL-18 through the formation of inflammasomes [137, 141, 142].

Non-dectin-1 β-glucan receptors (other than TLR2/TLR6)

Immune cells like NK cells and non-immune cells like EC, alveolar epithelial cells and
fibroblasts, do not express dectin-1 but have an important role in antifungal immunity [143]
and in mediating the protective effects of β-glucans. These cells express complement receptor
3 (CR3, CD11b/18), lactosylceramide (LacCer) and scavenger receptors (ScR) (fig. 3 and 4)
such as CD36, which can recognize certain carbohydrates. Except for the scavenger receptors,
these receptors signal through NF-κB to activate innate immunity and mainly recognize β-
glucan as ligands [144]. The NK cells of the innate immune response are classified as non-
phagocytic large granular lymphocytes, which without activation can damage various target
cells.

T cell responses to fungi

Innate sensing mechanisms are aimed to activate distinct T cells with protective and non-
protective functions against fungi in cooperation with highly adaptive DC subsets [145, 146]
through their PRRs, which lead to different T cell immune responses in infection [119].
Inflammatory DCs initiate antifungal TH17 and TH2 cell responses, whereas tolerogenic DCs
activate TH1 and Treg cells. The different signaling pathways in the DC subsets regulate the
balance between CD4+ effector T cells and Treg cells [119].
A dominant TH1 cell response correlates with protective immunity against fungi [147-150]
and effective fungal vaccines [151]. TH1 cell activation is determined by the DC response to
the combination of TLR- and CLR-specific signals provided by the fungi. The combination of
the synthesis of IFNγ and opsonizing antibodies contribute to the TH1 cell-induced activation
of phagocytes at inflammatory sites [119].

25
 The initial differentiation of naïve T cells to TH2 cells is mainly dependent on IL-4 and IL-
13. The TH17 cells have an important function in the host response against extracellular
pathogens, but they are also associated with the pathogenesis of many autoimmune and
allergic disorders. TH17 cell activation occurs in fungal infections, mainly through the SYK-
CARD9, MyD88 and MR signaling pathways in DCs and macrophages. Inhibition of TH17
cells is mediated by the RAF and TRIF-type I IFN signaling pathways. The activation and
inhibition of TH17 cells are believed to be presented downstream of both CLRs and TLRs
[119].
IDO is a metabolic enzyme that affects the Treg/TH17 cell balance during fungal infections,
by diminishing inflammatory responses through induction of Treg cells and inhibition of TH17
cell development [152, 153].

Intestinal absorption of β-glucans

Mammals enzymatically digest α- but not β-glucans. Therefore, orally administered β-glucans
can reach the small intestine without being degraded. The general notion is that carbohydrates
larger than monosaccharides are not absorbed from the human gut. However, orally
administered β-glucans in rodents have been shown to interact with innate immune cells in the
gastrointestinal tract via two mechanisms [70]. The first mechanism involves microfold cells
(M cells) between the mucosal enterocytes that are responsible for the transportation of the β-
glucans to lymphoid tissue known as Peyers patches, where innate immune cells reside [115,
154]. M cells (see fig. 6) pinocytose particles and transport luminal, soluble macromolecules,
particles, and whole microorganisms to the Payers patch. In situ, macrophages or DCs
recognize transported β-glucans that are internalized and fragmented in the endosome. The β-
glucan fragments are released from the macrophages and taken up by other immune cells,
leading to a cascade of immune responses. The second mechanism involves the intraluminal
pseudopods of DCs [155], where they capture luminal particles like β-glucans, which then are
presented and processed in Peyers patches as already described.
Many medicinal mushroom β-D-glucans have been shown to induce the biological
responses through the lectin-binding site of complement receptor type three (CR3
(CD11b/CD18)) on immune effector cells [156]. β-glucans also interact following binding to
an additional β-glucan receptor dectin-1 in neutrophils, macrophages, DCs and some T-cells,
but not in NK cells [157, 158], in which CR3 probably is the key glucan-receptor. Binding of

26
β-glucans to dectin-1 activates phagocytosis, ROS production, and release of inflammatory
cytokines [158].
Following a single oral dose in rodents of three structurally distinct soluble β-(1→3)-
glucans of molecular weight less than 103 kDa, the glucans were probably internalized by M
cells and DCs prior to rapid detection in the circulation [159]. Based on this study the
possibility for a similar uptake of β-glucans in humans is reasonable to assume. The
internalization of soluble β-glucans is mediated by TLR2 and dectin-1 in granulocytes,
macrophages and DCs, of which the two latter cells also are integrated in gut-associated
lymphoid tissue. This uptake is dependent on TLR2 on IECs that lack dectin-1 [115].
Despite challenges associated with low bioavailability, due to limited intestinal barrier
penetration and possible gastrointestinal degradation, oral administration is still the obvious
and safe delivery route of β-glucans.

Cytokines

Cytokines are a category of small proteins (about 25 kDa) secreted by immune cells, usually
in response to an activating stimulus, and that induce responses through binding to specific
receptors. It is more than 60 different cytokines which can act in an autocrine, paracrine or
endocrine manner [160], important in cell signaling and cross-talk. Cytokines include
chemokines, interferons, interleukins, (growth factors), and tumor necrosis factors. A broad
range of cells produces cytokines, including immune cells like macrophages, B-lymphocytes,
T-lymphocytes and mast cells, as well as endothelial cells, fibroblasts and various stromal
cells. In the following, the 17 different cytokines from studies in this thesis are presented.
Unless otherwise referred to in the text, the following description of different cytokines is based on the textbook
“Janeway´s Immunobiology. 9th ed.” [160].

Interleukin 1ß
IL-1β is a member of the interleukin 1 family of cytokines. IL-1β is a pro-inflammatory
cytokine intensely produced by tissue macrophages, monocytes, fibroblasts and DCs, but also
expressed by B lymphocytes, NK cells and epithelial cells. The exposure of the innate
immune cells to alarmins, which are endogenous molecules, that signal tissue and cell
damage, together with NF-κB, also induces the expression of IL-1β. They form an important
part of the inflammatory response, often in synergy with TNFα [161]. Immunologically
activated T-cells, immune complexes, complement fragment 5a (C5a) and IFNγ can stimulate
IL-1β production. Importantly, IL-1β induce the release of IL-2 that stimulate proliferation of

27
CD4+ T cell, promote growth and differentiation of B-cells, stimulate synthesis of IL-6 and
enhance adhesion between leukocytes and endothelial cells. IL-1β increases the expression of
adhesion factors on endothelial cells to enable transmigration of immunocompetent cells,
including phagocytes and lymphocytes, to inflammatory sites. It also affects the
hypothalamus, raising the body temperature, and thereby acts as an endocrine pyrogen. IL-1β
also causes hyperalgesia, myalgia, arthralgia, vasodilation and hypotension [162]. IL-1β
induces, after binding to the IL-1 receptor (IL-1RI), several transcription factors, especially
NF-κB. IL-1β also stimulates synthesis of acute-phase proteins by the liver [160].

Tumor necrosis factor α

The pro-inflammatory cytokine TNFα is produced mainly by activated macrophages,
monocytes, NK cells and T cells, but also by a broad variety of other cell types, such as CD4+
lymphocytes, mast cells, endothelial cells, and neurons. The primary role of TNFα is in the
regulation of immune cells, where it acts synergistically with IL-1, and exhibits mainly
overlapping effects. TNFα can induce fever, apoptosis, cachexia, inflammation as well as
inhibit carcinogenesis and viral replication. A local increase of TNFα will cause the cardinal
signs of inflammation to occur; calor, rubor, tumor, dolor and functio laesa [160].
Macrophages and T lymphocytes produce large amounts of TNFα that plays a pivotal role in
the pathogenesis of IBD [163]. When TNFα binds to its corresponding receptors (TNFR1 and
TNFR2), three different signaling pathways can be initiated; activation of NF-κB, activation
of the MAPK pathways, and induction of programmed cell death. Furthermore, TNF signaling
in colitis can drive pleiotropic pro-inflammatory effects, including augmented angiogenesis,
the induction of Paneth cell death via necroptosis, the production of matrix metalloproteinases
by myofibroblasts, the activation of macrophages and effector T cells, and the direct damage
of intestinal epithelial cells (IECs) via myosin light chain kinase activation [164-168]. TNFα-
inhibitors, such as infliximab and adalimumab, are crucial in the treatment of IBD.

Interleukin-6
IL-6 is a pleiotropic cytokine with the possibility to act in both a pro- and anti-inflammatory
way. IL-6 is produced by activated monocytes, macrophages, endothelial cells, activated T
cells and liver cells in response to IL-1 and TNFα. This cytokine is important for i) synthesis
of acute-phase proteins in the liver, ii) mucosal IgA production, iii) pathogen-induced
clearance of neutrophils, and iv) end-stage differentiation of B cells. IL-6 can exert pro-

28
inflammatory functions by activating multiple target cells, including APCs and T cells. In
particular, the IL-6-sIL-6R complex prevents programmed cell death (apoptosis) of mucosal
T cells and activates pro-inflammatory cytokine production by these cells [169]. However,
IL-6 may also have important homeostatic functions by stimulating the proliferation and
expansion of IECs [170]. The anti-inflammatory effect of IL-6 can be explained by inhibition
of the pro-inflammatory cytokines TNFα and IL-1, as well as stimulation of the anti-
inflammatory cytokines IL-10 and IL-1 receptor antagonist.

Chemokines
Chemokines (IL-8, MIP-1ß, MCP-1) are inflammatory products with chemotactic and other
leukocyte-activating properties, including trafficking of different leukocyte subsets between
blood, tissues and lymphatics.
IL-8, a member of the CXC (cysteine-x-cysteine) chemokine family, is produced by
macrophages and other cell types such as epithelial and endothelial cells and is an important
mediator in the innate immune response. There are many receptors capable of binding IL-8, in
which TLRs are of importance in the innate immunity. IL-8 induces chemotaxins in target
cells, primarily neutrophils but also other granulocytes, causing them to migrate toward the
site of infection. In addition, IL-8 also induces phagocytosis once they have arrived. It is also
known to be a potent promoter of angiogenesis, together with stimulating the activation and
mobility of T cells, eosinophils, basophils and monocytes. In B cells, IL-8 inhibits IL-4-
induced IgE production.
The CC (cysteine-cysteine) chemokines, such as MIP-1β (macrophage inflammatory
protein-1ß, CCL4) and MCP-1 (monocyte chemotactic protein-1, CCL2), induce the
migration of monocytes and other cell types such as NK cells and DCs and are primarily
produced by activated T cells and DC. MIP-1ß also selectively attracts CD4+ cells but not
CD8+ cells. In addition, MIP-1ß also binds to the chemokine receptor CCR5 on TH1 cells and
macrophages. MCP-1, by binding to CCR2, induces monocytes to leave the bloodstream and
enter the surrounding tissue to become tissue macrophages, together with TH2 cells acting on
T cells, NK cells, basophils and immature DCs. In general, neutrophils are unresponsive to
the group of CC chemokines.

Interleukin 2
The existence of IL-2 has been recognized for over 35 years, and it remains one of the most
extensively studied cytokines. IL-2 exerts a wide spectrum of effects on the immune system

29
and plays crucial roles in regulating both immune activation and homeostasis [171]. IL-2 is
primarily produced by T cells upon activation of the immune system. IL-2 binds to IL-2
receptors and promotes the differentiation of T cells into effector T cells and memory T cells
when the initial T cell is also stimulated by an Ag and stimulated with IL-1, thus helping the
body to fight off infections. The primary function of IL-2 is to stimulate the proliferation of
activated TH1 cells (CD4+/CD8+). This cytokine is also chemotactic on T cells and stimulates
NK- and B-cell proliferation, including antibody production. Unlike other cytokines, IL-2 is
crucial in discriminating between foreign (“non-self”) and “self”, i.e. induction of self-
tolerance.

Interleukin 17
IL-17 is a pro-inflammatory cytokine produced by T-helper cells, TH17 cells, and NK cells,
and is induced by IL-23. The TH17 cells also produce IL-21 and IL-22, which together with
IL-17, acts on epithelial cells and endothelial cells in order to stimulate secretion of IL-6, IL-8
and G-CSF. IL-17 serves as a potent mediator in delayed-type reactions by increasing
chemokine production in various tissues, thereby recruiting monocytes and neutrophils to the
site of inflammation, similar to IFNγ. IL-17 has been demonstrated to act synergistically with
TNF and IL-1 [172]. This activity can also be redirected towards the host and result in
autoimmune disorders that involve chronic inflammation. High levels of IL-17 has for
example been found in the synovial tissue of patients with rheumatoid arthritis. On the other
hand, inadequate levels of TH17 cytokines (IL-17 included) can give excessive inflammation
as seen in autoimmune diseases like rheumatoid arthritis and IBD [173]. Studies in mouse
models of experimental colitis have shown that the absence or neutralization of IL-17A or IL-
17F alone had no effect, or even aggravated disease activity, in a T cell transfer model of
colitis [174]. To date, clinical targeting of TH17 cells in patients with Crohn´s disease (CD)
has been restricted to the use of secukinumab, an IL17A-specific neutralizing antibody.
However, secukinumab treatment has been reported to be ineffective in treating CD and is
associated with higher rates of adverse events than placebo therapy [175].

Interferon γ
IFNγ is mainly produced by NK cells and natural killer T cells of the innate immunity, as
well as by CD4+ TH1 and CD8+ cytotoxic T cells of antigen-specific immunity. Moreover,
IFNγ activates macrophages and induce expression of class II molecules of the major
histocompatibility complex (MHC) on APCs. IFNγ has antiviral, immunoregulatory and

30
antitumor properties [176]. It alters transcription in up to 30 genes producing a variety of
physiological and cellular responses. Regardless the types of interferon, the two major
functions are antiviral activity and antiproliferative activity. It activates macrophages to
synthesize inflammatory cytokines such as TNF, IL-1ß, IL-12 and is also responsible for the
intracellular generation of antimicrobial nitric oxide and ROS. Increased expression of MHC
class I and II are mediated by INF-γ on several cell types. The IFN-γR is expressed in all cell
types except erythrocytes. IFNγ also has a key role in granuloma formation in different
infectious or inflammatory diseases, such as CD, where it activates macrophages so that they
can be more powerful in killing intracellular organisms. Chemokine IL-8 participates in
recruiting monocytic cells to the site of infection. A granuloma is the host´s way of dealing
with substances it cannot remove or sterilize. This is a process, including TH1 cells, IL-1, and
IL-12 that ultimately results in aggregation of macrophages that transform into fibroblast-like
cells walling off the infection or substance. Patients with steroid-refractory ulcerative colitis
(UC) have been treated with recombinant IFNβ1a without therapeutic benefit [177]. In
contrast, several similar patients were successfully treated with a CpG (bacterial motif)-
containing oligonucleotide, which indicates that immunostimulatory approaches to induce
IFNγ production might be useful in IBD therapy [178].

Interleukin 10
IL-10 is an anti-inflammatory cytokine with multiple, pleiotropic, effects in
immunoregulation and inflammation. The cells that produce most of the IL-10 are activated
monocytes, macrophages and TH2 cells. In addition, other cells like DCs, B cells,
eosinophils, mast cells and hepatocytes also synthesize IL-10. The major role of IL-10 is to
reduce inflammatory responses by affecting mainly monocytes, macrophages, neutrophils,
eosinophils and mast cells. The anti-inflammatory effect of IL-10 is for a large part
effectuated by inhibiting pro-inflammatory cytokines by inhibiting NF-ĸB-activated
transcription of genes encoding particularly for TNF, IL-1β, IL-6, IL-8 and IL-12. It also
enhances B cell survival, proliferation, and antibody production. Accordingly, IL-10 has a
crucial role in the reduction of the cytokine release during sepsis as well as inhibition
leukocyte-mediated ROS-dependent killing of microbes. Regarding the immunostimulatory
effect of IL-10, a TH2 response is promoted through inhibition of IFNγ and IL-2 secretion by
TH1 cells. There have been several clinical trials with recombinant human IL-10 treatment in
patients suffering from autoimmune diseases but with no significant effects in patients with
CD [179] or rheumatoid arthritis [180]. This treatment has also shown pro-inflammatory

31
results in another study with CD patients [181]. The two receptors for IL-10, IL-10R1 and -
R2, are expressed mainly on hematopoietic cells.

Interleukin 4
IL-4 induces the differentiation of TH0 into TH2 cells, and TH2 cells subsequently produce
additional IL-4 in a positive feedback loop. Moreover, IL-4 produced by activated TH2 cells
may amplify TH2 development from naive T-cell precursors. IL-4 is a strong pleiotropic TH2
cytokine, synthesized by CD4+ T cells and to a lesser degree by basophils and mast cells.
This cytokine influences all types of cells through interaction with IL-4R. IL-4 stimulate the
differentiation of B cells into plasma cells and the proliferation T-cells. IL-4 induces B-cell
synthesis of IgE and IgG1 and up-regulates MHC class II production. IL-4 also stimulates
IL-12 synthesis in macrophages and DCs, which functions as a negative feedback mechanism
for the TH2 response. IL-4 decreases the production of TH1 cells, inhibiting production of
IFNγ and IL-1β. Macrophages play a major role in chronic inflammation and wound repair.
Increased tissue IL-4 shifts the differentiation of macrophages from M1 into M2 cells, also
called repair macrophages. Combined with increased synthesis of IL-10 and TGFβ, the M2
cells reduce pathological inflammation and promote wound repair and fibrosis. IL-4 upon
stimulation exerts degranulation and proliferative effects on mast cells. Generally, the effects
of IL-4 are antagonistic to those of IFNγ.

Interleukin 12
It is primarily synthesized by macrophages, but to some extent also by neutrophils, DCs,
monocytes and B cells. IL-12 plays a key role in the immune response by linking
macrophages and DCs and thereby activating them for microbial ingestion. The innate
response is activated through an NK cell activation, which further differentiates naive TH0
cells into TH1 cells. The increased levels of these cytokines are important to exhibit an
adequate host defense against intracellular pathogens (e.g. mycobacteria). IL-12 also reduces
IL-4-mediated suppression of IFNγ, which contributes to an antiangiogenic effect. The result
of the IL-12-mediated TH1-cell response is increased levels of antibodies IgG2a, IgG2b and
IgG3, but not the TH2 associated antibodies IgG1 and IgE. Since CD has been considered a
TH1-cell disorder, IL-12 may contribute to the pathogenesis of this disease. The IL-12
receptor (IL-12R) is more densely expressed on activated T- and NK-cells, compared with
DCs and B cells.

32
Other cytokines (IL-5, IL-7, IL-13)
IL-5 is especially pertinent for the differentiation, activation and chemotaxis of eosinophils.
Other crucial functions of IL-5 is i) histamine release from mast cells, ii) IgA synthesis in B
cells, and iii) development of cytotoxic T lymphocytes. This cytokine is primarily
synthesized by TH2 cells, but in a smaller scale by activated mast cells, eosinophils, NK cells
and B cells.
IL-7 stimulates the development of B- and T-cells and also contributes to the formation of
memory T cells. The main source of IL-7 production is from stromal cells in the bone
marrow and thymus, but also by keratinocytes, DCs, epithelial cells and neurons, but not by
normal lymphocytes.
Activated T cells produce IL-13, in particular, TH2 cells, but also basophils and mast cells.
Except for the differentiation of TH0 into TH2 cells, IL-13 exhibits similar activities as IL-4,
but the response is smaller in magnitude. It inhibits synthesis of pro-inflammatory cytokines
(IL-1ß, IL-6, TNFα and IL-8) in macrophages, but phagocytosis is not blocked. B cell
proliferation and change of isotype to increased synthesis of IgE are stimulated by this
cytokine.

Colony stimulating factors

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-monocyte colony-
stimulating factor (GM-CSF) are mainly produced by stromal cells, mononuclear phagocytes,
endothelial cells, activated T cells and fibroblasts. They are glycoproteins that bind to receptor
proteins on the surfaces of hematopoietic stem cells, thereby activating intracellular signaling
pathways to proliferate and differentiate into a specific kind of blood cell. G-CSF promotes
the development and recruitment of leukocytes and their migration from bone marrow to
blood. GM-CSF promotes development from pluripotent hematopoietic stem cells, into
subsets of leukocytes as well as megakaryocytes and erythrocytes.

33
Inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by a chronic and relapsing inflammation

of the gastrointestinal tract that leads to structural damage of the bowel wall [163, 182, 183].
The main forms of IBD are Crohn´s disease (CD) and ulcerative colitis (UC). About 10% of
the IBD cases are called indeterminate colitis, with an overlapping disease presentation that
makes the CD and UC diagnosis difficult to distinguish. The target in UC is the colonic
mucosa while CD affects all layers of the intestinal wall. In UC, the continuous inflammation
usually originates in the distal colon or rectum, with disease progression in the proximal
direction. In CD, healthy and diseased patches of bowel are often interspersed. The small
bowel can give stenosis with malabsorption and subileus, and the transmural inflammation
can also cause fistulation to other epithelial lined organs. Clinically, IBD patients can suffer
from chronic diarrhea, malabsorption, weight loss, rectal bleeding, abdominal pain, stenosis,
abscesses, and fistula formation, and many patients require surgery over time [184]. In
addition, both CD and UC can be complicated with various extraintestinal manifestations such
as primary sclerosing cholangitis, ankylosing spondylitis, iridocyclitis, pyoderma
gangrenosum, erythema nodosum [184]. Finally, there is an increased risk of colitis-
associated neoplasias in IBD, particularly in patients with ulcerative pancolitis and colonic
CD [185, 186].
The highest occurrence of IBD is reported in Northern Europe and North America, and in
Norway, the adult incidence has been found to be 5.8/105 and 13.6/105 for CD and UC,
respectively [187, 188]. IBD affects children, adolescents, and adults, with a peak incidence
between 15 and 34 years [187, 188]. The diagnoses are confirmed by a combination of
specific clinical, endoscopic, histological, and radiological criteria [187, 188]. The disease
course is highly individual and variable, often with unpredictable periods of symptom flares
and remission. A high proportion of patients are on lifelong medication and in need of
frequent contact with the healthcare system. Many patients do not respond to medical
treatment, where the key treatment goal is mucosal healing on endoscopy [189, 190], and may
also be associated with adverse drug reactions, highlighting the relevance of finding new
targets for IBD therapy. Several studies have also shown a lower health-related quality of life
(HRQoL) [191, 192] and more fatigue [193] in IBD patients when related to the background
population.

34
The pathogenesis behind both CD and UC are multifactorial comprising environmental
changes, more than 200 gene variants, abnormal gut microbiota together with a dysregulated
immune response. Despite a considerable research effort and improved treatment of these
patients, the pathogenesis of IBD is still far from resolved [194].

Immunopathogenesis in IBD
The adaptive immune response has traditionally been considered to play a significant role in
the pathogenesis of IBD. However, advances in genome-wide association studies (GWAS)
and immunological studies have recently moved the focus of IBD pathogenesis on to mucosal
innate immune responses, such as epithelial barrier integrity, innate microbial sensing,
autophagy and unfolded protein response, as central pathogenic pathways in IBD [195]. With
the new knowledge from GWAS, the overall pathogenesis of IBD must be interpreted in a
broader context in which genetics, the microbiota and environmental factors contribute to the
pathological immune response [194]. The “hygiene hypothesis” indicates a correlation
between the decrease in infectious diseases, lack of parasites, use of antibiotics, vaccination
and general improvement in food, water and sanitary conditions with an increase in the
incidence of autoimmune and chronic inflammatory disorders [196]. This finding supports the
fact that the microbiota is fundamental to the “education” of the immune system after birth. A
balanced gut microbiota promotes the differentiation of naive gut DCs into tolerogenic DCs
followed by generation of regulatory (Treg) T cells and the establishment of immune
homeostasis. This understanding is supported by epidemiological studies of IBD,
demonstrating a link between IBD immunopathogenesis and dysbiosis of the gut microbiota
[197].

The gut microbiota and epithelial barrier

The gastrointestinal tract harbours a microbial community estimated to contain more than
1000 bacterial species and a microbial load of about 1013–1014 microorganisms, and
metagenomic studies have revealed an impressive number of microbial genes that powerfully
influence host gene expression [198]. Early environmental exposures, including delivery
mode, milk, food, hygiene and several other factors exert a fundamental effect on shaping the
intestinal microbiota in childhood [199], while in adulthood, the gut microbiota is more
stable. Microbe-associated molecular patterns (MAMPs; including pathogen-associated
molecular patterns, PAMPs) are sensed by innate immune receptors, such as TLRs and
cytosolic NLRs, a process essential to intestinal homeostasis [200, 201]. Abnormalities of the

35
gut microbiota (dysbiosis) are present in both forms of IBD, either quantitatively or
qualitatively [202], and evidence of abnormal gut microbiota in patients with UC has also
been documented, but to a somewhat lesser degree than for CD [203]. The microbial
dysbiosis is a result of complex interactions with environmental factors such as diet, smoking,
infections, and geographical regions as well as genetic modifications associated with IBD
susceptibility gene pathways that include microbiota recognition (CARD15/NOD2 and
TLR4), microbial clearance (autophagy genes - ATG16L1, IRGM), immune response (IL-
23R, JAK2, TNFSF15), and mucosal barrier function (IBD5) [204]. The impaired bacterial
recognition and clearance due to defects in the innate immune response, including neutrophil
dysfunction, allows the entry of microbial species into the epithelial cells and a breach in the
mucosal integrity forming an important hypothesis for IBD pathogenesis [205].
Inadequate acute inflammatory response to intestinal MAMPs could impair innate removal
of antigens and trigger a compensatory adaptive immune response, eventually leading to a
damaging chronic inflammation [206]. Patients with IBD have a compromised mucus layer
and an epithelial surface that is densely coated with bacteria, which may contribute to the
barrier defect observed in IBD [207].
To date, human microbiome studies have mostly focused on bacterial components of the
microbiome, though emerging data suggest that the viral components of the microbiome
(virome) can have a profound impact on the host. The functionality of the virome is not well
defined although recent studies on mice suggest the virome does, in fact, play an important
role [208]. New evidence demonstrates that disease-specific alterations exist in the gut virome
in IBD [209]. This study showed that the IBD gut virome is abnormal. Importantly, the
expansion and diversification of the gut virome were found to be independent of alterations in
bacterial populations.
The research on the human fungal microbial populations (mycobiome) in IBD is of novel
date. Fungal DNA accounts for approximately 0.2% of the mucosa-associated microbiota and
0.3% of the fecal microbiota [210]. Only a few studies have investigated the mycobiome for
IBD patients were data revealed a skewed mycobiome but of unknown clinical relevance
[211, 212].

Genetics
At present (2017), genome-wide association studies (GWAS) have identified 215 IBD-
associated loci that have substantially expanded understanding of the biology underlying these
diseases [213]. In 2012, 163 genetic IBD-loci had been found, and 110 were associated with

36
both forms of IBD, 30 and 23 specific to CD and UC, respectively [214]. In addition, multiple
interactions among gene variants are probably involved in defective microbial processing in
IBD [215]. The concordance rate in monozygotic twins of 10-15% in UC compared with 30-
35% in CD suggests that non-genetic factors may have an even more important role in UC
than in CD [216]. Studies on dysregulated genes in IBD have demonstrated aberrations
concerning intestinal barrier function, epithelial restitution, microbial defense, innate and
adaptive immune regulation, autophagy and endoplasmatic reticulum (ER) stress [217].
A few epigenome-wide association studies (EWAS) on IBD have been performed since
2011, and are believed to play a role in IBD pathogenesis, as in the regulation of the TNFα
and IL-1ß gene expression with different sensitivity to DNA methylation [218]. Epigenetics
refers to the mechanisms that modulate gene expression without altering the DNA sequence.
Epigenetic processes involve DNA methylation, histone modifications that modulate
chromatin structure or noncoding RNAs (ncRNAs) such as microRNAs or PIWI-interacting
(pi) RNAs that are involved in the regulation of the posttranscriptional steps. All these
mechanisms can be affected by exposure to environmental factors and are transmissible from
cell generation to cell generation, but can also be reversed [219].

Innate immunity
The innermost intestinal barrier (fig. 5) is formed primarily of mucus glycoproteins, trefoil
peptides, IgA and antimicrobial peptides (AMPs). Goblet cells generate the mucus layer,
which is essentially devoid of microbes. In IBD patients the mucus layer is compromised and
contain an increased load of mucolytic bacteria [220]. Antimicrobial peptides (AMPs), that
prevents microbial invasion and control the composition of the gut microflora, are secreted by
Paneth cells in the crypts of the small intestine. Also the mucosal epithelial cells produce
AMPs, cytokines and chemokines for mucosal protection against invading microbes [221].
The AMPs are small and mainly cationic proteins [222] with antimicrobial activity against
bacteria, fungi, viruses and protozoa [223]. In mammals, defensins and cathelicidins are the
two major classes of AMPs [224, 225], produced by granulocytes, Paneth cells of the small
intestine (α-defensins) and enterocytes (β-defensins). Paneth cells produce large amounts of
α-defensins and other antimicrobial peptides, such as lysozymes and secretory phospholipase
A2 (sPLA2) [221, 222]. The intestinal barrier integrity in IBD is genetically affected,
including the structure of tight and adherens junctions and desmosomes [226-229], thereby
suggesting that epithelial barrier defects represent a primary pathogenetic mechanism [207].

37
Defective epithelial barrier and increased intestinal permeability have long been observed in
patients with both CD and UC [230].
Behind the pre-epithelial physical defense are key players of the cellular innate immune
system, such as macrophages releasing cytokines for recruitment of neutrophils to generate an
inflammation for the elimination of the pathogen.

Figure 5. Innate immune responses in the gut

Mucous layer and epithelial barrier represent the first line of defense against bacterial invasion. Epithelial cells,
stromal cells and innate immune cells, such as macrophages and dendritic cells (DCs) can sense invading
bacteria through extracellular and intracellular pattern recognition. Innate lymphoid cells (ILCs) are also found
in the human lamina propria where they may contribute to cytokine production and inflammatory cell
recruitment. Reprinted with permission. Copyright © 2013 Elsevier B.V.

The contribution of neutrophils in IBD pathogenesis includes oxidative and proteolytic

damage of epithelial barrier function, tissues and the continuous release of mediators
maintaining inflammation [231]. The first line of cellular defense for killing bacteria and
fungi are neutrophils [232]. Interestingly, neutrophils may also reduce inflammation, for
example by synthesizing lipoxin A4. Impaired secretion of the anti-inflammatory lipoxin A4
has been demonstrated in gut mucosa from patients with [207].
Pivotal functions of dendritic cells are monitoring of the microenvironment, sampling of
antigens to create immune tolerance or develop a host defense pro-inflammatory response.

38
This dual function puts DCs in the unique position of controlling the interaction between
innate and adaptive immunity [233]. Mucosal DCs through interactions with T- and B cells,
intestinal epithelium and stromal cells, are enabled to maintain mucosal homeostasis or induce
inflammation [234]. In IBD, the DCs express increased levels of TLR2 and TLR4 compared
with normal control mucosa [235].
Intestinal epithelial cells (IECs) perform both barrier and signal-transduction functions by
sensing luminal contents through surface receptors and consequently secrete regulatory
products that can create an appropriate immune response in the underlying lamina propria
[236]. IBD-associated IECs are impaired in their capacity of inducing CD8+ T suppressor
cells, suggesting a possible defect in mucosal immunoregulation predisposing to IBD [236].
Autophagy contributes to the degradation and recycling of cytosolic contents and
organelles, as well as to resistance against infection and removal of intracellular microbes
[237]. Defects in autophagy in IBD patients can reduce clearance of bacteria and result in an
increased immune response [238]. Closely related to autophagy and innate immunity,
dysregulation of the unfolded protein response (UPR) may also contribute to IBD
pathogenesis. The unfolded protein response is induced by endoplasmic reticulum (ER) stress
due to an accumulation of misfolded or unfolded proteins within the ER [239].
When there is a mucosal injury or inflammation of the gut wall, surrounding IECs migrate
to the lesion to initiate wound healing, a process is called “epithelial restitution” [240]. This
process is followed by additional steps in wound healing such as cell proliferation, expansion,
migration, and differentiation, and ultimately leads to closure of erosions and ulcerations. This
process is supported by antimicrobial peptides and products released by Paneth cells such as
defensins and REG (regenerating protein family) proteins [183, 241] as well as by mucins that
prevent bacterial translocation and immune cell activation. Several transcription factors are
involved in epithelial regeneration, which controls crypt cell proliferation and IEC
differentiation [242-244].

39
Figure 6. The Intestinal Immune System

Mucus from goblet cells, antimicrobial peptides from Paneth cells, and production of IgA provide additional
protection from luminal microbiota. Innate microbial sensing by epithelial cells, dendritic cells, and
macrophages is mediated through PRRs such as TLRs and NOD proteins. Dendritic cells present antigens to
naive CD4+ T cells in secondary lymphoid organs (Peyers patches and mesenteric lymph nodes), where factors
such as the phenotype of the antigen-presenting cells and the cytokine milieu (TGF-β, and IL-10) modulate
differentiation of CD4+ T-cell subgroups with characteristic cytokine profiles (regulatory T cells, e.g., Treg, and
helper T cells, e.g., TH1, TH2, and TH17), and enterotropic molecules (e.g., α4β7) are induced that provide for gut
homing of lymphocytes from the systemic circulation. These activated CD4+ T cells then circulate to the
intestinal lamina propria, where they carry out effector functions.
Reproduced with permission from [245], Copyright © Massachusetts Medical Society.

