Soluble beta-1,3/1,6-glucan from yeast inhibits experimental periodontal disease in Wistar rats.

Abstract

We have investigated whether a purified immunomodulatory water soluble beta-1,3/1,6-glucan isolated from the cell wall of Bakers yeast, Saccharomyces cerevisiae, would influence the progression of ligature-induced periodontal disease, and to modulate accompanying cytokine and hypothalamic-pituitary-adrenal (HPA) axis responses to a lipopolysaccharide (LPS) challenge.
beta-1,3/1,6-glucan (10 mg/kg/day) was given in the drinking water to Wistar rats during the entire experiment, starting 14 days before disease induction, while control rats were given tap water only. Periodontal disease was assessed when the ligatures had been in place for 35 days.
Orally administered soluble beta-1,3/1,6-glucan significantly reduced periodontal bone loss as measured on digital X-rays (p=0,026). Glucan-treated rats also showed a significantly enhanced plasma level of the HPA axis-driven hormone corticosterone (p=0.047), and of the cytokine transforming growth factor-1beta (p=0.032), as well as a tendency to enhanced IL-10 (p=0.106), induced by intra-peritoneally administered LPS.
Soluble beta-1,3/1,6-glucan administered by the oral route diminishes ligature-induced periodontal bone loss in this model. This effect may be attributable to the well documented ability of beta-1,3/1,6-glucan to stimulate macrophage phagocytosis and to skew the T helper (Th)1/Th2 balance towards Th1 and T regulatory responses. The HPA axis may play a significant role in beta-1,3/1,6-glucan induced immune modulation.

Full text

Page 1

Soluble b-1,3/1,6-glucan from
yeast inhibits experimental
periodontaldiseaseinWistarrats
Breivik T, Opstad PK, Engstad R, Gundersen G, Gjermo P, Preus H. Soluble b-1,3/
1,6-glucan from yeast inhibits experimental periodontal disease in Wistar rats. J Clin
Periodontol 2005; 32: 347–352. doi: 10.1111/j.1600-051X.2005.00672.x. r
Blackwell Munksgaard, 2005.
Abstract
Objective: We have investigated whether a purified immunomodulatory water
soluble b-1,3/1,6-glucan isolated from the cell wall of Bakers yeast, Saccharomyces
cerevisiae, would influence the progression of ligature-induced periodontal disease,
and to modulate accompanying cytokine and hypothalamic–pituitary–adrenal (HPA)
axis responses to a lipopolysaccharide (LPS) challenge.
Material and Methods: b-1,3/1,6-glucan (10mg/kg/day) was given in the drinking
water to Wistar rats during the entire experiment, starting 14 days before disease
induction, while control rats were given tap water only. Periodontal disease was
assessed when the ligatures had been in place for 35 days.
Results: Orally administered soluble b-1,3/1,6-glucan significantly reduced
periodontal bone loss as measured on digital X-rays (p50,026). Glucan-treated rats
also showed a significantly enhanced plasma level of the HPA axis-driven hormone
corticosterone (p50.047), and of the cytokine transforming growth factor-1b
(p50.032), as well as a tendency to enhanced IL-10 (p50.106), induced by intra-
peritoneally administered LPS.
Conclusion: Soluble b-1,3/1,6-glucan administered by the oral route diminishes
ligature-induced periodontal bone loss in this model. This effect may be attributable to
the well documented ability of b-1,3/1,6-glucan to stimulate macrophage phagocytosis
and to skew the T helper (Th)1/Th2 balance towards Th1 and T regulatory responses.
The HPA axis may play a significant role in b-1,3/1,6-glucan induced immune
modulation.
Key words: b-1,3/1,6-glucan; cytokines;
hypothalamo–pituitary–adrenal axis;
periodontal disease; macrophages
Accepted for publication 21 July 2004
Periodontal disease is a tissue destruc-
tive immune and inflammatory condition
triggered by increased colonization of
pathogenic microorganisms, termed per-
iodontopathogens, in sub-gingival dental
plaques. The disease leads to destruction
of the tooth supporting tissues, includ-
ing destruction of periodontal attach-
ment fibres and resorption of the alveolar
bone. The disease may progress to
periodontal pocket formation, increased
tooth mobility and tooth loss in the most
severe cases (Lindhe 1995).
The increased colonization of perio
dontopathogens, including some anae-
robic Gram-negative species, motile
rods, spirochetes, as well as virus
(Socransky & Haffajee 1992, Sela
2001, Kamma & Slots 2003) may be a
result of a too weak specific T helper 1
(Th1)-mediated immune response (Was-
senaar et al. 1998, Bartova et al. 2000,
Breivik & Thrane 2001), while the
periodontal fibre destruction may be
caused by reactive oxygen species
(ROS) and matrix metallo-proteinases
(MMPs) released from immune system
cells belonging to the innate immune
system, suchas
phagocytes (PMNs) when they fight
and destroy periodontopathogens (Bir-
kedal-Hansen 1993, Ding et al. 1997,
polymorphonuclear
Lohinai et al. 1998). The bone resorption
by osteoclast seems to be a secondary
process that prevents the bone from
being infected (Breivik & Thrane 2001).
Thus, in patients with periodontal dis-
ease, the immune response may be
biased towards a too strong T-helper 2
(Th2) mediated immunity, and immune
responses that accelerate cell-mediated
immunity, which include macrophage
activation and Th1 dominance, as well
as T regulatory (Treg) responses, may
inhibit the tissue destruction observed in
periodontal disease.
Severe periodontal disease has been
found to be associated with genetics,
Torbjørn Breivik1,2, Per Kristian
Opstad2, Rolf Engstad3,
Glenn Gundersen3, Per Gjermol
and Hans Preusl
lDepartment of Periodontology, Faculty of
Dentistry, University of Oslo,
Defence Research Establishment, Division
for Protection and Material, Kjeller,
Pharmacon ASA, Tromsø, Norway
2Norwegian
3Biotec
347
J Clin Periodontol 2005; 32: 347–352 doi: 10.1111/j.1600-051X.2005.00672.x
Copyright r Blackwell Munksgaard 2005

