Production of interleukin-10 in asthmatic children after
E. Sarinhoa,?, D. Medeirosb, D. Schorc, A. Rego Silvaa, V. Salesd, M.E. Mottaa,
A. Costab, A. Azoubele, J.A. Rizzof
aMaternal and Child Department of Universidade Federal de Pernambuco, Brazil
bUniversidade Federal de Pernambuco, Brazil
cAllergy and Immunology Research Centre of Universidade Federal de Pernambuco, Brazil
dPharmacy Department of Universidade Federal do Rio Grande do Norte, Brazil
eUniversidade Federal de Sa ˜o Paulo, Brazil
fInternal Medicine of the Clinical Medicine Department of Universidade Federal de Pernambuco, Brazil
Received 31 December 2008; accepted 15 February 2009
Available online 4 August 2009
Background: Beta-1-3 Glucan is a polysaccharide extracted from Saccharomyces cerevi-
siae with a possible immunomodulating action that may have a favourable action on
asthma symptoms and other allergic diseases. An experimental study carried out using a
murine respiratory model detected a decrease in pulmonary tissue eosinophilia, as well as
an increase in Interleukin-10 (IL-10) after glucan use.
Methods: This open, exploratory study with blind outcome evaluation included asthmatic
children between 6 and 12 years of age with mild to moderate persistent asthma and
inadequate disease control (rescue medication needed more than twice a week) in spite of
inhaled budesonide 400mg/day. After a four week run-in period, subcutaneous Beta-1-3-
glucan injections were given weekly for the first four weeks and then every two weeks for
the last four weeks. IL-10 levels, measured by the immunoenzymatic method (ELISA), were
compared before and after glucan administration.
Results: Twenty patients (14 male and 6 female) were included. Mean IL-10 levels were
6.4pg/ml and 11.3pg/ml before and after glucan, respectively (p ¼ 0.02). There was also
a reduction of asthmatic symptoms score at the end of study.
Conclusions: This is the first study which shows that subcutaneous particulate Beta-1-3-
glucan increases serum IL-10 levels in asthmatics. The possibility of glucan being able to
modulate allergic sensitisation and having a beneficial action in restoring Th2 function
should be assessed by means of properly planned controlled clinical trials, as it may
represent a new therapeutic strategy.
& 2008 SEICAP. Published by Elsevier Espan ˜a, S.L. All rights reserved.
ARTICLE IN PRESS
0301-0546/$-see front matter & 2008 SEICAP. Published by Elsevier Espan ˜a, S.L. All rights reserved.
E-mail address: email@example.com (E. Sarinho).
Allergol Immunopathol (Madr). 2009;37(4):188–192
Allergic diseases, including asthma, have reached epidemic
proportions in several countries around the world. Epide-
miological studies have shown an inverse association
between infections and allergies, an observation that
influenced the formulation of the ‘‘hygiene hypotheses’’
and still arouses a great interest in the understanding of the
pathogenic mechanism of these diseases.1–4
These diseases are characterized by the development of
specific IgE against certain allergens. A new contact with
these allergens activates mastocytes and results in inflam-
mation; symptoms due to the release of mediators; and
infiltration of mucosa by cells, particularly eosinophils. One
of the proposed mechanisms that might facilitate the typical
allergic inflammation response is the immune imbalance
consequent to Th2 lymphocyte hyperfunction due, among
other factors, to a low production of interleukin 10 (IL-10),
which results in altered T lymphocyte regulation.5–7
Therapeutic strategies to change the natural history and
progress of asthma and other allergic diseases are required.
Perhaps drugs able to modulate the immune system could
influence the cytokine profile aiming towards decreasing the
exaggerated Th2 response of allergic patients and replacing
it for an efficient and balanced Th1 response.8–10
In the respiratory and intestinal mucosa, antigen proces-
sing and presentation by dendritic cells and/or macrophages
is associated with a Th1 immune pattern that can suppress
allergic symptoms.11,12This Th1 immune response may also
be triggered by some bacteria and/or their products, such as
Beta-1-3-glucan is a polyglucose from fungi and yeast
cells’ walls. As it is not present in animal cells, it is
recognized by the immune system as non-self, leading to a
widespread stimulus for the innate immune system, a fact
that could be explored in restoring human health or even in
the prophylaxis of diseases.13
Depending on the origin, there are several kinds of glucan,
with different biological actions.14Beta-1-3-glucan, derived
from Saccharomyces cerevisiae, has been prepared and used
in basic research and clinical trials for several years now.13
The ability to activate the innate immune response resides in
the insoluble fungus wall fraction, known as Zymosan; its
main component is Beta-1-3-glucan, a polysaccharide con-
tained in the inner part of yeast cell walls.15,16
An experimental trial carried out in a murine model of
respiratory disease has detected a decrease in pulmonary
tissue eosinophilia, as well as a functional increase of
regulatory T cells and IL-10 after glucan administration,17a
result that may be attributed to macrophage stimulation. Our
research was carried out in order to test the hypothesis that
the subcutaneous use of Beta-1-3-glucan in mild to moderate
asthmatic children could modify interleukin-10 serum levels.
