Immunological Effect of Active Hexose Correlated Compound (AHCC) in Healthy Volunteers: A Double-Blind, Placebo-Controlled Trial

Abstract

The aim of this study was to evaluate the effects of active hexose correlated compound (AHCC) intake on immune responses by investigating the number and function of circulating dendritic cells (DCs) in healthy volunteers. Twenty-one healthy volunteers were randomized to receive placebo or AHCC at 3.0 g/day for 4 wk. The number of circulating cluster of differentiation (CD)11c(+) DCs (DC1) and CD11c(-) DCs (DC2) were measured. Allogeneic mixed-leukocyte reaction (MLR) was performed. Natural killer (NK) cell activity and the proliferative response of T lymphocytes toward mitogen (phytohemagglutinin [PHA]) were measured. We also measured cytokine production stimulated by lipopolysaccharide [interleukin (IL)-2, IL-4, IL-6, IL-10, interferon gamma-gamma, tumor necrosis factor-alpha). The AHCC group (n = 10) after AHCC intake had a significantly higher number of total DCs compared to that at baseline and values from control subjects (n = 11). The number of DC1s in the AHCC group after intake was significantly higher than at baseline. DC2s in the AHCC group were significantly increased in comparison with controls. The MLR in the AHCC group was significantly increased compared to controls. No significant differences in PHA, NK cell activity, and cytokine production were found between groups. AHCC intake resulted in the increased number of DCs and function of DC1s, which have a role in specific immunity.

Full text

Nutrition and Cancer, 60(5), 643–651
Copyright © 2008, Taylor & Francis Group, LLC
ISSN: 0163-5581 print / 1532-7914 online
DOI: 10.1080/01635580801993280
Immunological Effect of Active Hexose Correlated
Compound (AHCC) in Healthy Volunteers: A Double-Blind,
Placebo-Controlled Trial
Naoyoshi Terakawa, Yoichi Matsui, Sohei Satoi, Hiroaki Yanagimoto,
Kanji Takahashi, Tomohisa Yamamoto, Jun Yamao, Soichiro Takai, A-Hon Kwon,
and Yasuo Kamiyama
Department of Surgery, Kansai Medical University, Osaka, Japan
The aim of this study was to evaluate the effects of active hex-
ose correlated compound (AHCC) intake on immune responses
by investigating the number and function of circulating dendritic
cells (DCs) in healthy volunteers. Twenty-one healthy volunteers
were randomized to receive placebo or AHCC at 3.0 g/day for
4 wk. The number of circulating cluster of differentiation (CD)11c+
DCs (DC1) and CD11c−DCs (DC2) were measured. Allogeneic
mixed-leukocyte reaction (MLR) was performed. Natural killer
(NK) cell activity and the proliferative response of T lymphocytes
toward mitogen (phytohemagglutinin [PHA]) were measured. We
also measured cytokine production stimulated by lipopolysaccha-
ride [interleukin (IL)-2, IL-4, IL-6, IL-10, interferon gamma-γ,
tumor necrosis factor-α). The AHCC group (n=10) after AHCC
intake had a significantly higher number of total DCs compared
to that at baseline and values from control subjects (n=11). The
number of DC1s in the AHCC group after intake was significantly
higher than at baseline. DC2s in the AHCC group were signif-
icantly increased in comparison with controls. The MLR in the
AHCC group was significantly increased compared to controls. No
significant differences in PHA, NK cell activity, and cytokine pro-
duction were found between groups. AHCC intake resulted in the
increased number of DCs and function of DC1s, which have a role
in specific immunity.
INTRODUCTION
Recently, the incidence of malignant tumor has been in-
creasing consistently in Japan (1). The development of imaging
modalities has enabled the diagnosis of malignant tumor at an
early stage with relative ease. However, it is still difficult to
control disease progression of advanced cancer.
Although some current cancer treatments can induce remis-
sion, most of these tumors ultimately relapse and cannot be
cured. Many attempts have been made to treat cancer by stimu-
lating the patient’s immune system. Several biological response
Submitted 31 May 2007; accepted in final form 17 January 2008.