Page 2

aging, and environmental factors, includ-
ing smoking, poorly controlled diabetes,
and poorly developed psychological
coping strategies to traumatic life events,
such as the loss of a spouse by death
(Breivik & Thrane 2001, Hugoson et al.
2002). All these factors, which predis-
pose to periodontal disease, are asso-
ciated with increased activation of the
hypothalamic–pituitary–adrenal
axis (Breivik & Thrane 2001), which is
one of the major overarching immuno-
regulatory mechanisms controlled by the
brain (Ader et al. 1995, Chrousos 1995,
Breivik et al. 1996). HPA axis activation
by pathogenic microorganisms or other
danger signals, and the subsequent
release of glucocorticoids (predomi-
nantly cortisol in man and corticosterone
in rodents), are known to down-regulate
cell-mediated immunity
immune responses towards Th2 and T
regulatory (Treg) responses (Rook 1999,
Chen et al. 2004), partly by down-
regulating IL-12 and increasing IL-10
release from antigen-presenting cells
(Vieira et al. 1998). Thus, selected
periodontal risk factors may predispose
to disease development and progression
by their effect on the HPA axis (Breivik
& Thrane 2001).
Thisrelationship
axis reactivity, immune and glucocor-
ticoidresponses,
disease has been demonstrated clearly
ina rat model
2000a,b,c, 2001a,b, 2002a,b). Indivi-
duals that exhibit circadian dysregula-
tion of HPA axis, and thus show
inappropriate glucocorticoid responses
(whether it is genetically determined,
age-related, or environmentally stress-
induced) to inflammatory signals, may
be more susceptible to periodontal
disease. For example, postnatal treat-
ment that permanently modulates sub-
sequent excitability of the HPA axis
leadtocorresponding
immune responses and susceptibility
to periodontal disease (Breivik et al.
2002b). Thus, treatment strategies that
could lead to restoration of a proper
balance between Th1, Th2 and Treg
responses, which have been generated
when the immunity has been affected
by immune-suppressing factors, may
be favourable for the treatment of
periodontal disease. This effect has
recently demonstrated by vaccination
withan immunomodulatory
developed from killed Mycobacterium
vaccae (Breivik & Rook 2000, 2002,
2003).
(HPA)
and bias
between HPA
andperiodontal
(Breiviketal.
changesin
agent
b-1,3-glucan is a polyglucose struc-
ture in the cell wall of fungi, certain
bacteria and plants, and has be found to
have immunomodulatory effects in ani-
mals and humans (Williams et al. 1996).
For example, it enhances immune func-
tion by activating macrophages and
establishesTh1dominance,
induces cell-mediated immunity (Inoue
et al. 2002, Lee et al. 2002). Moreover,
b-glucan enhances resistance to infec-
tions caused by bacteria and parasites,
certain tumours, as well as stimulates
wound healing (Brown & Gorden 2001,
Yun et al. 2003).
Inability tomount
immune and stress response to control
the colonization of pathogenic micro-
organisms in sub-gingival dental pla-
ques may play a significant role in
periodontal disease development and
progression (Breivik et al. 1996, Brei-
vik & Thrane 2001). A consequential
objective for periodontal
would thus be to find a well-tolerated
substance that can stimulate protective
immune responses and thus control and
prevent the growth of periodonto-
pathogens. In this study we have used
a purified soluble carbohydrate poly-
mer known as b-1,6-branched-1,3-glu-
canextractfrom
(Saccharomycescerevisiae),
has been demonstrated to be a potent
inducer of innate immune mechanisms
both in animal models as well as in
human cell cultures (Engstad 1994,
Engstad et al. 2002).
The purpose of this study was to
examine whether soluble b-1,3/1,6-
glucancould influence
destructionina
experimental model of periodontal
disease in Wistar rats.
which
asuitable
research
Bakersyeast
which
thetissue
well-established
Materials and Methods
Animals
Thirty male Wistar rats, weighing
260–300g, were obtained from Mo ¨lle-
gaard Breeding Center (Ejby, Den-
mark), and used after 2 weeks of
acclimatization.Standard
pellets and tap water were available
ad libitum. The animals were housed in
groups of five under a 12/24h light/
dark cycle (light on 7:00 14:00 hours)
with temperature and humidity at 221C
and 40–60%, respectively, and grouped
in two at random. The experiments
were registered and approved by the
rat chow
Norwegian Experimental Animal Board
(NEAB).
Soluble b-1,6-glucan and treatment of rats
Water soluble b-1,3/1,6-glucan was
provided by Biotec Pharmacon ASA
(Troms?, Norway) at a concentration of
2% in water (pH 4.5) and stored at
141C. The b-1,3/1,6-glucan (10mg/kg/
day) was administered in the drinking
water to 15 rats, starting 14 days before
application of the ligatures. Fifteen
control rats received tap water only.
Experimental periodontal disease
Fourteen days after b.1,3/1,6-glucan
induction, all animals were anaesthe-
tised by subcutaneous injection in the
neck with Hypnorm-Dormicum (fena-
tyl/fluanizone, midazolam), 0.2ml/100g
body weight. Two rats in each group
died during the anaesthesia. A sterile
silk ligature (Ethicon Perma-handssize
3/0, Norderstedt, Germany) was tied
around the neck of the maxillary right
2nd molar tooth in the gingival sulcus.
The ligature served as a retention device
for oral microorganisms. The left 2nd
molar served as a non-ligated internal
control tooth. Thirty-five days after
application of the ligatures, all animals
were killed by decapitation. The max-
illae were excised and fixed in 4%
formaldehyde.
Radiographic examination
The specimens were stabilised with
dental wax on a Sidexis digital X-ray
sensor, orientated with the axis of the
teeth parallel to the sensor surface by
using ? 4 magnification loupe glasses
(Zeiss, Aalen, Germany). The distance
between the cemento-enamel junction
(CEJ) and bone (B) on mesial and distal
surfaces of the 2nd molars were dis-
played digitally. The examiner was
unaware whether the specimens came
from experimental or control animals.
Each X-ray was read three times, and the
mean of the three readings calculated.
The reliability of the method has been
tested earlier (Breivik & Rook 2000).
Theaveragepercentage
between individual readings and the
mean of the respective triplicate was
3.48 ? 5.12%.
difference
Lipopolysaccharide (LPS) challenge
The peripheral blood monocytes of
humans with periodontal disease release
348
Breivik et al.

