Post on 30-Apr-2023
Poster Discussion Sessions
Poster Discussion Session 1
Childhood and adolescent and asthma
211
IgE-sensitisation to food allergens relates
to increased airways as well as systemic
inflammation in asthmatic children
Malinovschi, A1; Janson, C2; Berthold, M3; Borres, M3;
Alving, K4; Nordvall, L4
1Department of Medical Sciences: Clinical Physiology,
Uppsala University, Uppsala, Sweden; 2Department of
Medical Sciences: Respiratory Medicine and
Allergology, Uppsala University, Uppsala, Sweden;3Immunodiagnostics, Thermo Fischer Scientific,
Uppsala, Sweden; 4Department of Women’s and
Children’s Health, Uppsala University, Uppsala, Sweden
Background: Food allergy is common
among children with allergic asthma and
has been suggested to be linked to asthma
severity. However, the relation between IgE
sensitisation to food allergens and local air-
ways inflammation or systemic inflamma-
tion in subjects with allergic asthma has
been little studied.
Method: Within the frame of an industry-
academy collaboration on minimally-inva-
sive diagnostics (MIDAS), measurements
of the fraction of NO in exhaled air
(FeNO), serum eosinophil cationic protein
(ECP) and IgE against aeroallergen or food
allergen mix were done in 151 asthmatic
children aged 10–18 years. Three asthma
groups were defined: non-atopic (n = 31,
median age 15 years), atopic with IgE sen-
sitisation to only aeroallergens (n = 59,
median age 15 years) and atopic with IgE
sensitisation to both aero- and food aller-
gens (n = 61, median age 14 years).
Result: FeNO levels were 8.9 ppb (7.1,
11.1) in non-atopic asthma group,
14.2 ppb (11.7, 17.2) in atopic asthma
group with sensitisation to aeroallergens
(P = 0.01 compared to non-atopic
asthma) and 23.4 ppb (19.4, 28.3) in atopic
asthma group with sensitisation to both
aero- and food allergens (P £ 0.001 com-
pared to each of the other two groups).
Corresponding serum ECP levels for the
three groups were: 9.6 ng/ml (7.9, 11.8),
11.9 ng/ml (9.7, 14.5) (P = 0.55 compared
to non-atopic asthma), 21.9 ng/ml (18.6,
25.7) (P < 0.001 compared to each of the
other two groups). Asthmatic subjects sen-
sitized to both aero- and food allergens
had higher levels of FeNO and serum ECP
than asthmatics that were non-sensitised or
sensitised only to aeroallergens, after
adjustments for gender, age, height, lung
function, total IgE titers.
Conclusion: Sensitisation to food allergens
is common among children with allergic
asthma and is related to increased local
airways inflammation as well as systemic
inflammation. The clinical implications of
these findings warrant further studies.
212
Tolerance to egg proteins in
egg-sensitised infants without previous
consumption
Garcia, M; Alvaro, M; Giner, M; Chapman, E; Piquer, M;
Martin, M; Plaza, A
Pediatric Allergy and Clinical Immunology Section,
Hospital Sant Joan de Deu, Barcelona, Spain
Background: Egg allergy is one of the most
frequent allergy in infants. Positive skin
prick test (SPT) and specific IgE (sIgE) to
egg proteins has been described in infants
at high risk of atopy who have never eaten
egg. Although sensitisation can be asymp-
tomatic, clinical reactions are observed
when some of these infants eat eggs for the
first time. Our aim was: (i) To evaluate egg
tolerance in sensitised infants without pre-
vious consumption. (ii) To investigate the
relationship between food challenge (FC),
SPT and sIgE to egg proteins.
Method: Ninety-four infants with atopic
dermatitis and/or cow’s milk allergy. Mean
age 6.9 months (1–16), sensitised to egg
(positive SPT and/or sIgE), without previ-
ous consumption. SPT and sIgE to egg
proteins were performed in the first visit
and within 3 months previous to FC. All
infants underwent FC with cooked yolk
and egg white and raw egg between 12 and
18 months of age.
Result: Twenty-seven patients tolerated
cooked and raw egg (28.7%). Sixty-seven
patients had a positive FC (71.3%): 29 with
cooked egg (30.8%) and 38 with raw egg
(40.4%). Altogether, 65 patients tolerated
cooked egg (69%). Egg allergy: between the
first and the previous visit to FC, SPT to
egg yolk, white, ovalbumin and ovomucoid
had raised (P < 0.05). In three infants
(4.7%) sIgE were negative before FC. In
cooked egg allergic patients SPT to ovomu-
coid raised from a median of 3–8 mm
(P < 0.001), whereas infants allergic to raw
egg maintained a negative SPT to ovomu-
coid. SPT and sIgE to ovomucoid were
higher in cooked egg allergic patients before
FC than raw egg allergic patients
(P = 0.001). Egg tolerant: SPT became neg-
ative in 14 patients (52%). In the rest (13
patients, 48%) the trend was to maintain or
decrease SPT. Eighteen patients had nega-
tive sIgE before FC. SPT and sIgE in egg
allergic patients were higher than in tolerant
patients before FC (P < 0.01)
Conclusion: A high percent of sensitised
infants tolerated cooked egg. FC should be
performed safetly and early in order to
avoid unnecessary restrictive diets. SPT
changes help in the prediction of allergy or
tolerance to egg.
213
Modified skin prick testing to cod is a
diagnostic marker for Bangladeshi fish
allergy
Bakshi, D; Zoeteman, J; Minshall, E; Noimark, L
Paediatric General & Allergy, The Royal London
Hospital, London, United Kingdom
Background: The local population served
by the Royal London Hospital, UK,
consists largely of people of Bangladeshi
origin, where fish is a traditional compo-
nent of the diet. Currently there are no
commercial allergy tests for Bangladeshi
fish. Diagnosis is dependent on clinical
symptoms and modified skin prick testing.
We aimed to study the profile of fish
allergy in Bangladeshi children, and to
look for an association between the aller-
genicity of different species of fish, Ban-
gladeshi and native, using modified skin
prick testing (SPT).
Method: Modified SPT to three commonly
available fish in the UK (cod, salmon and
tuna) and four types of Bangladeshi fish
(ilish, boal, rui and ayre), was done in 32
children of Bangladeshi origin, with clinical
symptoms of IgE-mediated fish allergy.
98 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Result: There was significant cross-reactiv-
ity between Bangladeshi and UK fish.
Most children with positive reactions to
one or more types of Bangladeshi fish,
showed a positive reaction to cod 27
(84%) and salmon 23 (72%) on modified
SPT. The most common symptoms were
urticaria/rash in 23 (72%), angioedema in
15 (47%) and throat itch in 13 (41%). Epi-
pen (MEDA UK) had been prescribed in 7
(22%) children. Eczema 29 (94%) was the
most commonly associated co-morbidity,
followed by asthma 7 (22%) and hay fever
6 (19%). The most commonly associated
food allergies were tree nuts 14 (44%), egg
9 (28%) and lentils 9 (28%). Almost all
the children had a negligible reaction to
tuna on modified SPT.
Conclusion: Our study establishes that mod-
ified skin prick testing to cod, can be used as
a guide for diagnosing allergy to Banglade-
shi fish. The high degree of cross-reactivity
also suggests that parvalbumins are likely to
be the major allergen in Bangladeshi fish.
Tuna is likely tolerated in most patients due
to its paucity of parvalbumin content.
214
Serum immunoglobulin free light chain
levels in children are higher in females
than in males with eosinophilic
oesophagitis
Knipping, K1; Soulaines, P2; Groot Kormelink, T3;
Redegeld, F3; Knippels, L1; Garssen, J4; Dupont, C2
1Danone Research Centre for Specialised Nutrition,
Wageningen, The Netherlands; 2Hospital Necker,
University Paris-Descartes, Paris, France; 3Division of
Pharmacology, Utrecht University, Utrecht, The
Netherlands; 4Danone Research Centre for Specialised
Nutrition, Utrecht, The Netherlands
Background: Eosinophilic esophagitis (EoE)
is an emerging disease worldwide, charac-
terized by an eosinophilic infiltration of the
esophageal wall. EoE is closely associated
with male gender and allergic disorders,
such as food allergy, eczema and asthma.
The objective was to investigate a cohort
of 28 children with EoE for the distribu-
tion of established and newly explored
allergy markers, including immunoglobulin
free light chains (Ig-fLC).
Methods: Serum cow’s milk-specific IgE,
thymic stromal lymphopoietin (TSLP), thy-
mus- and activation-regulated chemokine
(TARC/CCL17) and Ig-fLC were analysed
in a cohort of 21 boys (age 6.7 years ± 4.2
SD) and seven girls (age 8.2 years ± 5.6
SD) suffering from EoE as diagnosed by
esophageal biopsy (eosinophils >30/high
power field).
Results: Cow’s milk specific IgE levels were
elevated when compared to clinical refer-
ence values in 9/21 (42.9%) males and 4/7
(57.1%) females. TARC was elevated in 1/
21 males (4.8%) and TSLP in 4/21 males
(20%) which was not the case in any
female within this cohort (ns). Kappa Ig-
fLC was elevated in 2/21 (9.5%) males vs
4/7 (57.1%) females (P = 0.008) and
lambda Ig-fLC was elevated in 1/21 (4.8%)
males vs 5/7 (71.4%) females, (P £ 0.001).
Conclusions: Gender is a key factor in the
biology of EoE. Serum Ig-fLC appeared
clearly increased in females as compared to
males, thereby adding another gender dif-
ference in the biology of EoE to the
already described non synonymous poly-
morphism in the TSLP receptor (TSLPR)
in males. The precise role for Ig-fLC in
EoE remains to be investigated.
215
Interleukin-5 and transforming growth
factor-beta levels in infants with cow
milk protein sensitisation
Sentsova, T; Revyakina, V; Denisova, S; Vorozhko, I;
Monosova, O; Pavlovskaya, E; Kirillova, O
Department of Allergology, Research Institute of
Nutrition, Moscow, Russian Federation
Background: Cow-milk protein sensitisat-
ion is common in infants, and allergic
reaction depends on ratio of interleukin-5
(IL-5) and transforming growth factor beta
(TGF-b) in the food allergy.
Method: Fifty-six infants (boys – 32, girls
– 24) aged 1–23 months with atopic derma-
titis were examined. Twenty-five age-
matched healthy infants were included in
the control group. Total IgE, allergen-spe-
cific IgE antibodies to cow-milk protein
and its fractions, IL-5, TGF-b serum levels
were measured by immunoenzyme method.
Result: High levels of cow-milk protein and
its fractions sensitisation were revealed in 51
infants (91.07%). IL-5 level was significantly
higher (P < 0.05) in infants with moderate
and severe course of the disease (62.17 ±
14.2 and 100.07 ± 13.4 pg/ml, respectively)
vs control group (2.77 ± 0.22 pg/ml); TGF-b
level was lower (9.3 ± 0.61 and 7.5 ±
0.71 pg/ml vs (18.02 ± 0.7 pg/ml). The
reverse correlation (r = -0.72; P = 0.01)
between IL-5 and TGF-b levels was found.
Conclusion: Cow milk protein sensitisation
in infants with atopic dermatitis is accompa-
nied by the high IL-5 level and insufficient
production of the growth factor TGF-b.
216
Mycoplasma pneumoniae infection affects
the serum levels of vascular endothelial
growth factor and interleukin-5 in atopic
children
Oh, J1; Kim, J1; Lee, H2
1Pediatrics, Hanyang University Guri Hospital, Guri,
Korea; 2Hanyang University Seoul Hospital, Seoul, Korea
Background: A number of studies has out-
lined mechanisms by which mycoplasma
infection may promote allergic lung inflam-
mation and airway remodeling. In addi-
tion, there is increasing evidence from
human studies suggesting that atypical bac-
terial infections contribute to asthma exac-
erbations, chronic asthma, and disease
severity with the change of cytokines. The
present study evaluated the change of
serum vascular endothelial growth factor
and interleukin-5 in atopic children with
Mycoplasma pneumonia.
Method: We recruited 25 atopic children
with mycoplasma pneumonia (Group 1),
24 non-atopic children with mycoplasma
pneumonia (Group 2), 15 atopic children
with viral pneumonia (group 3), 13 non-
atopic children with viral pneumonia
(Group 4). The change of serum levels of
interleukin (IL)-5, IL-13, vascular endothe-
lial growth factor (VEGF), tumor necrosis
factor-a, serum eosinophil cationic protein
concentration, were measured at admission
and at recovery for each group by using an
enzyme linked immunosorbent assay kits.
Result: The serum levels of VEGF and IL-5
from Group 1 increased compared with the
other groups. In addition, the serum level of
VEGF and IL-5 at admission was increased
at recovery in group 1 (VEGF: 876.0¡¾60
3.1 pg/ml at admission, 1103.2¡¾582.2 pg/
ml at recovery, IL-5: 114¡¾51.1 pg/ml at
admission, 143.2¡¾68.4 pg/ml at recovery).
Mean eosinophil cationic protein concentra-
tion were significantly increased at clinical
recovery compared to the serum concentra-
tion at admission.
Conclusion: The outcomes of the present
study implied the increased changes of
VEGF and IL-5 during Mycoplasma infec-
tion may be associated with the mechanism
by which the Mycoplasma pneumoniae
contribute to the development of hypersen-
sitivity during acute mycoplasma pneumo-
nia. There remained to evaluate the
pathophysiological mechanism of VEGF
and IL-5 during mycoplasma infection.
217
Risk factors for infections in early
childhood: a prospective birth cohort
study
Hawwa Vissing, N; Chawes, B; Rasmussen, M;
Bisgaard, H
Copenhagen Prospective Studies on Asthma in
Childhood, Health Sciences, University of Copenhagen
and Copenhagen University Hospital, Gentofte,
Denmark
Background: Young children experience
numerous simple infectious episodes partic-
ularly in the first years of life. There is a
considerable variation between children in
disease frequencies but evidence explaining
this variation is sparse. Some risk factors
have been identified but few have been
replicated.
Poster Discussion Session 1 – Childhood and adolescent and asthma
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 99
Aims: To identify risk factors associated
with incidence of common childhood infec-
tions during the first 3 years of life in a
clinical birth cohort study.
Material and methods: The Copenhagen
Study on Asthma in Childhood is a pro-
spective clinical study of a birth cohort of
411 children born of mothers with asthma
followed closely with planned and acute
visits, including 6-monthly interview ses-
sion on child’s infections during the first
3 years of life. Risk factor analysis was
performed including 112 endogenous and
environmental risk factors, using both tra-
ditional statistics and the data-driven
Sparse Principal Component Analysis.
Result: Three hundred and thirty-four chil-
dren had complete follow-up for this anal-
ysis. Children experienced an average of
15.0 infectious episodes in 3 years, most
commonly respiratory tract infections
(10.6 pr child/3 years). Tobacco exposure,
cesarean section, duration of breastfeeding,
older siblings and ORMDL3 genetic vari-
ant was associated with increased the risk
of lower respiratory tract infections.
Conclusion: Otherwise healthy children
experience a mean frequency of five epi-
sodes annually from 0 to 3 years, the
majority being respiratory tract infections
(approximately 70%). Individual variation
could in part be associated with tobacco
exposure, cesarean section, duration of
breastfeeding, older siblings and ORMDL3
genetic variant. However, these risk factors
only explain a small fraction of the inter-
individual variation of lower respiratory
infections and the major determinants of
childhood infections remains unknown.
218
The relationship between exhaled
leukotriene and 8-isoprostane levels with
the severity of asthma, asthma control
level and asthma control test score
Keskin, O1; Balaban, S2; Keskin, M2; Kucukosmanoglu, E1;
Bulent, G3; Ozkars, M1; Kul, S4; Bayram, H3; Co·kun, Y2
1Pediatric Allergy and Immunology, Gaziantep
University Hospital, Gaziantep, Turkey; 2Pediatrics,
Gaziantep University Hospital, Gaziantep, Turkey;3Pulmonology, Gaziantep University Hospital,
Gaziantep, Turkey; 4Biostatistics, Gaziantep University
Hospital, Gaziantep, Turkey
Background: Inflammation and oxidative
stress are essential parts of asthma patho-
physiology. A variety of methods are used
to measure intense inflammation in the air-
ways of asthmatics. Exhaled breath con-
densate (EBC) is a completely noninvasive
method for the collection of airway secre-
tions. Recently, it has been shown that
asthma control test (ACT) is a good mea-
sure which may be used in the evaluation
of asthmatic patients. It is not well known
if ACT score and asthma control level
correlates with the airway inflammation in
asthma. There is no study evaluating the
relationship between exhaled cysteinyl leu-
kotrienes (Cys-LTs) and 8-isoprostane lev-
els with the severity of asthma, asthma
control level and ACT score in children
with asthma.
Method: Thirty children with asthma were
evaluated with ACT score, pulmonary func-
tion tests and asthma severity and asthma
control level were assesed according to
GINA. EBC was collected and Cys-LTs and
8-isoprostane concentrations were deter-
mined using a specific immunoassay kit.
Result: Exhaled 8-isoprostane levels in
patients with moderate persistent asthma
[114 (55–146) pg] was higher than mild per-
sistent group [52 (21–91)] (P = 0.05,
MWU). EBC 8-isoprostane measures in
children with 1–4 asthma exacerbation/year
[52 (16.80) pg] was significantly lower than
children with >5 asthma exacerbation/year
[114 (57.129)] (P < 0.05, MWU). We could
not detect any significant relation between
exhaled 8-isoprostane, Cys-LTs and ACT
score, and asthma control level. There was a
positive correlation between exhaled Cys-
LTs and serum IgE levels (P = 0.028,
r = 0.467). In addition, exhaled 8-isopros-
tane levels correlated negatively with bron-
chodilator response (P = 0.045, r = -0.37).
Conclusion: Exhaled 8-isoprostane, as an
oxidative stress specifier, was found to be
increased in relation with asthma exacerba-
tion frequency. When the asthmatic children
had more asthma exacerbation they may be
faced to more oxidative stress. In addition,
oxidative stress increases with the severity of
asthma. Because most of our patients were
using inhaled corticosteroids we could not
show any relation between exhaled Cys-LTs
and asthma severity. Furthermore, ACT
score and asthma control level may not
reflect airway inflammation in asthma.
219
Pre- and postnatal administration of
Lactobacillus reuteri reduces TLR2
responses in infants
Forsberg, A1; Abrahamsson, T1; Jimenez, E1; Bjorksten,
B2; Jenmalm, M1
1Department of Clinical and Experimental Medicine,
Faculty of Health Sciences, Linkoping University,
Linkoping, Sweden; 2Karolinska Institutet, Institute of
Environmental Medicine, Stockholm, Sweden
Background: Mice models indicate that
intact Toll like receptor (TLR) signaling
may be essential for the allergy protective
effects of diverse bacterial exposure
observed in clinical and epidemiological
studies. We have previously shown that
supplementation with the Gram positive
probiotic strain Lactobacillus reuteri from
pregnancy week 36 and to the infant
through the first year of life decreased the
prevalence of IgE-associated eczema at
2 years. We explored the possibility that
the supplementation affected innate
immune responses to bacterial products
and the expression of associated TLRs.
Method: Blood mononuclear cells were
collected at birth, 6, 12 and 24 months
from 61 infants and cultured with the
ligands for TLR2, 4 and 9, i.e. lipoteichoic
acid (LTA) from Gram positive and lipo-
polysaccharide (LPS) from Gram negative
bacteria and unmethylated bacterial CpG
DNA. Cytokine and chemokine secretion
was determined using Luminex and mRNA
expression of TLR2, 4 and 9 by real time
RT-PCR.
Result: Probiotic supplementation was
associated with reduced LTA induced
chemokine (CCL4, P < 0.001, and CXCL8
P < 0.05) and cytokine (IL-1b, P < 0.001,
and IL-6, P < 0.05) responses at 12 months.
The levels of CCL4, trend, P = 0.094, and
IL-1b, P < 0.05, were also lower in the pro-
biotic group at 24 months of age. The
TLR4 and TLR9 responsiveness and the
mRNA expression of TLR2, 4 and 9 were
similar in the probiotic and the placebo
groups.
Conclusion: Reduced responses to TLR2,
which is the main receptor for LTA from
Gram positive bacteria, seem to be depen-
dent on factors downstream of TLR mRNA
expression. Since L. reuteri is Gram positive,
the reduced LTA responsiveness in the
probiotic group may reflect induction of a
tolerogenic immune response towards
Lactobacillus-associated TLR ligands. Pro-
biotic supplementation may be associated
with an increased immunoregulatory capac-
ity during infancy, in line with our previous
findings showing allergen hyporesponsive-
ness in the probiotic treated children.
220
Establishment and role of the
gastrointestinal microbiota in infantile
eczema: a randomised trial with bacterial
lysates
Penders, J1; Gerhold, K2; Rossberg, S2; Witt, I2;
Zimmermann, K3; Wahn, U2; Lau, S2; Hamelmann, E4
1NUTRIM School of Nutrition, Toxicology and
Metabolism, Maastricht University Medical Centre,
Maastricht, The Netherlands; 2Pediatric Pneumology
and Immunology, Charite Universitatsmedizin, Berlin,
Germany; 3Symbiopharm Herborn, Herborn, Germany;4Ruhr-University Bochum, University Children’s
Hospital, Bochum, Germany
Background: One of the explanations for
the allergic epidemic is that perturbations in
the gastrointestinal (GI) microbiota composi-
tion, as a result of changed lifestyles in west-
ernized countries, may have disrupted
mechanisms involved in the development of
immunological tolerance. The aim of the pres-
ent study was to examine the establishment of
Poster Discussion Session 1 – Childhood and adolescent and asthma
100 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
the infant gut microbiota and its association
to the development of atopic dermatitis
(AD).
Method: The present study was conducted
within the context of a randomized pla-
cebo-controlled trial (n = 606) on the pri-
mary prevention of atopic dermatitis by
oral supplementation of a bacterial lysate
containing heat-killed gram-negative E. coli
Symbio� and gram- positive Enterococcus
faecalis Symbio�. Fecal samples collected
at ages of 5 weeks (n = 594, start of inter-
vention) and of 7 months (n = 500, end of
intervention) were subjected to real-time
PCRs for the enumeration of bifidobacte-
ria, bacteroides, lactobacilli, Escherichia
coli, Clostridium difficile and Clostrium
cluster I. Children were followed up until
3 years of age and clinically examined on a
regular basis for signs of AD.
Result: Beside birth mode and duration of
breastfeeding, the presence of older siblings
had a strong effect on the GI microbiota
composition. With increasing number of
older siblings (0, 1, >2) the colonization
rates, at age 5 weeks, of lactobacilli (P for
trend <0.001) and bacteroides (P for
trend = 0.03) increased, whereas rates of
clostridia decreased (P for trend <0.001).
Colonization with clostridia at 5 weeks of
age was also associated with an increased
risk of developing AD in the subsequent
6 months of life in both the intervention
group and the placebo group. However, this
association persisted only in the placebo
group until the age of 2 years (ORadjusted
= 2.32; 95% confidence interval 1.18–4.56)
and 3 years (1.97; 1.01–3.86), respectively.
These findings were further supported by
survival analysis (Hazard Ratioadjust-
ed = 1.79; 95% CI 1.10–2.92). None of the
other bacteria under study at 5 weeks of age
or at 7 months were associated with subse-
quent AD development.
Conclusion: The results of this study are sup-
portive for a role of the indigenous microbi-
ota in the development of AD. Moreover, the
‘beneficial’ influence of older siblings suggests
that microbiota composition may be one of
the biological mechanisms underlying the
well-known ‘sibling effect’.
221
Impact of docosahexaenoic acid/arachidonic
acid supplementation on allergic
manifestations in the first year of life
Lapillonne, A1; Pastor, N2; Mitmesser, S3; Harris, C3;
Scalabrin, D3
1Paris Descartes University, APHP, Necker Enfants
Malades Hospital, Paris, France; 2Department of Medical
Affairs, Mead Johnson Nutrition, Madrid, Spain;3Department of Medical Affairs, Mead Johnson
Nutrition, Clinical Research, Evansville, IN, United States
Background: Infant formulas supplemented
with docosahexaenoic acid (DHA) and
arachidonic acid (ARA) provide visual and
cognitive benefits. Clinical and epidemio-
logic studies suggest that supplementation
with DHA/ARA may also provide protec-
tion against the incidence of allergic mani-
festations.
Method: In a multi-center, observational,
prospective study, participants received
marketed cow’s milk-based formula with
DHA and ARA (0.32% and 0.64% of
total fatty acids, respectively) supplementa-
tion (DHA/ARA, n = 233) or without
supplementation (Control, n = 92) from
the 1st or 2nd month through the 12th
month of age. At each of five study visits,
physicians collected information on the
incidence of allergic events.
Result: There was no difference between
groups for family history of allergy or
smoking in the home. Rate of study comple-
tion did not differ between groups [DHA/
ARA, n = 204 (88%); Control, n = 78
(85%)]. The number and proportion of par-
ticipants who had at least one episode of
atopic dermatitis (AD) were 49 (21%) in the
DHA/ARA and 28 (30%) in the Control
group; P = 0.083. The proportion of par-
ticipants who had at least one episode of
wheezing (defined as bronchiolitis or bron-
chitis with wheezing) [DHA/ARA, n = 35
(15%); Control, n = 24 (26%); P = 0.025],
wheezing/AD [DHA/ARA, n = 73 (31%);
Control, n = 46 (50%); P = 0.002], or
recurrent wheezing (>3 episodes) [DHA/
ARA, n = 3 (1%); Control, n = 5 (5%);
P = 0.044] was significantly lower in the
DHA/ARA vs Control. The odds ratio
(OR) of having an increased number of epi-
sodes of AD, wheezing, or wheezing/AD
were significantly lower for the DHA/ARA
vs Control (Table). The OR of having at
least one episode and the hazard ratio (HR)
for shorter time to first episode of wheezing
or wheezing/AD were significantly lower for
the DHA/ARA vs Control. Table. Allergic
manifestations in the first year of life in the
DHA/ARA group compared to Control
group
Conclusion: Early nutrition with DHA/
ARA supplementation was associated with
lower incidence, reduced number of epi-
sodes, and increased time to first diagnosis
of allergic manifestations in the first year
of life.
222
Safety of pediatric vaccinations in
children with mastocytosis
Pucino, V; Magliacane, D; Petraroli, A; Loffredo, S;
Marone, G; Triggiani, M
Division of Allergy and Clinical Immunology and Center
for Basic and Clinical Immunology (CISI), University of
Naples Federico II, Naples, Italy
Background: Mastocytosis is a rare disease
characterized by abnormal accumulation of
mast cells in the skin and internal organs.
Mastocytosis frequently appears in new-
borns and children. Symptoms of pediatric
mastocytosis are mainly due to the release
of mast cell-derived mediators, can be lim-
ited to the skin or systemic and include
pruritus, flushing, abdominal pain, diar-
rhea, tachycardia, hypotension and recur-
rent anaphylaxis. In pediatric patients
triggers are often unidentified although in
isolated cases vaccination has been consid-
ered as cause of anaphylaxis. In this pro-
spective study we analyzed the occurrence
of adverse reactions after mandatory vacci-
nations in a population of children with
mastocytosis.
Methods: Thirty-two children with masto-
cytosis, were prospectively followed in the
outpatient clinic of the Division of Allergy
and Clinical Immunology of the University
of Naples Federico II between 2003 and
2011. Mastocytosis was diagnosed accord-
ing to WHO criteria. Patients were vacci-
nated according to the current Italian
immunization program. Reactions to vacci-
nation were analyzed at our center between
1 and 72 h after vaccine administration.
Results: Cutaneous mastocytosis was clini-
cally evident and diagnosed before first
vaccination in 17 of the 32 patients. In the
remaining 15 patients, skin lesions
appeared between 4 and 6 months in 6 pts,
between 7 and 12 months (4 pts) and after
12 months (5 pts). A total of 265 vaccine
Diagnosis
OR (95% CI)*
of having at least
one episode� P-value
OR (95% CI)*
of having an
increased number
of episodes� P-value
HR (95% CI)*
for shorter time
to first episode§
P-value
AD 0.60 (0.34–1.04) 0.067 0.57 (0.33–0.98) 0.043 0.70 (0.44–1.11) 0.127
Wheezing 0.51 (0.27–0.96) 0.036 0.50 (0.27–0.92) 0.027 0.50 (0.28–0.87) 0.015
Wheezing/AD 0.44 (0.26–0.73) 0.002 0.42 (0.25–0.69) <0.001 0.59 (0.40–0.86) 0.006
*Adjusted for covariates.�Multiple logistic regression.�Ordinal analysis.§Cox proportional hazard–Defined as bronchiolitis or bronchitis with wheezing.
Poster Discussion Session 1 – Childhood and adolescent and asthma
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 101
doses were administered throughout the
study period. An adverse reaction was
detected in five out of the 32 patients
(15%) at the first dose of mandatory vacci-
nation that was in all cases an hexavalent
formulation (diphtheria, tetanus, pertussis,
poliovirus, Haemophilus influenzae type b
and hepatitis B). Clinical manifestations
appeared within 1 and 6 h and included
urticaria (3 pts) and bollous lesions (2 pts);
in one of these patients mild and transient
bronchospasm also developed.
Conclusions: Our study indicate that chil-
dren with mastocytosis have a slightly
higher rate of adverse reactions at the first
dose of mandatory vaccination than gen-
eral population. These reactions are gener-
ally mild and transient, do not recur on
booster administrations and do not require
treatment. Thus, it is advisable to adminis-
ter the first dose of vaccine to children
with mastocytosis in a monitored clinical
setting and to continue monitoring for at
least 6 h after vaccine injection.
223
Effect of intranasal corticosteroid
treatment on allergic rhinitis in patients
with asthma
Reyes, M1; Andaya, A1; Andaya, P2
1Section of Allergology and Clinical Immunology,
Department of Pediatrics, University of Santo Tomas
Hospital, Manila, Philippines; 2Pediatrics, University of
Santo Tomas Hospital, Espana, Manila, Philippines
Background: Allergic rhinitis (AR) and
asthma are the most common allergic air-
way diseases with epidemic proportions.
Studies attest to the interrelationship
between AR and asthma as evidenced by a
bidirectional relationship between nasal
and bronchial inflammation. Treating the
inflammation associated with AR may
have a beneficial effect on the control of
asthma, while the failure to treat rhinitis
may impair asthma control.
Method: This is a double-blind, random-
ized placebo-controlled trial that was done
in an out-patient department in a tertiary
university hospital. Sixty-six subjects (M/F:
32/34; age: 23 ± 7 years; mean ± SD)
with AR and mild asthma were random-
ized to a 30-day treatment period with
either nasal fluticasone furoate 27.5 lg,
two puffs per nostril twice a day, or pla-
cebo. Asthma control test (ACT) question-
naires and spirometry evaluations were
performed before and after intervention.
Result: Patients in the medication group
showed significant increase in lung function
in all parameters from baseline (FVC
P = 0.006, FEV1 P £ 0.001, FEV1/FVC
P = 0.006, FEF25–75% P £ 0.001, and
PEFR P £ 0.001) on repeat spirometry after
intervention. In comparing the increase in
placebo vs increase in the medication group
in the spirometry results, the increase in the
FVC (P = 0.002), FEV1 (P < 0.001),
FEV1/FVC (P = 0.048), FEF25–75%
(P < 0.001), and PEFR (P = 0.002) are all
significantly better in the active group than
in the placebo group. The ACT scores
increased significantly after treatment in the
medication group (22.35 ± 0.48 vs
16.65 ± 0.85, P £ 0.001; post- vs pre-treat-
ment). There was no significant difference in
all lung function parameters and ACT
scores after intervention in the placebo
group.
Conclusion: Intranasal corticosteroid treat-
ment resulted in significant improvement in
lung function parameters and significant
increase in asthma control scores in
patients with AR and intermittent asthma.
Physicians should keep in mind that treat-
ing the nose may strongly contribute to
control of asthma symptoms.
224
Attention deficit and hyperactivity
symptoms in children with allergic
rhinitis
Sogut, A1; Eres, M2; Beyhun, N3; Kalkandelen, S4;
Yilmaz, O5; Yuksel, H5
1Department of Pediatric Allergy, Regional Training and
Research Hospital, Erzurum, Turkey; 2Pediatrics,
Regional Training and Research Hospital, Erzurum,
Turkey; 3Department of Public Health, School of
Medicine, Ataturk University, Erzurum, Turkey;4Otolaryngology and Head & Neck Department,
Regional Training and Research Hospital, Erzurum,
Turkey; 5Department of Pediatric Allergy and
Pulmonology Unit, School of Medicine, Celal Bayar
University, Manisa, Turkey
Background: Allergic rhinitis (AR) may
cause neurobehavioral symptoms and
impair quality of life (QoL). We evaluated
efficacy of combination of fluticasone furo-
ate (FF) nasal sprey and oral levocetirizine
dihydrochloride (LD) on QoL and neuro-
behavioral health of seasonal allergic rhini-
tis patients.
Method: Twenty five children aged 8–
16 years with grass pollen-sensitized sea-
sonal AR were enrolled. They completed
the Total 4-Symptom Score (T4SS), and
Pediatric Rhinitis Quality of Life Question-
naire (PRQLQ). Conners’ Parent Rating
Scale-48 (CPRS) questionnaire was com-
pleted by the mothers to identify attention
deficit and hyperactivity symptoms in chil-
dren with AR. The patients were given FF
nasal spray 27.5 lg once daily and levoce-
tirizine dihydrochloride 5 mg teblet once
daily for 6 weeks. After the treatment for
6 weeks, the T4SS, PRQLQ, and the CPRS
were repeated. Thirty five healthy children
aged 8–16 years completed CPRS question-
naire as controls.
Result: There were no significant differ-
ences for age or sex between the AR and
control groups. After treatment, T4SS and
PRQLQ scores improved significantly in
the AR group (P = 0.001). Pre treatment
attention deficit and hyperactivity scores in
the AR group were significantly higher
than those in the control group
(P = 0.001, 0.009, respectively). After
treatment, attention deficit scores improved
in AR group, and there were no differences
in attention deficit scores between the two
groups, and hyperactivity scores in AR
group were lower than those in the control
(P = 0.785, 0.007, respectively).
Conclusion: AR may show a negative effect
on QoL and neurobehavioral health of the
patients. The combination treatment of FF
nasal sprey and LD tablet may improve
nasal symptoms, QoL, and neurobehavio-
ral symptoms in children with AR.
225
The relevance of patient-reported out-
comes in a grass pollen immunotherapy
trial in children with rhinoconjunctivitis
Roder, E1; Berger, M2; Hop, W3; de Groot, H4;
Gerth van Wijk, R1
1Section of Allergology, Department of Internal Medi-
cine, Erasmus MC, Rotterdam, The Netherlands;2Department of General Practice, University Medical
Center Groningen, Groningen, The Netherlands;3Department of Biostatistics, Erasmus MC, Rotterdam,
The Netherlands; 4Department of Pediatric Allergology,
Reinier de Graaf Groep, Delft, The Netherlands
Background: Patient-reported outcomes
(PROs) are the only instruments available
to assess the efficacy of an intervention in
patients with allergic rhinoconjunctivitis.
As allergic rhinoconjunctivitis is a systemic
disease, it is now recommended to use not
only PROs focusing at classical symptoms,
but also health related quality of life
(HRQL) instruments in immunotherapy
trials.
Method: A previously published immuno-
therapy trial in youngsters (6–18 years) with
hay fever provided us with data to assess the
relevance of two of these additional out-
come measures, the disease-specific Rhino-
conjunctivitis Quality of Life Questionnaire
(RQLQ) and the generic COOP/WONCA-
charts (CWC). We hypothesised that a rele-
vant PRO would have to be responsive to
pollen exposure and would at least have a
moderate correlation with the classical
symptoms of allergic rhinoconjunctivitis.
Furthermore, we evaluated a post-season
PRO, i.e. a global assessment of symptoms
(GAS). This assessment is used in clinical
trials as a tool for selecting participants with
sufficient symptoms and in daily practice to
evaluate the patient’s complaints during the
preceding season. We assessed the correla-
tion of this retrospective score with the
actual symptoms during the previous pollen
season.
Poster Discussion Session 1 – Childhood and adolescent and asthma
102 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Result: Data from 36 children and 63 ado-
lescents were analysed. Based on the total
scores of the paediatric and adolescent ver-
sion of the RQLQ, both questionnaires
were considered relevant, as they were
responsive to exposure and showed a mod-
erate to strong correlation with the rhino-
conjunctivitis symptoms. However, in both
children and adolescents 40% of the
RQLQ items were not relevant according
to our definition. The CWC as a whole
and the separate charts appear less relevant
because of the weak correlations with the
daily symptom score from the diary. The
correlation between our post-season GAS
and the in-season daily symptom score was
weak.
Conclusion: The paediatric and adolescent
RQLQ are relevant, but could be short-
ened as they contain a substantial number
of irrelevant items. The CWC are not rele-
vant in the monitoring of youngsters with
allergic rhinoconjunctivitis due to grass
pollen. The retrospective GAS does not
sufficiently reflect the actual symptoms
during the preceding season.
Poster Discussion Session 1 – Childhood and adolescent and asthma
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 103
Poster Discussion Session 2
Improving the diagnosis and management of allergies and
immune disorders
226
Serum specific IgE response to
staphylococcal super antigen may be a
potential biomarker for predicting the
progression from allergic rhinitis to
bronchial asthma
Shin, Y; Liu, J; Yoo, H; Hwang, E; Nam, Y; Jin, H; Ye, Y;
Nahm, D; Park, H
Department of Allergy and Clinical Immunology, Ajou
University School of Medicine, Suwon, Korea
Background: Allergic rhinitis (AR) is the
most common co-morbid condition in
allergic asthma (BA) and both of them are
considered as two different features of the
same airway disease. However, it is unclear
which factors could contribute to progress
from AR to BA. Several studies reported
higher prevalence of serum IgE to Staphy-
lococcal superantigens (sAg) in both AR
and BA patients. We hypothesized that
specific IgE response to sAg may contrib-
ute to develop BA from AR patients sensi-
tized to house dust mite (HDM).
Method: One hundred and two AR, 103
BA and 88 healthy controls (HC) were
enrolled. All AR and BA patients clinically
diagnosed by physicians were sensitized to
HDM. Serum total and specific IgE anti-
bodies to HDM and three sAgs (SEA, SEB
and TSST-1) were measured by immuno-
CAP system, and other clinical parameters
were retrospectively analyzed.
Result: The prevalence of serum specific
IgE to SEA, SEB, and TSST-1 were the
highest in BA patients (21.6%, 20.6%,
26.5%) followed by AR (11.7%, 14.6%,
21.3%) and HC (6%, 4%, 3%) with statis-
tical significances (P < 0.05 respectively).