Address correspondence to N. Terakawa, MD, Kansai Medical Uni-
versity, 10–15 Fumizono-cho, Moriguchi, Osaka, 570-8507, Japan.
E-mail: terakawn@takii.kmu.ac.jp
modifiers (BRMs) have been developed—such as BCG, Pi-
cibanil, polysaccharide-K (PSK), lentinan, interferon (IFN), and
interleukin (IL)-12—but the clinical efficacy of these substances
has not been clearly confirmed (2–5).
Active hexose correlated compound (AHCC; Amino UP
Chemical Co., Ltd., Sapporo, Japan) is a functional food that is
extracted from several species of Basidiomycetes mushrooms
(6,7). We have shown clinically that AHCC intake resulted in
improved liver function, prevented the recurrence of hepatocel-
lular carcinoma (HCC) after resection, and prolonged survival
of postoperative HCC patients without any adverse effects (8).
However, there has been no report on the functional effect of
AHCC on the immune response in humans.
Dendritic cells (DCs) are the most potent antigen-presenting
cells (9) capable of priming tumor-specific T cells, and their
use in cancer immunotherapy appears to be a promising way to
elicit and expand efficient antitumor immune responses (10,11).
Herein, we report the results of a randomized controlled trial
to evaluate the effects of AHCC intake on immune responses
by investigating the number and function of circulating DCs in
healthy volunteers.
METHODS
This preliminary study in a double-blinded randomized fash-
ion was approved by the Institutional Review Board at the Kan-
sai Medical University, Osaka, Japan. Informed consent was
obtained from each healthy volunteer in accordance with the
provisions of the Declaration of Helsinki. Volunteers were ex-
cluded if they had malignant tumor, viral hepatitis, uncontrolled
diabetes mellitus, and chronic heart dysfunction. Before screen-
ing physical and blood examinations, subjects were randomized
to receive placebo or AHCC at 3.0 g/day for 4 wk. Blood sam-
ples were collected in heparinized syringes in the morning after
an overnight fast, and various values were determined at base-
line and 4 wk later. The number of circulating CD11c+DCs
(myeloid DC population; DC1), CD11c−DCs (lymphoid DC
population; DC2), natural killer (NK) cells, and CD4+/CD8+
643

644 N. TERAKAWA ET AL.
T lymphocytes were measured in each sample by flow cytomet-
ric analysis. To assess immune function, the allogeneic (allo-)
mixed-leukocyte reaction (MLR; allo-MLR) was determined.
NK cell activity and the proliferative response of T lymphocytes
toward mitogen (phytohemagglutinin [PHA]) were measured.
We also measured serum hormone levels (thyroid-stimulating
hormone, 3,5,3-triiodothyronine, thyroxine, and estradiol) and
cytokine concentrations (IL-2, IL-4, IL-6, IL-10, IFN-γ, tumor
necrosis factor [TNF]-α). The duration of the study was 7 mo.
Reagents
The culture medium for all experiments consisted of RPMI
1640 supplemented with 2 mM l-glutamine, 100 U/ml penicillin,
100 µg/ml streptomycin, 50 µM 2-mercaptoethanol (Sigma, St.
Louis, MO), and heat-inactivated 10% fetal bovine serum.
The phenotypes of peripheral blood mononuclear cells
(PBMCs) were determined by two- and three-color flow cyto-
metric analysis using monoclonal antibodies (mAbs) that were
directly conjugated to fluorescein isothiocyanate (FITC), R-
phycoerythrin (PE), or PE cyanin 5.1 (PE-Cy5).
Cells were stained with the following mAbs: PE-Cy5-
conjugated anti-human leukocyte antigen(HLA)-DR; a mix-
ture of FITC-conjugated anti-cluster of differentiation (CD)3,
CD14, CD15, CD16, CD19 so-called lineage cocktail (Lin) and
PE-conjugated anti-CD11c mAbs for DCs; PE-Cy5-conjugated
anti-CD3, FITC conjugated anti-CD4, and PE-conjugated anti-
CD8 for T lymphocytes; and PE-conjugated anti-CD14 and
FITC-conjugated anti-CD56 for NK cells.
All antibodies were obtained from PharMingen (San Diego,
CA). The isotype controls, anti-immunoglobulin G1 was also
obtained from PharMingen.