40
Adaptive immunity
In the cellular adaptive immune response, TH0 cells can become activated and either
differentiate into TH1, TH2 or TH17 cells. This is an essential process, which contributes to the
clearance of specific pathogens. In particular TH1 cells are essential for the elimination of
intracellular pathogens, TH2 cells are protective against parasites and mediate allergic
reactions, and TH17 may contribute to the clearance of extracellular bacteria and fungi [246,
247]. However, a dysregulated T cell response with abnormal development of activated T cell
subsets may lead to the onset of inflammation by an excessive release of cytokines and
chemokines, which have multiple pathogenic effects on the components of both the adaptive
and the innate branches of the immune system.
On activation, CD4+ T cells secrete a particular set of cytokines that determine the type of
ensuing immune response and the phenotype of the mature effector T cells. CD4+ T cells
activated in the presence of IL-12 and the absence of IL-4 acquire a T helper 1 (TH1)
phenotype, resulting in IFNγ-producing cells effective in the control of intracellular
pathogens. CD4+ T cells activated in the presence of IL-4 acquire a TH2 phenotype,
generating IL-4-, IL-5- and IL-13-producing cells effective in the clearance of parasitic
infections and allergic responses. CD4+ T cell activation in the presence of IL-6, TGF-ß, and
IL-23, results in TH17 phenotype (i.e., IL-17 and IL-6 producing T cells responsible for acute
inflammation and recruitment of granulocytes).
Regulatory T cells (Treg), characterized by the expression of Foxp3, may be defined as T
cells able to suppress TH0 cell proliferation both in vitro and in vivo [248]. Treg cells are
crucially involved in the maintenance of gut mucosal homeostasis by suppressing abnormal
immune responses against the commensal flora or dietary antigens. In particular, Treg cells
exert their function by producing the anti-inflammatory cytokines IL-10 and TGF-β and by
preventing both the activation and the effector function of T cells that have escaped other
mechanisms of tolerance [249]. IBD immunopathogenesis is due, at least in part, to
insufficient suppressor function [250]. Boosting Treg cell function may have beneficial effects
as a target for IBD treatment, a possibility supported by finding that TNF-blockade restores
the suppressive function of Treg cells [249].
CD4+ T cell on activation by bacterial antigen recognition interacts with B cells (CD40)
through their cell-surface molecule CD40 ligand. This interaction results in the activation of B
cells leading to their proliferation and initiation of antibody secretion of IgA and IgM that
protect the epithelial barrier from commensal and pathogenic bacteria [251]. These IgA can
bind and coat the pathogens to neutralize them, thus protecting the intestine against bacterial

41
penetration and infection [252]. Homing pathways coordinate the movement of lymphocytes
into the site of intestinal inflammation. Chemokine receptor and ligand interactions are
essential for the process of migration of lymphocytes from the vasculature to the tissue
microenvironment in inflammation [253].

Cytokines in IBD
The cytokine responses characterizing IBD are key pathophysiologic elements that govern the
initiation, evolution and, ultimately, the resolution of these forms of inflammation [254].
Cytokines not only drive intestinal inflammation and associated symptoms, such as diarrhea,
but many also regulate extraintestinal disease manifestations (for example, arthralgia or
arthritis) and systemic effects in IBD [255, 256]. Furthermore, cytokines seem to have a
crucial role in the pathogenesis of progressive and destructive forms of IBD that are
associated with complications such as intestinal stenosis, rectal bleeding, abscess- and fistula
formation [183, 184].
Until less than a decade ago, CD was designated as a TH1 condition based on an elevated
production of IL-12 and IFNγ, whereas UC was characterized as an atypical TH2 condition
based on an enhanced production of IL-5 and IL-13, but low levels of IL-4. This TH1/TH2
paradigm was later revised with the discovery of IL-17-producing TH17 cells and the interplay
among TH1, TH2, TH17 and Treg cells [257]. The mucosal lymphocytes in CD produce both IL-
17 and IFNγ, redefining this form of IBD as a mix TH1 and TH1/TH17 condition [258],
although the TH1 response is believed to be quantitatively greater and thus more likely to be
the driving force of inflammation [170]. Increased IL-17 expression is found in the mucosa
and serum of most patients with IBD, but is consistently higher in those with CD than those
with UC [259].
Cytokine expression profiles from both forms of IBD mucosa have found increased levels
of IL-1ß, IL-6, and TNFα, together with decreased levels of IL-4. Furthermore, CD and UC
show different cytokine profiles from the mucosa, with elevated levels of IL-2, IFNγ, IL-12
and IL-18 in CD, and IL-5 and IL-13 for UC.
Most data on cytokine profiles in IBD are from mucosal samples. However, in a recent
study by Korolkova et al. (2015), 23 out of 38 cytokines were measurable in serum samples
from 25 and 28 patients with UC and CD, respectively, and with a control group of 30
persons. They found significantly increased levels in serum for the cytokines eotaxin, GRO
and TNFα for UC, and IFNγ, IL-6 and IL-7 for CD, compared with the control group. IL-8

42
was increased for both conditions [260]. Other studies have detected increased serum levels of
IL-6 [87] and TNFα [261] in IBD, while increased MIP-1ß was only found in UC [262].
A general notion is that the extent and degree of disease (acute or chronic) affects the
cytokine levels in the course of IBD [170]. Moreover, the pathological mechanisms driving
the mucosal inflammation are likely to differ between patients, and, in addition, a blockade of
a single cytokine in patients with IBD may lead to the development of alternative
compensatory pro-inflammatory cytokine pathways. This may explain why the attempts of
targeting of a single pro-inflammatory cytokine are not effective in many cases [170].

Other factors contributing to the pathogenesis of IBD

Intestinal fibrosis. The excessive deposition of extracellular matrix (ECM), constitutes a
common complication of IBD leading to stricture formation and obstruction [263]. Fibrosis in
CD is more pronounced and usually transmural [264, 265], but fibrosis also occurs in UC, in
which ECM deposits in the superficial layers of the intestinal wall [266]. Multiple
metalloproteinases (MMPs) are highly expressed in IBD tissues [267], the breakdown of
collagen fibers being mediated by interstitial collagenase (MMP-1) and MMP-2, whereas
fistula formation in CD has been associated with increased expression of MMP-3 and MMP-9
[268].
Angiogenesis is also thought to contribute to the IBD pathogenesis [269] because mucosal
and plasma levels of vascular endothelial growth factor (VEGF)-A are upregulated in IBD
patients. The gut microbiota is also believed to have potential stimulating and inhibiting
contributions in the angiogenesis [270-272], and thereby a therapeutic potential in IBD.
Lymphangiogenesis. The lymphatic vasculature is essential to immune regulation as it is
responsible for draining and removal of antigens and leukocytes from sites of inflammation
[273]. Chronic inflammation may lead to leukocyte retention and reduced antigen processing,
which then promotes lymphangiogenesis and inflammation [274]. In mice, it has been shown
that administration of the pro-lymphangiogenic factor VEGF-C protected against acute and
chronic colitis. This effect was explained by increased recruitment and bacterial clearance by
leukocytes from inflamed tissue to draining lymph nodes [275].
The redox state. The reduction and oxidation (redox) state of the gut depends on the
equilibrium between oxidants, such as free radicals, ROS or reactive nitrogen species, and
antioxidant mechanisms [276]. This redox state affects many signal-transduction pathways,
such as NF-kB signaling and AMP activity [276]. ROS have important antimicrobial activity,
and contribute to intracellular signaling, promoting the production of pro-inflammatory

43
cytokines. Genes within several IBD-associated loci may either regulate ROS production or
protect against oxidative stress. In addition to pro-inflammatory pathways, ROS are also
involved in Treg cell polarization and function [277, 278].

44
Methodological considerations

Ethics
The actual study was approved on April 8, 2011, by the regional ethics committee (REC—
South East Norway, ref. 2011/404) and followed the guidelines of the Helsinki declaration.
The participants were informed and signed a written consent for participation, including the
option of study withdrawal. The patients were recruited and followed up at the Department of
Medicine, Oslo University Hospital, Ullevål, Norway, in the period of June 2012 to May
2014. The study was registered with unique protocol ID AbM2012-IBD and clinical trials gov
ID NCT 01496053 (December 15, 2011). The authors confirm that all ongoing and related
trials for AndoSanTM are registered.

Competing Interests
Two of the authors (GH and EJ) have patent/patent applications and financial interests
relating to the material (AndoSanTM) pertinent to studies in this thesis: i) WO2005065063 A2,
Appl. No.: 10/ 585600, NO- and PCT-filed Jan 2004 and Jan 2005, respectively, by Inventor
Geir Hetland, and ii) NO20090003383, Appl. No.: NO20090003383 20091119, by Inventors
Geir Hetland and Egil Johnson and filed by Applicant Immunopharma AS in Nov 2009 and
financial interest of Geir Hetland as a shareholder in Immunopharma AS of Norway,
commercializing AndoSanTM.

Criteria for inclusion and exclusion of patients

173 patients with CD and 210 patients with UC were phone-interviewed, and those with
simple Crohn´s disease activity index (SCDAI-) and clinical activity index (CAI) score of at
least 3 were given the opportunity to join the study. At the first attendance, SCDAI and CAI
were re-recorded, and criteria for exclusion were pregnancy, biological treatment with
antibodies to TNFα (Adalimumab, Infliximab), daily use of more than 5 mg of prednisolone,
change of medication and/or consumption of mushroom products from two weeks before till
end of the study. (Additional reasons for exclusions of patients are explained in flow charts in
both paper I and II).

Experimental Design and Randomization

This is a single-center randomized two-armed patient-blinded study designed to determine in

45
this setting whether daily oral intake of a mushroom extract, AndoSanTM, in addition to
measuring clinical symptoms, fatigue and quality of life in patients with CD or UC during the
21-day study period, influenced the level of 17 different cytokines, with emphasis on the pro-
inflammatory cytokines. Patient evaluation, fecal tests and blood samples were taken before
(visit 1, day 0), during (visit 2, day 14) and after (visit 3, day 21) consumption of AndoSanTM
(30 ml twice daily), and cytokine assays from visit 1 and 3 were analyzed. The placebo group
was evaluated likewise but received an equal volume of color-like drink with ionized water
containing 0.5 ml per liter of caramel color (E150c) with salt.
Block-randomization was done after the phone interview, with uneven and even numbers
given for AndoSanTM or placebo, respectively. The patients, one by one, were placed in one
pile, and the group affiliations were placed in another pile. The randomization was performed
by combining one selection from each pile, both anonymized. The first author performed the
randomization, enrolled the participants, and assigned participants to interventions. A few
patients were excluded throughout the study by not attending or because of intercurrent
incidents (disease, unexpected life events). Accordingly, a slight imbalance of the study
groups occurred that was corrected for in the latter rounds of randomization. Patients in the
AndoSanTM group and the placebo group self-reported, in writing, at visit 1, 2 and 3 regarding
symptoms, fatigue and health-related quality of life. Patient-derived blood samples and fecal
calprotectin from these visits were also analyzed. All data were stored in a secure database
(Access–Microsoft Office) at a server at Oslo University Hospital, Ullevål, Norway. A study
number anonymized the patients. 50 patients with CD and UC were randomized, respectively,
into 25 and 24 patients in the AndoSanTM group and 25 and 26 patients in the placebo group.

Symptom score
The recruitment of IBD patients was in collaboration with the Department of Medicine, Oslo
University Hospital, Ullevål, and the gastroenterologists recommended the symptom score
questionnaires we chose to use in our studies.
The patient-reported symptom score was for CD the SCDAI, a.k.a. the Harvey-Bradshaw
index [279]. This index was devised in 1980 as a simpler version of the Crohn´s disease
activity index (CDAI) for data collection purposes. Multiple studies have demonstrated that
the SCDAI show a poor correlation with mucosal inflammation [280, 281], but is well
correlated with both fatigue and HRQoL [193, 282]. The simple index is based on five graded
items; general well-being (very well=0, slightly below par = 1, poor = 2, very poor = 3,
terrible = 4), abdominal pain (none = 0, mild = 1, moderate = 2, severe = 3), number of liquid

46
stools per day (1 = 0, 2 = 1, 3–4 = 2, 5–6 = 3, 7–9 = 4, > 9 = 5), abdominal mass (this item
was not examined) and extraintestinal manifestations (arthralgia, uveitis, erythema nodosum,
aphthous ulcers, pyoderma gangrenosum, anal fissure, new fistula, abscess (score 1 per item)).
The symptom score ranges from 0–21. Scores 3–5 meant mild, 6–9 moderate and over 9
severe disease activity. A criterion for inclusion was a score beyond 2. In a review from 2011,
the simple Crohn´s Disease Activity Index was found to be a valid, reliable and responsive
tool for the measurement of CD activity, when compared to the original CDAI [283].
In patients with UC, patient-reported symptom score was a modified version of the Clinical
Activity Index (CAI), including only the four clinical items and adding one item defining
stool consistency (normal = 0, soft = 1, watery = 2) [284]. The modified CAI contained four
self-reported items concerning abdominal pain (score 0–3) and stool with regard to frequency
(0–4), consistency (0–2) and blood (0–3). The fifth item evaluated by the physician dealt with
general well-being (0–3) of the patient. The symptom score ranged from 0–15. There are very
limited studies on the modified CAI, making it somewhat difficult to compare our results with
previous studies done on this parameter. Other symptom scorings on UC, like Simple Clinical
Colitis Activity Index (SCCAI), have shown a good correlation with mucosal inflammation,
fatigue and HRQoL [193, 282]. In a review article by D´Haens et al. [285], investigating six
symptom-based activity scores for UC, including the CAI, they found that virtually none of
the instruments have been validated. All of the activity scores included bowel frequency and
blood, four out of six included abdominal pain and general well-being, and three included
stool consistency. The five items in the modified CAI represent the most frequently used
items in symptom-based activity scores for UC [285].
The symptom score questionnaires, constituting a total of 1500 questions for both CD and
UC in this study, were answered correctly without any missing values.

Health-related quality of life, HRQoL

The short form 36 (SF-36) is a generic HRQoL questionnaire designed to assess functional
status, well-being and general perception of health. The questionnaire has been shown to have
high validity and reliability [286, 287] and is one of the most frequently used generic HRQoL
instruments.
Self-reported health-related quality of life (HRQoL) was assessed with the SF-36 (IQOLA
SF-36 Norwegian version 1.2), which consists of 36 items, of which 35 are grouped into the
following eight health domains: (1) physical functioning (PF), (2) social functioning (SF), (3)
role limitations due to physical problems (RP), (4) role limitation due to emotional problems

47
(RE), (5) mental health (MH), (6) vitality (VT), (7) bodily pain (BP) and (8) general health
perception (GH). Each domain is graded on a scale of 0–100, and the higher the score, the
better the HRQoL. The validity and reliability of the SF-36 form have been demonstrated for
a number of countries including Norway (version 1) [288]. The data were compared with
published norms from 2323 individuals in the general population [288].
In the CD patients, only 30 out of 5400 HRQoL questions were unanswered, and
accordingly, 17 out of 1200 dimensions were lacking. Using a scoring algorithm for missing
data outlined in the SF 36 survey manual, still, 5 out of 17 dimensions involving 3 patients in
the placebo group, could not be included in the results.
In the UC patients, only 11 out of 5400 HRQoL questions were unanswered, and
accordingly, 10 out of 1200 dimensions were lacking. Using a scoring algorithm for missing
data outlined in the SF 36 survey manual, these 10 dimensions could also be included in the
results.

Fatigue
Fatigue, which is expressed as a “persistent feeling of tiredness, reduced energy levels,
reduced muscle strength and cognitive impairment” [289], is recognized as a major concern
for IBD patients [290]. The fatigue questionnaire (FQ) [291] is translated to Norwegian and
validated in a Norwegian reference population [292], and consists of total fatigue (11 items of
graded questions with score 0–3 per question), which is the sum of physical fatigue (7 items)
and mental fatigue (4 items). The respective scores for total, mental and physical fatigue are
0–33, 0–21 and 0–12, and the higher score, the more fatigue. The items of physical (1–7) and
mental (8–11) fatigue were: 1) Do you have problems with tiredness? 2) Do you need to rest
more? 3) Do you feel sleepy or drowsy? 4) Do you have problems with starting things? 5) Are
you lacking in energy? 6) Do you have less strength in your muscles? 7) Do you feel weak? 8)
Do you have difficulty concentrating? 9) Do you have problems thinking clearly? 10) Do you
make slips of the tongue when speaking? 11) How is your memory? Criteria for chronic
fatigue syndrome was a dichotomized score >4 and duration>6 months [291, 292].
The fatigue questionnaires, constituting a total of 3300 questions for both CD and UC in
this study, were answered correctly without any missing values. The data were compared with
published norms from 2323 individuals in the general population [292].

48
Multiplex cytokine assay
We used the multiplex bead-based sandwich immunoassay performed using Bio-Plex xMAP
technology (Bio-Rad, Austin Texas, USA) with a Luminex IS 100 instrument (Bio-Rad,
Hercules, California, USA), powered using Bio-Plex Manager (version 6.0.1) software for
analysis of 17 different cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-
13, IL-17, G-CSF, GM-CSF, IFNγ, MCP-1, MIP-1β and TNFα) following manufacturers’
instructions and detailed elsewhere [293]. These particular cytokines were chosen because
they cover typical pro- (TNFα, IL-1β, -6, - and anti-inflammatory (IL-6, -10), TH1- (IFNγ, IL-
2, -12), TH2 (IL-4, -5, -13) and TH17 (IL-17) type of cytokines and growth factors (IL-7, G-
and GM-SCF) and chemokines (IL-5, -8, MCP-1, MIP-1β) for leukocytes. Briefly, samples
were thawed on ice, vortexed, and then spun down at 10.000xg for 10 min at 4 ◦C before
dilution and further processing. The StatLIA software package (ver. 3.2, Brendan Scientific
Carlsbad, California, USA), incorporating a weighted, five-parameter logistic curve-fitting
method, was used to calculate sample cytokine concentrations. Controls were used in order to
validate interassay variation. One test of each sample (visit 1 and 3) was run on the same
plate, thereby avoiding intra-assay variation. In order to optimize the assay for low-level
detection, the cytokine analysis process included i) a standard point in addition to the vendor’s
recommendation and ii) use of a magnetic plate washer to yield more reliable results. There
was an interassay coefficient of variation, usually from 10-30%, but more than 50% in one
plate in CD. Therefore, the results on within group and intergroup cytokine levels were also
presented as a relative index, using median and range values for each cytokine at visit 3
relative to visit 1 (baseline) where the index was defined as 1 for each cytokine from each
participant. In addition, in order to validate the results the analyses on cytokine levels in
patients with UC were performed a second time, yielding comparable results. Accordingly,
we used the data from the initial cytokine-analyses. The baseline for upper out of range levels
was given as the highest measurable cytokine concentration, while undetectable levels were
set to 0 pg/ml that excluded the possibility of comparing the results by using relative indices.
The number of cytokines in upper out of range levels at baseline for both CD (n=50) and UC
(n=50) were 30% for MIP-1β, but less than 2% for the remaining 16 cytokines.
The lower detection limits (pg/ml) for the 17 cytokines were; IL-1β (0.39), IL-2 (1.79), IL-4
(0.17), IL-5 (0.16), IL-6 (8.25), IL-7 (0.23), IL-8 (33.1), IL-10 (1.79) IL-12 (0.87), IL-13
(0.33), IL-17 (7.61), G-CSF (6.12), GM-CSF (2.32), IFNγ (2.36), MCP-1 (31.24), MIP-1β
(95.1) and TNFα (5.24).

49
Statistical analysis
In paper I and II, the data are presented as the mean and standard deviation or as median and
range values. Paired sample t-test and Wilcoxon test are used for within-group analysis. The
judgment of whether the distributions of the main efficacy variables were so close to the
normal distribution that normality-based significance tests might be used, is based on the
finding in a paper by Fagerland and Sandvik [294]. Mixed models corrected for baseline
values were used for measuring P values between the AndoSanTM and placebo groups, using
visit 1, 2 and 3 with time as a continuous variable. P values below 0.05 were considered
statistically significant. The SPSS statistical program for the social sciences, version 22
(IBM), was used in the analyses.
In Paper III, data are presented as median and range values. Wilcoxon test and Mann-
Whitney test were used for within-group- or inter-group analysis, both within and between
each disease, respectively. P values below 0.05 were considered statistically significant. The
SPSS statistical program for the social sciences, version 23 (IBM) was used in the analyses.
This study on clinical outcome is a follow-up of a previous pilot study [66] in which there
was a reduction of pro-inflammatory cytokines and chemokines in patients receiving the same
daily dose of AndoSanTM, but for 12 days. Prospective differences of 20% between the
experimental and placebo group and assumed standard deviation of 20% for the different
parameters with a significance level of 5% and a power of 90% (ß = 0.10), demanded about
25 patients per randomized arm (calculated in cooperation Oslo Center for Biostatistics and
Epidemiology, Oslo University Hospital).

Aims of the study

Inflammatory bowel diseases like ulcerative colitis (UC) and Crohn´s disease (CD) are
bothersome conditions of unknown etiology. We have shown in a previous pilot study anti-
inflammatory effects by ingestion of AndoSanTM in IBD patients as measured by reduction in
pro-inflammatory cytokines and also fecal calprotectin [66]. On this background, we wanted
to examine whether AndoSanTM, in a placebo-controlled randomized study, improved clinical
parameters and level of cytokines in these patients.

50
General summary

Paper I:
Ingestion of AndoSanTM has previously been shown to exhibit anti-inflammatory effects as
shown by 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 AndoSanTM also resulted in clinical effects.
50 patients with symptomatic ulcerative colitis were block-randomized and blinded for oral
daily intake of AndoSanTM 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
AndoSanTM 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 significantly better scores for the AndoSanTM-patients. HRQoL for
dimensions bodily pain, vitality, social functioning and mental health improved in the
AndoSanTM group. There were no alterations in general blood samples and fecal calprotectin.
Beneficiary effects on symptoms, fatigue and HRQoL from AndoSanTM 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 AndoSanTM in this study.

Paper II:
50 patients with symptomatic CD were randomized for oral daily consumption of AndoSanTM
or placebo for a 21-day experimental period, in this per-protocol study. Patients reported
validated 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
AndoSanTM group (n=25) symptoms improved from baseline (day 0) to days 14 and 21, with
respective mean scores (95% CI) of 5.52 (4.64-6.40), 4.48 (3.69-5.27) and 4.08 (3.22-4.94)
(p<0,001). We found significant improvements in symptom score for both genders in the
AndoSanTM group and no significant changes in the placebo (n=25) group. There were,

51
however, no significant differences between the groups (p=0.106), although a marginal effect
in symptom score for men (p=0.054). There were comparable improvements in physical,
mental and total fatigue for both groups. HRQoL versus baseline were at day 21 improved for
bodily pain and vitality in the AndoSanTM group and for vitality and social functioning in the
placebo group. No crucial changes in general blood samples and fecal calprotectin were
detected. The results from this single-blinded randomized clinical trial show significant
improvement in symptoms, for both genders, in the AndoSanTM group, but no significant
differences between the study groups. The results on fatigue, HRQoL, fecal calprotectin and
blood samples were quite similar compared with placebo. The patients did not report any
harms or unintended effects of AndoSanTM. CD patients with mild to moderate symptoms
may have beneficiary effects of AndoSanTM as a safe supplement in addition to conventional
medication.

Paper III:
Ingestion of the mushroom extract AndoSanTM has been shown in randomized placebo-
controlled studies to improve symptoms of 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 AndoSanTM 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 AndoSanTM 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, IFNγ, MCP-1, MIP-1ß and TNFα.
Baseline cytokine levels were similar in CD and UC. In CD cytokine levels at visit 1 versus
visit 3 were unaltered within the AndoSanTM- and the placebo groups. Only IL-2 was
significantly reduced at visit 3 in the AndosanTM- compared with the placebo group. However,
when combining IL-1ß, IL-6 and G-CSF in the patients with CD, the cytokine levels at visit 3
were significantly lower in the AndosanTM group – versus the placebo group. In UC levels of
IL-2, IL-5 and MIP-1ß were reduced within the AndoSanTM group. IL-5 was also reduced at
visit 3 compared with placebo. Generally, the effect on reduction in systemic cytokine levels
by consumption of AndoSanTM 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 antiallergic and
antioxidant activities.

52
General discussion

Inflammatory bowel diseases like ulcerative colitis (UC) and Crohn´s disease (CD) are
bothersome conditions of unknown etiology. We have shown in a previous pilot study anti-
inflammatory effects by ingestion of AndoSanTM in IBD patients as measured by reduction in
pro-inflammatory cytokines and also fecal calprotectin [66]. On this background, the aim of
this thesis was to determine whether the effect of AndoSanTM could be reproduced clinically
and by measurement of cytokines in a prospective, placebo-controlled randomized study. This
thesis is based on three single-blinded randomized studies on 50 symptomatic UC and CD
patients who ingested 60 ml/day of the AbM-based mushroom extract AndoSanTM or placebo
for a three-week study period. The major findings were improvements in clinical parameters,
especially for the UC patients, both within the AndoSanTM group and when compared with
placebo. However, AndoSanTM did largely not influence the plasma levels of the 17 different
cytokines analyzed throughout the study period. At best, a marginal anti-inflammatory effect
for the CD patients consuming AndoSanTM may be interpreted from the cytokine analysis.
All of the patients, 210 with UC and 173 with CD, interviewed to join this study had been
diagnosed with IBD by a gastroenterologist, at the Department of Medicine at Oslo University
Hospital, Ullevål. The included patients in this per-protocol study were randomized, resulting
in comparable patient characteristics at baseline between the placebo and AndoSanTM groups,
for both CD and UC patients. The randomization was not blinded for the authors, leaving a
possible bias of the result. This is especially true for the first author who was responsible for
the inclusion and randomization of participants, the implementation of the practical aspects of
and in meeting with the patients, and also in the analysis of the results. Reduced compliance
in carrying out the study, with missing or incorrect oral intake of AndoSanTM or placebo, may
be a possible source of error, even though this was not the impression in conversation with
patients during and after the study period of three weeks.
The moderate reductions in the patient-reported symptom scores were significant for
patients in the AndoSanTM group for both CD and UC, but only significant in UC patients
when compared with placebo. However, there was a close to significant p-value (0.054) when
comparing the symptom score for men among CD patients. The reductions in symptom score
in the AndoSanTM group were 23% and 26% in UC and CD patients, respectively.
Interestingly, in the subgroup analysis, there was a significant reduction in stool frequency for
both CD and UC, together with a patient-reported improved stool consistency in UC patients,

53
all in the AndoSanTM groups. No such effects were found in the placebo groups. Deterioration
in stool consistency is probably one of the most frequent and bothersome concerns of
symptomatic IBD.
The second major finding was significant improvements in total, physical and mental
fatigue for the UC patients when compared with placebo. Such effects were not present in CD
patients, where the fatigue scores actually improved more in the placebo group than in the
AndoSanTM group. Obviously, the clinical improvement due to the intake of this mushroom
extract in patients with CD was more limited vs. those with UC. This may partly be explained
by the fact that CD is pan-intestinal and characterized by transmural inflammation
complicated by stenosis and/or development of fistulas, together with a higher rate of
extraintestinal manifestations than in UC. The suggestion that environmental rather than
genetical factors are more important in UC than in CD [216] may partly also explain why
AndoSanTM had a more clinical impact in the former disease.
We compared the fatigue scores with a Norwegian background population [292] with
significant lower fatigue scores for both CD and UC in our study. We also found chronic
fatigue syndrome (CFS) at 40% and 30%, in CD and UC, respectively, compared with about
11% in the background population [292]. Previous studies are inconsistent on this matter
[295-298], with different findings regarding fatigue scores, CFS, and fatigue scores compared
with a background population. Fatigue is particularly problematic during active disease with
prevalence rates reported as high as 86% [299]. Some studies report that between 40% to 83%
of patients continue to experience high rates of fatigue, even during remission [295, 296,
300], that are comparable with levels experienced by oncology patients [301]. Huppertz-
Hauss et al. [193] published in 2017 fatigue scores in a Norwegian population-based cohort of
patients with IBD 20 years after diagnosis (the IBSEN study). The main findings from this
study were significantly higher fatigue scores for both CD and UC patients with active disease
than those with quiescent disease, and CFS was more frequent among IBD patients than in the
reference population [292]. Factors associated with fatigue included self-perceived disease
activity, poor sleep quality, anxiety and depression. Women had higher fatigue scores and
experienced more frequent CFS than men, and they found no statistically significant
difference in mean fatigue scores between IBD patients with subjectively perceived inactive
disease and the reference population. This study was a 20-year follow-up of IBD patients, and
one may speculate if the impact of IBD on fatigue might decrease over time, e.g. due to
improved coping abilities [292].

54
 The third major finding was significant improvements in four out of eight dimensions in the
health-related quality of life questionnaire (SF-36) in the AndoSanTM group in UC patients.
The dimensions were bodily pain, vitality, social functioning and mental health. In addition,
four out of eight dimensions were significantly improved when AndoSanTM was compared
with placebo in UC patients, and these dimensions were physical functioning, role limitation
physical, bodily pain and social functioning. The results were unaltered or worsened in the
placebo group in UC patients. The HRQoL in CD patients showed significant improvements
in bodily pain and vitality in the AndoSanTM group, together with significant improvements in
vitality and social functioning in the placebo group. When comparing the intervention groups,
we found no significant p-values, which consequently led to our conclusion that the effect of
AndoSanTM on HRQoL in CD patients was not different from placebo. It was though
interesting that the dimension bodily pain was significantly improved in the AndoSanTM
group in CD patients, as this is a major concern in active CD.
We compared the HRQoL scores in our study with a Norwegian background population
[292] and found significantly lower scores in seven out of eight health dimensions, for both
CD and UC. The dimensions vitality and general health had high Z-scores (data not shown)
for both CD and UC, indicating a large difference compared with the background population,
which is in line with previous studies [191, 192].
In paper III, we analyzed the cytokine levels for the included patients. We chose to study
cytokine levels in blood instead of gut mucosa for several reasons. In our study protocol, we
planned to do sigmoidoscopy for biopsies and endoscopic evaluation of treatment for the UC
patients, but this was not feasible mainly for practical reasons. Secondly, granted that findings
in blood reflect disease activity, the representativity of a blood sample would surpass the
challenge of hitting the hot spots of disease within the gut mucosa.
The fourth, and final, major finding in this thesis is that oral intake of AndoSanTM
influenced cytokine levels marginally for UC and moderately for CD, supporting a weak
systemic anti-inflammatory effect (paper III). Therefore, the reported beneficial clinical
effects mediated by AndoSanTM for these patients probably also involve other anti-
inflammatory compounds, not examined. In more detail, there was an all-over trend in CD of
lower pro-inflammatory cytokines, chemokines and growth factor analytes after AndoSanTM
compared with consumption of placebo. Since CD is more generalized disease than UC, one
may speculate that the AndoSanTM impact in CD, although clinically less than in UC, also was
more systemic as shown by greater changes in serum cytokines. With the exception of IL-2 in
CD and IL-2, IL-5 and MIP-1β in UC, all other within or intergroup comparisons were

55
insignificant, also when comparing between the diseases. Despite the fact that a moderate
anti-inflammatory effect was present in CD, the more pronounced clinical effect, comprising
symptoms, fatigue and HRQoL, was found in patients with UC. Thus, we were not able to
demonstrate the same degree of anti-inflammatory effect as previously indicated by the
decline in plasma cytokines in the two similar pilot studies [66, 85], without control groups.
Moreover, there was neither a positive nor a negative correlation between dichotomized
symptom scores (≤5 versus ≥6) and cytokine levels. This supported that the anti-inflammatory
effect in CD and UC, translated into the reported clinical effects in these IBD patients [88,
89], also must be explained by other mechanisms, which may not affect the cytokine levels in
blood. Some of these relevant mechanisms are previously described in the literature, and will
be discussed more precisely in the following, and include among others: antioxidant effects
including reduction in ROS, antiallergic effects, local immunological effects in the gut by the
mushroom extract, and beneficiary changes in the gut microbiota.
In a study on healthy volunteers ingesting AndoSanTM [112], there was a reduction in vivo
of ROS mainly reflecting superoxide ions, and again pointing to an anti-inflammatory effect.
However, this result was not demonstrated in the CD patients in the pilot study mentioned
above (data not shown). One explanation for the reduced superoxide anions may be related to
reduction of IL-1ß, because ROS inhibitors have been shown to reduce the synthesis of this
cytokine in macrophages [302].
In addition, a water-soluble polysaccharide isolated from AbM that was given orally for
eight weeks to ovariectomized and osteopenic rats markedly decreased ICAM-1,
cyclooxygenase-2, inducible nitric oxide synthetase and total antioxidant status [303].
Moreover, AbM contains absorbable low molecular weight antioxidant substances [50],
which downregulate the levels of ROS in vitro [304].
Substances such as β-glucan may further be transported by dendritic cells to lymphocytes in
GALT and induce local immune responses there, or systemic if circulated in blood [305, 306].
There also was an antiallergic effect in mice sensitized to ovalbumin (OVA), as demonstrated
by a reduction of specific anti-OVA IgE antibodies, both when AndoSanTM was given before
or after the OVA immunization [110]. Additionally, in this allergy model, there was an
increase in TH1 relative to TH2 cytokines in spleen cell cultures ex vivo obtained from the
animals treated with AndoSanTM. Although it has been speculated that CD is a TH1 (IL-2,
IFNγ, IL-12) – and that UC is a TH2 ((IL-4), IL-5, IL-13)-related disease based on the
TH1/TH2 dichotomy [307], the cytokine data presented in paper III actually show comparable
baseline cytokine levels and therefore do not clearly support this paradigm. This is also in line

56
with previous [66] cytokine measurements where the same cytokines were elevated (TNFα,
IFNγ, IL-2, IL-6, IL-8, IL-12, IL-17, MCP-1, GM-CSF, MIP-1ß) or reduced (IL-7) in CD and
UC compared with healthy individuals. On the other hand, regarding the TH1/TH2 profile,
there actually was a reduction, only, in the TH1 cytokine IL-2 in CD and the TH2 cytokine IL-
5 in UC in the AndoSan™ group, respectively. Since 3 weeks of AndoSan™, in fact, helped
ameliorate symptoms, the TH1/TH2 dichotomy may eventually have merit in IBD.
The inhibitory effect of an isolated carbohydrate fraction of AndoSanTM [106] on the tissue
degrading pro-inflammatory and tumor-associated enzyme, legumain (asparaginyl
endopeptidase) in vitro, which probably activates proMMP and processing of cathepsins, may
also be valid in vivo and also contribute to less pro-inflammatory activity in the patients with
IBD. In fact, in an intestinal tumor model in Min/+ mice, AndoSanTM did both reduce tumor
load and legumain expression in the intestines [109]. Alkaline and aqueous substances
isolated from AbM [51] had when given orally to rats for 1–2 weeks, several anti-
inflammatory effects. These included improved healing of stress-induced ulcers, reductions in
paw edema in the presence of nystatin or Freund's adjuvant, as well as reduced neutrophil
migration to the peritoneal cavity that in part was supposedly related to down-regulating the
immune system by interactions with ß-glucans of the extract [51].
In line with several human studies [55, 66, 85-87], we also demonstrated the safety of
AndoSanTM when taken orally. In these studies, there were no subjective side effects or
adverse effects on hematological parameters, electrolyte balance, liver-, pancreatic- and renal
function. Regarding possible drug interactions, AndoSanTM did less than green tea inhibit the
transmembrane efflux P-glycoprotein (P-gp) pump present in intestines and liver and hence
important for drug absorption and excretion [76]. However, possible interactions may occur
with P-gp substrates, e.g. some anticancer, diarrhea (loperamide) and cardiac (digoxin)
agents, and P-gp inhibitors, e.g. verapamil. When testing AndoSanTM on cytochrome P-450
metabolism, the extract inhibited it, but far less than green tea and clinically relevant systemic
interactions were therefore considered unlikely [78]. In our clinical study, none of the IBD
patients were concomitantly treated with the above-mentioned anticancer-, heart-, or diarrhea
drugs.
Moreover, contrary to a previous pilot study in which 10 UC patients received the same
amount of AndoSanTM for 12 days with a significant reduction of fecal calprotectin, we did
not find such effect. One reason for lack of reduction of fecal calprotectin in our studies could
be the large variability of baseline calprotectin levels (range 10–6000) that was not seen in the
previous small pilot study (128–1683) [66].