Page 3

an altered profile of mediators when
stimulated by LPS (Fokkema et al.
2002), and the immune and HPA
systems are mutually regulatory (Turn-
bull & Rivier 1999, Eskandari & Stern-
berg 2002). The animals were therefore
injected with LPS (Escherichia coli
serotype 0111:B4, Sigma Chemical, St.
Louis, MO, USA) shortly (2h) before
ending the experiment to assess whether
the treatment regime influenced on the
corticosterone response to LPS.
After decapitation of the rats, the
blood samples were collected (6–10ml
from each animal) in vacutainer tubes
(10ml without additives) and allowed
to clot on ice for 1h. Thereafter, the
samples were centrifuged for 20min at
2000 ? g,
removed, and stored at ?201C prior
to analysis ofcorticosterone
cytokines.
andtheserum samples
and
Assay of corticosterone
Corticosterone was measured with125I
radioimmunoassay (RIA) coat-A-count
kit from Diagnostic Products Corpora-
tion (Los Angeles, CA, USA), catalog
number TKRC1. The detection limit
was 5.7ng/ml.
Assay of serum IL-10, TGF-1b and TNF-a
The levels of IL-10, TGF-1b, and TNF-
a in the serum samples were measured
by means of enzyme-linked immuno-
sorbent assay (ELISA) kits from R&D
systems, Inc. (MN, USA), with catalo-
gue numbersR1000,
RTA00, respectively. The minimum
detectable dose for IL-10 and TGF-1b
is less than 31.2pg/ml, and less than
12.5pg/ml for TNF-a.
MB100 and
Statistics
Values are expressed as mean ? Std.,
and differences between the b-glucan-
treated rats and control rats were
examined using
Because some of the data were not
normally distributed,
metric Mann–Whitney test was used
for between-group comparisons.
Student’st-test.
thenon-para-
Results
The treatment had no effect on the
weight of the animals. The controls and
glucan-treated animals
319 ? 20.8 and 322.2 ? 23.0g, respec-
tively, when the ligatures were applied,
and 388.6 ? 30.0 and 382.5 ? 29.3g,
weighed
respectively, at the termination of the
experiment, 35 days later.
The animals were sacrificed 35 days
after application of the ligature. Radio-
graphicallythe
measured as the distance between the
cemento-enamel junction (CEJ) and the
most coronal bone (CEJ-B) in the
experimental sites in Wistar rats that
had been treated with oral b-glucan 12
days before application of the ligature
was 0.94 ? 0.11mm. In the control rats
the same distance was 1.05 ? 0.12mm
(Table 1). The bone loss in the treatment
group was significantly reduced com-
pared with that seen in the untreated
controls (p50.026, Student’s t-test)
(Fig. 1).
Treatment with oral b-glucan lead
to a significantly stronger corticosterone
response 2h after LPS injection (glu-
can-treated 1371.0 ? 308.0nm/l; con-
trols 1067.4 ? 421.6nm/l; p50.0047;
Table1).Theglucan-treated
showed a higher TGF-1b (34.04 ?
5.83pg/ml) compared with controls
(27.78 ? 8.02pg/ml; p50.032), and a
tendency to a higher serum level of
IL-10 (70.00 ? 30.02pg/ml) compared
with controls(51.54 ? 25.79pg/ml;
p50.106). There was no difference
between the groups in TNF-a plasma
levels (3513.54 ? 3400.77pg/ml versus
3733.23 ? 4766.91pg/ml; p50.89).
meanboneloss
rats
Discussion
The present study shows that orally
administrated soluble b-1,3/1,6-glucan
resulted in enhanced resistance to liga-
ture-induced periodontal
Wistar rats. In addition, the b-glucan-
treated animals showed an increased
HPA axis and TGF-1b responses to a
robust LPS challenge, as well as a
disease in
Table1. Differences in weight, ligature-induced alueolar bone loss, and glucocorticoid hormone (corticosterone) and cytokine responses to LPS in
b-glucan and tap water drinking Wistar rats
Treatment Mann–Whitney,
p-value
b-glucan (n513) Tap water (n513)
Weight at glucan induction (g)
Weight at ligature placement (g)
Weight at sacrifice (g)
Weight change (g)
Bone loss, X-ray (mm)
Corticosterone (nm/l–2h after i.p. LPS, day of sacrifice)
TGF-1b (pg/ml 2h after i.p. LPS, day of sacrifice)
IL-10 (pg/ml 2h after i.p. LPS, day of sacrifice)
TNF-a (pg/ml 2h after i.p. LPS, day of sacrifice)
269.8 ? 12.9
318.4 ? 20.8
388.6 ? 30.0
118.6 ? 21.8
0.92 ? 0.10
1371.00 ? 308.0
34.04 ? 5.83
70.00 ? 30.02
3513.54 ? 3400.77
267.8 ? 9.8
322.2 ? 20.0
382.5 ? 29.3
114.7 ? 24.1
1.03 ? 0.09
1067.4 ? 421.6
27.78 ? 8.02
51.54 ? 25.79
3733.23 ? 4766.91
0.659
0.750
0.601
0.649
0.016
0.047
0.032
0.106
0.890
All data are shown as means ? standard deviation.
TGF-1b, transforming growth factor-1b; IL-10, interleukin-10; TNF-a, tumour necrosis factor-a; i.p., intraperitoneally; LPS, lipopolysaccharide.
Fig.1. Digital X-rays of maxillary right upper molar teeth in Wistar rats illustrating
differences in alveolar bone loss in orally b-glucan treated (left picture) and control rats (right
picture) given tap water only.
b-1,3/1,6-glucan inhibits periodontal disease
349