The subjects having high serum specific
IgE to any sAg showed significantly higher
serum total and specific IgE levels to
HDM, and also significantly higher periph-
eral eosinophil count than those without
them (P < 0.05, respectively). Close corre-
lations were found among total and spe-
cific IgE level to sAgs (P < 0.05).
Conclusion: Specific IgE response to sAg in
AR patients may augment IgE production
to HDM and eosinophilic inflammation,
which may be a risk factor for progression
to BA.
227
IgE anti-Ascaris lumbricoides seems to
influence the specific IgE response to
indoor allergens in pediatric asthmatic
patients
Mendoza, D1; Lozano, S2; Garavito, G3; Navarro, E3;
Egea, E3
1Universidad del Magdalena, Santa Marta, Colombia;2Universidad del Norte, Barranquilla, Colombia;3Division ciencias de la Salud, Universidad del Norte,
Barranquilla, Colombia
Background: Dust mite Df, Dpt, Bt and
cockroach Pa are important sources of
allergens in the tropic. Host immune
response against Ascaris lumbricoides (Al)
could contribute to understand the influ-
ence of helminth antigenic proteins to
modulate the allergic response to mites in
patients. The objective of this study was to
establish the serological IgE Immune
response to Bt, Df and Pa, and to analyse
its possible association with the IgE spe-
cific response.
Method: Methods this study was an open
study conducted between January of 2010
and October 2011. Sixty asthmatic patients
were enrolled. Level of total IgE were deter-
minated by a commercial ELISA test. Spe-
cific IgE was determinated by a in house
indirect ELISA assay. Native extracts pro-
duced by us were characterized previously
by SDS-PAGE and enzymatic activity test
(APY ZYM). The IgE antibody reaction
was identified by Western blotting.
Result: SDS-PAGE showed the presence of
21 major bands in the extract of Al, 12 in Df
and 10 in Bt. All native extracts were posi-
tive for enzymatic activity. 4/60 (6.66%)
patients showed low levels of total IgE
(<40 IU/ml) (average = 23, 025 SD = 8,
60 634); 20 of 60 (33.33%) had normal
serum IgE level (40–150 IU/ml) aver-
age = 37, 90; SD = 34, 19); and 36/60
(60%) showed high IgE levels (>150 IU/
ml) SD = 814.8 SD = 1532, 9). ELISA
assay results show: 34/60 (56.66%) had a
positive IgE response to the Ascaris sp.
Average = 0.325357; SD = 0.219055. 46/
60 (76.66%) were positive to Bt and Df. Of
them 13 had a positive response for both
mite species. (21.66%), 30/60 were IgE posi-
tive to Bt. Seventeen patients (28.33%) were
IgE positive for Pa; average = 0.149824;
SD = 0.0648327. The statistical analysis
between total IgE, specific IgE to mites and
ascaris sp displays proportional significance.
Bt: P = 0, 007.df: P = 0, 027. Western
Blot results, identified proteins fractions in
extracts from Bt, Df, Ascaris sp and Pa with
nearby similar molecular weights between
200 and 45 kDa.
Conclusion: The specific IgE response
against Ascaris sp appear to influence the
IgE serological response to Bt, Df, and Pa
(CR) allergens in patients from the tropic.
Total IgE response seems to be inversely to
a specific IgE response against mite aller-
gens but those serological patter is not the
same when its compare total IgE and anti
Ascaris specific IgE in this group of
patients.
228
Comparative results of skin testing using
the preparation with recombinant pro-
teins CFP-10-ESAT-6 and interferon-
gamma release assay in tuberculosis in
children
Slogotskaya, L1; Litvinov, V1; Ivanova, D1; Kochetkov,
Y1; Ovsyankina, E1; Seltsovsky, P1; Koudlay, D2;
Nikolenko, N2; Filippov, A1
1Clinical Research, Scientific and Clinical Anti-tubercu-
losis Center, Moscow, Russian Federation; 2Pharmstan-
dart, Research, Moscow, Russian Federation
Background: Until recently, the tuberculin
skin test was the only test for detecting
tuberculosis and latent TB infection
(LTBI), but after decoding M. tuberculosis
genome and discovering genes of specific
proteins CFP-10 and ESAT-6 new in vitro
interferon-c release assays (IGRA) are
commercially licensed. So, QuantiFERON-
TB Gold in tube (QFT-GIT) measure
INF-c production to three specific antigens
– CFP-10, ESAT-6 and TB 7.7. But appli-
cation of laboratory tests in children is sig-
nificantly restricted by expensive costs,
need for equipped laboratories and intrave-
nous manipulations. The solution was
found in use of skin testing with DIA-
SKINTEST (DST) – recombinant protein
CFP-10-ESAT-6. Aim:
to compare sensitivity of DST and QFT-
GIT in children and adolescents with
active TB or latent TB infection (LTBI).
104 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Method: We studied 122 children and ado-
lescents aged 6–17 (average age
12.9 ± 3.34, median 12). DST revealed
pulmonary TB in 111 subjects (intratho-
racic lymph nodes TB – 44%, focal TB –
16%, primary TB complex – 23%, infiltra-
tive TB – 12%); LTBI in 11. All the chil-
dren received chemotherapy for at least
2 months (three drugs for active TB and
two drugs for LTBI). Initially we collected
3 ml of blood for QFT-GIT; at the same
day all the children received intradermal
injections with recombinant protein CFP-
10-ESAT-6 (DIASKINTEST) 0.2 lg in
0.1 ml.
Result: Positive DST was observed in 108
out of 122 subjects (88.5% 95%CI 81.5–
93.6%). The mean positive reaction was
16.6 ± 4.56 mm. Positive QFT-GIT was
registered in 109 (89.3% 95% CI 82.5–
94.2%) subjects. Agreement of DST and
QFT-GIT measured by kappa coefficient
was 0.709 (95% CI 0.605–0.813;
P = 0.000). Discordance was registered in
seven cases (5.7%, 95% CI 2.3–11.5%)
with threshold values of QFT-GIT and
reversion of early positive DST after treat-
ment.
Conclusion: DST and QFT-GIT have com-
parable high sensitivity; the discordance of
tests in seven out of 122 subjects possibly
results by near-threshold variability of
QFT-GIT values and dynamics of DST
during chemotherapy.
230
Variation of immunological parameters
in patients with chronic myeloid
leukemia
Humlova, Z1,2; Klamova, H3; Janatkova, I1,4; Hindos, M1,4;
Petrackova, M5; Nemeckova, S5; Vonka, V5
11st Medical Faculty, Charles University, Prague, Czech
Republic; 2Department of Immunology and
Microbiology, General Teaching Hospital in Prague,
Prague, Czech Republic; 3Clinical Department, Institute
of Hematology and Blood Transfusion, Prague, Czech
Republic; 4Department of Clinical Biochemistry and
Laboratory Medicine, General Teaching Hospital in
Prague, Prague, Czech Republic; 5Department of
Experimental Virology, Institute of Hematology and
Blood Transfusion, Prague, Czech Republic
Background: It is the purpose of the pres-
ent study to construct immunological pro-
files of a number of CML patients by
testing several parameters of their innate
and specific immunity early after diagnosis,
i.e. prior to the start of any therapy and
then to follow the influence of different
therapeutic regimens on these parameters
and the association of their changes with
the clinical condition. Finally, find parame-
ters which could be helpful in process of
vaccination treatment for CML.
Method: We have investigated 27 patients
(17 men and 10 women; median 56 years;
range 21–75) in chronic phase of CML
before starting any therapy and follow-up
five patients during therapy. In these
patients, we monitored a wide spectrum of
immunological parameters including immu-
noglobulin levels, presence of autoantibod-
ies, complement components, C- reactive
protein, parameters of cellular immunity,
intracellular cytokines and serum level of
selected cytokines and chemokines.
Result: Level of IgG was increased in three
patients and was normalised after therapy,
similarly as increased levels of IgM and
IgA. The level of C3 complement com-
pound was decreased in nine patients
(34.6%). C4 complement compound was
decreased during therapy in four patients.
Most important changes were observed in
CRP level which was increased in 12
patients (46%). Investigating autoantibod-
ies we have observed positivity of cANCA
targeted to proteinase-3 which belongs to
tumors antigens described in CML. The
most frequent were anti-desmosomal anti-
bodies which were detected in six cases
(22%). We have also investigated the
amount of Treg cells. Before the therapy
the percentage of positive cells was 1.84%.
And amount was increased after any ther-
apy up to 2.28%. We have also observed
decrease of intracellular cytokines. Produc-
tion of IFN gamma and IL-2 was
decreased in eight patients (42%), TNF
alpha in 9 (47%) and IL-4 in four patients
(21%).
Conclusion: The present results indicate
that the altered parameters of innate
immunity found in the CML patients prior
to the start of any therapy tended to nor-
malize in the course of therapy leading to
remission.
231
Immunohistochemical evaluation of p53
gene expression and survivin level in
children with non-Hodgkin’s lymphoma
ElBaz, H1; Fouda, M1; Yahya, R2
1Biochemistry Department, National Research Centre &
King Abdul Aziz University, Cairo, Egypt; 2Clinical
Pathology Labs, Children Hospital, Mansoura, Egypt
Background: This study was conducted to
evaluate the prognostic significance of the
p53 gene expression and serum survivin in
non-Hodgkin’s lymphoma (NHL) and its
correlation to patients’ outcome.
Methods: Forty five children newly diag-
nosed with NHL were treated with chemo-
therapy and followed up for 18 months or
until death. Twenty apparently healthy
volunteers with matched age and sex were
taken as control. P53 expression was
measured by Flow cytometry and Survivin
protein was measured by quantitative sand-
wich enzyme immunoassay at diagnosis and
at complete remission.
Results: A highly significant elevation
(P < 0.001) was found in survivin protein
and p53 in NHL children patients at diag-
nosis compared to controls. At complete
remission a significant decrease of the two
indices were found in NHL patients com-
pared to those at diagnosis (P < 0.001).
Survivin protein and p53 was significantly
higher in non-survived compared to sur-
vived group. A positive correlation was
found between survivin level and p53 level
in children with NHL.
Conclusion: P53 and surviving expression
have a synergetic bad prognostic indicator,
inde-pendent of age and sex, in children
with NHL. Survivin protein is related to
anti-apoptotic proteins and its high expres-
sion lead to unsuccessful treatment of
NHL.
232
Efficacy of intravenous immunoglobulin
therapy on steroid-resistant cardiac
insufficiency in patients with Churg
Strauss syndrome
Masami, T; Kazuo, A; Chihiro, M; Yuma, F; Noritaka, H;
Hidenori, T; Kiyoshi, S; Kenji, M
Sagamihara National Hospital, Sagamihara, Japan
Background: Cardiac insufficiency is the
most important prognostic factor in
patients with Churg-Strauss syndrome
(CSS). From our preliminary study (Aller-
gol Int 2007), we consider that intravenous
immunoglobulin (IVIG) therapy is a poten-
tial candidate for second-line treatment of
CSS patients, particularly in those with
cardiomyopathy, which are resistant to
conventional therapy. However, there is lit-
tle evidence supporting the effectiveness of
IVIG in CSS patients.
Methods: We determined the effectiveness
of IVIG therapy for steroid-resistant car-
diomyopathy in Japanese CSS patients.
Because cardiac involvement in CSS
patients is sometimes fatal and our preli-
minary study showed a marked improve-
ment of severe cardiac dysfunction after
IVIG therapy, we evaluated the effective-
ness of IVIG therapy in all new-onset CSS
patients with cardiomyopathy in an open
protocol without placebo. Fifteen Japanese
new-onset CSS patients showing cardiac
involvement with a low left ventricular
ejection fraction (LVEF), that is lower
than 50%, were enrolled in this study. We
treated these patients firstly with conven-
tional therapy (corticosteroid and cylo-
phosphamide) alone for 8 weeks, and then,
we added two cycle of IVIG therapy (Tei-
jin Kenketsu-Venilon I 2 g/kg body weight
once a month for 2 months). After 8 weeks
of IVIG therapy, we assessed the effect of
this therapy on LVEF % and serum and
BNP concentration.
Poster Discussion Session 1 – Childhood and adolescent and asthma
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 105
Results: There was a significant but slight
increase in LVEF % from 34 ± 6 to
38 ± 7% (P < 0.05) after conventional
therapy alone. However, we found a
marked and significant increase in LVEF%
from 38 ± 7 to 59 ± 11% after IVIG
therapy (P < 0.001). Moreover, BNP con-
centrations decreased significantly after
IVIG therapy (P < 0.01). No serious
adverse effect was observed in all patients.
Conclusion: IVIG may be a potential rescue
treatment of steroid-resistant cardiomyopa-
thy in CSS patients.
233
Efficacy of rupatadine in the treatment
of cutaneous and indolent systemic
mastocytosis
Siebenhaar, F; Fortsch, A; Maurer, M
Dermatology and Allergy, Charite – Universitatsmedizin
Belrin, Berlin, Germany
Background: The vast majority of adult
patients with mastocytosis suffer from
cutaneous or indolent systemic mastocyto-
sis, which is characterised by symptoms
resulting from mast cell mediator release,
including pruritus, flushing, diarrhoea, and
abdominal pain, up to severe and life-
threatening anaphylaxis. Therefore, many
patients require continuous and emergency
medications. Non-sedating antihistamines
(nsAHs) are the first line therapeutic strat-
egy, and nsAHs are routinely used for the
treatment of mastocytosis. However, data
on the efficacy of nsAH therapy in masto-
cytosis from controlled clinical trials is
missing. Therefore, rupatadine (RUP), a
nsAH and PAF receptor anatagonist, was
tested for efficacy in the treatment of cuta-
neous and indolent systemic mastocytosis.
Method: An exploratory, randomised, dou-
ble-blind, placebo-controlled, cross-over
trial was performed to assess the efficacy
of RUP 20 mg in the treatment of masto-
cytosis symptoms. Thirty adult patients
were enrolled, and efficacy was assessed by
volumetric and thermographic measure-
ments of lesional skin responses to standar-
dised mechanical irritation prior (elicitation
of Darier’s sign) before and after 28 days
of treatment. In addition, patients docu-
mented their symptoms using a visual ana-
log scale (VAS), and quality of life
impairment was assessed by a symptom
specific questionnaire (ItchyQoL).
Result: The development of wheal and
flare type skin reactions was significantly
reduced in subjects treated by RUP 20 mg
as assessed by volumetric analyses
(P = 0.01). Patients treated with RUP
exhibited significantly reduced symptom
severity compared to baseline as assessed
by VAS (P = 0.003), whereas placebo
failed to improve symptoms. In addition,
RUP treatment, but not placebo, resulted
in pronounced quality of life improvement
(RUP vs placebo: P < 0.001).
Conclusion: These data, for the first time,
demonstrate the efficacy of nsAH treat-
ment in mastocytosis in a randomised con-
trolled trial. RUP was found to be well
tolerated, controlled skin symptoms, and
improved quality of life of patients. RUP
should be considered a first line treatment
option for symptomatic patients with cuta-
neous or indolent systemic mastocytosis.
Further studies should determine the role
of PAF in mastocytosis and the contribu-
tion of RUP’s action on PAF receptors to
the overall treatment effects of RUP in
mastocytosis.
234
Efficacy and safety of the anti-interleukin-
1 beta antibody canakinumab in patients
with Schnitzler’s syndrome
de Koning, H1; Schalkwijk, J2; van der Ven-Jongekrijg,
J3; Stoffels, M3; van der Meer, J3; Simon, A3
1General Internal Medicine and Dermatology, Radboud
University Nijmegen Medical Centre, Nijmegen,
The Netherlands; 2Dermatology, Radboud University
Nijmegen Medical Centre, Nijmegen, The Netherlands;3General Internal Medicine, Radboud University
Nijmegen Medical Centre, Nijmegen, The Netherlands
Background: Schnitzler syndrome is a
chronic disabling autoinflammatory disor-
der, characterized by chronic urticaria,
paraproteinemia and systemic inflamma-
tion. The interleukin-1 receptor antagonist
(IL-1Ra) anakinra has been reported to be
effective, but requires daily injection
because of its short half-life, and is associ-
ated with injection site reactions. Cana-
kinumab is a fully human monoclonal
selective anti-IL-1b antibody with a half-
life of approximately 23 days. Here we
report the first trial of canakinumab in
patients with Schnitzler syndrome treated
with monthly injections.
Method: Eight adults with active classical
or variant type Schnitzler syndrome who
previously responded to anakinra, entered
a 9-month trial comprising 6 months of
open-label 150 mg canakinumab sc injec-
tions every 4 weeks and 3 months follow-
up. Patients were evaluated clinically using
a 5-point disease activity scale and serolog-
ically by C-reactive protein (CRP) levels.
The primary endpoint was the percentage
of patients with complete or clinical remis-
sion at Day 14. Complete remission was
defined as clinical remission with normal
CRP and clinical remission was defined as
having absent or minimal disease activity
and >70% improved, but still elevated,
CRP.
Result: Upon discontinuation of anakinra,
all patients developed active Schnitzler syn-
drome characterized by general malaise,
non-pruritic to burning urticaria, fever and
arthralgia or arthritis. In all patients, com-
plete or clinical remission was achieved at
Day 14. Mean CRP concentrations
decreased from 162 at baseline to <10 mg/
l on Day 14 and remained low or undetect-
able afterwards. One patient discontinued
on Day 39 because of return of symptoms
while all others remained in complete or
clinical remission for the remainder of the
6-month treatment period. Relapse in the
follow-up period occurred in four patients
and ranged between 1.5 and 3.5 months
after the last canakinumab dose. For two
patients, remission is continuing
>6 months post-study to date. Five
patients reported at least one adverse
event, predominantly a mild respiratory
tract infection, and one local site reaction
was reported. One patient died in a traffic
accident at Day 185.
Conclusion: In this 9-month study, monthly
150 mg canakinumab injection was effective
and well-tolerated in Schnitzler syndrome.
AEs were manageable. Data suggest that
IL-1b plays a pivotal role in this disease
and further study is needed to better define
canakinumab treatment.
235
Food consumption and nutritional
status evaluation of children with atopic
dermatitis
Penterich, V1; Yang, A1; Pereira, R2; Takayama, L2;
Kalil, J1; Castro, F1
1Allergy and Immunology Department, University of
Sao Paulo, Sao Paulo, Brazil; 2Rheumatology Division,
University of Sao Paulo, Sao Paulo, Brazil
Background: Atopic dermatitis (AD) is an
inflammatory allergic skin disease that
often requires glucocorticosteroid therapy.
Due to recurrent association with food
allergy, dietary intake of these children
might be an important concern. The aim
of this study was to evaluate food inges-
tion and nutritional status in children with
moderate to severe AD compared with a
control group.
Method: We evaluated 60 children with
atopic dermatitis and 54 healthy controls
(HC), aged from 4 to 12 years old. Food
ingestion was evaluated within 3 days 24-h
food recalls. The dietary intake was evalu-
ated according to the standards set by the
American Institute of Medicine (2010),
Dietary References Intake – DRIs, for cal-
cium and vitamin D, and by the National
Academy of Sciences Research Council
(1989), named Recommended Dietary
Allowance – RDA, for proteins and calo-
ries. The nutritional status was evaluated
according to height to age z-score, weight
for age z-score and body mass index
(BMI) z-score, folowing the World Health
Poster Discussion Session 2 – Improving the diagnosis and management of allergies and immune disorders
106 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Organization child growth standards
(2006).
Result: Results are described in Table 1.
Dietary intake evaluation showed a ten-
dency of less consumption of calories in
the AD group (P = 0.061), although both
groups had an adequate consumption of
calories. It was interesting to notice that in
both groups there was an exceeding
amount of proteins consumed compared to
the standard recommendations, even
though there was no statistical difference
between groups. There was a statistically
significant difference considering fat con-
sumption (P < 0.05), children with AD
consumed more fat than HC. Most of the
AD patients consumed <80% of the die-
tary recommendations for calcium and
vitamin D. In the subgroup of children
who had also cow’s milk allergy the con-
sumption of calcium was lower than 27%.
Children with AD were smaller than HC
children, according to the height for age z-
score for nutritional status evaluation.
Conclusion: Children with AD had a high
ingestion of proteins and a low consump-
tion of calcium and vitamin D. The AD
children had a decrease in the Z-score of
height for age.
236
Patient treatment years used as
denominator in specific immunotherapy
safety surveillance
Dige, E; Riis, B; Walmar, M
ALK, Global Clinical Development, Horsholm, Denmark
Background: Allergen products used for
specific immunotherapy treatment are typi-
cally based on natural allergen sources.
The biological activity is expressed in vari-
ous potency units and products come in
different administration forms, e.g. subcu-
taneous or sublingual, and with different
dosing regimens. Evaluation of safety in
agreement with the requirements from the
authorities and across units of strength,
dose schedules and administration forms
requires a meaningful denominator for fre-
quency estimation of adverse reactions. A
method is presented here in terms of
patient treatment years (TYs). The concept
is based on the theoretical number of
patients who could have been treated for
1 year provided the product was adminis-
tered as recommended in the label and is
thus not the number of individual patients
exposed.
Method: The number of TYs is calculated
based on the time period for use of the
available pack sizes for the products:
TYs = number of packs sold · time per-
iod for use of one pack [in weeks]/52 weeks
The time period for use of each pack
size is estimated in a conservative manner
(i.e. assuming 100% treatment compli-
ance), since taking a lower compliance into
consideration could bias the data in favour
of the manufacturer by suggesting a higher
patient exposure. This would lead to a
lower frequency for any adverse reaction
evaluated. In case of multiple pack sizes
for a product, the TYs for each type of
pack are added together for an overall
number.
Result: For sublingual grass allergy immu-
notherapy tablets (i.e. Grazax, ALK, Den-
mark), the recommended daily dosage is
one tablet; thus the time period for use of
a pack size of 100 tablets is 14.3 weeks and
every 100 packs sold correspond to
100·14.3/52 = 27.5 TYs. For subcutane-
ous immunotherapy (e.g. Alutard, ALK
Denmark) the maintenance dose is 1 ml
every 6 ± 2 weeks; thus the time period
for use of one vial is approximately
26 weeks and every 100 vials sold corre-
spond to 100·26/52 = 50 TYs. These
numbers are then used as denominator
when calculating the frequency of adverse
reactions for each product.
Conclusion: The number of TYs is an esti-
mate of exposure to be used as the denom-
inator in safety surveillance. By use of TYs
it is possible to express exposure in patient
time, and thus report safety profiles of dif-
ferent specific immunotherapy products in
a way that would not have been meaning-
ful had the denominator been number of
injections or dosages.
237
Improving anaphylaxis prevention and
management in healthcare settings:
lessons from analysing patient safety
incident reports
Worth, A1; Panesar, S1; Healy, L2; Sheikh, A1
1The University of Edinburgh, Centre for Population
Health Sciences, Edinburgh, United Kingdom;2University of Glasgow, Glasgow, United Kingdom
Background: Anaphylaxis is an important
cause of mortality. A review of fatal ana-
phylaxis reactions in the United Kingdom
found that almost half were iatrogenic
(Pumphrey 2000). Anaesthetic agents, anti-
biotics and contrast media were particu-
larly implicated. In England and Wales,
patient safety incidents are collected via
the National Patient Safety Agency from
all healthcare settings for the purpose of
identifying risks and finding practical solu-
tions to prevent harm to patients.
Atopic dermatitis (n = 60) Healthy Control (n = 54) P
Demographic data
Age (years) 7.8 (2.2) 8.1 (2.3) 0.374
Gender (M/F) 26/34 28/26 0.363
Anthropometric characteristics
Weight (kg) 29.2 (9.0) 30.6 (9.2) 0.453
Height (m) 1.25 (0.15) 1.29 (0.14) 0.108
Food intake
Calories
Mean (SD) (Kcal) 1515 (498) 1726 (508) 0.061
<80 n (%) 38 (64.0) 32 (60.5)
80–120 n (%) 20 (34.0) 13 (23.7)
>120 n (%) 2 (2.0) 9 (15.8)
Proteins
Mean (SD) (g) 67.1 (24.3) 76.1 (30) 0.165
<80 n (%) – –
80–120 n (%) 6 (10.0) –
>120 n (%) 54 (90.0) 54 (100)
Fat
Mean (SD) (g) 44.4 (18.3) 53.2 (21.5) 0.032
Calcium
Mean (SD) (mg) 461 (303.4) 533.7 (340.5) 0.306
<80 n (%) 50 (84.3) 46 (84.2)
80–120 n (%) 8 (13.7) 4 (7.9)
>120 n (%) 2 (2.0) 4 (7.9)
Vitamin D
Mean (SD) (lg) 2.3 (2.3) 2.6 (2.2) 0.470
<80 n (%) 52 (87.5) 40 (73.7)
80–120 n (%) 4 (6.3) 11 (21.1)
>120 n (%) 4 (6.3) 3 (5.3)
Nutritional status (z-score)
Weight for age 0.33 (1.02) 0.56 (1.08) 0.373
Height for age -0.85 (1.13) -0.21 (1.20) 0.007
Body Mass Index 0.95 (1.32) 0.81 (1.32) 0.605
Poster Discussion Session 2 – Improving the diagnosis and management of allergies and immune disorders
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 107
Method: We analysed patient safety inci-
dents from 2005 to 2010 where anaphylaxis
was mentioned (n = 1858). Two research-
ers examined reports and drew up a typol-
ogy of incidents, categorising events as
follows: anaphylaxis as a result of exposure
to an allergen in a healthcare setting; inap-
propriate response to anaphylaxis in a
healthcare setting; no actual harm, but
incidents which potentially compromised
patient safety. In order to identify recom-
mendations to improve patient safety, we
focussed on incidents where anaphylaxis
occurred as a result of treatment or investi-
gation when it could reasonably have been
prevented. We also analysed ‘near misses’.
Results: Incidents were reported in a wide
range of healthcare settings, predominantly
operating theatres, radiology, medical
wards, Accident and Emergency/medical
assessment units and obstetrics, but also
community, mental health, and patient
transport. The most frequent incidents
resulting in potentially preventable anaphy-
laxis involved patients being given drugs to
which they were known to be (and were
documented as being) allergic to, typically
antibiotics, local/general anaesthetics, anal-
gesics and contrast media. Lack of knowl-
edge of appropriate dose of drugs and
route of administration during anaphylaxis
was also common, as was equipment fail-
ure, failure to recognise and treat anaphy-
laxis and documentation/communication
errors.
Conclusion: The number of apparently pre-
ventable incidences of anaphylaxis and
less-than-optimum treatment of anaphy-
laxis in healthcare settings is disturbing,
particularly as clear anaphylaxis guidelines
exist (WAO 2011). Access to appropriate
care, including drugs, equipment and ade-
quate levels of trained staff is vital in both
emergency care and careful monitoring of
patients in the aftermath of anaphylaxis.
There is a need for a systems approach to
patient safety in order to overcome these
concerns.
238
Allergy alerts in hospital electronic
medical records
Lopez, R1; Gonzalez, R1; Hernandez, D1; Hervas, D2;
Campos, A3; Diaz, M3; Nieto, A3
1Allergy Department, La Fe University Hospital,
Valencia, Spain; 2IISS La Fe, Valencia, Spain; 3La Fe
University Hospital, Valencia, Spain
Background: Electronic medical records
(EMR) are used to register important
health related information as allergic con-
ditions and contribute to both safety and
quality of medical assistance. Allergy alerts
are mandatory in EMR especially in the
hospital setting. In our center EMR
include a new tool to identify allergic con-
ditions. Objective:
To evaluate the use of allergy alert
entries in EMR and to establish the allergy
profile of in-patients in a tertiary hospital.
Methods: A cross-sectional observational
descriptive study of allergy alerts recorded
in the EMR of all patients admitted during
the period between January and July 2011.
The allergic status of patients was evalu-
ated in four categories: No allergy, Allergy,
Intolerance and Adverse reactions. Aller-
gies and intolerances (A/I) were analyzed
as a whole due to its indistinct activation
in some cases. Statistical analysis was per-
formed with usual software.
Results: Fifteen thousand five hundred and
thirty-four patients were admitted in our
hospital during the study period. The rate
of use of the new tool was 64.45% (allergy
information was provided in 10 013 of the
EMR). In 2106 patients (21.03%) the
allergy resource was used to confirm aller-
gies, intolerances and adverse reactions.
Drugs were the most frequent responsible
agents (75.63%) followed by foods
(11.23%) and materials (4.92%). Entries
for drug A/I were more common in women
1285 (64.76%) than males 699 (35.23%)
with a significant statistical difference
(P < 0.05) and increased proportionally
with age. Entries for food A/I were also
more frequent in women 170 (58.02%)
than males 123 (41.90%) but this trend
was reversed in the age group between 0
and 15 years. By contrast, the entries for
food A/I decreased proportionally with
age. In 7907 cases (78.96%) the allergy
resource was used to rule out allergic con-
ditions.
Conclusion: Electronic allergy records are a
useful tool to identify the allergic back-
ground of hospitalized patients and pro-
vide information about the A/I profile of
patients in order to reach optimum quality
in the hospital care.
239
Developing a decision support system for
the management of allergy in primary
care: system requirements as reported by
general practitioners
Brakel, T1; Flokstra-de Blok, B1; van der Molen, T1;
Dubois, E2
1Department of General Practice, University Medical
Center Groningen, Groningen, The Netherlands;2Department of Pulmonology and Pediatric Allergy,
University Medical Center Groningen, Groningen,
The Netherlands
Background: The dramatic increase in
prevalence of allergy has increased the
necessity of expanding and improving care
of allergic patients in primary care. Gen-
eral practitioners (GPs) feel inadequately
prepared to manage food-allergic patients.
Although several management guidelines
exist for allergy, they have a limited effect
on clinical practice and alternative methods
are needed to implement in practice. We
aim to develop a decision support system
for the management of allergy in primary
care. This study describes the first step of
the development process.
Method: GPs were interviewed on prob-
lems in their current allergy management
and their expectations regarding an allergy
support system. The semi-structured inter-
views were audio taped and transcribed
verbatim. The transcribed interviews were
analysed by three researchers separately to
generate themes to describe patterns of
allergy management problems and expecta-
tions of the GPs concerning such a new
support system for allergy.
Result: Five GPs were interviewed and the
duration of the interviews ranged from 55
to 110 min (mean 78 min). The age of the
GPs ranged from 29 to 63 years (mean
50 years) and their experience in primary
care ranged from 1 to 40 years (mean
22 years). Emerging themes concerning
problems in allergy management were: dis-
crepancies between history and IgE test
result and the diagnosis and management
of food allergy, insect allergy, and drug
allergy. Emerging themes concerning
expected features of a new support system
for allergy were: integration in current
(computerized) information system for
GPs, minimal time expenditure, ease of use
and maintenance of autonomy of GPs.
Suggestions were to include flowcharts and
decision trees to guide GPs through the
management especially with uncommon or
difficult allergy topics.
Conclusion: GPs reported most problems
with diagnosing and managing anaphylac-
tic allergies. They perceived less problems
with diagnosing and managing patients
with allergic rhinitis and asthma, although
opportunities exist for improvement. GPs
were interested in a decision support sys-
tem for the management of allergy if it
would be easy to use, rapidly available and
compatible with daily management and
(computerized) GP information systems.
240
Allergy to ortho-phthalaldehyde in the
healthcare setting: the forefront of a new
epidemic?
Pala, G; Pignatti, P; Moscato, G
Allergy and Clinical Immunology Unit, Fondazione
‘Salvatore Maugeri’, Institute of Care and Research,
Scientific Institute of Pavia, Pavia, Italy
Introduction: Fighting infection transmis-
sion provides an exciting and instructive
piece of allergy history. The main lesson is
the epidemic allergy to latex in subjects
Poster Discussion Session 2 – Improving the diagnosis and management of allergies and immune disorders
108 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
occupationally exposed and children with
spina bifida or urological abnormalities,
observed during the 1980s and the 90s, that
decreased when powder-free, latex-poor
gloves were introduced. Another lesson
regards glutaraldheyde, which has been the
primary choice for disinfecting heat-sensi-
tive medicals for 40 years. Due to concerns
related to its irritative and sensitizing prop-
erties, an increasing number of hospitals are
now adopting Ortho-Phthalaldehyde
(OPA), which is not volatile and is much less
irritable to workers. At present, it is reason-
able to assume that in U.S. more than
300 000 workers might be exposed. Along
with approval for disinfecting medical
devices, OPA is also approved for use as
indoor antimicrobial pesticide; as intermedi-
ate for synthesis of pharmaceuticals, medi-
cines, and other organic compounds; as
reagent for drinking water analysis; and as
diagnostic for urea nitrogen test system.
Case description: We report on a 60-year-
old man, with a clinical history of laryn-
gectomy for larynx carcinoma in 2004.
After surgery he underwent several laryn-
goscopies, without any adverse effect until
2008, when he experienced face and neck
oedema, and generalized urticaria after
three different laringoscopies. Upon admis-
sion at our Unit, skin prick test with com-
mon sensitizers and latex were negative,
and he was asked to obtain from the hos-
pital the disinfectant used for sterilization
of endoscopic equipments, which resulted
to be OPA. Patch test with OPA resulted
in nonvesicular erythema. A significant
basophil activation after stimulation with
OPA was demonstrated by the basophil
activation test.
Discussion: Since 2004 few cases of OPA
allergy following endoscopic procedures
and one case of occupational allergy in a
medical worker have been reported. Our
further case confirms that the spreading of
OPA for sterilization of endoscopic equip-
ments may be dangerous to patients. To
our knowledge, no regulations exist regard-
ing the proper use and safe exposure levels
of OPA, in spite of the potential exposure
of a large number of both healthcare work-
ers and patients. Since the allergy history
has already provided lessons on similar
events, should we rethink about our sterili-
zation strategies before facing a new epi-
demic?
Poster Discussion Session 2 – Improving the diagnosis and management of allergies and immune disorders
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 109
Poster Discussion Session 3
Leukocyte activation in allergy
241
EP3 negatively regulates TLR3-dependent
eosinophil infiltration of allergic
conjunctivitis
Ueta, M1; Matsuoka, T2; Narumiya, S3; Akira, S4;
Kinoshita, S1
1Department of Ophthalmology, Kyoto Prefectural
University of Medicine, Kyoto, Japan; 2Tenri Yorozu
Hospital, Nara, Japan; 3Department of Pharmacology,
Faculty of Medicine, Kyoto University, Kyoto, Japan;4Department of Host Defense, Research Institute for
Microbial Diseases, Osaka University, Osaka, Japan
Background: We previously reported that
conjunctival eosinophilic infiltration in
murine experimental allergic conjunctivitis
(EAC) was significantly more marked in
ptger3 KO (knock out) mice, and signifi-
cantly less marked in TLR3 KO mice than
in wild-type mice. Considering the opposite
roles of Ptger3 and TLR3 genes in allergic
conjunctivitis, we speculate the possibility
of unknown functional interaction between
EP3 (protein of Ptger3 gene) and TLR3. In
this study, we examined whether EP3 nega-
tively regulate the TLR3 dependent eosino-
philic infiltration of allergic conjunctivitis.
Moreover, we examined whether EP3 ago-
nist could suppress polyI:C (TLR3 ligand)-
induced TSLP and RANTES production
in conjunctival epithelial cells.
Method: We compared conjunctival eosin-
ophil infiltration in wild-type, TLR3KO,
Ptger3KO and TLR3/Ptger3DKO mice in
our experimental allergic conjunctivitis
model. We also performed ELISA and
quantitative RT-PCR using primary
human conjunctival epithelial cells.
Result: Although sensitisation of RW with-
out challenge (RW eyedrop) did not affect
the number of eosinophils, after sensitiza-
tion and challenge their number in the
lamina propria mucosae of the conjunctiva
was significantly increased in all of them,
and the number after sensitization and
challenge in Ptger3KO mice was signifi-
cantly larger, and significantly lower in
TLR3KO than in wild-type mice, as we
have reported previously. Furthermore, in
TLR3/Ptger3DKO, the number of eosin-
ophils in the lamina propria mucosae of
the conjunctiva was decreased to a level
similar to that in TLR3KO mice, and was
significantly lower than that in not only
Ptger3 KO mice but also in wild-type mice.
Furthermore, EP3 agonist suppressed the
production and mRNA expression of
TSLP and RANTES, which were known
to recruit eosinophils.
Conclusion: Our results suggest that EP3
negatively regulates the TLR3 dependent
eosinophilic infiltration in allergic conjunc-
tivitis, which causes reduced eosinophilic
conjunctival inflammation in TLR3/Ptger3
DKO mice despite the pronounced eosino-
philic conjunctival inflammation in Ptger3
KO mice.
242
The role of granulocyte-macrophage
colony stimulating factor on activation/
inhibitory receptors on eosinophils
Nissim Ben Efraim, A; Ben Zimra, M; Karra, L;
Levi-Schaffer, F
The Institute of Drug Research, The Hebrew University
of Jerusalem, Jerusalem, Israel
Background: Eosinophils are key effector
cells in allergy and are involved in several
inflammatory processes. It was shown that
functions of eosinophil may be regulated
by activating or inhibitory receptors such
as CD48 and CD300a. IL-3, and IL-5 and
granulocyte-macrophage colony stimulat-
ing factor (GM-CSF) are considered
important cytokines in eosinophil develop-
ment, survival and activation. The purpose
of the present work was to study the effect
of these cytokines on regulatory receptor
expression on eosinophils.
Method: Human peripheral blood eosin-
ophils were cultured in medium alone or
with GM-CSF, IL-3 or IL-5 (20 ng/ml) in
different time points. CD300a and CD48
expression was evaluated by flow cytome-
try.
Result: After overnight incubation, GM-
CSF significantly increased CD300 and
CD48 expression. A weaker effect was
observed with IL-3, while IL-5 has no
effect on their expression. The specific
ERK1/2 inhibitor PD98059 partially inhib-
ited the enhancing effect of GM-CSF on
CD300a, but not on CD48. Short term
exposure (3 and 6 h) to GM-CSF also
strongly increased CD300a expression but
not the one of CD48. Brefeldin A had no
effect on this short term increase of
CD300a.
Conclusion: GM-CSF is one of the most
important factors involved in eosinophil
biology. We found that GM-CSF increases
regulatory receptors, particularly CD300a
and involves signal transduction trough
ERK1/2 phosphorylation. A short term
increase in membrane CD300a expression
following treatment with GM-CSF would
imply the presence of an intracellular pull
of the receptor that is ready to be external-
ized. This pathway does not involve trans-
port from the ER as it was not inhibited
by Brefeldin A. These results are important
for the understanding of eosinophils
behavior during allergic inflammation.
243
Is eosinophilic oesophagitis the atopic
dermatitis of the oesophagus?
Burwinkel-Kramer, K1; Doucet-Ladeveze, R2; Holvoet,
S2; Nutten, S2; Blanchard, C2
1Cincinnati Children’s Hospital, Nutrition and Health,
Cincinnati, OH, United States; 2Centre de Recherche
Nestle, Nutrition and Health, Lausanne, Switzerland
Background: As its prevalence increases,
the awareness of eosinophilic esophagitis
(EoE) disease is growing around the world.