Flow Cytometry (FCM)
PBMCs were prepared by Lymphoprep (Nycomed Pharma,
Oslo, Norway) gradient centrifugation of heparinized periph-
eral blood and then washed in phosphate-buffered solution
supplemented with 1% fetal bovine serum and 0.1% NaN3.
PBMCs were incubated for 30 min at 4◦C with the mAbs.
The stained cells, as mentioned above, were analyzed using
a FACScan R
(Becton Dickinson, Sunnyvale, CA). At least
100,000 events were counted for each mononuclear fraction
by FACScan. The typical forward and side scatter gates for
DCs and lymphocytes in combination with FITC-, PE-Cy5-,
and PE-conjugated mAbs were set to exclude any dead or con-
taminating cells from the analysis. The following DCs and lym-
phocyte subsets were analyzed by 2- and 3-color FCM: DC1,
myeloid-lineage dendritic cells (CD11c+/lin−/DR+); DC2,
lymphoid-lineage DCs (CD11c−/lin−/DR+); helper T lympho-
cytes (CD3+/CD4+); cytotoxic T lymphocytes (CD3+/CD8+);
and NK cells (CD14−/CD56+). The number of PBMCs per mm2
was counted under a microscope, and viable cells were deter-
mined by the trypan-blue dye exclusion test. Absolute numbers
of DCs and lymphocytes were calculated from the number of
PBMCs per milliliter of blood multiplied by the percentage of
DCs and lymphocytes.
Typical FCM profiles in the AHCC group are shown in
Fig. 1. Region R1 includes lymphocytes and monocytes but
excludes debris. DCs were detected in region R2 as the popu-
lation of Lin−/HLA-DR+cells. Two subsets of DCs were iden-
tified within the Lin−/HLA-DR+population, which was based
on differential expression of CD11c: DC1 (CD11c+population;
region R3) and DC2 (CD11c−population; region R4). The NK
cell fraction was gated in the CD14−/CD56+population (region
R5). CD3+/CD4+T lymphocytes were detected in region R6,
and CD3+/CD8+T lymphocytes were detected in region R7.
Cell Surface Staining
For surface marker analysis, PBMCs were incubated for 30
min with 4◦C with FITC, PE, PE-Cy5, or ECD mAbs conjugated
to Lin, HLA-DR, CD11c, and CD40 or CD86. The stained cells,
as mentioned above, were analyzed using an EPICS R
XL-MCL
(Coulter, Hialeah, FL).
DCs Isolation From Peripheral Blood
DCs from peripheral blood were enriched as described else-
where (12–15). Briefly, PBMCs were incubated with anti-CD3
and anti-CD14 mAbs for 30 min on ice, and cells binding to these
mAbs were removed using sheep antimouse Ig-coated magnetic
beads (M-450; Dynal, Oslo, Norway). The CD3–/CD14–cells
were further incubated with CD4-conjugated microbeads (Mil-
tenyi Biotec., Bergisch Gladbach, Germany), and the CD4+
cells were then enriched by passing them through a Mini
MACS R
magnetic separation column (Miltenyi Biotec.). By
using this protocol, the percentage of DCs (originally <1% of
total PBMCs) increased up to 20–50%, which was dependent
on the individuals.
The resultant DC-enriched population (CD4+/CD3−/CD14−
cells) was stained with PE-conjugated anti-CD11c mAb, FITC-
conjugated lineage cocktail, and PE-Cy5-conjugated anti-HLA-
DR mAb. The stained cells were then analyzed and sorted by an
EPICS ELITER R
flow cytometer (Coulter, Hialeah, FL). Purity
of the sorted cells was always greater than 96% by reanalysis
using a FACScan (Becton Dickinson). Consequently, two phe-
notypically distinct fractions of DC1s and DC2s were collected
and used in MLR.
Allo-MLR of Circulating DC1
The cDC1s isolated from peripheral blood were examined for
their stimulating capacity against allogeneic T lymphocytes in
a standard MLR (13). DC1s were irradiated at 15 Gy (Gamma
Cell, Nordion, Ontario, Canada). Graded doses of DC1 were
cocultured with 2 ×105allogeneic T lymphocytes (collected
by magnetic beads as CD3+cells) in 200 µl of culture medium
in 96-well culture plates for 4 days.