Material and methods
The research was approved by the institutional Ethics
Committee and all parents or guardians signed the informed
The study was carried out at the Research Center for
Allergy and Immunology at Hospital das Clı ´nicas from
Universidade Federal de Pernambuco, Recife, Brazil. It
included outpatients from 6 to 12 years of age with mild
or moderate persistent asthma and inadequately controlled
disease (characterized by rescue medication needed more
than twice per week) after a four week run-in treatment
period of inhaled corticosteroid (budesonide 400mg/day).
Thus, patients included were non-controlled asthmatics
despite previous treatment with inhaled corticosteroids
and none had any change in their prescribed therapeutic
scheme during treatment with glucan.
All 20 children had blood exams (total serum IgE, IL-10,
haemogram with platelets counts, aspartate and alanine
aminotransferases, urea, creatinine) and peak expiratory
flow rate (PEF) before glucan treatment period as shown in
the research flow diagram (Fig. 1).
Study design and sample estimate
This is an open label study with a blind outcome evaluation
(quasi-experimental) designed as an exploratory project to
assess changes in IL-10 serum levels after eight weeks
treatment with subcutaneous Beta-1-3-glucan in mild to
moderate asthmatic children.
For sample estimation we used the work of Ceyhan
el al.18who observed IL-10 serum levels in asthmatics of
4.373.8pg/ml. Twenty patients were needed in order to be
able to detect a 3.5pg/ml difference after treatment with a
and b errors of 0.05 and 0.20 (two-tailed) respectively.
Treatment with Beta- 1-3-glucan and adverse
During the eight weeks glucan (Imunoglucans) treatment
period patients continued to have their asthma medication.
For the first four weeks 0.5mg (0.25ml) of glucan was given
by subcutaneous injections once a week; each application
was carried out in a different limb in order to assess for
any local side effects. For the last four weeks the interval
between injections was increased to every two weeks. At
each new visit the patient was examined before receiving
the glucan dose in order to check for hyperaemia, pain,
nodulation or abscess on the injection area.
Parents or guardians filled a diary of asthma symptoms
(Visual Analogue Scale) and use of rescue medication
(salbutamol sulphate canister consumption). Side effects
such as fever, headache, asthenia, myalgia and hyperaemia,
pain, nodulation or abscess at injection site or any other
complaints were also recorded, as well as morning and
bedtime peak expiratory flow rate. One week after the last
injection, blood samples were drawn for IL-10 dosage and to
repeat blood tests. The follow-up period continued for the
ensuing three weeks in order to assess whether there were
any side effects after medication was stopped.
Serum was separated from whole peripheral blood by
centrifugation and stored at ?201C in coded vials. IL-10
determinations were blindly done and concealment was
ARTICLE IN PRESS
Production of interleukin-10 in asthmatic children after Beta-1-3-glucan189
achieved as the researcher was not able to identify
the patients or either if the sample was from before or
after treatment. IL-10 was quantified by means of the
immunoenzymatic method (ELISA) using the IL-10 Human,
Biotrak Easy ELISA – RPN5962 commercial kit (Amersham
Biosciences UK Limited, Buckinghamshire, UK), all the
manufacturer’s recommendations were followed and the
exam was carried out at the laboratory of Clinical
Immunology of the College of Pharmacy of Universidade
Federal do Rio Grande do Norte, Brazil. In brief, the
technique involved the following: serum samples and
standards with known concentrations of IL-10 were placed
in duplicates in well plates sensitised with anti-IL-10 human
monoclonal antibody and containing biotin coupled anti-IL-
10, sample diluent and lyophilised streptavidin-HRP. They
were incubated for 3h at 181C [64.41F] in a microplate
rocker; plates were then washed and the substrate solution
(tetramethyl benzidine-TMB) was added. After 15min
incubation under agitation 1M phosphoric acid was pipetted
in so as to interrupt the reaction. A reading of the
calorimetric reaction was taken at 450nm using a micro-
plate reader (Expert Plus-Asys, Eugendorf, Aus) and results
expressed in pg/mL.
As IL-10 log-transformation showed a fair adjustment to
normal distribution, Student’s paired t test was carried out
to compare means.
The study also had the secondary endpoint of check-
ing for adverse events and changes in symptoms. The
the 4th week of run-in period to the last week of treat-
ment period by Friedman nonparametric test for paired
was done comparing only
ARTICLE IN PRESS
Patients with mild or
Do not includeInclude
Blood tests, PEF, IL10
sc 0.25 ml/week
sc 0.25 ml/fifteen days
Blood test, PEF, IL10
Patients’ flow during the study.
E. Sarinho et al 190
The sample included 20 patients with persistent mild
(10 patients) and moderate (10 patients) asthma who were
followed up for 12 weeks. There were 14 male and 6 female,
with age median of 8 years (6–12 years). The research
included only those patients with partly controlled asthma
(use of rescue medication more than twice a week) despite
the use of 400mcg/day of inhaled budesonide in the first
four weeks (run-in period).