EoE is defined as a chronic, immune/anti-
gen-mediated disease characterised clini-
cally by symptoms related to esophageal
dysfunction and histologically by eosino-
phil-predominant inflammation.
Aim and objectives: To investigate and
identify the molecular differences between
this food allergy-related disease EoE, and
other well known Th2 diseases such as ato-
pic dermatitis (AD) and acute asthma
patients (AA).
Methods: Transcriptomic analyses were
performed in human disease of EoE, AD
and AA, as well as in Th2-related murine
models and in vitro in IL-13-stimulated
cells, using publically available and newly
generated data. In vitro and in vivo analy-
ses of gene expression were performed by
qPCR.
Results: As expected, many known Th2
genes were found dysregulated in EoE, AD
and AA such as eotaxin-3, MMPs and
many genes from the epidermal differentia-
tion complex (EDC). We also identified
transcripts of genes consistently dysregulat-
ed in the three diseases in human but also
in cells stimulated with IL-13 and in vivo in
models of the diseases, thus identifying a
new subset of Th2/IL-13 related genes such
as TSG6 a gene involved in immune cell
110 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
recruitment and tissue remodeling. Indeed,
we demonstrated in vivo and in vitro that
TSG6 mRNA was induced by IL-13 and
IL-4 and this induction required the tran-
scriptional factor STAT6. Finally, despite
numerous tissues specific transcripts found
dysregulated independently in the different
diseases, a strong and sticking overlap
between EoE and AD transcriptomic pro-
files was observed.
Conclusion: All together, these data empha-
sise the involvement of IL-13 in the EoE
and AD disease and suggest a stronger over-
lap of the human eosinophilic esophagitis
and atopic dermatitis rather than with other
Th2 related diseases or models.
245
Aeroallergen basophil activation in local
allergic rhinitis
Gomez, E1; Rondon, C2; Herrera, R1; Campo, P2;
Huertas, J2; Garcia, J2; Torres, M2; Blanca, M2
1Research Laboratory, Carlos Haya Hospital-Fundacion
IMABIS, Malaga, Spain; 2Allergy Service, Carlos Haya
Hospital, Malaga, Spain
Background: Local allergic rhinitis (LAR)
is characterized by local production of
sIgE, a Th2 nasal inflammatory pattern
and positive response to nasal allergen
provocation test (NAPT) in absence of sys-
temic atopy (negative skin prick test and
serum specific-IgE). However the presence
of surface-bound IgE of basophils had not
been investigated yet. In this study we was
evaluated the aeroallergen specific activa-
tion of basophil in patients with LAR to
D. pteronyssinus (DP).
Method: The study group constituted by
10 LAR patients to DP was compared with
two controls groups: 11 allergic rhinitis
(AR) patients to DP and 10 healthy con-
trols. Skin prick test, serum specific-IgE,
NAPT, and BAT with DP (5, 20, 50 ng/
ml) were performed. The local ethics com-
mittee approved the study, and informed
consent was given by all participants.
Result: NAPT, gold standard diagnostic
test in this study, was positive in all LAR
and AR patients and negative in healthy
controls. BAT was positive in 50% of LAR
patients, in 72.7% of AR patients, and in
20% of healthy controls. The study of sensi-
tivity and specificity in AR patients and
healthy controls showed that BAT with DP
showed a sensitivity of 72.2%, a specificity
of 80%, and a significant concordance with
NAPT (kappa index 0.525, P = 0.016).
Conclusion: The 50% of LAR patients to
DP showed specific basophil activation.
These preliminary results suggest that after
local production of specific-IgE, basophils
may be the first target cells for specific-IgE,
prior to the detection of free serum specific-
IgE and skin mast-cells sensitization.
246
IgE-dependent stimulation regulates
transmembrane RANK ligand expression
in human basophils
Huber, C; Fux, M; Dahinden, C
University Instiute of Immunology, University Hospital
Bern, Inselspital, Bern, Switzerland
Background: Receptor activator of NFjB
Ligand (RANKL) is expressed either as
transmembrane or soluble isoform by cells
of the skeletal and the immune system and
has several biological roles such as differ-
entiation of osteoclasts and activation of
dendritic cells. Thus, RANKL constitutes
a link between an activated immune sys-
tem and abnormal bone physiology in con-
ditions of chronic inflammation like
rheumatoid arthritis. Recent publications
show that basophils are not only the
major effector cells in allergic inflamma-
tion but also play immunoregulatory roles.
Here we investigate whether and under
which conditions human basophils express
RANKL.
Method: Purified human blood basophils
were cultured with different stimuli known
to affect their survival, activation and
degranulation. The amount of RANKL
mRNA was analyzed by microarrays after
4 and 18 h of stimulation. Transmembrane
protein expression of RANKL was studied
by flow cytometry after 24, 48 and 72 h of
culturing.
Result: Microarray analysis shows that
RANKL mRNA expression increases up to
hundredfold in IL-3 stimulated human ba-
sophils compared to non-stimulated ones.
Our flow cytometry data demonstrate that
IL-3 induces de novo RANKL surface
expression in a time and dose dependent
manner. None of the tested IgE-indepen-
dent stimuli known to result in exocytosis
had an impact on RANKL surface expres-
sion. On the contrary, an anaphylactogenic
anti-FceRIa antibody reduced the IL-3
induced upregulation of RANKL in a dose
dependent manner. This could either
indicate that IgE-dependent exocytosis stim-
ulates ectodomain shedding of transmem-
brane RANKL or that it induces alternative
splicing resulting in the soluble isoform of
RANKL.
Conclusion: So far, we can conclude that
IL-3 induces the transmembrane isoform
of RANKL in a selective manner and that
this upregulation of RANKL is negatively
regulated by IgE-dependent but not by
IgE-independent triggers. In future studies
we investigate in more details if IgE-depen-
dent downregulation of transmembrane
RANKL is dependent on its shedding from
the membrane or on the induction of the
soluble splicing form of RANKL.
247
Implications of nasopharynx-associated
lymphoid tissue in the development of
allergic responses
Wee, J1; Kim, D1; Lee, C1; Kiyono, H2
1Department of Otorhinolaryngology, Seoul National
University College of Medicine, Seoul, Korea;2Department of Microbiology and Immunology, The
Institute of Medical Science, The University of Tokyo,
Tokyo, Japan
Background: Nasopharynx-associated lym-
phoid tissue (NALT) serves as an impor-
tant inductive site for mucosal immunity in
the upper respiratory tract. Despite its
importance in the mucosal immune system,
little is known regarding the role of NALT
in airway allergic immune responses. We
aimed to elucidate the role of NALT in the
induction of upper airway allergic
responses in a mouse model.
Method: Inhibitor of DNA binding/differ-
entiation 2 (Id2)-/- and Id2+/- mice were
exposed to the ovalbumin (OVA)-induced
allergic rhinitis model, since the former
resulted in the NALT deficiency. The aller-
gic parameters, such as allergic symptoms,
serum OVA-specific IgE levels, eosinophil
infiltration, and cytokine profiles in the
nasal mucosa were compared between
Id2-/- and Id2+/- groups.
Result: NALT-null, Id2-/- mice displayed
significantly lower allergic responses com-
pared with Id2+/- mice, as demonstrated by
lower levels of allergic symptoms, serum
OVA-specific IgE, eosinophilic infiltration,
and local Th2 cytokine transcriptions. To
determine which of two factors, i.e., the
absence of NALT or the alteration of
immunocompetent cell populations caused
by the Id2 deficiency, has a larger effect on
the attenuated allergic immune responses in
Id2-/- mice, lethally irradiated Id2-/- mice
were engrafted with C57BL/6 wild-type
bone marrow cells and showed still signifi-
cantly lower allergic immune responses
compared with equally treated Id2+/- mice.
In addition, IgE class switch recombination
(CSR)-associated molecules, such as ¥aGLT,
¥a mRNA and AID mRNA, were detected
in NALT from OVA-sensitised wild type mice.
Conclusion: These results show the critical
role of NALT for the induction of allergic
responses in the upper airway at least in part
by means of class switching to IgE in situ.
Poster Discussion Session 3 – Leukocyte activation in allergy
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 111
248
HLA-phenotypes in IgE-dependent and
IgE-independent atopic dermatitis vs
glomerulonephritis with nephrotic syn-
drome
Drannik, G1; Kurchenko, A1; Petrina, O1; Drijanska, V1;
DuBuske, L2
1National Medical University, Kiev, Ukraine;2Immunology Research Institute of New England,
Gardner, MA, United States
Background: Atopic dermatitis (AD) is
among the most common systemic allergic
diseases. HLA associations have been sug-
gested in patients with AD and also
patients with glomerulonephritis with
nephrotic syndrome (GN, NS), perhaps
determining risk of acquisition of these
conditions
Methods: The distribution patterns of
HLA-A, B, DR antigens in 160 adult
patients suffering from IgE-dependent (80
persons – group I), IgE-independent (80 –
group 2) forms of AD and from GN, NS
in adults (208 – group 3) and in children
(255 – group 4) were analyzed. The HLA
antigens were assessed by a microlympho-
cytotoxic test on the Terasaki‘s planchette
using panels of anti-HLA serums (20 anti-
gens of locus A, 31 of locus B and nine of
locus DR). The control group consisted of
270 healthy donors.
Results: In Group 1 the relative risk for the
disease to develop was statistically greater in
carriers of HLA-A24 expression (RR =
2.21). For class II loci showed the attribu-
tive risk for development of IgE-dependent
AD for individuals having antigens B7
(RR = 2.08), B22 (RR = 2.02), B27
(RR = 2.42), DR2 (RR = 2.18). In the
patients with IgE-independent AD form no
associations were seen. The associations of
HLA-A, B, DR antigens in the patients hav-
ing GN, NS include for Group 3: A24
(RR = 2.31), A28 (RR = 2.0); B8 (RR =
2.38), B41 (RR = 3.05); DR1 (RR = 2.14),
DR4 (RR = 6.36), w52 (RR = 4.04) and
for Group 4: B21 (RR = 2.28), B27
(RR = 2.01), B41 (RR = 2.79); DR 4
(RR = 2.25), and DR5 (RR = 2.37).
Conclusion: IgE-dependent AD is associ-
ated with HLA-antigens including A 24,
B7, B22, and B27 as in GN, NS patients,
and DR 2 while IgE-independent AD has
no HLA associations consistent with the
varied causality of this form of AD.
249
The chemokine CCL18 prevents allergic
airway inflammation and hyperrespon-
siveness in a humanised mouse model of
allergy
Heydenreich, B1; Reuter, S2; Martin, H2; Maxeiner, J2;
Grabbe, S1; Taube, C3; Saloga, J1; Bellinghausen, I1
1Department of Dermatology, University Medical Center
of the Johannes Gutenberg-University Mainz, Mainz,
Germany; 2III Medical Department, University Medical
Center of the Johannes Gutenberg-University Mainz,
Mainz, Germany; 3Department of Pulmonology, Leiden
University Medical Center, Leiden, The Netherlands
Background: The dendritic cell (DC)-
derived chemokine CCL18 is constitutively
expressed in human serum and upregulated
in several inflammatory diseases including
allergic asthma. However, in absence of a
murine counterpart, in vivo studies regard-
ing the effects of CCL18 in mouse asthma
models are not possible. This study was set
out to investigate the role of CCL18 not
only in vitro but also in vivo using a
recently developed humanized mouse
model of allergy.
Method: For in vitro experiments, CCL18
was added to co-cultures of CD4+ T cells
from grass pollen, birch pollen or house
dust mite allergic donors and autologous
monocyte-derived allergen-pulsed mature
DC. For in vivo studies, human PBMC
from allergic donors together with the
respective allergen in the presence or
absence of CCL18 were injected intraperi-
toneally in NOD-Scid-cc-/- mice.
Result: Addition of CCL18 inhibited the
allergen-specific production of IL-4 and IL-
5 by DC-stimulated CD4+ T cells, while
IFN-gamma, IL-10 and T cell proliferation
were not affected. In vivo, CCL18 inhibited
airway hyperresponsiveness and lung
inflammation measured 48 h after intrana-
sal allergen challenge, while addition of
another Th2-associated chemokine, CCL17,
had no effect. Chemotaxis assays revealed
that CCL18 preferentially attracted Treg
and less efficiently T effector cells.
Conclusion: These data demonstrate that
CCL18 may represent a molecule of significant
importance in immunoregulation with ther-
apeutic potential in allergic inflammation.
250
Th2, Th1 and Th17 cytokine profiles in
pollen and mould allergic patients and
healthy subjects
Maslova, L1; Hancharou, A2; Titov, L2; DuBuske, L3
1Belarusian Medical Academy of Postgraduate
Education, Minsk, Belarus; 2Republican Scientific and
Practical Center for Epidemiology and Microbiology,
Minsk, Belarus; 3Immunology Research Institute of New
England, Gardner, MA, United States
Background: Production of cytokines
involved in the allergic inflammation is
crucial for the development of respiratory
allergy as well as for worsening and of ato-
pic respiratory disease. This study esti-
mates the intracellular cytokine profile in
patients with pollen and mold induced
respiratory allergy.
Methods: CD4+T-cells from patients with
mold induced respiratory allergy (M)
(n = 21), grass and tree pollen (P) respira-
tory allergy (n = 35) and healthy controls
(C) (n = 19) were assayed for intracellular
IFN-c, IL-4, IL-5, IL-13 and IL-17 after
stimulation for 6 h with PMA+ionomycin.
Results: The number of IL-4+ cells was
significantly elevated in P, but not in M
compared to C. IL-13+ cells were signifi-
cantly increased in both groups of patients
with respiratory allergy. No differences
between groups in the numbers of IFN-
gamma and IL-5 cells were detected, while
CD4+Th17+ cells were increased signifi-
cantly in both groups of patients.
Conclusions: Increase of IL-13+ and IL-
17+ cells in patients with respiratory
allergy supports a role of these cytokine in
allergic inflammation. CD4+IL4+ cells,
increased only in the patients with pollen-
induced allergy, suggesting a more impor-
tant role of Th-17 vs Th-2 immune
responses in mold allergy.
251
Distribution of allergen specific cells in
diverse T-cell subsets for seasonal and
non-seasonal allergens
Brodie, T1; Brenna, E1; Kolla, R2; Sette, A2;
Lanzavecchia, A3; Sallusto, F1
1Cellular Immunology, Institute for Research in
Biomedicine, Bellinzona, Switzerland; 2Allergy and
Asthma, La Jolla Institute for Allergy and Immunology,
La Jolla, CA, United States; 3Institute for Research in
Biomedicine, Immune Regulation, Bellinzona,
Switzerland
Background: Phenotypic and functional
heterogeneity is the hallmark of effector
and memory T cells. Upon antigenic stimu-
lation, naıve CD4+ T cells make choices
to become TH1 (T helper 1), TH2, or
TH17 cells, or even regulatory T cells. In
addition to differences in cytokine reper-
toire, memory CD4+ T cells exhibit diver-
sity in homing: central memory cells patrol
lymphoid organs while effector memory
cells act as sentinels in peripheral tissues
such as the skin and the gut.
Method: To link the phenotype and func-
tion of the different memory T cell subsets
with their antigen specificity, we have
developed a high throughput cellular
screening method and are applying this
method, called T cell library, to dissect the
T cell response to allergens.
Result: We will report data on the distribu-
tion and responsiveness of allergen-specific
T cells in the different subsets in allergic and
Poster Discussion Session 3 – Leukocyte activation in allergy
112 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
non-allergic individuals and how the
response changes with season.
Conclusion: Understanding the dynamics
of allergen-specific T cells in naıve, effector
and memory Th2 subsets can give insight
to the mechanisms of pathology for allergic
individuals and tolerance for non-allergic
individuals.
252
Bifidobacterium infantis activated
plasmacytoid dendritic cells induce
regulatory T-cells
Konieczna, P; Ziegler, M; Frei, R; Ferstl, R; O’Mahony, L
Swiss Institute of Allergy and Asthma Research,
University of Zurich, Davos, Switzerland
Background: The commensal microbiota is
required for optimal immune development
and for ongoing intestinal homeostasis,
which involves an inter-dependence
between microbes and immunity. Bifidobac-
terium infantis is a commensal bacterium
which has been previously shown to pro-
tect against inflammatory diseases in mur-
ine models e.g. colitis and respiratory
allergy. The protective host immune
response in murine models was demon-
strated to include the induction of T regu-
latory cells. However, the molecular basis
for the Foxp3 regulatory T cells induction
by specific microbes is poorly understood.
The aims of this study are: (i) To evaluate
the plasmacytoid dendritic cell response to
Bifidobacterium infantis; (ii) To determine
the effect of activated plasmacytoid den-
dritic cells on naive T cell priming.
Method: Firstly, plasmacytoid dendritic
cells were stimulated with bacteria and
cytokine secretion was measured by lumin-
ex. Moreover gene expression was quanti-
fied using qRT-PCR. Pattern recognition
receptors were blocked using antibodies or
oligonucleotides. Bacterial binding was
visualized using multispectral flow cytomet-
ric imaging. Bacterial activated plasmacy-
toid dendritic cells were cultured with
autologous CD4+ T cells and Foxp3
expression was analysed by flow cytometry.
Result: Bifidobacterium infantis stimulated
plasmacytoid dendritic cells expressed IL-
10 and indoleamine 2, 3-dioxygenase
(IDO), with low levels of IL-12p70 and
IFN-alpha. The cytokine response was
TLR-9, but not TLR-2, dependent. Co-cul-
ture of naive T cells with bacterial primed
plasmacytoid dendritic cells resulted in
enhanced expression of Foxp3 in
CD4+CD25+ cells. Inhibition of IDO
activity with 1-methyl-tryptophan blocked
induction of T regulatory cells.
Conclusion: These results demonstrate that
Bifidobacterium infantis-stimulated plasma-
cytoid dendritic cells induce an immuno-
regulatory response, suggesting that this
microbe may have therapeutic utility in
patients with inflammatory disease.
253
Transforming growth factor b1 (TGF-b1)
and Forkhead Box Protein 3 (FOXP3)
genes polymorphisms in allergic rhinitis
Ghaffari, J1; Abediankenari, S1; Hassannia, H1;
ghaffari, J2
1Mazandaran University of Medical Sciences, Sari,
Islamic Republic of Iran; 2Allergy and Immunology,
Mazandaran University of Medical Scinces, Sari, Islamic
Republic of Iran
Background: Regulatory CD4+T (Treg)
cells are effective in maintaining immune
tolerance.
Objective: To investigate single nucleotide
polymorphisms (SNPs) of Transforming
Growth Factor a-1 (TGF-a1) and Fork-
head Box Protein 3 (FOXP3) genes in Ira-
nian patients with allergic rhinitis (AR).
Methods: Variations at codons 10 and 25
of TGF-a1 and FOXP3 at positions -3279
A>C and -924 A>G were evaluated in
AR patients and compared with controls.
In a case-control study, 155 AR patients
and 163 allergy-free controls were geno-
typed using polymerase chain reaction
sequence-specific primer (PCR-SSP) tech-
nique.
Results: The analysis of the frequency of
these SNPs showed that the haplotype
formed by FOXP3 -3279 A allele occurred
significantly more frequently in patients
than controls (odds ratio = 1.44, 95%
CI = 1.312–2.66; P = 0.001).
Conclusion: Our results suggest that poly-
morphism in FOXP3 gene is associated
with susceptibility to AR.
254
FoxP3+CD4+CD25-high T-cells in foals
and their possible role in the modulation
of prevalence of allergic disease of the
horse
Hamza, E; Marti, E
Clinical Research Department, Vetsuisse Faculty, Bern
University, Bern, Switzerland
Background: Equine insect bite hypersensi-
tivity (IBH) is an IgE-mediated dermatitis
caused by bites of Culicoides spp. IBH
does not occur in Iceland where Culicoides
are absent. However, following importa-
tion of adult Icelandic horses (1st genera-
tion) into continental Europe where
Culicoides are present, ‡50% of the horses
develop IBH within 2 years from import.
In contrast, only £10% of their offspring
born in Europe (2nd generation) develop
IBH. Furthermore, preliminary data indi-
cate that the prevalence of IBH is £10%
when horses are imported from Iceland
within their first year of life. We have
recently demonstrated the regulatory func-
tion of equine FoxP3+CD4+CD25high
cells and shown that upon stimulation with
Culicoides extract, the proportion of
FoxP3+CD4+CD25high cells was signifi-
cantly lower in IBH 1st generation horses
compared to healthy 2nd generation
horses. Our hypothesis is that exposure of
the horses to the allergens causing IBH
during maturation of the immune system is
required for establishment of Treg toler-
ance. Our aim was to characterize circulat-
ing FoxP3+CD4+ CD25high cells in
foals and their induction in vitro compared
to adult horses.
Method and results: Freshly isolated
PBMC from nine foals (age range, 3–
12 weeks), their mothers and from six year-
lings (1 year old) were examined for the
presence of circulating
FoxP3+CD4+CD25high cells using flow-
cytometry. The proportion of FoxP3+
cells within circulating CD4+CD25high
population was significantly higher in foals
(47%) compared to their mothers (18%)
and to the yearlings (26%). Interestingly,
upon stimulation with a combination of
ConA, IL-2 and TGF-b1, the proportion
of induced FoxP3+ cells within
CD4+CD25high population was signifi-
cantly higher in foals (88%) than in their
mothers (67%) and in the yearlings (62%).
Conclusion: Our results show the existence
of natural FoxP3+CD4+CD25high cells
in the peripheral blood of foals during the
first 3 months of life. The proportion of
these cells diminishes significantly by the
age of 1 year. Importantly, the ability to
induce FoxP3+CD4+CD25high cells in
vitro is higher in foals than in adult horses.
The suppressive function of these cells in
foals and their role in the prevention of
sensitization to environmental allergens
remains to be determined
255
Modeling and exploring the ‘hygiene
hypothesis’ in a barn mouse model of
allergy prevention
Frossard, C; Eigenmann, P
Departments of Pediatrics and Internal Medicine,
Geneva University Hospital, Geneva, Switzerland
Background: Allergy in a given individual is
determined by both environmental as well
as genetic pressure. Among the environmen-
tal factors, it has been demonstrated that
exposure to common pathogens early in life
is highly protective for allergy development.
These findings have been largely become
known under the name of ‘Hygiene hypoth-
esis’. Interestingly, this protection effect has
in particular been observed in children born
and raised in Alpine farming households
who are protected from asthma. Animal
Poster Discussion Session 3 – Leukocyte activation in allergy
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 113
experiments suggest that this protective
effect is dependant of a correlation between
environmental micro-organisms and the
innate immune response.
Objectives: We aimed to establish a mouse
model of allergy prevention by breeding
and raising mice in an Alpine barn in the
presence of cattle, and to compare the phe-
notype of the systemic immune system to
mice housed in a clean university animal
facility.
Methods: Mice from both environments
were bled every 4 weeks, five times, and
the frequency of major lymphoide cells was
quantified by flow cytometry. Serum cyto-
kines levels were assayed by Bioplex and
antibody titers were measured by ELISA.
Subsequently, both groups of mice were
immunized for a antigen-specific contact
hypersensitivity in order to investigate the
reactivity of the skin to an allergic inflam-
mation.
Results: First, sensitized mice raised in the
barn developed a reduce hypersensitivity
reaction of the skin after allergen challenge
allergen when compared to mice housed in
a clean environment, confirming the allergy
protective effect of the barn. Second, the
barn environment induced an increase in
the frequency of NK cells, CD4+/
CD25+/FoxP3- and CD4+/CD25+/
FoxP3+ T cells. We measure in the blood
of barn mice an increase of cytokines
related to the innate system (TNFalpha,
IL-1 and IL-6), Th1 cytokines (gam-
maIFN), Th2 cytokines (IL-4 and IL-5)
IL-10 and IL-17. Moreover, titers of IgG1,
IgG2a and IgE were higher in barn mice
whereas IgG3 titers were reduced. These
data suggest a large, but allergy protective
inflammatory response in barn raised mice.
256
Invariant natural killer T-cells frequency
in peripheral blood from allergic patients
to house dust mite is higher than in
tolerant controls
Lopez, S1; Pozo, D1; Fernandez, T2; Rodrıguez-Bada, J2;
Melendez, L2; Mayorga, C2; Blanca, M3
1Department of Cell Therapy CABIMER-Andalusian
Center for Molecular Biology and Regenerative
Medicine, University of Seville, Sev, Seville, Spain;2Research Laboratory, Carlos Haya Hospital-Fundacion
IMABIS, Malaga, Spain; 3Allergy Service, Carlos Haya
Hospital, Malaga, Spain
Background: Human invariant natural
killer T cells (iNKT cells) are a unique
population of T-cells that express a semi-
invariantly rearranged T cell receptor
(TCR) and play an important role in regu-
lating the development of allergy.
Objective: To characterize the iNKT popu-
lation in peripheral blood from allergic
patients to house dust mite compared with
the iNKT population from tolerant controls.
Method: Peripheral blood mononuclear
cells (PBMC) were separated by gradient
density from 15 allergic patients to house
dust mite and 20 tolerant controls. We
assessed the frequency of peripheral blood
iNKT cells. These cells were measured as
CD3+ cells co-expressing TCRVa24 and
TCRVb11, which recognize specifically
their invariant TCR rearrangement by six
colours flow cytometry.
Results: We have compared the pheno-
types of iNKT cells and our results showed
that the absolute number of iNKT cells
was 5194 in allergic patients and 661 in
controls (CD3+/TCRVa24+/
TCRVb11+) iNKT cells per millilitre of
blood. The relative iNKT cell count was
found statistically significant differences
being 0.312% in allergic patients and
0.063% in tolerant controls (P = 0.033).
Because alteration in the T cell numbers
may affect the iNKT cells count, we deter-
mined the percentage of the CD3+ T cell,
CD4+, CD8+ T cell subsets and the
CD4/CD8 ratio in PBMC from both aller-
gic patients and tolerant controls. The
allergic patients to house dust mite popula-
tion did not differ significantly from the
investigated tolerant controls. No statistical
relationships were found between the fre-
quency or number of iNKT cells and with
the age or sex of the patients or the serum
levels of total and specific Der p 1 and Der
p 2 IgE.
Conclusion: These results show that iNKT
frequency is significantly elevated in aller-
gic patients to house dust mite. However,
additional work is needed to characterize
the iNKT cells and their function in this
pathology, as well as to understand the
relationship between the iNKT cells, anti-
gen presenting cells (dendritic cells, B cells)
and conventional CD4+ T cells.
257
Quantification of antigen-specific IgE
antibodies in mouse models of allergy:
problems and solutions
Huitema, C; Ferstl, R; Rhyner, C; Schawaller, M;
Crameri, R
Swiss Institute of Allergy and Asthma Research, Univer-
sity of Zurich, Davos, Switzerland
Background: When using mouse models to
study allergy and asthma it is important to
quantitate allergen-specific IgE. Enzyme-
linked immunosorbent assay (ELISA) is a
simple method used to measure specific
IgE, although results still take several
hours to generate. The allergen Ovalbumin
(OVA), derived from chicken egg is
commonly used to immunize mice and the
concentration of OVA specific IgE is an
important criterion for monitoring the
development of allergic complications,
including asthma, in this in vivo experimen-
tal model. However, in common immuni-
zation procedures, large amounts of OVA
specific IgG is also generated which could
potentially interfere with IgE quantifica-
tion.
Method: There are currently two wide-
spread, commercially available ELISA-
based methods for measuring OVA specific
IgE, the forward assay captures IgE using
an OVA coating and detects with labelled
anti-IgE, and the reverse assay captures
anti-IgE specifically with an anti mouse
IgE immobilised monoclonal with detec-
tion using labelled OVA. Using monoclo-
nal OVA-specific IgE we examined its
recovery in both ELISA formats from ser-
ums of both immunized and non-immu-
nized animals. As an improvement of
ELISA, we have developed new instrumen-
tation based on real time Total Internal
Reflection Fluorometry (TIRF), which
allows rapid quantitative determination of
antigen specific IgE in a fraction of time
compared to ELISA.
Result: We demonstrate that IgG present
in immunized mice interferes with the
quantification of specific IgE serum for
ELISAs using OVA coating and does not
interfere in the reverse assay. We show that
our TIRF instrument, using a method sim-
ilar to anti-IgE coat ELISAs, gives a result
in 15 min and is also insensitive to high
IgG concentrations.
Conclusion: Here we have demonstrated
recovery problems with the OVA-coating
ELISA method due to interference from
excess OVA specific IgG present in serum
samples. We have developed a new real
time TIRF–based detection method where
IgG does not interfere with specific IgE
quantification. Furthermore, using this new
system, time-to-result is <15 min using
less mouse serum than ELISA-based
methods.
258
Haplotypes and diplotypes in the
tryptase gene (TPSAB1 gene), a highly
polymorphic gene
Hernandez-Hernandez, L; Sanz, C; Garcıa-Sanchez, M;
Pascual, M; Lorente, F; Davila, I
Allergy Department, Complejo Asistencial Universitario
de Salamanca, Salamanca, Spain
Background: TPSAB1 gene codes for tryp-
tase, a serine protease stored in the secre-
tion granules of mast cells. It has been
described that this is a polymorphic gene
with several described SNPs along this.
TPSAB1 gene, which encodes the alpha-
tryptase and beta-tryptase, is the most
studied from the functional point of view.
It has a homology of 90% with TPSB2.
This high degree of homology makes
Poster Discussion Session 3 – Leukocyte activation in allergy
114 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
difficult the real characterization of the
SNPs. The aim of this study is to analyze
the TPSAB1 gene SNPs in a population of
control individuals.
Method: A total of 110 healthy adult indi-
viduals were included in this study with
normal serum tryptase levels. DNA frag-
ments were amplified by PCR and subse-
quently sequenced. The haplotype analysis
was carried out by the SNPAnalyzer on-
line software. EM, Clark and Pseudo
Gibbs Sampler (PGS) algorithms were
used, EM estimates haplotype frequencies
within the given population, Clark is
appropriate for homozygous genotypes
and PGS uses conditional probabilities. In
order to confirm the haplotypic character-
ization and the resulting PCR fragments
were cloned in three representative cases.
Finally, the cloned DNA fragments were
sequenced.
Result: Combinations of the 17 mutations
provided four haplotype combinations
obtained by EM algorithm with a fre-
quency >0.05, four with Clark algorithm
and three with PGS. The most frequent
haplotype is the haplotype 2 (ACGACGA
TGCTCCGGGT) and being the most
common in the population (8.5%). In our
analysis the diplotype combination
obtained with higher frequency is GCGAC
GACGCTCCGGTG/GCGACGCCACTCC
GGGT with a frequency of 0.025. Cloning
allow us to confirm six haplotypes and to
obtain two new ones All cloned samples
provided more than two alleles.
Conclusion: Given the large number of
combinations obtained with the employed
programs and considering that more than
one gene could be simultaneously analyzed;
cloning of PCR product is needed to avoid
biased results.
Poster Discussion Session 3 – Leukocyte activation in allergy
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 115
Poster Discussion Session 4
Exploring food allergy: bench to bedside
259
Dehydrated egg-white, a future
treatment for egg allergy
Ruiz-Garcia, M; Haroun Diaz, E; Landivar Encalada, M;
Torres Hernandez, J; Sastre Domınguez, J
Allergy Department, Fundacion Jimenez Dıaz, Madrid,
Spain
Background: To evaluate OVO-DES NM
(dehydrated egg white), a commercialised
treatment, for egg allergy desensitisation
and specific oral challenge.
Method: Sixteen children (10 boys: six girls)
and three adults (two women: one man)
with a previous diagnose of egg allergy were
enrolled. Children’s mean age at desensitisa-
tion was 7.88 ± 2.55 years-old. Adult’s
mean age 28.33 ± 9.50 years-old. OVO-
DES NM consists of nine doses: 4, 20, 50,
100, 225, 450, 900, 1800 and 3600 mg. Dose
1–6 comes as capsules and from 7 to 9 in
sackets. Usually this was mixed with, either
juice, yoghurt or milkshake in order to hide
the flavour and texture. 4/17 had oral chal-
lenge with OVO-DES NM, 11/17 had oral
challenge with liquid egg and two patients
didn’t have oral challenge prior to desensiti-
sation. All patients had total IgE levels,
mean value 339.75 ± 298.66 UI/ml and
specific IgE levels either to whole egg and/or
ovomucoid, 21.8 ± 19.3 and 18.82 ±
29.32 KU/l respectively. Two patients had
asthma so were started on Budesonide
200 lg during the whole desensitisation.
Patients attended our Clinic on a weekly
base in order to up dose if the previous dose
was well tolerated, before updosing, all
patients had espirometric and exhaled nitric
oxide control. All patients had signed
informed consent
Result: 12/17 started desensitisation at dose
1 (4 mg) the rest started desensitisation at a
mean dose of 408 ± 779 mg (dose 4). Only
two patients quit the protocol for personal
reasons. Only one patient needed dose
adjustment therefore the treatment con-
sisted of 11 doses. Mean time to achieve
total egg desensitisation (3600 mg = dose
9) was 7.81 ± 2.97 weeks. Three patients
needed premedication with Desloratadine
5 mg/day. The most frequent symptoms
were oral itching and abdominal pain. Only
one patient needed adrenaline 0.15 mg
intramuscular at dose 6 (urticaria, bron-
cospasm, dyspnea and abdominal pain) but
was able to complete desensitisation.
Conclusion: The main problem was updos-
ing from 4 mg (dose1) to 20 mg (dose 2) as
this is more than double and most patients
presented abdominal pain. OVO-DES NM
is a clean, sterilised and easy to handle
treatment that can be safely use for egg de-
sensitisation or oral challenge prior to this
which shortens time of desensitisation.
260
Partially hydrolysed formulas in cow’s
milk allergic children
Koulias, C; Tsilochristou, O; Chliva, C; Zeliou, C;
Chatzipetrou, A; Aggelides, X; Makris, M
Allergy Unit, 2nd Department of Dermatology and
Venereology, Paediatric Allergy Outpatient Clinic,
Attikon University Hospital, Athens, Greece
Background: Cow’s milk protein allergy
(CMPA) dictates a very restrictive diet for
infants since elimination of cow’s milk pro-
teins (CMP) is mandatory. Use of partially
hydrolysed formulas (pHF) is currently
restricted to the prevention of CMPA. The
aim of the study was to explore the possi-
ble tolerance to pHF in IgE-mediated
CMP allergic children.
Method: Study subjects were recruited
among infants and young children referred
to the Paediatric Allergy Outpatient Clinic
during 1 year period with suspected CMPA
due to which they underwent CMP restric-
tive diet. The eligibility criteria were: 1)
confirmed IgE-mediated CMPA via a posi-
tive open oral food challenge (OFC), or by
a clear-cut case history of anaphylactic
reaction to CMP within 6 months before
study entry, along with the detection of
cow milk (CM) specific IgE levels or skin
prick tests (SPT) highly predictive for clini-
cal reactivity and 2) Wheal diameter on
prick to prick skin test to pHF £ 4 mm.
PHF milk challenges were performed
openly under physician supervision. An
informed consent was also obtained before
the OFC.
Result: Among 21 subjects 7 (five male/
two female, mean age 18.1 months, range
8–34) fulfilled the inclusion criteria and
were included in study analysis. The first
anaphylactic reaction in the study popula-
tion occured at the mean age of
6.8 months, range 4–11. Six out of seven
cases had atopic dermatitis, and 1/7 had
asthma. The mean serum total IgE was
461 IU/ml and the mean serum specific
IgE was 32.3 for CM, 19.8 for a-lactalbu-
min, 19.2 for b-lactoglobulin and 22.7 for
casein. The mean wheal size of the CM
SPT was 9.3 mm.
Starting dose for the OFC protocol was
0.01 ml pHF, gradually increased to a total
dose of 132 ml.
All challenge tests with pHF were nega-
tive for immediate and late onset reactions.
Notably 4 and 7 months after the negative
pHF OFC, two out of seven children expe-
rienced an anaphylactic reaction due to
accidental exposure to CM.
Conclusion: The results of our study indi-
cate that the use of pHF is safe in the
management of selected cases of IgE-medi-
ated CMP allergic patients. Advantages of
the use of pHF include lower cost and bet-
ter taste. Further studies should be held to
examine the role of pHF in the diet of
some infants allergic to cow’s milk pro-
teins. Such studies should also include a
cost/benefit analysis of the use of hydro-
lyzed formulas and the possible effect on
the natural course of CMPA.
261
Evaluation of the hypoallergenicity of
donkeys’ milk in 92 highly-problematic
cow’s milk allergic children
Monti, G1; Viola, S1; Liguori, S1; Castagno, E1; Tovo, P1;
Baro, C2; Cresi, F3; Bertino, E3; Ferrero, M4; Conti, A2
1Department of Paediatric and Adolescence Science,
Regina Margherita Children’s Hospital, Turin, Italy;2CNR-Institute of Science of Food Production, Turin,
Italy; 3Neonatal Unit, Department of Paediatric and
Adolescence Science, University of Turin, Turin, Italy;4Koelliker – Missionari della Consolata Hospital, Turin,
Italy
Background: Children with cows’ milk pro-
tein allergy (CMPA) who are not exclu-
sively breast-fed require a formula or
alternative diet, but there is no agreement
among the international scientific commu-
nity concerning the foodstuffs of choice.
Use of the currently-available formulas is
hampered by their extremely unpleasant
taste. Recent studies have investigated the
use of donkey’s milk (DM), with good
results in terms of clinical tolerability and
palatability.
Method: Our last prospective study on a
series of 92 children (55 boys; age-range
7.5–121.5 months, median 19 months,
116 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
mean 27 months) with proven CMPA
reports on the tolerability and nutritional
adequacy of DM. Maternal milk was
unavailable and current CM substitutes
could not be used. Moreover, 89% were
affected by multiple FA, and subjected to
very restricted diets. The series is larger
than those previously reported and com-
prised subjects with mild-to-severe clinical
forms of CMPA. Native electrophoresis
and immunoblotting were used to identify
CM and DM cross-reactive proteins. Z-
scores of weight and length/stature for age
were calculated at DM food challenge (T0)
and during DM assumption.
Result: Eighty-three children (90.2%) both
liked and tolerated DM, at the challenge
and for the entire duration of follow-up
(mean 15.48 months, median 12 months).
Throughout this period there was an
increase in Z-score both for weight and for
length/stature. Nutritional parameters
(hemoglobin, serum iron, saturated trans-
ferrin, 1,25-OH2 vitamin D and serum pre-
albumin) evaluated in 24 subjects showed a
significant improvement after 6–12 months’
DM ingestion (median 8 months). Bovine
beta-lactoglobulin was identified as the
cross-reacting protein among the DM aller-
gic patients
Conclusion: DM was found to be clinically
tolerated by at least 90% of subjects with
CMPA, both in our study both in the most
recent previous ones and if this result will
be confirmed in a multi-centre study, DM
could be considered a natural ‘hypoaller-
genic’ food for treatment of CMPA,
according to American Accademy od Pae-
diatrics Committee on Nutrition (AAP)
guidelines. A final but not less important
aspect is the psychological advantage of
being able to use ‘proper’ milk as an alter-
native to CM, that the children consumed
either as such, or in the form of derivates
(e.g. ice-cream) or of other milk-based
foods (e.g. cakes, puddings, home-made
biscuits).