Page 4

tendency to stronger IL-10 response.
There are several possible modes of
action that could explain these effects.
b-1,3/1,6-glucan is known to act as
an immunostimulant enhancing host-
mediated immune responses to patho-
gens as well as tumour cells, especially
by activating macrophages, and stimu-
lating differentiation of T-cells towards
the Th 1 cellular subset (Brown &
Gorden 2001, Suzuki et al., 2001).
These effects are likely to be mediated
by Toll-like receptors (TLRs) and the
newly discovered Dectin-1 receptor
(Brown et al. 2003). These cell surface
pattern recognition receptors, which are
highly expressed on dendritic cells and
also on macrophages, lead to stimula-
tion of cytokine production such as
interleukin (IL)-12 and tumour necrosis
factor-a (TNF-a) (Gantner et al. 2003).
The cytokines (together with a number
of other immune signals) orchestrate an
immune and inflammatory response by
Th cells that skew the Th1/Th2 balance
towardsaTh1-dominated
(Suzuki et al. 2001). For example,
b-glucan stimulates TNF-a production
and macrophage phagocytosis (Lee et
al. 2002), and switches the balance from
IgG1 antibodies (which are Th2-depen-
dent antibody subclasses) towards a
Th1-dependent IgG2a response (Suzuki
et al. 2001). b-glucan also induces the
production of Th1-stimulating cytokine
interferon-g (IFN-g), but suppresses IL-4
that is inducing Th2 responses (Inoue et
al. 2002). Experiments also show that b-
glucan can modify immune responses by
stimulating the production of the Th1-
skewing cytokine IL-12 versus IL-6,
IL-10 and prostaglandin E2 (PGE2) by
macrophages (Murata et al. 2002).
In addition, soluble b-glucan has
been foundto alter
response to LPS as shown by suppres-
sion of the production of the cytokine
TNF-a from monocytes and lympho-
cytes stimulated by LPS (Soltys &
Quinn 1999). LPS consists of potent
antigens and immunostimulators located
on the outer membrane on all Gram-
negative bacteria. In addition, soluble
b-glucan protects against LPS-induced
shock in rats (Rasmussen & Seljelid
1991, Vereschagin et al. 1998). This can
be explained by its capability to sup-
press LPS-induced TNF-a production.
The immune response to b-glucan has
further been shown to be different from
the response to LPS. The latter causes
high recruitment of PMNs (Thorn et al.
2001), whereas
b-glucan
response
theimmune
primarily
seems to induce recruitment of macro-
phages (J?rgensen et al. 1993).
The ability of b-glucan to stimulate
macrophages may
the result found in this experiment. In
the gingival tissues from patients with
untreated advanced periodontitis, there
is an apparent failure of the recruitment
and activation of macrophages com-
pared with that found in gingival tissues
of patients with gingivitis (Chaple et al.
1998). Moreover, the cytokine pattern in
gingival tissue from cases of periodontal
disease reveals a relative increase in
Th2 cytokines (IL-4, IL-5 and IL-13)
whether studied by in situ hybridization
(Tokoro et al. 1997) or immuno-
histochemistry (Yamazaki et al. 1995).
Increased expression of IL-13 has been a
consistent finding whereas IL-4 mRNA
has been elusive (Fujihashi et al. 1996,
Yamamoto et al. 1997, Takeichi et al.
2000). However, difficulties in demon-
strating mRNA encoding IL-4 by RT-
PCR are not unusual because of its
extremely low mRNA copy number,
and the very short half-life of this mRNA
(Breivik & Rook 2003). The presence of
a Th2-biased response to the period-
ontopathogens is further supported by the
