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Article
Agaricus blazei-Based Mushroom Extract
Supplementation to Birch Allergic Blood Donors:
A Randomized Clinical Trial
Faiza Mahmood 1,2, Geir Hetland 1, 3, * , Ivo Nentwich 1, Mohammad Reza Mirlashari 1,
Reza Ghiasvand 4and Lise Sofie Haug Nissen-Meyer 1
1Department of Immunology and Transfusion Medicine, Oslo University Hospital, 0407 Oslo, Norway;
faiza.mahmood@ahus.no (F.M.); ivo.nentwich@ous-hf.no (I.N.); uxmoir@ous-hf.no (M.R.M.);
lise.sofie.haug.nissen-meyer@ous-hf.no (L.S.H.N.-M.)
2Department of Immunology and Transfusion Medicine, Akershus University Hospital,
1478 Lørenskog, Norway
3Department of Immunology, Institute of Clinical Medicine, University of Oslo, 0318 Oslo, Norway
4Department of Biostatistics, Institute of Basic Medical Sciences, University of Oslo, 0372 Oslo, Norway;
reza.ghisvand@medisin.uio.no
*Correspondence: geir.hetland@medisin.uio.no; Tel.: +47-2211-8878
Received: 30 July 2019; Accepted: 19 September 2019; Published: 2 October 2019
Abstract:
Since Agaricus blazei Murill (AbM) extract reduced specific IgE and ameliorated a skewed
Th1/Th2 balance in a mouse allergy model, it was tested in blood donors with self-reported,
IgE-positive, birch pollen allergy and/or asthma. Sixty recruited donors were randomized in a
placebo-controlled, double-blinded study with pre-seasonal, 7-week, oral supplementation with the
AbM-based extract Andosan
TM
. Before and after the pollen season, questionnaires were answered for
allergic rhino-conjunctivitis, asthma, and medication; serum IgE was measured, and Bet v 1-induced
basophil activation was determined by CD63 expression. The reported general allergy and asthma
symptoms and medication were significantly reduced in the AbM compared to the placebo group
during pollen season. During the season, there was significant reduction in specific IgE anti-Bet
v 1 and anti-t3 (birch pollen extract) levels in the AbM compared with the placebo group. While
the maximal allergen concentrations needed for eliciting basophil activation before the season,
changed significantly in the placebo group to lower concentrations (i.e., enhanced sensitization) after
the season, these concentrations remained similar in the Andosan
TM
AbM extract group. Hence,
the prophylactic effect of oral supplementation before the season with the AbM-based Andosan
TM
extract on aeroallergen-induced allergy was associated with reduced specific IgE levels during the
season and basophils becoming less sensitive to allergen activation.
Keywords:
Agaricus blazei mushroom; Andosan
TM
; asthma; basophil activation; birch pollen allergy;
clinical trial
1. Introduction
Agaricus blazei Murill (AbM) is a Brazilian relative of champignon, and Hericium erinaceus and
Grifola frondosa from Asia are all medicinal Basidiomycetes mushrooms used as health food and
against cancer [
1
–
3
]. Their antitumor properties are partly immunomodulatory via stimulation by,
e.g.,
β
-glucans of monocytic, natural killer (NK), and T helper 1 cells [
4
–
7
]. The Th1 immune response
is reciprocal to the Th2 response, which promotes allergy and asthma [
8
]. An AbM extract given orally
to asthma-induced and tumor-bearing mice ameliorated their skewed Th1/Th2 balance and reduced
specific IgE and tumor load [
9
]. Andosan
TM
is a Japanese extract of 82% AbM, 15% H. erinaceus, and
Nutrients 2019,11, 2339; doi:10.3390/nu11102339 www.mdpi.com/journal/nutrients
Nutrients 2019,11, 2339 2 of 15
3% G. frondosa that gave anti-allergic effects in a mouse allergy model, as demonstrated by reduced
serum IgE and IgG1 anti-ovalbumin levels and shift from Th2 to Th1 predominant cytokine profiles in
spleen cell cultures [
10
]. We have recently found that Andosan
TM
in drinking water has antitumor
effects in a mouse model for colorectal cancer [
11
]. Thus, both anti-allergic/-asthmatic and antitumor
properties have been demonstrated in vivo for AbM given enterally.
Atopic diseases like rhino-conjunctivitis and allergic bronchial asthma affect 20%–25% of the
population in developed countries [
12
], but prophylactic measures have limited effect, particularly
on aeroallergy [
13
]. The aims for both atopy prophylaxis and treatment are improvement of
clinical symptoms by reversing Th2-dominant atopic phenotype to nonatopic Th1 phenotype.
Immunomodulation by food supplements is a simple treatment for atopy with few side effects,
as previously documented for β-glucans on symptoms of pollen allergy [14,15].
Allergic and asthmatic blood donors can give blood if they are not strongly affected and/or have
not had anaphylactic reactions or taken corticosteroids. Since donors with pollen allergy represent
a select group with regard to symptoms and allowed medication, standard recommendations of
clinical outcome for allergic rhino-conjunctivitis [
16
] cannot be readily used in such a clinical study
of pollen-induced allergy. Therefore, in a blood bank setting, we chose to use a modified (in-house)
questionnaire that also evaluates asthma. In southeastern Norway, the birch pollen season is from
mid-April to mid-May.