For the maintenance of DCs, GM-CSF was added to the
culture medium for DC1s. Cells were pulsed with 1 µCi of
3H-thymidine during the last 16 h of the culture period. They

IMMUNOLOGICAL EFFECT OF AHCC IN HEALTHY VOLUNTEERS 645
FIG. 1. Flow cytometric analyses of peripheral blood mononuclear cells (PBMCs) by FACScan. In each sample, a total of 300,000 cells were analyzed. Typical
profiles of 1 subject in the active hexose correlated compound (AHCC) group are shown. Using light scatter properties, region R1 was defined to include
lymphocytes and monocytes and exclude debris. Dendritic cells (DCs) were detected in region R2 as the population of lineage cocktail anti-human leukocyte
antigen(Lin)-DR (Lin−/HLA-DR+) and divided into 2 fractions by the expression of cluster of differentiation (CD)11c [region R3: CD11c+DC (DC1); and
region R4: CD11c−DC (DC2)]. The natural killer (NK) cell fraction was gated in the CD14−/CD56+population (region R5). CD3+/CD4+T lymphocytes were
detected in region R6, and CD3+/CD8+T lymphocytes were detected in region R7. SS, slide scatter; FS, forward scatter; FITC, fluorescein isothiocyanate; PE,
R-phycoerythrin; PE-Cy5, PE cyanin 5.1.

646 N. TERAKAWA ET AL.
TABLE 1
Characteristics of Study Subjectsa
AHCC Group (n=10) Control Group (n=11) PVal u e
Age (yr) 59.3 ±4.4 60.2 ±5.5 0.621
Gender (Male:Female) 3:7 5:6 0.659
Height (cm) 158.6 ±4.4 159.5 ±8.1 0.901
Weight (kg) 57.1 ±4.8 60.5 ±7.3 0.385
BMI (kg/m2) 22.7 ±1.0 23.9 ±2.8 0.099
PNI 54.1 ±2.9 53.1 ±5.1 0.870
aContinuous variables are expressed as mean ±SD. Abbreviations are as follows: AHCC,
active hexose correlated compound. BMI, body mass index; PNI, prognostic nutritional index.
BMI equals a person’s weight in kilograms divided by height in meters squared (BMI =kg/m2).
PNI =10 ×Serum Alb (g/dl) +0.005 ×total lymphocyte count (/µl).
were harvested onto glass fiber filter papers using an automated
harvester, and cell-bound radioactivity was counted in a liquid
scintillation counter.
Proliferative Response of T Lymphocytes Toward Mitogen
(PHA)
The in vitro proliferative capacity of lymphocytes toward
mitogen (PHA) was quantified using standardized assay for-
mats (BAG, Lich, Germany). Lymphocytes were incubated with
1µCi of 3H-thymidine during the last 6.5 h of the culture period.
Cell-bound radioactivity was counted in a liquid scintillation
counter. Results were presented as the mean counts per minute
(cpm) of triplicate cultures.
NK Cell Activity
NK cell activity was measured with the standard 4-h 51Cr-
release assay. Each 100 µl of PBMCs (effector) and K562 cells
(target) was combined at an effector-to-target (E:T) ratio of 50:1
in 96-well microtiter plates. Maximum release was determined
by the addition of detergent to K562 cells. Spontaneous release
was measured by culturing K562 cells without PBMCs. Af-
ter a 4-h incubation at 37◦C in air with 5% CO2, supernatants
were harvested, and radioactivity was assessed using a gamma
counter (Aloka, Tokyo, Japan). All assays were performed in
triplicate, and the value was calculated as the mean of triplicate
cultures. The percentage cytotoxicity was determined as (ex-
perimental cpm −spontaneous cpm)/(maximum cpm −spon-
taneous cpm) ×100. Spontaneous release was less than 10% of
the maximum release in all experiments. Because of the limited
number of samples, only the measurement at an E:T ratio of
50:1 was performed in this experiment.