262
Specific oral tolerance induction at the
onset of IgE-mediated food allergy
Badina, L; Matarazzo, L; Berti, I; Longo, G
Pediatric Department, Institute for Maternal and Child
Health-IRCCS ‘Burlo Garofolo’-University of Trieste,
Trieste, Italy
Background: DRACMA guidelines recom-
mend elimination of cow’s milk (CM) from
the diet in case of IgE-mediated reactions
and performance of an oral food challenge
after almost 9 months for assessment of
tolerance onset. Nevertheless sensitisation
to CM usually starts during the first year
of life and reaches its height during the
2nd or 3rd half-year period; thereafter a
decrease is seen in most children. Therefore
infants with CM IgE-mediated allergy usu-
ally remain on the elimination diet for
some years and in a considerable number
of case CM allergy became persistent. On
the other hand recent evidences suggest
that a prerequisite for the development of
tolerance is repeated exposure to antigen.
Aim: To verify the hypothesis that low-
dose controlled milk intake started soon
after the diagnosis of CM allergy could
accelerate tolerance development.
Methods: We performed a low-dose oral
food challenge (starting from 1 ml to a
maximum of 10 ml of CM) in 32 infants
(20 male) with a history of recent immedi-
ate reaction to CM and resulted sensitised
to milk by a positive skin-prick test (SPT).
The tolerate dose was maintained daily at
home with periodical increasing in dosage
up to free milk diet.
Results: 29/32 children (91%), median age
8.38 (IQ range 6.6–11.17) months at diag-
nosis, underwent a successful desensitiza-
tion program and achieved a free milk diet
in a median time of 120 days (IQ range
97.5–187.5). The mean SPT wheal diameter
at diagnosis were 6.57 mm (SD 2.89) and
median time for normalization of milk spe-
cific-IgE detected with SPT was 130 days
(IQ range 106–163). An average of 0.5 (SD
0.68) adverse reaction for child occurred
during desensitisation program, but all
were mild-moderate (grade £ 3 on the
Clark’s scale).
Conclusions: Specific oral tolerance induc-
tion (SOTI) started at the onset of IgE-
mediated CM allergy seems to be a prom-
ising approach, able to modify the natural
increasing pattern of sensitisation to CM
during the 2nd-3rd half-year period. Fur-
thermore it seems to be easier, faster and
safer to perform in infant age in respect to
other analogue experiences in older chil-
dren.
263
Treatment of non-IgE-mediated food
hypersensitivity reactions due to nickel
sensitisation
Ricciardi, L; Arena, E; Loschiavo, G; D’Angelo, A;
Tigano, V; Saitta, S
Allergy and Clinical Immunology Division, Human
Pathology, University of Messina, Messina, Italy
Background: Patients with allergic contact
dermatitis to nickel can present systemic
manifestations correlated to the ingestion
of nickel containing foods such as cutane-
ous symptoms, gastrointestinal symptoms
and other signs and systemic symptoms;
these symptoms are referred as ‘Systemic
nickel allergy’ (SNA). A clinical suspicion
of SNA is confirmed by an improvement
of the symptoms after a low nickel diet.
Methods: In the period between June 2006
and June 2011, 173 (165f – 8m) (165f –
8m; mean age, 31.26 + 13.04 years; range,
18–64 years) because of a clinical suspicion
of SNA underwent a nickel patch test
according to the International Contact
Dermatitis Research Group guidelines.
Therefore, after confirming cutaneous sen-
sitization to nickel, a low nickel diet was
prescribed with foods which are generally
tolerated by patients with SNA while foods
which usually cause exacerbation of SNA
had to be avoided. The low nickel diet
caused clinical improvement of symptoms
correlated to SNA in 173 patients (165f –
8m; mean age, 31.26 + 13.04 years; range,
18–64 years), who underwent an oral
nickel provocation test in our Allergy out-
patient Unit. The test was positive in 130
patients (125f and 5m) and negative in 21
(20f – 1m). NOHT was started in 126
patients, following an up-dosing schedule
from 0.1 to 500 ng. The maintenance treat-
ment consisted in the administration of
500 ng capsules three times a week.
Results: NOHT overall improved patient’s
quality of life, reducing drug consumption,
inducing recovery from symptoms and tol-
erance to nickel containing foods.
Conclusion: SNA being an allergic disease
due to nickel contained in foods and
requiring the need for a special diet should
be taken into account by health profession-
als and dieticians when evaluating food
related problems in order to reach a cor-
rect diagnosis and treatment with NOHT.
264
Daily calcium intake and bone strength
in children with persistent cow’s milk
allergy
Singer, D1; Prais, D2; Ziv-Busani, D3; Levy, Y1
1Schneider Children’s Medical Center, Kipper Institute
of Immunology, Petach Tikva, Israel; 2Schneider
Children’s Medical Center, Institute of Pulmonology,
Petach Tikva, Israel; 3Nutrition and Diet Unit, Schneider
Children’s Medical Center, Petach Tikva, Israel
Background: Cow milk and milk products
are the main source for calcium intake in
childhood. The peak bone mass is attained
during adolescence. Therefore, children
and adolescents with persistent milk allergy
might be affected by the low calcium
intake in their diet and have decreased
bone strength.
Method: Children with persistent milk
allergy, aged >3 years, underwent clinical
and nutritional evaluations which included
history of bone fractures, anthropometric
measurements, questionnaire for daily cal-
cium intake during the preceding 6 months
and blood tests for calcium, phosphorus,
alakaline phosphatase, parathyroid hor-
mone (PTH) and vitamin D levels. Bone
Poster Discussion Session 4 – Exploring food allergy: bench to bedside
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 117
strength was evaluated using quantitative
ultrasound of the radius (Omnisense
7000P; Sunlight Medical, Petach Tikva,
Israel); z- score value of 2SD and more
below the mean according to age and sex
was considered pathological. As control
group served children with asthma without
milk allergy.
Results: The study group included 81
patients [38 (47%) of them with asthma,
35 (43.2%) girls]. The control group
included 27 patients [8 (29.6%) girls].
Mean age of the study group was
7.01 ± 3.08 years, compared to
11.59 ± 4.17 years of the control group
(P < 0.001). No significant differences in
BMI percentiles were identified. The mean
daily calcium intakes of the cow milk aller-
gic children and control group were
42.41 ± 48.02% and 49.92 ± 38.36% of
the recommended daily calcium intake
(P = 0.141). History of bone fractures was
found in six of 69 (8.7%) children with
milk allergy compared to eight out of 26
(30.8%) in the control group (P = 0.016).
In the milk allergic group, two patients
had pathological z-score, in contrast to
none in the control group; the mean z-
score values were 0.02 ± 0.86 and
0.12 ± 0.86 respectively (P = 0.930). A
negative correlation between z-scores and
serum PTH levels was found in boys with
persistent milk allergy.
Conclusions: The daily calcium intake of
children and adolescents with persistent
milk allergy was lower than the recom-
mended intake, but similar to the results in
the control group. Z-scores were lower in
the milk allergic patients compared to the
control group, but without statistical sig-
nificance. The control patients had a higher
rate of fractures, most probably due to
their older age. Children and adolescents
should be encouraged to increase their cal-
cium intake. Special attention should be
given to those with persistent milk allergy,
and a long term follow-up of their bone
strength is needed.
265
The impact of elimination diets on
nutritional status of allergic children
Nunes, M1; Moreira, A2; Morais-Almeida, M3; Placido,
J2; Coimbra, A2; Moreira, P1; Barros, R1
1Faculty of Nutrition and Food Sciences, University of
Porto, Porto, Portugal; 2Immunoallergology Department,
Hospital Sao Joao and Faculty of Medicine, University of
Porto, Porto, Portugal; 3Immunoallergology Department,
Hospital CUF Descobertas, Lisbon, Portugal
Background: Suspicion of food allergy,
confirmed or not, often drives children to
avoidance of culprit foods. Our study
aimed to investigate the nutritional status
and dietary intake adequacy of children on
elimination diets.
Method: In a cross-sectional manner mea-
surements of anthropometry (weight,
height, skinfold thickness, waist circumfer-
ence, and classification according to CDC
Growth Chart percentiles and z-scores), die-
tary intake (nutrient intake determined by
24 h recall interview, and compared with
dietary reference intakes – DRI), Food
Allergy Quality of Life Questionnaire scores
for children’s parents – FAQLQ – PF, and
teenagers – FAQLQ-TF and clinical, demo-
graphic and physical activity were taken
from children on elimination diets based on
a self reported food allergy diagnosis.
Result: Forty seven children, mean (SD)
age 5.9 (3.8), were included. Egg and cow’s
milk were the main avoided foods. Two
(4.3%) children had short stature and 5
(10.6%) children were underweight. Body
mass index for age percentiles classified 15
(31.9%) children as overweight or obese.
Total carbohydrates (84.2% of intake to
DRI), vitamin A (91.9%) vitamin D
(25.5%), vitamin E (64.8%), calcium
(87.1%) and potassium (64.7%) fell below
dietary reference intake. Children’s quality
of life was slightly impaired, mean (SD)
score 1.9 (1.6).
Conclusion: Regular nutritional status eval-
uation and dietary counseling is essential
as complementary approach to food
allergy treatment, to ensure adequate nutri-
tional intake, normal grow and health of
children on food elimination diets.
266
Multicentre validation of a mouse model
for cow’s milk allergy to assess the
allergenicity of hydrolysed cow’s milk
based infant formulae
van Esch, B1; van Bilsen, J2; Jeurink, P3; Garssen, J1;
Verhoeckx, K2; Smit, J4; Pieters, R4; Knippels, L3
1Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands; 2TNO Earth
Environmental and Life Sciences, Quality and Safety,
Zeist, The Netherlands; 3Immunology, Danone
Research Centre for Specialised Nutrition, Wageningen,
The Netherlands; 4Institute for Risk Assessment
Sciences, Utrecht University, Utrecht, The Netherlands
Background: The EC-directive 2006/141/E
requires objective and scientifically verified
data to claim hypoallergenicity of hydroly-
sed formulae. However, no validated animal
models are currently available to assess the
residual sensitising capacity, although gui-
nea pig assays are frequently used. This
study is part of a multi-phase project which
aims to validate a recently developed mouse
model to assess the potential allergenicity of
hydrolysed cow’s milk based infant formu-
lae. The discriminatory power of a mouse
allergy model for cow’s milk allergy to
distinguish sensitizing properties of cow’s
milk products was evaluated in four
independent research centers.
Method: C3H/HeOuJ mice were sensitised
by oral administration of whey (WPC80)
or extensively hydrolysed cow’s milk
(eWH) at weekly intervals for 5 weeks.
One week after the last sensitisation, the
acute allergic skin response (ear swelling at
1 h), anaphylactic symptoms and tempera-
ture were determined upon intradermal ear
injection of whey. Subsequently, mice were
challenged orally with 50 mg whey and
blood samples were taken after 30 min.
Serum was analyzed for whey-specific im-
munoglobulins and mMCP-1. All proto-
cols, test substances and procedures were
standardised.
Result: All participating research laborato-
ries detected elevated levels of whey-spe-
cific IgE/IgG1/IgG2a, serum mMCP-1
(reflection of mast cell degranulation) and
acute allergic skin responses in whey sensi-
tised animals. Anaphylactic symptoms and
temperature drop were scored in three out
of four research centers. In contrast, none
of the evaluated parameters was altered in
eWH-sensitized groups as compared to the
vehicle control group.
Conclusion: All four independent research
centers were able to discriminate between
the sensitising properties of whey protein
and extensively hydrolysed cow’s milk. The
results demonstrate the discriminatory
power of the mouse model and thereby the
suitability of the mouse model to evaluate
the allergenicity of hydrolysed cow’s milk
formulae. In the next phase of the multi-
center validation process, partially hydroly-
sates will be included to assess the
sensitivity of the discriminatory power of
the mouse model.
267
Frequency of food allergy in an allergy
department in Portugal using the
Portuguese Allergy Society case-report
form over 29 months
Lozoya-Ibanez, C1; Rodrigues, A2; Lobo, C3;
Taborda-Barata, L4
1Allergy Department, Unidade Local de Saude de
Castelo Branco, EPE, CICS, Health Sciences Research
Centre, University of Beira Interior, Castelo Branco,
Portugal; 2EPE, Emergency Department, Unidade Local
de Saude de Castelo Branco, Castelo Branco, Portugal;3Clinical Pathology Department, Unidade Local de
Saude de Castelo Branco, EPE, CICS, Health Sciences
Research Centre, University of Beira Interior, Castelo
Branco, Portugal; 4Department of Allergy & Clinical
Immunology, Centro Hospitalar Cova da Beira, CICS,
Health Sciences Research Centre, University of Beira
Interior, Covilha, Portugal
Background: Since the implementation in
2009 of a new Case-Report Form (CRF)
by the Portuguese Allergy Society (SPAIC)
Food Allergy Interest Group, no studies of
food allergy (FA) prevalence have been
carried out in Portuguese hospitals. There-
fore, the aim of the present study was to
determine the prevalence of FA in patients
Poster Discussion Session 4 – Exploring food allergy: bench to bedside
118 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
attending an Allergy Outpatient Depart-
ment, using the new CRF.
Method: Clinical histories of 1597 patients
aged between 1 and 74 years, seen at the
Allergy department of the Local Health
Unit in Castelo Branco (Portugal) between
June 2009 and November 2011, were ana-
lyzed. These patients were subdivided
according to age: children and adults if age
was under 18 or 18 or more years-old,
respectively. Descriptive statistical analysis
was carried out.
Results: FA was diagnosed in 53 (3.32%)
patients (mean age: 26.81 years, median
age: 26.5 years, 62.26% female). Twenty
patients were children (mean age 9.1 years,
50% female) and 33 adults (mean age
37.42 years, 69.7% female). Foodstuffs
most frequently implicated in FA in chil-
dren were fish (17.5%), dry fruits (15%),
latex-related fruits (15%) and egg (13.5%)
whereas in adults, it was seafood (37%),
fresh fruits (13%), latex-related fruits (13%)
and dry fruits (9.25%). Fifty percent of chil-
dren and 27% of adults were diagnosed with
FA to more than one foodstuff. Both
groups reported urticaria as the most fre-
quent FA reaction (FAR) upon ingestion of
seafood, fish, egg and dry fruits, whereas
oral allergic syndrome (OAS) was most fre-
quently reported in the case of fresh fruits,
latex-related fruits, dry fruits and peanut.
Most FAR developed within 30 min of
ingestion (83% of cases), followed by 11%
of cases in which symptoms arose 2–24 h
after food ingestion. Most patients (60%)
reported more than five FAR episodes, with
seafood as the most frequently implicated
foodstuff (34%). Around 34% of cases
required medical treatment (50% at a Hos-
pital Emergency Department and 27% at a
Health Care Centre within the first 24 h).
Most patients had personal (83%) or family
(64%) history of atopy.
Conclusions: In this first study of the prev-
alence of FA using the new SPAIC tool in
Portugal, the prevalence of FA is similar
to that observed in other allergy depart-
ments reported in the literature. Urticaria
and OAS were the most frequent symp-
toms. Seafood, fresh fruits and latex-
related fruits in adults and fish, dry fish
and latex-related fruits in children were the
most frequently implicated foodstuffs
268
Quality of life in children with food
allergy
Indinnimeo, L; Del Greco, P; Celani, C; Occasi, F;
Ragusa, G; Duse, M; De Vittori, V
Pediatric Immunology and Allergology Department,
Rome Sapienza University Hospital, Rome, Italy
Background: There are few studies about
QoL in the Italian children with food
allergy. Our research therefore aims to
assess QoL in children with food allergies
who carry out exclusion diets.
Methods: Children with an elimination diet
for one or more foods were enrolled.
A questionnaire on the Quality of Life
(Food Allergy Quality of Life Question-
naire – Parent Form FAQLQ-PF 0–
12 years, DunnGalvin et all) was adminis-
tered to parents by a psychologist. FA-
QLQ-PF considers critical values >3 and
consists of specific questions which differ
depending on age of children (0–3, 4–6, 7–
12 years).
Results: Ninety-six children were enrolled,
59 (61.4%) males. The average age of the
sample to the administration of the ques-
tionnaire is 3.92 years (SD + 2.67, range
1 month–11 years). Twenty-four children
(25%) had mild or moderate anaphylactic
shock. All participants underwent TPO.
Those who have experienced anaphylactic
shock, had elevated levels of anxiety
related to food and generally a worse QoL
than all others (P < 0.02). Children aged
between 3 and 6 years and those aged
between 6 and 12 years, showed a greater
degree of anxiety and emotional impact for
food and a worse QoL (P < 0.007) com-
pared to 43 children <3 years. As
expected, those who carried out a greater
number of exclusion diets were more con-
cerned about the food (P < 0.006) and in
general pay more attention to food
(P < 0.06) than those who carried out a
diet alone. Finally we found in children
who carried out the diets of longer dura-
tion for cow’s milk (49–120 months), a
worse QoL than those who had made diets
shorter (P < 0.001). Our children showed
a bad QoL in the social relationships with
no significant differences between the
groups studied.
Conclusions: Children with dietary restric-
tions are primarily limited in their social
relationships: the environmental control is
a preventive measure taken by the families
of all children with food allergy. Those
who have experienced episodes of anaphy-
laxis have a worse QoL because they are
concerned about accidental ingestion of the
food and the occurrence of a new episode.
Older children, 6 and 12 years old, report
a significant deterioration of QoL. Finally,
a longer duration of diet for milk, signifi-
cantly worse QoL. We can assume that the
avoidance of milk, widely represented in
our foods, involves a great number of
restrictions which, if continued for long
periods, greater stress the children and
their families.
269
Analysis of cow’s milk-specific IgE and
components IgE antibodies in infants and
young children with atopic dermatitis
Lee, J; Jeon, S; Lee, S
Department of Pediatrics, Ajou University School of
Medicine, Suwon, Korea
Background: Cow’s milk protein is one of
the most common sensitized allergen in
children with atopic dermatitis. Allergen
components studies have become available
recently, thus this study is aimed to investi-
gate distributions and clinical symptoms
according to the specific IgE levels to
whole cow’s milk and milk component in
young children.
Method: Cow’s milk (CM), a-lactalbumin
(ALA), b-lactoglobulin (BLG), casein-spe-
cific IgE (ImmunoCAP, Phadia, Sweden)
levels from sera of the patients with clinical
history during January 2004–December
2010 were collected with medical records.
Patients >3 years old of age were
excluded.
Result: Total 950 patients, median age 2,
diagnosed as atopic dermatitis enrolled.
Four hundred and seventy-one (49.6%)
were sensitized to whole cow’s milk (CM-
IgE > 0.35 kU/l). Among them, 349
(74.1%) were sensitized to casein (Cas-
IgE > 0.35 kU/l), 283 (60.1%) sensitized
to ALA (ALA-IgE > 0.35 kU/l), and 245
(52.5%) were sensitized to BLG (BLG-
IgE > 0.35 kU/l). Four hundred and
twelve were £2 years of age and 139
patients had positive predictive level to
CM (CM-IgE ‡ 5 kU/l) according to the
diagnostic decision points. In this group,
the proportion of patients sensitized to
each allergen are as follows; Casein
(n = 139, 100%), BLG (n = 115, 82.7%),
and ALA (n = 113, 81.3%). Fifty-nine
were >2 years of age and eight had posi-
tive predictive level to CM (CM-
IgE ‡ 15 kU/l). All eight were sensitized to
ALA, BLG, and casein. In addition, 95
patients had at least two times of follow-
up study to milk. Eighty of them remained
positive results to CM and casein was the
main sensitized allergen in these patients
(n = 65, 81.3%). Meanwhile, 35 (7.5% of
466) patients were not sensitized to whole
cow’s milk (CM-IgE £ 0.35 kU/l) but sen-
sitized to components allergens. Telephone
survey was accessible in 21 cases. Eight out
of 21 (38%) had adverse reactions after
consuming milk or other dairy foods. Also,
positive correlation between CM and ALA
(r = 0.679, P < 0.01), CM and BLG (r =
0.666, P < 0.01), and CM and casein (r =
0.847, P < 0.01) was observed in this
study.
Conclusion: Casein is the most common
sensitized allergen in CM-IgE positive
Poster Discussion Session 4 – Exploring food allergy: bench to bedside
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 119
patients and follow-up studies indicate
same results. Negative predictive value dif-
ference between patients whose CM-IgE,
ALA-IgE, BLG-IgE, Cas-IgE <0.35 kU/l
and patients whose CM-IgE <0.35 kU/l
but are sensitized either to ALA, BLG or
casein need further evaluation.
270
Cross-reactive carbohydrate determinants
in peanut allergy in Ghana: high
peanut-specific IgE sensitisation without
skin reactivity or reported symptoms
Amoah, A1; Obeng, B1; Larbi, I2; Versteeg, S3;
Aryeetey, Y2; Fernandez-Rivas, M4; Hartgers, F1;
van Ree, R3; Boakye, D2; Yazdanbakhsh, M1
1Department of Parasitology, Leiden University Medical
Center, Leiden, The Netherlands; 2Department of
Parasitology, Noguchi Memorial Institute for Medical
Research, Accra, Ghana; 3DepartmentS of Experimental
Immunology and Otorhinolaryngology, Academic
Medical Center, Amsterdam, The Netherlands; 4Servicio
de Alergia, Hospital Clinico San Carlos, Madrid, Spain
Background: Recent studies point to an
increase in the prevalence of peanut allergy
particularly among children in western
countries. Few studies on peanut allergy
have been conducted in parts of the world
where peanut consumption is known to be
high. Our aim was to examine the preva-
lence of peanut allergy and the role of
cross-reactivity in peanut sensitization
among children in Ghana, a country where
peanuts are frequently consumed.
Method: We conducted a cross-sectional
study among urban and rural Ghanaian
children aged 5–16 years. A questionnaire
was administered to collect data on
reported peanut adverse reactions and pea-
nut consumption patterns. Each child was
skin prick tested for peanut allergic sensiti-
zation and blood was drawn to quantify
peanut specific IgE (sIgE) antibodies. Stool
and urine samples were collected to detect
helminth infections while a blood film slide
was prepared for malarial parasite identifi-
cation. In a subset of participants, IgE
reactivity to recombinant peanut allergens
(rAra h 1–3) and to the cross-reactive car-
bohydrate determinant (CCD) marker bro-
melain was determined. ImmunoCAP
inhibition assays were performed to
explore inhibition of peanut sIgE by bro-
melain as well as inhibition by helminth
antigens.
Result: In 877 enrolled participants, the
prevalence of reported adverse reactions to
peanut was 1.3%; peanut skin reactivity
was 1.8% while peanut sIgE (‡0.35 kU/l)
was 17.9%. About 19.2% of subjects were
positive for at least one geohelminth while
the prevalence of S. haematobium infection
was 6.4%. Reported peanut reactions were
not associated with either peanut SPT or
peanut sIgE in crude and multivariate
analyses. Although elevated peanut sIgE
was associated with peanut skin reactivity,
only 7.6% of IgE sensitized subjects were
peanut SPT positive. This discrepancy can
be attributed to the cross-reactivity of pea-
nut sIgE with carbohydrate moieties as
inhibition assays revealed 87.7% inhibition
of peanut sIgE by bromelain and 82.4%
inhibition by S. haematobium soluble egg
antigen.
Conclusion: Our study is one of the first to
explore peanut allergy among African chil-
dren. We observed poor correlation between
reported peanut adverse reactions and
peanut IgE sensitization markers. In our
study population, cross-reactivity induced
by helminth and plant N-glycans resulted in
elevated peanut sIgE that did not seem to be
functional as it did not lead to peanut skin
reactivity or reported symptoms.
271
Immunoglobulin E reactivity to
carbohydrate
Lee, K1; Hong, J2; Kim, M3; Heo, W3; Kim, K4; Kim, K4
1Department of Pediatrics and Institute of Allergy,
Severance Biomedical Science Institute, Yonsei
University College of Medicine, Seoul, Korea;2Department of Pediatrics and Institute of Allergy, Brain
Korea 21 Project for Medical Science, Yonsei University
College of Medicine, Seoul, Korea; 3Brain Korea 21
Project for Medical Science, Yonsei University College
of Medicine, Seoul, Korea; 4Yonsei University College
of Medicine, Seoul, Korea
Background: Wheat is one of the major
crops consumed by humans and is associ-
ated with respiratory allergy and dietary
allergy. Soybean is also important source
of food allergy and respiratory allergy in
those handling the grain. Carbohydrate
moieties of plant and invertebrate glyco-
proteins are very abundant environmental
immune determinants and induce extensive
cross-reactivity. Thus, we investigated pos-
sible participation of the carbohydrate epi-
topes from salt soluble protein and alcohol
soluble protein of wheat in their IgE-bind-
ing capacity and cross-reactivity with those
of soybean.
Method: Salt soluble protein and alcohol
soluble protein (gliadin) were extracted
with PBS buffer and gliadin extraction buf-
fer, respectively. Constitutive proteins were
separated by SDS-PAGE and the reactivity
of IgE with extracts, before and after
periodate oxidation treatment, were evalu-
ated by immunoblotting. The specific bind-
ing of lectins to carbohydrate moieties was
used to identify these structures. Sera of
patients with positive IgE-reactivity against
wheat and soybean by CAP test were
selected (over 0.35 kU/l).
Result: (i) By using specific lectins to car-
bohydrate moieties, it was determined that
glycoprotein of wheat (salt soluble protein
and alcohol soluble protein) and soybean
(salt soluble protein) has high mannose N-
glycan chains. (ii) After periodate oxida-
tion, in case of wheat, glycoproteins ranged
between 37 and 60 kD including gliadin
were less recognized by specific IgE from
allergic patients. In addition, the IgE rec-
ognition of 12–16 kD protein known as
alpha-amylase/trypsin inhibitor was lost
upon deglycosylation. (iii) Even though
glycans were removed from soybean
extract, most proteins still maintained IgE
reactivity.
Conclusion: Our results suggest that glyco-
proteins harboring carbohydrate moieties in
wheat and soybean may play a role in some
hypersensitive reactions though their struc-
tural basis as cross-reactive carbohydrate
determinants must carefully be considered.
272
Human dendritic cells stimulated with a
novel peanut protein express high levels
of retinaldehyde dehydrogenase 2 and
induce RA-sensitive genes in naıve
T-cells
Ruiter, B1; Grishina, G2; den Hartog Jager, S3; Knol, E3;
Ozias-Akins, P4; Sampson, H2; Shreffler, W1
1Division of Rheumatology, Allergy & Immunology,
Department Medicine, Massachusetts General Hospital,
Boston, MA, United States; 2Pediatric Allergy &
Immunology, Mount Sinai School of Medicine, New
York, NY, United States; 3Dermatology & Allergology,
University Medical Center, Utrecht, The Netherlands;4Department of Horticulture, University of Georgia,
Tifton, GA, United States
Background: Dendritic cells instruct naıve
T cells to differentiate into various effector
cells determining immune responses such
as allergy or tolerance. Our objective was
to identify peanut extract (PE)-induced
changes in gene expression in human mye-
loid dendritic cells (mDC), and assess the
role of differentially expressed genes in the
induction of T cell differentiation.
Method: mDC (CD11c+BDCA1+) and
naıve Th cells (CD4+CD45RA+) were
isolated from blood donors. mDC were
incubated for 24 h with medium alone, PE,
or control stimulants (lipopolysaccharide,
cholera toxin). mRNA was isolated for use
in expression array and qRT-PCR. To
assess T cell differentiation, mDC were co-
cultured for 6 day with autologous naıve T
cells and the retinaldehyde dehydrogenase
2 (RALDH2) substrate retinal.
Result: PE induced a unique expression
profile in mDC, including the gene that
encodes RALDH2, the rate-limiting reti-
noic acid (RA)-producing enzyme. qRT-
PCR confirmed the ~20-fold induction of
this gene in PE-treated mDC. PE-treated
mDC also demonstrated a 7-fold increase
in enzymatic activity of RALDH2. Naıve
T cells cocultured with PE-treated mDC
showed a 3-fold increase in production of
IL-5 and in expression of the RA-sensitive
Poster Discussion Session 4 – Exploring food allergy: bench to bedside
120 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
surface markers CD38 and a4b7-integrin.
Size exclusion purification and mass spec-
trometry suggest the RALDH2-inducing
constituent to be a member of the plant
glycine-rich proteins superfamily, which is
not recognised as a major allergen.
Conclusion: A previously undescribed pea-
nut protein induces RALDH2 in mDC,
leading to RA production which can act
on T cells to induce gut-homing integrin
and IL-5 production. RA has been impli-
cated in potentiating gut-homing T cell dif-
ferentiation. RALDH2 induction by PE
could be an important factor determining
allergic or tolerant responses to peanut.
273
Mesenteric lymph nodes and Peyer’s
patch in food allergy: participant or
essential?
Vinuesa, M1; Bassan, N2; Martinez, A1; Batle, R1;
Giacomozzi, F1; Torres, V1
1Lab. Inmunohistoquimica. Cat. Histologia.,
Universidad Nacional de Rosario, Rosario, Argentina;2Cat. Histologia. Cat. Genetica, Universidad Abierta
Intermamericana, Rosario, Argentina
Background: We already demonstrated
that subcutaneous sensitisation with Oval-
bumin (OVA) induce generation of specific
IgE antibodies and modifications of
immune cells populations in different
mucosal sites in rabbit. The aim of the
study was the evaluation of OVA specific
lymphoproliferation in mesenteric lymph
nodes (MLNs), spleen and Peyer’s patch
from OVA sensitised and challenged rab-
bits.
Method: New Zealand white rabbits were
divided into six groups: G1 (n = 8): orally
sensitised and challenged with OVA; G2
(n = 10): subcutaneous sensitised with
OVA and oral challenged (OVA); G3
(n = 10): subcutaneous sensitised and oral
challenged with PBS (phosphate buffer sal-
ine). G4-G5 and G6 (n = 9 each) controls.
Four hours after challenge animals were
sacrificed and obtained samples were pro-
cessed for lymphoproliferation studies: iso-
lated cells from MLNs, spleen and Peyer’s
patch from the different groups were sus-
pended in culture media containing OVA
or Concanavaline A and were cultured for
48, 72 and 120 h. Proliferation was mea-
sured as incorporation of radioactive ele-
ment as counts per minute (CPM).
Result: Spleen derived lymphocytes showed
important proliferation at subcutaneous
sensitised groups when incubated with
OVA. Meanwhile, proliferation was signifi-
cantly higher in lymphocytes originated in
MLNs from subcutaneous OVA sensitized
and oral challenged rabbits at 48, 72 and
120 h of incubation. No significant prolif-
eration was observed in Peyer’s Patch
derived lymphocytes.
Conclusion: We conclude that proliferation
of OVA-specific spleen originated lympho-
cytes was successful after systemic sensiti-
zation but after oral challenge with the
antigen, only OVA incubated MLNs-origi-
nated lymphocytes showed proliferation as
compared with Peyer’s patch. This evi-
dence a main participation of MLNs in
this model of food allergy.
Poster Discussion Session 4 – Exploring food allergy: bench to bedside
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 121
Poster Discussion Session 5
Immunotherapy: vaccines and mechanisms
274
Efficiency of a DNA vaccine on allergic
asthma
Tissot, A; Beilvert, F; Chesne, J; Langelot, M; Lair, D;
Muller, M; Pitard, B; Magnan, A
Inserm UMR 915 Institut du Thorax, Nantes, France
Background: Asthma is a chronic respira-
tory disease with a predominant allergic
origin and the main allergens incriminated
are from house dust mites (HDM). Specific
immunotherapy which consists in repeated
administration of increasing doses of aller-
gen induces hyposensitivity, reduces symp-
toms and is an attractive therapeutic
option. Nevertheless the efficiency of
immunotherapy remains limited and it may
cause anaphylactic adverse effects when
administrated subcutaneously. One of the
new modality of immunotherapy is based
on DNA vaccination which consists in the
administration of DNA sequences encod-
ing an antigen. It enables a targeted immu-
nogenicity a Th1 bias and a good tolerance
profile.
Method: In this study we used a DNA
plasmid encoding Der f 1, a major allergen
from the house dust mite dermatophago-
ides farinae, formulated with a synthetic
vector which enhances its intra cytoplasmic
transfer. This vaccine was tested on a mur-
ine model of allergic asthma based on
cutaneous sensitization followed by airway
challenges with total HDM extract. Mice
were vaccinated twice before the sensitiza-
tion period. We evaluated the response to
treatement by airways hyperresponsiveness
(AHR) measurements by plethysmography,
and by systemic (Ig assays) and in situ
(cytokine assay) immunological responses.
Result: Vaccinated mice showed a
decreased AHR. The cellularity of the
bronchio-alveolar lavage (BAL) was also
decreased in the vaccinated group with a
decrease of neutrophils and eosinophils.
IgG2a/IgG1 ratio was increased in the vac-
cinated group in favor of Th1 bias con-
firmed by increase of IFN-c secretion by
splenocytes restimulated in vitro with puri-
fied Der f 1. Analyses of cytokines in BAL
showed a decrease of Th2 and Th17 cyto-
kines (IL-4, IL-5, IL-13, IL-17A and IL-6)
and an increase of IL-10 and IFN-c. Total
IgE levels were not modified.
Conclusion: This formulation of DNA
vaccination induces cellular and humoral
specific response with a pro-Th1 bias
accompanied by a decrease of the Th17
and Th2 responses. It enables an improve-
ment of lung cellular infiltrate and airway
hyperresponsiveness. DNA vaccination
seems therefore promising to prevent
allergy.
275
Towards an integrated characterisation
of allergen extracts in support of safe
and efficacious immunotherapy
Batard, T; Nony, E; Chabre, H; Bouley, J; Brier, S;
Lemoine, P; Jain, K; Mascarell, L; Bodo, V; Moingeon, P
Scientific Department, Stallergenes, Antony, France
Background: As an etiologic treatment of
allergy, allergen extract-based immunother-
apy (AIT) is a clinically documented cen-
tury-old therapy that will remain the main
approach for many years. Allergen extracts
consist in complex mixtures of molecules.
Development of a safe and efficacious AIT
product rely upon (i) definition of the tar-
get product profile, (ii) selection of the
most appropriate source material(s), and
(iii) selection of a robust manufacturing
process. Herein, we illustrate how an
extensive characterization benefiting from
up-to-date technologies can support those
three critical development steps, with the
examples of grass pollens and house dust
mites (HDM).
Method: Methods used for the character-
ization of allergen sources and extracts are
divided into three categories, depending
upon readouts: (i) molecular structure
(proteomics such as LC-MS/MS and 2D-
DIGE); (ii) immunogenicity (ImmunoSol-
id-phase Allergen Chip, or ISAC; flow
cytometry-based T cell response assays;
…); (iii) biological activity (desensitizatizon
of mice with allergic asthma; activation of
TLR-expressing cells; …).
Result: (i) Extensive LC-MS/MS character-
ization of purified relevant allergens indi-
cates numerous inter-species structural
differences, for grass pollens as well as
HDM. Many of them are recognized by
the immune system (IgE, T cells) of allergic
patients. This implies the target product to
be a mix extract, for both grass pollens
and HDM. For the latter, this is further
supported by desensitization experiments
in a murine model of allergic asthma. (ii)
As exemplified by HDM, the most appro-
priate source material can be selected on
the basis of immunochip-based seroepi-
demiology data obtained in large cohorts
of patients, combined with a comprehen-
sive profiling (both qualitative and quanti-
tative) of allergen sources. (iii) Analytical
methods such as flavonoid quantification
and LC-MS/MS-based species identifica-
tion in five-grass pollen extracts have been
successfully applied to select manufacturing
parameters yielding safe extracts, and to
confirm the robustness of manufacturing
processes.
Conclusion: Benefiting from modern tech-
nologies, extensive characterization pro-
vides a critical support for the
development of allergen extracts intended
for safe and efficacious AIT. A multi-
pronged integrated approach is needed for
the definition of the target product profile,
as well as for the selection of the most
appropriate source material and manufac-
turing process.
276
Immunological responses of fast updosed
subcutaneous immunotherapy with an
optimised allergen/adjuvant ratio and
traditional subcutaneous immunotherapy
Kleine-Tebbe, J1; Jung, K2; Pfaar, O3; Wustenberg, E4;
Birk, A5; Grønager, P5; Frew, A6
1Allergy & Asthma Center Westend, Berlin, Germany;2Private practice, Erfurt, Germany; 3Center for
Rhinology and Allergology Wiesbaden, University
Hospital Mannheim, Mannheim, Germany; 4ALK,
Wedel, Germany; 5ALK, Hørsholm, Denmark; 6Brighton
and Sussex University Hospitals NHS Trust, Brighton,
United Kingdom
Background: Subcutaneous immunotherapy
(SCIT) traditionally includes an updosing
phase injecting increasing doses of allergen
over a period of several weeks, followed by
a maintenance phase. A fast updosed SCIT
formulation with an optimised allergen/
adjuvant (aluminium) ratio has been devel-
oped to establish shorter and more conve-
nient updosing schedules with the same
clinical benefits as traditional immunother-
apy. Immunological changes after treatment
with the optimised SCIT formulation and
traditional SCIT have been compared.
Method: Immunological results from two
randomised, controlled, parallel-group trials
(AF-H-01, Wolf et al, Allergy 2010, 65
(Suppl 92): 135 and UK-22, Shamji et al,
122 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Allergy 2012, 67 (2)) in patients with grass-
pollen induced rhinoconjunctivitis with/
without asthma were obtained. Patients
included in this analysis were treated with
either the optimised SCIT formulation
(15 000 SQ+) or traditional SCIT (100 000
SQ-U). The optimised SCIT formulation
was given as five weekly updosing injections
followed by two maintenance injections; tra-
ditional SCIT was given as 15 updosing
injections over 8 weeks followed by mainte-
nance injections every 6 ± 2 weeks for
approximately 8 months. Immunological
parameters (Phleum pratense specific IgE
and IgE-blocking factor) were measured by
ADVIA Centaur assays and analysed inde-
pendently at the end of each trial. The
parameters were measured at initiation of
treatment and at end of trial/after two main-
tenance injections (10 weeks) for the opti-
mised SCIT formulation and at initiation of
treatment, after 1st maintenance injection
(week 8), at a seasonal visit (week 22) and at
end of trial (week 32) for traditional SCIT.
Result: The level of the immunological
responses (change from baseline in Log
sIgE and IgE-blocking factor) observed
after 8–10 weeks of treatment in the two
trials were comparable.