57
 In paper I with UC patients consuming AndoSanTM, the improvements in addition to
symptoms irrefutably were also evident for fatigue and HRQoL. Obviously, the clinical
improvement due to the intake of this mushroom extract in patients with CD (paper II) was
more limited compared with UC. This may partly be explained by the fact that CD is a more
severe disease than UC. Higher dosage of AndoSanTM and longer treatment may have given
more results.
There are several studies of AbM, He and Gf, isolated or in the mixture together as
AndoSanTM, showing beneficial immunomodulatory, antioxidant, antihyperglycemic, and
antitumor effects [36, 38, 92, 112, 308]. However, most data, to date, were produced using
rodents or cell cultures, there are a very limited number of studies measuring their effects in
humans. Even though AbM is the dominating mushroom (82%) of the mushroom extract
compared with He (15%) and Gf (3%), biologic activities exerted by the latter two
mushrooms isolated or in synergy must be kept in mind. All of these mushrooms are
described in the literature as being rich in polysaccharides, where especially β-glucans have
been credited the biological effects observed. However, since AndoSanTM, which is an
extract of the mushrooms´ mycelium and not their fruit bodies, recently was shown to contain
less β-glucan than anticipated from the published data of ß-glucan content in the fruiting
bodies, action also of other yet not identified immunomodulating substances in the extract
must part-take to render the observed effects [106]. Although, despite these findings, we still
believe β-glucan is a major contributor to the observed biological effects, as it is found to be a
potent immunomodulator in several studies [70, 91, 92].

Conclusion
In conclusion, this thesis demonstrates clinically significant effects by consumption of
AndoSanTM for symptoms in both UC and CD, as well as improvement in quality of life and
fatigue for the patients with UC. A relevant interpretation of the results supports the use of
this mushroom extract as an adjuvant to routine medical treatment of IBD patients with
moderate disease. However, since there only was a trend in reduction of pro-inflammatory
cytokines and chemokines in CD and basically no effect in UC, this study could not unmask
the main mechanisms behind the complex AndoSanTM-mediated improved clinical effects.

58
Future perspectives

When AndoSanTM is used as an adjuvant in the treatment of IBD patients in the near future,
we recommend the use of validated patient-reported outcomes (PROs) combined with
classical inflammatory parameters (leukocytes, C-reactive protein, fecal calprotectin).

References
1. Kerrigan RW (2005) Agaricus subrufescens, a cultivated edible and medicinal
mushroom, and its synonyms. Mycologia 97: 12-24. doi: PMID: 16389952
2. Hawksworth DL (2001) Mushrooms: The Extent of the Unexplored Potential. 3: 5.
doi: 10.1615/IntJMedMushr.v3.i4.50
3. Hawksworth DL (2012) Global species numbers of fungi: are tropical studies and
molecular approaches contributing to a more robust estimate? Biodivers Conserv 21:
2425-33. doi: 10.1007/s10531-012-0335-x
4. Kirk PM CP, David JC, Stalpers JA. Ainsworth and Bisby's Dictionary of the Fungi.
Tenth Edition . Edited by Paul M. Kirk, Paul F. Cannon, David W. Minter, and Joost
A. Stalpers. Wallingford (United Kingdom) and Cambridge (Massachusetts): CABI
Publishing. $140.00. xi + 771 p.; ill.; no index. ISBN: 978-0-85199-826-8. 2008.
(Book review). 2010. p. 113-4.
5. Mizuno T (1995) Bioactive biomolecules of mushrooms: Food function and medicinal
effect of mushroom fungi. Food Reviews International 11: 5-21. doi:
10.1080/87559129509541017
6. Wasser SP, Weis AL (1999) Therapeutic effects of substances occurring in higher
Basidiomycetes mushrooms: a modern perspective. Crit Rev Immunol 19: 65-96.
doi: PMID: 9987601
7. Wasser SP (2014) Medicinal mushroom science: Current perspectives, advances,
evidences, and challenges. Biomedical journal 37: 345-56. doi: 10.4103/2319-
4170.138318 PMID: 25179726
8. Reshetnikov SV, Tan K-K (2001) Higher Basidiomycota as a Source of Antitumor and
Immunostimulating Polysaccharides (Review). 3: 34. doi:
10.1615/IntJMedMushr.v3.i4.80
9. Van Griensven LJ (2009) Culinary-Medicinal Mushrooms: Must Action Be Taken?
11: 281-6. doi: 10.1615/IntJMedMushr.v11.i3.70
10. Wasser SP (2010) Medicinal Mushroom Science: History, Current Status, Future
Trends, and Unsolved Problems. 12: 1-16. doi: 10.1615/IntJMedMushr.v12.i1.10
11. Hyodo I, Amano N, Eguchi K, Narabayashi M, Imanishi J, Hirai M, et al. (2005)
Nationwide survey on complementary and alternative medicine in cancer patients in
Japan. J Clin Oncol 23: 2645-54. doi: 10.1200/jco.2005.04.126 PMID: 15728227
12. NBJ (Ed.) (2014) NBJ´s global supplement & nutrition industry report. Nutritional
Business Journal Penton Media Inc., Boulder 39.
13. Sarma N, Giancaspro G, Venema J (2016) Dietary supplements quality analysis tools
from the United States Pharmacopeia. Drug testing and analysis 8: 418-23. doi:
10.1002/dta.1940 PMID: 26857794

59
14. Wasser SP, Sokolov D, Reshetnikov SV, Timor-Tismenetsky M (2000) Dietary
Supplements from Medicinal Mushrooms: Diversity of Types and Variety of
Regulations. 2: 19. doi: 10.1615/IntJMedMushr.v2.i1.10
15. Chang S-T, Wasser SP (2012) The Role of Culinary-Medicinal Mushrooms on Human
Welfare with a Pyramid Model for Human Health. 14: 95-134. doi:
10.1615/IntJMedMushr.v14.i2.10
16. De Silva DD, Rapior S, Sudarman E, Stadler M, Xu J, Aisyah Alias S, et al. (2013)
Bioactive metabolites from macrofungi: ethnopharmacology, biological activities and
chemistry. Fungal Diversity 62: 1-40. doi: 10.1007/s13225-013-0265-2
17. Bentley R (2005) The development of penicillin: genesis of a famous antibiotic.
Perspect Biol Med 48: 444-52. doi: PMID: 16089021
18. Calne R (2004) Cyclosporine as a milestone in immunosuppression. Transplant Proc
36: 13S-5S. doi: 10.1016/j.transproceed.2004.01.042 PMID: 15041300
19. Novak M, Vetvicka V (2008) Beta-glucans, history, and the present:
immunomodulatory aspects and mechanisms of action. J Immunotoxicol 5: 47-57.
doi: 10.1080/15476910802019045 PMID: 18382858
20. Peacock SJ, Paterson GK (2015) Mechanisms of Methicillin Resistance in
Staphylococcus aureus. Annu Rev Biochem 84: 577-601. doi: 10.1146/annurev-
biochem-060614-034516 PMID: 26034890
21. Kim SP, Moon E, Nam SH, Friedman M (2012) Hericium erinaceus mushroom
extracts protect infected mice against Salmonella Typhimurium-Induced liver damage
and mortality by stimulation of innate immune cells. J Agric Food Chem 60: 5590-6.
doi: 10.1021/jf300897w PMID: 22624604
22. Liu JH, Li L, Shang XD, Zhang JL, Tan Q (2016) Anti-Helicobacter pylori activity of
bioactive components isolated from Hericium erinaceus. J Ethnopharmacol 183: 54-8.
doi: 10.1016/j.jep.2015.09.004 PMID: 26364939
23. Chen P, Yong Y, Gu Y, Wang Z, Zhang S, Lu L (2015) Comparison of antioxidant
and antiproliferation activities of polysaccharides from eight species of medicinal
mushrooms. Int J Med Mushrooms 17: 287-95. doi: PMID: 25954912
24. Sheu S-C, Lyu Y, Lee M-S, Cheng J-H (2013) Immunomodulatory effects of
polysaccharides isolated from Hericium erinaceus on dendritic cells. Process
Biochemistry 48: 1402-8. doi: http://dx.doi.org/10.1016/j.procbio.2013.06.012
25. Kim YO, Lee SW, Oh CH, Rhee YH (2012) Hericium erinaceus suppresses LPS-
induced pro-inflammation gene activation in RAW264.7 macrophages.
Immunopharmacol Immunotoxicol 34: 504-12. doi: 10.3109/08923973.2011.633527
PMID: 22126451
26. Kim SP, Kang MY, Kim JH, Nam SH, Friedman M (2011) Composition and
mechanism of antitumor effects of Hericium erinaceus mushroom extracts in tumor-
bearing mice. J Agric Food Chem 59: 9861-9. doi: 10.1021/jf201944n PMID:
21846141
27. Friedman M (2015) Chemistry, Nutrition, and Health-Promoting Properties of
Hericium erinaceus (Lion's Mane) Mushroom Fruiting Bodies and Mycelia and Their
Bioactive Compounds. J Agric Food Chem 63: 7108-23. doi:
10.1021/acs.jafc.5b02914 PMID: 26244378
28. Ma B-J, Shen J-W, Yu H-Y, Ruan Y, Wu T-T, Zhao X (2010) Hericenones and
erinacines: stimulators of nerve growth factor (NGF) biosynthesis in Hericium
erinaceus. Mycology 1: 92-8. doi: 10.1080/21501201003735556
29. Fu H-Y, Shieh D-E, Ho C-T (2002) ANTIOXIDANT AND FREE RADICAL
SCAVENGING ACTIVITIES OF EDIBLE MUSHROOMS. Journal of Food Lipids
9: 35-43. doi: 10.1111/j.1745-4522.2002.tb00206.x

60
30. Zhang Z, Lv G, Pan H, Pandey A, He W, Fan L (2012) Antioxidant and
hepatoprotective potential of endo-polysaccharides from Hericium erinaceus grown on
tofu whey. Int J Biol Macromol 51: 1140-6. doi: 10.1016/j.ijbiomac.2012.09.002
PMID: 22982810
31. Mayell M (2001) Maitake extracts and their therapeutic potential. Altern Med Rev 6:
48-60. doi: PMID: 11207456
32. Shomori K, Yamamoto M, Arifuku I, Teramachi K, Ito H (2009) Antitumor effects of
a water-soluble extract from Maitake (Grifola frondosa) on human gastric cancer cell
lines. Oncol Rep 22: 615-20. doi: PMID: 19639212
33. Kubo K, Aoki H, Nanba H (1994) Anti-diabetic activity present in the fruit body of
Grifola frondosa (Maitake). I. Biol Pharm Bull 17: 1106-10. doi: PMID: 7820117
34. Shigesue K, Kodama N, Nanba H (2000) Effects of maitake (Grifola frondosa)
polysaccharide on collagen-induced arthritis in mice. Jpn J Pharmacol 84: 293-300.
doi: PMID: 11138730
35. Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ, et al. (2003) Biological
activities of the polysaccharides produced from submerged culture of the edible
Basidiomycete Grifola frondosa. Enzyme Microb Technol 32: 574-81. doi:
http://dx.doi.org/10.1016/S0141-0229(03)00026-7
36. Lee JS, Park SY, Thapa D, Choi MK, Chung IM, Park YJ, et al. (2010) Grifola
frondosa water extract alleviates intestinal inflammation by suppressing TNF-alpha
production and its signaling. Exp Mol Med 42: 143-54. doi:
10.3858/emm.2010.42.2.016 PMID: 20054232
37. Kawagishi H, Inagaki R, Kanao T, Mizuno T, Shimura K, Ito H, et al. (1989)
Fractionation and antitumor activity of the water-insoluble residue of Agaricus blazei
fruiting bodies. Carbohydr Res 186: 267-73. doi: PMID: 2736561
38. Firenzuoli F, Gori L, Lombardo G (2008) The Medicinal Mushroom Agaricus blazei
Murrill: Review of Literature and Pharmaco-Toxicological Problems. Evid Based
Complement Alternat Med 5: 3-15. doi: 10.1093/ecam/nem007 PMID: 18317543
39. Fujimiya Y, Suzuki Y, Oshiman K, Kobori H, Moriguchi K, Nakashima H, et al.
(1998) Selective tumoricidal effect of soluble proteoglucan extracted from the
basidiomycete, Agaricus blazei Murill, mediated via natural killer cell activation and
apoptosis. Cancer Immunol Immunother 46: 147-59. doi: PMID: 9625538
40. Hetland G, Sandven P (2002) beta-1,3-Glucan reduces growth of Mycobacterium
tuberculosis in macrophage cultures. FEMS Immunol Med Microbiol 33: 41-5. doi:
PMID: 11985967
41. Hetland G, Johnson E, Lyberg T, Bernardshaw S, Tryggestad AM, Grinde B (2008)
Effects of the medicinal mushroom Agaricus blazei Murill on immunity, infection and
cancer. Scand J Immunol 68: 363-70. doi: 10.1111/j.1365-3083.2008.02156.x
PMID: 18782264
42. Fujimiya Y, Suzuki Y, Katakura R, Ebina T (1999) Tumor-specific cytocidal and
immunopotentiating effects of relatively low molecular weight products derived from
the basidiomycete, Agaricus blazei Murill. Anticancer Res 19: 113-8. doi: PMID:
10226531
43. Kawagishi H, Nomura A, Yumen T, Mizuno T, Hagiwara T, Nakamura T (1988)
Isolation and properties of a lectin from the fruiting bodies of Agaricus blazei.
Carbohydr Res 183: 150-4. doi: PMID: 3233595
44. Takaku T, Kimura Y, Okuda H (2001) Isolation of an antitumor compound from
Agaricus blazei Murill and its mechanism of action. J Nutr 131: 1409-13. doi:
PMID: 11340091

61
45. Wisitrassameewong K, Karunarathna SC, Thongklang N, Zhao R, Callac P, Moukha
S, et al. (2012) Agaricus subrufescens: A review. Saudi J Biol Sci 19: 131-46. doi:
10.1016/j.sjbs.2012.01.003 PMID: 23961172
46. Kimura Y, Kido T, Takaku T, Sumiyoshi M, Baba K (2004) Isolation of an anti-
angiogenic substance from Agaricus blazei Murill: its antitumor and antimetastatic
actions. Cancer Sci 95: 758-64. doi: PMID: 15471563
47. Itoh H, Ito H, Hibasami H (2008) Blazein of a new steroid isolated from Agaricus
blazei Murrill (himematsutake) induces cell death and morphological change
indicative of apoptotic chromatin condensation in human lung cancer LU99 and
stomach cancer KATO III cells. Oncol Rep 20: 1359-61. doi: PMID: 19020714
48. Endo M, Beppu H, Akiyama H, Wakamatsu K, Ito S, Kawamoto Y, et al. (2010)
Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against
leukemic cells. Biochim Biophys Acta 1800: 669-73. doi:
10.1016/j.bbagen.2010.03.016 PMID: 20347942
49. Oh TW, Kim YA, Jang WJ, Byeon JI, Ryu CH, Kim JO, et al. (2010) Semipurified
fractions from the submerged-culture broth of Agaricus blazei Murill reduce blood
glucose levels in streptozotocin-induced diabetic rats. J Agric Food Chem 58: 4113-9.
doi: 10.1021/jf9036672 PMID: 20196600
50. Izawa S, Inoue Y (2004) A screening system for antioxidants using thioredoxin-
deficient yeast: discovery of thermostable antioxidant activity from Agaricus blazei
Murill. Appl Microbiol Biotechnol 64: 537-42. doi: 10.1007/s00253-003-1467-4
PMID: 14593506
51. Padilha MM, Avila AA, Sousa PJ, Cardoso LG, Perazzo FF, Carvalho JC (2009) Anti-
inflammatory activity of aqueous and alkaline extracts from mushrooms (Agaricus
blazei Murill). J Med Food 12: 359-64. doi: 10.1089/jmf.2008.0177 PMID:
19459738
52. Tsai WJ, Yang SC, Huang YL, Chen CC, Chuang KA, Kuo YC (2013) 4-Hydroxy-17-
methylincisterol from Agaricus blazei Decreased Cytokine Production and Cell
Proliferation in Human Peripheral Blood Mononuclear Cells via Inhibition of NF-AT
and NF-kappaB Activation. Evid Based Complement Alternat Med 2013: 435916.
doi: 10.1155/2013/435916 PMID: 23533483
53. Faccin LC, Benati F, Rincao VP, Mantovani MS, Soares SA, Gonzaga ML, et al.
(2007) Antiviral activity of aqueous and ethanol extracts and of an isolated
polysaccharide from Agaricus brasiliensis against poliovirus type 1. Lett Appl
Microbiol 45: 24-8. doi: 10.1111/j.1472-765X.2007.02153.x PMID: 17594456
54. Sorimachi K, Ikehara Y, Maezato G, Okubo A, Yamazaki S, Akimoto K, et al. (2001)
Inhibition by Agaricus blazei Murill fractions of cytopathic effect induced by western
equine encephalitis (WEE) virus on VERO cells in vitro. Biosci Biotechnol Biochem
65: 1645-7. doi: 10.1271/bbb.65.1645 PMID: 11515550
55. Grinde B, Hetland G, Johnson E (2006) Effects on gene expression and viral load of a
medicinal extract from Agaricus blazei in patients with chronic hepatitis C infection.
Int Immunopharmacol 6: 1311-4. doi: 10.1016/j.intimp.2006.04.005 PMID:
16782544
56. Liu Y, Fukuwatari Y, Okumura K, Takeda K, Ishibashi K, Furukawa M, et al. (2008)
Immunomodulating Activity of Agaricus brasiliensis KA21 in Mice and in Human
Volunteers. Evid Based Complement Alternat Med 5: 205-19. doi:
10.1093/ecam/nem016 PMID: 18604247
57. Kim YW, Kim KH, Choi HJ, Lee DS (2005) Anti-diabetic activity of beta-glucans and
their enzymatically hydrolyzed oligosaccharides from Agaricus blazei. Biotechnol Lett
27: 483-7. doi: 10.1007/s10529-005-2225-8 PMID: 15928854

62
58. Hsu CH, Liao YL, Lin SC, Hwang KC, Chou P (2007) The mushroom Agaricus
Blazei Murill in combination with metformin and gliclazide improves insulin
resistance in type 2 diabetes: a randomized, double-blinded, and placebo-controlled
clinical trial. J Altern Complement Med 13: 97-102. doi: 10.1089/acm.2006.6054
PMID: 17309383
59. Chen L, Shao HJ, Su YB (2004) Coimmunization of Agaricus blazei Murill extract
with hepatitis B virus core protein through DNA vaccine enhances cellular and
humoral immune responses. Int Immunopharmacol 4: 403-9. doi:
10.1016/j.intimp.2003.12.015 PMID: 15037217
60. Chen L, Shao H (2006) Extract from Agaricus blazei Murill can enhance immune
responses elicited by DNA vaccine against foot-and-mouth disease. Vet Immunol
Immunopathol 109: 177-82. doi: 10.1016/j.vetimm.2005.08.028 PMID: 16213597
61. Tryggestad AM, Espevik T, Forland DT, Ryan L, Hetland G. The Medical Mushroom
Agaricus blazei Murill Activates NF-κB via TLR2. The Congress of Immunology;
Rio de Janeiro2007. p. P1193.
62. Niu YC, Liu JC, Zhao XM, Wu XX (2009) A low molecular weight polysaccharide
isolated from Agaricus blazei suppresses tumor growth and angiogenesis in vivo.
Oncol Rep 21: 145-52. doi: PMID: 19082455
63. Stamets PE, Wasser SP, da Eira AF, Didukh MY, de Amazonas MAL (2002) Is a
Widely Cultivated Culinary-Medicinal Royal Sun Agaricus (the Himematsutake
Mushroom) Indeed <i>Agaricus blazei</i> Murrill? 4: 24. doi:
10.1615/IntJMedMushr.v4.i4.10
64. Lee EW, Shizuki K, Hosokawa S, Suzuki M, Suganuma H, Inakuma T, et al. (2000)
Two novel diterpenoids, erinacines H and I from the mycelia of Hericium erinaceum.
Biosci Biotechnol Biochem 64: 2402-5. doi: 10.1271/bbb.64.2402 PMID: 11193408
65. Adachi Y, Okazaki M, Ohno N, Yadomae T (1994) Enhancement of cytokine
production by macrophages stimulated with (1-->3)-beta-D-glucan, grifolan (GRN),
isolated from Grifola frondosa. Biol Pharm Bull 17: 1554-60. doi: PMID: 7537572
66. Forland DT, Johnson E, Saetre L, Lyberg T, Lygren I, Hetland G (2011) Effect of an
extract based on the medicinal mushroom Agaricus blazei Murill on expression of
cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease.
Scand J Immunol 73: 66-75. doi: 10.1111/j.1365-3083.2010.02477.x PMID:
21129005
67. Chen J, Seviour R (2007) Medicinal importance of fungal beta-(1-->3), (1-->6)-
glucans. Mycological research 111: 635-52. doi: 10.1016/j.mycres.2007.02.011
PMID: 17590323
68. Kitajma Y (2000) [Structural and biochemical characteristics of pathogenic fungus:
cell walls, lipids and dimorphism, and action modes of antifungal agents]. Nihon
Ishinkin Gakkai Zasshi 41: 211-7. doi: PMID: 11064317
69. Leung MY, Liu C, Koon JC, Fung KP (2006) Polysaccharide biological response
modifiers. Immunol Lett 105: 101-14. doi: 10.1016/j.imlet.2006.01.009 PMID:
16554097
70. Lee DH, Kim HW (2014) Innate Immunity Induced by Fungal Beta-Glucans via
Dectin-1 Signaling Pathway. 16: 1-16. doi: 10.1615/IntJMedMushr.v16.i1.10
71. Hetland G, Therkelsen SP, Nentvich I, Johnson E (2015) Andosan - An Anti-Allergic
and Anti-Inflammatory Ingredient Prepared from Agaricus blazei Mushroom. J Clin
Cell Immunol 2015. doi:
72. Karppinen PK. Search for biologically active compounds in AndoSanTM, a medicinal
mushroom extract. Norwegian University of Life Sciences, Ås; 2013.

63
73. Ingvaldsen IA. Proteiner i det medisinske soppekstraktet AndoSanTM. Norwegian
University of Life Sciences, Ås; 2014.
74. Volman JJ, Helsper JP, Wei S, Baars JJ, van Griensven LJ, Sonnenberg AS, et al.
(2010) Effects of mushroom-derived beta-glucan-rich polysaccharide extracts on nitric
oxide production by bone marrow-derived macrophages and nuclear factor-kappaB
transactivation in Caco-2 reporter cells: can effects be explained by structure?
Molecular nutrition & food research 54: 268-76. doi: 10.1002/mnfr.200900009
PMID: 19885842
75. Sorimachi K, Akimoto K, Ikehara Y, Inafuku K, Okubo A, Yamazaki S (2001)
Secretion of TNF-alpha, IL-8 and nitric oxide by macrophages activated with
Agaricus blazei Murill fractions in vitro. Cell Struct Funct 26: 103-8. doi: PMID:
11482452
76. Engdal S, Nilsen OG (2008) Inhibition of P-glycoprotein in Caco-2 cells: effects of
herbal remedies frequently used by cancer patients. Xenobiotica 38: 559-73. doi:
10.1080/00498250801986969 PMID: 18570158
77. Shapiro LE, Shear NH (2002) Drug interactions: Proteins, pumps, and P-450s. J Am
Acad Dermatol 47: 467-84; quiz 85-8. doi: PMID: 12271287
78. Engdal S, Nilsen OG (2009) In vitro inhibition of CYP3A4 by herbal remedies
frequently used by cancer patients. Phytother Res 23: 906-12. doi: 10.1002/ptr.2750
PMID: 19170155
79. Rendic S, Di Carlo FJ (1997) Human cytochrome P450 enzymes: a status report
summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab Rev 29:
413-580. doi: PMID: 9187528
80. Hajslova J, Hajkova L, Schulzova V, Frandsen H, Gry J, Andersson HC (2002)
Stability of agaritine - a natural toxicant of Agaricus mushrooms. Food Addit Contam
19: 1028-33. doi: 10.1080/02652030210157691 PMID: 12456273
81. Toth B, Nagel D, Patil K, Erickson J, Antonson K (1978) Tumor induction with the
N'-acetyl derivative of 4-hydroxymethyl-phenylhydrazine, a metabolite of agaritine of
Agaricus bisporus. Cancer Res 38: 177-80. doi: PMID: 563287
82. Stijve T, Pittet A, Andrey D, Amazonas MALD, Goessler W (2003) Potential toxic
constituents of Agaricus brasiliensis (A-blazei ss. Heinem.), as compared to other
cultivated and wild-growing edible mushrooms. Dtsch Lebensm-Rundsch 99: 475-81.
doi:
83. Nagaokaa MH, Nagaoka H, Kondo K, Akiyama H, Maitani T (2006) Measurement of
a Genotoxic Hydrazine, Agaritine, and Its Derivatives by HPLC with Fluorescence
Derivatization in the <i>Agaricus</i> Mushroom and Its Products. Chem Pharm Bull
(Tokyo) 54: 922-4. doi: 10.1248/cpb.54.922
84. Shephard SE, Schlatter C (1998) Covalent binding of agaritine to DNA in vivo. Food
Chem Toxicol 36: 971-4. doi: PMID: 9771560
85. Johnson E, Forland DT, Saetre L, Bernardshaw SV, Lyberg T, Hetland G (2009)
Effect of an extract based on the medicinal mushroom Agaricus blazei murill on
release of cytokines, chemokines and leukocyte growth factors in human blood ex
vivo and in vivo. Scand J Immunol 69: 242-50. doi: 10.1111/j.1365-
3083.2008.02218.x PMID: 19281536
86. Ahn WS, Kim DJ, Chae GT, Lee JM, Bae SM, Sin JI, et al. (2004) Natural killer cell
activity and quality of life were improved by consumption of a mushroom extract,
Agaricus blazei Murill Kyowa, in gynecological cancer patients undergoing
chemotherapy. Int J Gynecol Cancer 14: 589-94. doi: 10.1111/j.1048-
891X.2004.14403.x PMID: 15304151

64
87. Hsu CH, Hwang KC, Chiang YH, Chou P (2008) The mushroom Agaricus blazei
Murill extract normalizes liver function in patients with chronic hepatitis B. J Altern
Complement Med 14: 299-301. doi: 10.1089/acm.2006.6344 PMID: 18370584
88. Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016) Effect of a
Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSan, on Symptoms,
Fatigue and Quality of Life in Patients with Ulcerative Colitis in a Randomized
Single-Blinded Placebo Controlled Study. PLoS One 11: e0150191. doi:
10.1371/journal.pone.0150191 PMID: 26933886
89. Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016) Effect of the
Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSanTM, on
Symptoms, Fatigue and Quality of Life in Patients with Crohn's Disease in a
Randomized Single-Blinded Placebo Controlled Study. PLoS One 11: e0159288.
doi: 10.1371/journal.pone.0159288 PMID: 27415795
90. Tangen JM, Tierens A, Caers J, Binsfeld M, Olstad OK, Troseid AM, et al. (2015)
Immunomodulatory effects of the Agaricus blazei Murrill-based mushroom extract
AndoSan in patients with multiple myeloma undergoing high dose chemotherapy and
autologous stem cell transplantation: a randomized, double blinded clinical study.
BioMed research international 2015: 718539. doi: 10.1155/2015/718539 PMID:
25664323
91. Hetland GB, Rafael; Hetland, Aleksander; Badmaev, Vladimir. "AndoSanTM: An
Optimal Biological Response Modifier and Health Promoting Ingredient Prepared
from Agaricus blazei Mushroom". Nova Science Publishers, Inc. 2014.
92. Hetland G, Johnson E, Lyberg T, Kvalheim G (2011) The Mushroom Agaricus blazei
Murill Elicits Medicinal Effects on Tumor, Infection, Allergy, and Inflammation
through Its Modulation of Innate Immunity and Amelioration of Th1/Th2 Imbalance
and Inflammation. Adv Pharmacol Sci 2011: 157015. doi: 10.1155/2011/157015
PMID: 21912538
93. Bernardshaw S, Hetland G, Ellertsen LK, Tryggestad AM, Johnson E (2005) An
extract of the medicinal mushroom Agaricus blazei Murill differentially stimulates
production of pro-inflammatory cytokines in human monocytes and human vein
endothelial cells in vitro. Inflammation 29: 147-53. doi: 10.1007/s10753-006-9010-2
PMID: 17091395
94. Weitberg AB (2008) A phase I/II trial of beta-(1,3)/(1,6) D-glucan in the treatment of
patients with advanced malignancies receiving chemotherapy. J Exp Clin Cancer Res
27: 40. doi: 10.1186/1756-9966-27-40 PMID: 18803849
95. Forland DT, Johnson E, Tryggestad AM, Lyberg T, Hetland G (2010) An extract
based on the medicinal mushroom Agaricus blazei Murill stimulates monocyte-
derived dendritic cells to cytokine and chemokine production in vitro. Cytokine 49:
245-50. doi: 10.1016/j.cyto.2009.09.002 PMID: 20036142
96. Liu Y, Zhang L, Zhu X, Wang Y, Liu W, Gong W (2015) Polysaccharide Agaricus
blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to
M1 type, which mediates inhibition of tumour immune-evasion via the Toll-like
receptor 2 pathway. Immunology 146: 379-91. doi: 10.1111/imm.12508 PMID:
26194418
97. Tryggestad AMA, Espevik T, Ryan L., Hetland G. (2013) The medicinal mushroom
Agaricus blazei Murill promotes NF-KB activation via stimulation of TLR2 and
inhibits its activation via TLR4. J Pharm Biomed Sci 77: 753-61. doi:
98. Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM (2003)
Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor
2. J Exp Med 197: 1107-17. doi: 10.1084/jem.20021787 PMID: 12719479

65
99. Vetvicka V, Thornton BP, Ross GD (1996) Soluble beta-glucan polysaccharide
binding to the lectin site of neutrophil or natural killer cell complement receptor type 3
(CD11b/CD18) generates a primed state of the receptor capable of mediating
cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98: 50-61. doi:
10.1172/jci118777 PMID: 8690804
100. Ben Nasr A, Haithcoat J, Masterson JE, Gunn JS, Eaves-Pyles T, Klimpel GR (2006)
Critical role for serum opsonins and complement receptors CR3 (CD11b/CD18) and
CR4 (CD11c/CD18) in phagocytosis of Francisella tularensis by human dendritic cells
(DC): uptake of Francisella leads to activation of immature DC and intracellular
survival of the bacteria. J Leukoc Biol 80: 774-86. doi: 10.1189/jlb.1205755 PMID:
16857732
101. Zimmerman JW, Lindermuth J, Fish PA, Palace GP, Stevenson TT, DeMong DE
(1998) A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-
glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes. J
Biol Chem 273: 22014-20. doi: PMID: 9705343
102. Kennedy AD, Willment JA, Dorward DW, Williams DL, Brown GD, DeLeo FR
(2007) Dectin-1 promotes fungicidal activity of human neutrophils. Eur J Immunol
37: 467-78. doi: 10.1002/eji.200636653 PMID: 17230442
103. Miller RA, Britigan BE (1997) Role of oxidants in microbial pathophysiology. Clin
Microbiol Rev 10: 1-18. doi: PMID: 8993856
104. Ellertsen LK, Hetland G, Johnson E, Grinde B (2006) 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. Int Immunopharmacol 6: 133-43. doi:
10.1016/j.intimp.2005.07.007 PMID: 16399618
105. Huang TT, Ojcius DM, Young JD, Wu YH, Ko YF, Wong TY, et al. (2012) The anti-
tumorigenic mushroom Agaricus blazei Murill enhances IL-1beta production and
activates the NLRP3 inflammasome in human macrophages. PLoS One 7: e41383.
doi: 10.1371/journal.pone.0041383 PMID: 22844468
106. Berven L, Karppinen P, Hetland G, Samuelsen AB (2015) The polar high molecular
weight fraction of the Agaricus blazei Murill extract, AndoSan, reduces the activity of
the tumor-associated protease, legumain, in RAW 264.7 cells. J Med Food 18: 429-
38. doi: 10.1089/jmf.2014.0018 PMID: 25136950
107. Ebina T, Fujimiya Y (1998) Antitumor effect of a peptide-glucan preparation
extracted from Agaricus blazei in a double-grafted tumor system in mice. Biotherapy
11: 259-65. doi: PMID: 9950102
108. Yu CH, Kan SF, Shu CH, Lu TJ, Sun-Hwang L, Wang PS (2009) Inhibitory
mechanisms of Agaricus blazei Murill on the growth of prostate cancer in vitro and in
vivo. The Journal of nutritional biochemistry 20: 753-64. doi:
10.1016/j.jnutbio.2008.07.004 PMID: 18926679
109. Hetland G, Eide DM, Tangen JM, Haugen MH, Mirlashari MR, Paulsen JE (2016)
The Agaricus blazei-Based Mushroom Extract, Andosan, Protects against Intestinal
Tumorigenesis in the A/J Min/+ Mouse. PLoS One 11: e0167754. doi:
10.1371/journal.pone.0167754 PMID: 28002446
110. Ellertsen LK, Hetland G (2009) An extract of the medicinal mushroom Agaricus
blazei Murill can protect against allergy. Clin Mol Allergy 7: 6. doi: 10.1186/1476-
7961-7-6 PMID: 19416507
111. Takimoto H, Kato H, Kaneko M, Kumazawa Y (2008) Amelioration of skewed
Th1/Th2 balance in tumor-bearing and asthma-induced mice by oral administration of
Agaricus blazei extracts. Immunopharmacol Immunotoxicol 30: 747-60. doi:
10.1080/08923970802279092 PMID: 18720167