Basophilic granulocytes (“basophils”) and tissue mast cells [
17
] are involved in the immediate
type-I allergic reaction, where allergen-specific IgE antibodies in plasma are bound to high-affinity
Fc
ε
receptor (R) I (Fc
ε
RI) on the effector cells. They are then activated by allergen cross-linking of the
bound IgE, which results in degranulation of the cells and release of mediators like histamine and
arachidonic acid metabolites, e.g., prostaglandin D
2
after activation of cyclooxygenase-2 (COX-2), and
proinflammatory cytokines, e.g., IL-6 [18–20].
Basophil activation test (BAT) is a validated functional test for allergy in which basophils isolated
from blood samples of people with allergies are stimulated ex vivo with the particular allergen the
individual is allergic to, here, birch pollen allergen. When relevant allergen is added, it will cross-link
the Fc
ε
RI-bound allergen-specific IgE on the cells and thus activate the basophils to degranulation and
COX-2 activation that incite an allergic reaction. This activation can be detected
in vitro
when cells
after allergen co-incubation are analyzed by flow cytometry with fluorochrome-labelled IgG antibodies
specific to activation markers such as CD63 on basophil surface [
21
]. In highly allergic individuals,
even very low concentrations of the particular allergen will provoke basophil activation in contrast
to the situation for less allergic individuals where higher allergen concentrations are needed. If no
activation is achieved regardless of allergen concentration, the individual is not allergic to the allergen
being tested. However,
≥
5% of clinically allergic individuals have a negative BAT [
22
], which probably
is due to aberrant intracellular signaling after FcεRI engagement. They are called nonreactives.
Since Andosan
TM
has an anti-inflammatory effect in blood donors and inflammatory bowel disease
(IBD) patients [
23
–
26
] and aeroallergy and asthma can be defined as local inflammatory conditions in
the airways, it was pertinent to investigate this mushroom extract in an allergy and asthma intervention
study. We examined whether the AbM-based mushroom extract Andosan
TM
, taken orally before the pollen
season, could reduce allergy and asthma symptoms, medication, and specific IgE basophil sensitization
to t3 (birch pollen extract) and Bet v 1 (major component from birch) and could influence the Th1/Th2
cytokine balance in otherwise healthy humans as found in mouse models for allergy and asthma [9,10].
2. Materials and Methods
2.1. Trial Design
This is a single-center, randomized, two-armed, double-blinded study designed to determine
whether daily oral intake of Andosan
TM
mushroom extract before birch pollen season improved clinical
symptoms and reduced medication and specific IgE and basophil sensitization in otherwise healthy
Nutrients 2019,11, 2339 3 of 15
blood donors during and after the study period. The donors were evaluated before, during, and after
Andosan
TM
or placebo consumption (60 mL once daily). This dose, but not 30 mL/day (unpublished),
reduced levels of pro-inflammatory cytokines in healthy volunteers [
23
] and patients with IBD [
24
] or
multiple myeloma undergoing high-dose chemotherapy with stem cell transplantation [
27
]. Treatment
for 7 weeks in the latter gave no serious side effects.
2.2. Study Subjects
These were voluntary blood donors at Oslo Blood Bank (OBB), Oslo University Hospital (OUH), with
self-reported birch pollen-induced allergy and/or asthma. Among 68 blood donors at OBB assessed for
eligibility with self-reported birch pollen-induced allergy or asthma, 60 donors with positive specific IgE
were recruited to the clinical trial through a letter. Criteria of exclusion were undergoing or planning to
undergo allergen immune therapy (AIT), anti-IgE therapy, or cortisone injection. Twenty-nine donors were
randomized stochastically to Andosan
TM
intervention and 31 were randomized to placebo intervention
(Figure 1). They were not travelling long distances during birch pollen season, giving all participants
similar birch pollen exposure. Study groups were comparable, with insignificant differences with respect
to details of demographic data and study subjects characteristics regarding allergy symptoms, comorbidity
from other allergies and asthma, and medication for such conditions (Table 1).
Figure 1. CONSORT 2010 Flow Diagram
Assessed for eligibility (n=68)
Excluded (n=8)
Not meeting inclusion criteria (n=3 )
Declined to participate (n=4 )
Other reasons (n=1): could not meet
Analysed (n=27)
Excluded from analysis (give reasons) (n=0)
Lost to follow-up (give reasons) (n=1)
Discontinued intervention (did not like taste)
(n=1)
AndosanTM group (n=29)
Received allocated intervention (n=29)
Did not receive allocated intervention (n=0)
Lost to follow-up and discontinued intervention:
did not take medication (n=2), very ill from
allergy, hospitalized for other reasons (n=1)
Discontinued intervention (give reasons) (n=0)
Placebo group (n=31)
Received allocated intervention (n=31)
Did not receive allocated intervention (n=0)
Analysed (n=28)
Excluded from IgE analysis (preseason
specific IgE values > upper reference values)
(n=1)
Allocation
Analysis
Follow-Up
Randomized (n=60)
specific IgE positives
Enrollment of
blood donors with
birch pollen
allergy
BAT non-reactors (n=1)
BAT non-reactives (n=2)
Eligible for comparison of BAT outcomes
(n=19)
BAT non-reactors (n=1)
BAT non-reactives (n=7), missed BAT after (1)
Eligible for comparison of BAT outcomes
(n=24)
Follow-Up
Follow-Up
Figure 1.