Conclusion: Immunological results
obtained from two trials with a fast up-
dosed SCIT formulation with an optimised
allergen/adjuvant ratio (15 000 SQ+) and
traditional SCIT (100 000 SQ-U) indicated
comparable immunological response levels
within a similar timeframe. This may be
explained by an enhancing effect on the
immune system resulting from the altered
allergen/adjuvant ratio in the optimised
SCIT formulation.
277
A Dermatophagoides pteronyssinus 2
derivative peptide vaccine in allergic
asthma
Lair, D1; Vrtala, S2; Cheminant, M3; Sagan, C4; Magnan,
A3
1INSERM UMR 1087/CNRS 6291, Universite de Nantes,
IRT-UN, L’institut du thorax, Nantes, France; 2Division
of Immunopathology, Department of Pathophysiology,
Center for Physiology and Pathophysiology, Medical
University of Vie, Vienna, Austria; 3INSERM UMR 1087/
CNRS 6291, Universite de Nantes, IRT-UN, l’institut du
thorax, Nantes, France; 4Service d’anatomie et
cytologique pathologiques, Hopital G.- et R.-Laennec,
Nantes, France
Background: Allergic asthma requires the
discovery of new therapeutic strategies,
particularly to prevent exacerbations.
House dust mite allergic sensitization rep-
resents a major cause of this form of
asthma. We propose a strategy for immu-
notherapy with recombinant derivative
peptide of the major allergen of dermato-
phagoides pteronyssinus (Der p), Der p 2.
Method: We developed a mouse model of
Der p-induced allergy mimicking asthma
and allergen-driven exacerbation. A recom-
binant Der p 2 derivative peptide exhibit-
ing less in vivo allergenic activity and
allergenicity than the native Der p 2 aller-
gen with preserved immunogenicity was
injected sub-cutaneously to mice at time of
allergic sensitization. The vaccine effect
was assessed by lung function measurement
and broncho-alveolar lavage (BAL) cytol-
ogy.
Result: Vaccination of asthmatic mice
inhibited Der p-induced airway hyperre-
sponsiveness to metacholine and pulmo-
nary inflammation.
Conclusion: Vaccination with the recombi-
nant Derp 2 derivative peptide seems to be
a suitable new therapeutic strategy to pre-
vent asthma and asthma exacerbations.
278
Comparison of antibody-based assay
with physicochemical assays for
monitoring the stability of alum-
adsorbed mite allergoid
Luykx, D; Kerkvliet, E; de Bruijn, J; Sinnige, N; van den
Hout, R
Development Department, HAL Allergy BV, Leiden, The
Netherlands
Background: An IgG inhibition assay has
been developed previously to monitor the
stability of alum-adsorbed mite allergoids
in a mite allergy vaccine (ALU-M). In
addition, two physicochemical methods
have been developed to monitor the struc-
tural stability of the mite vaccine. These
methods concern circular dichroism (CD)
and front-face fluorescence spectroscopy
(FF-fluorescence). In order to find out
whether there is a relation between the
antibody-based assay and physicochemical
methods, temperature-stressed samples
were analysed with both types of methods
and the results were compared.
Method: ALU-M: D. pteronyssinus and D.
farinae mite extracts were modified with
glutaraldehyde and adsorbed onto alumin-
ium hydroxide. After adsorption the species
were mixed. IgG inhibition assay: Mite
allergoid specific IgG antibodies were used
to determine the 50% IgG inhibition value.
CD: Far-UV CD spectra were recorded
from 195 to 260 nm while stirring the sam-
ple suspension. FF-fluorescence: Fluores-
cence emission spectra were recorded from
290 to 400 nm (excitation at 280 nm).
Result: Storage of the ALU-M vaccine at
various elevated temperatures resulted in
increased IgG inhibition values and spec-
tral alterations with CD and FF-fluores-
cence, meaning structural alterations of the
adsorbed protein. The increase of the 50%
IgG inhibition value indicates a reduction
of IgG binding to the epitopes. The spec-
tral alterations observed with CD indicate
partial loss of a-helices and increase of b-
structure at higher temperatures. The spec-
tral alterations observed with FF-fluores-
cence indicate formation of a more rigid
protein structure at higher temperatures.
When the temperature stress stability data
obtained with the three methods were com-
pared, a relation was observed between the
IgG inhibition assay and CD assay by
means of the CD-ratio 207/222 nm. Sam-
ples stored at elevated temperatures
showed a similar trend with respect to IgG
inhibition and CD ratio; both values
increased in time.
Conclusion: By performing stress stability
studies on ALU-M it was shown that the
IgG inhibition assay and physicochemical
assays were stability indicating. The results
obtained with the IgG inhibition assay and
CD assay relate well suggesting a relation
between the IgG binding and secondary
protein structure of the adsorbed mite
allergoid
279
Sublingual immunotherapy in mice
induces regulatory cells and systemic
tolerance
Brimnes, J1; Rask, C1; Lund, K2; Wagtmann, V1
1Pharmacology Department, ALK-Abello, Horsholm,
Denmark; 2ALK-Abello, Horsholm, Denmark
Background: Antigens introduced at muco-
sal surfaces can induce a state of immuno-
logical unresponsiveness to subsequent
systemic challenge with the same antigen.
This phenomenon has been termed oral or
mucosal tolerance and been demonstrated
for nasal and intestinal surfaces. In the
present project, we have investigated the
sublingual tissue as a site for induction of
systemic tolerance.
Method: Naıve BALB/c mice were treated
sublingually with Phleum pratense extract
(Phl p) or ovalbumin (OVA) for 2 weeks
followed by an intraperitoneal challenge
with alum-adsorbed Phl p or OVA. In
some experiments this was followed by an
Table 1.
Optimised SCIT,
15 000 SQ+, AF-H-01 trial
Traditional SCIT,
100 000 SQ-U, UK-22 trial
DLog sIgE (log kU/ml) 0.75 (N = 191) 1.08 (N = 106)
DIgE-blocking factor 0.32 (N = 164) 0.37 (N = 106)
Poster Discussion Session 5 – Immunotherapy: vaccines and mechanisms
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 123
intranasal challenge to induce airway hyp-
erreactivity.
Result: Our results show that sublingual
treatment of naıve mice leads to the induc-
tion of systemic tolerance. This is mea-
sured by decreased systemic T-cell
reactivity after the intraperitoneal challenge
as well as decreased clinical signs of rhini-
tis, such as airway hyperresponsiveness and
eosinophilia, when mice are challenged
intranasally. In sublingually treated mice,
both T-cell proliferation and secretion of
IFN-gamma, IL-4 and IL-5 are signifi-
cantly reduced compared to buffer-treated
mice, in a dose and duration dependent
manner. We show that sublingual adminis-
tration is more efficient in generating sys-
temic T-cell tolerance compared to peroral
and intranasal administration. Transfer of
CFSE-labeled OVA specific DO11.10 cells
into wild type mice, demonstrate in vivo
proliferation of antigen-specific T-cells in
the cervical lymph nodes of sublingually
treated mice, and these cells display a regu-
latory phenotype. Furthermore, spleen cells
from sublingually treated mice are able to
transfer the sublingually induced tolerance
to naıve mice.
Conclusion: These findings show that sub-
lingual treatment of naıve mice is an effi-
cient way of inducing systemic tolerance,
which most likely is mediated by regula-
tory T-cells.
280
Allergen uptake by skin dendritic cells in
epicutaneous immunotherapy is
modulated by the size of the patch
Dioszeghy, V1; Mondoulet, L1; Dhelf, V1; Ligouis, M1;
Puteaux, E1; Dupont, C2; Benhamou, P1
1Research & Development, DBV-Technologies,
Bagneux, France; 2Universite Paris Descartes – Hopital
Necker, Paris, France
Background: In previous studies, we estab-
lished that epicutaneous application of
allergen in sensitised mice resulted in rapid
uptake by skin dendritic cells. The uptake
reached a plateau with 80% of epidermal
DCs capturing allergen within 6 h. This
study evaluated the effect of the dose of
allergen and the size of the patch on cap-
ture and migration of skin DCs.
Method: BALB/c mice were sensitised
orally to peanut protein extract (PPE). Dif-
ferent concentrations (40, 100 or 250 lg) of
Fluoroprobe488 conjugated PPE (F488-
PPE) were applied to the back of the mice
inside 1, 2.5 or 5 cm2 occlusive patch for 2,
6 or 24 h. Draining lymph nodes (dLNs)
were harvested and treated to obtain single
cell suspensions for flow cytometry analysis.
Result: After 2 h of application, no aller-
gen was observed in dLNs whatever the
dose of allergen or size of patch confirming
the absence of passive passage through the
skin. As already observed, application of
100 lg with 1 cm2 patches induced the
migration of skin DCs with 5.87 ± 0.87%
of DCs positive for F488-PPE after 6 h
and 15.05 ± 2.91% after 24 h. With a
lower dose (40 lg), DCs migration was
slower with no detection after 6 h and
11.03 ± 2.08% of DCs positive for F488-
PPE after 24 h. Increasing the dose
(250 lg) within patches same size did not
modify the DCs migration (7.10 ± 0.45%
and 14.64 ± 1.33% of positive DCs after
6 and 24 h respectively). Using larger
patches (100 lg in 2.5 cm2, 100 lg in
5 cm2 or 250 lg in 2.5 cm2) induced higher
number of migrating DCs after 24 h
(26.42 ± 2.81%, 30.94 ± 4.27% and
33.46 ± 2.32% of DCs positive for F488-
PPE respectively).
Conclusion: In this model, 100 lg of aller-
gen is sufficient to reach all the skin DCs
that could capture allergen and migrate to
the dLNs, with no better delivery using
higher dose. However, increasing the size of
the patch allowed higher uptakes of allergen
through the involvement of a higher number
of cells. The quantity of allergen presented
by DCs in dLNs could then be modulated
by the size of the patch in order to obtain
the profile adapted to the optimal modula-
tion of immune responses.
281
Design of a dendrimeric structure
containing Ole e 1 and CpG to modulate
an in vivo anaphylactic response
Blazquez, A1; Aranda, A1; Mascaraque, A2; Torres, M3;
Mayorga, C1; Rojo, J2; Blanca, M3
1Research Laboratory-Allergy Department, IMABIS
Foundation-Carlos Haya Hospital, Malaga, Spain;2Instituto de Investigaciones Quımicas, CSIC, Sevilla,
Spain; 3Allergy Service-Carlos Haya Hospital, Malaga,
Spain
Rationale: Polymers containing immuno-
genic peptides bonded to dendrimeric
structures have been developed to be used
for vaccines in cancer, infectious diseases,
and allergy. Our aim is to design a dendri-
meric structure containing Ole e 1 and
CpG, in order to modulate an allergic
immune response towards Th1, in an
experimental model of anaphylaxis.
Methods: C56BL/6 mice were sensitized by
intranasal administration of olive
extract + cholera toxin B, for 6 weeks.
Then, mice received immunotherapy (IT)
treatment by subcutaneous (sc) injection of
dendrimer-Ole1 ± 50 lg of CpG, for
8 weeks. Seven days after the last sc injec-
tion, mice were challenged with 100 lg of
olive extract by intraperitoneal injection
(ip). Severity of anaphylaxis was measured
by drop in body temperature and the
humoral response by ELISA.
Results: Olive sensitized mice treated with
the dendrimer-Ole1 without CpG developed
a drop in body temperature similar to ana-
phylactic mice (35.02 ± 1.39� vs 34.48 ±
0.82�, respectively), indicating that Ole e 1
within a dendrimeric structure is recognized
by the immune system in vivo. On the other
hand, 8 weeks after immunotherapy, mice
receiving the dendrimer-Ole1 + CpG were
significantly protected from the develop-
ment of systemic anaphylaxis (37.18 ± 1.58
vs 34.48 ± 0.82, P < 0.05, respectively).
IgE and IgG2a levels decreased after 1–2
weeks of treatment and remained stable
over the time; however IgG1 were normal.
Conclusion: Our results indicate that the
dendrimer-Ole1 is recognized in vivo by the
immune system. Furthermore, we show
that sc administration of dendrimer-
Ole1 + CpG protects sensitized mice from
the onset of anaphylaxis, and the decrease
in IgE levels during the IT treatment may
be responsible from that protection.
282
Component resolved individually
adapted treatment: immunotherapy of
polysensitised profilin positive patients
Gay-Crosier Chabry, F
Private Practice, Immunotherapy, Carouge, Switzerland
Background: Poly-sensitised patients and
associated OAS pollinosis are usually not
an indication for subcutaneous immuno-
therapy (SIT). The clinical response to
allergen specific immunotherapy does not
correlate well with allergen-specific IgE lev-
els analysed by standard immunoassay. In
this study, we analyse the clinical response
to SIT for profilin positive patients suffer-
ing from OAS and pollinosis. Clinical
response is correlated with allergen-specific
IgE levels, IgG4 levels measured by a new
microarray-based assay (Immuno Solid
phase Allergen Chip, Isac 112) allowing a
component resolved individually diagnosis
leading to a Component Resolved Individ-
ually Adapted Treatment (CRIAT).
Method: Efficacy of SIT for seven poly-sen-
sitised patients for a total of 18 different
related food associated allergies was analy-
sed. All patients are positive to profilin with
OAS to 2–9 food related allergies per
patient. All have rhinoconjonctivitis and
four of them have asthma. Patients’ evalua-
tion score (VAS), rescue medication and
physician ‘s assessment score have been cal-
culated before and after a one to a 3 years
course of SIT. Allergen-specific IgE and
IgG4 levels were measured before, during
and after SIT by Isac 112 assays. CRIAT
during the course of SIT is described.
Result: The median of related food
improvement score was 71.42% for the
physician and 95% for the patient. The
Poster Discussion Session 5 – Immunotherapy: vaccines and mechanisms
124 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
median decrease of patients’score (VAS)
for pollinosis was 85%. The median
improvement of pollinosis symptoms’score
evaluated by the physician was 76.2%.
Melon was the most frequently implicated
food (71.4%). OAS to melon persists after
a complete schedule of SIT in the only
patient treated by a unique SIT extract.
The frequency where the 18 related food
allergies were implicated was 32 before SIT
and nine after SIT. Two patients together
collect 8–9 persistent food allergies, the 9th
being related to an uncomplete schedule of
SIT. The specific IgE levels and also the
IgE/IgG4 ratio measured by Isac were sig-
nificantly decreased whereas allergen spe-
cific IgG4 levels were significantly
increased during SIT.
Conclusion: Poly-sensitised profilin patients
with pollinosis and associated OAS can
benefit from an individual SIT. Component
Resolved Individually Adapted Treatment
(CRIAT) based on biological evaluation of
IgE and IgG4 level is possible. Larger
studies are needed to confirm the extend of
this therapeutic success and their biological
relationships.
283
Patterns of specific IgG4 to allergen
components in normal healthy
individuals as measured with a
microarray immunoassay
Elfverson, G; Harlin, A; Ghasemzadeh, N; Kober, A;
Nystrand, M
ThermoFisher Scientific, Uppsala, Sweden
Background: When monitoring patients
undergoing allergen specific immunother-
apy (SIT) increases in levels of specific
IgG4 are generally seen and may have a
role in immune deviation and development
of tolerance. However specific IgG4 anti-
bodies against some allergen components
also occur in normal healthy individuals.
Method: One hundred normal healthy
adult individuals divided into two sub-
groups, 50 selected on total IgE level
<10 kU/l and specific IgE <0.1 kUA/l,
and 50 normal healthy individuals without
allergy symptoms regardless of IgE levels.
The microarray system used for analysis
was ImmunoCAP ISAC which consists of
a selected panel of 112 allergen compo-
nents, recombinant or purified, immobi-
lized onto a chemically modified glass slide
in an arrayed fashion. The detection limits
of the test system are 0.1 ISU G4 and 0.3
ISU E. All samples were analysed with
ImmunoCAP ISAC IgG4 and IgE respec-
tively. Thirty microlitre of serum or
plasma, diluted 1:50 were used for analysis
of IgG4 antibodies in the microarray while
IgE antibodies were measured in 30 ll
undiluted sample.
Result: The analysis revealed that IgG4
antibodies to milk (Bos d 5, Lactoferrin),
egg (Gal d 1 and Gal d 2) and Kiwi com-
ponent (Act d 1) were quite common with
a prevalence of more than 50%. On the
contrary no IgG4 concentrations above the
detection limit were detected for mite or
grass components. It could also been seen
that the subgroup selected on very low
total IgE level had a lower prevalence of
IgG4 antibodies to any component then
the other group. Only five of 100 individu-
als completely lacked detectable IgG4 anti-
bodies. For specific IgE 99.99% of all
results were negative.
Conclusion: The microarray test for specific
IgG4 is a useful tool for semi-quantitative
measurements of IgG4 antibodies to aller-
gen components. Healthy individuals are
more likely to have IgG4 antibodies
against common food allergens such as egg
and milk than to inhalant allergens such as
pollen and mites. The unexpected high
prevalence of IgG4 antibodies to Act d 1
need further studies for explanation.
284
Long-term maintenance of Foxp3+
regulatory T-cells induced by
epicutaneous immunotherapy in mice
sensitised to peanut
Dioszeghy, V1; Mondoulet, L1; Dhelf, V1; Ligouis, M1;
Puteaux, E1; Dupont, C2; Benhamou, P1
1DBV-Technologies, Research & Development,
Bagneux, France; 2Universite Paris Descartes – Hopital
Necker, Paris, France
Background: Previous studies have estab-
lished that epicutaneous immunotherapy
(EPIT) of sensitized mice decrease both clin-
ical responses to allergen exposure and aller-
gen specific TH2 responses, and increase
local and peripheral Foxp3+ regulatory T
cells (Tregs). The aim of this study was to
confirm the role of Tregs in EPIT and their
maintenance following the end of EPIT.
Method: Tregs were investigated using in
vivo depletion or adoptive transfer. Mice
sensitized orally to peanut (PPE) were trea-
ted by EPIT during 8 weeks, with or with-
out injection of anti-mouse CD25 antibody
(aCD25). EPIT+aCD25 was compared to
Sham treatment, EPIT alone, and naıve
mice. CD25+CD4+ Tregs were isolated
from spleen of mice 1/sham treated, 2/just
after EPIT or 3/8 weeks after the end of
EPIT, then transferred into new PPE-sensi-
tized mice. The recruitment of eosinophils
and cytokine expression in esophagus after
oral exposure to PPE was measured in recei-
ver mice and compared to sensitized not
transferred (nT) mice. The allergen specific
response of splenocytes was also evaluated.
Result: Induction of Foxp3+ CD25+
CD4+ cells in spleen of EPIT mice
(1.39 ± 0.10% vs 0.61 ± 0.10% in Sham,
P < 0.05) and Foxp3 expression in esopha-
gus (2.04 ± 0.49 vs 085 ± 0.15 rmRNA,
P < 0.05) was abrogated by aCD25
(0.52 ± 0.10%, and 0.71 ± 0.18 Foxp3
rmRNA, respectively, P < 0.05 vs EPIT).
This resulted in levels of PPE-induced eosin-
ophilic infiltration in esophagus comparable
to Sham and significantly higher than EPIT
(38.93 ± 7.13 and 36.39 ± 5.61 vs 13.30 ±
4.07 eosinophils per mm2 respectively,
P < 0.05). Transferring Tregs recovered
just after EPIT prevented eosinophil infiltra-
tion and eotaxin expression, and induced
Foxp3 in esophagus whereas transfer of cell
from Sham treated mice demonstrated no
effect. Transfer of Tregs isolated 8 weeks
after stopping EPIT did suppress allergen
specific Th2 cytokine production as effi-
ciently than Tregs isolated just after EPIT
(IL-5: 33.2 ± 14.1 and 32.1 ± 9.6 vs
142.1 ± 55.6 pg/ml in nT; IL-13: 39.6 ±
19.1 and 28.9 ± 11.3 vs 197.1 ± 106.9 pg/
ml in nT; IL-10: 22.1 ± 7.7 and 15.2 ± 4.3
vs 54.3 ± 27.1 pg/ml in nT; P < 0.05), and
similarly increased Foxp3+ CD25+ CD4+
cells percentages in spleen (4.25 ± 0.11 and
3.90 ± 0.16 vs 3.13 ± 0.08% in nT; P <
0.01).
Conclusion: These results confirm the Treg
mediated mechanism of EPIT and demon-
strate the persistence of efficient Tregs dur-
ing a long period after stopping treatment.
285
Optimisation and comparison of four
different immunoassays for the detection
of anti-amyloid b IgGs in mouse plasma
Vukicevic, M; Pihlgren, M; Giriens, V; Paganetti, P;
Piorkowska, K; Pfeifer, A; Muhs, A
AC Immune, Lausanne, Switzerland
Background: ELISA is the most common
assay for antibody titer measurements in
mouse and human plasma. However, it
might be not sensitive enough for the
detection of low antibody titers, epitope
screening or in different matrices, such as
CSF or saliva. The aim of this work was
to develop and optimise different sensitive
immuno-assays and to evaluate their
potential to replace current ELISA for the
detection of anti-amyloid b (Ab) IgGs in
mouse plasma after induction of specific
antibody response by vaccination. There-
fore, four techniques: AlphaLISA, Meso-
scale discovery (MSD), Time-Resolved
Fluorescence Resonance Energy Transfer
(TR-FRET) and Luminex were developed.
Method: In AlphaLISA, activated donor
bead in the presence of the analyte transmits
the oxygen to the acceptor bead, which
emits the signal. Donor beads were bound
to streptavidin and the acceptor beads to anti-
mouse IgG. In MSD, electrically-activated
Poster Discussion Session 5 – Immunotherapy: vaccines and mechanisms
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 125
plate in the presence of the analyte leads to
the activation of a sulfo-tag, which then
emits the light. MSD plate was bound to
streptavidin and the sulfo-tag to anti-mouse
IgG. In TR-FRET, two dyes are used and
in the presence of the analyte, activation of
the donor dye with a light triggers a FRET
to the acceptor, which fluoresces. Donor
dye was bound to streptavidin and the
acceptor to anti-mouse IgG. In Luminex,
beads [dyed with fluorochrome(s)] are
bound via the analyte to another fluoro-
chrome. Each bead is analyzed by double
fluorescence. Lumavidin beads were used
with the phycoerythrin-bound anti-mouse
IgG. In all cases, biotinylated Ab peptide
was added to bind to (strept) avidin compo-
nent. Anti-Ab IgGs were detected via Ab
peptide on one side and anti-mouse IgG on
the other one.
Result: All methods were successfully opti-
mised and reproducible. They showed dif-
ferent sensitivities, detection range, easiness
to use and time savings. Limits of detec-
tion were the following: AlphaLISA:
approximately 40 pg/ml, MSD: approxi-
mately 35 pg/ml, TR-FRET: approxi-
mately 2000 pg/ml, and Luminex:
approximately 2500 pg/ml, compared to
approximately 2000 pg/ml in the standard
ELISA. MSD had the largest detection
range of more than four logs, compared to
the other techniques with 2–3 logs. TR-
FRET and AlphaLISA required few exper-
imental manipulations and no washing
steps. TR-FRET was the fastest (2 h), fol-
lowed by AlphaLISA, MSD and Luminex
(approximately 5 h).
Conclusion: In conclusion, AlphaLISA and
MSD are both suited to replace current
ELISA due to enhanced convenience, sen-
sitivity, dynamic range and high through-
put capability.
286
Foxp3+ regulatory T-cells during the first
year of preseasonal immunotherapy
Kepil Ozdemir, S1; Guloglu, D2; Sin, B1; Ikinciogullari,
A2; Misirligil, Z1
1Department of Immunology and Allergy, Ankara
University School of Medicine, Ankara, Turkey;2Department of Pediatric Immunology and Allergy,
Ankara University School of Medicine, Ankara, Turkey
Background: Stimulation of CD4+CD25+
regulatory T cells (Treg) expressing Foxp3
transcription factor has been shown to be
one of the important mechanisms of aller-
gen-specific immunotherapy. However, its
role in pre seasonal allergoid immunother-
apy is unclear. The aim of this study was
to evaluate the effect of pre seasonal grass
pollen allergoid immunotherapy on periph-
eral blood regulatory T cells.
Method: Twenty-one adult patients with
moderate-severe persistent allergic rhinitis
alone were enrolled into this double-blind,
placebo-controlled, prospective study. All
patients had grass pollen sensitization con-
firmed by skin prick tests and in vitro grass
pollen specific IgE measurements. They
also had compatible clinical history with
seasonal allergic rhinitis. The patients were
either received grass pollen allergen (Aller-
govit�, Allergopharma KG, Germany)
according to a pre seasonal immunother-
apy protocol (n = 10) or placebo injec-
tions (n = 11). The treatment cycles were
conducted off-season between February
and March. All patients were received sub-
cutaneously seven injections on a weekly
basis. Prior to the first injection, afterwards
the completion of seventh injection and at
the peak pollen season, CD4+CD25high-
Foxp3+ Treg cells were analysed by flow
cytometry (Beckman Coulter Cytomics
FC500, USA) in accordance with the sur-
face and intra cytoplasmic staining proto-
col. Symptom and medication free days,
and average visual analogue scores during
the pollen season were compared to evalu-
ate clinical effectiveness.
Result: CD4+CD25highFoxp3+ Treg cells
that determined before treatment, after
treatment and at the peak pollen season
were similar in the immunotherapy and
placebo groups (P > 0.05). Symptom free
days at the pollen season were significantly
higher in the immunotherapy group
(P < 0.05). There was no difference in
medication free days and average visual
analogue scores between the two groups
(P > 0.05).
Conclusion: The present study indicates
that pre seasonal grass pollen allergoid
immunotherapy does not lead to an impor-
tant change in the peripheral blood
CD4+CD25highFoxp3+ Treg cell levels
during the first year of therapy. However,
the clinical efficacy may be related to the
local regulatory T cells in the nasal mucosa
and/or allergen-specific regulatory T cells.
287
Characterisation of pollen allergoids with
physicochemical techniques
Luykx, D1; de Bruijn, J1; Cordewener, J2; America, T2;
van den Hout, R1
1Development Department, HAL Allergy BV, Leiden,
The Netherlands; 2Plant Research International,
Wageningen, The Netherlands
Background: Birch and grass allergoids are
drug substances for birch and grass allergy
vaccines, respectively. Until now these al-
lergoids have been poorly characterised
due to their complexity. Nevertheless, a
better characterisation is needed. This
includes identification of the relevant aller-
gens, determination of the degree of poly-
merisation and cross-linking, and
investigation of the protein structures. Sev-
eral physicochemical techniques were tested
for their suitability to analyse these charac-
teristics in the allergoid and corresponding
allergen extract: Mass spectrometry (MS),
SDS-PAGE, HPLC-SEC, lysine determina-
tion and fluorescence spectroscopy.
Method: Allergoids: Allergen extracts from
birch and grasses were treated with glutar-
aldehyde to obtain birch and grass allerg-
oids, respectively. MS: Preparation of
tryptic digests and peptide separation via
nano-LC before electrospray ionisation.
Ionised peptides were fragmented revealing
sequences. SDS-PAGE: 10–20% gels with
Coomassie Blue staining and reduced sam-
ples. HPLC: GF250 and GF450 SEC col-
umns were used combined with UV-
detection. Lysine determination: Samples
were hydrolysed into amino acids followed
by free lysine determination with HPLC.
Fluorescence: Emission spectra were
recorded from 290 to 400 nm, with excita-
tion at 280 nm.
Result: Several isoforms of relevant birch
allergen Bet v1 were identified in the birch
allergoid with MS. In the grass allergoid
relevant grass allergens group 1 and 5 were
identified. SDS-PAGE showed for both al-
lergoids the formation of various high
molecular weight molecules including
masses ‡250 kDa. HPLC-SEC showed for
both allergoids the formation of molecules
‡670 kDa. For both pollen extracts a
minority of HPLC peaks (6% and 9%)
was corresponding to proteins with a
molecular mass >44 kDa. For the allerg-
oids the majority of peaks (81 and 85%)
was corresponding to proteins with a
molecular mass (far) >44 kDa. Determina-
tion of free lysines showed that the major-
ity of the lysines were modified in both
pollen allergoids (73% and 74%). Fluores-
cence indicated hydrophobic tertiary pro-
tein structures after modification of the
pollen extracts.
Conclusion: Applying a combination of
physicochemical techniques was shown to
be a suitable approach to characterise the
pollen allergoids well: Identification of the
relevant allergens, determination of the
degree of polymerisation and cross-linking,
and investigation of the protein structures
were accomplished.
Poster Discussion Session 5 – Immunotherapy: vaccines and mechanisms
126 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Poster Discussion Session 6
Common hypersensitivity reactions
288
Diagnosis in oxaliplatin hypersensitivity:
a 2-year experience with skin tests and
specific IgE
Madrigal-Burgaleta, R1; Berges-Gimeno, M1;
Angel-Pereira, D1; Sanchez-Moreno, V1; Venemalm, L2;
Alvarez-Cuesta, E1
1Allergy Division, Ramon y Cajal University Hospital,
Madrid, Spain; 2Phadia AB Thermo Fisher, Uppsala,
Sweden
Background: Diagnostic tests for oxalipla-
tin hypersensitivity have not been vali-
dated. We evaluate skin testing and specific
IgE in our population.
Method: We retrospectively searched our
Desensitization Unit database. During the
2-year period from June 2009 to June
2011, 32 patients had been referred to our
Unit for assessment after suffering an acute
infusion reaction with oxaliplatin. We
searched for patients who met these inclu-
sion criteria: History of acute infusion
reaction to oxaliplatin, subjected to skin
testing (immediate lecture) with oxaliplatin
(prick test 5 mg/ml, intradermal test 0.5
and 5 mg/ml), subjected to in vitro test to
oxaliplatin (specific IgE), and conclusive
results (positive or negative) in their aller-
gological study. Gold Standard for conclu-
sive negative result was defined as negative
challenge; Gold Standard for conclusive
positive result, as positive challenge or
repeated breakthrough reactions during
desensitization. We evaluated sensitivity,
specificity, positive predictive value (PPV),
and negative predictive value (NPV) for
skin testing, specific IgE (cut-off of 0.35
and 0.10 KU/l). We evaluated elapsed time
from reaction to allergy tests and how it
might influence results.
Result: Fifteen patients were included. Sen-
sitivity was: 58% for skin testing, 50% for
specific IgE (cut-off 0.35 KU/l), 58% for
specific IgE (cut-off 0.10 KU/l). Specificity
was: 100% for skin testing and specific IgE
(cut-off 0.35 KU/l), and 66% for specific
IgE (cut-off 0.10 KU/l). PPV was: 100%
for skin testing and specific IgE (cut-off
0.35 KU/l), and 87.5% for specific IgE
(cut-off 0.10 KU/l). NPV was: 37.5% for
skin testing, 33% for specific IgE (cut-off
0.35 KU/l), 28.6% for specific IgE (cut-off
0.10 KU/l). All patients with positive tests
and conclusive positive study had been
evaluated within 1 month from their initial
reaction; however, 80% of patients with
negative tests, but conclusive positive study
had been evaluated at least 3 months after
their initial reaction.
Conclusion: In our selected population,
diagnostic tests are very specific but less sen-
sitive for oxaliplatin hypersensitivity, with
high PPV. Skin testing and specific IgE
could be useful for oxaliplatin diagnosis.
Selection bias may overestimate specificity
and underestimate sensitivity. Elapsed time
from initial reaction to allergy tests could be
relevant. Prospective well-designed studies
are necessary to validate these diagnostic
tools.
289
Hypersensitivity reactions to non-ionic
iodinated contrast media
Nikolaos, T; Vovolis, V; Mikos, N; Delyargiris, C;
Kompoti, E
Allergy and Clinical Immunology Department, Laiko
General Hospital Athens, Athens, Greece
Background: Hypersensitivity reactions to
nonionic iodinated contrast media (ICM)
represent a serious problem in modern
medicine and may be divided into immedi-
ate (<1 h of administration) and nonim-
mediate responses (>1 h).
Method: Twenty-one patients referred over
a 4-year period for ICM hypersensitivity
past reactions, either immediate or nonim-
mediate, were investigated by means of
skin prick, intradermal and patch tests
with a series of contrast media (iobitriol,
iodixanol, iomeprol, iopamidol, iopromide,
ioversol). Positive skin tests were defined
according to internationally accepted
guidelines. Allergological and general med-
ical history was obtained in each case.
Result: Positive skin tests were observed in
seven of 13 patients with immediate reac-
tion (53%) and in one of eight patients
with nonimmediate reaction (12.5%). The
scores for the immediate reactions were
Grade I in 4, Grade II in 4, Grade III in 3
and Grade IV in 2. The non-immediate
reactions were classified mainly as moder-
ate. In the skin positive group of immedi-
ate reactors severity scores were Grade I in
2, Grade III in 3 and Grade IV in 2.
Nearly 50% of positive tested immediate
reactors had not been exposed to ICM pre-
viously. Cross reactivity was extended to
57% in skin positive group of immediate
reactors. Respiratory allergy was more fre-
quent in immediate than in nonimmediate
reactors (4/13 vs 0/8). The reasons for
ICM use were CT scans in 16 patients, ou-
rography in three and angiography in one
(missing data for one patient).
Conclusion: These data suggest that at
least 50% of immediate hypersensitivity
reactions to contrast media are caused by
an immunological mechanism. Skin testing
appears to be a necessary tool for diagno-
sis of contrast medium allergy and may
play an important role in selection of a
safe product in previous reactors.
290
Dual haptenic presentation in carrier
molecules for the in vitro testing to
detect IgE-antibodies in patients allergic
to betalactams
Montanez, M1; Ruiz-Sanchez, A2; Ariza, A1; Mayorga,
C1; Perez-Inestrosa, E2; Rodriguez-Bada, J1; Blanca, M3;
Torres, M3
1Research Laboratory, IMABIS Foundation-Carlos Haya
Hospital, Malaga, Spain; 2Organic Chemistry, University
of Malaga, Malaga, Spain; 3Allergy Service, Carlos Haya
Hospital, Malaga, Spain
Background: Benzylpenicilloyl-dendrimer
conjugates are recognized by IgE specific to
benzylpenicillin. The use of dendrimers
compared to the conventional poly-L-lysine
carrier improves the reproducibility of
in vitro tests to diagnose allergy. The aim of
this study was to include other betalactam
hapten, amoxicillin, and the concept of hav-
ing two kinds of haptens in the same dendri-
mer carrier. Therefore, we compare the
ability of different dendrimers displaying
multiple presentation of only one hapten,
either benzylpenicillin or amoxicillin, and
mixed-dendrimer bearing both haptens, to
be recognized by specific IgE to betalac-
tams, for developing a universal in vitro
allergy diagnostic test to betalactams.
Method: Well-defined hapten-carrier conju-
gates were synthesized by haptenization of
4th generation PolyAMidoAMine dendri-
mers with either one kind of penicillin (ben-
zylpenicillin and amoxicillin) or both
penicillins. The chemical structures of the
resulting conjugates were successfully charac-
terized by means of nuclear magnetic
resonance experiments. The different conju-
gates were immunologically tested by RAST
(RadioAllergoSorbentTest) inhibition using
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 127
sera from four controls and seven patients
allergic to penicillins, both selective and
cross-reactive responder.
Result: The content of haptens in the
carrier dendrimer could be determined and
controlled by the developed fabrication
methodology. Regarding immunological
assays, at the maximum conjugate concen-
tration, all sera allergic to benzylpenicillin
showed 80% of RAST inhibition to both
benzylpenicilloyl-dendrimer and mixed-den-
drimer, and negative to amoxicilloyl-dendri-
mer. All sera allergic to amoxicillin showed
90% of inhibition to both amoxicilloyl-den-
drimer and mixed-dendrimer, with no inhi-
bition to benzylpenicilloyl-dendrimer. All
sera with cross-reactivity to both penicillins
inhibited above 70% to the three different
conjugates.
Conclusion: Immunological results suggest
that these conjugates inhibit IgE recognition
when containing any of the haptens to
which the patient is allergic to, reaching sim-
ilar values with monoantigenic and bianti-
genic conjugates. Thus, IgE recognition is
specific and the sensitivity does not vary
from biantigenic to monoantigenic conju-
gates. This provides information about the
structural chemical requirements to develop
a universal in vitro test, being the described
mixed-dendrimers potential candidates to
progress in allergy diagnosis to betalactams.
291
Anaphylaxis during general anesthesia:
10-year survey from a Belgian allergy
clinic
Leysen, J1; Bridts, C1; Sabato, V1; Vercauteren, M2;
Ebo, D1
1Immunology – Allergology – Rheumatology, Antwerp
University Hospital, Antwerp, Belgium; 2Anesthesiology,
Antwerp University Hospital, Antwerp, Belgium
Background: Anaphylactic reactions occur-
ring during anesthesia remain a major
cause of concern for anesthesiologists. The
authors report results of a 10-year survey
of such reactions observed during anesthe-
sia in Belgium.
Method: Between January 1, 2001, and
December 31, 2011, 344 patients who expe-
rienced perioperative anaphylaxis were
referred to our allergy center. Allergy was
documented on the basis of clinical history,
skin tests, specific immunoglobulin E assay
and/or the basophil activation test.
Result: This study describes 344 patients,
age ranged from 2 to 86 years (111 male/233
female). Grade I and II in 110 and grade III
and IV were observed in 176 patients,
respectively. IgE mediated allergic reactions
were documented in 246 cases (72%). The
most common elicitors were neuromuscular
blocking agents (NMBA) (40%), latex (25%),
antibiotics (12%) and chlorhexidine (7%).
In 71 out of 93 NMBA-related cases, rocu-
ronium was identified as the offending drug.
Eighty-seven percent of the rocuronium-
induced reactions were grade III or IV
reactions. Eighteen of the patients with an
IgE-mediated identified cause (7%) were
allergic to more than one compound. In 28%
of cases no allergic IgE-mediated cause could
be found.
Conclusion: In our region, NMBA are the
most frequent causal agents of anaphylaxis
during general anesthesia, particularly
rocuronium. Other important causes are
latex, antibiotics, chlorhexidine and masto-
cytosis/elevated serum tryptase
292
Detection of IgE- and IgG-antibodies to
local anaesthetics and dental materials.
What is the diagnostic value?
Lazarenko, L
Allergy Clinacal Immunology, Pirogov National Medical
Surgery Centre, Saint-Petersburg, Russian Federation
Background: Evaluation of allergic reac-
tions to local anaesthetics and dental mate-
rials is difficult, because of poor sensitivity
of in vivo testing, which controlled admin-
istration provocative tests in order to con-
firm the diagnosis. Contrary, in vitro
testing is a high sensitive and reliable diag-
nostic procedure, which avoid risks of an
in vivo testing. It is apparently clear, that
IgE – monitoring is a reliable method for
testing in cases of dental allergy, but it is
unclear the role of IgG-antibodies. The
aim of our study: determine the diagnostic
value of IgG-testing in cases of allergy to
local anaesthetics and dental materials.