66
112. Johnson E, Forland DT, Hetland G, Saetre L, Olstad OK, Lyberg T (2012) Effect of
AndoSan on expression of adhesion molecules and production of reactive oxygen
species in human monocytes and granulocytes in vivo. Scand J Gastroenterol 47: 984-
92. doi: 10.3109/00365521.2012.660544 PMID: 22564240
113. Lima CU, Souza VC, Morita MC, Chiarello MD, Karnikowski MG (2012) Agaricus
blazei Murrill and inflammatory mediators in elderly women: a randomized clinical
trial. Scand J Immunol 75: 336-41. doi: 10.1111/j.1365-3083.2011.02656.x PMID:
22010847
114. Latge JP (2010) Tasting the fungal cell wall. Cell Microbiol 12: 863-72. doi:
10.1111/j.1462-5822.2010.01474.x PMID: 20482553
115. Chan GC, Chan WK, Sze DM (2009) The effects of beta-glucan on human immune
and cancer cells. J Hematol Oncol 2: 25. doi: 10.1186/1756-8722-2-25 PMID:
19515245
116. Brown GD, Gordon S (2003) Fungal beta-glucans and mammalian immunity.
Immunity 19: 311-5. doi: PMID: 14499107
117. Bianchi ME (2007) DAMPs, PAMPs and alarmins: all we need to know about danger.
J Leukoc Biol 81: 1-5. doi: 10.1189/jlb.0306164 PMID: 17032697
118. Sorci G, Giovannini G, Riuzzi F, Bonifazi P, Zelante T, Zagarella S, et al. (2011) The
danger signal S100B integrates pathogen- and danger-sensing pathways to restrain
inflammation. PLoS Pathog 7: e1001315. doi: 10.1371/journal.ppat.1001315 PMID:
21423669
119. Romani L (2011) Immunity to fungal infections. Nat Rev Immunol 11: 275-88. doi:
10.1038/nri2939 PMID: 21394104
120. Figdor CG, van Kooyk Y, Adema GJ (2002) C-type lectin receptors on dendritic cells
and Langerhans cells. Nat Rev Immunol 2: 77-84. doi: 10.1038/nri723 PMID:
11910898
121. Cambi A, Netea MG, Mora-Montes HM, Gow NA, Hato SV, Lowman DW, et al.
(2008) Dendritic cell interaction with Candida albicans critically depends on N-linked
mannan. J Biol Chem 283: 20590-9. doi: 10.1074/jbc.M709334200 PMID:
18482990
122. Jouault T, El Abed-El Behi M, Martinez-Esparza M, Breuilh L, Trinel PA,
Chamaillard M, et al. (2006) Specific recognition of Candida albicans by macrophages
requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association
with TLR2 for signaling. J Immunol 177: 4679-87. doi: PMID: 16982907
123. Wells CA, Salvage-Jones JA, Li X, Hitchens K, Butcher S, Murray RZ, et al. (2008)
The macrophage-inducible C-type lectin, mincle, is an essential component of the
innate immune response to Candida albicans. J Immunol 180: 7404-13. doi: PMID:
18490740
124. Saijo S, Ikeda S, Yamabe K, Kakuta S, Ishigame H, Akitsu A, et al. (2010) Dectin-2
recognition of alpha-mannans and induction of Th17 cell differentiation is essential for
host defense against Candida albicans. Immunity 32: 681-91. doi:
10.1016/j.immuni.2010.05.001 PMID: 20493731
125. Brown GD (2008) Sensing necrosis with Mincle. Nat Immunol 9: 1099-100. doi:
10.1038/ni1008-1099 PMID: 18800160
126. den Dunnen J, Gringhuis SI, Geijtenbeek TB (2009) Innate signaling by the C-type
lectin DC-SIGN dictates immune responses. Cancer Immunol Immunother 58: 1149-
57. doi: 10.1007/s00262-008-0615-1 PMID: 18998127
127. Sancho D, Mourao-Sa D, Joffre OP, Schulz O, Rogers NC, Pennington DJ, et al.
(2008) Tumor therapy in mice via antigen targeting to a novel, DC-restricted C-type
lectin. J Clin Invest 118: 2098-110. doi: 10.1172/jci34584 PMID: 18497879

67
128. Brown GD, Taylor PR, Reid DM, Willment JA, Williams DL, Martinez-Pomares L, et
al. (2002) Dectin-1 is a major beta-glucan receptor on macrophages. J Exp Med 196:
407-12. doi: PMID: 12163569
129. Brown GD (2006) Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat
Rev Immunol 6: 33-43. doi: 10.1038/nri1745 PMID: 16341139
130. Reid DM, Montoya M, Taylor PR, Borrow P, Gordon S, Brown GD, et al. (2004)
Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ
correlates with its function in pathogen recognition and reveals potential roles in
leukocyte interactions. J Leukoc Biol 76: 86-94. doi: 10.1189/jlb.0104031 PMID:
15107454
131. Willment JA, Lin HH, Reid DM, Taylor PR, Williams DL, Wong SY, et al. (2003)
Dectin-1 expression and function are enhanced on alternatively activated and GM-
CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and
lipopolysaccharide. J Immunol 171: 4569-73. doi: PMID: 14568930
132. Brown GD (2011) Innate antifungal immunity: the key role of phagocytes. Annu Rev
Immunol 29: 1-21. doi: 10.1146/annurev-immunol-030409-101229 PMID:
20936972
133. Geijtenbeek TB, Gringhuis SI (2009) Signalling through C-type lectin receptors:
shaping immune responses. Nat Rev Immunol 9: 465-79. doi: 10.1038/nri2569
PMID: 19521399
134. Murray PJ (2006) Understanding and exploiting the endogenous interleukin-
10/STAT3-mediated anti-inflammatory response. Curr Opin Pharmacol 6: 379-86.
doi: 10.1016/j.coph.2006.01.010 PMID: 16713356
135. Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG (2011) Regulation and
functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev
Immunol 29: 71-109. doi: 10.1146/annurev-immunol-031210-101312 PMID:
21166540
136. Ferwerda G, Meyer-Wentrup F, Kullberg BJ, Netea MG, Adema GJ (2008) Dectin-1
synergizes with TLR2 and TLR4 for cytokine production in human primary
monocytes and macrophages. Cell Microbiol 10: 2058-66. doi: 10.1111/j.1462-
5822.2008.01188.x PMID: 18549457
137. van de Veerdonk FL, Kullberg BJ, van der Meer JW, Gow NA, Netea MG (2008)
Host-microbe interactions: innate pattern recognition of fungal pathogens. Curr Opin
Microbiol 11: 305-12. doi: 10.1016/j.mib.2008.06.002 PMID: 18602019
138. Bourgeois C, Majer O, Frohner IE, Tierney L, Kuchler K (2010) Fungal attacks on
mammalian hosts: pathogen elimination requires sensing and tasting. Curr Opin
Microbiol 13: 401-8. doi: 10.1016/j.mib.2010.05.004 PMID: 20538507
139. Blander JM, Medzhitov R (2006) On regulation of phagosome maturation and antigen
presentation. Nat Immunol 7: 1029-35. doi: 10.1038/ni1006-1029 PMID: 16985500
140. Moretti S, Bellocchio S, Bonifazi P, Bozza S, Zelante T, Bistoni F, et al. (2008) The
contribution of PARs to inflammation and immunity to fungi. Mucosal Immunol 1:
156-68. doi: 10.1038/mi.2007.13 PMID: 19079173
141. Joly S, Ma N, Sadler JJ, Soll DR, Cassel SL, Sutterwala FS (2009) Cutting edge:
Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome. J
Immunol 183: 3578-81. doi: 10.4049/jimmunol.0901323 PMID: 19684085
142. Said-Sadier N, Padilla E, Langsley G, Ojcius DM (2010) Aspergillus fumigatus
stimulates the NLRP3 inflammasome through a pathway requiring ROS production
and the Syk tyrosine kinase. PLoS One 5: e10008. doi:
10.1371/journal.pone.0010008 PMID: 20368800

68
143. Evans SE, Hahn PY, McCann F, Kottom TJ, Pavlovic ZV, Limper AH (2005)
Pneumocystis cell wall beta-glucans stimulate alveolar epithelial cell chemokine
generation through nuclear factor-kappaB-dependent mechanisms. Am J Respir Cell
Mol Biol 32: 490-7. doi: 10.1165/rcmb.2004-0300OC PMID: 15746433
144. Skaug B, Jiang X, Chen ZJ (2009) The role of ubiquitin in NF-kappaB regulatory
pathways. Annu Rev Biochem 78: 769-96. doi:
10.1146/annurev.biochem.78.070907.102750 PMID: 19489733
145. Bonifazi P, D'Angelo C, Zagarella S, Zelante T, Bozza S, De Luca A, et al. (2010)
Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways
protects from aspergillosis. Mucosal Immunol 3: 193-205. doi: 10.1038/mi.2009.130
PMID: 19924119
146. Bonifazi P, Zelante T, D'Angelo C, De Luca A, Moretti S, Bozza S, et al. (2009)
Balancing inflammation and tolerance in vivo through dendritic cells by the
commensal Candida albicans. Mucosal Immunol 2: 362-74. doi: 10.1038/mi.2009.17
PMID: 19421183
147. Romani L (2004) Immunity to fungal infections. Nat Rev Immunol 4: 1-23. doi:
10.1038/nri1255 PMID: 14661066
148. de Oliveira LL, Coltri KC, Cardoso CR, Roque-Barreira MC, Panunto-Castelo A
(2008) T helper 1-inducing adjuvant protects against experimental
paracoccidioidomycosis. PLoS Negl Trop Dis 2: e183. doi:
10.1371/journal.pntd.0000183 PMID: 18335066
149. Nesbit L, Johnson SM, Pappagianis D, Ampel NM (2010) Polyfunctional T
lymphocytes are in the peripheral blood of donors naturally immune to
coccidioidomycosis and are not induced by dendritic cells. Infect Immun 78: 309-15.
doi: 10.1128/iai.00953-09 PMID: 19901066
150. Zhang Y, Wang F, Tompkins KC, McNamara A, Jain AV, Moore BB, et al. (2009)
Robust Th1 and Th17 immunity supports pulmonary clearance but cannot prevent
systemic dissemination of highly virulent Cryptococcus neoformans H99. Am J Pathol
175: 2489-500. doi: 10.2353/ajpath.2009.090530 PMID: 19893050
151. Spellberg B, Ibrahim AS, Lin L, Avanesian V, Fu Y, Lipke P, et al. (2008) Antibody
titer threshold predicts anti-candidal vaccine efficacy even though the mechanism of
protection is induction of cell-mediated immunity. J Infect Dis 197: 967-71. doi:
10.1086/529204 PMID: 18419471
152. Zelante T, Fallarino F, Bistoni F, Puccetti P, Romani L (2009) Indoleamine 2,3-
dioxygenase in infection: the paradox of an evasive strategy that benefits the host.
Microbes and infection 11: 133-41. doi: 10.1016/j.micinf.2008.10.007 PMID:
19007906
153. Grohmann U, Volpi C, Fallarino F, Bozza S, Bianchi R, Vacca C, et al. (2007)
Reverse signaling through GITR ligand enables dexamethasone to activate IDO in
allergy. Nat Med 13: 579-86. doi: 10.1038/nm1563 PMID: 17417651
154. Hashimoto K, Suzuki I, Yadomae T (1991) Oral administration of SSG, a beta-glucan
obtained from Sclerotinia sclerotiorum, affects the function of Peyer's patch cells. Int J
Immunopharmacol 13: 437-42. doi: PMID: 1828793
155. Garrett WS, Gordon JI, Glimcher LH (2010) Homeostasis and inflammation in the
intestine. Cell 140: 859-70. doi: 10.1016/j.cell.2010.01.023 PMID: 20303876
156. Goodridge HS, Wolf AJ, Underhill DM (2009) Beta-glucan recognition by the innate
immune system. Immunol Rev 230: 38-50. doi: 10.1111/j.1600-065X.2009.00793.x
PMID: 19594628
157. Sun L, Zhao Y (2007) The biological role of dectin-1 in immune response. Int Rev
Immunol 26: 349-64. doi: 10.1080/08830180701690793 PMID: 18027205

69
158. Willment JA, Gordon S, Brown GD (2001) Characterization of the human beta -
glucan receptor and its alternatively spliced isoforms. J Biol Chem 276: 43818-23.
doi: 10.1074/jbc.M107715200 PMID: 11567029
159. Rice PJ, Adams EL, Ozment-Skelton T, Gonzalez AJ, Goldman MP, Lockhart BE, et
al. (2005) Oral delivery and gastrointestinal absorption of soluble glucans stimulate
increased resistance to infectious challenge. J Pharmacol Exp Ther 314: 1079-86.
doi: 10.1124/jpet.105.085415 PMID: 15976018
160. Murphy KWC. Janeway´s Immunobiology. 9th ed: Garland Science, Taylor & Francis
Group, LCC; 2017.
161. Opal SM, DePalo VA (2000) Anti-inflammatory cytokines. Chest 117: 1162-72. doi:
PMID: 10767254
162. Contassot E, Beer HD, French LE (2012) Interleukin-1, inflammasomes,
autoinflammation and the skin. Swiss Med Wkly 142: w13590. doi:
10.4414/smw.2012.13590 PMID: 22653747
163. Neurath MF, Finotto S, Glimcher LH (2002) The role of Th1/Th2 polarization in
mucosal immunity. Nat Med 8: 567-73. doi: 10.1038/nm0602-567 PMID: 12042806
164. Atreya R, Zimmer M, Bartsch B, Waldner MJ, Atreya I, Neumann H, et al. (2011)
Antibodies against tumor necrosis factor (TNF) induce T-cell apoptosis in patients
with inflammatory bowel diseases via TNF receptor 2 and intestinal CD14(+)
macrophages. Gastroenterology 141: 2026-38. doi: 10.1053/j.gastro.2011.08.032
PMID: 21875498
165. Su L, Nalle SC, Shen L, Turner ES, Singh G, Breskin LA, et al. (2013) TNFR2
activates MLCK-dependent tight junction dysregulation to cause apoptosis-mediated
barrier loss and experimental colitis. Gastroenterology 145: 407-15. doi:
10.1053/j.gastro.2013.04.011 PMID: 23619146
166. Di Sabatino A, Pender SL, Jackson CL, Prothero JD, Gordon JN, Picariello L, et al.
(2007) Functional modulation of Crohn's disease myofibroblasts by anti-tumor
necrosis factor antibodies. Gastroenterology 133: 137-49. doi:
10.1053/j.gastro.2007.04.069 PMID: 17631138
167. Gunther C, Martini E, Wittkopf N, Amann K, Weigmann B, Neumann H, et al. (2011)
Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis.
Nature 477: 335-9. doi: 10.1038/nature10400 PMID: 21921917
168. Meijer MJ, Mieremet-Ooms MA, van Duijn W, van der Zon AM, Hanemaaijer R,
Verheijen JH, et al. (2007) Effect of the anti-tumor necrosis factor-alpha antibody
infliximab on the ex vivo mucosal matrix metalloproteinase-proteolytic phenotype in
inflammatory bowel disease. Inflamm Bowel Dis 13: 200-10. doi:
10.1002/ibd.20051 PMID: 17206679
169. Atreya R, Mudter J, Finotto S, Mullberg J, Jostock T, Wirtz S, et al. (2000) Blockade
of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in
chronic intestinal inflammation: evidence in crohn disease and experimental colitis in
vivo. Nat Med 6: 583-8. doi: 10.1038/75068 PMID: 10802717
170. Neurath MF (2014) Cytokines in inflammatory bowel disease. Nat Rev Immunol 14:
329-42. doi: 10.1038/nri3661 PMID: 24751956
171. Gaffen SL, Liu KD (2004) Overview of interleukin-2 function, production and clinical
applications. Cytokine 28: 109-23. doi: 10.1016/j.cyto.2004.06.010 PMID:
15473953
172. Chiricozzi A, Guttman-Yassky E, Suarez-Farinas M, Nograles KE, Tian S, Cardinale
I, et al. (2011) Integrative responses to IL-17 and TNF-alpha in human keratinocytes
account for key inflammatory pathogenic circuits in psoriasis. J Invest Dermatol 131:
677-87. doi: 10.1038/jid.2010.340 PMID: 21085185

70
173. Ouyang W, Kolls JK, Zheng Y (2008) The biological functions of T helper 17 cell
effector cytokines in inflammation. Immunity 28: 454-67. doi:
10.1016/j.immuni.2008.03.004 PMID: 18400188
174. O'Connor W, Jr., Kamanaka M, Booth CJ, Town T, Nakae S, Iwakura Y, et al. (2009)
A protective function for interleukin 17A in T cell-mediated intestinal inflammation.
Nat Immunol 10: 603-9. doi: 10.1038/ni.1736 PMID: 19448631
175. Hueber W, Sands BE, Lewitzky S, Vandemeulebroecke M, Reinisch W, Higgins PD,
et al. (2012) Secukinumab, a human anti-IL-17A monoclonal antibody, for moderate
to severe Crohn's disease: unexpected results of a randomised, double-blind placebo-
controlled trial. Gut 61: 1693-700. doi: 10.1136/gutjnl-2011-301668 PMID:
22595313
176. Schroder K, Hertzog PJ, Ravasi T, Hume DA (2004) Interferon-gamma: an overview
of signals, mechanisms and functions. J Leukoc Biol 75: 163-89. doi:
10.1189/jlb.0603252 PMID: 14525967
177. Musch E, Andus T, Kruis W, Raedler A, Spehlmann M, Schreiber S, et al. (2005)
Interferon-beta-1a for the treatment of steroid-refractory ulcerative colitis: a
randomized, double-blind, placebo-controlled trial. Clin Gastroenterol Hepatol 3:
581-6. doi: PMID: 15952100
178. Musch E, Lutfi T, von Stein P, Zargari A, Admyre C, Malek M, et al. (2013) Topical
treatment with the Toll-like receptor agonist DIMS0150 has potential for lasting relief
of symptoms in patients with chronic active ulcerative colitis by restoring
glucocorticoid sensitivity. Inflamm Bowel Dis 19: 283-92. doi: 10.1002/ibd.23019
PMID: 22605641
179. Fedorak RN, Gangl A, Elson CO, Rutgeerts P, Schreiber S, Wild G, et al. (2000)
Recombinant human interleukin 10 in the treatment of patients with mild to
moderately active Crohn's disease. The Interleukin 10 Inflammatory Bowel Disease
Cooperative Study Group. Gastroenterology 119: 1473-82. doi: PMID: 11113068
180. van Roon J, Wijngaarden S, Lafeber FP, Damen C, van de Winkel J, Bijlsma JW
(2003) Interleukin 10 treatment of patients with rheumatoid arthritis enhances Fc
gamma receptor expression on monocytes and responsiveness to immune complex
stimulation. J Rheumatol 30: 648-51. doi: PMID: 12672180
181. Tilg H, van Montfrans C, van den Ende A, Kaser A, van Deventer SJ, Schreiber S, et
al. (2002) Treatment of Crohn's disease with recombinant human interleukin 10
induces the proinflammatory cytokine interferon gamma. Gut 50: 191-5. doi: PMID:
11788558
182. Danese S, Fiocchi C (2011) Ulcerative colitis. N Engl J Med 365: 1713-25. doi:
10.1056/NEJMra1102942 PMID: 22047562
183. Baumgart DC, Sandborn WJ (2012) Crohn's disease. Lancet 380: 1590-605. doi:
10.1016/s0140-6736(12)60026-9 PMID: 22914295
184. Peyrin-Biroulet L, Loftus EV, Jr., Colombel JF, Sandborn WJ (2011) Long-term
complications, extraintestinal manifestations, and mortality in adult Crohn's disease in
population-based cohorts. Inflamm Bowel Dis 17: 471-8. doi: 10.1002/ibd.21417
PMID: 20725943
185. Bernstein CN, Blanchard JF, Kliewer E, Wajda A (2001) Cancer risk in patients with
inflammatory bowel disease: a population-based study. Cancer 91: 854-62. doi:
PMID: 11241255
186. Foersch S, Waldner MJ, Neurath MF (2012) Colitis and colorectal cancer. Dig Dis
30: 469-76. doi: 10.1159/000341692 PMID: 23108302
187. Moum B, Vatn MH, Ekbom A, Aadland E, Fausa O, Lygren I, et al. (1996) Incidence
of ulcerative colitis and indeterminate colitis in four counties of southeastern Norway,

71
 1990-93. A prospective population-based study. The Inflammatory Bowel South-
Eastern Norway (IBSEN) Study Group of Gastroenterologists. Scand J Gastroenterol
31: 362-6. doi: PMID: 8726304
188. Moum B, Vatn MH, Ekbom A, Aadland E, Fausa O, Lygren I, et al. (1996) Incidence
of Crohn's disease in four counties in southeastern Norway, 1990-93. A prospective
population-based study. The Inflammatory Bowel South-Eastern Norway (IBSEN)
Study Group of Gastroenterologists. Scand J Gastroenterol 31: 355-61. doi: PMID:
8726303
189. Neurath MF, Travis SP (2012) Mucosal healing in inflammatory bowel diseases: a
systematic review. Gut 61: 1619-35. doi: 10.1136/gutjnl-2012-302830 PMID:
22842618
190. Schnitzler F, Fidder H, Ferrante M, Noman M, Arijs I, Van Assche G, et al. (2009)
Mucosal healing predicts long-term outcome of maintenance therapy with infliximab
in Crohn's disease. Inflamm Bowel Dis 15: 1295-301. doi: 10.1002/ibd.20927
PMID: 19340881
191. Hoivik ML, Moum B, Solberg IC, Cvancarova M, Hoie O, Vatn MH, et al. (2012)
Health-related quality of life in patients with ulcerative colitis after a 10-year disease
course: results from the IBSEN study. Inflamm Bowel Dis 18: 1540-9. doi:
10.1002/ibd.21863 PMID: 21936030
192. Hoivik ML, Bernklev T, Solberg IC, Cvancarova M, Lygren I, Jahnsen J, et al. (2012)
Patients with Crohn's disease experience reduced general health and vitality in the
chronic stage: ten-year results from the IBSEN study. Journal of Crohn's & colitis 6:
441-53. doi: 10.1016/j.crohns.2011.10.001 PMID: 22398064
193. Huppertz-Hauss G, Hoivik ML, Jelsness-Jorgensen LP, Opheim R, Henriksen M,
Hoie O, et al. (2017) Fatigue in a population-based cohort of patients with
inflammatory bowel disease 20 years after diagnosis: The IBSEN study. Scand J
Gastroenterol 52: 351-8. doi: 10.1080/00365521.2016.1256425 PMID: 27852169
194. de Souza HS, Fiocchi C (2016) Immunopathogenesis of IBD: current state of the art.
Nature reviews Gastroenterology & hepatology 13: 13-27. doi:
10.1038/nrgastro.2015.186 PMID: 26627550
195. Geremia A, Biancheri P, Allan P, Corazza GR, Di Sabatino A (2014) Innate and
adaptive immunity in inflammatory bowel disease. Autoimmunity reviews 13: 3-10.
doi: 10.1016/j.autrev.2013.06.004 PMID: 23774107
196. Rook GA (2012) Hygiene hypothesis and autoimmune diseases. Clin Rev Allergy
Immunol 42: 5-15. doi: 10.1007/s12016-011-8285-8 PMID: 22090147
197. Saidel-Odes L, Odes S (2014) Hygiene hypothesis in inflammatory bowel disease.
Annals of gastroenterology : quarterly publication of the Hellenic Society of
Gastroenterology 27: 189-90. doi: PMID: 24975779
198. Venema K (2010) Role of gut microbiota in the control of energy and carbohydrate
metabolism. Curr Opin Clin Nutr Metab Care 13: 432-8. doi:
10.1097/MCO.0b013e32833a8b60 PMID: 20531179
199. Dominguez-Bello MG, Blaser MJ, Ley RE, Knight R (2011) Development of the
human gastrointestinal microbiota and insights from high-throughput sequencing.
Gastroenterology 140: 1713-9. doi: 10.1053/j.gastro.2011.02.011 PMID: 21530737
200. Medzhitov R (2007) Recognition of microorganisms and activation of the immune
response. Nature 449: 819-26. doi: 10.1038/nature06246 PMID: 17943118
201. Hooper LV, Littman DR, Macpherson AJ (2012) Interactions between the microbiota
and the immune system. Science 336: 1268-73. doi: 10.1126/science.1223490
PMID: 22674334

72
202. Chassaing B, Darfeuille-Michaud A (2011) The commensal microbiota and
enteropathogens in the pathogenesis of inflammatory bowel diseases.
Gastroenterology 140: 1720-28. doi: 10.1053/j.gastro.2011.01.054 PMID: 21530738
203. Andoh A, Imaeda H, Aomatsu T, Inatomi O, Bamba S, Sasaki M, et al. (2011)
Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn's
disease using terminal restriction fragment length polymorphism analysis. J
Gastroenterol 46: 479-86. doi: 10.1007/s00535-010-0368-4 PMID: 21253779
204. Serban DE (2015) Microbiota in Inflammatory Bowel Disease Pathogenesis and
Therapy: Is It All About Diet? Nutr Clin Pract 30: 760-79. doi:
10.1177/0884533615606898 PMID: 26452390
205. Yadav V, Varum F, Bravo R, Furrer E, Bojic D, Basit AW (2016) Inflammatory
bowel disease: exploring gut pathophysiology for novel therapeutic targets. Transl Res
176: 38-68. doi: 10.1016/j.trsl.2016.04.009 PMID: 27220087
206. Sewell GW, Marks DJ, Segal AW (2009) The immunopathogenesis of Crohn's
disease: a three-stage model. Curr Opin Immunol 21: 506-13. doi:
10.1016/j.coi.2009.06.003 PMID: 19665880
207. Khor B, Gardet A, Xavier RJ (2011) Genetics and pathogenesis of inflammatory
bowel disease. Nature 474: 307-17. doi: 10.1038/nature10209 PMID: 21677747
208. Kernbauer E, Ding Y, Cadwell K (2014) An enteric virus can replace the beneficial
function of commensal bacteria. Nature 516: 94-8. doi: 10.1038/nature13960 PMID:
25409145
209. Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. (2015)
Disease-specific alterations in the enteric virome in inflammatory bowel disease. Cell
160: 447-60. doi: 10.1016/j.cell.2015.01.002 PMID: 25619688
210. Ott SJ, Kuhbacher T, Musfeldt M, Rosenstiel P, Hellmig S, Rehman A, et al. (2008)
Fungi and inflammatory bowel diseases: Alterations of composition and diversity.
Scand J Gastroenterol 43: 831-41. doi: 10.1080/00365520801935434 PMID:
18584522
211. Sokol H, Leducq V, Aschard H, Pham HP, Jegou S, Landman C, et al. (2016) Fungal
microbiota dysbiosis in IBD. Gut. doi: 10.1136/gutjnl-2015-310746 PMID:
26843508
212. Forbes JD, Van Domselaar G, Bernstein CN (2016) The Gut Microbiota in Immune-
Mediated Inflammatory Diseases. Frontiers in microbiology 7: 1081. doi:
10.3389/fmicb.2016.01081 PMID: 27462309
213. Luo Y, de Lange KM, Jostins L, Moutsianas L, Randall J, Kennedy NA, et al. (2017)
Exploring the genetic architecture of inflammatory bowel disease by whole-genome
sequencing identifies association at ADCY7. Nat Genet 49: 186-92. doi:
10.1038/ng.3761 PMID: 28067910
214. Jostins L, Ripke S, Weersma RK, Duerr RH, McGovern DP, Hui KY, et al. (2012)
Host-microbe interactions have shaped the genetic architecture of inflammatory bowel
disease. Nature 491: 119-24. doi: 10.1038/nature11582 PMID: 23128233
215. Stappenbeck TS, Rioux JD, Mizoguchi A, Saitoh T, Huett A, Darfeuille-Michaud A,
et al. (2011) Crohn disease: a current perspective on genetics, autophagy and
immunity. Autophagy 7: 355-74. doi: 10.4161/auto.7.2.13074 PMID: 20729636
216. Spehlmann ME, Begun AZ, Burghardt J, Lepage P, Raedler A, Schreiber S (2008)
Epidemiology of inflammatory bowel disease in a German twin cohort: results of a
nationwide study. Inflamm Bowel Dis 14: 968-76. doi: 10.1002/ibd.20380 PMID:
18253950
217. Momozawa Y, Mni M, Nakamura K, Coppieters W, Almer S, Amininejad L, et al.
(2011) Resequencing of positional candidates identifies low frequency IL23R coding

73
 variants protecting against inflammatory bowel disease. Nat Genet 43: 43-7. doi:
10.1038/ng.733 PMID: 21151126
218. Petronis A, Petroniene R (2000) Epigenetics of inflammatory bowel disease. Gut 47:
302-6. doi: PMID: 10896927
219. Fogel O, Richard-Miceli C, Tost J (2017) Epigenetic Changes in Chronic
Inflammatory Diseases. Adv Protein Chem Struct Biol 106: 139-89. doi:
10.1016/bs.apcsb.2016.09.003 PMID: 28057210
220. Fu J, Wei B, Wen T, Johansson ME, Liu X, Bradford E, et al. (2011) Loss of intestinal
core 1-derived O-glycans causes spontaneous colitis in mice. J Clin Invest 121: 1657-
66. doi: 10.1172/jci45538 PMID: 21383503
221. Bevins CL, Martin-Porter E, Ganz T (1999) Defensins and innate host defence of the
gastrointestinal tract. Gut 45: 911-5. doi: PMID: 10562592
222. Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev
Immunol 13: 61-92. doi: 10.1146/annurev.iy.13.040195.000425 PMID: 7612236
223. Papo N, Shai Y (2003) Can we predict biological activity of antimicrobial peptides
from their interactions with model phospholipid membranes? Peptides 24: 1693-703.
doi: 10.1016/j.peptides.2003.09.013 PMID: 15019200
224. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415: 389-
95. doi: 10.1038/415389a PMID: 11807545
225. Zanetti M (2004) Cathelicidins, multifunctional peptides of the innate immunity. J
Leukoc Biol 75: 39-48. doi: 10.1189/jlb.0403147 PMID: 12960280
226. Muise AM, Walters TD, Glowacka WK, Griffiths AM, Ngan BY, Lan H, et al. (2009)
Polymorphisms in E-cadherin (CDH1) result in a mis-localised cytoplasmic protein
that is associated with Crohn's disease. Gut 58: 1121-7. doi:
10.1136/gut.2008.175117 PMID: 19398441
227. Sabath E, Negoro H, Beaudry S, Paniagua M, Angelow S, Shah J, et al. (2008)
Galpha12 regulates protein interactions within the MDCK cell tight junction and
inhibits tight-junction assembly. J Cell Sci 121: 814-24. doi: 10.1242/jcs.014878
PMID: 18285450
228. Scharl M, Paul G, Weber A, Jung BC, Docherty MJ, Hausmann M, et al. (2009)
Protection of epithelial barrier function by the Crohn's disease associated gene protein
tyrosine phosphatase n2. Gastroenterology 137: 2030-40 e5. doi:
10.1053/j.gastro.2009.07.078 PMID: 19818778
229. Hassan SW, Doody KM, Hardy S, Uetani N, Cournoyer D, Tremblay ML (2010)
Increased susceptibility to dextran sulfate sodium induced colitis in the T cell protein
tyrosine phosphatase heterozygous mouse. PLoS One 5: e8868. doi:
10.1371/journal.pone.0008868 PMID: 20111595
230. Salim SY, Soderholm JD (2011) Importance of disrupted intestinal barrier in
inflammatory bowel diseases. Inflamm Bowel Dis 17: 362-81. doi:
10.1002/ibd.21403 PMID: 20725949
231. Brazil JC, Louis NA, Parkos CA (2013) The role of polymorphonuclear leukocyte
trafficking in the perpetuation of inflammation during inflammatory bowel disease.
Inflamm Bowel Dis 19: 1556-65. doi: 10.1097/MIB.0b013e318281f54e PMID:
23598816
232. Segal AW (2005) How neutrophils kill microbes. Annu Rev Immunol 23: 197-223.
doi: 10.1146/annurev.immunol.23.021704.115653 PMID: 15771570
233. Rossi M, Young JW (2005) Human dendritic cells: potent antigen-presenting cells at
the crossroads of innate and adaptive immunity. J Immunol 175: 1373-81. doi:
PMID: 16034072

74
234. Rescigno M, Di Sabatino A (2009) Dendritic cells in intestinal homeostasis and
disease. J Clin Invest 119: 2441-50. doi: 10.1172/jci39134 PMID: 19729841
235. Hart AL, Al-Hassi HO, Rigby RJ, Bell SJ, Emmanuel AV, Knight SC, et al. (2005)
Characteristics of intestinal dendritic cells in inflammatory bowel diseases.
Gastroenterology 129: 50-65. doi: PMID: 16012934
236. Mayer L, Eisenhardt D (1990) Lack of induction of suppressor T cells by intestinal
epithelial cells from patients with inflammatory bowel disease. J Clin Invest 86:
1255-60. doi: 10.1172/jci114832 PMID: 2145321
237. Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, et al. (2008) A key role
for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth
cells. Nature 456: 259-63. doi: 10.1038/nature07416 PMID: 18849966
238. Gardet A, Xavier RJ (2012) Common alleles that influence autophagy and the risk for
inflammatory bowel disease. Curr Opin Immunol 24: 522-9. doi:
10.1016/j.coi.2012.08.001 PMID: 23041451
239. Kaser A, Blumberg RS (2011) Autophagy, microbial sensing, endoplasmic reticulum
stress, and epithelial function in inflammatory bowel disease. Gastroenterology 140:
1738-47. doi: 10.1053/j.gastro.2011.02.048 PMID: 21530740
240. Iizuka M, Konno S (2011) Wound healing of intestinal epithelial cells. World J
Gastroenterol 17: 2161-71. doi: 10.3748/wjg.v17.i17.2161 PMID: 21633524
241. Wehkamp J, Salzman NH, Porter E, Nuding S, Weichenthal M, Petras RE, et al.
(2005) Reduced Paneth cell alpha-defensins in ileal Crohn's disease. Proc Natl Acad
Sci U S A 102: 18129-34. doi: 10.1073/pnas.0505256102 PMID: 16330776
242. Darsigny M, Babeu JP, Dupuis AA, Furth EE, Seidman EG, Levy E, et al. (2009) Loss
of hepatocyte-nuclear-factor-4alpha affects colonic ion transport and causes chronic
inflammation resembling inflammatory bowel disease in mice. PLoS One 4: e7609.
doi: 10.1371/journal.pone.0007609 PMID: 19898610
243. Cattin AL, Le Beyec J, Barreau F, Saint-Just S, Houllier A, Gonzalez FJ, et al. (2009)
Hepatocyte nuclear factor 4alpha, a key factor for homeostasis, cell architecture, and
barrier function of the adult intestinal epithelium. Mol Cell Biol 29: 6294-308. doi:
10.1128/mcb.00939-09 PMID: 19805521
244. Pabst O, Zweigerdt R, Arnold HH (1999) Targeted disruption of the homeobox
transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal
development of small intestine and spleen. Development 126: 2215-25. doi: PMID:
10207146
245. Abraham C, Cho JH (2009) Inflammatory bowel disease. N Engl J Med 361: 2066-
78. doi: 10.1056/NEJMra0804647 PMID: 19923578
246. Romagnani S (1994) Lymphokine production by human T cells in disease states. Annu
Rev Immunol 12: 227-57. doi: 10.1146/annurev.iy.12.040194.001303 PMID:
8011282
247. Korn T, Bettelli E, Oukka M, Kuchroo VK (2009) IL-17 and Th17 Cells. Annu Rev
Immunol 27: 485-517. doi: 10.1146/annurev.immunol.021908.132710 PMID:
19132915
248. O'Garra A, Vieira P (2004) Regulatory T cells and mechanisms of immune system
control. Nat Med 10: 801-5. doi: 10.1038/nm0804-801 PMID: 15286781
249. Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE
(2006) TNF downmodulates the function of human CD4+CD25hi T-regulatory cells.
Blood 108: 253-61. doi: 10.1182/blood-2005-11-4567 PMID: 16537805
250. Huibregtse IL, van Lent AU, van Deventer SJ (2007) Immunopathogenesis of IBD:
insufficient suppressor function in the gut? Gut 56: 584-92. doi:
10.1136/gut.2006.103523 PMID: 17047100