CONSORT (Consolidated Standards of Reporting Trials) 2010 flow diagram. Definitions:
specific IgE (IgE anti-Bet v 1), BAT (Basophil Activation Test).
Nutrients 2019,11, 2339 4 of 15
Table 1. Demographic and blood donor data.
Andosan™Placebo p-Value
Number 27 27 -
Age (years) 40
range 22–60
37
range 20–64 0.62
Gender (male, female) 15, 12 14, 13 -
Specific symptoms, pollen allergy 6
range 4–7
5
range 3–6 0.10
Comorbidity: allergy other than for birch 20 21 -
Comorbidity: asthma 7 5 -
Medication: types of allergy medication 1
range 1–3
3
range 1–3 0.14
Medication: frequency of medication during season 7
range 7–14
7
range 7–14 0.42
Values for age and medication are given as number or median (range 25%–75%). (Statistical analysis: Mann–Whitney
(M–W) rank sum test). The table describes participants included for analysis.
Study subjects were recruited at OBB by the principal investigator (author F.M.) and three part-time
nurses who handled information, study medicine, questionnaire, and blood sampling. Blood samples
for IgE and cytokine measurements were taken from recruited blood donors in conjunction with blood
donation (a) before the birch pollen season in Oslo 2016 (from end of April until mid-May), (b) during
the whole pollen season (throughout August) (only IgE), and (c) after the season (September–October).
2.3. Sample Size
It was determined from a similar randomized clinical trial with Andosan
TM
in 50 patients with
ulcerative colitis or Crohn’s disease that 50 patients were needed for significant differences at the
p=0.05 level in symptoms and plasma cytokine levels [25,26].
2.4. Randomization: Sequence Generation and Type, Allocation Concealment Mechanism, and Implementation
Putative participants were numbered continuously and randomized stochastically (Microsoft
Excel randomization table) by simple randomization into two treatment groups, Andosan
TM
yes or
no, thus coupling a number for each participant to a treatment before he/she signed up for the study.
Time points for available interviews were listed in advance numerically, thus allocating study number.
Study medicine (Andosan
TM
or placebo) of similar color and odor was contained in similar plastic
bottles marked with study numbers and given out to participants by study nurses. Only the principal
investigator knew the study key. An independent colleague generated the allocation sequence, and the
principal investigator enrolled participants and assigned participants to interventions.
2.5. Interventions—Study Medicine: AndosanTM and Placebo Blinding
The mushroom extract Andosan
TM
was provided by Immunopharma company AS (organization
no. 994924273), Oslo, Norway, after import as mushroom juice from ACE company, Gifuken, Japan.
The production (commercial information) process comprises fermentation and heat sterilization,
conforming with Japanese health food regulations. Lipopolysaccharide content was <0.5 pg/mL
(Limulus test, Chromogenix, Falmouth, MA, USA). Andosan
TM
was imported to Norway as mushroom
juice in accordance with Norwegian Food Safety Authorities and stored sterile at room temperature
in dark, glass bottles until transfer/refill in larger 1.5-L plastic containers and storage at 4
◦
C until
consumed (60 mL/day) by study participants—60 mL/75 kg is similar to the effective dose in allergic
mice (200
µ
L/25 g) [
10
]. The placebo solution, kept in plastic containers and used similarly, was
Nutrients 2019,11, 2339 5 of 15
composed of a color-like drink with ionized water, salt, and caramel color (E150c; 0.5 mL/L). Study
subjects, study nurses, data collectors, and outcome adjudicators were blinded.
2.6. Outcomes
Primary outcomes were analyzed by the following: (i) An in-house questionnaire for the
blood bank setting modified from recommendations for clinical outcomes in AIT trials for allergic
rhino-conjunctivitis [
16
] was given out at the beginning and collected at the end of study. It contained
questions regarding specific symptoms from conjunctiva: itchy, red, and watery eyes; itchy, runny,
and blocked nose; itchiness/irritation and blocking of the throat; blocking of the chest and dyspnea;
and eczema and skin irritation. Each symptom was scored 1, with the total possible symptom score
being 12. Additional scoring was based on the presence of general allergy symptoms (
yes =1
,
no =0
)
and change from last season in general allergy symptoms (more = +1, unchanged =0, less =
−
1).
Medication was not scored but listed separately as frequency (per week) and types of medication
(antihistamines, nasal corticoids, degranulation inhibitors, and asthma drugs) during the intervention
period relative to before the intervention period.
(ii) Levels of total IgE (kU/L), IgE anti-Bet v 1 (recombinant birch allergen), and IgE anti-t3 (common
silver birch extract) (kUA/L) were analyzed by ImmunoCAP
®
technology in an ImmunoCAP
®
250
instrument (ImmunoDiagnostics, Thermo Fisher Scientific Inc., Uppsala, Sweden) in serum from blood
sampled into glass tubes with gel or 15 IU heparin/mL for IgE and cytokine analyses, respectively.