Cytokine Production
Briefly, blood samples (2 ml) were drawn directly into hep-
arinized control tubes (15 U/ml final). One ml of blood from
each tube was transferred into an lipopolysaccharide (LPS) tube
containing 100 EU LPS per tube. The LPS was isolated from
Escherichia coli 055B5 (16,17). LPS tubes were incubated for
4 h at 37◦C in a programmable incubator on a rocking platform.
Cytokine concentrations were measured with the CBA kit
(BD PharMingen, Franklin Lakes, NJ), according to the manu-
facturer’s manual as previously described, with modification of
data analysis to use GraphPad Prism software (GraphPad Soft-
ware, San Diego, CA). These assays are multiplexed such that
numerous substances are measured simultaneously in a single
well.
The CBA assay consists of a mixture of 6 types of beads
uniform in size but containing different fluorescence intensities
of a red-emitting dye. Each series of beads is coated with a mAb
against a single cytokine (IL-2, IL-4, IL-6, IL-10, IFN-γ,or
TNF-α), and the mixture of beads detects 6 cytokines in 1 sam-
ple. The captured cytokines are detected via direct immunoassay
using 6 different antibodies coupled to phycoerythrin PE. The
calibrator’s standards ranging from 0 to 5,000 pg/ml for the as-
say system capture Ab-bead reagent; detector Ab–PE reagent are
mixtures of all 6 cytokines. After fluorescence intensity calibra-
tions and electronic color compensation procedures, standard
and test samples were analyzed with FACScan. Six standard
curves are thus obtainable from 1 set of calibrators; 6 results are
obtained from each calibration point. Data were collected using
EXPO analysis software (Beckman Coulter, Fullerton, CA).
Statistical Analysis
Numerical values are given as the mean ±SD. All data were
analyzed using the StatView 5.0 statistical software package
(Abacus Concepts, Inc., Berkeley, CA). Comparisons of some
parameters among AHCC and control groups were made us-
ing the Mann–Whitney test and Wilcoxon rank test. Statistical
significance was determined at P<0.05.
RESULTS
Twenty-one healthy volunteers underwent a screening phys-
ical and blood examination and were randomized to either
the placebo (n= 11) or AHCC (n= 10) group. There were
no significant differences in age or gender between the two
groups (Table 1). None of the subjects withdrew during the
study period. Results of blood examinations were within normal

IMMUNOLOGICAL EFFECT OF AHCC IN HEALTHY VOLUNTEERS 647
FIG. 2. Comparison of the number of dendritic cells (DCs) between control and active hexose correlated compound (AHCC) groups. The boxes show 75,
50(median), and 25 percentiles; horizontal bar shows 90 and 10 percentiles in box plots. The AHCC group at 4 wk after AHCC intake had a significantly higher
number of total DCs in comparison with baseline. A: There were significant differences in the number of total DCs between control and AHCC groups. Significantly
higher number of DC1s in the AHCC group after AHCC intake was observed compared with baseline. B: Moreover, number of DC1s in the AHCC group tended
to be higher than in controls. C: After AHCC intake for 4 wk, the AHCC group had significantly higher numbers of DC2s than controls.
reference values in both groups (data not shown). Baseline levels
of tumor markers (carcinoembryonic antigen, carbohydrateanti-
gen 19-9, alpha-fetoprotein, and protein induced vitamin K ab-
sence II) were also within normal reference values in all subjects.
Flow Cytometric Analysis
At baseline, there were no significant differences in the num-
bers of total DCs, DC1s, and DC2s between the AHCC group
and controls (Fig. 2A–2C).
The AHCC group at 4 wk after AHCC intake had a signifi-
cantly greater number of total DCs than at baseline and than did
controls (total DCs: pre-AHCC, 11,932 ±8,706 cells/ml; post-
AHCC, 18,528 ±7,891 cells/ml; post-controls, 11,116 ±5,293
cells/ml, Fig. 2A). The number of DC1s was significantly higher
in the AHCC group after AHCC intake than at baseline. More-
over, the number of DC1s in the AHCC group had a tendency
to be higher than in controls (DC1: pre-AHCC, 9,018 ±7,046
cells/ml; post-AHCC, 13,061 ±7,364 cells/ml; post-controls,

648 N. TERAKAWA ET AL.
FIG. 3. Comparison of the allogeneic (allo-) mixed -leukocyte reaction
(MLR; allo-MLR) of dendritic cells (DC1s) between control and active hex-
ose correlated compound (AHCC) groups. Freshly prepared DC1s from blood
samples were examined for stimulating capacity against allogeneic T lympho-
cytes in a standard MLR. The allo-MLR of DC1s after AHCC intake was
significantly higher than in controls (P=0.044). NS, nonsignificant.