75
251. Brandtzaeg P (2007) Induction of secretory immunity and memory at mucosal
surfaces. Vaccine 25: 5467-84. doi: 10.1016/j.vaccine.2006.12.001 PMID:
17227687
252. Yel L (2010) Selective IgA deficiency. J Clin Immunol 30: 10-6. doi:
10.1007/s10875-009-9357-x PMID: 20101521
253. Hamilton MJ, Snapper SB, Blumberg RS (2012) Update on biologic pathways in
inflammatory bowel disease and their therapeutic relevance. J Gastroenterol 47: 1-8.
doi: 10.1007/s00535-011-0521-8 PMID: 22215058
254. Strober W, Fuss IJ (2011) Proinflammatory cytokines in the pathogenesis of
inflammatory bowel diseases. Gastroenterology 140: 1756-67. doi:
10.1053/j.gastro.2011.02.016 PMID: 21530742
255. Strober W, Fuss IJ, Blumberg RS (2002) The immunology of mucosal models of
inflammation. Annu Rev Immunol 20: 495-549. doi:
10.1146/annurev.immunol.20.100301.064816 PMID: 11861611
256. Ruffolo C, Scarpa M, Faggian D, Basso D, D'Inca R, Plebani M, et al. (2010)
Subclinical intestinal inflammation in patients with Crohn's disease following bowel
resection: a smoldering fire. J Gastrointest Surg 14: 24-31. doi: 10.1007/s11605-
009-1070-9 PMID: 19902313
257. Weaver CT, Hatton RD (2009) Interplay between the TH17 and TReg cell lineages: a
(co-)evolutionary perspective. Nat Rev Immunol 9: 883-9. doi: 10.1038/nri2660
PMID: 19935807
258. Annunziato F, Cosmi L, Santarlasci V, Maggi L, Liotta F, Mazzinghi B, et al. (2007)
Phenotypic and functional features of human Th17 cells. J Exp Med 204: 1849-61.
doi: 10.1084/jem.20070663 PMID: 17635957
259. Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, et al. (2003) Increased
expression of interleukin 17 in inflammatory bowel disease. Gut 52: 65-70. doi:
PMID: 12477762
260. Korolkova OY, Myers JN, Pellom ST, Wang L, M'Koma AE (2015) Characterization
of Serum Cytokine Profile in Predominantly Colonic Inflammatory Bowel Disease to
Delineate Ulcerative and Crohn's Colitides. Clin Med Insights Gastroenterol 8: 29-44.
doi: 10.4137/CGast.S20612 PMID: 26078592
261. Mukai H, Watanabe T, Ando M, Katsumata N (2006) An alternative medicine,
Agaricus blazei, may have induced severe hepatic dysfunction in cancer patients. Jpn J
Clin Oncol 36: 808-10. doi: 10.1093/jjco/hyl108 PMID: 17105737
262. Sato K, Chiba T, Ohkusa T (2009) Serial changes of cytokines in active ulcerative
colitis: effects of antibiotic combination therapy. Hepatogastroenterology 56: 1016-
21. doi: PMID: 19760932
263. Rieder F, Fiocchi C (2009) Intestinal fibrosis in IBD--a dynamic, multifactorial
process. Nature reviews Gastroenterology & hepatology 6: 228-35. doi:
10.1038/nrgastro.2009.31 PMID: 19347014
264. Burke JP, Mulsow JJ, O'Keane C, Docherty NG, Watson RW, O'Connell PR (2007)
Fibrogenesis in Crohn's disease. Am J Gastroenterol 102: 439-48. doi:
10.1111/j.1572-0241.2006.01010.x PMID: 17156147
265. Rieder F, Zimmermann EM, Remzi FH, Sandborn WJ (2013) Crohn's disease
complicated by strictures: a systematic review. Gut 62: 1072-84. doi:
10.1136/gutjnl-2012-304353 PMID: 23626373
266. Gordon IO, Agrawal N, Goldblum JR, Fiocchi C, Rieder F (2014) Fibrosis in
ulcerative colitis: mechanisms, features, and consequences of a neglected problem.
Inflamm Bowel Dis 20: 2198-206. doi: 10.1097/mib.0000000000000080 PMID:
24892966

76
267. Heuschkel RB, MacDonald TT, Monteleone G, Bajaj-Elliott M, Smith JA, Pender SL
(2000) Imbalance of stromelysin-1 and TIMP-1 in the mucosal lesions of children
with inflammatory bowel disease. Gut 47: 57-62. doi: PMID: 10861265
268. Kirkegaard T, Hansen A, Bruun E, Brynskov J (2004) Expression and localisation of
matrix metalloproteinases and their natural inhibitors in fistulae of patients with
Crohn's disease. Gut 53: 701-9. doi: PMID: 15082589
269. Danese S, Sans M, de la Motte C, Graziani C, West G, Phillips MH, et al. (2006)
Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.
Gastroenterology 130: 2060-73. doi: 10.1053/j.gastro.2006.03.054 PMID: 16762629
270. Schirbel A, Kessler S, Rieder F, West G, Rebert N, Asosingh K, et al. (2013) Pro-
angiogenic activity of TLRs and NLRs: a novel link between gut microbiota and
intestinal angiogenesis. Gastroenterology 144: 613-23 e9. doi:
10.1053/j.gastro.2012.11.005 PMID: 23149220
271. Danese S, Sans M, Spencer DM, Beck I, Donate F, Plunkett ML, et al. (2007)
Angiogenesis blockade as a new therapeutic approach to experimental colitis. Gut 56:
855-62. doi: 10.1136/gut.2006.114314 PMID: 17170016
272. Danese S, de la Motte C, Sturm A, Vogel JD, West GA, Strong SA, et al. (2003)
Platelets trigger a CD40-dependent inflammatory response in the microvasculature of
inflammatory bowel disease patients. Gastroenterology 124: 1249-64. doi: PMID:
12730866
273. Alitalo K, Tammela T, Petrova TV (2005) Lymphangiogenesis in development and
human disease. Nature 438: 946-53. doi: 10.1038/nature04480 PMID: 16355212
274. Liao S, von der Weid PY (2014) Inflammation-induced lymphangiogenesis and
lymphatic dysfunction. Angiogenesis 17: 325-34. doi: 10.1007/s10456-014-9416-7
PMID: 24449090
275. D'Alessio S, Correale C, Tacconi C, Gandelli A, Pietrogrande G, Vetrano S, et al.
(2014) VEGF-C-dependent stimulation of lymphatic function ameliorates
experimental inflammatory bowel disease. J Clin Invest 124: 3863-78. doi:
10.1172/jci72189 PMID: 25105363
276. Schroeder BO, Wu Z, Nuding S, Groscurth S, Marcinowski M, Beisner J, et al. (2011)
Reduction of disulphide bonds unmasks potent antimicrobial activity of human beta-
defensin 1. Nature 469: 419-23. doi: 10.1038/nature09674 PMID: 21248850
277. Efimova O, Szankasi P, Kelley TW (2011) Ncf1 (p47phox) is essential for direct
regulatory T cell mediated suppression of CD4+ effector T cells. PLoS One 6:
e16013. doi: 10.1371/journal.pone.0016013 PMID: 21253614
278. Kraaij MD, Savage ND, van der Kooij SW, Koekkoek K, Wang J, van den Berg JM,
et al. (2010) Induction of regulatory T cells by macrophages is dependent on
production of reactive oxygen species. Proc Natl Acad Sci U S A 107: 17686-91. doi:
10.1073/pnas.1012016107 PMID: 20861446
279. Harvey RF, Bradshaw JM (1980) A simple index of Crohn's-disease activity. Lancet
1: 514. doi: PMID: 6102236
280. Peyrin-Biroulet L, Panes J, Sandborn WJ, Vermeire S, Danese S, Feagan BG, et al.
(2016) Defining Disease Severity in Inflammatory Bowel Diseases: Current and
Future Directions. Clin Gastroenterol Hepatol 14: 348-54 e17. doi:
10.1016/j.cgh.2015.06.001 PMID: 26071941
281. Zittan E, Kabakchiev B, Kelly OB, Milgrom R, Nguyen GC, Croitoru K, et al. (2016)
Development of the Harvey-Bradshaw Index-pro (HBI-PRO) Score to Assess
Endoscopic Disease Activity in Crohn's Disease. Journal of Crohn's & colitis. doi:
10.1093/ecco-jcc/jjw200 PMID: 27816934

77
282. Huppertz-Hauss G, Lie Hoivik M, Jelsness-Jorgensen LP, Henriksen M, Hoie O,
Jahnsen J, et al. (2016) Health-related Quality of Life in Patients with Inflammatory
Bowel Disease 20 Years After Diagnosis: Results from the IBSEN Study. Inflamm
Bowel Dis 22: 1679-87. doi: 10.1097/mib.0000000000000806 PMID: 27206016
283. Thia K, Faubion WA, Jr., Loftus EV, Jr., Persson T, Persson A, Sandborn WJ (2011)
Short CDAI: development and validation of a shortened and simplified Crohn's
disease activity index. Inflamm Bowel Dis 17: 105-11. doi: 10.1002/ibd.21400
PMID: 20629100
284. Rachmilewitz D (1989) Coated mesalazine (5-aminosalicylic acid) versus
sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. BMJ
298: 82-6. doi: PMID: 2563951
285. D'Haens GR, Sartor RB, Silverberg MS, Petersson J, Rutgeerts P (2014) Future
directions in inflammatory bowel disease management. Journal of Crohn's & colitis 8:
726-34. doi: 10.1016/j.crohns.2014.02.025 PMID: 24742736
286. Ware JE, Jr., Gandek B (1998) Overview of the SF-36 Health Survey and the
International Quality of Life Assessment (IQOLA) Project. J Clin Epidemiol 51: 903-
12. doi: PMID: 9817107
287. Ware JE, Jr., Sherbourne CD (1992) The MOS 36-item short-form health survey (SF-
36). I. Conceptual framework and item selection. Med Care 30: 473-83. doi: PMID:
1593914
288. Loge JH, Kaasa S (1998) Short form 36 (SF-36) health survey: normative data from
the general Norwegian population. Scand J Soc Med 26: 250-8. doi: PMID: 9868748
289. Loge JH, Abrahamsen AF, Ekeberg O, Kaasa S (1999) Hodgkin's disease survivors
more fatigued than the general population. J Clin Oncol 17: 253-61. doi:
10.1200/jco.1999.17.1.253 PMID: 10458240
290. Czuber-Dochan W, Ream E, Norton C (2013) Review article: Description and
management of fatigue in inflammatory bowel disease. Aliment Pharmacol Ther 37:
505-16. doi: 10.1111/apt.12205 PMID: 23311461
291. Chalder T, Berelowitz G, Pawlikowska T, Watts L, Wessely S, Wright D, et al. (1993)
Development of a fatigue scale. J Psychosom Res 37: 147-53. doi: PMID: 8463991
292. Loge JH, Ekeberg O, Kaasa S (1998) Fatigue in the general Norwegian population:
normative data and associations. J Psychosom Res 45: 53-65. doi: PMID: 9720855
293. Dahl J, Ormstad H, Aass HC, Malt UF, Bendz LT, Sandvik L, et al. (2014) The
plasma levels of various cytokines are increased during ongoing depression and are
reduced to normal levels after recovery. Psychoneuroendocrinology 45: 77-86. doi:
10.1016/j.psyneuen.2014.03.019 PMID: 24845179
294. Fagerland MW, Sandvik L (2009) Performance of five two-sample location tests for
skewed distributions with unequal variances. Contemp Clin Trials 30: 490-6. doi:
10.1016/j.cct.2009.06.007 PMID: 19577012
295. Minderhoud IM, Oldenburg B, van Dam PS, van Berge Henegouwen GP (2003) High
prevalence of fatigue in quiescent inflammatory bowel disease is not related to
adrenocortical insufficiency. Am J Gastroenterol 98: 1088-93. doi: 10.1111/j.1572-
0241.2003.07414.x PMID: 12809832
296. van Langenberg DR, Gibson PR (2010) Systematic review: fatigue in inflammatory
bowel disease. Aliment Pharmacol Ther 32: 131-43. doi: 10.1111/j.1365-
2036.2010.04347.x PMID: 20456309
297. Graff LA, Vincent N, Walker JR, Clara I, Carr R, Ediger J, et al. (2011) A population-
based study of fatigue and sleep difficulties in inflammatory bowel disease. Inflamm
Bowel Dis 17: 1882-9. doi: 10.1002/ibd.21580 PMID: 21830266

78
298. Romberg-Camps MJ, Bol Y, Dagnelie PC, Hesselink-van de Kruijs MA, Kester AD,
Engels LG, et al. (2010) Fatigue and health-related quality of life in inflammatory
bowel disease: results from a population-based study in the Netherlands: the IBD-
South Limburg cohort. Inflamm Bowel Dis 16: 2137-47. doi: 10.1002/ibd.21285
PMID: 20848468
299. Minderhoud IM, Samsom M, Oldenburg B (2007) Crohn's disease, fatigue, and
infliximab: is there a role for cytokines in the pathogenesis of fatigue? World J
Gastroenterol 13: 2089-93. doi: PMID: 17465453
300. Vogelaar L, van't Spijker A, Timman R, van Tilburg AJ, Bac D, Vogelaar T, et al.
(2014) Fatigue management in patients with IBD: a randomised controlled trial. Gut
63: 911-8. doi: 10.1136/gutjnl-2013-305191 PMID: 23884638
301. Stone PC, Minton O (2008) Cancer-related fatigue. Eur J Cancer 44: 1097-104. doi:
10.1016/j.ejca.2008.02.037 PMID: 18381237
302. O'Neill LA (2008) Immunology. How frustration leads to inflammation. Science 320:
619-20. doi: 10.1126/science.1158398 PMID: 18451288
303. Wang P, Li XT, Sun L, Shen L (2013) Anti-Inflammatory Activity of Water-Soluble
Polysaccharide of Agaricus blazei Murill on Ovariectomized Osteopenic Rats. Evid
Based Complement Alternat Med 2013: 164817. doi: 10.1155/2013/164817 PMID:
24348690
304. Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016) Cytokine levels after
consumption of a Medicinal Agaricus blazei Murill-based Mushroom Extract,
AndoSan , in Patients with Crohn's disease and Ulcerative Colitis in a Randomized
Single-Blinded Placebo Controlled Study. Scand J Immunol. doi: 10.1111/sji.12476
PMID: 27588816
305. Ikuzawa M, Matsunaga K, Nishiyama S, Nakajima S, Kobayashi Y, Andoh T, et al.
(1988) Fate and distribution of an antitumor protein-bound polysaccharide PSK
(Krestin). Int J Immunopharmacol 10: 415-23. doi: PMID: 3170055
306. Sakurai T, Hashimoto K, Suzuki I, Ohno N, Oikawa S, Masuda A, et al. (1992)
Enhancement of murine alveolar macrophage functions by orally administered beta-
glucan. Int J Immunopharmacol 14: 821-30. doi: PMID: 1512075
307. Romagnani S (1997) The Th1/Th2 paradigm. Immunol Today 18: 263-6. doi:
PMID: 9190109
308. Niwa A, Tajiri T, Higashino H (2011) Ipomoea batatas and Agarics blazei ameliorate
diabetic disorders with therapeutic antioxidant potential in streptozotocin-induced
diabetic rats. J Clin Biochem Nutr 48: 194-202. doi: 10.3164/jcbn.10-78 PMID:
21562638

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a11111
OPEN ACCESS
Citation: Therkelsen SP, Hetland G, Lyberg T,
Lygren I, Johnson E (2016) Effect of a Medicinal
Agaricus blazei Murill-Based Mushroom Extract,
AndoSan™, on Symptoms, Fatigue and Quality of
Life in Patients with Ulcerative Colitis in a
Randomized Single-Blinded Placebo Controlled
Study. PLoS ONE 11(3): e0150191. doi:10.1371/
journal.pone.0150191
Editor: John Green, University Hospital Llandough,
UNITED KINGDOM
Received: December 13, 2015
Accepted: February 9, 2016
Published: March 2, 2016
Copyright: © 2016 Therkelsen et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: The authors received no specific funding
for this work.
Competing Interests: Two of the authors (GH and
EJ) have patent/patent applications and financial
interests relating to material (AndoSan™) pertinent to
this article: i) WO2005065063 A2, Appl. No.:10/
585600, NO- and PCT-filed Jan 2004 and Jan 2005,
PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016
RESEARCH ARTICLE
Effect of a Medicinal Agaricus blazei Murill-
Based Mushroom Extract, AndoSan™, on
Symptoms, Fatigue and Quality of Life in
Patients with Ulcerative Colitis in a
Randomized Single-Blinded Placebo
Controlled Study
Stig Palm Therkelsen1*, Geir Hetland2,5, Torstein Lyberg3, Idar Lygren4, Egil Johnson1,5
1 Department of Gastrointestinal and Pediatric Surgery, Oslo University Hospital, Ullevål, Norway,
2 Immunology and Transfusion Medicine, Oslo University Hospital, Ullevål, Norway, 3 Medical
Biochemistry, Oslo University Hospital, Ullevål, Norway, 4 Medicine, Oslo University Hospital, Ullevål,
Norway, 5 Faculty of Medicine, University of Oslo, Norway
* stig.therkelsen@ous-hf.no
Abstract
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 sin-
gle-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.
1 / 17
 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

respectively, by Inventor Hetland Geir, and ii) Conclusions

NO20090003383, Appl. No.: NO20090003383
20091119, by Inventors Hetland Geir and Johnson Beneficiary effects on symptoms, fatigue and HRQoL from AndoSan™ consumption were
Egil and filed by Applicant Immunopharma AS in Nov demonstrated in this per-protocol study, supporting its use as a supplement to conventional
2009 and financial interest of Geir Hetland as medication for patients with mild to moderate symptoms from ulcerative colitis. The patients
shareholder in Immunopharma AS of Norway,
commercializing AndoSan™. This does not alter the
did not report any harms or unintended effects of AndoSan™ in this study.
authors’ adherence to PLOS ONE policies on sharing
data and materials.
Trial Registration
ClinicalTrials.gov NCT01496053

1. Introduction
Agaricus blazei Murill, a mushroom of the Basidiomycetes family, grows in the wild in the Pie-
dade area outside of São Paulo, Brazil, and the local population has for centuries utilized it as a
health food ingredient. Serious diseases such as atherosclerosis, hyperlipidemia, diabetes and
cancer were less prevalent in the Piedade population compared with counterparts in neighbor-
ing regions [1], presumably owing to consumption of AbM. The mushroom was brought to
Japan in 1966 and introduced to the health food market and effects of AbM (Himematsutake,
jp) and other Basidiomycetes mushrooms such as Hericium erinaceus (He) (Yamabushitake, jp)
[2] and Grifola frondosa (Gf) (Maitake, jp) [3] have received an increasing research effort.
AbM per se and the AbM based mushroom extract, AndoSan™, (ACE Co. Ltd., Gifu-ken,
Japan), composed of AbM (82.4%), He (14.7%) and Gf (2.9%), contain immunomodulatory ß-
glucans but also other biologically active substances like α-glucans [4], proteoglucans [5], lec-
tins [6], ergosterol (provitamin D2) [7], agaritine [8], isoflavonoids [9], anti-oxidant substances
[10], and anti-inflammatory substances such as isolated alkaline and aqueous extracts [11] and
the steroid 4-hydroxy-17-methylincisterol (4-HM) [12].
AbM per se and the AbM based extract, AndoSan™, have been shown to exhibit multiple bio-
logical effects including anti-tumor, anti-allergic and both pro-inflammatory and anti-inflam-
matory effects as reviewed [13, 14]. AbM stimulation in vitro of mononuclear phagocytes
induced secretion of nitric oxide [15] and pro-inflammatory cytokines IL-1ß, Il-6 and TNFα
and IL-8 using AndoSan™ [16], which in monocyte-derived dendritic cells also stimulated such
cytokine production as well as that of chemokine MIP-1ß [17]. One mechanism behind these
effects is probably mediated by binding of glucans in the extract to Toll-like receptor 2 [18] as
well as to the dectin-1 receptor [19], the lectin-binding site of CD11b/18 [20] and possibly CR4
CD11c/18 [21]. However, since AndoSan™, which is an extract of the mushrooms´ mycelium
and not their fruit bodies, recently was shown to contain less ß-glucan than anticipated from
the published data of ß-glucan content in the fruit bodies [22], action also of other yet not iden-
tified immunomodulating substances in the extract must part-take to render the observed
effects. The in vitro results above were supported by microarray expression analysis in AbM
stimulated promonocytic THP-1 tumor cells [23], demonstrating markedly upregulated genes
for IL-1ß, IL-8, moderately for TLR-2 and co-operative molecule MyD88, but not for TLR-4.
However, in another in vivo study, daily consumption of 60 ml of AndoSan™ for a week in
chronic hepatitis C patients [24] had no effect on expression of these genes in blood cells.
Ex vivo stimulation of whole blood with this AbM-based mushroom extract resulted in a
pronounced release, mainly from monocytes, of many cytokines being pro-inflammatory
(IL-1ß, IL-6, TNFα), anti-inflammatory (IL-10), chemokines (IL-8, MIP-1ß, MCP-1, leukocyte

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 2 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

growth factors (G-CSG, GM-CSF), pleiotropic (IL-7, IL-17) as well as of the Th1- (IFNγ, IL-2,
IL-12) and Th-2 types (IL-4, IL-5, IL-13) [25]. However, after daily consumption of 60 ml of
AndoSan™ for 12 days in 8 healthy volunteers, there was a significant reduction in cytokine lev-
els in plasma of IL-1ß, TNFα, IL-6, IL-2 and IL-17, whilst levels of the remaining 12 cytokines
in the kit were unaltered, thereby pointing to an anti-inflammatory effect in vivo, when given
by the oral route.
In patients with ulcerative colitis (UC), increased mucosal levels have been demonstrated
for MIP-1ß, MCP-1 and IL-8 [26], IL-1ß [27], IL-6 and TNFα [28]. Cytokine levels in serum
are less well studied but increased levels have been reported for IL-6 [29], TNFα [30, 31] and
MIP-1ß [32]. Moreover, in a recent extensive review [33] the cytokines eotaxin, GRO (chemo-
kine), TNFα and IL-8 were considered to be persistently elevated in blood of UC patients com-
pared with findings in healthy individuals.
In 10 patients with UC who likewise consumed the mushroom extract, there was an anti-
inflammatory cytokine effect [34] as demonstrated by reduction at day 12 from baseline values
of in vivo levels of one cytokine (MCP-1-ß) in untreated blood and of 7 other cytokines (MIP-
1ß, IL-6, IL-1ß, IL-8, G-CSF, MCP-1, GM-CSF) in LPS-stimulated blood ex vivo. The level of
fecal calprotectin was also reduced as a consequence of consumption of the mushroom extract.
Accordingly, the next step was to examine whether a decline in pathological levels of cytokines
mediated by the mushroom extract in vivo, does result in a putative beneficial clinical effect in
patients with UC.
The aim of the present study was to examine whether consumption of AndoSan™ for 21
days had a positive impact in patients with UC on clinical sympoms, fatique and quality of life
in a randomized single-blinded placebo controlled study. It should be noted that this trial also
includes Crohn's disease patients and that those results are being reported separately.

2. Materials and Methods

2.1. Reagents
The mushroom extract AndoSan™ was provided by the company Immunopharma AS (organi-
zation no. 994924273), Oslo, Norway. It was stored at 4°C in metal cans and used under sterile
conditions ex vivo and kept sterile until taken by volunteers for in vivo experiments. This
mushroom extract is a commercial product produced by the company ACE Co. Ltd., Gifu-ken,
Japan, and its extract contained a business secret, part of which has not been revealed until
recently. The AbM mixed powder contains per 100 g the following constituents: moisture 5.8
g, protein 2.6 g, fat 0.3 g, carbohydrates 89.4 g, of which ß-glucan constitutes 2.8 g, and ash 1.9
g. The AndoSan™ extract contains 82.4% of Basidiomycetes mushroom derived from AbM
(Himematsutake, jp), 14.7% from Hr (Yamabushitake) [2] and 2.9% from Gf (Maitake) [3],
and its final concentration was 340 g / l. The amount per liter of the extract was for sodium 11
mg, phosphorus 254 mg, calcium 35 mg, potassium 483 mg, magnesium 99 mg and zinc 60
mg. The LPS content of AndoSan™ was found, using the Limulus amebocyte lysate test (COA-
MATIC Chromo-LAL; Chromogenix, Falmouth, MA, USA) with detection limit 0.005 EU / ml
(1 EU = 0.1 ng / ml), to be a miniscule concentration of <0.5 pg / ml. The results from tests for
heavy metals were conformable with strict Japanese regulations for health foods. AndoSan™
had been heat-sterilized (124°C for 1 h) by the producer. Potential radioactivity in the extract
was not detected by examination of the Norwegian Food Safety Authorities.

2.2. Analyses
Blood was harvested from the antecubital vein into glass tubes containing 15 IU heparin per ml
or 10 mmol EDTA per ml. The EDTA blood was each time (days 0, 14 and 21) analyzed for

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 3 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

haemoglobin, haematocrite, mean cellular volume, mean cellular haemoglobin, reticulocytes,

immature reticulocytes, leukocytes including a differential count of neutrophils, basophils,
eosinophils, lymphocytes and monocytes, thrombocytes, C-reactive protein (CRP), urea, creat-
inine, bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase,
γ-glutamine transferase, alkaline phosphatase and pancreatic amylase. Fecal calprotectin con-
centrations (mg/kg) (normal value <50 mg/kg) at days 0, 14, 21 were determined in duplicates
as reported [25, 35].
The mainly patient-reported symptom score was a modified version of the Clinical Activity
Index (CAI), including only the 4 clinical items and adding one item defining stool consistency
(normal = 0, soft = 1, watery = 2) [36]. The modified CAI contained four self-reported items
concerning abdominal pain (score 0–3) and stool with regard to frequency (0–4), consistency
(0–2) and blood (0–3). The fifth item evaluated by the physician dealt with general well-being
(0–3) of the patient. The symptom score ranged from 0–15. Scores 0–2 meant patients in
remission and 3–15 gradually increasing disease activity. The modified CAI, rather than the
simple clinical colitis activity index (SCCAI) as described in the study-protocol, was used
because of recommendation from the participating gastroenterologist in this study.
Self reported health-related quality of life (HRQoL) was assessed with the short form 36
(IQOLA SF-36 Norwegian Version 1.2), which is a generic HRQoL questionnaire consisting
of 36 items, of which 35 are grouped into the following eight health domains: (1) physical
functioning (PF), (2) social functioning (SF), (3) role limitations due to physical problems
(RP), (4) role limitation due to emotional problems (RE), (5) mental health (MH), (6) vitality
(VT), (7) bodily pain (BP) and (8) general health perception (GH). Each domain is graded on
a scale of 0–100, and the higher the score the better the HRQoL. The validity and reliability of
the SF-36 form have been demonstrated for a number of countries including Norway (version
1) [37]. The data from our study were compared with published norms from 2323 individuals
in the general population. Only 11 out of 5400 HRQoL questions were unanswered and,
accordingly, 10 out of 1200 dimensions were lacking. Using a scoring algorithm for missing
data outlined in the SF 36 survey manual, these 10 dimensions could also be included in the
results.
Total fatigue score consists of 11 items of graded questions with score 0–3 per question,
which is the sum of physical fatigue (7 items) and mental fatigue (4 items). This score has been
validated in a Norwegian general population [38]. The respective scores for total, mental and
physical fatigue are 0–33, 0–21 and 0–12, and the higher score the more fatigue. The items of
physical (1–7) and mental (8–11) fatigue were: 1) Do you have problems with tiredness? 2) Do
you need to rest more? 3) Do you feel sleepy or drowsy? 4) Do you have problems with starting
things? 5) Are you lacking in energy? 6) Do you have less strength in your muscles? 7) Do you
feel weak? 8) Do you have difficulty concentrating? 9) Do you have problems thinking clearly?
10) Do you make slips of the tongue when speaking? 11) How is your memory?
Rectosigmoidoscopy with biopsies, as described in the study-protocol, were not performed
because of lack of resources.

2.3. Inclusion and Exclusion of Patients

210 patients with UC were phone interviewed and those with CAI score of at least 3 were given
the opportunity to join the study. At the first attendance CAI was re-recorded and criteria for
exclusion were pregnancy, biological treatment with antibodies to TNFα (Adalimumab, Inflixi-
mab), daily use of more than 5 mg of prednisolone, change of medication and/or consumption
of mushroom products from two weeks before till end of the study. A flow chart reveals addi-
tional reasons for exclusions (Fig 1).

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 4 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Fig 1. An algorithm showing the scheme for inclusion of the patients in the study.
doi:10.1371/journal.pone.0150191.g001

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 5 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

2.4. Experimental Design and Randomization

This is a single-center randomized two-armed patient-blinded study designed to determine
whether daily oral intake of a mushroom extract, AndoSan™, improved clinical symptoms,
fatigue and quality of life in patients with UC during the 21 days’ study period. The patients
were evaluated before (visit 1, day 0), during (visit 2, day 14) and after (visit 3, day 21) Ando-
San™ or placebo consumption (30 ml twice daily). This dose (60 ml/day) reduced levels of pro-
inflammatory cytokines and chemokines in healthy volunteres [25] and in patients with UC
and CD [34], whilst half dosage (30 ml/day) had no detectable effects (unpublished data). The
placebo group was evaluated likewise but received an equal volume of color-like drink with
ionized water containing 0.5 ml per liter of caramel color (E150c) with salt.
The 50 patients were divided into 13 groups and manually randomized with the overall allo-
cation ratio of one to one. Block-randomization was done after the phone interview, with uneven
and even numbers given for AndoSan™ or placebo, respectively. The patients, one by one, were
placed in one pile, and the group affiliations were placed in another pile. The randomization was
performed by combining one selection from each pile, both anonymized. The first author per-
formed the randomization, enrolled the participants, and assigned participants to interventions.
A few patients were excluded throughout the study by not attending or because of intercurrent
incidents (disease, unexpected life events). Accordingly, a slight imbalance of the study groups
occurred that was corrected in the latter rounds of randomization. More specifically, the 50 UC
patients were divided into 13 study groups (range 2–9 per group), each with a study period of 3
weeks. The included 50 symptomatic patients had no missing data and were randomized and
blinded for oral daily consumption (30 ml twice daily) of AndoSan™ or placebo for the 21 days’
experimental period. Patients in the AndoSan™ group and the placebo group self-reported, in
written, at visit 1 (day 0), visit 2 (day 14) and visit 3 (day 21) regarding symptoms, fatigue and
health-related quality of life. Patient derived blood samples and fecal calprotectin from these vis-
its were also analyzed. All data were stored in a secure database (Access—Microsoft Office) at a
server at Oslo University Hospital, Ullevål, Norway. A study number anonymized the patients.
This study is a follow up of a previous pilot study [34] in which there was a reduction of
pro-inflammatory cytokines and chemokines in patients receiving the same daily dose of
AndoSanTM, but for 12 days. We calculated for prospective differences of 20% between the
experimental and placebo group and assumed standard deviation of 20% for the different
parameters with a significant level of 5% and a power of 90% (ß = 0.10), demands about 25
patients per randomized arm (calculated in cooperation Oslo Center for Biostatistics and Epi-
demiology, Oslo University Hospital).

2.5. Patient Characteristics

Clinical disease data concerning duration, anatomic extent and activity were registered. The
study groups were comparable and with no significant differences with respect to details of
demographic data and patient characteristics (Table 1). Eight patients in the AndoSan™ group
used several medications, Azathioprine and 5-ASA in 4 and Azathioprine and Prednisolone in
1 patient(s), respectively. Three patients used oral 5-ASA combined with rectal enema with
Budesonide, Prednisolone and 5-ASA, respectively. In the placebo group 2 patients used Aza-
thioprine and 5-ASA and 5 patients used 5-ASA by oral and rectal route. The medication was
unaltered from baseline and throughout the study period, in both groups.

2.6. Statistical Analysis

Data are presented as mean and standard deviation or as median and range values. Paired sam-
ple t-test and Wilcoxon test are used for within-group analysis. The judgement of whether the

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 6 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Table 1. Demographic and patient data.

AndoSan™ Placebo P
Number 24 26 ns
Age (years) 40.5 (23–56) 40.0 (23–67) ns
Gender (male, female) 13, 11 12, 14 ns
Duration of diagnosis (months) 96 (4–324) 66 (4–260) ns
Anatomic extent
rectosigmoidal 5 7 ns
left colon 6 10 ns
entire colon 13 9 ns
Disease pattern
continous 4 2 ns
episodic 20 24 ns
Medication
5-ASA 13 17 ns
Azathioprin 2 0 ns
Several medications 8 7 ns
No medication 1 2 ns
Comorbidity
Arthralgia 8 6 ns
Sarcoidosis 0 1 ns
Diabetes mellitus type 2 1 0 ns
PSC, livercirrhosis and anemia 0 1 ns
Myalgic encephalomyelitis 1 0 ns

Values for age and duration of diagnosis are given as median (range).

doi:10.1371/journal.pone.0150191.t001

distributions of the main efficacy variables were so close to the normal distribution that nor-
mality-based significance tests may be used, is based on the finding in a paper by Fagerland
and Sandvik [39]. Mixed models corrected for baseline values were used for measuring P values
between the study groups. P values below 0.05 were considered statistically significant. The
SPSS statistical program for the social sciences, version 22 (IBM), was used in the analyses.