The levels of IgE anti-t3 (kU/L) analysed by Euroline atopy screen (IgE) test (Euroimmun AG, Lübeck,
Germany) that gives similar results as ImmunoCAP
®
, was also examined in 50 male and 50 female
unselected consecutive blood donors in mid-April of the previous year (2015).
(iii) BAT [
28
] for basophil sensitivity against t3 (common birch pollen extract) and rBet v 1 (gift from
Euroimmun AG) allergens was tested by a FlowCAST
®
kit (Bühlmann Laboratories AG, Schönenbuch,
Switzerland) in whole EDTA blood according to manufacturer’s instructions. Basophils were identified
by chemokine receptor type 3 (CCR3) [
29
] and their activation by CD63 surface expression by using a
Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) at 488 nm. Positive controls, anti-Fc EpsilonRI
and N-formyl-methionyl-leucyl-phenylalanine, were used to detect nonreactives. When the positive
control is negative, the test cannot be evaluated and such individuals are then called nonreactors.
A patient individual base value of 2.0%–2.5% of the activated basophils was regarded as negative and
used as the basis for determining allergen-specific cutoffs. An increase compared with negative control
of <10% in CD63-positive basophils was considered negative, and an increase of >10% was considered
a positive result.
In vitro
experiments were performed with 0–10% of Andosan
TM
incubated with
whole blood from untreated donors at 37 ◦C for 30 min and were subjected to BAT analysis.
The gating strategy for basophilic cells was performed according to the FlowCAST
®
kit (Bühlmann
Laboratories AG, Schönenbuch, Switzerland). Briefly, leukocyte population, which is separated
into three discrete populations, was gated on a side scatter versus forward scatter (SSC-FSC) plot.
The basophils are identified as CCR3
high
SSC
low
from the gated lymphocytes (SSC-CCR3-PE plot),
and eosinophils located on the high right side have been excluded due to their SSC high position.
Basophils are analyzed for activation (CD63
high
) in the CD63-FITC-CCR3-PE plot. The upregulation of
the activation marker CD63 was calculated by the percentage of the CD63-positive cells compared
to the total amount of basophilic cells (CCR3
high
SSC
low
). In each assay, at least 200 basophils were
assessed. We used stimulation buffer as the negative control and the two antibodies provided by the
kit as positive controls.
Secondary outcomes were shown by cytokine levels in plasma before vs. after intervention.
Multiplex bead-based sandwich immunoassay using Bio-Plex xMAP technology (Bio-Rad, Austin,
TX, USA) was employed with Luminex IS 100 instrument (Bio-Rad Laboratories, Hercules, CA, USA),
powered using Bio-Plex Manager (version 6.0.1) software (Bio-Rad Laboratories)) for analysis of
9 different cytokines (TNF
α
, IFN
γ
, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, and GM-SCF) following
manufacturers’ instructions [30].
Nutrients 2019,11, 2339 6 of 15
2.7. Study Subjects Flow, Losses, and Exclusions
Sixty specific IgE positive blood donors, 29 in the Andosan
TM
and 31 in the placebo groups, were
randomized for inclusion in this study (Figure 1). Six were excluded according to study protocol
criteria, 1 and 2 in the respective groups took no study medicine, 1 in the Andosan
TM
group missed
attendance, 1 in the placebo group was hospitalized for severe allergy, and another with the above
reference value (100 kUA/L) for specific IgE before pollen season was excluded from IgE evaluation,
thus resulting in 27 participants in each group (Figure 1).
Intervention was from January 2016 until the beginning of birch pollen season (varying from
mid-February until primo-April), and follow-up was during pollen season throughout August and,
again after season, in September–October.
2.8. Age and Gender and Numbers Analyzed
Median age for the 54 included blood donors with birch pollen allergy was 39 years (range 20–64).
There was similar number of men and women, ages, and additional allergies (approximately 75%) in
the AndosanTM and placebo groups (Table 1).
Subjects analyzed negative or below cutoffor lacking values before and during/after season were
excluded from evaluation of outcomes of the particular analysis; for IgE analyses, 0 to 7 participants
with negative or too low values and 5 to 8 with lacking values were excluded, and for cytokine analyses,
a large proportion was excluded. For evaluation of basophil response, 1 non-reactor in each group was
excluded and 2 and 7 non-reactives were respectively excluded in the Andosan
TM
and placebo groups
(Figure 1). Participants with negative IgE anti-Bet v 1 were also excluded from further evaluation
of symptoms and medication score in the questionnaire. The number of participants then eligible
for evaluation of analysis of total IgE, IgE anti-Bet v 1, and anti-t3 during the pollen season were 20
and 19, 18 and 14, and 19 and 15 for the Andosan
TM
and placebo groups, respectively. After season,
the respective numbers for total IgE were 16 and 18 and, for specific IgE, were 12 and 12. Participants
eligible for pre- and post-seasonal comparison of BAT were 24 and 19 in the Andosan
TM
and placebo
groups, respectively.
2.9. Statistical Analysis
Data are presented as mean and standard error or as median and range values, depending on
normal distribution. Parametric (t-test) and nonparametric (Mann–Whitney (M–W) and Wilcoxon
rank sum and test) tests were used between study groups for normally or abnormally distributed
data, respectively. Paired sample t-test and Wilcoxon signed rank test were used for within-group
analysis and Pearson’s r or Spearman rank order correlation were used for correlations. All tests were
two sided, and results were deemed to be statistically significant at p<0.05. pvalues at 0.10 were
considered to reflect a trend.