8,877 ±4,169 cells/ml, Fig. 2B). After AHCC intake for 4 wk,
DC2s in the AHCC group had significantly increased after 4 wk
compared to control values (DC2: pre-AHCC, 3,018 ±1,997
cells/ml; post-AHCC, 5,467 ±3,664 cells/ml; post-controls,
2,239 ±1,373 cells/ml, Fig 2C).
Allo-MLR
DCs isolated from peripheral blood were tested for the
ability to stimulate allogeneic T lymphocytes in a standard
MLR. At baseline, there was no significant difference in the
MLR of DC1s between the AHCC group and controls. Af-
ter AHCC intake for 4 wk, the MLR in the AHCC group
was significantly increased in comparison with the control
value (AHCC, 2.5 ±2.2 cpm: 1 ×103cells/DC1; controls,
1.1 ±1.1 cpm; P<0.05, Fig. 3). There was no signifi-
cant difference in MLR between baseline and after AHCC
intake.
Cell Surface Expression Levels of Costimulatory
Molecules in DC1s
Phenotypic analysis of Lin−DR+11c+(DC1) was per-
formed for both groups. Results are expressed as mean flu-
orescence intensity. The expression of CD40 and CD86 did
not differ significantly after AHCC intake in the AHCC
group and after 4 wk in the control group (data not
shown).
Other Parameters of Immune Function
There were no significant differences in the number of
PBMCs and lymphocyte subset distribution at baseline between
the AHCC group and controls. Changes in the count of each
fraction of PBMC, CD4+:CD8+ratio or DC1:DC2 ratio after
AHCC intake were not significant (Table 2). Also, no significant
differences in PHA and NK cell activity were found between
the AHCC group and controls (Table 2).
Cytokine Production and Serum Hormone Levels
All cytokine production stimulated by LPS did not differ
significantly between the AHCC group and controls before and
after AHCC intake (Table 3). No significant changes in thyroid
and adrenal gonadal hormones after AHCC intake were found.
DISCUSSION
In this double-blind, randomized controlled trial, we investi-
gated whether AHCC is as useful in improving immunological
competence as BRM. AHCC is an extract obtained from sev-
eral species of Basidiomycetes mushrooms. AHCC is a mixture
of polysaccharides, amino acids, lipids, and minerals derived
from fungi. It is obtained by hot water extraction after cultur-
ing mycelia of several Basidiomycetes in a liquid culture me-
dia and then treating them with some enzymes. The chemical
analysis has revealed that oligosaccharides are the major com-
ponents of AHCC, consisting about 74%, among which nearly
20% of the oligosaccharide are a-1, 4-glucan and their acety-
lated forms with an average molecular weight of approximately
5,000, which may be responsible for its biological activities.
In contrast to conventional active components such as the β-
1,3-glucan structural component found in PSK and lentinan,
the glucose oligomer in AHCC has an α-1,4-linkage structure
and some esterified hydroxy groups (18). However, AHCC may
function as a BRM in the same manner as PSK and lentinan. A
food is considered functional if it has been satisfactorily demon-
strated to have a beneficial effect on one or more target functions
in the body as a BRM in a way that is beyond adequate nutri-
tional effects and is relevant to either the state of well-being and
health or to a reduction in the risk of a disease (19).
We have reported that AHCC intake resulted in improved
liver function, the prevention of recurrence of HCC after resec-
tion, and the prolonged survival of postoperative HCC patients
without any adverse effects (8). Therefore, AHCC treatment
could be a valuable adjuvant therapy as a BRM in these pa-
tients. AHCC has been successfully used as a BRM in various
disorders, but little is known of its mechanism of action. There
have been only a few published, well-controlled studies of the
effect of AHCC on immune function. Therefore, we thought it
would be of interest to test AHCC for its immunomodulating
effects in a clinical trial.