2.7. Ethical Considerations

The study was approved on April 8, 2011, by the regional ethics committee (REC—South East
Norway, ref. 2011/404) and followed the guidelines of the Helsinki declaration. The partici-
pants were informed and signed a written consent for participation, including the option of
study withdrawal. The patients were recruited and followed up at the department of Medicine,
Oslo University Hospital, Ullevål, Norway, in the period of June 2012 to May 2014. The study
was registered with unique protocol ID AbM2012-IBD and clinical trials gov ID NCT
01496053 (December 15, 2011). The authors confirm that all ongoing and related trials for this
drug/intervention are registered.

3. Results
3.1. Exclusion of randomized patients
A total of 62 patients, 31 in both study groups, were randomized for inclusion in this study. 12
of these patients were excluded according to the criteria of the study protocol, because three

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 7 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

and two changed their medical treatment just prior to or during the study period, two in each
group had missing laboratory data, and two and one had missing attendance, in the AndoSan™
and placebo group, respectively. Thereby we ended up with 24 patients in the AndoSan™ group
and 26 in the placebo group.

3.2. Age and Gender

Median age for the 50 included patients with UC was 40.5 years (range 23–67). There were 13
men and 11 women in the AndoSan™ group and 12 men and 14 women in the placebo group.
Respective ages in the two groups were median 35 (range 23–64) and 41.5 (27–50) for men
(p = 0.611) and 44 (24–67) and 35.5 (23–56) for women (p = 0.075).

3.3. Symptom Score

The symptom scores using the CAI were similar at inclusion in the AndoSan™ and placebo
groups, with respective mean scores of 5.88 and 5.81. Compared with baseline only the patients
in the AndoSan™ group reported a significant reduction of symptoms that was also further
reduced from visit 2 (day 14) to visit 3 (day 21) after the mushroom extract intake (Table 2).
There were no significant differences in baseline symptom scores between male and females
within the two groups. When comparing the two groups using mixed models corrected for
baseline values, there also was a significant difference (p = 0.023) in favor of the AndoSan™
group.
Regardless of disease activity at inclusion the patients had a reduction of symptom score
(data not shown). Within the AndoSan™ group from visit 1 to 3, there was a significant reduc-
tion regarding stool frequency (p = 0.005), consistency (p = 0.02), and the doctor´s evaluation
of disease activity (p = 0.02). For blood in stool (p = 0.08) and abdominal pain (p = 0.07) there
was a trend favoring improvement. In the placebo group there were no significant improve-
ments in these parameters, but a trend in favor of reduction of stool frequency (p = 0.07). The
doctor’s evaluation of disease activity could be a source of bias in the CAI score because of lack
of blinding. Therefore we also examined the symptoms without this item, and the p values for
difference of scores were the same (data not shown). The patients did not report any harms or
unintended effects of AndoSan™ in this study.

3.4. Fatigue Score

Firstly, age-adjusted normative fatigue scores in the Norwegian population were compared
with such scores in the UC patients at inclusion in this study (Table 3). There were for both

Table 2. Symptom score for the UC patients.

Group V1 V2 V3 PV1V2 PV1V3 Pbetween groups

™
AndoSan 5.88 (4.92–6.83) 4.71 (3.90–5.52) 4.50 (3.70–5.30) 0.002 0.001 0.023
M (n = 13) 6.15 (4.70–7.61) 5.08 (3.78–6.37) 5.08 (3.93–6.22) 0.037 0.024
F (n = 11) 5.55 (4.09–7.00) 4.27 (3.19–5.36) 3.82 (2.66–4.97) 0.031 0.017
Placebo 5.81 (4.81–6.80) 5.58 (4.42–6.73) 5.27 (4.28–6.26) 0.471 0.114
M (n = 12) 6.33 (4.82–7.85) 6.42 (4.76–8.07) 5.83 (4.48–7.18) 0.878 0.309
F (n = 14) 5.36 (3.90–6.82) 4.86 (3.15–6.56) 4.79 (3.25–6.26) 0.205 0.252

V1; visit 1 (day 0), V2; visit 2 (day 14), V3; visit 3 (day 21). M; male, F; female.
Values are given as means and 95% confidence intervals. Paired sampled t-test for the p-values.
P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0150191.t002

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 8 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Table 3. Mean fatigue scale scores. Normative data in the Norwegian population compared to patients with UC on inclusion.

Normative data Ulcerative colitis P Normative data vs UC

M (n = 1112) F (n = 1175) M (n = 25) F (n = 25) M F

Total 11.9 (3.9) 12.6 (4.0) 16.32 (4.7) 16.56 (5.7) <0.0001 <0.0001
Physical 7.6 (3.0) 8.2 (3.2) 11.08 (3.9) 11.28 (4.2) <0.0001 <0.0001
Mental 4.3 (1.4) 4.4 (1.4) 5.24 (1.4) 5.28 (1.8) 0.0009 0.0021

Normative data from the general Norwegian population.

Values are given as mean and standard deviation (SD).
Paired sample t-test for the p-values.

doi:10.1371/journal.pone.0150191.t003

genders a significant increase of physical, mental and total fatigue scores in the UC patients
compared with the general population. This effect was more pronounced for physical fatigue
than for mental fatigue.
The scores for genders on inclusion were quite similar within and between the groups. In
the AndoSan™ group (Table 4) for both genders the UC patients reported a significant decline
in mental and total fatigue, that was more pronounced at visit 2 (day 14) vs. visit 3 (day 21).
When broken down into gender the reduction in mental and total fatigue was not significant
at visit 3. There was, however, a significant decline in physical fatigue at visit 2 (p = 0.007), but
not at visit 3 (p = 0.128). The improvement in physical fatigue at visit 2 was significant for men
(0.042) but not for women (0.055). In the placebo group the fatigue scores were unaltered
throughout the experimental period as a whole and by division into gender.
However, when comparing the AndoSan™ and placebo groups using mixed models cor-
rected for baseline values, there were significant improvements in the AndoSan™ group for the
three scores of total (p = 0.018), mental (p = 0.022) and physical (p = 0.037) fatigue (Table 4).

3.5. Quality of Life

HRQoL score (SF-36) for UC patients were compared with age-adjusted normative data for
the Norwegian population (Table 5). We found significantly much lower scores for HRQoL in

Table 4. Fatigue scores for the patients with (n = 24 AndoSan™ and n = 26 placebo) UC.

AndoSan™ group Placebo group

V1 V2 V3 PV1V2 PV1V3 V1 V2 V3 PV1V2 PV1V3 Pbetween groups

TF 16.6 (5.6) 14.1 (4.5) 15.1 (4.1) 0.001 0.023 16.3 (4.8) 16.3 (4.9) 16.9 (5.2) 1.000 0.507 0.018
M 16.38 14.46 15.31 0.002 0.141 16.25 15.00 15.83 0.325 0.764
F 16.91 13.73 14.91 0.039 0.098 16.29 17.36 17.79 0.297 0.248
PhF 11.1 (4.3) 9.4 (3.8) 10.4 (3.2) 0.007 0.128 11.2 (3.9) 11.2 (3.4) 11.7 (3.9) 0.949 0.564 0.037
M 10.85 9.77 10.38 0.042 0.436 11.33 10.42 11.00 0.366 0.765
F 11.45 9.00 10.45 0.055 0.199 11.14 11.86 12.21 0.325 0.289
MF 5.50 (1.8) 4.71 (1.3) 4.71 (1.1) 0.002 0.010 5.0 (1.3) 5.1 (1.9) 5.2 (1.6) 0.898 0.446 0.022
M 5.54 4.69 4.92 0.035 0.071 4.92 4.58 4.83 0.339 0.809
F 5.45 4.73 4.45 0.024 0.076 5.14 5.50 5.57 0.455 0.254

V1; visit 1 (day 0), V2; visit 2 (day 14), V3; visit 3 (day 21).
TF; total fatigue, PhF; physical fatigue, MF; mental fatigue.
Values are given as mean and standard deviation (SD). Paired sampled t-test for p-values.
P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0150191.t004

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 9 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Table 5. Mean SF-36 scale scores. Age-adjusted Normative Data from the Norwegian Population compared with patients with UC on inclusion.

Normative data Ulcerative colitis P Normative data vs UC

M (n = 977–1017) F (n = 1013–67) M (n = 25) F (n = 25) M F

PF 91.4 (16.2) 87.7 (17.5) 85.8 (17.5) 85.6 (14.9) 0.09 0.55
RP 83.3 (32.0) 79.2 (35.0) 59.0 (41.4) 49.0 (40.5) 0.0002 <0.0001
BP 78.1 (24.6) 74.5 (26.0) 59.3 (26.0) 57.2 (23.0) 0.0002 0.0011
GH 78.3 (21.0) 77.5 (22.1) 51.6 (21.3) 54.4 (24.9) <0.0001 <0.0001
VT 63.4 (18.2) 57.6 (21.0) 39.0 (18.6) 38.1 (19.7) <0.0001 <0.0001
SF 88.2 (20.5) 84.8 (22.8) 67.5 (27.2) 66.0 (20.6) <0.0001 <0.0001
RE 85.9 (28.5) 80.9 (33.1) 66.7 (41.9) 62.7 (43.4) 0.0010 0.0070
MH 79.7 (15.8) 77.6 (16.9) 63.5 (17.2) 68.6 (13.6) <0.0001 0.0082

Age-adjusted normative data from the general Norwegian population, age 19–69.
Values are given as mean and standard deviation (SD). Paired sampled t-test for the p-values.
SF-36; Short form 36, PF; physical functioning, RP; role limitations, physical, BP; bodily pain, GH; general heath perception, VT; Vitality, SF; social
functioning, RE; role limitations, emotional, MH; mental health.

doi:10.1371/journal.pone.0150191.t005

the UC patients for both genders regarding 7 out of 8 dimensions. Only for physical function-
ing (PF) there were no differences in both genders relative to the general population. The
reduction of quality life scores in the UC patients were for both genders most pronounced for
the dimensions; general health (GH), vitality (VT) and social functioning (SF) (p values <
0.0001) as compared with the general population.
In the AndoSan™ group as a whole the results were significantly improved (Table 6) for
bodily pain (BP), VT, SF and mental health (MH), of which BP and MH also scored signifi-
cantly but less pronounced at visit 2. Although not significant, there was an increase of scores
for the remaining four dimensions GH, PF, role limitation, physical (RP) and role limitation,
emotional (RE). In the placebo group, except from an improvement of BP (p = 0.036) at visit 2,
there were no significant alterations in the 8 quality of life dimensions throughout the study.
When comparing the two groups, using mixed models corrected for baseline values, we found
significant improvement of SF-36 scores for PF, RP, BP and SF in the AndoSan™ group.
Except for unaltered score in GH for females in the AndoSan™ group, there were improve-
ments of scores in all dimensions for both genders. In the placebo group, however, the corre-
sponding general pattern was a gender-like unchanged score during the three weeks study
period (Table 6).

3.6. Calprotectin in Feces

The patients delivered fecal tests at visits 1, 2 and 3 (Table 7). In the AndoSan™ group the
median (range) values for fecal calprotectin (mg/kg) were 439 (10–6000), 366 (10–6000) and
489 (18–6000). In the placebo group the values were 328 (16–5361), 521 (18–6000) and 563
(10–6000). There were no significant differences in levels of calprotectin within or between the
groups. However, for men there was rather a significant increase of calprotectin in the placebo
group from visit 1 to visit 3 (p = 0.019).

3.7. Effect on General Blood Parameters

The following blood samples were analyzed at visit 1 and 3: CRP, leukocytes, eosinophils, baso-
phils, neutrophils, lymphocytes, monocytes, hemoglobin, haematocrite, mean cellular volume,
mean cellular haemoglobin, immature reticulocytes, reticulocytes, thrombocytes, urea,

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 10 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Table 6. Mean SF-36 scale scores (n = 24 AndoSan™ and n = 26 placebo) UC.

AndoSan™ group Placebo group

V1 V2 V3 PV1V2 PV1V3 V1 V2 V3 PV1V2 PV1V3 P AvsP

PF 85.0 (16.9) 87.6 (13.7) 88.8 (13.4) 0.096 0.101 86.4 (15.5) 85.8 (14.6) 84.6 (15.5) 0.701 0.265 0.039
M 86.9 89.9 90.4 0.148 0.145 84.6 85.0 84.2 0.809 0.045
F 82.7 84.9 86.8 0.399 0.347 87.9 86.4 85.0 0.569 0.220
RP 55.2 (43.0) 59.4 (39.6) 64.6 (40.3) 0.426 0.059 52.9 (39.6) 47.1 (43.2) 47.1 (43.8) 0.265 0.265 0.028
M 61.5 71.2 69.2 0.096 0.219 56.3 60.4 58.3 0.551 0.586
F 47.7 45.5 59.1 0.810 0.176 50.0 35.7 37.5 0.055 0.169
BP 57.1 (26.1) 67.3 (24.0) 73.6 (21.5) 0.015 0.000 59.3 (23.0) 64.7 (25.0) 58.3 (62.0) 0.036 0.760 0.013
M 59.3 70.8 77.8 0.114 0.015 59.3 68.5 61.9 0.009 0.655
F 54.5 63.2 68.7 0.022 0.010 59.4 61.5 55.3 0.567 0.281
GH 53.0 (23.2) 56.5 (24.0) 54.8 (22.2) 0.174 0.536 52.9 (23.1) 50.7 (23.4) 48.7 (21.1) 0.299 0.093 0.062
M 49.5 55.8 53.5 0.137 0.374 53.8 55.7 53.6 0.343 0.936
F 57.3 57.4 56.4 0.972 0.789 52.1 46.5 44.6 0.107 0.046
VT 38.8 (19.9) 44.6 (18.9) 46.9 (17.9) 0.076 0.018 38.4 (18.5) 40.6 (19.4) 38.3 (17.0) 0.403 0.968 0.088
M 39.2 46.9 48.1 0.139 0.046 38.8 47.1 42.9 0.014 0.248
F 38.2 41.8 45.5 0.371 0.205 38.1 35.0 34.3 0.406 0.466
SF 65.1 (26.6) 75.5 (22.9) 75.0 (19.8) 0.013 0.015 66.3 (21.6) 65.9 (21.1) 67.8 (20.7) 0.597 0.885 0.014
M 67.3 81.7 75.0 0.050 0.180 67.7 75.0 68.8 0.306 0.870
F 62.5 68.2 75.0 0.096 0.041 68.8 58.0 67.0 0.061 0.583
RE 68.1 (39.9) 69.4 (40.4) 75.0 (39.6) 0.846 0.423 61.5 (44.9) 52.6 (44.4) 55.1 (43.1) 0.215 0.477 0.095
M 64.1 64.1 71.8 1.000 0.461 69.4 72.2 77.8 0.809 0.555
F 72.7 75.8 78.8 0.588 0.690 54.8 35.7 35.7 0.040 0.104
MH 65.3 (15.7) 69.5 (14.4) 71.0 (13.7) 0.032 0.005 66.8 (15.7) 69.2 (17.1) 66.0 (17.1) 0.285 0.671 0.123
M 63.1 67.4 68.0 0.141 0.075 64.0 70.7 66.3 0.041 0.385
F 68.0 72.0 74.5 0.137 0.036 69.1 68.0 65.7 0.723 0.165

Paired sampled t-test for the p-values.

P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0150191.t006

creatinine, and GFR (glomerular filtration rate), bilirubin, aspartate aminotransferase, alanine
aminotransferase, lactate dehydrogenase, γ-glutamine transferase, alkaline phosphatase and
pancreatic amylase. Significant changes in the blood samples were neither found in the Ando-
San™—nor the placebo group.

Table 7. Fecal test in the 50 UC patients.

Group V1 V2 V3 PV1V2 PV1V3 Pbetween groups

AndoSan™ (n = 24) 439 (10–6000) 366 (10–6000) 489 (18–6000) 0.673 0.808 0.705
M 358 382 527 0.727 0.600
F 517 317 366 0.314 0.959
Placebo (n = 26) 328 (16–5361) 521 (18–6000) 563 (10–6000) 0.298 0.551
M 234 570 1702 0.060 0.019
F 679 480 445 0.875 0.245

Values are given as median (range). Non-parametric Wilcoxon test for the p-values.
P between groups is measured with Mann-Whitney U test.

doi:10.1371/journal.pone.0150191.t007

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 11 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

The median and range haemoglobin (g/l), leukocyte counts (109/l) and CRP levels for visit 1
and 3 the AndoSan™ group were 13.55 (11.5–17.2) versus 13.55 (11.0–17.3), 6.20 (3.1–9.2) ver-
sus 5.95 (3.3–10.5) and 2.00 (0.6–30.4) versus 1.80 (0.6–16.9), respectively. Corresponding val-
ues in the placebo group were 13.7 (5.7–16.1) versus 13.5 (6.0–15.8) for haemoglobin, 5.85
(4,4–10.3) versus 6.55 (3.1–14.6) for leukocytes and 1.60 (0.6–63) versus 1.95 (0.6–58.0) for
CRP. Accordingly, there were no statistical changes of these parameters neither within nor
between the groups.

4. Discussion
The present study demonstrates for the first time, in a randomized patient-blinded placebo
controlled study, that the immunomodulatory Agaricus blazei Murill based mushroom extract
AndoSan™ [14] improved clinical symptoms as well as fatigue and quality of life in patients
with UC during a 3 weeks’ study period. Moreover, there was no effect in the placebo group,
whatsoever, and when comparing the two groups the difference was significant in favor of
AndoSan™. The moderate, but significant reduction (about 20%) in symptom score occurred
already after 2 weeks and persisted after 3 weeks.
Compared with the normal population the UC patients had considerably more fatigue
(women 32%, men 37%). However, the general effect on fatigue subsided after 3 weeks but still
was significant for mental fatigue. This may be due to a reduced effect of AndoSan™ over time,
but external factors influencing the patients’ physical status like intercurrent subclinical disease
must also be considered.
Regarding limitations of this study we need to adress some issues. The study was not blinded
for the authors leaving possible biases of the results. This is especially true for the first author who
was responsible for the inclusion and randomization of participants, the implementation of the
practical aspects of and in meeting with the patients, and also in the analysis of the results. This is
a relatively small study, allthough with significant results, with its limitations. The study was con-
ducted with participants from the Oslo (Norway) area, and the results may not be transferable to
other geographical areas with other patient characteristics. Reduced compliance in carrying out
the study, with missing or incorrect oral intake of AndoSan™ or placebo, may be a possible source
of error, even though this was not the impression in conversation with patients after the study
period of three weeks. The modified CAI is not used much in previous studies, making it some-
what difficult to compare our results with previous studies done on this parameter.
For HRQoL in the AndoSan™ group, which is a broad coverage of the patient’s well-being,
there was a persistent an even improved effect on bodily pain (29% reduction), vitality (21%),
social functioning (15%) and mental health (9%). Although no within-group significance for
physical functioning and role limitation, there was a significant effect in favor of the AndoSan™
compared with the placebo group.
In line with a previous study [34] in which 10 UC patients received the same amount of
AndoSan™ for 12 days, there were no alterations in general blood parameters including CRP.
However, contrary to this study there was a significant reduction of fecal calprotectin in those
patients during the study period. However, in the placebo group for men in the present study
there was, on the other hand, a significant increase in level of fecal calprotectin (p = 0,019),
implying a prospective stabilizing effect on calprotectin levels in the AndoSan™ group that was
not seen for the controls. One reason for lack of reduction of fecal calprotectin in this study
could be the large variability of baseline calprotectin levels (range 10–6000) that was not seen
in the previous small pilot study (128–1683).
The AndoSan™ and placebo groups had similar baseline values with respect to symptom
score, fatigue and quality of life. The groups were also comparable with regard to disease

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 12 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

duration, extent of disease, disease pattern, arthralgia and relevant comorbidity, and thereby
well-fitted for comparison. Hence, putatively increased symptoms tolerance in patients with
extensive or prolonged disease experience, should not affect the SF-36 results.
The patients did not report any obvious clinical side-effects from consumption of the mush-
room extract, which is similar to clinical studies in patients with chronic hepatitis B [40] or
hepatitis C infection [24] where liver function was either normalized or unaltered. However,
although other causative factors such as cancer chemotherapy and hepatitis virus could not be
ruled out, consumption of Agaricus blazei extract for days to months may have induced severe
hepatic dysfunction in three patients receiving concomitant chemotherapy for breast and ovar-
ian cancer [41]. In a phase I study different doses (1.8 g, 3.6 g and 5.8 g) of AbM granulated
powder was consumed in orally for 6 months in 78 patients with different cancers [42].
Adverse effects were observed in 12%, mainly nausea and diarrhea. Only in one case with ovar-
ian cancer receiving six cycles of chemotherapy (Paclitaxel/Carboplatin) after surgery, general-
ized urticaria with papulae and moderate liver dysfunction occurred after two months’ AbM
consumption, which was the definitive cause as judged by a positive lymphocyte AbM stimula-
tion test. However, in another recent clinical trial done with AndoSan™ or the same placebo as
used here, as supplementary treatment 60 ml/day over 7 weeks to high-dose chemotherapy and
bone marrow transplantation for 40 patients with multiple myeloma, there were no side effects,
neither during nor after the trial [43]. Hence, AndoSan™ is proven to be a safe product per se
for very different categories of patients. However, caution is advised for possible interaction
with some drugs as referred below.
Herb-drug interactions are associated with cytochrome P-450 metabolism in the liver and
the trans-membrane efflux pump P-glycoprotein (P-gp) that is present in normal intestinal
lumen where it may limit drug absorption, as well as in the liver, where it may increase excre-
tion of the drug [44]. In this respect, AndoSan™ (called Agaricus from Japan) has previously
been investigated by another independent researchers at the Department of Cancer Research
and Molecular Medicine, Norwegian University of Science and Technology, for in vitro inhibi-
tory potential on P-gp-mediated transport in an intestinal cell line. It was found that AndoSan™
inhibited P-gp in vitro in a similarly as did green tea. Because the mushroom may interact with
some drugs beeing P-gp substrates (i.e. vinblastine, loperamide, digitoxin, cyclosporine, verap-
amil) concomitant AbM should not be given to patients using these drugs. When tested in
vitro for inhibition potential on cytochrome P-450 (CYP3A4 isoform) the AndoSan™ extract
was found to inhibit it but far less than green tea [45]. The researchers [45] concluded that
“clinical relevant systemic or intestinal interactions with CYP3A4 were considered unlikely”.
In our clinical study, none of the UC patients were concomitantly treated with the above men-
tioned anticancer-, heart-, immunodepressive- or diarrhea drugs.
The notion of a potential anti-inflammatory effect after intake of AndoSan™ was a result of
the surprising finding of reduced serum pro-inflammatory cytokines (IL-1ß, IL-6, IL-8) and
chemokines (MCP-1, G-CSF, GM-CSF) in a pilot safety study with AndoSan™ in healthy indi-
viduals without a placebo control [25]. Recently, a steroid 4-hydroxy-17-methylincisterol
(4-HM) [12] isolated from AbM dose-dependently suppressed the synthesis in PHA-stimu-
lated peripheral blood mononuclear cells of cytokines IL-2, IL-4, IFNγ and TNFα by decreasing
both NF-AT (nuclear factor of activated T-cells), which belongs to a family of transcription fac-
tors required for activation and proliferation of T lymphocytes including production of the
first three aforementioned cytokines, and NF-κB—the latter being the “mother” of all inflam-
mation. Substances isolated from AbM had several anti-inflammatory effects in rats, related to
IL-1β, TNFα and IL-8 modulations [11,46]. In a study on healthy volunteers ingesting Ando-
San™ [47] there was a reduction in vivo of ROS mainly reflecting superoxide ions, and again
pointing to an anti-inflammatory effect. However, this result was not demonstrated in the UC

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 13 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

patients. The reason for reduced superoxide anions may be related to reduction of IL-1ß
because inhibitors of ROS reduce synthesis of this cytokine in macrophages [48].
There also was an anti-allergic effect in mice sensitized to ovalbumin (OVA), regarding
reduction of specific anti-OVA IgE antibodies, both when AndoSan™ was given before or after
the OVA immunization [49]. Additionally, in this allergy model there was an increase in Th1
relative to Th2 cytokines in spleen cell cultures ex vivo obtained from the animals treated with
AndoSan™ [49]. Moreover, the inhibitory effect of an isolated carbohydrate fraction of Ando-
San™ [22] on the tissue degrading enzyme legumain (aspariginyl endopeptidase), which proba-
bly activates proMMP and processing of cathepsins may also contribute to less pro-
inflammatory activity in the UC patients.
It is commonly believed that carbohydrates larger than monosaccharides are not absorbed
from the human gut. However, in murine models [13, 50], uptake of β-1,3 glucans across the
gut wall, probably by microfold cells (M cells) but also by dendritic cells (DC) [51], has been
demonstrated. The β-glucan may further be transported by DC to lymphocytes in GALT, but
also circulated in blood in rodents [52, 53]. Presumably, a similar mechanism is operating in
humans for intestinal absorption of small immunomodulatory bioactive β-glucan fragments
into the lymphoid system and blood. As mentioned, other yet unidentified small immunomod-
ulatory substances in the mixed mushroom extract are probably also playing a role in this con-
text. This assumption is supported by the fact that when molecules <12.5 kDa, and thus
smaller than β-glucans, were dialyzed away from the AndoSan™ extract prior to performing the
experiments in the mouse allergy model, the anti-allergic effect of the extract was diminished to
no longer statistically significant levels [49]. Hence, since the anti-allergic effect of AbM extract
seems to be owing to low-molecular-weight substances in the mouse allergy model, other
smaller and simpler substances than ß-glucans in AbM could very well be as important for the
mushroom’s biological effect in other settings such as UC. In addition, AbM also contains small
molecular anti-oxidant and anti-inflammatory substances that may be absorbed actively or by
diffusion through the enterocytes in the gut. A key to understanding the function of a changed
or down-regulated cytokine response locally in the gut-wall, is probably the signals mediated by
DC [54] after processing and presenting to CD4 T helper cells of native antigens from the
mushrooms or novel bacteria-derived antigens resulting from mushroom-microbiota interac-
tions. As seen from animal models [49, 55], the AbM extracts seem to drive the Th1/Th2 bal-
ance towards an increased Th1 response, which also inhibits Treg cells and Th17 cells [14].
In conclusion, the results support that AndoSan™ may be beneficial as a supplement to con-
ventional medication in UC patients with mild to moderate disease activity because of
improvements in symptoms, fatigue and quality of life. There is reason to assume that Ando-
San™ may stabilize these patients with subsequently less need for increased medical treatment,
which may be associated with potentially troublesome and harmful side effects.

Supporting Information
S1 Fig. Flow chart UC kopi.
(TIF)
S1 CONSORT Checklist. CONSORT 2010 Checklist.
(DOC)
S1 Protocol. Study Protocol Norwegian/English version.
(DOCX)
S2 Protocol. Study Protocol English version.
(DOCX)

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 14 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

Acknowledgments
The skillful statistical guidance by professor Leiv Sandvik, Oslo Center for Biostatistics and
Epidemiology, Oslo University Hospital, Norway, is gratefully acknowledged.

Author Contributions
Conceived and designed the experiments: SPT EJ. Performed the experiments: SPT EJ. Ana-
lyzed the data: SPT EJ. Contributed reagents/materials/analysis tools: SPT GH EJ. Wrote the
paper: SPT GH EJ. Inclusion of patients and review of manuscript: IL. Laboratory setup and
review of manuscript: TL.

References
1. Wasser SP, Weis AL (1999) Therapeutic effects of substances occurring in higher Basidiomycetes
mushrooms: a modern perspective. Crit Rev Immunol 19: 65–96. PMID: 9987601
2. Lee EW, Shizuki K, Hosokawa S, Suzuki M, Suganuma H, Inakuma T, et al. (2000) Two novel diterpe-
noids, erinacines H and I from the mycelia of Hericium erinaceum. Biosci Biotechnol Biochem 64:
2402–5. doi: 10.1271/bbb.64.2402 PMID: 11193408
3. Adachi Y, Okazaki M, Ohno N, Yadomae T (1994) Enhancement of cytokine production by macro-
phages stimulated with (1—>3)-beta-D-glucan, grifolan (GRN), isolated from Grifola frondosa. Biol
Pharm Bull 17: 1554–60. PMID: 7537572
4. Fujimiya Y, Suzuki Y, Katakura R, Ebina T (1999) Tumor-specific cytocidal and immunopotentiating
effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei
Murill. Anticancer Res 19: 113–8. PMID: 10226531
5. Kawagishi H, Inagaki R, Kanao T, Mizuno T, Shimura K, Ito H, et al. (1989) Fractionation and antitumor
activity of the water-insoluble residue of Agaricus blazei fruiting bodies. Carbohydr Res 186: 267–73.
PMID: 2736561
6. Kawagishi H, Nomura A, Yumen T, Mizuno T, Hagiwara T, Nakamura T (1988) Isolation and properties
of a lectin from the fruiting bodies of Agaricus blazei. Carbohydr Res 183: 150–4. PMID: 3233595
7. Takaku T, Kimura Y, Okuda H (2001) Isolation of an antitumor compound from Agaricus blazei Murill
and its mechanism of action. J Nutr 131: 1409–13. PMID: 11340091
8. Endo M, Beppu H, Akiyama H, Wakamatsu K, Ito S, Kawamato Y, et al. (2010) Agaritine purified from
Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells. Biochim Biophys Acta 1800:
669–73. doi: 10.1016/j.bbagen.2010.03.016 PMID: 20347942
9. Oh TW, Kim YA, Jang WJ, Byeon JI, Ryu CH, Kim JO, et al. (2010) Semipurified fractions from the sub-
merged-culture broth of Agaricus blazei Murill reduce blood glucose levels in streptozotocin-induced
diabetic rats. J Agric Food Chem 58: 4113–9. doi: 10.1021/jf9036672 PMID: 20196600
10. Izawa S, Inoue Y (2004) A screening system for antioxidants using thioredoxin-deficient yeast: discov-
ery of thermostable antioxidant activity from Agaricus blazei Murill. Appl Microbiol Biotechnol 64: 537–
42. doi: 10.1007/s00253-003-1467-4 PMID: 14593506
11. Padilha MM, Avila AA, Sousa PJ, Cardoso LG, Perazzo FF, Carvalho JC (2009) Anti-inflammatory
activity of aqueous and alkaline extracts from mushrooms (Agaricus blazei Murill). J Med Food 12:
359–64. doi: 10.1089/jmf.2008.0177 PMID: 19459738
12. Tsai WJ, Yang SC, Huang YL, Chen CC, Chuang KA, Kuo YC (2013) 4-Hydroxy-17-methylincisterol
from Agaricus blazei Decreased Cytokine Production and Cell Proliferation in Human Peripheral Blood
Mononuclear Cells via Inhibition of NF-AT and NF-kappaB Activation. Evid Based Complement Alternat
Med 2013: 435916. doi: 10.1155/2013/435916 PMID: 23533483
13. Firenzuoli F, Gori L, Lombardo G (2008) The Medicinal Mushroom Agaricus blazei Murrill: Review of
Literature and Pharmaco-Toxicological Problems. Evid Based Complement Alternat Med 5: 3–15. doi:
10.1093/ecam/nem007 PMID: 18317543
14. Hetland G, Johnson E, Lyberg T, Kvalheim G (2011) The Mushroom Agaricus blazei Murill Elicits
Medicinal Effects on Tumor, Infection, Allergy, and Inflammation through Its Modulation of Innate Immu-
nity and Amelioration of Th1/Th2 Imbalance and Inflammation. Adv Pharmacol Sci 2011: 157015. doi:
10.1155/2011/157015 PMID: 21912538
15. Sorimachi K, Akimoto K, Ikehara Y, Inafuku K, Okubo A, Yamazaki S (2001) Secretion of TNF-alpha,
IL-8 and nitric oxide by macrophages activated with Agaricus blazei Murill fractions in vitro. Cell Struct
Funct 26: 103–8. PMID: 11482452

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 15 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

16. Bernardshaw S, Hetland G, Ellertsen LK, Tryggestad AM, Johnson E (2005) An extract of the medicinal
mushroom Agaricus blazei Murill differentially stimulates production of pro-inflammatory cytokines in
human monocytes and human vein endothelial cells in vitro. Inflammation 29: 147–53. doi: 10.1007/
s10753-006-9010-2 PMID: 17091395
17. Forland DT, Johnson E, Tryggestad AM, Lyberg T, Hetland G (2010) An extract based on the medicinal
mushroom Agaricus blazei Murill stimulates monocyte-derived dendritic cells to cytokine and chemo-
kine production in vitro. Cytokine 49: 245–50. doi: 10.1016/j.cyto.2009.09.002 PMID: 20036142
18. Liu Y, Zhang L, Zhu X, Wang Y, Liu W, Gong W (2015) Polysaccharide Agaricus blazei Murill stimulates
myeloid derived suppressor cell differentiation from M2 to M1 type, which mediates inhibition of tumour
immune-evasion via the Toll-like receptor 2 pathway. Immunology 146: 379–91. doi: 10.1111/imm.
12508 PMID: 26194418
19. Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM (2003) Collaborative induction of
inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med 197: 1107–17. doi: 10.1084/
jem.20021787 PMID: 12719479
20. Vetvicka V, Thornton BP, Ross GD (1996) Soluble beta-glucan polysaccharide binding to the lectin site
of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state
of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98: 50–
61. doi: 10.1172/jci118777 PMID: 8690804
21. Ben Nasr A, Haithcoat J, Masterson JE, Gunn JS, Eaves-Pyles T, Klimpel GR (2006) Critical role for
serum opsonins and complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in phagocy-
tosis of Francisella tularensis by human dendritic cells (DC): uptake of Francisella leads to activation of
immature DC and intracellular survival of the bacteria. J Leukoc Biol 80: 774–86. doi: 10.1189/jlb.
1205755 PMID: 16857732
22. Berven L, Karppinen P, Hetland G, Samuelsen AB (2015) The polar high molecular weight fraction of
the Agaricus blazei Murill extract, AndoSan, reduces the activity of the tumor-associated protease,
legumain, in RAW 264.7 cells. J Med Food 18: 429–38. doi: 10.1089/jmf.2014.0018 PMID: 25136950
23. Ellertsen LK, Hetland G, Johnson E, Grinde B (2006) 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. Int Immunopharmacol 6: 133–43. doi: 10.1016/j.intimp.2005.07.007 PMID: 16399618
24. Grinde B, Hetland G, Johnson E (2006) Effects on gene expression and viral load of a medicinal extract
from Agaricus blazei in patients with chronic hepatitis C infection. Int Immunopharmacol 6: 1311–4.
doi: 10.1016/j.intimp.2006.04.005 PMID: 16782544
25. Johnson E, Forland DT, Saetre L, Bernardshaw SV, Lyberg T, Hetland G (2009) Effect of an extract
based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leu-
kocyte growth factors in human blood ex vivo and in vivo. Scand J Immunol 69: 242–50. doi: 10.1111/j.
1365-3083.2008.02218.x PMID: 19281536
26. Banks C, Bateman A, Payne R, Johnson P, Sheron N (2003) Chemokine expression in IBD. Mucosal
chemokine expression is unselectively increased in both ulcerative colitis and Crohn's disease. J
Pathol 199: 28–35. doi: 10.1002/path.1245 PMID: 12474223
27. Mahida YR, Wu K, Jewell DP (1989) Enhanced production of interleukin 1-beta by mononuclear cells
isolated from mucosa with active ulcerative colitis of Crohn's disease. Gut 30: 835–8. PMID: 2787769
28. Woywodt A, Ludwig D, Neustock P, Kruse A, Schwarting K, Jantschek G, et al. (1999) Mucosal cyto-
kine expression, cellular markers and adhesion molecules in inflammatory bowel disease. Eur J Gas-
troenterol Hepatol 11: 267–76. PMID: 10333199
29. Hyams JS, Fitzgerald JE, Treem WR, Wyzga N, Kreutzer DL (1993) Relationship of functional and anti-
genic interleukin 6 to disease activity in inflammatory bowel disease. Gastroenterology 104: 1285–92.
PMID: 7683293
30. Murch SH, Lamkin VA, Savage MO, Walker-Smith JA, MacDonald TT (1991) Serum concentrations of
tumour necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 32: 913–7. PMID:
1885073
31. McClane SJ, Rombeau JL (1999) Cytokines and inflammatory bowel disease: a review. JPEN J Paren-
ter Enteral Nutr 23: S20–4. PMID: 10483888
32. Sato K, Chiba T, Ohkusa T (2009) Serial changes of cytokines in active ulcerative colitis: effects of anti-
biotic combination therapy. Hepatogastroenterology 56: 1016–21. PMID: 19760932
33. Korolkova OY, Myers JN, Pellom ST, Wang L, M'Koma AE (2015) Characterization of Serum Cytokine
Profile in Predominantly Colonic Inflammatory Bowel Disease to Delineate Ulcerative and Crohn's Coli-
tides. Clin Med Insights Gastroenterol 8: 29–44. doi: 10.4137/CGast.S20612 PMID: 26078592
34. Forland DT, Johnson E, Saetre L, Lyberg T, Lygren I, Hetland G (2011) Effect of an extract based on
the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 16 / 17