Method: Fifty-four outpatients with symp-
toms of hypersensitivity (HS) to local anaes-
thetics (LA) – Group A and 264 outpatients
with hypersensitivity to dental materials
(DM) – Group B, were investigated for spe-
cific IgG-antibodies in serum. Assays were
arranged by ELISA method. We studied
patients with controversial result of
patient’s case history and prick (Group A)
or patch (Group B) skin tests. No drug
provocation tests were done because of risk
of immediate allergic reactions with a posi-
tive history of multi hypersensitivity. In par-
allel we determine IgE-antibodies by ELISA
method. Group A (n = 54) consist of 40
females, 14 males, mean age 39.1 years (6–
80), group B (n = 264) – of 201 females and
63 males, mean age 57 years (31–82). For in
vitro specific IgG EAST we used such local
anaesthetics allergens as – articaine, mepi-
vacain, prilocain, lidocain; and dental mate-
rials allergens – gold, platinum, palladium,
cuprum, cobalt, nickel, and chromium.
Result: We found positive IgG levels to LA
(Group A) to: articaine – 2.4%; mepivacain
– 1.84%; prilocain – 1%; lidocain – 4%. In
the second group, sensitive to dental materi-
als (Group B), IgG-specific antibodies were
positive for: cuprum – 13.4%, cobalt –
21.7%, nickel – 21.8%, palladium – 4.9%,
chromium – 20.3%, gold – 33.1%, platinum
– 20.7% of patients.
Conclusion: The result of IgG and IgE-test-
ing is not the same. When it was similar, we
decided that it was really allergic reactions
(confirmed by patient case history). Most
cases of positive results are correlated with
previous history of using those dental mate-
rials. We propose the parallel testing of IgE
and IgG antibodies in cases of allergy to
local anaesthetics and dental materials in
order to elevate diagnostic value of those in
vitro methods. Detail analysis of patient his-
tory is too useful.
293
Infliximab-specific T-cells in patients with
acute infusion reactions
Vultaggio, A1; Matucci, A1; Petroni, G2; Pratesi, S2;
Nencini, F2; Maggi, E2
1Immunoallergology Unit, Department of Biomedicine,
Careggi Hospital, Florence, Italy; 2Department of
Internal Medicine, University of Florence, Florence, Italy
Background: Infliximab (IFX), a chimeric
anti-TNFa monoclonal antibody, is an
established targeted therapy for immuno-
mediated inflammatory diseases. As other
biotherapeutics, it carries potential risks of
immunogenicity, with the production of
specific anti-IFX antibodies (ATI) that can
lead to loss of response or acute infusion
reactions. Our study was aimed to analyse
the memory T-cell response to IFX in
patients who developed ATI.
Methods: We investigated the presence of
IFX-specific T cells in 27 subjects (11 reac-
tive ATI+, six non responder ATI+, five
tolerant ATI- and five non exposed healthy
donors), using different T-cell proliferation
assays. A two-step procedure of stimula-
tion, in which peripheral blood mononu-
clear cells (PBMC) were initially cultured
with IFX for 7 days and then re-stimulated
with the drug for an additional 5 days,
resulted the best procedure to detect IFX-
specific memory T cells. Drug-specific T
cell clones (TCC) were also generated from
PBMC of two reactive patients.
Results: PBMC obtained from seven (five
reactive and two non responder) out of 17
(41.2%) ATI+ patients displayed a prolif-
erative to IFX, while no proliferation was
detectable in both ATI- patients and
healthy donors. Cytokine (IL-13, IFNc,
IL-17) production by PBMC, as well as by
TCC, upon in vitro re-stimulation with
IFX, was highly variable between the
patients. The analysis of Vb usage analysis
of drug-specific TCC revealed an oligoclo-
nal expansion. An high proportion of
Poster Discussion Session 6 – Common hypersensitivity reactions
128 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
IFX-specific TCC, showed a regulatory
phenotype and function. Cross-reactivity of
IFX-specific TCC with other chimeric
mAbs and murine IgG was also detected.
Conclusion: Overall, our study provide evi-
dence of both effector and regulatory
memory T cells specific for IFX and con-
tributes to the understanding of pathogenic
mechanisms of immunogenicity of biologi-
cal agents.
294
Hypersensitivity to non-steroidal
antiinflammatory drugs – does
premedication with antileukotriene and/
or antihistamine work?
Almeida, E; Ribeiro, F; Sousa, N; Faria, E;
Segorbe Luıs, A
Immunoallergology Department, Coimbra University
Hospitals, Coimbra, Portugal
Background: Hypersensitivity reactions to
nonsteroidal anti-inflammatory drugs
(NSAIDs) are currently responsible for 21–
25% of the reported adverse drug events.
In this study we tried a novel approach in
patients with no safe alternative NSAID.
Method: We performed the retrospective
study of the clinical charts of patients with
history of cutaneous hypersensitivity reac-
tions to NSAIDs in the last 2 years (2010–
2011). We analyzed the involved drugs,
symptoms, timing of reaction, underlying
disease and challenge with an alternative
drug. We enrolled those with positive oral
challenge test (OCT), all with mild symp-
toms. Then we proceed to OCT with
premedication with either cetirizine 10 mg
or montelukaste 10 mg the day before and
on the challenge day.
Result: Seventy-six patients were observed
in this period (52F/24M), mean age of
44.9 ± 16.5 years. Six patients (four female/
two male; mean age 50.3 ± 16.7 years) met
the criteria to be included in the study. Two
patients presented isolated angioedema, two
isolated urticaria and two anaphylaxis. The
most implicated drug was acetylsalicylic
acid (AAS). Three patients tolerated parac-
etamol while the others had no known alter-
native. In four patients the reaction did not
occurred in the first hour. The OCT per-
formed by each patient are in table 1. The
Patient 2 reacted to OCTs with cetirizine
and montelukate premedication so we pro-
ceed to an OCT with both premedication
with cetirizine and montelukaste.
Conclusion: As we can see in the table
above all patients with previous positive
reactions in OCT with an alternative
NSAID, tolerated the same drug with
premedication whether with an anti-hista-
mine or anti-leukotriene. Only one patient
required premedication with both cetirizine
and montelukaste to tolerate the NSAID.
295
Cutaneous hypersensitivity to
non-steroidal antiinflammatory drugs
Ribeiro, F; Almeida, E; Sousa, N; Faria, E;
Carrapatoso, I; Segorbe Luıs, A
Immunoallergology Department, Coimbra University
Hospitals, Coimbra, Portugal
Background: Hypersensitivity reactions to
nonsteroidal anti-inflammatory drugs
(NSAIDs) are currently responsible for 21–
25% of the reported adverse drug events.
This study aims to characterize the clinical
profile of patients with suspected cutaneous
hypersensitivity reactions to NSAIDs.
Method: We performed the retrospective
study of the clinical charts of patients fol-
lowed in a Drug Outpatient Clinic in a
2 year period (2010–2011). The investiga-
tion included the analysis of involved
drugs, symptoms, timing of reaction,
underlying disease and challenge with cul-
prit or alternative drug.
Result: Seventy-six patients were observed
in this period (52F/24M), mean age of
44.9 ± 16.5 years. Overall the most impli-
cated drugs were salicylates (41/28.5%) fol-
lowed by NSAIDs derived from propionic
acid (33/22.9%) and NSAIDs derived from
acetic acid (24/16.7%). Twenty-four
patients presented isolated angioedema, 24
isolated urticaria, 18 anaphylaxis, nine urti-
caria associated to angioedema and other
symptoms 1. The reaction was immediate
in 48 cases. Twenty-seven out of 76
patients were atopic. Of the 128 oral chal-
lenge test (OCT) performed, 19 were with
the culprit drug (four positive). To confirm
the safety of alternative drugs 109 OCT
were carried out, 42 with meloxicam (two
positive), 38 with coxibs (four positive), 23
with nimesulide (four positive), three with
metamizol and three with paracetamol.
Sixty-one patients presented hypersensitiv-
ity reactions to multiple NSAIDs and in
five patients the clinical history and the
diagnostic work-up suggests hypersensitiv-
ity to a single NSAID, however it was not
confirmed by means of OCT with culprit
drug. Five patients had NSAIDs-exacer-
bated urticaria. NSAIDs hypersensitivity
was excluded in five patients.
Conclusion: The salicylates were the most
implicated drugs in our series, but the
NSAIDs derived from propionic and acetic
acids were responsible for the most serious
reactions. Twenty-four patients reacted to
more than one strong COX-1 inhibitor
NSAID. In all patients we were able to
identify a safe drug that was in the major-
ity of cases a meloxicam and/or celecoxib/
etoricoxib.
296
Non-opioid analgesic, antipyretic and
non-steroidal antiinflammatory drug
hypersensitivity in children
Ponvert, C; Scheinmann, P; Delacourt, C; de Blic, J
Pediatric Pulmonology & Allergology Service, Paris
Descartes University, Sick Children’s Hospital, Paris,
France
Background: Although NOA-AP-NSAIDs
are the second cause of suspected drug HS,
a few studies have been performed in
children.
Aims: To confirm or rule out a suspected
diagnosis of NOA-AP-NSAID HS, to eval-
uate the diagnostic value of immediate and
non immediate responses in skin tests (ST)
with these drugs, to determine the fre-
quency of allergic (specific, immediate and
non immediate) and non allergic (intoler-
ance/cross-reactive) HS, and to identify
risk factors for HS to NOA-AP-NSAIDs
in children.
Methods: Pricks, intradermal (ID) and
patchs were performed with suspected
drugs (paracetamol, ibuprofen and acetyl-
salicylic acid [ASA]) diluted in saline, and
with non suspected drugs to determine
their specificity. Responses were assessed at
20 min (immediate, pricks and ID), 6–8 h
(semi-late, ID), 48–72 h (late, ID and
patchs), and 6–7 day (hyper-late, ID and
patchs). Challenge tests (CT, one-several
Table 1.
Oral Challenge Test Without premedication Cetirizine Montelukaste Cetirizine + Montelukaste
Patient 1 Meloxicam+ (urticaria) Meloxicam+ (urticaria) Meloxicam - ND
Patient 2 Meloxicam+ (facial angioedema) Meloxicam+
(facial angioedema)
Meloxicam+
(facial angioedema)
Meloxicam -
Patient 3 Paracetamol+ Paracetamol - ND ND
Patient 4 Celecoxib+ (urticaria) ND Celecoxib - ND
Patient 5 Celecoxib+ (cutaneous pruritus) ND Celecoxib - ND
Patient 6 Nimesulide+ (urticaria) Nimesulide - ND ND
Poster Discussion Session 6 – Common hypersensitivity reactions
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 129
days in the hospital or at home, based on
chronology and severity of the reactions)
were performed in children with negative
responses in ST, and with alternative drugs
in children with a highly suggestive/worry-
ing clinical history (CH) or positive CT
with suspected drugs.
Results: Most frequent reactions were urti-
caria and/or angioedema (n = 125/161,
77.6%). Twenty-one children reported
severe reactions. The specificity of ST was
excellent, except for immediate responses
in ID-ASA 25 mg/ml.
A complete work-up (ST + CT and/or
CH) was performed in 93 children. HS was
diagnosed in 27 children (29%), either
allergic (n = 21, 77.8%) or non allergic
(n = 6, 22.2%), based on positive
responses in immediate (n = 1, 3.7%) or
non immediate (n = 1, 3.7%) responses in
ST or CT (n = 18, 66.7%), or a highly
suggestive CH (n = 7, 25.9%) (Table 1).
Risk factors were immediate reactions
(P = 0.0002), age (m = 9.1 vs 6.5 years,
P = 0.003), and chronic urticaria in chil-
dren with non allergic HS (P = 0.03).
Discussion and conclusion: Our results, in
numerous children, show that about one
third of children with suspected allergy to
NOA-AP-NSAIDS have allergic (immedi-
ate or non immediate) or non allergic HS
to these drugs, immediate reactions being
the most suggestive. Except for a few cases
diagnosed allergic by means of ST, diagno-
sis of NOA-AP-NSADS HS was primarily
based on CT and on CH in highly sugges-
tive and severe reactions. Finally, although
most reactions in adults result from non
allergic HS, our results suggest that allergic
HS to NOA-AP-NSAIDs is frequent in
children.
297
Drug provocation tests with betalactam
in children presenting with rash
Iglesias-Souto, J; Gonzalez-Perez, R; Sanchez-Machın, I;
Poza-Guedes, P; Matheu, V
Allergy, Hospital del Torax (Ofra), Santa Cruz de
Tenerife, Spain
Background: Immediate mild skin reactions,
maculopapular rashes and exanthemas are
often observed in children treated with bet-
alactams. Many children are then labelled
as ‘allergic’ without a reliable testing. In
patients explored several years after a sus-
pected allergic reaction to betalactams,
International Guidelines recommend the
implementation of retest for the diagnosis of
penicillin allergy, increasing the time and
cost of the study.
Objective: We aimed to study the useful-
ness of a second allergological work-up
(retesting) in the diagnosis of penicillin
allergy in the period 2001–2010 in children
reporting immediate mild skin reactions,
maculopapular rashes and exanthemas.
Method: This retrospective study included
all 0–14 year-old patients reporting an
immediate mild skin reaction or maculo-
papular rash occurring within 12 h of a
treatment with a betalactam antibiotic.
Diagnosis was performed following Inter-
national Guidelines by skin tests (ST),
including Skin Prick Test (SPT) and Intra-
dermal Test (ID) with (i) Major and (ii)
Minor determinants of penicillin, (iii) ben-
zylpenicillin, (iv) Amoxicillin and (v) the
suspected drug. A drug provocation test
(DPT) was performed in children with neg-
ative responses in ST. This procedure was
repeated 2–4 weeks later in all inconclusive
or negative cases.
Result: Of 948 children (518 boys, 430
girls) with suspected drug hypersensitivity,
758 (80%) reported suspected allergic reac-
tions to betalactams, mainly amoxicillin.
ST with betalactams (n = 1494) were neg-
ative in 1470 cases. DPTs with betalactams
(n = 1475) resulted in negative response in
1470 cases. Betalactam allergy was diag-
nosed in 22 children (3%) in the first
round of tests, by means of ID tests in
most cases. Only three of the remaining
736 patients (0.4%) were diagnosed allergic
in the second round of tests (retest).
Conclusion: In agreement with previous
studies, our results strongly suggest that
retesting with betalactams is not necessary
in children reporting immediate and accel-
erated mild skin reactions and exanthemas.
However, these results should be confirmed
by multicenter studies.
298
Risk factors for developing immediate
hypersensitivity to fluoroquinolones
Torres, M1; Salas, M1; Dona, I1; Gomez, F1; Garcia, I1;
Blanca-Lopez, N2; Ruiz, M1; Guerrero, M1; Blanca, M1
1Allergy Service, Carlos Haya Hospital, Malaga, Spain;2Allergy Service, Infanta Leonor Hospital, Madrid, Spain
Background: Immediate hypersensitivity
reactions to fluoroquinolones, especially
moxifloxacin and ciprofloxacin, have
increased. The aim of the study was to
identify risk factors, including previous
confirmed allergy to betalactams, for devel-
oping hypersensitivity to quinolones.
Method: We evaluated all consecutive
patients attending our allergy department
between January 2009 and December 2010
due to a reaction associated with fluoroqui-
nolones administration. The diagnosis was
confirmed by drug provocation tests or a
clinical history of well-defined repeated epi-
sodes. The patients were classified as aller-
gic or non-allergic.
Results: Of the 108 patients evaluated, 72
(66.7%) were confirmed allergic and 36
(33.3%) non-allergic. Comparisons between
groups made by Mann-Whitney and Chi-
square tests showed significant differences
for the presence of allergy to betalactams
(P = 0.03), type of reaction (immediate or
non-immediate, P = 0.001) and the quino-
lone involved (being higher for moxifloxacin
in the group of allergics, P = 0.021). There
were no differences in age, gender, con-
firmed allergy to non-betalactam drugs and
time between reaction and study. Logistic
regression analysis detected that the risk of
being allergic to quinolones was 17.18 times
higher in those previously diagnosed of
allergy to betalactams, 53.37 times higher in
those developing immediate reactions and
9.36 higher when moxifloxacin was the cul-
prit drug.
Conclusion: In patients developing a reac-
tion associated with quinolones, hypersensi-
tivity is more frequently confirmed in those
previously diagnosed as allergic to betalac-
tams, those with immediate reactions and
when moxifloxacin is the drug involved.
Reasons for this association require further
study. The study was funded by FIS-The-
matic Networks (RIRAAF/RD07/0064),
Junta de Andalucia (CTS 06603, PI-0545-
2010) and FIS (09/01768).
Table 1.
Diagnosis (n) by means of n ST CT CH
Non-allergic HS to all drugs 4 4
Non-allergic HS with tolerance of paracetamol 2 2
Immediate-type HS to ASA 2 1 (confirmed by CT) 1
Delayed-type HS to ASA 3 2 1
Immediate-type HS to ibuprofen 2 2
Delayed-type HS to ibuprofen 3 3
Immediate-type HS to paracetamol 9 7 2
Delayed-type HS to paracetamol 2 1 (confirmed by CH) 1
Total 27 2 18 7
Poster Discussion Session 6 – Common hypersensitivity reactions
130 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
299
The prevalence of type 1 hypersensitivity
drug reactions in school children –
preliminary results
Erkocoglu, M1; Kaya, A1; Civelek, E1; Banu, C2;
Kocabas, C1
1Department of Pediatric Allergy and Immunology,
Ankara Children’s Hematology Oncology Education and
Research Hospital, Ankara, Turkey; 2Hacettepe
University Medical School, Ankara, Turkey
Background: Poorly documented self-
reported drug allergy (DAll) is a frequent
problem in daily clinical practice and has a
considerable impact on prescription
choices. The goal of this study is to find
out the self reported and documented
prevelance of type 1 drug hypersensitivity
reactions amongst the school children aged
between 12 and 15 in Ankara.
Method: This study included three phases:
(i) A cross-sectional survey of students
with a questionnaire. (ii) Phone survey
with ENDA for children whose parents
reported drug allergy at questionnaire. (iii)
Clinical evaluation that took place at the
allergy clinic and included children whose
history was compatible with type 1 drug
hypersensitivity after ENDA survey. The
sample number of students has been calcu-
lated 9096 out of a total of 210 000 stu-
dents at the second stage (6th, 7th and 8th
grades) State Elementary Schools in the
metropolitan counties of Ankara province
with the assumption of type 1 drug hyper-
sensitivity reaction prevelance (P) = 1%
and a Delta value = 0.2 (a < 0.05,
b = 0.8). The participation in the ques-
tionnaire has been assumed 70%, therefore
approximately an additional 2700 other
students have been identified as substitutes.
Results: Ten thousand and ninety-six
(89.4%) out of a total of 11 233 question-
naires distributed at 34 schools have been
collected in the study between 1 March
and 27 May 2011. While the prevelance of
life-time self-reported drug related type 1
hypersensitivity reaction was 7.9%, it was
reduced to 1.2% after ENDA survey with
their parents. Third phase of study is still
ongoing.
Conclusion: The prevelance of self-reported
type 1 drug hypersensitivity reaction
(7.9%) was much more higher than the
real drug hypersensitivity prevelance (1%)
in literature.
300
The strength of the relationship between
specific human leukocyte antigens and
severe cutaneous allergic reactions
Leong, K1; Shen, M2; Thong, B1; Tan, N3; Chng, H4;
Chan, G1; Tan, S1; Chia, F1; Tab, T1; Tan, J1; Loh, N3;
Ren, E2
1Department of Rheumatology, Allergy and
Immunology, Tan Tock Seng Hospital, Singapore,
Singapore; 2Department of Microbiology, National
University of Singapore, Singapore, Singapore;3Department of Neurology, National Neuroscience
Institute, Singapore, Singapore; 4Department of
Neurology, Tan Tock Seng Hospital, Singapore,
Singapore
Background: HLA-B*1502 is associated with
carbamazepine- and phenytoin-induced severe
cutaneous allergic reactions (SCARs),
HLA-B*5801 with allopurinol-induced reac-
tions and HLA-B*5701 with abacavir-
induced ones. The association is believed to
be very strong in certain populations, with
odds ratio of 500–800.
Method: We calculated the odds ratio for
the relevant genotypes using data from our
patients with carbamazepine-, phenytoin-
and allopurinol-induced SCARs and the
drug-tolerant controls, recruited from Tan
Tock Seng Hospital and the National Neu-
roscience Institute. Sequence-based typing
was performed for the HLA class I loci
using primers for exons 2–4; a similar
method was used for the sequencing of
HLA-DRB1.
Result: For carbamazepine, there were 21
patients and 18 controls; for phenytoin,
five cases and 15 controls and for allopuri-
nol 11 patients and nine control. For car-
bamazepine, the odds ratio of SCAR in
the presence of HLA-B*1502 was 5.688
(95% CI 1.378–23.480), for phenytoin the
odds ratio was 3.2 (0.516–19.843). For
allopurinol the odds ratio was 21.333
(1.811–251.264).
Conclusion: In our study group, the associ-
ation between specific HLAs and SCAR
does not appear to be as strong as sug-
gested. Further studies are warranted as
this has implications for wide implementa-
tion of pre-treatment genotyping.
Poster Discussion Session 6 – Common hypersensitivity reactions
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 131
Poster Discussion Session 7
Immunotherapy: progress in clinical management
301
Preventing progression of allergic rhinitis
into asthma in school children treated
with Salsola kali immunotherapy
Arifhodzic, N; Al-Ahmed, M; Al-Ahmed, N; Paniker, R;
Ali, A
Allergy Department, Al Rashed Allergy and Chest
Centre, Kuwait, Kuwait
Background: Allergen immunotherapy
(AIT) has the potential to reduce the pro-
gression of seasonal rhinoconjunctivitis
(SAR) into asthma in children.
Objective: To compare development of
asthma and new sensitization in Kuwaiti
schoolchildren diagnosed as having Salsola
kali SAR, treated with subcutaneous
immunotherapy (SCIT), sublingual immu-
notherapy (SLIT) or drugs only.
Methods: 97/142 children, age 9–15, both
gender, randomly selected to receive SCIT
(n = 34), SLIT (n = 25) or drugs only
(n = 38) finished 3 years treatment course.
Inclusion criteria: a moderate – severe SAR
for two or more seasons, positive skin prick
test (SPT) or specific IgE to Salsola kali
only, and absence of comorbid asthma.
Asthma symptoms, FEV1 and asthma res-
cue medication were assessed yearly at the
peak of pollination. New sensitization was
assessed at the end.
Results: FEV1 value was more stable in both
AIT groups than in control (P < 0.001).
Throughout the treatment, asthma medica-
tion was used less frequently in AIT groups if
compared with control (P < 0.002), but of
the two AIT groups, SCIT showed better
results (P < 0.02). Both SCIT and SLIT
reduced appearance of new sensitization
(P < 0.001), although SCIT was slightly
more protective than SLIT (P < 0.07).
Conclusion: Both SCIT and SLIT have
preventive role in children with SAR. SCIT
seems superior to SLIT.
302
Early effect of high-dose hypoallergenic
house dust mite preparation in the
treatment of allergic asthma
Rudert, M; Kettner, J; Haefner, D; Narkus, A
Medical Department, Allergopharma Joachim Ganzer
KG, Reinbek, Germany
Background: In order to increase the prob-
ability of superiority in the presence of a
large placebo effect particularly during
early treatment phases, controlled studies
in house dust mite (HDM) allergy com-
monly include clinical outcome measure-
ments 2 or 3 years after initialization of
SCIT. Patients’ expectations and compli-
ance, however, warrant efficacy assess-
ments at the earliest possible time point.
Efficacy in HDM-SCIT studies can only be
assesed once a year during the winter in
the absence of flowering trees, grasses and
weeds, to which many HDM-allergic
patients are co-sensitized. In the present
study – following a baseline assessment –
efficacy was assessed 9 months after the
start of SCIT.
Method: One hundred and twenty-one
HDM allergic patients with controlled
bronchial asthma (GINA II, III) aged 6–
40 years, requiring treatment with inhaled
corticosteroids (ICS) in daily doses of
‡2 · 50 lg fluticasone propionate (FP)
were randomised in 1:1 ratio using a con-
trolled study design and could be evaluated
for efficacy. All patients received basic
pharmacotherapy by FP and salbutamol
p.r.n. as a rescue medication. One group of
patients (n = 60) received perennial SCIT
using a high hose hypoallergeneic allergen
preparation additionally. In the control
group (n = 61), blinded placebo injections
were given in patients ‡18 years, whereas
treatment in children and adolescents was
by FP and salbutamol only. The minimum
FP-dose required for asthma control
according to GINA criteria was assessed
before and after 9 months of treatment.
Result: FP-dose at asthma control in
patients hyposensitized for 9 months with
the HDM allergoid was significantly
reduced by more steps compared to the non-
hyposensitized control group (P = 0.0105).
In the SCIT-group the improvement rate in
means of at least one dose step amounted to
55.0% compared to 32.8% in the control
group. Although FP dose was reduced, lung
function (mean morning PEF) in the SCIT
group was maintained stable after 9 months
of SCIT. Serum specific IgG1 and IgG4
antibody levels significantly increased dur-
ing SCIT vs control patients (P < 0.0001).
Conclusion: SCIT with the high-dose hypo-
allergenic HDM preparation showed an
early onset of clinical efficacy resulting in a
reduced need for inhaled corticosteroids in
the treatment of bronchial asthma. In vivo
immunogenic activity was in accordance
with clinical outcomes.
303
Sublingual immunotherapy in patients
with house dust mite induced allergic
rhinitis: a retrospective study of clinical
outcome
Casia, S; Thalayasingam, M; Shek, L; Chao, S
Paediatrics, National University Hospital, Singapore,
Singapore
Background: To evaluate the efficacy of
house dust mite (HDM) sublingual immu-
notherapy (SLIT) on nasal symptoms and
quality of life (QoL), in children and adults
with allergic rhinitis (AR), over a period of
2 years.
Method: This study was a retrospective
analysis of records of children and adults
with a diagnosis of AR secondary to HDM
sensitisation to the local house dust mites
[Dermatophagoides pteronyssinus (DP)
and/or Dermatophagoides farinae (DF)
and/or Blomia tropicalis (BT)], who were
treated with SLIT at a tertiary hospital in
Singapore from 2008 to 2010. Symptom
scores, calculated to obtain the daily Total
Nasal Symptom Score (TNSS) and a mini-
rhinoconjunctivitis quality of life question-
naire (m-RQLQ) were administered before
commencement of treatment, and at 1 and
2 years of treatment respectively.
Result: Forty-three patients, of which 29
were children, were analysed. Patients who
required SLIT with extract for
DP + DF + BT, or DP + DF, demon-
strated significant improvement in their
TNSS scores at 1 and 2 years of treatment
[DP + DF + BT: Initial score 8.61 fi 6.83
at 1 year (P = 0.006) fi 6.16 at 2 years
(P = 0.001); DP + DF: Initial score
9.62 fi 8.07 at 1 year (P = 0.001) fi 5.71
at 2 years P < 0.001)]. The DP + BT
group showed significant improvement only
at 2 years into treatment [Initial score
7.28 fi 5.42 at 1 year (P = 0.066) fi 4.42
at 2 years, (P = 0.018)]. The m-RQLQ also
showed significant improvement for the
entire cohort, with the score decreasing
from 31.4 initially to 14.3 at 1 year, and 8.7
at 2 years into treatment.
Conclusion: SLIT is efficacious at improv-
ing symptoms and QoL in patients with
HDM induced AR, the effects becoming
132 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
significant in most as early as 1 year after
treatment.
304
Adverse reactions to subcutaneous
allergen-specific immunotherapy in
patients with atopic dermatitis with a
rush buildup
Sanchez, J1; Juliana, B2; Lopez, E1; Cardona, R1
1University of Antioquia, Medellın, Colombia;2Allergology Service, University of Antioquia, Medellın,
Colombia
Background: Subcutaneous allergen-specific
immunotherapy is a proven, highly effec-
tive treatment for IgE–mediated diseases.
However, rush immunotherapy is pre-
scribed infrequently because of the percep-
tion that accelerated immunotherapy
buildup leads to a higher rate of systemic
reactions and few studies have explored the
safety of rush immunotherapy in patients
with atopic dermatitis.
Objective: To evaluate the frequency of
adverse reactions in patients with atopic
dermatitis receiving rush immunotherapy
with house dust mites (HDM).
Methods: A retrospective, observational
review was conducted for patients with ato-
pic dermatitis receiving HDM allergen-spe-
cific immunotherapy between January 2009
and august 2011. Subcutaneous Immuno-
therapy with depigmented polymerized
mites extract was administered monthly.
Mite allergen extracts were administered in
two refracted doses of 0.2 and 0.3 ml at first
injection, and in single 0.5 ml doses in sub-
sequent monthly injections. A 30 min obser-
vation time was required after each
injection, for observing and counteracting
possible side effects. Patients or patients’
parents were instructed to identify and
report any delayed local or systemic reac-
tion. Systemic reactions were graded using
the World Allergy Organization Subcutane-
ous Immunotherapy Systemic Reaction
Grading System.
Results: Data from 102 patients with ato-
pic dermatitis (SCORAD 34 range 23–67)
receiving HDM rush immunotherapy were
collected. One thousand three hundred
forty-seven injections were registered; 124
buildup and 1223 maintenance injections.
Three patients (2.9%) experienced 11 local
reactions (hives <5 mm). The frequency of
reactions per injections was 0.8%. There
were no fatalities or systemic reactions.
Conclusion: Mites immunotherapy for
patients with atopic dermatitis using a de-
pigmented polymerized mites extract with
a rush buildup, has a lower frequency of
systemic reactions than that seen in allergic
respiratory diseases. Accelerated buildup
could improve patients’ adherence to treat-
ment and reduce dropout rates.
305
Measuring allergen-specific bronchial
hyperreactivity after specific
immunotherapy to detect responder and
non-responder states
Rosewich, M1; Arendt, S1; Schulze, J1; Schubert, R1;
Bohle, B2; Zielen, S1
Allergy, Pneumology and Cystic Fibrosis,1Goethe-University – Children’s Hospital, Frankfurt/M,
Germany; 2Department of Pathophysiology/Division of
Immunopathology, Medical University of Vienna –
Center for Physiology and Pathophysiology, Vienna,
Austria
Background: There is now a growing body
of evidence that clinical efficacy of aller-
gen-specific immunotherapy (SIT) is vary-
ing in patients. In addition the efficacy of
SIT is allergen dependent (bee venom
allergy 90%, grass pollen 80%, mite 70%).
The primary question was whether bron-
chial allergen provocation (BAP) was an
appropriate method to assess efficacy of
SIT.
Method: In 42 children (average age:
8.6 years; 22 male and 20 female) with
allergic asthma/rhinoconjunctivitis and
house dust mite allergy we analysed to
which extent a standardized mite allergoid
given subcutaneously led to clinical and
objective improvement. All patients under-
went two BAPs, before SIT and 1 year
after SIT (average 12.9 months). Surpris-
ingly, 14 patients did not start SIT
although recommended, due to different
reasons. These patients represented the
control group. Before SIT total-IgE, spe-
cific IgE-mite and cumulative IgE, and
after SIT additionally specific IgG-mite
and specific IgG4-mite were analysed.
Result: As expected in patients receiving a
SIT, allergen-specific bronchial hyperreac-
tivity (BHR) improved significantly:
PD20FEV1-median before SIT 34.4 AE
(range 5.5–357.9) and after SIT 63.3 AE
(range 2.6–1200); P < 0.01, 1.8-fold
increase), whereas no difference was found
in the control group. Although SIT signifi-
cantly improved allergen-specific BHR, we
could differentiate between responders
(n = 17 – improvement in the BAP) and
non-responders (n = 11 – without improve-
ment in the BAP). The results of the immu-
nological analysis showed that the
responders had significant lower mite spe-
cific IgE after SIT compared to non-
responders; P = 0.001. The specific IgG-
mite and IgG4-mite was not appropriate to
discriminate between responders and non-
responders. Interestingly both groups stated
that the SIT had led to a subjective improve-
ment of symptoms, whereas the control
group without SIT showed no subjective
improvement at all. In contrast only
responders defined by BAP needed less med-
ications than non-responders (P = 0.002).
Conclusion: Our results showed that SIT is
an effective therapy to treat mite allergic
asthma. One year after SIT 60.7% of the
patients improved significantly. However
non-responder can be defined by BAP.
Thus, BAP is a useful objective method to
estimate the effectiveness of SIT and is – in
contrast to the subjective rating – not influ-
enced by the placebo effect.
306
Tolerability of the grass allergen
immunotherapy tablets and patient
satisfaction for intra-seasonal start of
treatment
Wolf, H1; Schwab, J2; Schnitker, J3; Wustenberg, E4
1Clinical Development, ALK-Abello Arzneimittel GmbH,
Wedel, Germany; 2ENT-Physician, Munich, Germany;3Institut fur angewandte Statistik GmbH, Bielefeld,
Germany; 4ALK-Abello Arzneimittel GmbH, Medical &
Regulatory Affairs, Wedel, Germany
Background: For specific immunotherapy
to pollen allergy a pre-seasonal start of
treatment is recommended by international
guidelines. Intra-seasonal start of therapy
with the SQ-standardised grass allergy
immunotherapy tablet (AIT) was well tol-
erated in a randomised placebo-controlled
clinical trial with adult patients. In our
study tolerability in relation to an intra-
seasonal start of grass AIT was recorded
during routine application.
Method: In a non-interventional, non-con-
trolled, non-randomised open-label obser-
vational study with the grass AIT (Phleum
pratense, 75 000 SQ-T/2800 BAU), treat-
ment was planned to be started during the
grass pollen season and the tolerability was
recorded for the first intake in the clinic
and for the subsequent 1–3 months of
treatment. Adverse events (AEs) were
recorded by the patients in a diary for the
first 14 days of treatment.
Result: Data for 662 patients aged 5–78 years
(median 29.0, 75 patients <18 years) were
recorded by 286 physicians in Germany. In
620 patients the initiation of treatment was
intra-seasonal and in 42 patients post-sea-
sonal. Diaries could be evaluated for 77%
of patients. AEs judged to be related to
treatment were recorded in 49.8% of
patients with throat irritation (21.3% of
patients), oral paraesthesia (19.6%), oral itch-
ing (14.0%), itching of the ears (10.0%) and
mouth oedema (6.8%) as the most frequent
reactions. Other AEs occurred in <5.0% of
patients). The overall tolerability was
assessed by 87.2% of patients and 91.4% of
physicians to be very good or good. Compli-
ance was assessed ‡80% by the physician in
88.1% patients, >90% patients were very
satisfied or satisfied with therapy.
Conclusion: The tolerability of an intra-
seasonal start of grass AIT during routine
Poster Discussion Session 7 – Immunotherapy: progress in clinical management
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 133
treatment observed in this study confirms
the results from the randomised placebo-
controlled clinical trial in adults. Intra-
seasonal start of grass AIT was well tolerated,
the tolerability was assessed as good by
patients and physicians and satisfaction
with therapy and compliance was high.
307
Maintenance allergen immunotherapy
prevents increase in FeNO induced by
bronchial allergen challenge in seasonal
allergic rhinitis patients
Swiebocka, E1; Siergiejko, G1; Siergiejko, Z2
1Pediatrics, Gastroenterology and Allergology
Department, University Children Hospital, Bialystok,
Poland; 2Respiratory System Diagnostic and
Bronchoscopy Department, Medical University of
Bialystok, Bialystok, Poland
Background: The aim of this study was to
compare the effects of two protocols of
allergenic immunotherapy (maintenance vs
pre-seasonal) on nitric oxide concentration
in exhaled air (FeNO) determined prior to
and 22 h after bronchial allergen challenge
(BAC) in seasonal allergic rhinitis (SAR)
patients sensitive to grass pollen.
Method: Studies were carried out on 57
patients subjected to 3-year subcutaneous
immunotherapy (SCIT). The patients were
randomly assigned to desensitisation by
pre-seaasonal (n = 28) or maintenance
(n = 29). Bronchial allergen challenge was
performed before initiating SCIT and
yearly thereafter, between November and
January. FeNO was measured prior to
BAC, with follow-up measurements taken
up to 72 h after the challenge.
Result: In the maintenance group, bron-
chial responsiveness to BAC decreased
gradually during subsequent years of
immunotherapy with significant changes
noted after 1 and 3 years of SCIT, while in
pre-seasonal group significant reduction in
the bronchial reactivity was documented
no earlier than after 3 years of immuno-
therapy. After 3 years of of maintenance
SCIT, FeNO determined 22 h after BAC
was significantly lower compared to previ-
ous season, in contrast to pre-seasonal
group where no significant differences in
BAC-induced FeNO levels were observed
through the course of this study.
Conclusion: Maintenance allergenic immu-
notherapy better than pre-seasonal pre-
vents increase in FeNO induced by BAC in
SAR patients.
308
Patient benefits of treatment with 500IR
and 300IR sublingual tablets of house
dust mite allergen extract
Bergmann, K1; Demoly, P2; Roux, M3; Cadic, V3; Furrer,
M3; Zeldin, R3
1Allergy-Centre-Charite, Charite – Universitatsmedizin
Berlin, Berlin, Germany; 2Allergy Division, Pneumology
Department, INSERM U657, Hopital Arnaud de
Villeneuve, University Hospital of Montpellier,
Montpellier, France; 3Stallergenes, Antony, France
Background: The efficacy and safety of
500IR and 300IR sublingual tablets of
house dust mite (HDM) allergen extract
have been demonstrated. Here, we focus
on the patient reported outcome measures:
quality of life (QoL) and patient global
evaluation of treatment efficacy.
Method: Adults, with HDM-induced aller-
gic rhinitis were included in a 2-year, dou-
ble-blind, study. Patients were randomised
to treatment with 500IR or 300IR sublin-
gual tablets of HDM allergen extract or pla-
cebo for 1 year and were followed for the
subsequent treatment-free year. The Rhino-
conjunctivitis Quality of Life Questionnaire
(RQLQ), consisting of 28 items on a 7-point
Likert scale (lower score indicating better
quality of life), was completed at baseline
and at the end of each year. The patient glo-
bal evaluation of treatment efficacy, 5 point
Likert scale (5 = marked improvement to
1 = marked worsening), was completed at
the end of the treatment year, relative to the
previous year. The percentages of patients
reporting treatment success (ratings of
‘Marked improvement’ or ‘Slight to moder-
ate improvement’) and non-success (ratings
of ‘No change’, ‘Slight to moderate worsen-
ing’ or ‘Marked worsening’) were compared
between groups. The overall RQLQ score
and the change from baseline of overall
RQLQ score were analysed using an AN-
COVA model. The patient global evaluation
of treatment efficacy was analysed by using
a Cochran Mantel-Haenszel method.