2.10. Registration and Protocol
The study was registered with unique protocol ID: ClinicalTrials.gov Identifier: NCT03198455.
The authors confirm that all ongoing and related trials for this drug (food)/intervention are registered.
See CONSORT Clinical Trials record website and CONSORT 2010 Checklist (Supplementary Materials
1 and 2).
2.11. Statement of Ethics
The study was approved on May 28, 2015 by the regional ethics committee (REC—South East
Norway, ref. 2015/716) and followed the guidelines of the Helsinki declaration. All study subjects were
informed and signed a written consent for participation, including the option of study withdrawal.
Blood donors were recruited and followed up at the OBB, OUH, Oslo, Norway, from November 2015
through October 2016.
Nutrients 2019,11, 2339 7 of 15
3. Results
3.1. Symptom and Medication Score Reduction
Scores for 12 specific allergy-related symptoms from the questionnaire tended to be higher (p=0.10)
at inclusion in the Andosan
TM
than placebo group (Table 1) but were equalized in the mid-intervention
season (p=0.97) (Table 2). Then, general allergy symptoms also tended to be relatively less (p=0.10)
in the Andosan
TM
group. Moreover, when compared with the season prior (2015) to the intervention
(2016), general allergy-related symptoms were significantly reduced (score:
−
1) in the Andosan
TM
compared to the placebo group (p=0.02) (Table 2) with no change (score: 0). However, there was no
statistical difference between the groups in the specific symptom scores (2 and 1 less, respectively,
p=0.51
). The included individuals with pollen allergies were allowed to use their regular medication,
which was similar before intervention (Table 1). None-the-less, types of medication used was reduced
in the Andosan
TM
relative to placebo group (p=0.03) and the frequency of medication showed a
similar tendency (p=0.11) (Table 2). During pollen season, there was no difference in the number of
blood donations between the groups (p=0.87).
Table 2. Intervention data: allergy.
Andosan™Placebo p-Value
Allergy symptoms
General allergy symptoms mid-season 0
(0–1)
1
(0–1) 0.10
Specific allergy symptoms mid-season 5.5
(4–7.7)
5.0
(3–7) 0.97
Change in general allergy symptoms (2016 vs. 2015) −1
(−1–0)
0
(−1–0) 0.02
Change in specific allergy symptoms (2016 vs. 2015) −2
(−4–0)
−1
(−3–0) 0.51
Allergy medication
Types of drugs
(antihistamines, nasal corticoids, and degranulation inhibitors)
1
(1–1)
1
(1–3) 0.03
Frequency of medication 7
(3–7)
7
(1–14) 0.11
Number of blood donations April–August 1
(0.5–1)
1
(0–1) 0.87
Values are given as median (range 25%–75%). (M–W rank sum test). Scores: General allergy symptoms (
yes =1
,
no =0
), change in general allergy (more = +1, no =0, less =
−
1); specific symptoms total score (n=12, 1 or 0 for
each), and change =difference in the total score. The table describes results from participants included for analysis.
At inclusion, 26% and 18.5% of participants suffered from pollen-induced asthma in the Andosan
TM
and placebo groups, respectively (Table 1). Compared with the placebo group, Andosan
TM
-treated
asthmatics experienced less asthma symptoms (median score:
−
3 (range:
−
5–0) for Andosan
TM
vs.
−
1 (
−
3.5–0) for placebo; p=0.01) and used fewer types of medication (
−
2 (
−
2–
−
1) vs. 0 (
−
0.5–0);
p=0.02
) at lesser frequency (
−
7 (
−
14–
−
4) vs. 0 (0–0); p=0.009) for their asthma and allergies during
the intervention pollen season compared to the previous one.
3.2. Specific IgE Level Reduction
Before intervention, there were positive but low correlations between symptom score and total
IgE (Spearman rank order correlation
ρ
=0.31, p=0.02), IgE anti-t3 (
ρ
=0.30, p=0.02), and IgE anti-Bet
v 1 (
ρ
=0.25, p=0.06). Changes in specific IgE anti-Bet v 1 levels during the pollen season relative to
before were significantly reduced in the Andosan
TM
compared to the placebo group (Table 3;
p=0.007
).
Nutrients 2019,11, 2339 8 of 15
There was a similar finding for IgE anti-t3 levels (p=0.01) but not for total IgE (p=0.14; Table 3).
The reduction in relative values (indices) during season in the Andosan
TM
vs. placebo group was
36%–37% for specific IgE anti-Bet v 1 and anti-t3, 16% for total IgE, and still 22% (indices 0.97 vs. 1.25)
after season for IgE anti-Bet v 1 (Table 3). The current selected blood donors with self-reported pollen
allergy and specific IgE-verified sensitization (placebo group examined) had significantly higher IgE
anti-t3 levels than 100 unselected donors (median 10.2 vs. 0.0 kU/L, respectively; p<0.001) that were
examined the previous pollen season (mid-April 2015).
Table 3. Intervention data: IgE measurements.