observation that peripheral blood cells of
patients, or T-cell clones derived from
them, release more Th2 cytokines in
response to periodontopathogens in vitro
(Wassenaar et al. 1998, Bartova et al.
2000). Interestingly, the peripheral blood
monocytes of patients with periodontal
disease release less IL-12 and more
PGE2 in response to LPS than do
monocytes from normal controls, and
this is a shift that would drive responses
to periodontopathogens towards Th2
(Fokkema et al. 2002).
It is also significant that the factors
that predispose to periodontal disease,
such as smoking (Martinez-Canut et al.
1995), age (Burt 1994), diabetes (Thor-
stensson & Hugoson 1993), depressive
mood stated caused by experiences of
negative life events and poor psycholo-
gical coping (Hugoson et al. 2002), all
lead to increased secretion of glucocor-
ticoids (Breivik & Thrane 2001), which
are known to bias responses towards
Th2 (Ramirez et al. 1996, Vieira et al.
1998, Visser et al. 1998). This relation-
ship between HPA axis activation,
glucocorticoid activity and periodontal
disease has been demonstrated clearly
inthe ratmodel
2000a,b,c, 2001a,b, 2002a,b).
In this experiment, b-1,3/1,6-glucan
showed a significant protective effect on
in partexplain
(Breivik et al.
the development of ligature-induced
periodontitisand
resulted in a stronger HPA axis response
to an acute and robust LPS challenge, as
demonstrated by higher corticosterone
levels. At the first glance, this result
may appear contradictory. However, the
HPA axis serves as a peripheral limb via
which the brain is suppressing pro-
inflammatoryTh1
boostinganti-inflammatory
responses (Elenkov & Chrousos 1999).
There are also indications that immune-
induced HPA activation may skew Th1
and Th2 effector responses towards Treg
responses (Chen et al. 2004), leading to
stronger IL-10 and TGF-b cytokine
responses (McGuirk & Mills 2002).
Thus, the continuous b-1,3/1,6-glucan
treatment may have lead to an enhanced
HPA axis response in order to control
the Th1 responses, and these rats may
be more capable of suppressing LPS-
induced immune responses. The immu-
nomodulatory effects of b-glucans on
LPS challenges may, thus, in part be a
result of its effect on the HPA axis.
In conclusion, these data suggest that
the enhanced periodontal disease resis-
tance induced by the soluble b-1,3/1,6-
glucan treatment, could be explained by
its action on the immune system and the
HPA axis. The relationships between
HPA axis activation and immunity,
tissue destruction induced by immune
mediators, and periodontal
development and progression are com-
plex. However, some of these issues can
be addressed by further experimental
animal studies, and b-1,3/1,6-glucan
would be an obvious candidate to be
tested as a therapeutic agent in clinical
trials for periodontal disease.
simultaneously
responses,while
Th2
disease
Acknowledgements
We are grateful to the Norwegian
Defence Research Establishment, Divi-
sion of Protection and Material, Kjeller,
Norway, for supporting this work.
References
Ader, R., Cohen, N. & Felten, D. (1995)
Psychoneuroimmunology: interactions bet-
ween the nervous system and the immune
system. Lancet 345, 99–103.
Bartova, J., Kratka, O. Z., Prochazkova, J.,
Krejsa, O., Duskova, J., Mrklas, L., Tlaska-
lova, H. & Cukrowska, B. (2000) Th1 and
Th2 cytokine profile in patients with early
onset periodontitis and their healthy siblings.
Mediators of Inflammation 9, 115–120.
350
Breivik et al.