 Clinical Effects of AndoSan™ on Patients with Ulcerative Colitis

with ulcerative colitis and Crohn's disease. Scand J Immunol 73: 66–75. doi: 10.1111/j.1365-3083.
2010.02477.x PMID: 21129005
35. Roseth AG, Fagerhol MK, Aadland E, Schjonsby H (1992) Assessment of the neutrophil dominating
protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 27: 793–8. PMID: 1411288
36. Rachmilewitz D (1989) Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treat-
ment of active ulcerative colitis: a randomised trial. BMJ 298: 82–6. PMID: 2563951
37. Loge JH, Kaasa S (1998) Short form 36 (SF-36) health survey: normative data from the general Norwe-
gian population. Scand J Soc Med 26: 250–8. PMID: 9868748
38. Loge JH, Ekeberg O, Kaasa S (1998) Fatigue in the general Norwegian population: normative data and
associations. J Psychosom Res 45: 53–65. PMID: 9720855
39. Fagerland MW, Sandvik L (2009) Performance of five two-sample location tests for skewed distribu-
tions with unequal variances. Contemp Clin Trials 30(5): 490–6. doi: 10.1016/j.cct.2009.06.007 PMID:
19577012
40. Hsu CH, Hwang KC, Chiang YH, Chou P (2008) The mushroom Agaricus blazei Murill extract normal-
izes liver function in patients with chronic hepatitis B. J Altern Complement Med 14: 299–301. doi: 10.
1089/acm.2006.6344 PMID: 18370584
41. Mukai H, Watanabe T, Ando M, Katsumata N (2006) An alternative medicine, Agaricus blazei, may
have induced severe hepatic dysfunction in cancer patients. Jpn J Clin Oncol 36: 808–10. doi: 10.
1093/jjco/hyl108 PMID: 17105737
42. Ohno S, Sumiyoshi Y, Hashine K, Shirato A, Kyo S, Inoue M (2011) Phase I Clinical Study of the Die-
tary Supplement, Agaricus blazei Murill, in Cancer Patients in Remission. Evid Based Complement
Alternat Med 2011: 192381. doi: 10.1155/2011/192381 PMID: 21584278
43. Tangen JM, Tierens A, Caers J, Binsfeld M, Olstad OK, Troseid AM, et al. (2015) Immunomodulatory
effects of the Agaricus blazei Murrill-based mushroom extract AndoSan in patients with multiple mye-
loma undergoing high dose chemotherapy and autologous stem cell transplantation: a randomized,
double blinded clinical study. BioMed research international 2015: 718539. doi: 10.1155/2015/718539
PMID: 25664323
44. Engdal S, Nilsen OG (2008) Inhibition of P-glycoprotein in Caco-2 cells: effects of herbal remedies fre-
quently used by cancer patients. Xenobiotica 38: 559–73. doi: 10.1080/00498250801986969 PMID:
18570158
45. Engdal S, Nilsen OG (2009) In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer
patients. Phytother Res 23: 906–12. doi: 10.1002/ptr.2750 PMID: 19170155
46. Wang P, Li XT, Sun L, Shen L (2013) Anti-Inflammatory Activity of Water-Soluble Polysaccharide of
Agaricus blazei Murill on Ovariectomized Osteopenic Rats. Evid Based Complement Alternat Med
2013: 164817. doi: 10.1155/2013/164817 PMID: 24348690
47. Johnson E, Forland DT, Hetland G, Saetre L, Olstad OK, Lyberg T (2012) Effect of AndoSan on expres-
sion of adhesion molecules and production of reactive oxygen species in human monocytes and granu-
locytes in vivo. Scand J Gastroenterol 47: 984–92. doi: 10.3109/00365521.2012.660544 PMID:
22564240
48. O'Neill LA (2008) Immunology. How frustration leads to inflammation. Science 320: 619–20. PMID:
18451288
49. Ellertsen LK, Hetland G (2009) An extract of the medicinal mushroom Agaricus blazei Murill can protect
against allergy. Clin Mol Allergy 7: 6. doi: 10.1186/1476-7961-7-6 PMID: 19416507
50. Chan Y, Chang T, Chan CH, Yeh YC, Chen CW, Shieh B, et al. (2007) Immunomodulatory effects of
Agaricus blazei Murill in Balb/cByJ mice. J Microbiol Immunol Infect 40: 201–8. PMID: 17639159
51. Coombes JL, Powrie F (2008) Dendritic cells in intestinal immune regulation. Nat Rev Immunol 8: 435–
46. doi: 10.1038/nri2335 PMID: 18500229
52. Ikuzawa M, Matsunaga K, Nishiyama S, Nakajima S, Kobayashi Y, Andoh T, et al. (1988) Fate and dis-
tribution of an antitumor protein-bound polysaccharide PSK (Krestin). Int J Immunopharmacol 10:
415–23. PMID: 3170055
53. Sakurai T, Hashimoto K, Suzuki I, Ohno N, Oikawa S, Masuda A, et al. (1992) Enhancement of murine
alveolar macrophage functions by orally administered beta-glucan. Int J Immunopharmacol 14: 821–
30. PMID: 1512075
54. Sartor RB (2010) Genetics and environmental interactions shape the intestinal microbiome to promote
inflammatory bowel disease versus mucosal homeostasis. Gastroenterology 139: 1816–9. doi: 10.
1053/j.gastro.2010.10.036 PMID: 21029802
55. Takimoto H, Kato H, Kaneko M, Kumazawa Y (2008) Amelioration of skewed Th1/Th2 balance in
tumor-bearing and asthma-induced mice by oral administration of Agaricus blazei extracts. Immuno-
pharmacol Immunotoxicol 30: 747–60. doi: 10.1080/08923970802279092 PMID: 18720167

PLOS ONE | DOI:10.1371/journal.pone.0150191 March 2, 2016 17 / 17

II

a11111
OPEN ACCESS
Citation: Therkelsen SP, Hetland G, Lyberg T,
Lygren I, Johnson E (2016) Effect of the Medicinal
Agaricus blazei Murill-Based Mushroom Extract,
AndoSanTM, on Symptoms, Fatigue and Quality of
Life in Patients with Crohn’s Disease in a
Randomized Single-Blinded Placebo Controlled
Study. PLoS ONE 11(7): e0159288. doi:10.1371/
journal.pone.0159288
Editor: John Green, University Hospital Llandough,
UNITED KINGDOM
Received: March 9, 2016
Accepted: June 30, 2016
Published: July 14, 2016
Copyright: © 2016 Therkelsen et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by grants from
the Faculty of Medicine, University of Oslo.
Competing Interests: Two of the authors (Geir
Hetland and Egil Johnson) have patent/patent
applications and financial interests relating to material
(AndoSan™) pertinent to this article: i)
WO2005065063 A2, Appl. No.:10/585600, NO- and
PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016
RESEARCH ARTICLE
Effect of the Medicinal Agaricus blazei Murill-
Based Mushroom Extract, AndoSanTM, on
Symptoms, Fatigue and Quality of Life in
Patients with Crohn’s Disease in a
Randomized Single-Blinded Placebo
Controlled Study
Stig Palm Therkelsen1*, Geir Hetland2, Torstein Lyberg3, Idar Lygren4, Egil Johnson1,5
1 Department of Gastrointestinal and Pediatric Surgery, Oslo University Hospital, Ullevål, Oslo, Norway,
2 Immunology and Transfusion Medicine, Oslo University Hospital, Ullevål, Oslo, Norway, 3 Medical
Biochemistry, Oslo University Hospital, Ullevål, Oslo, Norway, 4 Medicine, Oslo University Hospital, Ullevål,
Oslo, Norway, 5 Faculty of Medicine, University of Oslo, Oslo, Norway
* stig.therkelsen@ous-hf.no
Abstract
Background
Ingestion of AndoSanTM, based on the mushroom Agaricus blazei Murill, has previously
shown an anti-inflammatory effect through reduction of pro-inflammatory cytokines in
healthy individuals and patients with Crohn’s disease (CD). In this randomized single-
blinded placebo-controlled study we examined whether intake of AndoSanTM also resulted
in clinical effects.
Methods and Findings
50 patients with symptomatic CD were randomized for oral daily consumption of Ando-
SanTM or placebo for a 21-day experimental period, in this per-protocol study. Patients
reported validated 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 AndoSanTM group (n = 25) symptoms improved from baseline (day 0) to days 14 and
21, with respective mean scores (95% CI) of 5.52 (4.64–6.40), 4.48 (3.69–5.27) and 4.08
(3.22–4.94) (p<0,001). We found significant improvements in symptom score for both gen-
ders in the AndoSanTM group, and no significant changes in the placebo (n = 25) group.
There were however no significant differences between the groups (p = 0.106), although a
marginal effect in symptom score for men (p = 0.054). There were comparable improve-
ments in physical, mental and total fatigue for both groups. HRQoL versus baseline were at
day 21 improved for bodily pain and vitality in the AndoSanTM group and for vitality and
social functioning in the placebo group. No crucial changes in general blood samples and
fecal calprotectin were detected.
1 / 17

PCT-filed Jan 2004 and Jan 2005, respectively, by
Inventor Hetland Geir, and ii) NO20090003383, Appl.
No.: NO20090003383 20091119, by Inventors
Hetland Geir and Johnson Egil and filed by Applicant
Immunopharma AS in Nov 2009 and financial interest
of Geir Hetland as shareholder in Immunopharma AS
of Norway, commercializing AndoSan™. This does
not alter the authors’ adherence to PLOS ONE
policies on sharing data and materials.
WO2005065063 – “Use of the mushroom Agaricus
blazei Murill for the production of medicaments
suitable for treating infections and allergies”.
PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016
Clinical Effects of AndoSan on Patients with Crohn's Disease
Conclusions
The results from this single-blinded randomized clinical trial shows significant improvement
on symptoms, for both genders, in the AndoSanTM group, but no significant differences
between the study groups. The results on fatigue, HRQoL, fecal calprotectin and blood sam-
ples were quite similar compared with placebo. The patients did not report any harms or unin-
tended effects of AndoSanTM. CD patients with mild to moderate symptoms may have
beneficiary effects of AndoSanTM as a safe supplement in addition to conventional medication.
Trial Registration
ClinicalTrials.gov NCT01496053
1. Introduction
A mushroom Agaricus blazei Murill (AbM) has for centuries been utilized as a health
food ingredient by the local population in the Piedade area in Brazil, where prevalence of ath-
erosclerosis, hyperlipidemia, diabetes and cancer was lower than in neighboring regions [1],
presumably owing to AbM consumption. In 1966 the mushroom was brought to Japan and
introduced to the health food market. Since then, AbM and other Basidiomycetes mushrooms
[2, 3] have been subjected to an increasing research effort regarding their effects.
AbM per se and the AbM-based mushroom extract, AndoSanTM (ACE Co. Ltd., Gifu-ken,
Japan), composed of AbM (82.4%), Hericium erinaceus (He) (14.7%) [2] and Grifola frondosa
(Gf) (2.9%) [3], contain immunomodulatory ß-glucans and other biologically active substances
like α-glucans [4], proteoglucans [5], lectins [6], ergosterol (provitamin D2) [7], agaritine [8],
isoflavonoids [9], anti-oxidant [10], and anti-inflammatory substances [11] including the
4-HM steroid [12].
Depending on the experimental set up, AbM or the AbM-based extract AndoSanTM, com-
prise as reviewed [13, 14] anti-tumor, anti-allergic, and anti-inflammatory effects in vivo.
AndoSanTM is an extract of the mushrooms´ mycelium and not their fruiting bodies and
was recently shown to contain less ß-glucan [15] than anticipated from the published data on
AbM fruiting body. Therefore, action also of other yet not identified immunomodulating sub-
stances in this particular extract must part-take to render the observed effects. An example is
an isolated fraction of AndoSanTM that was found to inhibit the production in macrophages of
the tumor-associated and pro-inflammatory protease, legumain [15].
In patients with Crohn’s disease (CD) increased mucosal levels have been demonstrated for
MIP-1ß, MCP-1 and IL-8 [16], IL-1ß [17], IL-6 and TNFα [18]. Cytokine levels in serum are
less well studied but increased levels have been reported for IL-6 [18] and TNFα [19, 20].
Moreover, in a recent extensive review [21] the cytokines IFNγ, IL-6, IL-7 and IL-8 were con-
sidered to be persistently elevated in blood of CD patients compared with findings in healthy
individuals.
In 11 patients with CD who consumed the mushroom extract AndoSanTM for 12 days [22]
cytokine levels were reduced in untreated (IL-2, IL-8, IL-17) and in LPS-stimulated blood ex
vivo (IL-1ß, MIP-1ß, MCP-1, IL-8, IL-17, G-CSF and GM-CSF). Then, the next step was to
examine whether a decline in pathological levels of cytokines mediated by the mushroom
extract in vivo, did result in a putative beneficial clinical effect in patients with CD.
We have in a recent single-blinded randomized placebo controlled study, in which patients
with ulcerative colitis received this mushroom extract (AndoSanTM) for three weeks [23],
2 / 17
 Clinical Effects of AndoSan on Patients with Crohn's Disease

demonstrated improvements in symptoms, fatigue and HRQoL compared with patients in the
placebo group.
On this background, it was pertinent to study whether consumption of AndoSanTM had
similar effects in patients with CD.

2. Materials and Methods

2.1. Reagents
The mushroom extract AndoSanTM was provided by the company Immunopharma AS (orga-
nization no. 994924273), Oslo, Norway. It was stored at 4°C in metal cans and used under ster-
ile conditions ex vivo and kept sterile until taken by volunteers for in vivo experiments. This
mushroom extract is a commercial product produced by the company ACE Co. Ltd., Gifu-ken,
Japan, for Immunopharma AS. The AbM mixed powder contains per 100 g the following con-
stituents: moisture 5.8 g, protein 2.6 g, fat 0.3 g, carbohydrates 89.4 g, of which ß-glucan consti-
tutes 2.8 g, and ash 1.9 g. The AndoSanTM extract contains 82.4% of Basidiomycetes mushroom
derived from AbM, 14.7% from He [2] and 2.9% from Gf [3], and its final concentration was
340 g / l. The amount per litre of the extract was for sodium 11 mg, phosphorus 254 mg, cal-
cium 35 mg, potassium 483 mg, magnesium 99 mg and zinc 60 mg. The LPS (lipopolysaccha-
ride) content of AndoSanTM was found, using the Limulus amebocyte lysate test (COAMATIC
Chromo-LAL; Chromogenix, Falmouth, MA, USA) with detection limit 0.005 EU / ml (1
EU = 0.1 ng / ml), to be a miniscule concentration of <0.5 pg / ml. AndoSanTM had been heat-
sterilized (124°C for 1 h) by the producer. Several tests from Japan Food Research Laboratories
(authorized by the Japanese Government) were done in March 2012, December 2013, October
2014, April 2015 and February 2016. The tests were for pH, arsenic, lead, cadmium, tin, aerobic
plate count, coliform bacteria (MPN), viable molds count, viable yeasts count, mesophilic aero-
bic spores, refractometric brix degree and specific gravity (15°C)–and all of the results were
within the quantitation limits. AndoSanTM also passed the water quality test (no bacteria,
acceptable level of ions, pH, taste, color and odor). An accelerated aging test (up to four
months) with almost unchanged character of the mushroom drink. AndoSanTM were also
tested for radioactivity, with no detection of Cesium-137, Cesium-134 and Iodine-131 (Meiji
Co, Japan). In addition, the Norwegian Food Safety Authorities found no radioactivity.

2.2. Analyses
Blood was harvested from the antecubital vein into glass tubes containing 15 IU heparin per ml
or 10 mmol EDTA per ml. The EDTA blood was each time (days 0, 14 and 21) analyzed for
haemoglobin, haematocrite, mean cellular volume, mean cellular haemoglobin, reticulocytes,
immature reticulocytes, leukocytes including a differential count of neutrophils, basophils,
eosinophils, lymphocytes and monocytes, thrombocytes, C-reactive protein (CRP), urea, creat-
inine, bilirubin, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase,
γ-glutamine transferase, alkaline phosphatase and pancreatic amylase. Fecal calprotectin con-
centrations (mg/kg) (normal value <50 mg/kg) at days 0, 14 and 21 were determined in dupli-
cates as reported [24, 25].
The patient-reported symptom score was the simple Crohn’s disease Activity Index
(SCDAI) also denounced the Harvey-Bradshaw index [26]. The simple index is based on five
graded items; general well-being (very well = 0, slightly below par = 1, poor = 2, very poor = 3,
terrible = 4), abdominal pain (none = 0, mild = 1, moderate = 2, severe = 3), number of liquid
stools per day (1 = 0, 2 = 1, 3–4 = 2, 5–6 = 3, 7–9 = 4, > 9 = 5), abdominal mass (this item was
not examined) and extraintestinal manifestations (arthralgia, uveitis, erythema nodosum,
aphthous ulcers, pyoderma gangrenosum, anal fissure, new fistula, abscess (score 1 per item)).

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 3 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

The symptom score ranges from 0–21. Scores 3–5 meant mild, 6–9 moderate and over 9 severe
disease activity. A criterion for inclusion was a score beyond 2.
Self reported health-related quality of life (HRQoL) was assessed with the short form 36
(IQOLA SF-36 Norwegian version 1.2), which is a generic HRQoL questionnaire consisting of
36 items, of which 35 are grouped into the following eight health domains: (1) physical func-
tioning (PF), (2) social functioning (SF), (3) role limitations due to physical problems (RP), (4)
role limitation due to emotional problems (RE), (5) mental health (MH), (6) vitality (VT), (7)
bodily pain (BP) and (8) general health perception (GH). Each domain is graded on a scale of
0–100, and the higher the score the better the HRQoL. The validity and reliability of the SF-36
form have been demonstrated for a number of countries including Norway (version 1) [27].
The data were compared with published norms from 2323 individuals in the general popula-
tion. Only 30 out of 5400 HRQoL questions were unanswered, and accordingly, 17 out of 1200
dimensions were lacking. Using a scoring algorithm for missing data outlined in the SF 36 sur-
vey manual, still 5 out of 17 dimensions involving 3 patients in the placebo group, could not be
included in the results.
Fatigue consists of total fatigue (11 items of graded questions with score 0–3 per question),
which is the sum of physical fatigue (7 items) and mental fatigue (4 items), which has been vali-
dated in a Norwegian general population [28]. The respective scores for total, mental and phys-
ical fatigue are 0–33, 0–21 and 0–12, and the higher score the more fatigue. The items of
physical (1–7) and mental (8–11) fatigue were: 1) Do you have problems with tiredness? 2) Do
you need to rest more? 3) Do you feel sleepy or drowsy? 4) Do you have problems with starting
things? 5) Are you lacking in energy? 6) Do you have less strength in your muscles? 7) Do you
feel weak? 8) Do you have difficulty concentrating? 9) Do you have problems thinking clearly?
10) Do you make slips of the tongue when speaking? 11) How is your memory? Criteria for
chronic fatigue syndrome was a dichotomized score >4 and duration>6 months.

2.3. Inclusion of Patients

173 patients with CD were phone interviewed and those with SCDAI score of at least 3 were
given the opportunity to join the study. At the first attendance SCDAI was re-recorded and cri-
teria for exclusion were pregnancy, biological treatment with antibodies to TNFα (Adalimu-
mab, Infliximab), daily use of more than 5 mg of prednisolone, change of medication and/or
consumption of mushroom products from two weeks before till end of the study. A flow chart
reveals additional reasons for exclusions (Fig 1). The 23 excluded patients in the initial screen-
ing mentioned as “other reasons” were: 10 could not participate because of not able to attend, 5
had moved to a different part of the country, 3 never showed up, and 1 proved to be pregnant,
and one each had an ileostomy with high output, drug abuse, severe comorbidity or language
difficulties.

2.4. Experimental Design and Randomization

This is a single-center randomized two-armed patient-blinded study designed to determine
whether daily oral intake of a mushroom extract, AndoSanTM, improved clinical symptoms,
fatigue and quality of life in patients with CD during the 21 days’ study period. The patients
were evaluated before (visit 1, day 0), during (visit 2, day 14) and after (visit 3, day 21) con-
sumption of AndoSanTM (30 ml twice daily). This dose (60 ml/day) reduced levels of pro-
inflammatory cytokines and chemokines in healthy volunteers [24] and in patients with UC
and CD [34], whilst half dosage (30 ml/day) had no detectable effects (unpublished data). The
placebo group was evaluated likewise but received an equal volume of color-like drink with
ionized water containing 0.5 ml per litre of caramel color (E150c) with salt.

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 4 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

Fig 1. An algorithm showing the scheme for inclusion of the patients in the study.
doi:10.1371/journal.pone.0159288.g001

Block-randomization was done after the phone interview, with uneven and even numbers
given for AndoSanTM or placebo, respectively. The patients, one by one, were placed in one
pile, and the group affiliations were placed in another pile. The randomization was performed
by combining one selection from each pile, both anonymized. The first author performed the
randomization, enrolled the participants, and assigned participants to interventions. A few
patients were excluded throughout the study by not attending or because of intercurrent inci-
dents (disease, unexpected life events). Accordingly, a slight imbalance of the study groups

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 5 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

occurred that was corrected for in the latter rounds of randomization. More specifically, the 50
CD patients were divided into 13 groups (range 1–9 per group), each with a study period of 3
weeks. The included 50 symptomatic patients had almost no missing data (only for 3 patients
with missing a total of 5 out of 1200 dimensions for the SF-36 results) and were randomized
and blinded for oral daily consumption (30 ml twice daily) of AndoSanTM or placebo for the 21
days’ experimental period. Patients in the AndoSanTM group and the placebo group self-
reported, in written, at visit 1 (day 0), visit 2 (day 14) and visit 3 (day 21) regarding symptoms,
fatigue and health-related quality of life. Patient derived blood samples and fecal calprotectin
from these visits were also analyzed. All data were stored in a secure database (Access–Micro-
soft Office) at a server at Oslo University Hospital, Ullevål, Norway. A study number anon-
ymized the patients.
This study on clinical outcome is a follow up of a previous pilot study [22] in which there
was a reduction of pro-inflammatory cytokines and chemokines in patients receiving the same
daily dose of AndoSanTM, but for 12 days. Prospective differences of 20% between the experi-
mental and placebo group and assumed standard deviation of 20% for the different parameters
with a significant level of 5% and a power of 90% (ß = 0.10), demands about 25 patients per
randomized arm (calculated in cooperation Oslo Center for Biostatistics and Epidemiology,
Oslo University Hospital).

2.5. Patient Characteristics

Disease duration for the CD patients was 9.7 years (range 0.5–46) and 8.0 years (range 0.5–42)
in the AndoSanTM and placebo groups, respectively. Disease location and behavior, number
and type of resections as well as proportions of patients subjected to surgery were registered
(Table 1). There were 30 and 35 extra-intestinal manifestations in 21 patients in the Ando-
SanTM group and 22 patients in the placebo group, respectively. Comorbidities, exclusively in
the AndoSanTM group, were Mb Bechterew in 3, diabetes mellitus in 1 and chronic obstructive
lung disease in 1 patient(s). Combinations of 5-ASA, azathioprine and budesonide or predniso-
lone were consumed in 3 and 2 patients, respectively. Topical treatment with 5-ASA was used
in 3 patients in the AndoSanTM group.

2.6. Statistical Analysis

Data are presented as mean and standard deviation or as median and range values. Paired sam-
ple t-test and Wilcoxon test were used for within-group analysis. The judgment of whether the
distributions of the main efficacy variables were so close to the normal distribution that nor-
mality-based significance tests may be used, also for each individual index at baseline that com-
pose the SCDAI, is based on the finding in a paper by Fagerland and Sandvik [29]. Mixed
models corrected for baseline values were used for measuring P values between the AndoSanTM
and placebo groups, using V1, V2 and V3 with time as a continuous variable. P values below
0.05 were considered statistically significant. The SPSS statistical program for the social sci-
ences, version 22 (IBM) was used in the analyses.

2.7. Ethical Considerations

The study was approved on April 8, 2011, by the regional ethics committee (REC–South East
Norway, ref. 2011/404) and followed the guidelines of the Helsinki declaration. The partici-
pants were informed and signed a written consent for participation, including the option of
study withdrawal. The patients were recruited and followed up at the Department of Medicine,
Oslo University Hospital, Ullevål, Norway, in the period of June 2012 to May 2014. The
study was registered with unique protocol ID AbM2012-IBD and clinical trials gov ID NCT

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 6 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

Table 1. Demographic and patient data.

AndoSanTM Placebo P
Number 25 25 1.00
Age (years) 41.0 (25–74) 42.0 (22–70) 0.59
Gender (male, female) 11, 14 10, 15 0.78
Duration of diagnosis (years) 9.7 (0.5–46) 8.0 (0.5–42) 0.68
Disease location
L1 terminal ileum 11 8 0.38
L2 colon 5 5 1.00
L3 ileocolon 9 12 0.39
Behavior
B1 inflammatory 12 19 0.04
B2 stricturing 12 5 0.04
B3 perforating 0 1 0.31
Resection
Ileal 6 0 0.01
Ileocolic 13 5 0.02
Colic 1 4 0.16
Total (number of resections (patients)) 20 (9) 9 (6) 0.002 (0.35)
Medication during the study period
None 7 11 0.24
5-ASA/Azathioprin 9 8 0.77
Low dose of steroids 4 4 1.00
Several medications 3 2 0.64
Topical treatment 3 0 0.07
Extra-intestinal manifestations
None 4 3 0.68
Arthralgia 17 17 1.00
Anal fissures 7 7 1.00
Aphtous ulcers 3 6 0.27
Erythema nodosum 2 2 1.00
Anal fistula 0 2 0.15
Pyoderma gangrenosum 1 0 0.32
Enteric fistula 1 0 0.32

Values for age and duration of diagnosis are given as median (range). P values between groups is measured with unadjusted chi square test, and Mann-
Whitney U test for age and gender.

doi:10.1371/journal.pone.0159288.t001

01496053 (December 15, 2011). The authors confirm that all ongoing and related trials for this
drug/intervention are registered.

3. Results
3.1. Exclusion of randomized patients
A total of 76 patients, 37 in the AndoSanTM group and 39 in the placebo group, were random-
ized for inclusion in this study. 26 of these patients were excluded according to the criteria of
the study protocol, because of missing data on symptom score, laboratory data and not attend-
ing in 20, 4 and 1, patient(s), respectively. Thereby we ended up with 25 patients in the Ando-
SanTM group and 25 in the placebo group (Fig 1).

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 7 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

Table 2. Symptom score (SCDAI) for the CD patients.

V1 V2 V3 PV1V2 PV1V3 Pbetween groups
AndoSanTM 5.52 (4.64–6.40) 4.48 (3.69–5.27) 4.08 (3.22–4.94) 0.001 <0.001 0.106
M (n = 11) 5.05 (4.07–6.11) 3.64 (2.72–4.55) 3.27 (1.99–4.55) 0.014 0.011
F (n = 14) 5.85 (4.41–7.30) 5.14 (3.95–6.34) 4.71 (3.52–5.90) 0.035 0.014
Placebo 5.04 (4.49–5.59) 4.52 (3.72–5.32) 4.68 (3.92–5.44) 0.119 0.327
M (n = 10) 4.70 (4.11–5.29) 4.50 (2.74–6.26) 4.80 (3.23–6.37) 0.764 0.885
F (n = 15) 5.27 (4.37–6.17) 4.53 (3.65–5.42) 4.53 (3.62–5.44) 0.044 0.085

V1; visit 1 (day 0), V2; visit 2 (day 14), V3; visit 3 (day 21).
Values are given as means and 95% confidence intervals. Paired sampled t-test for the p-values.
P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0159288.t002

3.2. Age and Gender

Median age for the 50 included patients with CD was 41 years (range 22–74). There were 11
men and 14 women in the AndoSanTM group and 10 men and 15 women in the placebo group.
Respective ages in the two groups were median 42 (range 22–70) and 44.5 (28–74) for men
(p = 0.611) and 43 (26–69) and 38 (25–61) for women (p = 0.36).

3.3. Symptom Score

The symptom scores were similar at inclusion in the AndoSanTM and placebo groups, with
respective mean scores of 5.52 and 5.04. There were no significant differences in baseline symp-
tom scores between male and females within the two groups. Compared with baseline there
were in the AndoSanTM group increasing reductions of symptom score for both genders from
visit 2 (day 14) to visit 3 (day 21) (Table 2). In the placebo group, only for women there was a
reduction of symptom score from baseline to visit 2 but not visit 3. When comparing the two
groups using mixed models corrected for baseline values for both genders there was no differ-
ence (p = 0.106). However, for men, there was a close to significant difference (p = 0.054) in
favor of the mushroom group.
Within the AndoSanTM group there was for both genders a significant improvement in
stool frequency with scores 2.04, 1.56 (p = 0.01) and 1.20 (p<0.01) at visit 1–3, respectively.
There also was a trend for both genders towards reduction of abdominal pain with scores 1.12
and 0.88 (p = 0.06) at visit 1 vs. 3, with significant values for men (p = 0.04). When using
mixed models corrected for baseline values there was a significant difference between the study
groups for stool frequency (p = 0.011), in favor of the AndoSanTM group, but not for abdomi-
nal pain (p = 0.36).

3.4. Fatigue Score

Firstly, the normative fatigue scores in the Norwegian population were compared with scores
in the CD patients at inclusion in this study (Table 3). There were for both genders significant
decreases of physical, mental and total fatigue scores in the CD patients compared with the
general population. This effect was much more pronounced for physical fatigue than for men-
tal fatigue.
Twenty of the 50 patients (40%) had chronic fatigue at visit 1 vs. about 11% in the normative
population [28]. The scores for genders on inclusion were quite similar within and between the
groups (data not shown). In the AndoSanTM group (Table 4) for both genders the CD patients
reported a significant decline in total and physical fatigue, but not mental fatigue, from baseline

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 8 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

Table 3. Mean fatigue scale scores. Normative data in the Norwegian population compared with the included CD patients.
Normative data CD PNormative data vs UC
M (n = 1112) F (n = 1175) M (n = 21) F (n = 29) M F
Total 11.9 12.6 16.43 17.76 <0.0001 <0.0001
(3.9) (4.0) (4.48) (5.87)
Physical 7.6 8.2 11.38 12.14 <0.0001 <0.0001
(3.0) (3.2) (3.69) (4.14)
Mental 4.3 4.4 5.05 5.62 0.0152 0.0052
(1.4) (1.4) (1.40) (2.16)

Normative data from the general Norwegian population, age 19–80.

Values are given as mean and standard deviation (SD). Independent sample t-test for the p-values.

doi:10.1371/journal.pone.0159288.t003

to visit 3 (day 21). Mental fatigue was transiently improved at visit 2 but not at visit 3. In the
placebo group, however, the three aspects of fatigue was improved both at visit 2 and 3.
Moreover, when comparing the AndoSanTM and placebo groups using mixed models cor-
rected for baseline values there were no differences between the two groups regarding the three
fatigue scores.

3.5. Quality of Life

HRQoL scores (SF-36) for CD patients were compared with age-adjusted normative data for
the Norwegian population (Table 5). Men had significant reduction in scores of all 8 dimen-
sions whilst women had similar results with the exception of physical functioning (PF), which
was within the normal range. The reductions of quality life scores in the CD patients were for
both genders most pronounced for the dimensions vitality (VT), general health (GH) and
role limitations, physical (RP) (p values < 0.0001) as compared with the general population.
In the AndoSanTM group as a whole the HRQoL score were at visit 3 significantly improved
(Table 6) for bodily pain (BP) and VT, whilst VT and SF were improved in the placebo group.
When broken into gender the respective improvements at visit 3 were BP for men and SF
for women in the AndoSanTM group versus none in the placebo group (data not shown). There
were no significant differences when comparing the two groups, using mixed models corrected
for baseline values.