IgE Ab Indices nAndosanTM nPlacebo p-Value
Total IgE
During/Before
20 0.93
(0.81–1.06)
19 1.11
(0.82–1.55) 0.14
After/Before 16 1.02
(1.86–1.21)
18 1.31
(0.98–1.62) 0.14
Bet v 1
During/Before
18 0.82
(0.72–1.15)
14 1.30
(0.94–2.14) 0.007
After/Before 12 0.97
(0.81–1.31)
12 1.25
(0.92–1.45) 0.21
t3
During/Before
19 0.86
(0.76–1.07)
15 1.35
(0.95–2.33) 0.01
After/Before 12 1.02
(0.82–1.24)
12 1.14
(0.80–1.50) 0.46
Wilcoxon signed rank test was used. Levels before pollen season were for total immunoglobulin E (IgE): mean
107.7 ±25.6
standard error of the mean (SEM)/median 61 (range 2–892); for Bet v 1: 10.3
±
1.6/6.4 (0–37); and for
t3: 10.3
±
1.6/6.6 (0–36) kUA/L. Participants with negative (n=0–7) or missing (n=5–8) values were excluded.
The indices represent IgE levels during or after the pollen season divided by the respective IgE levels before the
season. Test for difference in Andosan
TM
before (median 9.2) vs. placebo before (median 8.9): p=0.85 (M–W rank
sum test).
3.3. Basophil Sensitization Reduction
Blood samples for basophil activation were taken before and after pollen season, together with
samples for IgE and cytokine measurements, and determined by CD63 expression by flow cytometry.
Those allocated to placebo treatment tended to have basophils that were less sensitive to pollen
activation (p=0.062) (Table 4). In the placebo group, the maximal (peak) allergen concentrations
needed for eliciting basophil activation before the season mostly changed to lower peak concentrations
after the season (p=0.004; Table 4; Figure 2a). In contrast, for patients with allergies allocated to
Andosan
TM
treatment, most BAT peak profiles did not change from before to after the pollen season
(p=0.312
; Table 4; Figure 2b). When comparing the resultant changes observed during the season
within each group, there was a relative and significant (p=0.028) shift in the Andosan
TM
group
to less changes in allergen sensitivity (Table 5; Figure 2c). In fact, whereas the mean peak Bet v 1
concentration for basophil activation was reduced 10-fold in the placebo group, it was doubled in the
Andosan
TM
group (Table 4). Hence, Andosan
TM
treatment before the pollen season rendered basophils
less sensitive to allergen activation during the season as compared with placebo.
Nutrients 2019,11, 2339 9 of 15
Nutrients 2019, 11, x FOR PEER REVIEW 9 of 15
(a)
(b)
(c)
Figure 2. Basophil reactivity in the intervention groups. Basophil reactivity is shown before and after
Placebo (a) or AndosanTM (b) intervention, and after relative to before the respective interventions (c).
Also see Table 4 for depicted data in (a,b), and Table 5 for (c).
Figure 2.
Basophil reactivity in the intervention groups. Basophil reactivity is shown before and after
Placebo (
a
) or Andosan
TM
(
b
) intervention, and after relative to before the respective interventions (
c
).
Also see Table 4for depicted data in (a,b), and Table 5for (c).
Nutrients 2019,11, 2339 10 of 15
When participants whose blood samples were analyzed after the tree pollen season (August)
were separated from those analyzed later (September–October), there were similar findings as above
compared with pre-seasonal BAT measurements for both cohorts, a significantly lower activation in
the Andosan
TM
(change in peak Bet v 1 conc. needed: 0 (
−
0.25–0)) than the placebo group (
−
1 (
−
2–0))
(p=0.046). Also, for participants examined for basophil activation later in September–October, there
tended to be similar findings in favor of AndosanTM treatment (p=0.06).
Table 4.
Comparisons before and after intervention of maximal (peak) Bet v 1 concentration needed for
basophil activation.
Bet v 1 conc.
Placebo Andosan
Peak before Peak after Peak before Peak after
n(%)
100 1
(5.2)
1
(4.1)
10 7
(36.8)
4
(16.6)
1
(4.1)
111
(57.8)
17
(89.4)
20
(83.3)
19
(79.1)
0.1 2
(10.5)
3
(12.5)
Mean ng/mL 9.52 ±5.12 0.90 ±0.06 2.50 ±0.69 5.38 ±4.12
Total 19
(100)
19
(100)
24
(100)
24
(100)
Test for difference
p=0.004 p=0.312
Test for difference: p=0.004, p=0.312 (Wilcoxon signed rank test). Test for difference in placebo before vs.
Andosan
TM
before intervention: p=0.062. (M–W rank sum test). Values represent number of individuals and
percentages of total. Concentration (conc.).
Table 5.
Shift in maximal Bet v 1 concentration needed for basophil activation from before until
after intervention.
Diffin (Bet v 1) 1 ×10nPlacebo Andosan
logarithmic Power n
−2 2 1
−1 7 3
0 10 19
1 0 1
Total 19 24
Test for difference p=0.028
Test for difference: p=0.028 (M–W rank sum test). Values represent number of individuals.