As is shown in Fig. 2, mean serum IL-10 rose from 6.4pg/ml
to 11.3pg/ml (p ¼ 0.02).
The commonest complaint reported by the patients after
subcutaneous injection of Beta-1-3-glucan was short-term
local pain but there was no report of any painful nodulation
or abscess. There was no adverse event demanding treat-
ment interruption or any other intervention.
Despite not being designed as a trial to assess clinical
treatment response, symptoms showed a significant im-
provement in diary scores in wheezing and day and night
cough but not in salbutamol sulphate rescue use (Fig. 3).
Beta-1-3-glucan is a polyglucose without lateral ramifica-
tions. It has no primary toxic action and shows wide
immunologic activity since it does not exist in animal cells.
Due to its capacity of coupling with the Toll Like Receptor 2
(TLR2), Dectin-1 (type C Lectin) and other receptors, Beta-
1-3-glucan is a powerful stimulant of the innate immunolo-
gic system and of the phagocytose macrophage defence
mechanisms.13,19–22The particulate form of glucan derived
from Saccharomyces cerevisiae was capable of flagging
activation of innate immunity directly by means of Dectin-
1 without any need to activate reactive linking TLR-2.23
Likewise, it has been shown that Beta-1-3-glucan is capable
of stimulating tumoricidal activity of Polymorphonuclears,
Macrophages and Natural-Killer Cells, and these effects are
mediated by its capacity to link with lecithin in the
complementary receptor 3 (CR3), thus making it work as
an immunomodulator for several infectious diseases and
neoplasias.13,22,24On the other hand, there are almost no
studies on a possible effect of Beta-1-3-glucan on allergic
Glucan action mechanism is dependent on the way it is
administered (oral, venous, intramuscular, subcutaneous)
and of some characteristics, including the source, solubility,
molecular mass, purity and structural conformation. An
improvement in the neutrophyle function, the oxidative
burst, bacteria death and increase in nuclear transcription
factors after the use of yeast glucan have been reported.25
This study is the first to demonstrate that injectable
particulate Beta-1-3-glucan is capable of increasing serum
IL-10 levels in asthmatics. Moreover, diary symptoms records
showed an improvement in wheezing and day and night
cough in these not well-controlled patients in spite of
inhaled budesonide 400mg/day.
Asthma inflammatory process is the result of a complex
set of innate and acquired immune system interactions. As
for innate immunity, the participation of the antigen-
presenting cell in the genesis of asthma has been increas-
ingly highlighted, since it is the starting point of Th2 bias
immune arrangement. Beta-1-3-glucan has been proving
itself to be a medication with a powerful action on
interferon-gamma production, in stimulating macrophages
and in its differentiation to antigen-presenting cells.13
Macrophages are able of modulating the immune response
because they secrete anti-inflammatory mediators such as
Prostaglandin E2 (PGE-2), Tumor Growth Factor (TGF-a) and
IL-10.13As such, Beta-1-3-glucan can act as a macrophage
stimulant and prevent the appearance of a Th2 response.
ARTICLE IN PRESS
*p = 0.02
IL10 (pg/ml) (logaritmic scale)
subcutaneous. Injections of Beta1-3-glucan. Bars represent
Serum IL10 both before and after 8 weeks of
in period and the 12th week of Beta1-3-glucan administration.
There was an improvement in wheezing (p ¼ 0.022), day
coughing (p ¼ 0.018), nocturnal coughing (p ¼ 0.044) and there
was no significant decrease in Salbutamol Sulphate rescue use
(p ¼ 0.722).
Mean diary symptom score of the fourth week of run-
Production of interleukin-10 in asthmatic children after Beta-1-3-glucan191
In an animal model a single high dose of Beta 1-3-glucan
has been related to an improvement in asthma and
pulmonary function abnormalities.17Likewise, it has been
reported that intradomiciliary exposure to high levels-
concentration above 60mg/g-of 1–3-b-D-glucan was asso-
ciated with a low risk of wheezing in infants born from
atopic parents; this effect was more emphatic in allergen
In a clinical trial using oral glucan, Yamada et al.27have
found that patients with allergic rhinitis showed an
improvement in seasonal and perennial symptoms after
the use of that medication. Besides, some patients
continued to show improved symptoms up to 6 months after
the treatment was interrupted, which might suggest the
effect was sustained.27
An improvement in oxidative burst, increase in nuclear
transcription factors and in neutrophil and macrophage
functions have been reported after the use of particulate
glucan from Saccharomyces cerevisiae,28a fact that had not
been described with the orally-administered soluble glucan.
Despite our study’s design not being adequate to assess an
improvement in asthma symptoms and the small patient
number, there was a symptom improvement. This may be
due to a placebo effect, but the increase of IL-10 in these
patients’ serum may point to the possibility of a therapeutic
effect of the medication. The possibility that Glucan may
favourably interfere on allergic diseases and exert a
beneficial action in restoring Th2 function, modulating
allergic sensitisation, should be assessed by means of
properly planned and controlled clinical trials, as it may
become a new therapeutic strategy.
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E. Sarinho et al192