DCs are highly specialized antigen-presenting cells able to
efficiently induce immune responses. During migration, they
acquire professional antigen presenting capacity, upregulate the

IMMUNOLOGICAL EFFECT OF AHCC IN HEALTHY VOLUNTEERS 649
TABLE 2
Immunological Parametersa
Baseline 4 Wk
AHCC Control PValue AHCC Control Pvalue
PBMC (×106/ml) 2.35 ±1.03 2.15 ±0.71 0.820 2.95 ±0.81 2.35 ±0.76 0.184
CD4 (%) 32.2 ±12.2 20.2 ±12.8 0.137 32.7 ±6.4 26.2 ±15.7 0.184
CD8 (%) 8.5 ±4.3 8.3 ±8.6 0.470 8.9 ±3.5 8.5 ±6.4 0.790
CD4:8 4.2 ±1.1 3.9 ±2.6 0.271 4.0 ±1.4 4.2 ±3.3 0.382
NK (%) 6.9 ±3.1 9.2 ±7.4 0.704 7.6 ±3.9 8.9 ±5.3 0.514
NK activity (%) 30.8 ±15.8 35.7 ±14.8 0.526 31.3 ±12.5 35.3 ±13.0 0.704
PHA (cpm) 39,304 ±16,570 51,221 ±20,812 0.324 41,768 ±13,517 56,117 ±18,783 0.157
aAbbreviations are as follows: PBMC, peripheral blood mononuclear cell; CD, cluster of differentiation; CD4:8, CD4:8 subset ratio; NK,
natural killer; PHA, phytohemagglutinin; cpm, counts per minute. Continuous variables are expressed as mean ±SD. PHA =proliferative
response of T lymphocytes towards mitogen.
major histocompatibility complex and costimulatory molecules,
and become competent to activate both T and B cells (9). DCs
play a central role in the initiation and modulation of immune
system responses.
We have investigated that both OK432 and KP-40, as BRMs,
were found to upregulate the activity of DC1 (13). The immuno-
logical monitoring of DCs may be a useful therapeutic strategy
in the treatment of pancreatic cancer (14,15). Two major sub-
sets of DC precursors have been identified in human peripheral
blood (20): the CD11c+subset belongs to the myeloid lineage,
whereas the CD11c−plasmacytoid subset (21–24) is of lym-
phoid lineage. Both subsets express high levels of HLA-DR and
lack the lineage markers CD3, CD14, CD15, CD16, and CD19.
Two DC subsets were shown to regulate immune responses via
the polarization of Th1, Th2, or even Th3/Tr1 differentiation
through the production of cytokines (25). DC1s are an essential
part of protection against cancer through the strong stimulation
of naive T lymphocytes. When tissues are damaged by malig-
nant transformation, DCs migrate to these sites. After capturing
antigens there, DCs produce a high amount of IL-12 as they
mature and migrate into the draining lymph nodes where they
present processed antigens to T lymphocytes to initiate an im-
mune response against the tumor (26).
In our study, the AHCC group had a significantly higher
number of total DCs than did controls, and the number of DC1s
in the AHCC group had a tendency to be higher than in con-
trols. Moreover, the allo-stimulatory activity of DC1s was also
increased. These results suggest that AHCC could be an ef-
fective modulator of immunological function in patients with
cancer. AHCC is as useful in improving immunological com-
petence as BRM. It might be that AHCC has an influence to
immune function from DCs. AHCC was reported to enhance
the activity of NK cells in cancer patients (27). Also, AHCC
reduced the metastasis rate of rat mammary adenocarcinomas
(18). Therefore, this AHCC effect may be mediated by natural
host immunity, which is restored or activated by AHCC. These
results suggest that AHCC acts as a promising BRM.