Page 5

Birkedal-Hansen, H. (1993) Role of cytokines
and inflammatory mediators in tissue destruc-
tion. Journal of Periodontal Research 28,
500–510.
Breivik, T., Opstad, P. K., Gjermo, P. &
Thrane, P. S. (2000a) Effects of hypothala-
mic–pituitary–adrenal
periodontal tissue destruction in rats. Euro-
pean Journal of Oral Science 108, 115–122.
Breivik, T. & Rook, G. A. (2000) Prevaccina-
tion with SRL172 (heat-killed Mycobacter-
ium vaccae)inhibits
periodontal disease in Wistar rats. Clinical
andExperimental
463–467.
Breivik, T. & Rook, G. A. (2002) Treatment
with SRL172 (heat-killed Mycobacterium
vaccae) inhibits progression of established
experimental periodontal disease in Wistar
rats. Journal of Periodontal Research 37,
210–214.
Breivik, T. & Rook, G. A. (2003) Oral
treatment with SR299 (killed Mycobacterium
vaccae) inhibits experimental periodontal
disease in Wistar rats. Journal of Clinical
Periodontology 30, 931–936.
Breivik, T., Sluyter, F., Hof, M. & Cools, A.
(2000b) Differential susceptibility to perio-
dontitis in genetically selected Wistar rat
lines that differ in their behavioral and
endocrinological response to stressors. Beha-
viour Genetics 30, 123–130.
Breivik, T., Stephan, M., Brabant, G. E., Straub,
R. H., Pabst, R. & von Horsten, S. (2002b)
Postnatal lipopolysaccharide-induced illness
predisposes to periodontal disease in adult-
hood. Brain, Behaviour and Immunity 16,
421–438.
Breivik, T. & Thrane, P. S. (2001) Psychoneur-
oimmune interactions in periodontal disease.
In Ader, R., Felten, L. & Cohen, N. (eds).
Psychneuroimmunology,
627–644. San Diego, CA: Academic Press
(chapter 63).
Breivik, T., Thrane, P. S., Gjermo, P., Cools, A.
& Myhrer, T. (2002a) Effects of hippocampal
lesioning on experimental periodontal dis-
ease in Wistar rats. Journal of Periodontal
Research 37, 360–365.
Breivik, T., Thrane, P. S., Gjermo, P. &
Fonnum, F. (2001b) Postnatal glutamate-
induced central nervous system lesions alter
periodontal disease susceptibility in adult
Wistar rats. Journal of Clinical Perio-
dontology 28, 904–909.
Breivik, T., Thrane, P. S., Gjermo, P. & Opstad,
P.K.(2000c) Glucocorticoid
antagonist RU-486 treatment reduces perio-
dontitis in Fischer 344 rats. Journal of
Periodontal Research 35, 285–290.
Breivik, T., Thrane, P. S., Gjermo, P., Opstad,
P. K., Pabst, R. & von Horsten, S. (2001a)
Hypothalamic–pituitary–adrenal axis activa-
tion by experimental periodontal disease in
rats. Journal of Periodontal Research 36,
295–300.
Breivik, T., Thrane, P. S., Murison, R. &
Gjermo, P. (1996) Emotional stress effects on
immunity, gingivitis and periodontitis. Eur-
axis reactivityon
experimental
Immunology
120,
3rdedition, pp.
receptor
opean
327–334.
Brown, G. D. & Gorden, S. (2001) Immune
recognition. A new receptor for beta-glucans.
Nature 413, 36–37.
Brown, G. D., Herre, J., Williams, D. L.,
Willment, J. A. & Underhill, D. M. (2003)
Dectin-1 mediates the biological effects of
beta-glucans. Journal of Experimental Med-
icine 197, 1119–1124.
Burt, B. A. (1994) Periodontitis and aging:
reviewing recent evidence. Journal of Amer-
ican Dental Association 125, 273–279.
Chaple, C. C., Srivastrava, M. & Hunter, N.
(1998) Failure of macrophage activation in
destructive periodontal disease. Journal of
Pathology 186, 281–286.
Chen, X., Murakami, T., Oppenheim, J. J. &
Howard, O. M. (2004) Differential response
of murine CD41CD51and CD41CD241to
dexamethasone-induced cell death. European
Journal of Immunology 34, 859–869.
Chrousos, G. P. (1995) The hypothalamic–
pituitary–adrenal axis and immune-mediated
inflammation. New
Medicine 332, 1351–1361.
Ding, Y., Haapasalo, M., Kerosuo, E., Lounat-
maa, K., Kotiranta, A. & Sorsa, T. (1997)
Release and activation of human neutrophil
matrix metallo-and serine proteinases. during
phagocytosis of Fusobacterium nucleatum,
Porphyromonas gingivalis and Treponema
denticola. Journal of Clinical Periodontology
24, 237–248.
Elenkov, I. J. & Chrousos, G. P. (1999) Stress
hormones, Th1/Th2 patterns, pro/anti-inflam-
matory cytokines