Result: At the end of the treatment year,
the differences in Least Square (LS) means
vs placebo of the overall RQLQ score in
the 500IR and 300IR groups were )0.15
[95% CI ()0.37, 0.06), P = 0.1666] and
)0.31 [95% CI ()0.53, 0.10), P = 0.0040],
respectively. The treatment effects corre-
spond to relative LS mean differences vs
placebo in the two active treatment groups
of 11% and -23%, respectively. Marked
improvement was reported by 33.1% of
patients in the 500IR group, 36.9% of
patients in the 300IR group, and 18.0%
of those receiving placebo. The proportion
of patients reporting treatment success was
significantly higher in both the active treat-
ment groups than in the placebo group
(P = 0.0206 for the 500IR group and
P = 0.0001 for the 300IR group).
Conclusion: The improvements in the
patient reported outcome measures of
quality of life and global evaluation of
treatment efficacy substantiate the clinical
meaningfulness of the efficacy of house
dust mite sublingual immunotherapy tablets.
309
Non-compliance to specific
immunotherapy: a 28- year retrospective
study
Yesillik, S; Kartal, O; Demirel, F; Baysan, A; Gulec, M;
Musabak, U; Sener, O
Division of Immunology and Allergy, Gulhane Military
Medical School, Ankara, Turkey
Background: Subcutaneous allergen immu-
notherapy (SCIT) alters the natural course
of allergic diseases and has been practiced
since 1911. SCIT is an effective treatment
for allergic rhinitis, asthma and severe
venom allergy. Noncompliance to SCIT is
one of the important reasons of the ther-
apy failure.
Method: A retrospective chart review was
conducted of all the patients who received
SCIT in our clinic between 1983 and 2011.
Patients were analyzed with respect to
causes of SCIT discontinuation.
Result: Of the 1982 patients, 474 (23.9%)
patients discontinued SCIT. Four hundred
and seventy-four patients (286 male;
60.3%, 188 female; 39.7%; mean age
42.25 years) were studied. Mean duration
of cessation of SCIT was 20.2 months. The
main causes of cessation were assignment,
moving, pregnancy and loss of social secu-
rity in 310 (%65.2). The other reasons
were; no response to SCIT in 10 (2.1%),
systemic reactions in 46 (9.7%), recurrent
large local reactions in 7 (1.5%), and sev-
eral diseases (cardiopulmonary, autoim-
mune, psychiatric, neurological,
hemathologic, liver and inflammatory
bowel diseases) occured in 9 (%1.8) during
the course of SCIT. Any reason were not
found in 92 patients (19.4%). Of the 54
patients, four patients (7.4%) discontinued
venom immunotherapy due to assignment-
moving (3) and myocardial infarction (1).
Conclusion: SCIT is a safe and effective
treatment in appropriately selected
patients. The optimum duration of SCIT is
3–5 years. Benefits of therapy will be seen
for about 6–12 months after starting SCIT
and clinical benefits may persist for several
years. In our study, mean cessation time
was 20.2 months after starting, and the
most common causes were assignment and
moving. Pre-treatment evaluation is one of
the important factors for effectiveness of
SCIT. In addition to medical conditions,
patients’ social status should be taken into
account such as occupation, assignment,
and moving.
Poster Discussion Session 7 – Immunotherapy: progress in clinical management
134 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
310
Specific immunotherapy – vaccination or
elimination of allergy?
Muehlmeier, G1; Maier, H2
1Military Hospital of Ulm, Center of Allergic Diseases,
Ulm, Germany; 2ENT, Military Hospital of Ulm, Ulm,
Germany
Background: Before regulatory effects were
described, the main mechanism of specific
immunotherapy (SIT) seemed to be the
Th2/Th1 shift. Increases in IgG4 as an
effect of vaccination and decreases in IgE
antibody counts in the sense of beginning
elimination did not prove significant mark-
ers of success so far. In numerous studies
success rates in SIT against different pollen
species and dust mites rated up to 85%.
However, it still remains unclear, why
other patients fail.
Method: Courses of SIT were surveyed in
150 consecutive patients with allergic rhini-
tis (AR) and/or controlled allergic asthma
(AA). According to EAACI guidelines SIT
was performed perennially in mite allergy
(35%) and in a pre-seasonal regimen in
case of pollen allergy (74%), 15 patients
(10%) received both. Symptom and medi-
cation scores as well as specific IgE (sIgE)
and IgG4 antibodies were evaluated at dif-
ferent points of time before starting SIT,
during and after therapy.
Result: IgE and IgG4 levels showed no con-
cordant distribution, however revealed a
large variety of constellations. Whilst about
32% had significant decreases in sIgE
counts, 69% showed specific IgG4 antibody
level increases. No immune response in
sense of vaccination or elimination could be
detected in 19%. Significant immune
responses correlated well to reductions in
SMS scores. Quality of immune responses
turned out different in pollen and mite
allergy.
Conclusion: Continuous IgE and IgG4
counts combined with the observation of
allergy symptoms lead to early detection of
non-responders to SIT. A consecutive
change of therapeutic approaches should
be discussed. Factors of modification could
be the way of processing of the allergens
(native or allergoid), the route of applica-
tion or supplementation of unspecific
immune stimulant substances.
311
Relationship between disease severity
and post-treatment, long-term efficacy of
300IR 5-grass pollen sublingual tablets
Didier, A1; Malling, H2; Worm, M3; Amistani, Y4; Le Gall,
M4; Zeldin, R4
1Respiratory Diseases Department, Rangueil-Larrey
Hospital, Toulouse, France; 2National University
Hospital, Copenhagen, Denmark; 3Allergie-Centrum-
Charite, Klinik fur Dermatologie, Venerologie und
Allergologie, Universitatsmedizin Berlin, Berlin,
Germany; 4Stallergenes, Antony, France
Background: The sustained efficacy of dis-
continuous treatment with a 300IR sublin-
gual tablet of 5-grass pollen allergen
extract has been demonstrated over three
consecutive pollen seasons. Here, we inves-
tigate the relationship between disease
severity and clinical efficacy over the first
grass pollen season post-treatment.
Method: Adults with grass pollen-induced
allergic rhinoconjunctivitis were rando-
mised to treatment with a 300IR sublingual
tablet or placebo for three pollen seasons.
Treatment was initiated 4 months (4M) or
2 months (2M) before the expected start of
each pollen season and continued for its
duration. Patients were followed during the
subsequent (treatment-free) pollen season.
The primary efficacy criterion was the
Average Adjusted Symptom Score
(AAdSS, range: 0–18), which adjusts the
Rhinoconjunctivitis Total Symptom Score
for rescue medication use, and was analy-
sed by ANCOVA. To evaluate the rela-
tionship between efficacy and disease
severity, pooled centres were ranked from
the lowest to the highest mean AAdSS in
the placebo group and then divided into
three tertiles. The AAdSSs during the pol-
len period of the first season post-treat-
ment were analysed descriptively by tertile.
Result: A total of 633 patients were rando-
mised. Overall, in Year 4, the difference
between the 300IR (4M) and the 300IR
(2M) groups and the placebo group in
AAdSS Least Square (LS) means during
the pollen period was statistically signifi-
cant (LS mean difference -1.14, 95%
CI [-2.03, -0.26], P = 0.0114 in the 4M
group and LS mean difference 1.43, 95%
CI [-2.32, -0.53], P = 0.0019 in the 2M
group). The treatment effect corresponds
to a relative LS Mean difference from pla-
cebo of -22.9% and -28.5%, respectively.
The relative mean differences vs placebo in
the high, medium and low severity tertiles
were -34.0%, 28.3% and +14.3%, respec-
tively for the 300IR (4M) patients, and
38.3%, 24.6% and -6.3%, respectively for
the 300IR (2M) patients.
Conclusion: During the first treatment-free
grass pollen season after 3 years of pre-
and co-seasonal treatment, the 300IR sub-
lingual tablet of 5-grass pollen allergen
extract showed greater efficacy at centres
with higher symptom severity in the pla-
cebo group.
312
Clinical efficacy and risk factors
associated with adverse reactions in
children with asthma or allergic rhinitis
receiving subcutaneous allergen
immunotherapy
Yilmaz, O1; Toprak Kanik, E1; Yangin, E2; Turkeli, A1;
Yuksel, H1
1Pediatric Allergy and Pulmonology, Celal Bayar
University Medical Faculty, Manisa, Turkey; 2Pediatrics,
Celal Bayar University Medical Faculty, Manisa, Turkey
Background: Subcutaneous allergen immu-
notherapy (SIT) improves clinical outcome
in children with allergic rhinitis (AR) or
asthma, however, it is associated with sys-
temic and local adverse reactions. The aim
of this study was to assess the clinical effi-
cacy and risk factors associated with local
or systemic reactions in children with AR
or asthma receiving SIT.
Method: This is a retrospective cohort
study. All children with asthma or allergic
rhinitis who received SIT in our Depart-
ment of Pediatric Allergy were enrolled.
This preliminary analysis describes the
results of the first 248 cases that had been
reviewed up to date. Thirty cases were
excluded because they were lost to follow
up before 3 years of SIT was completed.
Results of skin prick tests, ages at initia-
tion of SIT, diagnosis, presence of addi-
tional allergic manifestations such as
urticaria, atopic dermatitis or food allergy
as well as parameters indicating severity of
AR and asthma during the first and third
years of SIT were recorded. Moreover,
presence and severity of local and systemic
reactions were also reviewed.
Result: Mean age of the children enrolled
in the study (n = 218) was 10.9 ± 3.3 and
63.8% were males. Among all children
29.8% had only asthma, 42.7% had AR
and 27.5% had both asthma and AR. Skin
prick tests have revealed that 39.9% of the
children were sensitized to more than one
group of allergens while only grass pollen
and dermatophagoid sensitization was the
most common results following this with
37.2% and 15.6% respectively. Systemic or
local adverse reaction was detected in 4%
and 8.3% of the children respectively.
Number of asthma exacerbations, days of
hospitalization and number of emergency
visits had decreased significantly during the
third year of SIT compared to the first
year after initiation of SIT (P £ 0.001 for
all). Frequency of systemic or local reac-
tions did not change significantly between
children with AR and asthma (P > 0.05).
Similarly, IgE levels or type of allergen
Poster Discussion Session 7 – Immunotherapy: progress in clinical management
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 135
sensitized were not significantly different
between children with and without and
adverse reaction during the course of IT.
Conclusion: The preliminary results of this
retrospective cohort study has shown that
SIT is associated with significant improve-
ment in clinical findings of asthma in chil-
dren. The frequency of systemic reactions
is 4%. Neither the diagnosis nor the type
of allergen sensitized is a risk factor for
development of adverse reactions during
the course of SIT.
313
Optimised allergen:adjuvant formulation
potentiates the immunogenicity and
reduces allergenicity of a novel
subcutaneous immunotherapy product
for treatment of grass pollen allergy
Rask, C1; Lund, L2; Lund, G1; Heydenreich, B3;
Wurtzen, P1; Bellinghausen, I3; Saloga, J3; Lund, K4
1Pharmacology, ALK-Abello A/S, Hoersholm,
Denmark; 2ALK-Abello, CMC Research, Hoersholm,
Denmark; 3Universitatsmedizin der Johannes
Gutenberg-Universitat, Hautklinik und Poliklinik, Mainz,
Germany; 4ALK-Abello A/S, Global Innovation
Management, Hoersholm, Denmark
Background: Subcutaneous immunother-
apy (SCIT) has proven sustained clinical
efficacy against grass-pollen induced
allergy. Gradual updosing over a period of
weeks is the recommended regimen for
SCIT. Commonly, in commercial products
for SCIT, the allergen extract is formulated
with an adjuvant, most often in the form
of aluminium hydroxide (AlOH). The
mechanism behind the therapeutic effect of
SIT is not completely known. However, it
has been shown that allergen-specific IgG
antibodies are induced by SCIT. Here we
investigate the possibility of optimising the
formulation of AlOH-based grass-pollen
allergy vaccines for SCIT in a way that
allows for shorter updosing regimens for
better compliance while maintaining the
immunogenicity of the vaccine.
Method: Mice were immunised with vari-
ous concentrations of Phleum pratense (Phl
p) allergen extract and AlOH or with SCIT
products that were differently formulated
with regard to allergen and adjuvant con-
tents. In vivo kinetics of Phl p specific IgG
antibody responses and T cell responses
were monitored. Allergenicity, measured as
the ability of the formulations to activate
human basophils, was also determined.
Additionally, human T cell responses and
the expression of dendritic cell surface
markers after vaccine challenge in vitro
were analyzed.
Result: Phl p-specific IgG antibody
responses were shown to depend on the
AlOH-concentration, but not on the aller-
gen concentrations within the dilution
range tested. There were no differences in
Phl p-specific IgG1 antibody or T cell
responses between the groups immunised
with either 20 000SQ Phl p + 0.228 mg
AlOH or the new formulation with
3000SQ Phl p + 0.114 mg AlOH. In con-
trast, the allergenicity of the new formula-
tion was 5–20 fold reduced as compared to
the 20 000SQ Phl p + 0.228 mg AlOH
formulation. In addition immunisation
with 3000 SQ Phl p extract + 0.114 mg
gave rise to significantly higher antibody
and T cell responses as compared to the
group immunised with 3000SQ Phl p
extract + 0.034 mg AlOH.
Conclusion: The optimisation of the formu-
lation between allergen and adjuvant
allows for administration of a lower dose
of allergen while maintaining the immuno-
genicity of the product and at the same
time reducing allergenicity. These non-clin-
ical data has led into clinical evaluations of
a SCIT product with an optimised aller-
gen:adjuvant ratio with the aim to investi-
gate if the formulation benefits the safety/
efficacy profile and allow for shorter up-
dosing.
314
An evidence-based consideration of
treatment of multiple-allergen and
single-allergen immunotherapy in
polysensitised patients
Calderon, M1; Cox, L2; Casale, T3; Moingeon, P4;
Demoly, P5; Lheritier-Barrand, M4
1Section of Allergy and Clinical Immunology, Imperial
College-NHLI, Royal Brompton Hospital, London,
United Kingdom; 2Nova Southeastern University
School of Osteopathic Medicine, Davie, United States;3Division of Allergy and Immunology, Department of
Medicine, Creighton University – Allergy & Asthma,
Omaha, United States; 4Stallergenes, Antony, France;5Allergy Division, Pneumology Department, INSERM
U657, Hopital Arnaud de Villeneuve, University Hospital
of Montpellier, Montpellier, France
Background: There is debate as to whether
polysensitised patients are best treated with
multiple-allergen immunotherapy or a sin-
gle-allergen immunotherapy with the most
clinically bothersome allergens. The former
approach predominates in North America,
whereas the latter is more popular in
Europe.
Method: We considered the literature on
the treatment of polysensitised and mono-
sensitised patients with single and multiple
allergens.
Result: Polysensitisation is more prevalent
(range: 50%-80%) than monosensitisation
in patients seeking treatment for moderate-
to-severe respiratory allergies; this is the
case in both Europe and North America.
Inevitably, single-allergen preparations will
almost certainly have been clinically tested
in polysensitised patients in Europe
and, more recently, in the USA in robust,
large-scale clinical trials of grass pollen
subcutaneous allergen immunotherapy
(SCIT) and sublingual allergen immuno-
therapy (SLIT). The efficacy results of two
such single-allergen trials showed that
polysensitised subjects benefited at least as
much as monosensitised patients did. The
safety profile did not appear to differ
according to the patient’s sensitisation sta-
tus. A recent comparative trial in the USA
of single-allergen (timothy) SLIT and
multi-allergen SLIT in mainly polysensi-
tised patients concluded that the co-admin-
istration of multiple allergens may have
interfered with the effectiveness of the sin-
gle-allergen extract. Findings from conven-
tional vaccine research (antigen
competition) suggest that higher allergen
doses may be required in multi-allergen
SCIT. Other clinical studies have suggested
that multi-allergen immunotherapy is less
effective in polysensitised patients than sin-
gle-allergen allergen immunotherapy is in
monosensitised patients.
Conclusion: Our evidenced-based review of
robust clinical trials found that single-aller-
gen grass pollen extracts) show clear efficacy
and safety in polysensitised participants.
Multi-allergen immunotherapy (whether
SLIT or SCIT) in polysensitised patients
requires further supporting data in large
clinical trials. It also remains to be seen
whether single- and multi-allergen immuno-
therapy protocols elicit different immune
responses in monosensitized and (in
comparison) polysensitised patients.
315
Subcutaneous immunotherapy and
factors affecting patient compliance
Cigerci Gunaydin, N; Demir, E; Gulen, F; Tanac, R
Department of Allergy and Immunology, Ege University
Faculty of Medicine, Izmir, Turkey
Background: Allergen immunotherapy (IT)
is a long term treatment modality. As well
as the need to appropriate dose and regu-
lar intervals, the compliance of patient is
also important for successful treatment.
During the long duration of therapy
(5 years), almost half of patients can not
continue the treatment.
Method: The purpose of this study is to
evalute the compliance of patients and the
factors affecting it.
Results: Two hundred-thirtythree patients
(91 female, 142 male) were included into
the study. Patients having subcutaneous
immunotherapy shorter than 6 months
were excluded from the study. The mean
age of the patients at the beginning of ther-
apy was 10.9 (5–18.8) years. Allergic rhini-
tis was the most common indication for IT
(72.5%). Nine percent of the patients left
IT during the course of treatment, and
43.2% of them couldn’t follow the regular
Poster Discussion Session 7 – Immunotherapy: progress in clinical management
136 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
intervals, mostly due to delay in vaccine
supply, respiratory system infections and
lack of interest. Common causes of drop-
ping out the treatment were allergic reac-
tions (54.8%), lack of interest and
unresponsiveness to IT. Most of the com-
plications were local reactions (79.8%);
anaphylaxis was rare (3.4%). IT was suc-
cesful with a rate of over 75% in 132
patients. There was no statistical correla-
tion between the type and frequency of
reactions seen and the kind of allergen,
therapy center, sex, additional therapy
need, compliance and the manufacturer.
Also there were no relation between the
causes of incompliance and the allergens,
diagnosis, therapy center and the rate of
success. The ratio of incompliance was
negatively correlated with the rate of suc-
cess (P = 0.00). No relation were observed
between the dropping out the treatment
and the type of allergen, place of living,
place of IT performed, incompliance, addi-
tional therapy, gender and the manufac-
turer. Dropping out the therapy was seen
more frequently in incompliant patients
(P = 0.00) and patients with allergic
rhinitis (P = 0.02), whereas it was lower in
asthmatic patients (P = 0.03).
Conclusion: Taking into consideration that
there is no correlation between the adverse
reactions of IT and incompliance with the
therapy, and knowing that most common
reasons for dropping out the therapy were
irregular visits and belief of patient that he
could not gain benefit from treatment,
comprimises the importance of regulary
follow-up in IT.
Poster Discussion Session 7 – Immunotherapy: progress in clinical management
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 137
Poster Discussion Session 8
Cutaneous allergy
316
Hereditary angioedema due to
C1-inhibitor deficiency: early vs delayed
treatment with icatibant
Bork, K1; Hardt, J2
1Department of Dermatology, Johannes Gutenberg
University Mainz, Mainz, Germany; 2Department of
Medical Psychology and Medical Sociology, Johannes
Gutenberg University Mainz, Mainz, Germany
Background: To compare the efficacy of i-
catibant in early vs late treated attacks of
hereditary angioedema.
Method: Thirty five patients received
30 mg icatibant subcutaneously at various
times for 149 swelling attacks. The time
periods between onset of attacks and icati-
bant injection, icatibant injection and the
first symptom relief, and icatibant injection
and resolution of symptoms or, in some
attacks, the start of a rebound attack were
compared in three patient groups with dif-
ferent times to injection.
Result: Data are reported as mean ± SD.
In 74 attacks treated at two or less hours
after attack onset, the time to first relief
was 0.9 ± 0.9 h and the time to symptom
resolution was 11.6 ± 11.2 h. In 52 attacks
treated at more than 2–5 h, the time to
first relief was 0.7 ± 1.1 h and the time to
resolution was 17.5 ± 15.6 h. In 23 attacks
treated later than 5 h after attack onset,
time to first relief was 1.0 ± 1.1 h and
time to resolution was 17.5 ± 16.9 h. The
percentages of attacks with first symptom
relief within 30 min in the three groups
were 64.9%, 70.2%, and 60.9%, respec-
tively. There were no statistically signifi-
cant differences between the groups. In a
subgroup of 23 attacks treated within 1 h
after attack onset, the time to first relief
was 0.7 ± 0.6 h and the time to symptom
resolution was 9.3 ± 11.4 h. The only
adverse events were injection site reactions
that all resolved without intervention.
Conclusion: Icatibant is equally effective in
early and delayed treatment of acute HAE
attacks, with an early onset of relief.
317
Rebound or relapse rates in hereditary
angioedema attacks treated with
ecallantide in an open-label,
repeat-treatment study
Soteres, D1; MacGinnitie, A2; Craig, T3; Bernstein, J4;
Lumry, W5; Li, H6; Riedl, M7; Iarrobino, R8; Chyung, Y8;
Shea, E8; Pullman, W8
1Asthma & Allergy Associates, P.C., Colorado Springs,
CO, United States; 2Children’s Hospital Boston, Boston,
MA, United States; 3Penn State University, Hershey,
PA, United States; 4University of Cincinnati, Cincinnati,
OH, United States; 5Allergy & Asthma Specialists,
Dallas, TX, United States; 6Institute for Asthma and
Allergy, Wheaton, MD, United States; 7UCLA–David
Geffen School of Medicine, Los Angeles, CA, United
States; 8Dyax Corp., Burlington, MA, United States
Background: Hereditary angioedema
(HAE) is a rare autosomal dominant disor-
der resulting in recurring, unpredictable,
and variable attacks of swelling. Ecallan-
tide is a potent, selective, and reversible
inhibitor of plasma kallikrein that is
approved for treatment of HAE attacks
affecting any anatomic site. Because eca-
llantide has a 2 ¡A0.5 h half-life, we exam-
ined the potential for attack rebound or
relapse following ecallantide treatment.
Method: DX-88/19 (Continuation) was an
open-label, repeat-treatment study assess-
ing efficacy and safety of ecallantide for
the treatment of acute attacks of HAE
(patients were treated for ¡Y1 attack). Dose
B was allowed between 4 and 24 h;
patients who received a Dose B were
excluded from this analysis. Efficacy assess-
ments included Mean Symptom Complex
Severity (MSCS) score, Treatment Out-
come Score (TOS), and global response
assessment. Treatment episodes showing
improvement in all three assessments at
4 h after dosing, followed by worsening in
any assessment at 24 h, were identified as
potential cases for further characterization.
Rebound was defined as worsening beyond
baseline severity, whereas relapse was
worsening that was not beyond baseline.
All potential rebound or relapse cases were
characterized as likely, possible, or unlikely
based on the number of assessments show-
ing worsening and the quantitative magni-
tude of change in each assessment.
Result: A total of 147 patients were trea-
ted. Out of 587 treated attacks with avail-
able data, 456 (77.7%) had improvement
in all three assessments at 4 h. Of these
456 attacks, 58 (12.7%) were identified as
potential cases for further characterization.
Potential rebound was seen in 3 (0.7%)
treated attacks; all of which were consid-
ered likely. Potential relapse was seen in 55
treated attacks: 9 (2.0%) were considered
likely relapse, 11 (2.4%) possible, and 35
(7.7%) unlikely. Among the potential
rebound/relapse cases, three required medi-
cal intervention. The 58 cases of potential
rebound/relapse occurred in 44 patients: 36
experienced potential rebound/relapse in
one attack; seven experienced rebound/
relapse in two attacks; and one patient
experienced it in >2 attacks.
Conclusion: Rates of rebound and relapse
were low among HAE attacks treated with
ecallantide.
318
Angiotensin-converting enzyme
inhibitor-induced angioedema attacks
triggered by dental care procedures
Popescu, S1; Popescu, F2
1Department of Oral Rehabilitation and Medical and
Surgical Emergencies, University of Medicine and
Pharmacy Craiova, Craiova, Romania; 2Allergology
Department, University of Medicine and Pharmacy
‘Carol Davila’, Bucharest, Romania
Background: Life-threatening and even fatal
bradykinin-induced angioedema (AE) attacks
due to C1-inhibitor esterase deficiency may
be triggered by oral trauma, including den-
tal surgery or tooth extraction. Similarly,
direct facial or intraoral trauma may also be
a trigger factor for angiotensin-converting
enzyme inhibitor (ACEI)-related AE. Acute
trauma causes activation of bradykinin
through contact system activation.
Methods: From adult hypertensive patients
consecutively presented in a 24-month
period to a dentistry clinic in Craiova, we
Table 1.
HAE attacks treated with
ecallantide that showed
improvement at 4 h in TOS,
MSCS, and global response N = 456 (%)
Total potential rebound 3 (0.7)
Likely 3 (0.7)
Possible 0
Unlikely 0
Total potential relapse 55 (12.1)
Likely 9 (2.0)
Possible 11 (2.4)
Unlikely 35 (7.7)
138 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
selected those treated with renin-angiotensin
system (RAS) inhibitors, ACEI or angioten-
sin receptor blockers, needing dental care
procedures provoking oral trauma, includ-
ing tooth extraction, scaling and root plan-
ning, surgical debridement procedures,
dental caries treatment, endodontic treat-
ment, teeth preparations and dental impres-
sions for prosthetic appliances like crowns,
bridges, partial or complete dentures.
Because an unmasking of C1-esterase inhib-
itor (C1-INH) deficiency is possible, we
determined serum C1-INH and C4 values
by immunonephelometry in patients with AE.
Results: From 138 hypertensive patients,
from which 69.56% had controlled blood
pressure with pharmacotherapeutic regi-
mens including ACEI, we detected two
patients in treatment with ACEI presenting
episodes of perioral AE and unilateral ton-
gue AE, several hours after dental impres-
sions for prosthetic appliances. ACEI
involved were benazepril and perindopril,
used as antihypertensive drugs in these
patients for 3, and 8 years respectively. In
both patients complete resolution of symp-
toms was obtained in maximum 24 h, with-
out bradykinin B2 receptor antagonist
icatibant use. Non-steroidal anti-inflamma-
tory drugs were not found to potentiate
AEattack. C4 and C1-INH determination
results were within the normal range.
Conclusion: General dentists, endodontists,
oral surgeons, periodontists, prosthodon-
tists, and allergists need to be familiar with
this rare, potentially fatal and unpredictable
adverse drug reaction triggered by dental
care procedures in patients treated with
ACEI, because early recognition and proper
management of AE attack are important,
along with instructions for patients to
replace the RAS inhibitor pharmacotherapy
with other cardiovascular drugs.
319
The influence of mometasone furoate
0.1% cream vs an emollient cream on
cytokine gene expression in the skin and
peripheral blood
Filimonova, T; Elisutina, O; Fedenko, E; Boldureva, M
Institute of Immunology, Moscow, Russian Federation
Background: To investigate the influence of
mometasone furoate 0.1% cream on cyto-
kine gene expression in the skin and
peripheral blood of atopic dermatitis (AD)
patients comparing with control.
Material and methods: Forty AD patients
were included in the study and divided into
two groups. Group 1 patients were given
continuous course of mometasone furoate
0.1% cream treatment for 14 days. Group 2
patients were given indifferent emollient elo-
base cream for 14 days. Clinical efficacy of
the treatment was assess on the following
features: SCORAD index, Investigators’
Global Assessment (IGA) score and subjec-
tive assessment of itch and dryness of the
skin according to skin area to be studied.
Skin samples and peripheral blood of atopic
dermatitis were used as material for immu-
nological study. Interleukin (IL)1B, IL2,
IL2r, IL4, IL5, IL6, IL7, IL8, IL10, IL 12A,
IL12B, IL15 (total), IL15, IL17A, IL18,
IL23, IL28, IL29, Interferon (IFN)a, Tumor
necrosis factor (TNF), Transforming
growth factor beta 1 (TGFB1), forkhead
box P3 (FOXP3) gene expression were
defined in the skin and peripheral blood of
AD patients by real-time reverse transcrip-
tion polymerase chain reaction (RT-PCR).
Cytokine gene expression were defined
twice: before and after topical treatment.
Results: Positive clinical effect was found
with all AD patients on the mometasone
furoate 0.1% cream therapy background
for 14 days and also dryness, rushes and
skin itch decreased. Statistical significant
decrease of proinflammatory cytokines
IL2, IL2r, IL5, IL8, IL12A, IL23, IFNa
gene expression was marked. They are the
markers of chronic inflammation and Th1
immune response. Studying peripheral
blood after mometasone furoate 0.1%
cream treatment increase of TGFB1,
FOXP3 gene expression level was found.
There were no significance changes of cyto-
kine gene expression in AD patients, who
got elobase cream was found.
Conclusion: Antiinflammatory activity of
mometasone furoate 0.1% cream was
shown by its influence on proinflammatory
cytokines IL2, IL2r, IL5, IL8, IL12A,
IL23, IFNa gene expression in the skin
and mechanisms of immune response in
moderate and severe AD patients.
320
Association of IL-4, IL-10 and TGF-beta
cytokine gene polymorphisms in children
with atopic dermatitis
Stavric, K
University Children’s Hospital, Department of immunol-
ogy, Skopje, Macedonia, Fyrom
Background: The balance between Tr1 and
IL4 are crucial in the development of
allergy. The aim of this study was to exam-
ine the association of IL-4, IL-10 and TGF
beta cytokine gene polymorphism in the
Macedonian population with atopic der-
matitis (AD).
Method: Single nucleotide polymorphisms
in IL-4, IL-4R, IL-10 and TGF beta genes
were investigated by PCR and sequence
specific primers in 67 children with AD and
301 healthy controls. Result Susceptible
odds ratio was found for IL-4 -33/T
(P < 0.001, OR = 6.438, 95% CI 3.602–
11.509), IL-4 -1098/T (P < 0.001, OR
= 5.22, 95% CI 1.85–14.72) and TGFbeta1
cdn25/C allele (P < 0.001, OR = 4.94,
95% CI 2.81–8.67). IL-4-33 T allele is pres-
ent in 56% and IL-4-1098 T allele in 87% of
children with AD. Protective association for
AD was found for the following alleles: IL-4
-33/C (P < 0.001, OR = 0.15, 95% CI
0.09–0.28), IL-4 -1098/G (P < 0.001,
OR = 0.19, 95% CI 0.07–0.54) and TGF
beta1 cdn25/G (P < 0.001, OR = 0.2,
95% CI 0.11–0.36). IL-4-33/C allele is pres-
ent in 84% while TGF beta1 cdn25 G allele
in 97% in healthy population. Susceptible
association was found between patients with
atopic dermatitis and following genotypes:
IL-4 -33/T:T (P < 0.001, OR = 18.17,
95% CI 7.33–55.05), IL-4 -1098/T:T
(P < 0.001, OR = 5.518, 95% CI 2.16–
14.1), TGFbeta1 cdn25/C:G (P < 0.001,
OR = 5.65, 95% CI 3–10.65) and IL-10 -
1082/A:G (P < 0.039, OR = 2.05, 95% CI
1.02–4.11). IL-4-33 T:T genotype is present
in 52%, while IL-4-1098 T:T in 78% of chil-
dren with AD. Protective association
between AD and following genotypes was
found: IL-4 -1098/G:T (P < 0.001, OR =
0.15, 95% CI 0.05–0.4), IL-4 -33/C:C
(P < 0.001, OR = 0.25, 95% CI 0.11–
0.57), TGFbeta1 cdn25/G:G (P < 0.001,
OR = 0.17, 95% CI 0.09–0.31), and IL-10 -
1082/A:A. (P < 0.043, OR = 0.45, 95%
CI 0.21–0.99). TGF beta1 cdn25 G:G geno-
type is present in 89%, G:T of IL-4-1098 in
60% and C:C genotype of IL-4-33 in 73%
of control group. Genotype IL-10 -819/T:T
was present only in normal Macedonian
population, while only patients with AD
had TGFbeta1 cdn25/C:C genotypes.
Conclusion: IL-4-33, -1098, TGF beta1
cdn25 and IL-10-1082 polymorphism were
associated with AD in Macedonian popu-
lation. The frequency of alleles and geno-
types of IL-4 are increased in patients with
AD, while they are present in small fre-
quency in control group. Polymorphisms
with protective association are present in
significantly higher frequency in healthy
population. The genetic backgrounds can
explain the low prevalence of AD in Mace-
donian population.
321
The temporal and spatial dynamics of
Treg cell-mediated suppression during
contact hypersensitivity responses in a
murine model
Fyhrquist, N1; Lehtimaki, S1; Savinko, T1; Lahl, K2;
Sparwasser, T3; Wolff, H1; Lauerma, A4; Alenius, H1
1Finnish Institute of Occupational Health, Helsinki,
Finland; 2Stanford University School of Medicine,
Stanford, CA, United States; 3Centre for Experimental
and Clinical Infection Research, Hannover, Germany;4University of Helsinki, Helsinki, Finland
Background: Regulatory T (Treg) cells
suppress contact hypersensitivity (CHS)
Poster Discussion Session 8 – Cutaneous allergy
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 139
responses, but the dynamics, mode and site
of their action is not well characterised. We
studied Foxp3+ Treg cells during the CHS
response in conditional Foxp3 knock-out
DEREG mice, where Foxp3+ cells can be
transiently deleted by diphtheria toxin.
Method: The mice were sensitised and
challenged by oxazolone, and Foxp3+
cells were depleted by diphteria toxin treat-
ment either during sensitisation or elicita-
tion. Inflammatory parameters including
ear swelling, the expression of cytokines
and chemokines, and the recruitment of
inflammatory cells, were analysed at differ-
ent time points after challenge.
Result: Treg cell depletion prior to sensiti-
sation led to significantly exacerbated and
prolonged CHS responses. In contrast,
depleting Treg cells during elicitation had
no effect on the 24 h response, but the
response was significantly prolonged. In
WT mice, the gradual resolution of the
CHS response was accompanied by a simi-
larly gradual, accumulation of Foxp3+
Treg cells in the skin. The recruitment of
Treg cells to the skin was impaired in
the Treg depleted mice, although Treg cell
numbers in the LNs had been restored,
suggesting that skin-seeking Treg cells are
implicated in the resolution phase.
Conclusion: Together, our results demon-
strate that endogenous Foxp3+ Treg cell
function is important in the LNs during
the sensitisation phase and in the skin dur-
ing the resolution phase, but seem redun-
dant during the first 24 h after challenge.
322
Establishing normal values for newborn
transepidermal water loss using the Cork
BASELINE birth cohort study
Kelleher, M1; O’ Carroll, M1; Murray, D1; Irvine, A2;
Hourihane, J1; BASELINE, T1
1Department of Paediatrics and Child Health, University
College Cork, Cork, Ireland; 2Clinical Medicine, Trinity
College Dublin, Dublin, Ireland
Background: Trans-Epidermal Water Loss
(TEWL) is a simple non-invasive measure-
ment of inside-out skin barrier function.
TEWL increases when skin barrier is dam-
aged. Skin barrier dysfunction is implicated
in many dermatological disorders including
Atopic Dermatitis (AD). We, and others,
have shown how increased changes in
TEWL in infancy predate and predict devel-
opment of AD. We report on the results of
1636 infants who have had TEWL measured
in the first 96 h of life and establish normal
values for Newborn TEWL.
Method: Infants recruited into the Cork
BASELINE Birth Cohort Study between
July 2009 and October 2011 had a TEWL
measurement within the first 96 h of life.
This was carried out in an environmentally
controlled room in the maternity hospital.
Measurements were taken on the volar sur-
face of the forearm after skin had been
exposed and acclimatised. TEWL was mea-
sured using an open chamber device. Three
measurements were taken and the average
recorded. Parent’s completed a comprehen-
sive questionnaire. It was also recorded
whether infants had been washed prior to
TEWL measurement.
Result: One thousand six hundred and
thirty-six neonates had TEWL measure-
ment within 96 h of birth. The mean
TEWL reading was 7.38 ± 3.64 g water/
m2/h. The mean age of reading was
44.57 ± 20.87 h. There was no correlation
between TEWL measurement and gesta-
tional or post natal age at measurement.
Seven hundred and ninety-five neonates
had been washed prior to measurement
(48.6%) with no significant difference in
readings between those who had or had
not been washed, nor what they were
washed with (water only/soap/emollient).
Forty-six percent of mothers and 34% of
fathers reported atopic symptoms (asthma/
eczema/rhinitis/food allergy), none of
which affected neonatal TEWL readings.
Even when analysing values based on spe-
cific Maternal and Paternal history of Ato-
pic Dermatitis, there was no significant
difference between infants whose mother
or father reported a history of AD and
infants whose parents did not.
Conclusion: We have produced the largest
cohort of newborn TEWL values to date.
This data has given us a normal value of
TEWL for use in this and other studies.
323
Skin resident cells can be induced to
produce the endogenous inflammation
limiting molecule IL-18 binding protein
Doble, R1; Werfel, T2; Bachmann, M3; Muehl, H3;
Wittmann, M1
1Faculty of Biological Sciences, Institute of Molecular
and Cellular Biology, University of Leeds, Leeds, United
Kingdom; 2Devision of Immunodermatology and
Allergy Research, Department of Dermatology,
Hannover Medical School, Hannover, Germany;3University Hospital Goethe-University Frankfurt,
Pharmazentrum Frankfurt/ZAFES, Frankfurt, Germany
Background: IL-18 is an important media-
tor involved in chronic inflammatory con-
ditions such as chronic eczema. An
imbalance between IL-18 and its endoge-
nous antagonist IL-18 binding protein (BP)
may account for increased IL-18 activity.
IL-27 is a cytokine with dual function dis-
playing pro- and anti-inflammatory proper-
ties. Here we provide evidence for a yet
not described anti-inflammatory mode of
action on skin resident cells.
Method: We investigated the role of IL-27
on the expression and production of
IL-18BP by fibroblasts and keratinocytes.
Real time polymerase chain reaction (RT-
PCR) was used to investigate mRNA
expression and enzyme-linked immunosor-
bent (ELISA) was used to investigate pro-
tein production. Western blotting and
luciferase assays were used to determine
levels and phosphorylation of signalling
molecules.
Result: Human keratinocytes and surpris-
ingly also fibroblasts (which do not pro-
duce any IL-18) show a robust, dose-
dependent and highly inducible mRNA
expression and secretion of IL-18BP upon
IL-27 stimulation. Other IL-12 family
members failed to induce IL-18BP. The
production of IL-18BP peaked between 48
and 72 h after stimulation and was sus-
tained for up to 96 h. Investigation of the
signalling pathway showed that IL-27 acti-
vates STAT1 in human keratinocytes and
that a proximal GAS site at the IL-18BP
promoter is of importance for the func-
tional activity of IL-27.