3.4. Basophil Activation: IgE and Symptoms (Allover Allergy Ailments) Negative Correlations
There were negative and significant correlations between basophil activation (Cmax) values and
IgE anti-Bet v1 (Pearson’s r
−
0.43, p=0.006), IgE anti-t3 (correlation coefficient (corr. Coeff.):
−
0.44,
p=0.004
), and total IgE (corr coeff
−
0.34, p=0.032). This agrees with basophils being activated by less
allergens (becoming more sensitized) in people with allergies with high specific IgE values and vice
versa. Also, for changes in basophil activation values and symptoms, there was a significant negative
correlation (p=0.015) (corr coeff−0.38).
Nutrients 2019,11, 2339 11 of 15
3.5. Basophils Are Not Activated In Vitro by AndosanTM Stimulation
When basophil activation was analyzed before participants entered the study, there was no
difference in BAT between basophils pre-incubated
in vitro
with Andosan
TM
(1%–10%) or saline
(p>0.05), indicating no direct effect of AndosanTM on basophils.
3.6. Plasma Cytokines Changes Not Measurable
When cytokines TNF
α
, IFN
γ
, IL-2, IL-4, IL-5, IL-10, IL-12p70, IL-13, and GM-SCF were measured
in plasma and examined before and after intervention in the two groups, there were either no differences
or the levels were too low (0 or below lowest standard value) to be analyzed.
3.7. No Harm
One participant in the Andosan
TM
and two participants in the placebo groups did not take the
study medicine because of taste. Otherwise, no harm was recorded.
4. Discussion
This randomized, double-blinded, and placebo-controlled study in birch-allergic blood donors
suggests that add-on Andosan
TM
pretreatment before birch pollen season generally improves allergy
ailments—although not shown by specific symptom scores—and reduces need for medication. One
major challenge in this study was to see the effects of Andosan
TM
supplementation on top of regular
drugs taken for allergy and asthma. The different results regarding changes from previous season
between twelve specific allergy symptoms and general allergy symptoms may reflect that memory
of specific symptoms the year before is less precise than global memory of change in allergy-related
quality of life.
The clinical outcome agrees with preclinical findings showing that Agaricus blazei extracts protected
against allergen sensitization and asthma in mice [
9
,
10
]. Based on the positive albeit low correlation
between specific IgE and allergy symptoms before intervention, the close to 40% reduction in specific
IgE levels in the Andosan
TM
group during pollen season is assumingly associated with the clinical
findings. This is supported by the negative correlation between change in BAT resultsand change in
allergic ailments, indicating that when basophils became less sensitized to allergen by Andosan
TM
supplementation, the symptoms decreased. However, it could not be explained by alterations in
plasma cytokines that were difficult to measure.
Plasma cytokine levels were either too low to measure or there were no changes before compared
to after the season, when the cytokine concentrations most probably had reverted back to pre-seasonal
levels. Preferably, cytokines should have been measured again during the season to see putative
changes induced by Andosan
TM
intervention as seen for change in specific IgE (Table 3). Although
Andosan
TM
given orally has an effect on cytokines in plasma [
11
,
23
,
24
,
26
,
27
], plasma samples are not
ideal for analyses of putative topical changes in cytokines caused by local inflammations, e.g., in asthma
and aeroallergy, but should rather be measured locally in bronchial aspirates and nasal secrete/tears,
respectively. In the mouse allergy model, amelioration of the Th1/Th2 imbalance was found in spleen
cells harvested from animals treated with an AbM extract or Andosan
TM
[
9
,
10
]. Andosan
TM
also had
anti-inflammatory effects both in blood donors and IBD patients [23,24].
After the pollen season, specific IgE levels in the Andosan
TM
group rose again towards pre-seasonal
levels (Table 3), suggesting that the extract did not affect IgE beyond the season but stabilized IgE
levels that normally peak mid-season. However, the finding that basophil activation, in contrast to
specific IgE levels, was reduced significantly after the pollen season suggests that the Andosan
TM
effect on basophil sensitization lasted longer. The density of expressed basophil Fc
ε
RI correlates with
levels of serum IgE, which stabilizes the receptor at the cell surface [17]. Hence, the lower circulating
specific IgE during pollen season in Andosan
TM
-treated individuals likely rendered their basophils
less sensitive to allergen cross-binding and activation by reducing specific IgE binding to Fc
ε
RI on the
Nutrients 2019,11, 2339 12 of 15
cells. The negative correlations between changes in BAT results and change in specific and total IgE
levels agrees with the notion that decreas in IgE levels during pollen season stabilizes less Fc
ε
RI on
basophils and thus decreases the cells’ sensitivity to pollen allergen.
Presumably,
in vivo
Andosan
TM
supplementation for people with allergies had a similar indirect
effect on mast cells as on basophils because an Agaricus blazei extract has been shown to have both
anti-inflammatory and anti-allergic effects in bone marrow-derived mast cells
in vitro
[
31
]. These effects
were suggested to occur through blocking of COX-2 expression and protein kinase B activation, which
are associated with release of inflammatory and allergic mediators in mast cells such as prostaglandin
D2and leukotriene C4[32].
The preferred method for evaluating basophil sensitivity to allergen is calculation of 50% of the
effective concentration (EC50) [
22
]. However, since the needed wide variation of eight declining 10-fold
concentrations to calculate EC50 was missing in most patients, peak shift within three overlapping
concentrations between analyses before and after intervention in each group (A or P) was used instead.