disease. Trends in Endocrinology and Meta-
bolism 10, 359–368.
Engstad, C. S., Engstad, R. E., Olsen, J. O. &
Osterud, B. (2002) The effect of soluble beta-
1,3-glucan and lipopolysaccharide on cyto-
kine production and coagulation activation in
whole blood. International Immunopharma-
cology 2, 1585–1597.
Engstad, R. E. (1994) Yeast b-glucan as an
immunostimulant in Atlantic salmon (Salmo
salar L.): Biological effects, recognition and
structural aspects. Troms?: University of
Troms?.
Eskandari, F. & Sternberg, E. M. (2002) Neural
immune interaction in health and disease.
Annals of the New York Academy of Sciences
966, 20–27.
Fokkema, S. J., Loos, B. G., Slegte, C. & van
der Velden, U. (2002) A type 2 response in
lipopolysaccharide (LPS)-stimulated whole
blood cell cultures
patients. Clinical and Experimental Immu-
nology 127, 374–378.
Fujihashi, K., Yamamoto, M., Hiroi, T., Bam-
berg, T. V., McGhee, J. R. & Kiyono, H.
(1996) Selected Th1 and Th2 cytokine
mRNA expression by CD4(1) T cells
isolated from inflamed human gingival tis-
sues. Clinical and Experimental Immunology
103, 422–428.
Gantner, B. N., Simmons, R. M., Canavera, S.
J., Akira, S. & Underhill, D. M. (2003)
Collaborativeinduction
Journal of OralSciences
104,
England Journal of
andsusceptibilityto
fromperiodontitis
ofinflammatory
responses by dectin-1 and Toll-like receptor
2. Journal of Experimental Medicine 197,
1107–1117.
Hugoson, A., Ljungquist, B. & Breivik, T.
(2002) The relationship of some negative
events and psychological factors to perio-
dontal disease in an adult Swedish population
50 to 80 years of age. Journal of Clinical
Periodontology 29, 247–253.
Inoue, A., Kodama, N. & Nanba, H. (2002)
Effect of maitake (Grifola frondosa) D-
fraction of control of the T lymph node Th-
1/Th-2 proportion. Biological and Pharma-
cological Bulletin 25, 536–540.
J?rgensen, J. B., Lunde, H. & Robertsen, B.
(1993) Peritoneal and head kidney cell
response to intraperitoneally injected yeast
glucan in Atlantic salmon, Salmo salar L.
Journal of Fish Diseases 16, 313–325.
Kamma, J. J. & Slots, J. (2003) Herpervirus-
bacterial interaction in aggressive perio-
dontits. Journal of Clinical Periodontology
30, 420–426.
Lee, D-Y., Ji, I. H., Chang, H. I. & Kim, C. W.
(2002) High-level
and macrophage activity with soluble b-
glucansfrom Saccharomyces
Bioscience Biotechnology and Biochemistry
66, 233–238.
Lindhe, J. (1995) Textbook of Clinical Perio-
dontology. Copenhagen: Munksgaard.
Lohinai, Z., Benedek, P., Feher, E., Gyorfi, A.,
Rosivall, L., Fazekas, A., Salzman, A. L. &
Szabo, C. (1998) Protective effects of
mercaptoethylguanidine, a selective inhibitor
of inducible nitric oxide synthase, in ligature-
induced periodontitis in the rat. British
Journal of Pharmacology 123, 353–360.
Martinez-Canut, P., Lorca, A. & Magan, R.
(1995) Smoking and periodontal disease
severity. Journal of Clinical Periodontology
22, 743–749.
McGuirk, P. & Mills, H. G. (2002) Pathogen-
specific regulatory T cells provoke a shift in
the Th1/Th2 paradigm in immunity to
infectious diseases. Trends in Immunology
23, 450–455.
Murata, Y.,Shimamura,
Kobayashi, T., Takatsuki, F. & Hamuro, J.
(2002) The skewing to Th1 by lentinan is
directed through the distinctive cytokine
production by macrophages with elevated
intracellular glutathione content. Interna-
tional Immunopharmacology 2, 673–689.
Ramirez, F., Fowell, D. J., Puklavec, M.,
Simmonds, S. & Mason, D. (1996) Gluco-
corticoids promote a Th2 cytokine response
by CD41T cells in vitro. Journal of
Immunology 156, 2406–2412.
Rasmussen, L. T. & Seljelid, R. (1991) Novel
immunomodulators with pronounced in vivo
effects caused by stimulation of cytokine
release. Journal of Cell Biochemistry 46,
60–68.
Rook, G. A. (1999) Glucocorticoids and
immune function. Baillie `re’s Best Practice
& Researchs. Clinical Endocrinology &
Metabolism 13, 567–581.
Sela, M. N. (2001) Role of Treponema
denticola in periodontal disease. Critical
TNF-a
secretion
cerevisiae.
T., Tagami, T.,
b-1,3/1,6-glucan inhibits periodontal disease
351