Results of an in vivo study showed that AHCC restores the
NK cell activity that was depressed by an anticancer agent and
stimulated peritoneal macrophage cytotoxicity and nitric oxide
TABLE 3
Cytokine Production Stimulated by LPSa
Baseline 4 Wk
AHCC Control PValue AHCC Control PVa lu e
IFN-γ(pg/ml) 774.3 ±329.7 915.8 ±299.4 0.205 844.3 ±395.6 701.5 ±283.7 0.481
TNF-α(ng/ml) 64.4 ±44.6 63.4 ±28.4 0.573 60.8 ±43.0 61.7 ±21.8 0.725
IL-10 (pg/ml) 281.1 ±87.9 317.7 ±252.6 0.944 288.0 ±142.5 251.0 ±162.4 0.526
IL-6 (ng/ml) 148.1 ±111.8 140.8 ±71.2 0.833 142.7 ±91.8 150.0 ±75.5 0.778
IL-4 (pg/ml) 601.6 ±246.3 744.1 ±382.1 0.204 598.6 ±225.2 455.7 ±457.5 0.205
IL-2 (pg/ml) 290.0 ±72.7 420.8 ±197.1 0.058 285.4 ±94.2 192.2 ±219.0 0.398
aAbbreviations are as follows: LPS, lipopolysaccharide; AHCC, active hexose correlated compound; IFN, interferon; TNF, tumor necrosis
factor; IL, interleukin. Continuous variables are expressed as mean ±SD.

650 N. TERAKAWA ET AL.
and cytokine production (18). The ratio of NK cells to total
lymphocytes increased after intake of AHCC for 3 mo in patients
with solid cancer (26). Uno et al. (27) reported that the basal
level of NK cell activity in cancer patients with solid tumors was
lower than in normal controls, but NK cell activity increased
to normal levels after AHCC intake at 6.0 g/day for over 4
mo. There was no obvious change in the ratio of CD4+:CD8+
after AHCC intake (26). We also examined PHA and NK cell
activity to assess the function of T lymphocytes and NK cells.
Impaired NK activity or PHA was observed in patients with lung,
esophageal, head and neck, or breast cancer (28–30). However
we found no significant difference in the number of NK cells or
CD4+:CD8+T lymphocytes or NK cell activity or PHA after
AHCC intake. Further study will be needed to determine the
effect of longer and higher doses of AHCC on PHA and NK
cells.
It was reported that IFN-γand IL-12 production in patients
with solid cancers was lower than in normal controls, and both
cytokines were increased to normal levels after AHCC intake
(27). IL-12 preferentially induced Th1 cells from na¨
ıve T cells
(31). It was suggested that AHCC might induce Th1 differen-
tiation. However, in our small study, there were no significant
differences in Th1 cell product cytokines; IL-2, IFN-γ, and Th2
cell product cytokines; and IL4, 6, and 10.
AHCC intake might improve subjective symptoms such as
lack of sleep, poor appetite, and feeling unwell in patients with
advanced cancer (32). Adrenocortical hormone has been used to
relieve some symptoms in many patients with advanced cancer.
It could be speculated that AHCC has adrenocortical hormonal
effects and psychotropic action.However, we found no alter-
ations in adrenocortical and thyroid hormones and those stim-
ulating hormones in healthy volunteers after 4 wk of intake of
AHCC.
In conclusion, AHCC intake for 4 wk in healthy volunteers
resulted in the improved number of DCs and function of DC1s,
which is a part of specific immunity but not innate immunity
such as NK cell activity and PHA. AHCC may be useful to
protect against cancer progression as well as microbial infection.
These observations need to be confirmed in longer, randomized-
controlled, double-blind trials. In addition, the dose-response
relationship should be investigated, and more detailed studies
are required to elucidate the mechanisms responsible for the
effects of AHCC.
ACKNOWLEDGMENTS
We thank Mr. Kohji Wakame and Kenichi Kosuna (Amino
Up Chemical Co. Ltd) for providing the AHCC and placebo.
Also, we thank Dr. M. Inaba (First Department of Pathology,
Kansai Medical University) for his skillful technical assistance,
Ms. S. Miura (First Department of Pathology, Kansai Medical
University) for sorting cells on a FACStar, and Ms. A. Kihara
(Department of Surgery, Kansai Medical University) and Ms. K.
Amamori (Department of Surgery, Kansai Medical University)
for manuscript preparation. There are no sources of funding,
grants, or contracts for the whole study with any companies
including Amino Up Chemical Co. Ltd.
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