Table 4. Fatigue scores for the patients with (n = 25 AndoSanTM and n = 25 placebo) CD.
AndoSanTM group Placebo group
V1 V2 V3 PV1V2 PV1V3 V1 V2 V3 PV1V2 PV1V3 PBetween groups
TF 16.40 15.28 14.00 0.128 0.032 18.00 16.28 15.36 0.011 0.011 0.813
(5.07) (4.86) (5.10) (5.55) (4.97) (4.65)
PhF 11.08 10.48 9.04 0.289 0.016 12.56 11.32 10.64 0.029 0.016 0.187
(3.51) (3.74) (3.66) (4.26) (4.00) (4.01)
MF 5.32 4.80 4.96 0.045 0.265 5.44 4.96 4.72 0.008 0.031 0.824
(1.97) (1.56) (1.77) (1.83) (1.43) (1.10)

V1; visit 1 (day 0), V2; visit 2 (day 14), V3; visit 3 (day 21).
TF; total fatigue, PhF; physical fatigue, MF; mental fatigue.
Values are given as mean and standard deviation (SD). Paired sampled t-test for p-values.
P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0159288.t004

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 9 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

Table 5. Mean SF-36 scale scores. Age-adjusted Normative Data from the Norwegian Population compared with patients with CD on inclusion.
Normative data Crohn´s disease PNormative data vs CD
M (n = 977–1017) F (n = 1013–67) M (n = 21) F (n = 29) M F
PF 91.37 (16.19) 87.72 (17.53) 83.97 (15.33) 84.48 (15.49) 0.0383 0.3250
RP 83.27 (31.98) 79.18 (34.99) 51.19 (42.92) 31.90 (35.92) 0.0028 <0.0001
BP 78.06 (24.61) 74.46 (26.00) 58.86 (21.36) 55.07 (20.04) 0.0004 <0.0001
GH 78.34 (20.98) 77.46 (22.14) 46.19 (23.70) 46.98 (21.92) <0.0001 <0.0001
VT 63.36 (18.19) 57.57 (21.00) 34.52 (21.56) 32.26 (14.74) <0.0001 <0.0001
SF 88.23 (20.52) 84.78 (22.79) 68.45 (27.28) 62.05 (22.94) 0.0036 <0.0001
RE 85.89 (28.47) 80.94 (33.10) 66.67 (38.99) 65.52 (43.17) 0.0361 0.0144
MH 79.74 (15.75) 77.64 (16.85) 71.24 (17.46) 65.57 (19.30) 0.0148 0.0002

Age-adjusted normative data from the general Norwegian population, age 19–69.
Values are given as mean and standard deviation (SD). Independent sample t-test for the p-values.
SF-36; Short form 36, PF; physical functioning, RP; role limitations, physical, BP; bodily pain, GH; general heath perception, VT; Vitality, SF; social
functioning, RE; role limitations, emotional, MH; mental health.

doi:10.1371/journal.pone.0159288.t005

3.6. Calprotectin in Feces and Effect on General Blood Parameters

The patients delivered fecal tests at visits 1, 2 and 3. In the AndoSanTM group (n = 25) the
median (range) values for fecal calprotectin (mg/kg) were 394 (17–6000), 398 (20–2244) and
472 (36–1623), respectively. In the placebo group the corresponding values were 293 (21–
2783), 515 (12–6000) and 342 (10–4659). There were no significant differences in levels of cal-
protectin within or between the groups, also when broken into gender (data not shown).
The following blood samples were analyzed at visit 1and 3: CRP, leukocytes, eosinophils,
basophils, neutrophils, lymphocytes, monocytes, hemoglobin, haematocrite, mean cellular

Table 6. Mean SF-36 scale scores (n = 25 AndoSanTM and n = 25 placebo) CD.

AndoSanTM group Placebo group
V1 V2 V3 PV1V2 PV1V3 V1 V2 V3 PV1V2 PV1V3 PAvsP
PF 85.80 84.60 83.80 0.417 0.470 82.73 85.89 87.20 0.126 0.072 0.083
(14.8) (14.4) (14.7) (15.9) (13.7) (14.4)
RP 41.00 54.00 49.00 0.040 0.088 39.00 39.00 42.00 1.000 0.671 0.120
(43.2) (39.3) (44.2) (36.9) (37.6) (40.7)
BP 53.96 58.88 63.16 0.108 0.028 59.54 58.50 60.71 0.805 0.764 0.292
(20.3) (18.6) (22.0) (20.8) (17.3) (18.0)
GH 50.64 48.52 51.08 0.396 0.886 42.66 45.08 46.32 0.328 0.156 0.454
(23.4) (23.6) (26.1) (21.1) (19.5) (18.5)
VT 32.53 40.00 39.20 0.008 0.045 33.96 36.32 42.92 0.400 0.019 0.777
(20.0) (21.5) (22.9) (15.6) (17.8) (18.7)
SF 65.50 74.00 72.00 0.021 0.152 64.06 66.67 72.40 0.447 0.032 0.537
(25.8) (20.7) (21.1) (24.3) (22.3) (24.7)
RE 69.33 65.33 73.33 0.372 0.503 62.50 56.94 58.33 0.405 0.588 0.358
(41.9) (41.4) (40.8) (40.9) (43.4) (42.0)
MH 69.28 71.04 69.76 0.440 0.836 66.67 71.00 71.00 0.057 0.164 0.392
(17.1) (13.3) (18.0) (20.3) (17.5) (19.0)

Paired sampled t-test for the p-values.

P between groups is measured with mixed models corrected for baseline values.

doi:10.1371/journal.pone.0159288.t006

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 10 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

volume, mean cellular haemoglobin, immature reticulocytes, reticulocytes, thrombocytes, urea,

creatinine, and GFR (glomerular filtration rate), bilirubin, aspartate aminotransferase, alanine
aminotransferase, lactate dehydrogenase, γ-glutamine transferase, alkaline phosphatase and
pancreatic amylase. Significant changes were for reduction of bilirubin (μmol/L) from 11.4 to
9.2 (p = 0.02) in the AndoSanTM group and for increase of thrombocytes (109/L) from 289 to
302 (p = 0.03) in the placebo group.
The median and range of blood samples of special interest were haemoglobin (g/l), leuko-
cyte counts (109/l) and CRP levels for visit 1 and 3 the AndoSanTM group (n = 25) were 13.4
(12.0–16.2) versus 13.3 (11.4–16.0), 6.1 (3.1–12.2) versus 6.8 (2.7–13.0) and 2.9 (0.6–30.9) ver-
sus 2.1 (0.6–25.3), respectively. Corresponding values in the placebo group (n = 25) were 13.7
(10.5–15.5) versus 13.6 (10.7–15.5) for haemoglobin, 6.6 (4.2–13.8) versus 6.7 (3.9–11.0) for
leukocytes and 2.2 (0.6–41.6) versus 2.1 (0.6–34.8) for CRP. Accordingly, there were no statisti-
cal changes of these parameters neither within nor between the groups.

4. Discussions
The main finding in this placebo-controlled patient blinded prospective study was that the immu-
nomodulatory Agaricus blazei Murill-based mushroom extract AndoSanTM [14] increasingly
improved clinical symptoms in patients of both genders during a 3 weeks’ study period. In the pla-
cebo group there was an improvement of symptoms exclusively for women, although less than in
the AndoSanTM group and not significant. There were no significant differences between the study
groups, but a close to significant p-value (0.054) when comparing the symptom score for men.
Of the four items (general well-being, abdominal pain, number of liquid stools per day,
complications) of the SCDAI [26] there were improvements of key symptoms such as of num-
ber of liquid stools for both genders and pain for men. Interestingly, despite the trend in the
AndoSanTM group towards more stricturing disease (p = 0.04), less inflammatory disease
(p = 0.04), significantly more ileal (p = 0.01) and ileocolic resections (p = 0.02) (Table 1), which
implied increased disease activity vs. the placebo group, the improvement of symptoms as a
whole was only evident in the former group.
Compared with the normal population the CD patients had considerably more fatigue,
especially physical fatigue probably owing to the somatic manifestations of this chronic granu-
lomatous disease. However, we were not able to demonstrate any advantage using AndoSanTM
vs. placebo on outcome of fatigue. At visit 3 there was as a whole an improvement in mental-,
physical- and total fatigue in the placebo group vs. only the two latter fatigue scores in the
AndoSanTM group (Table 4). Thus, despite external factors that presumably could influence
mental fatigue due to intercurrent subclinical and mental disease, we conclude that the mush-
room extract vs. placebo had no demonstrable effect on fatigue in these patients.
For HRQoL in the AndoSanTM group as a whole there was an improvement in bodily pain
at visit 3, which is interesting because pain was the second item of the SCDAI that was signifi-
cantly reduced for men at visit 3. Common to the AndoSanTM and placebo groups were the
improvements of vitality, whilst social functioning also was improved in the latter group. With
the exception of improvement in bodily pain, which is a crucial factor in patients with CD suf-
fering from intestinal and other symptoms, we conclude that the effect of AndoSanTM on
HRQoL was not different from placebo. Besides a reduction of bilirubin in the AndoSanTM
group and an increase of thrombocytes in the placebo group there was as reported [22] in a
one-armed pilot study with 12 days’ AndoSanTM consumption in CD patients, no alterations
in general blood parameters including CRP as well as fecal calprotectin.
In a recent similar placebo-controlled study [23] of patients with UC consuming Ando-
SanTM, the improvements in addition to symptoms irrefutably also were evident for fatigue

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 11 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

and HRQoL. Obviously, clinical improvement due to intake of this mushroom extract in
patients with CD was more limited vs. those with UC. This may partly be explained by the fact
that CD is pan-intestinal and characterized by transmural inflammation complicated by steno-
sis and/or development of fistulas in addition to more systemic manifestations (e.g. joints and
anal fissures).
The AndoSanTM and placebo groups had similar baseline values with respect to symptom
score, fatigue and quality of life. The groups were also quite comparable with regard to disease
duration, arthralgia and relevant comorbidity. However, there were significantly more resec-
tions in the AndoSanTM group (20 vs. 9) and, accordingly, more stricturing and severe disease.
Analyses of symptom score and fecal calprotectin were similar when we did calculations com-
paring the patients with inflammatory and stenotic presentation (data not shown).
The patients did not discontinue consumption of the mushroom extract throughout the
study, since there were no side effects and normal blood samples, including liver function. As
recently outlined [30], AndoSanTM and AbM per se have been well tolerated by the patients in
several clinical studies for hepatitis B [31] and C [30] and different cancers (breast, ovarian,
myeloma) [32–34]. AbM induced temporary urticaria and moderate liver dysfunction devel-
oped after two months of AbM consumption [33], in one out of 78 patients only, which also
received chemotherapy for ovarian cancer. With our knowledge of AndoSanTM and AbM, and
review of the literature, we believe it is safe as a supplement to patients with different types of
disease.
The patients were not blinded for the authors leaving possible bias with respect to different
attitude towards patients in the two groups. This is especially true for the first author who was
responsible for the inclusion and randomization of participants, the implementation of the
practical aspects of and in meeting with the patients, and also in the analysis of the results. This
is a relatively small study, although with some significant results, with its limitations. Reduced
compliance in carrying out the study, with missing or incorrect oral intake of AndoSanTM or
placebo, may be a possible source of error, even though this was not the impression in conver-
sation with patients during and after the study period of three weeks.
Regarding possible drug interactions, AndoSanTM did less than green tea inhibit the trans-
membrane efflux P-gp pump present in intestines and liver and hence important for drug
absorption and excretion [35]. However, possible interactions may occur with P-gp substrates,
e.g. some anti-cancer, diarrhea (loperamide) and cardiac (digoxin) agents, and P-gp inhibitors,
e.g. verapamil. When testing AndoSanTM on cytochrome P-450 metabolism, the extract inhib-
ited it, but far less than green tea and clinically relevant systemic interactions were therefore
considered unlikely [36]. In our clinical study, none of the CD patients were concomitantly
treated with the above-mentioned anticancer-, heart-, or diarrhea drugs.
There are several studies of Agaricus blazei Murill, Hericium erinaceus and Grifola frondosa,
isolated or in the mixture together as AndoSanTM, showing beneficial immunomodulatory,
antioxidant, antihyperglycemic and anti-tumor effects [13, 14, 37–39]. However, most data, to
date, were produced using rodents or cell cultures, and there are a very limited number of stud-
ies measuring their effects in humans.
In a placebo-controlled study in patients with gynecological cancer, AbM treatment in addi-
tion to chemotherapy was reported to increase NK cell activity in blood and improved the
patients quality of life [40].
A randomized double-blind placebo-controlled study on 30 critically ill ICU patients, fol-
lowed for 7 days, found significant increases in NK cell activities when given immune-enhanc-
ing enteral nutrition (IMHP) enriched with ß-glucan from the baseline and significantly
greater increase than the control group [41]. The authors suggest that ß-glucan can be an
attractive candidate to add to IMPH for stimulation of protective immunity without enhanced

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 12 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

inflammation in critically ill patients. This finding is in line with previous studies in which ß-
glucan enhanced NK cell activation in mice [42, 43].
A randomized double-blinded clinical trial in 57 elderly females found no immunomodula-
tory effect as measured by unaltered plasma levels of IL-6, TNFα and IFNγ after ingesting
dried AbM capsules for a 60-day study period [44].
The notion of a potential anti-inflammatory effect of AndoSanTM intake was a result of the
surprising finding of reduced serum pro-inflammatory cytokines (IL-1ß, IL-6, IL-8) and che-
mokines (MCP-1, G-CSF, GM-CSF) in a pilot safety study with AndoSanTM in healthy individ-
uals without a placebo control [24].
In a study on healthy volunteers ingesting AndoSanTM [37] there was a reduction in vivo of
ROS mainly reflecting superoxide ions, and again pointing to an anti-inflammatory effect.
However, this result was not demonstrated in the CD patients in the aforementioned pilot
study (data not shown). The reason for reduced superoxide anions may be related to reduction
of IL-1ß because inhibitors of ROS reduce synthesis of this cytokine in macrophages [45].
Oral administration of AndoSanTM is associated with low bioavailability due to polysaccha-
rides, like ß-glucan, that is normally not taken up from the GI-tract as it is a non-degradable
cellulose (the human tract normally only takes up monosaccharides). However, Rice et al [46]
showed that soluble ß-glucans are able to bind directly and undergo internalization by intesti-
nal epithelial cells and gut associated lymphoid tissue (GALT) cells. The internalization of solu-
ble ß-glucan by intestinal epithelial cells is not dependent on dectin-1, however in GALT cells
dectin-1 and TLR-2 participate in uptake of soluble ß-glucan.
Recently, a steroid 4-hydroxy-17-methylincisterol (4-HM) [12] isolated from AbM dose-
dependently suppressed the synthesis in PHA-stimulated peripheral blood mononuclear cells
of cytokines IL-2, IL-4, IFNγ and TNFα by decreasing both NF-AT (nuclear factor of activated
T-cells), which belongs to a family of transcription factors required for activation and prolifera-
tion of T lymphocytes including production of the first three aforementioned cytokines, and
NF-κB—the latter being the “mother” of all inflammation.
Alkaline and aqueous substances isolated from AbM [11] had, when given orally to rats for
1–2 weeks, several anti-inflammatory effects such as improved healing of stress-induced ulcers
and reductions of paw edema in the presence of nystatin or Freund’s adjuvant, as well as
reduced neutrophil migration to the peritoneal cavity. It was speculated that these effects in
part were related to modulations of cytokine levels for TNFα and IL-8. In another recent study
[47] a water soluble polysaccharide isolated from AbM was given orally for 8 weeks to ovarec-
tomized and osteopenic rats, and it markedly decreased serum levels of IL-1β, TNFα, ICAM-1
and total antioxidant status. AbM contains absorbable low-molecular weight anti-oxidant sub-
stances [10] that down-regulate the levels of reactive oxygen species (ROS) in vitro. Type 1 dia-
betes has similarities to CD because it has been regarded both as an autoimmune and as an
innate inflammatory disease affecting the pancreas [48]. Anecdotes of possible benefits of AbM
for diabetes in folk medicine have been supported by findings in a rodent model for diabetes
[9]. Since the hypoglycemic effect of AbM seems to result from its suppression of oxidative
stress and pro-inflammatory cytokine production [38], similar therapeutically mechanisms
probably play a role in CD patients who responded positively on the AndoSanTM treatment in
the current study.
There also was an anti-allergic effect in mice sensitized to ovalbumin (OVA), as demon-
strated by reduction of specific anti-OVA IgE antibodies, both when AndoSanTM was given
before or after the OVA immunization [49]. In this allergy model also there was an increase in
Th1 relative to Th2 cytokines in spleen cell cultures ex vivo obtained from the animals treated
with AndoSanTM [49]. In addition, the inhibitory effect of an isolated carbohydrate fraction of
AndoSanTM [15] on the tissue degrading pro-inflammatory and tumor-associated enzyme,

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 13 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

legumain (aspariginyl endopeptidase), that probably may also contribute to less inflammatory
activity in the CD patients.
Pharmaceutical investigation of AndoSanTM revealed the carbohydrate content to be only
2% of the ~5mg dry material/ml, and that it was concentrated in the polar high molecular
weight fraction of AndoSanTM [15]. It was this fraction that was the most potent inhibitor of
legumain [15]. The mushroom extract also contains some not yet characterized proteins (per-
sonal communication, prof G Vegarud at The Norwegian University of Life Sciences). As to
the pharmacokinetics of AndoSanTM that is a mixed water extract of mycelium to the afore-
mentioned three Basidiomycetes mushrooms, it has biological effects when taken orally both in
mice and men. Ingredients in AndoSanTM may execute their effects directly after absorption
through intestinal mucosa enterocytes to the blood and processing in the liver, where Kuppfer
cells and endothelial cells may be stimulated similar to our previous findings in monocyte/mac-
rophage and endothelial cell cultures [50, 51]. However, substances in the mushroom extract
may have a greater indirect influence on the body by inducing changes in the microbiota and
production of analytes by bacteria after their uptake of sugar moieties for energy etc.. More-
over, substances such as β-glucan my further be transported by dendritic cells to lymphocytes
in GALT and induce local immune responses there, or systemic if circulated in blood [52, 53].
In conclusion, we found increasingly improved symptom score in the AndoSanTM group, of
both genders, foremost regarding stool frequency and abdominal pain. Compared with placebo
there was a significant reduction in stool frequency, favoring the AndoSanTM group. For
fatigue, quality of life, calprotectin in feces and blood samples there were comparable results
between the study groups. We suggest that AndoSanTM may be used as a safe supplement to
conventional medication to relive symptoms in these patients. At present, the effects on cyto-
kine levels from AndoSanTM consumption in these patients are being studied.

Supporting Information
S1 Table. CONSORT 2010 Checklist.
(DOC)
S1 Text. Study Protocol Norwegian version.
(DOCX)
S2 Text. Study Protocol English version.
(DOCX)

Acknowledgments
This work was supported by grants from the Faculty of Medicine, University of Oslo.
The skillful statistical guidance by professor Leiv Sandvik, Oslo Center for Biostatistics and
Epidemiology, Oslo University Hospital, Norway, is gratefully acknowledged.

Author Contributions
Conceived and designed the experiments: EJ GH TL IL. Performed the experiments: SPT EJ.
Analyzed the data: SPT EJ. Contributed reagents/materials/analysis tools: SPT EJ GH TL.
Wrote the paper: SPT EJ GH. Recruitment of patients: IL SPT.

References
1. Wasser SP, Weis AL (1999) Therapeutic effects of substances occurring in higher Basidiomycetes
mushrooms: a modern perspective. Crit Rev Immunol 19: 65–96. doi: PMID: 9987601

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 14 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

2. Lee EW, Shizuki K, Hosokawa S, Suzuki M, Suganuma H, Inakuma T, et al. (2000) Two novel diterpe-
noids, erinacines H and I from the mycelia of Hericium erinaceum. Biosci Biotechnol Biochem 64:
2402–5. doi: 10.1271/bbb.64.2402 PMID: 11193408
3. Adachi Y, Okazaki M, Ohno N, Yadomae T (1994) Enhancement of cytokine production by macro-
phages stimulated with (1—>3)-beta-D-glucan, grifolan (GRN), isolated from Grifola frondosa. Biol
Pharm Bull 17: 1554–60. PMID: 7537572
4. Fujimiya Y, Suzuki Y, Katakura R, Ebina T (1999) Tumor-specific cytocidal and immunopotentiating
effects of relatively low molecular weight products derived from the basidiomycete, Agaricus blazei
Murill. Anticancer Res 19: 113–8. PMID: 10226531
5. Kawagishi H, Kanao T, Inagaki R, Mizuno T, Shimura K, Ito H, et al. (1990) Formolysis of a potent anti-
tumor (1 ! 6)-β-d-glucan-protein complex from Agaricus blazei fruiting bodies and antitumor activity of
the resulting products. Carbohydrate Polymers 12: 393–403. http://dx.doi.org/10.1016/0144-8617(90)
90089-B
6. Kawagishi H, Nomura A, Yumen T, Mizuno T, Hagiwara T, Nakamura T (1988) Isolation and properties
of a lectin from the fruiting bodies of Agaricus blazei. Carbohydr Res 183: 150–4. PMID: 3233595
7. Takaku T, Kimura Y, Okuda H (2001) Isolation of an antitumor compound from Agaricus blazei Murill
and its mechanism of action. J Nutr 131: 1409–13. PMID: 11340091
8. Endo M, Beppu H, Akiyama H, Wakamatsu K, Ito S, Kawamoto Y, et al. (2010) Agaritine purified from
Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells. Biochim Biophys Acta 1800:
669–73. doi: 10.1016/j.bbagen.2010.03.016 PMID: 20347942
9. Oh TW, Kim YA, Jang WJ, Byeon JI, Ryu CH, Kim JO, et al. (2010) Semipurified fractions from the sub-
merged-culture broth of Agaricus blazei Murill reduce blood glucose levels in streptozotocin-induced
diabetic rats. J Agric Food Chem 58: 4113–9. doi: 10.1021/jf9036672 PMID: 20196600
10. Izawa S, Inoue Y (2004) A screening system for antioxidants using thioredoxin-deficient yeast: discov-
ery of thermostable antioxidant activity from Agaricus blazei Murill. Appl Microbiol Biotechnol 64: 537–
42. doi: 10.1007/s00253-003-1467-4 PMID: 14593506
11. Padilha MM, Avila AA, Sousa PJ, Cardoso LG, Perazzo FF, Carvalho JC (2009) Anti-inflammatory
activity of aqueous and alkaline extracts from mushrooms (Agaricus blazei Murill). J Med Food 12:
359–64. doi: 10.1089/jmf.2008.0177 PMID: 19459738
12. Tsai WJ, Yang SC, Huang YL, Chen CC, Chuang KA, Kuo YC (2013) 4-Hydroxy-17-methylincisterol
from Agaricus blazei Decreased Cytokine Production and Cell Proliferation in Human Peripheral Blood
Mononuclear Cells via Inhibition of NF-AT and NF-kappaB Activation. Evid Based Complement Alternat
Med 2013: 435916. doi: 10.1155/2013/435916 PMID: 23533483
13. Firenzuoli F, Gori L, Lombardo G (2008) The Medicinal Mushroom Agaricus blazei Murrill: Review of
Literature and Pharmaco-Toxicological Problems. Evid Based Complement Alternat Med 5: 3–15. doi:
10.1093/ecam/nem007 PMID: 18317543
14. Hetland G, Johnson E, Lyberg T, Kvalheim G (2011) The Mushroom Agaricus blazei Murill Elicits
Medicinal Effects on Tumor, Infection, Allergy, and Inflammation through Its Modulation of Innate Immu-
nity and Amelioration of Th1/Th2 Imbalance and Inflammation. Adv Pharmacol Sci 2011: 157015. doi:
10.1155/2011/157015 PMID: 21912538
15. Berven L, Karppinen P, Hetland G, Samuelsen AB (2015) The polar high molecular weight fraction of
the Agaricus blazei Murill extract, AndoSan, reduces the activity of the tumor-associated protease,
legumain, in RAW 264.7 cells. J Med Food 18: 429–38. doi: 10.1089/jmf.2014.0018 PMID: 25136950
16. Banks C, Bateman A, Payne R, Johnson P, Sheron N (2003) Chemokine expression in IBD. Mucosal
chemokine expression is unselectively increased in both ulcerative colitis and Crohn's disease. J
Pathol 199: 28–35. doi: 10.1002/path.1245 PMID: 12474223
17. Mahida YR, Wu K, Jewell DP (1989) Enhanced production of interleukin 1-beta by mononuclear cells
isolated from mucosa with active ulcerative colitis of Crohn's disease. Gut 30: 835–8. PMID: 2787769
18. Woywodt A, Ludwig D, Neustock P, Kruse A, Schwarting K, Jantschek G, et al. (1999) Mucosal cyto-
kine expression, cellular markers and adhesion molecules in inflammatory bowel disease. Eur J Gas-
troenterol Hepatol 11: 267–76. PMID: 10333199
19. Murch SH, Lamkin VA, Savage MO, Walker-Smith JA, MacDonald TT (1991) Serum concentrations of
tumour necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 32: 913–7. PMID:
1885073
20. McClane SJ, Rombeau JL (1999) Cytokines and inflammatory bowel disease: a review. JPEN J Paren-
ter Enteral Nutr 23: S20–4. PMID: 10483888
21. Korolkova OY, Myers JN, Pellom ST, Wang L, M'Koma AE (2015) Characterization of Serum Cytokine
Profile in Predominantly Colonic Inflammatory Bowel Disease to Delineate Ulcerative and Crohn's Coli-
tides. Clin Med Insights Gastroenterol 8: 29–44. doi: 10.4137/CGast.S20612 PMID: 26078592

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 15 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

22. Forland DT, Johnson E, Saetre L, Lyberg T, Lygren I, Hetland G (2011) Effect of an extract based on
the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients
with ulcerative colitis and Crohn's disease. Scand J Immunol 73: 66–75. doi: 10.1111/j.1365-3083.
2010.02477.x PMID: 21129005
23. Therkelsen SP, Hetland G, Lyberg T, Lygren I, Johnson E (2016) Effect of a Medicinal Agaricus blazei
Murill-Based Mushroom Extract, AndoSan, on Symptoms, Fatigue and Quality of Life in Patients with
Ulcerative Colitis in a Randomized Single-Blinded Placebo Controlled Study. PLoS One 11:
e0150191. doi: 10.1371/journal.pone.0150191 PMID: 26933886
24. Johnson E, Forland DT, Saetre L, Bernardshaw SV, Lyberg T, Hetland G (2009) Effect of an extract
based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leu-
kocyte growth factors in human blood ex vivo and in vivo. Scand J Immunol 69: 242–50. doi: 10.1111/j.
1365-3083.2008.02218.x PMID: 19281536
25. Roseth AG, Fagerhol MK, Aadland E, Schjonsby H (1992) Assessment of the neutrophil dominating
protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 27: 793–8. PMID: 1411288
26. Harvey RF, Bradshaw JM (1980) A simple index of Crohn's-disease activity. Lancet 1: 514. PMID:
6102236
27. Loge JH, Kaasa S (1998) Short form 36 (SF-36) health survey: normative data from the general Norwe-
gian population. Scand J Soc Med 26: 250–8. PMID: 9868748
28. Loge JH, Ekeberg O, Kaasa S (1998) Fatigue in the general Norwegian population: normative data and
associations. J Psychosom Res 45: 53–65. PMID: 9720855
29. Fagerland MW, Sandvik L (2009) Performance of five two-sample location tests for skewed distribu-
tions with unequal variances. Contemp Clin Trials 30: 490–6. doi: 10.1016/j.cct.2009.06.007 PMID:
19577012
30. Grinde B, Hetland G, Johnson E (2006) Effects on gene expression and viral load of a medicinal extract
from Agaricus blazei in patients with chronic hepatitis C infection. Int Immunopharmacol 6: 1311–4.
doi: 10.1016/j.intimp.2006.04.005 PMID: 16782544
31. Hsu CH, Hwang KC, Chiang YH, Chou P (2008) The mushroom Agaricus blazei Murill extract normal-
izes liver function in patients with chronic hepatitis B. J Altern Complement Med 14: 299–301. doi: 10.
1089/acm.2006.6344 PMID: 18370584
32. Mukai H, Watanabe T, Ando M, Katsumata N (2006) An alternative medicine, Agaricus blazei, may
have induced severe hepatic dysfunction in cancer patients. Jpn J Clin Oncol 36: 808–10. doi: 10.
1093/jjco/hyl108 PMID: 17105737
33. Ohno S, Sumiyoshi Y, Hashine K, Shirato A, Kyo S, Inoue M (2011) Phase I Clinical Study of the Die-
tary Supplement, Agaricus blazei Murill, in Cancer Patients in Remission. Evid Based Complement
Alternat Med 2011: 192381. doi: 10.1155/2011/192381 PMID: 21584278
34. Tangen JM, Tierens A, Caers J, Binsfeld M, Olstad OK, Troseid AM, et al. (2015) Immunomodulatory
effects of the Agaricus blazei Murrill-based mushroom extract AndoSan in patients with multiple mye-
loma undergoing high dose chemotherapy and autologous stem cell transplantation: a randomized,
double blinded clinical study. BioMed research international 2015: 718539. doi: 10.1155/2015/718539
PMID: 25664323
35. Engdal S, Nilsen OG (2008) Inhibition of P-glycoprotein in Caco-2 cells: effects of herbal remedies fre-
quently used by cancer patients. Xenobiotica 38: 559–73. doi: 10.1080/00498250801986969 PMID:
18570158
36. Engdal S, Nilsen OG (2009) In vitro inhibition of CYP3A4 by herbal remedies frequently used by cancer
patients. Phytother Res 23: 906–12. doi: 10.1002/ptr.2750 PMID: 19170155
37. Johnson E, Forland DT, Hetland G, Saetre L, Olstad OK, Lyberg T (2012) Effect of AndoSan on expres-
sion of adhesion molecules and production of reactive oxygen species in human monocytes and granu-
locytes in vivo. Scand J Gastroenterol 47: 984–92. doi: 10.3109/00365521.2012.660544 PMID:
22564240
38. Niwa A, Tajiri T, Higashino H (2011) Ipomoea batatas and Agarics blazei ameliorate diabetic disorders
with therapeutic antioxidant potential in streptozotocin-induced diabetic rats. J Clin Biochem Nutr 48:
194–202. doi: 10.3164/jcbn.10-78 PMID: 21562638
39. Lee JS, Park SY, Thapa D, Choi MK, Chung IM, Park YJ, et al. (2010) Grifola frondosa water extract
alleviates intestinal inflammation by suppressing TNF-alpha production and its signaling. Exp Mol Med
42: 143–54. doi: 10.3858/emm.2010.42.2.016 PMID: 20054232
40. Ahn WS, Kim DJ, Chae GT, Lee JM, Bae SM, Sin JI, et al. (2004) Natural killer cell activity and quality
of life were improved by consumption of a mushroom extract, Agaricus blazei Murill Kyowa, in gyneco-
logical cancer patients undergoing chemotherapy. Int J Gynecol Cancer 14: 589–94. doi: 10.1111/j.
1048-891X.2004.14403.x PMID: 15304151

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 16 / 17

 Clinical Effects of AndoSan on Patients with Crohn's Disease

41. Lee JG, Kim YS, Lee YJ, Ahn HY, Kim M, Kim M, et al. (2016) Effect of Immune-Enhancing Enteral
Nutrition Enriched with or without Beta-Glucan on Immunomodulation in Critically Ill Patients. Nutrients
8. doi: 10.3390/nu8060336 PMID: 27271657
42. Pelizon AC, Kaneno R, Soares AM, Meira DA, Sartori A (2005) Immunomodulatory activities associ-
ated with beta-glucan derived from Saccharomyces cerevisiae. Physiol Res 54: 557–64. PMID:
16238470
43. Yatawara L, Wickramasinghe S, Nagataki M, Takamoto M, Nomura H, Ikeue Y, et al. (2009) Aureobasi-
dium-derived soluble branched (1,3–1,6) beta-glucan (Sophy beta-glucan) enhances natural killer
activity in Leishmania amazonensis-infected mice. Korean J Parasitol 47: 345–51. doi: 10.3347/kjp.
2009.47.4.345 PMID: 19967081
44. Lima CU, Souza VC, Morita MC, Chiarello MD, Karnikowski MG (2012) Agaricus blazei Murrill and
inflammatory mediators in elderly women: a randomized clinical trial. Scand J Immunol 75: 336–41.
doi: 10.1111/j.1365-3083.2011.02656.x PMID: 22010847
45. O'Neill LA (2008) Immunology. How frustration leads to inflammation. Science 320: 619–20. doi: 10.
1126/science.1158398 PMID: 18451288
46. Rice PJ, Adams EL, Ozment-Skelton T, Gonzalez AJ, Goldman MP, Lockhart BE, et al. (2005) Oral
delivery and gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious
challenge. J Pharmacol Exp Ther 314: 1079–86. doi: 10.1124/jpet.105.085415 PMID: 15976018
47. Wang P, Li XT, Sun L, Shen L (2013) Anti-Inflammatory Activity of Water-Soluble Polysaccharide of
Agaricus blazei Murill on Ovariectomized Osteopenic Rats. Evid Based Complement Alternat Med
2013: 164817. doi: 10.1155/2013/164817 PMID: 24348690
48. Skog O, Korsgren S, Melhus A, Korsgren O (2013) Revisiting the notion of type 1 diabetes being a T-
cell-mediated autoimmune disease. Curr Opin Endocrinol Diabetes Obes 20: 118–23. doi: 10.1097/
MED.0b013e32835edb89 PMID: 23422243
49. Ellertsen LK, Hetland G (2009) An extract of the medicinal mushroom Agaricus blazei Murill can protect
against allergy. Clin Mol Allergy 7: 6. doi: 10.1186/1476-7961-7-6 PMID: 19416507
50. Bernardshaw S, Hetland G, Ellertsen LK, Tryggestad AM, Johnson E (2005) An extract of the medicinal
mushroom Agaricus blazei Murill differentially stimulates production of pro-inflammatory cytokines in
human monocytes and human vein endothelial cells in vitro. Inflammation 29: 147–53. doi: 10.1007/
s10753-006-9010-2 PMID: 17091395
51. Forland DT, Johnson E, Tryggestad AM, Lyberg T, Hetland G (2010) An extract based on the medicinal
mushroom Agaricus blazei Murill stimulates monocyte-derived dendritic cells to cytokine and chemo-
kine production in vitro. Cytokine 49: 245–50. doi: 10.1016/j.cyto.2009.09.002 PMID: 20036142
52. Ikuzawa M, Matsunaga K, Nishiyama S, Nakajima S, Kobayashi Y, Andoh T, et al. (1988) Fate and dis-
tribution of an antitumor protein-bound polysaccharide PSK (Krestin). Int J Immunopharmacol 10:
415–23. PMID: 3170055
53. Sakurai T, Hashimoto K, Suzuki I, Ohno N, Oikawa S, Masuda A, et al. (1992) Enhancement of murine
alveolar macrophage functions by orally administered beta-glucan. Int J Immunopharmacol 14: 821–
30. PMID: 1512075

PLOS ONE | DOI:10.1371/journal.pone.0159288 July 14, 2016 17 / 17