Page 6

Review of Oral Biology and Medicine 12,
399–413.
Socransky, S. S. & Haffajee, A. D. (1992) The
bacterial aetiology of destructive periodontal dis-
ease. Journal of Periodontology 63, 322–331.
Soltys, J. & Quinn, T. (1999) Modulation of
enditoxin-and enterotoxin-induced cytokine
release by in vivo treatment with b-(1,3)-
glucan.Infections
244–252.
Suzuki, Y., Adachi, Y., Naohito, O. & Yado-
mae, T. (2001) Th1/Th2-balancing immuno-
modulating activity of gel-forming (1-3)-b-
glucans from fungi. Biological Pharmacolo-
gical Bulletin 24, 811–819.
Takeichi, O., Haber, J., Kawai, T., Smith, D. J.,
Moro, I. & Taubman, M. A. (2000) Cytokine
profiles of T-lymphocytes from gingival
tissues with pathological pocketing. Journal
of Dental Research 79, 1548–1555.
Thorn, J., Beijer, L. & Rydlander, R. (2001)
Effects after inhalation of (1-3)-beata D-
glucan in healthy humans. Mediators of
Inflammation 10, 173–178.
Thorstensson, H. & Hugoson, A. (1993)
Periodontal disease experience
long-duration insulin-dependent
JournalofClinical
352–358.
Tokoro, Y., Matsuki, Y., Yamamoto, T.,
Suzuki, T. & Hara, K. (1997) Relevance of
local Th2-type cytokine mRNA expression in
immunocompetent infiltrates in inflamed
andImmunology
67,
in
diabetics.
adult
Periodontology
20,
gingival tissue to periodontal diseases. Clin-
icalExperimental
166–174.
Turnbull, A. V. & Rivier, C. L. (1999)
Regulation of the hypothalamic–pituitary–
adrenal axis by cytokines: actions and
mechanisms of action. Physiological Reviews
79, 1–71.
Vereschagin, E. I., Lambalgen, A. A., Dushkin,
M. I., Schwartz, Y. S., Polyascov, L. &
Heemskerk, A. (1998) Soluble glucan pro-
tects against endotoxin shock in the rat: the
role of scavenger receptor. Shock 9, 193–198.
Vieira, P. L., Kalinski, P., Wierenga, E. A.,
Kapsenberg, M. L. & de, J. E. (1998)
Glucocorticoids inhibit bioactive IL-12p70
production by in vitro-generated human
dendritic cells without affecting their T cell
stimulatory potential. Journal of Immunology
161, 5245–5251.
Visser, J., Blauw, B., Hinloopen, B., Brommer,
E. R. d. K. E., Kluft, C. & Nagelkerken, L.
(1998) CD4 T lymphocytes from patients
withchronicfatigue
decreased interferon-gamma production and
increased sensitivity to dexamethasone. Jour-
nal of Infectious Disease 177, 451–454.
Wassenaar, A., Reinhardus, C., Abraham, I. L.,
Snijders, A. & Kievits, F. (1998) Character-
istics ofPrevotella
CD41T cell clones from peripheral blood
of a chronic adult periodontitis patient.
Immunology
107,
syndrome have
intermedia-specific
Clinical and Experimental Immunology 113,
105–110.
Williams, D. L., Mueller, A. & Browder, W.
(1996) Glucan-based macrophage stimulators
– A review of their anti-infective potential.
Clinical Immunotherapy 5, 392–399.
Yamamoto, M., Fujihashi, K.,
McGhee, J. R., Van Dyke, T. V. D. &
Kiyono, H. (1997) Molecular and cellular
mechanisms for periodontal diseases: role of
Th1 and Th2 type cytokines in induction of
mucosal inflammation. Journal of Perio-
dontal Research 115–119.
Yamazaki, K., Nakajima, T. & Hara, K. (1995)
Immunohistological analysis of T cell func-
tional subsets in chronic inflammatory perio-
dontal disease. Clinical and Experimental
Immunology 99, 384–391.
Yun, CH., Estrada, A., Van Kessel, A., Park, B.
C. & Laarveld, B. (2003) Beta-glucan,
extracted from oat, enhances disease resis-
tance against bacterial and parasitic infec-
tions.FEMS Immunology
Microbiology 35, 67–75.
Hiroi,T.,
and Medical
Address:
Torbj?rn Breivik
Department of Periodontology
P.O.Box 1109 Blindern
N-0317 Oslo
Norway
E-mail: tbreivik@odont.uio.no
352
Breivik et al.