This is generally accepted in BAT examinations [
22
]. When comparing BAT peaks before intervention
in the two groups, there was no statistical difference but there was a tendency (p=0.062) to be less
sensitized for participants in the placebo than Andosan
TM
group. Despite the randomization, this
suggests that those allocated to later Andosan
TM
supplementation tended to be more allergy prone
during the pollen season.
Based on improved Th1/Th2 balance, most studies seem to agree that
β
-glucan has a potential as
adjunct treatment of patients with allergies [
33
]. However, since the
β
-glucan content of Andosan
TM
is
low [
34
], the biological properties of Andosan
TM
must be attributed also to other glycans detected,
which inhibited activity of the proinflammatory and tumor-associated endopepetidase legumain
in macrophages [
34
]. Legumain may play a role in atopic dermatitis [
35
], and there was reduced
expression of this enzyme in intestines of mice given Andosan
TM
orally [
11
]. In addition, the mushroom
extract contains low protein but higher lipid concentrations (not shown) that may explain some of
its effects.
How Andosan
TM
interacts with antihistamines, corticosteroids, and other drugs for allergy and
asthma is unknown in contrast to its interference with cytochrome P-450 metabolism and transmembrane
efflux pump P-glycoprotein (P-gp), which governs pharmacokinetics and drug concentrations [
36
].
The AbM extract Agaricus Gold Label from Japan, later named Andosan
TM
, inhibited P-gp transport
and cytochrome P450 (CYP3A4) metabolism in similar or lower concentrations than did green tea [
37
,
38
],
indicating that clinical or intestinal interactions of Agaricus with CYP3A4 were unlikely [
38
]. This
agrees with the lack of side effects observed in clinical trials with IBD [
25
] and multiple myeloma
patients [27] after AndosanTM supplementation to regular treatments for 3–7 weeks.
Andosan
TM
probably has effects against various allergens because of its nonspecific anti-allergic
effect as shown against both food- (ovallergen) [
10
] and birch pollen-induced sensitization. Since
inhibitory effects of AbM has been shown on mast cell-mediated anaphylaxis reactions in mice [
39
],
Andosan
TM
may also exhibit similar effects. Another interesting aspect is whether Andosan
TM
could
be used as an adjuvant for AIT similar to experimental use of AbM and
β
-glucan in vaccines for viral
infections in animal models [
40
,
41
]. Since the extract also had antibacterial effects in murine sepsis
models [
42
,
43
], it would have been interesting to examine the anti-allergic effects of Andosan
TM
in
patients with combined allergic and infectious conditions as often seen in asthma/bronchiolitis and
atopic dermatitis in infants and children.
The protection observed against basophil activation in pollen-allergic patients after Andosan
TM
supplementation before pollen season seems to taper offafter August. At this time, the season is
long gone for birch pollen and mostly over for grass pollen in southern Norway. This suggests
that a prophylactic regimen similar to the current one with this mushroom extract could reduce
pollen-induced allergy problems. The supplementation most probably has to be repeated to have
positive effects in the subsequent pollen season, but this was not examined. Also, we do not know
Nutrients 2019,11, 2339 13 of 15
whether the anti-allergy effect of Andosan
TM
will be diminished over time if people with allergies
develop tolerance to it. Further studies are warranted for reaffirmation of the present results.
5. Conclusions
This randomized placebo-controlled clinical study suggests that pre-seasonal oral supplementation
with Andosan
TM
mushroom extract can have anti-allergic effects in pollen-induced allergy by protecting
against basophil sensitization during pollen season. Most probably, prevented mastocyte sensitization,
albeit not examined, is the main mechanism behind the reduction in general allergy symptoms and
medication in the participants of this study.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2072-6643/11/10/2339/s1,
Supplementary Material 1: CONSORT Clinical Trials record, Supplementary Material 2: CONSORT 2010 Checklist.
Author Contributions:
All authors made substantial contributions to the conception and design, acquisition of
data, or analysis and interpretation of data. F.M. is the principal investigator and was in charge of data acquisition
that was done with M.R.M. for BAT. I.N. is an allergologist and BAT expert. R.G. did the statistical analysis, and
L.S.H.N.-M. is the project leader. G.H. drafted the article, and all authors reviewed it critically for important
intellectual content and gave final approval for publishing.
Funding:
Andosan
TM
was provided by Immunopharma company, which had no other role in this study, and
the funds for analyses were provided by the University of Oslo and OUH, which also employs the authors and
study nurses.
Acknowledgments:
This work was supported by the Department of Immunology and Transfusion Medicine,
OBB, OUH and the University of Oslo, Norway. We are grateful for the recruiting, interviewing, and following-up
of participants by study nurses Trude Betten, Jon-Kristian Hvila, and Lisbeth Malerstuen at Section for Blood
Donation, OBB. Skillful analysis of cytokines was performed by Hans Christian D. Aass at the Department of
Medical Biochemistry. The sponsor was Immunopharma R&D. The allergen molecule Bet v 1 was kindly provided
by Euroimmun AG.
Conflicts of Interest:
Geir Hetland is a cofounder and shareholder of Immunopharma, Oslo, Norway. The other
authors declare no commercial or financial conflict of interest. Immunopharma had no other role than providing
AndosanTM free of charge for the study.
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