Conclusion: The data support a significant
anti-inflammatory effect of IL-27 on skin
resident cells. An important novel property
of IL-27 in skin pathobiology may be to
counter-regulate IL-18 activities by acting
on keratinocytes and importantly also on
dermal fibroblasts. Thus, the induced
expression of endogenous IL-18BP by tis-
sue resident cells may prove an interesting
treatment strategy for chronic inflamma-
tory skin diseases.
324
Effects of Lactobacillus plantarum
CJLP133 on pediatric atopic dermatitis:
a double-blind, placebo-controlled trial
Kim, J1; Han, Y1; Kim, B2; Ban, J1; Lee, J1; Kim, B3;
Choi, B4; Kim, H5; Lee, H6; Ahn, K1
1Department of Pediatrics, Samsung Medical Center,
Seoul, Korea; 2CJ CheilJedang Corporation, CJ Foods
R & D, Seoul, Korea; 3Department of Dermatology,
Chung-Ang University College of Medicine, Seoul,
Korea; 4Department of Preventive Medicine, Chung-
Ang University College of Medicine, Seoul, Korea;5Department of Pediatrics, College of Medicine, Pusan
National University, Pusan, Korea; 6Department of
Pediatrics, Cheil General Hospital, Seoul, Korea
Background: There have been considerable
disagreements regarding the therapeutic
effects of probiotics in atopic dermatitis
(AD). We performed this study to examine
whether the oral administration of Lacto-
bacillus plantarum CJLP133 improved clini-
cal and immunological parameters in
children with mild to moderate AD.
Method: In a randomized, double-blind,
placebo-controlled study, children aged
12 months to 13 years presenting AD with a
SCORing of Atopic Dermatitis (SCORAD)
index between 20 and 50. At the end of the
initial 2-week placebo washout period, a
dosage of 0.5 · 1010 colony forming units of
Poster Discussion Session 8 – Cutaneous allergy
140 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
L. plantarum CJLP133 or placebo was given
twice a day for 12 weeks. Changes in SCO-
RAD indexes, the use of topical corticoste-
roid (TCS), eosinophil count, total serum
IgE, cytokines, and microbial colonization
of the gut were evaluated.
Result: Eighty-three patients (44 in probi-
otic group, 39 in placebo group) completed
this study. The SCORAD index at week 14
was significantly lower in the probiotic
group than in the placebo group
(P = 0.022). The mean change in the
SCORAD index from baseline at week 14
was 9.1 in the probiotic group, which was
greater than the mean change of 1.2 in the
placebo group (P = 0.002). No statistical
differences in the total use of TCS were
found between two groups (P = 0.876). In
the probiotic group, the total eosinophil
count was significantly lower at the end of
the intervention compared to the baseline
measurements (P = 0.006). Logarithmic
IFN-c had significantly decreased by the
end of the intervention compared to the
baseline in the probiotic and placebo
groups (P = 0.001 and 0.042). From the
real time PCR data, L. plantarum was not
detected in 10 fecal DNA samples
extracted from the placebo group but was
detected with having tiny amounts (the
median score was 0.0001%) at week 2 in
the probiotic group. L. plantarum popula-
tion increased at week 8 and 14, compared
to those at week 2 (P = 0.043 and 0.007,
respectively). Whereas the L. plantarum
population decreased at week 16 compared
to those at week 14 (P = 0.037).
Conclusion: Supplementation of probiotic
L. plantarum CJLP133 was associated with
an improvement in the clinical severity of
eczema and a decrease of eosinophil in
pediatric patients with AD suggesting a
beneficial role of this microorganism in the
management of AD.
325
The time window of intervention with a
L. lactis differently regulates the tissue
specific response to allergen challenge in
mice
Holvoet, S1; Doucet-Ladeveze, R2; Moennoz, D2; Joubin,
A2; Nutten, S1; Blanchard, C1
1Nutrition and Health, Centre de Recherche Nestle,
Lausanne, Switzerland; 2Centre de Recherche Nestle,
Lausanne, Switzerland
Background: Probiotics have been sug-
gested to play an important role in control-
ling the homeostasis of the immune system
in the context of the hygiene hypothesis.
Conflicting results regarding the benefit of
probiotics in the context of allergy have
been observed suggesting that only specific
stains at specific doses may be effective.
Aim: We aimed at identifying in different
tissues, the beneficial effects of diverse
timings of oral administration of the
Lactococcus lactis NCC2287 strain in a
skin and airway allergic inflammation
mouse model.
Method: We used a murine model of ato-
pic dermatitis induced by two epicutaneous
sensitizations with Aspergillus (ASP) fol-
lowed by an intranasal allergen challenge.
Transepidermal water loss and skin score
were measured. Lung, esophageal and skin
eosinophilia was assessed as well as skin
thickness. L. lactis NCC2287 was added to
the drinking water of the mice during the
first epicutaneous sensitization (preven-
tion), the second skin exposure (manage-
ment) or all along the experiments.
Result: No difference in the total cell counts
in the bronchoalveolar lavage (BAL) was
observed across the different groups. Eosin-
ophils were absent in the BAL of saline
(SAL)-sensitized/ASP-challenged animals
but were significantly increased in ASP-sen-
sitized/ASP-challenged animals. Significant
decreases of the eosinophil counts were
observed in mice treated with the L. lactis
strain during management phase. The skin
scores and TEWL in the positive control
group (ASP/ASP) were increased compared
to the negative control group (SAL/ASP)
after the second epicutaneous sensitization.
A decrease of the skin score was only
observed after the second skin sensitization
with the L. lactis strain used for ‘preven-
tion’. No change in the IgE levels was
observed in the different L. lactis-treated
groups compared to the positive control
group suggesting that observed differences
in clinical symptoms and biological readouts
may rather involve a regulation of the tissue
specific Th2 inflammation.
Conclusion: These results suggest that L.
lactis NCC2287 can be beneficial to reduce
both skin and airway allergic inflammation
but the time window of administration will
favor an effect in a specific tissue.
326
Nature and outdoor activity improves
atopic dermatitis in children
Kim, W1; Kim, H2; Yoon, H2
1Pediatrics, Allergy & Respiratory Research Laboratory,
Inje University, Seoul, Korea; 2Allergy & Respiratory
Research Laboratory, Inje University, Seoul, Korea
Background: Vitamin D has also been
inconsistently associated with atopic dis-
eases, although large-scale prospective and
randomised studies are lacking. Among the
existingstudiesweregiving thesupplements of
vitamin D. And, there is no research on
these outdoor activities (forest). The role of
vitamin D in atopic dermatitis is uncertain.
We have analysed the association between
serum 25(OH)-D3 levels in atopic dermatitis
children.
Method: A total of 35 children were
enrolled for this study, being referred from
local health centers in Republic of Korea,
from April to October 2009. They were
conveniently assigned to either ‘forest
group’ participating in the forest therapy
program or control group. Measurements
of salivary cortisol level and QoL were
done at initial visits and at 8-week final
visits. And also the serum samples were
collected and check for 25(OH)-D3 levels.
From the blood samples we measured
eosinophil counts, total IgE, specific IgE of
D. pteronyssinus, D. farinae, Milk, Egg,
Cat, Dog, and Cockroach. (Pharmacia &
Upjohn Uni-CAP FEIA system)
Result: Both groups did not differ signifi-
cantly in baseline clinical characteristics.
The salivary cortisol level change at week 4
and week 8 did not differ between the two
groups. But SCORAD index reduction was
significantly and QoL measures slightly
improved in the forest group at week 8 com-
pared with the control group. The mean
25(OH)2-D3 levels significantly increased
after the outdoor activities, the level
were25.82i¾4.81 and 49.90i¾19.77 pg/ml
(P = 0.000).
Conclusion: The forest therapy program
did not induce salivary cortisol level reduc-
tion. However, considering the significant
decrease in SCORAD index andimprove-
ment in vitamin D level, this may be a use-
ful model of atopic dermatitis management
program.
327
Positive autologous serum skin test and
high titres of antinuclear antibodies
could be a marker of ‘autoimmune
urticaria’
Bettoni, L; Ghidini, K; Manisco, L
Allergology Unit, Hospital of Manerbio, Manerbio, BS,
Italy
Background: The autologous serum skin
test (ASST) is widely adopted internation-
ally in patients with chronic urticaria (CU)
to demonstrate circulating endogenous
weal-inducing factors i.e. autoantibodies.
We analize a group of CU patients to find
a clinical relationship between ASST
positivity and the presence of antinuclear
antibodies.
Method: One hundred and seventy-five con-
secutive patients (130 F; 45 M;) affected by
non-physically induced CU underwent
ASST. Antihistamines, Doxepin and corti-
costeroids were stopped at least 2 weeks
before. Venous blood was collected into
sterile glass tubes without anticoagulant and
centrifugated for 10 min at 3000 g. ASST
was performed intradermally associated to a
negative control (sterile physiological saline)
and to negative and positive skin prick test
Poster Discussion Session 8 – Cutaneous allergy
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 141
controls. A 30 min reading was performed.
Antinuclear antibodies (ANA) with pattern
and titer were also analized in all patients by
IIF. Negative patients were treated with
antihistamines. Positive ones with corticos-
teroids and/or immunosuppressants (Cyclo-
sporine)
Result: 133/175 patients showed positive
reaction to ASST. Of this positive group in
59/133 patients was demonstrated the pres-
ence of ANA with significant titer
(>1:160) and pattern speckled in most of
them (93%). No ENA specificity was
detected. All positive patients to ASST
with the concomitant presence of ANA
were more refractory to corticosteroids
therapy. Increased doses and/or Cyclospor-
ine (3 mg/kg/day) were necessary to obtain
a control of the itchy weals.
Conclusion: Despite ASST can be consid-
ered a test for autoreactivity rather than a
specific test for autoimmune urticaria, the
concomitant detection of high titer ANA
appears to determine a subset of patients
in which higher doses of immunosuppres-
sants are needed. A pathogenetic autoim-
mune mechanism is strongly suspected.
328
Anxiety and depression are less common
in patients with autoreactive chronic
spontaneous urticaria
Weller, K1; Koti, I2; Makris, M2
1Charite – Universitatsmedizin Berlin, Berlin, Germany;2Attikon University Hospital, Athens, Greece
Background: Patients with chronic sponta-
neous urticaria (csU) frequently exhibit
psychiatric comorbidities such as anxiety
and depression. Autoreactive csU is a com-
mon subform of csU that is caused by cir-
culating histamine-releasing serum factors.
It is readily identified by positivity in the
autologous serum skin test (ASST), i.e.
inflammatory reactions to the patients own
serum after intracutaneous injection. As of
yet, it is not known whether the burden of
disease as well as the frequency of psychi-
atric comorbidities varies in different sub-
forms of csU, particularly between ASST
positive and negative patients.
Method: For this joint project, data of 209
csU patients was collected in tertiary refer-
ral centers in Berlin and Athens. During
their visits, most of the patients were sub-
jected to the ASST (n = 164). In addition,
all patients were asked to complete the
Urticaria Activity Score for four consecu-
tive days (UAS4), the Dermatology Life
Quality Index (DLQI) and the Hospital
Anxiety and Depression Scale (HADS).
Result: The populations in Berlin (n = 137)
and Athens (n = 72) were not found to be
different in terms of age, gender distribu-
tion, urticaria activity, quality of life impair-
ment and frequency of ASST positive
patients (36.4% vs 34.3%). Accordingly,
both populations were pooled for the fur-
ther comparison of autoreactive and non-
autoreactive csU patients. In both groups,
age and gender distribution was not differ-
ent, whereas the mean UAS4 (9.8 vs 8.6)
and DLQI total scores (7.7 vs 6.6) showed a
non-significant trend towards higher values
in ASST positive patients. In contrast, the
proportion of patients with suspected anxi-
ety and depression (¡Y 8 points in the
HADS) was significantly higher in ASST
negative patients (20.0% vs 36.9%,
P < 0.05 and 7.3% vs 20.4%, P < 0.05).
Conclusion: Patients with autoreactive csU
are less likely to exhibit anxiety and depres-
sion although they tend to show a higher
disease activity and quality of life impair-
ment. These results suggest that autoreac-
tive csU is different from other subtypes of
csU and that psychiatric comorbidity plays
a minor role in these patients.
Poster Discussion Session 8 – Cutaneous allergy
142 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Poster Discussion Session 9
Asthma: from bench to real life
329
Expression of CD206, CD124 and CD210
on peripheral blood monocyte
subpopulations of house dust mite
allergic patients during allergen
challenge
Kowal, K1; Moniuszko, M2; Dabrowska, M1;
Bodzenta-Lukaszyk, A1
1Allergology and Internal Medicine, Medical University
of Bialystok, Bialystok, Poland; 2Allergology and
Internal Medicine, Medcial University of Bialystok,
Bialystok, Poland
Background: Alternatively activated mono-
nuclear phagocytes participate in tissue
remodeling. They are characterised by
expression of the mannose receptor
CD206. The aim of this study was to eval-
uate the expression of CD206, CD124 and
CD210 on peripheral blood monocytes of
house dust mite allergic patients (HDM-
APs) before and after allergen challenge.
Method: Blood samples were collected
from 40 HDM-APs before (T0), 6 h
(TLAR) and 24 h (T24) after intrabronchi-
al allergen challenge. The samples were
stained with labeled monoclonal antibodies
against CD14, CD16, CD124, CD206 or
CD210 and flow cytometry was performed
using FACSCalibur cytometer.
Result: CD210 was expressed on majority
of circulating monocytes (90.4 ± 10.4%),
while CD206 and CD124 only on
9.1 ± 7.1% and 13.6 ± 12% respectively.
However, CD206 was significantly more fre-
quently expressed on CD14++CD16+
monocytes (25.3 ± 13.6%) than on
CD14++CD16- cells (1.8 ± 1.9%;
P < 0.0001) or on CD14+CD16+ cells
(10.9 ± 10%; P = 0.008). The expression
of CD206 evaluated as mean fluorescence
intensity (MFI) correlated with expression
of CD124 (r = 0.487; P = 0.0063) and
CD210 (P = 0.684; P < 0.0001). Allergen
challenge resulted in decrease in the number
of circulating CD206+ monocytes (to
7.4 ± 5% of CD14+ cells at TLAR;
P = 0.03). The greatest fall was found in
the CD14++CD16+ subpopulation.
Conclusion: In HDM-APs CD206 is pre-
dominantly expressed on CD14++CD16+
monocytes. Allergen challenge results in
mobilisation of circulating CD14++CD16+
CD206+ cells.
330
ST2 requires Th2-, but not Th17-type
airway inflammation in epicutaneously
antigen-sensitised mice
Morita, H1; Matsumoto, K1; Nakae, S2
1Department of Allergy and Immunology, National
Research Institute for Child Health and Development,
Tokyo, Japan; 2Frontier Research Initiative, The
Institute of Medical Science, The University of Tokyo,
Tokyo, Japan
Background: IL-33 is known to induce Th2-
type cytokine production by various types
of cells through its receptors, ST2 and
IL-1RAcP. Polymorphism in the ST2 and/
or IL-33 genes was found in patients with
atopic dermatitis and asthma, implying that
the IL-33/ST2 pathway is closely associated
with susceptibility to these diseases. Expo-
sure to allergens through damaged skin is
suspected to be a trigger for allergen sensiti-
sation, resulting in development of such
allergic disorders as asthma and atopic der-
matitis. However, the role(s) of the IL-33/
ST2 pathway in allergic airway inflamma-
tion in individuals who had been epicutane-
ously sensitised to an antigen remains
unclear. We attempted to elucidate its
role(s) by studies in ST2-deficient mice.
Method: Wild-type and ST2-/- mice were
epicutaneously sensitised with ovalbumin
(OVA) and then were intranasally chal-
lenged with OVA. The degree of airway
inflammation, the number of leukocytes
and the activities of myeloperoxidase
(MPO) and eosinophil peroxidase (EPO) in
bronchoalveolar lavage fluids (BALFs),
The levels of cytokines and chemokines in
lungs and OVA-specific IgE levels in sera
were determined by histological analysis, a
hemocytometer, colorimetric assay, quanti-
tative PCR or ELISA, respectively.
Result: The number of eosinophils in
BALFs, the levels of Th2 cytokines (i.e.
IL-4, IL-5 and IL-13) and chemoattrac-
tants (i.e., CCL11 and CCL22) in the lungs
and OVA-specific IgE in sera from ST2-/-
mice were significantly reduced compared
with wild-type mice. Although the number
of neutrophils in BALFs and the pulmo-
nary levels of IL-17 were comparable in
both mice, the levels of MPO activity in
BALFs and neutrophil chemoattractants
(i.e. CXCL1 and CXCL2) in the lung were
reduced in ST2-/- mice.
Conclusion: The IL-33/ST2 pathway is cru-
cial for Th2-cytokine-mediated eosinophilic,
rather than Th17-cytokine-mediated neutro-
philic, airway inflammation in mice that had
been epicutaneously sensitised with antigens
and then challenged with antigen. Our find-
ings may provide a clue for development of
novel therapeutics for asthma.
331
Transient potential channel A1 and
mast cell activation in bronchial
hyperreactivity caused by hypochlorite
and OVA in mice
Hox, V1; Vanoirbeek, J2; Aguiar Alpizar, Y3; Voedisch, S4;
Bobic, S1; Callebaut, I1; De Vooght, V2; Sharify, A5;
Van Gerven, L1; Ceuppens, J1; De Vries, A6; Braun, A4;
Hoet, P2; Talavera, K3; Nemery, B2; Hellings, P7
1Laboratory of Clinical Immunology, University of
Leuven, Leuven, Belgium; 2Research Unit of Lung
Toxicology, University of Leuven, Leuven, Belgium;3Department of Molecular Cell Biology, University of
Leuven, Laboratory for Ion Channel Research, Leuven,
Belgium; 4Department of Immunology, Allergology and
Immunotoxicology, Fraunhofer Institute of Toxicology
and Experimental Medicine (ITEM), Hannover, Germany;5Laboratory of Autoimmune Disease, University of
Leuven and VIB, Leuven, Belgium; 6Laboratory of
Gastroenterology, University of Leuven, Leuven,
Belgium; 7Department of Otorhinolargyngology,
University Hospital Leuven, Leuven, Belgium
Background: Some epidemiologic studies
link attendance to chlorinated swimming
pools with airway hyperreactivity (AHR)
and non-allergic asthma. In previous exper-
iments, we showed that a single nasal
instillation of low concentration of hypo-
chlorite (ClO-) prior to ovalbumin (OVA)
induces AHR in response to methacholine
without airway inflammation. In this study
we investigate the pathophysiological
mechanisms of this phenomenon.
Method: The role of substance P (SP) in the
induction of AHR was investigated by pre-
treating animals with an antagonist of its
specific receptor RP67580 and by immuno-
fluorescent staining of large airways of trea-
ted mice. The involvement of the transient
receptor potential (TRP) A1 channel was
checked by the use of TRPA1 knockout
mice, by replacing the ClO--instillation by
an instillation of the TRPA1-agonist cin-
namaldehyde and in vitro, by Ca2+-imaging
experiments of hTRPA1-expressing cells.
The role of mast cells was evaluated by
repeating the experiment in mast cell defi-
cient KitW-sh/KitW-sh mice and by in vitro
stimulation of bone marrow cultured mast
cells (BMMCs) with incremental concentra-
tions of OVA.
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 143
Result: AHR induced by ClO- + OVA
was reduced after pretreatment with
RP67580. However, no increased SP-
expression could be shown in the airways
of treated mice. Low concentration of ClO-
induces a Ca2+-influx in hTRPA1-trans-
fected cells and its effect in vivo can be
mimicked by replacing ClO- by the
TRPA1-agonist cinnamaldehyde.
ClO- + OVA did not induce AHR in
TRPA1-/- mice or in mast cell deficient
KitW-sh/KitW-sh mice. OVA directly stim-
ulates BMMCs in vitro to produce and
release TNFa, IL13 and IL6 in dose-
dependent way.
Conclusion: Our findings show that nasal
exposure to ClO- and OVA induces a non-
inflammatory AHR by a neuro-immune
interaction mechanism.
332
Airway regulatory T-cells: a feature of
asthma severity or induced by daily
treatment?
Seys, S1; Decraene, A2; Grabowski, M3; Adriaensen, W1;
Dilissen, E1; Ceuppens, J1; Dupont, L4; Bullens, D5
1Microbiology and Immunology, Lab of Clinical
Immunology, University of Leuven, Leuven, Belgium;2Clinical and Experimental Medicine, Lab of
Pneumology, University of Leuven, Leuven, Belgium;3Physiology, Wroclaw Medical University, Wroclaw,
Poland; 4Pneumology, University Hospital Leuven,
Leuven, Belgium; 5Microbiology and Immunology,
Pediatric Immunology, University of Leuven, Leuven,
Belgium
Background: Regulatory T cells (Tregs)
maintain immune homeostasis and immune
tolerance in the human body. The signal
transduction molecule Foxp3 is thought to
be unique for regulatory cells and neces-
sary for their function. Recent data showed
that the percentages of CD4(+) FoxP3(+)
Tregs are increased in the BAL fluid of
patients with moderate to severe asthma
and hence indicate a role for these cells in
asthma pathophysiology.
Method: Lower airway cells were obtained
by sputum induction in 79 stable asthmatic
(steroid naive n = 30; treated with inhaled
steroids, n = 36; treated with oral steroids:
n = 11; unknown: n = 2) and 54 healthy
individuals. Sputum Foxp3 and CD3a
mRNA levels were measured by real-time
PCR. Sputum Foxp3 protein expression
was stained intracellularly and analysed by
flow cytometry.
Result: Sputum Foxp3 mRNA levels were
significantly higher in the total group of
asthmatics compared to healthy subjects.
Sputum Foxp3/CD3a mRNA ratios,
reflecting the proportion of Treg cells
amongst the recruited airway T cells, how-
ever tended to be lower in steroid naive
asthmatics compared to healthy subjects
(P = 0.11). Sputum Foxp3/CD3a mRNA
ratios were on the other hand significantly
higher in patients treated by inhaled corti-
costeroids than steroid-naive asthmatics
(P < 0.05) and controls (P = 0.03). The
percentage of CD4(+) Foxp3(+) cells was
significantly lower in asthmatics compared
to healthy subjects (gated on CD4;
P = 0.03).
Conclusion: Sputum Foxp3/CD3a mRNA
ratios were found to be higher in patients
treated with glucocorticosteroids compared
to steroid naive asthmatics and healthy
subjects. Our data favour the hypothesis
that inhaled steroids, rather than moderate
to severe asthma by itself, are responsible
for the presence of Treg cells in the asth-
matic airways. Whether the induction of
these cells also play a role in the beneficial
effects of corticosteroid treatment, has to
be studied.
333
Interleukin-21 promotes T helper type 2
allergic airway responses by inhibition of
Foxp3+ T regulatory cells
Pawelski, H1; Sonar, S1; Tortola, L2; Yadava, K2;
Schneider, C2; Sparwasser, T3; Kopf, M2
1ETH, Moleculare Biomedicine, Zurich, Switzerland;2ETH, Zurich, Switzerland; 3TWINCORE, Centre for
Experimental and Clinical Infection Research, Hannover,
Germany
IL-21 plays an important role in the devel-
opment of Th2-driven airway inflamma-
tion. Previous studies performed in IL-21
receptor deficient (IL-21R-/-) mice revealed
a key role of IL-21 in TH2 cell driven
inflammatory responses such as lung eosin-
ophilia, airway hyperresponsiveness, blood
basophils and type 2 granuloma in asthma
and nematode infection. In this study we
sought to identify IL-21 dependent mecha-
nisms in the course of allergic asthma. For
this, we immunized mice intraperitoneally
and challenged them intratracheally with
OVA. Mice were analysed 3 days after the
last challenge. IL-21 has been previously
shown to inhibit development of inducible
T regulatory cells (iTregs) in vitro. Here,
we found a significantly increased popula-
tion of Tregs in the lung of IL-21R-/- as
compared to WT mice upon airway chal-
lenge with OVA. To further elucidate the
role of Treg cells in asthma we utilized
DEREG mice carrying a BAC transgene
encoding for the diphtheria toxin receptor
(DTR) under the Foxp3 promoter. Here
specific depletion of Tregs by administra-
tion of diptheria toxin (DT) is possible.
Depletion of Tregs led to an enhanced
influx of Th2 cells into the lung and exag-
gerated features of asthma in both IL-
21R+/+xDEREG and IL21R-/-xDEREG
mice; indicating that Tregs are potent sup-
pressors of asthma. To identify the time
point when Tregs are imperative for the
outcome of asthma, administration of DT
was limited to either the sensitization or
the challenge phase. Tregs were seen to be
essential in both phases since depletion in
either phase affected the outcome of
asthma, suggesting an additive effect of
Treg action. Taken together, these results
suggest that IL-21R signaling negatively
regulates Treg cell population and conse-
quently the severity of OVA-induced lung
inflammation.
334
IgE- and IgG4-autoantibodies in allergic
asthma
Konishcheva, A; Gervazieva, V
Allergy Diagnosis Department, Mechnikov Research
Institute for Vaccines and Sera, Moscow, Russian Fed-
eration
Background: Current evidence suggests
that autoreactivity may underlie the patho-
genesis of chronic allergic diseases. It still
needs to be clarified whether detection of
autoAbs (autoantibodies) can define a dis-
tinct immunological phenotype of BA
(bronchial asthma). We aimed to estimate
the clinical and immunological features of
BA and AR (allergic rhinitis) depending
upon the intense of IgE- and IgG4-
autoreactivity.
Method: IgE and IgG4-autoAbs against
some lung tissue Ags (antigens): keratin,
III and VI collagen types, elastin and myo-
sin were detected using modified ELISA in
serum samples of 83 adult patients with
different severity of BA, 11 patients with
AR and 30 healthy people. Serum content
of total IgE, IL-4 and IL-10 was measured
by ELISA.
Result: The levels of IgE-autoAbs to all
listed Ags were raised in 81% of patients
with persistent moderate and severe BA
[2.8 ± 0.7 IU/ml (M ± m)] in comparison
with mild intermittent BA (1.4 ± 0.1 IU/
ml), AR (1.5 ± 0.08 IU/ml) and healthy
controls (1.2 ± 0.01 IU/ml) with highest
content of IgE-autoAbs to myosin
(3.92 ± 0.7 IU/ml) in severe BA. The dura-
tion of asthma more than 5 years was
attended with most strong elevation of IgE-
autoAbs to myosin (3.3 ± 0.8 IU/ml) and
keratin (3.2 ± 0.5 IU/ml), vs those
patients, who displayed asthma symptoms
within nearest 6 months (1.9 ± 0.02,
1.3 ± 0.05 IU/ml, accordingly). It were
direct correlations between IgE-autoAbs
and elevated serum levels of total IgE and
IL-4 (r = 0.57). Conversely, the content of
IgG4-autoAbs to all Ags considerably
raised during easy asthma – 4934 (2880;
5560) ng/ml [Me (25%; 75%)] and AR,
especially to keratin [6659 (4300;
16 620) ng/ml] as compared with severe BA
[3750 (2960; 5100) ng/ml] and healthy [2700
Poster Discussion Session 9 – Asthma: from bench to real life
144 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
(2460; 4460) ng/ml]. Patients with BA,
receiving IGS (inhaled corticosteroids) dis-
played significantly higher levels of IgG4-
Abs [4860 (3720; 7000) ng/ml] and lower
content of IgE-autoAbs vs those, who take
only beta-2 agonists [2940 (2640; 4480) ng/
ml]. The levels of IgG4-and IgE-autoAbs
correlated negatively (r = -0.61) and it was
direct association between IgG4-autoAb
and IL-10 (r = 0.55).
Conclusion: Our data suggests that severe
BA is frequently accompanied by synthesis
of IgE-autoAbs against some lung tissue
Ags, especially myosin, in the case of dura-
ble clinical course of disease, whereas
IgG4-autoAbs prevails during AR and
intermittent BA. Besides, in patients with
BA the levels of IgG4 – autoAbs can indi-
cate therapeutic benefits of IGS therapy.
335
Pathological features of the upper respi-
ratory tract in patients with uncontrolled
bronchial asthma
Iashyna, L; Polianska, M; Ignatieva, V; Gumenuk, G;
Zagrebelnyi, R
Department of Differential Diagnosis, Treatment and
Clinical Pharmacology, F.G. Yanovsky Institute of
Phthysiology and Pulmonology, Kiev, Ukraine
Background: We examined the pathological
and functional features of the upper respi-
ratory tract in patients with uncontrolled
asthma (BA). The study involved 60
patients (27 men and 33 women aged from
24 to 83 years) with uncontrolled asthma
(FEV1 – (56.4 + 2.0) Asthma control test
was (16.3 + 0.9) points.
Method: Clinical, radiological, functional –
front active rhinomanometry, spirometry,
statistics.
Result: Forty-six (76.7%) surveyed had dif-
ficulty in nasal breathing. Twenty-two
(36.7%) of them were diagnosed with aller-
gic rhinitis and 24 (40.0%) – vasomotor.
Chronic rhinitis in 18 (30.0%) patients com-
bined with the distortion of the nasal mem-
brane. Six (10.0%) patients have previously
conducted intranasal corrective surgery to
normalize the nasal passages. Chronic
antrioetmoiditis observed in 4 (6.7%)
patients. Total nasal flow on inspiration
(FSUMI) after halazolinum was (388.2 ±
58.0) ml/sec after test – (582.1 ± 71) ml/
sec, P < 0.05, for expiration (FSUME)
increased from (345.1 ± 48.3) ml/sec to
(541.0 ± 72.1) ml/sec, P < 0.05, which tes-
tified to the pronounced vegetative-vascular
disorders. After tests with bronchodilators
there was a significant increase in total nasal
flow – FSUMI from (388.2 ± 58.0) ml/sec
to (664.1 ± 98.0) ml/sec, P < 0.05, and
FSUME from (345. 1 ± 48.3) ml/sec to
(700.5 ± 118.6) ml/sec, P < 0.05 and evi-
dence of pronounced bronchonasal reflex.
Conclusion: Allergic and vasomotor rhinitis
in patients with uncontrolled asthma is
accompanied by pronounced vegetative-
vascular disorders that directly contributes
to the strengthening of broncho-obstructive
syndrome through nasobronchial reflex.
336
Both alveolar and central airways are
subjected to extracellular matrix
remodelling linked to inflammation in
uncontrolled asthma
Weitoft Lundstrom, M1; Andersson, C2; Bjermer, L2;
Erjefalt, J1; Westergren-Thorsson, G1
1Department of Experimental Medical Sciences, Lund
University, Lund, Sweden; 2Department of Clinical
Sciences, Lund University, Lund, Sweden
Background: Conventional asthma thera-
pies, such as inhaled corticosteroids, do
not prevent airway remodelling and we
have previously reported on distal airway
inflammation as a prominent feature in
both mild and uncontrolled asthma.
Whether distal inflammation in asthmatics
also involve structural changes remain
poorly examined, especially in uncontrolled
asthmatics. This study performs a detailed
characterization of peripheral connective
tissue alterations in uncontrolled asthmat-
ics and how these are correlated to infil-
trating leukocytes and clinical data.
Method: Bronchial and transbronchial
biopsies from controls, patients with mild
to moderate uncontrolled asthma were pro-
cessed for immunohistochemical analysis
of and extracellular matrix molecules; vers-
ican, biglycan, decorin and collagen. Regu-
larity of collagen fibrils were examined
using transmission electron microscopy.
Result: The density of collagen (trichrome
staining) was significantly increased in dis-
tal lung tissue of patients with uncontrolled
asthma compared to controls
(P = 0.0095). In addition, there was a neg-
ative correlation between collagen in bron-
chial biopsies and FEV1 (rs = -0.66,
P = 0.04) in asthmatic patients. Also, the
expression of decorin was increased in dis-
tal lung of asthmatics compared to con-
trols (P = 0.03). Furthermore, there was a
positive correlation between collagen and
decorin in distal lung of asthmatic subjects
(rs = 0.79, P = 0.009). Expression of vers-
ican and biglycan was not altered in cen-
tral vs distal airways when comparing
controls to asthmatic patients. The density
of connective tissue mast cells (MCTC) cor-
related positively to the density of collagen
(rs = 0.71 P = 0.027) in distal airways of
asthmatic patients. Expression of a scaven-
ger receptor for clearance of unwanted
self-molecules (fasciclin-like hyaluronan
receptor homolog) was significantly
increased in distal lung both in controls
(P = 0.0006) and asthmatic patients
(P < 0.0001) compared to central localiza-
tions. Collagen fibrils were altered resulting
in irregular profiles in some asthmatic
patients.
Conclusion: Our data suggest that there is
an ongoing remodeling in the distal lung
already in milder stages of allergic asthma.
This altered structure and resulting change
in tissue elasticity leads to abnormal
mechanical properties, which could be an
important factor for lung function impair-
ment. These findings strengthen the
rational to target also the alveolar com-
partment in uncontrolled asthma.
337
Filarial immunomodulator alters grass
allergic responses
Danilowicz-Luebert, E1; Lucius, R2; Hamelmann, E3;
Hartmann, S4
1Department of Molecular Parasitology, Humboldt
University Berlin and Free University Berlin, Berlin,
Germany; 2Department of Molecular Parasitology,
Humboldt University Berlin, Berlin, Germany;3University Children’s Hospital, Ruhr University
Bochum, Bochum, Germany; 4Institute of Immunology,
Free University Berlin, Berlin, Germany
Background: The eradication of helminth
infections over the last 30 years in industri-
alized countries was very successful, while
the prevalence of allergic diseases is increas-
ing dramatically. Possibly reduction of viral,
bacterial or helminth infections, in return
contribute to a disregulated immune system
that leads to allergic and autoimmune disor-
ders. It is known that parasitic helminths
have developed complex mechanisms to
evade and modulate host immune
responses. As a bystander effect, worm-
derived immunomodulators temper
responses to non-helminth antigens, like
environmental allergens. Previous studies in
our group characterized a single filarial mol-
ecule: a secreted cysteine protease inhibitor
(AvCystatin), potent modulator of macro-
phages that had a suppressive effect on
allergic and inflammatory responses in an
ovalbumin (OVA)-induced mouse model of
asthma.
Method: The aim of this study was to test
the preventive potential of filarial cystatin in
a clinically relevant model of airway hyp-
erreactivity induced by timothy grass
(Phleum pratense) and to translate the
results into a human system in vitro of
PBMCs from timothy grass allergic
subjects. Animals were sensitized and chal-
lenged with timothy grass pollen. AvCysta-
tin was applied during the sensitization
phase. In the translational approach,
PBMCs from allergic subjects and healthy
controls were treated in vitro with AvCysta-
tin prior stimulation with timothy grass
pollen extract.
Poster Discussion Session 9 – Asthma: from bench to real life
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 145
Result: Administration of AvCystatin sup-
pressed allergen-specific Th2-related inflam-
mation, inhibited local recruitment of
eosinophils into the lungs, reduced serum
levels of allergen-specific IgE, local and
systemic production of IL-4, IL-5 and IL-
13. Moreover, ex vivo restimulation with
AvCystatin significantly induced IL-10 pro-
duction by spleen cells. In the human
model, AvCystatin changed the allergy bal-
ance towards Th1 immune response by
increasing production of IFN gamma.
Conclusion: The results show that AvCyst-
atin down-regulates allergic immune
responses and improves murine airway dis-
ease after sensitization and challenges with
a clinically relevant aeroallergen and pro-
vides a Th1-type shift in in vitro human
model. The impact of AvCystatin might be
exploited for the treatment of allergic dis-
eases, as it selectively mimics the beneficial
properties of a chronic nematode immune
regulation.
338
IL-33 regulates lung in situ
eosinophilopoiesis by affecting their
proliferation, survival and migration
Lu, Y; Sjostrand, M; Radinger, M; Malmhall, C; Lotvall,
J; Bossios, A
Sahlgrenska Academy, University, Krefting Research
Centre, Gothenburg, Sweden
Background: Allergic asthma is character-
ised, among others, by eosinophilic-associ-
ated lung inflammation. We have
previously shown that anti-IL-33 markedly
attenuated the number of eosinophil-line-
age-committed progenitors
(CD45+CD34+IL-5Ra+CCR3+Sca-
1+SSClow) and immature eosinophils,
including early (CD45+CD34+IL-
5Ra+CCR3+Sca-1+SSChigh), intermediate
(CD45+CD34+IL-5Ra+CCR3+Sca-
1-SSChigh) and late (CD45+CD34-IL-
5Ra+CCR3+Sca-1-SSChigh), in the lungs
without changing the number of Th2 cells,
suggesting a direct effect on them. Thus
our purpose here was to detect the possible
mechanisms through which IL-33 regulates
lung in situ eosinophilia.
Method: C57BL/6 mice were sensitized and
exposed to ovalbumin (OVA), lung CD45+
cells separated with MACS and Bone Mar-
row (BM) cells were harvested 24 h after the
last exposure. Lung CD45+ cells were
labeled with CFSE and cultured with rmIL-
5, rmEotaxin-2 and rmIL-33 with or with-
out anti-IL-33 for 60 h to assess the prolifer-
ation of eosinophils. The role of IL-33 in
eosinophil survival was evaluated in CD45+
cells cultured with rmIL-5, rmEotaxin-2 and
H2O2 with or without IL-33 and analysed
by Annexin-V/7-AAD. BM cells were
applied in an in vitro transmigration assay
to investigate the migration of eosinophil in
response to IL-33 alone or in combination
with eotaxin-1 and -2. The proliferation,
apoptosis and migration of eosinophil were
evaluated by flow cytometry.
Result: Three generations were found in
both conditions on CD45+CCR3+ mono-
nuclear cells and CD45+CCR3+ granulo-
cytes. Blockage of IL-33 did not affect the
first and second generations; however, it sig-
nificantly decreased cells on the third gener-
ation in CD45+CCR3+ mononuclear
population (P < 0.05). IL-33 protected late
immature eosinophils from H2O2 induced
apoptosis (P < 0.05). Finally, IL-33
together with eotaxin-2 significantly
increased the migration of BM eosinophil
progenitors compared to either IL-33 or eo-
taxin-2 alone and to control, while IL-33
together with eotaxin-1 prominently
increased the migration of intermediate
immature eosinophils compared to IL-33 or
eotaxin-1 alone. Finally, in late immature
eosinophils, both eotaxin-1 and eotaxin-2
increased the migration, but not IL-33.
Conclusion: Above data argues that IL-33
regulates lung in situ eosinophilopoiesis
though several different mechanisms as
affect eosinophil progenitors proliferation,
survival and migration.
339
Th17 cytokines induce profibrotic
cytokine release from human eosinophils
Al-Muhsen, S1; Halwani, R2; Hamid, Q3
1Pediatrics, College of Medicine, Prince Naif Center for
Immunology Research, King Saud University, Riyadh,
Saudi Arabia; 2Prince Naif Center for Immunology
Research, College of Medicine, King Saud University,
Riyadh, Saudi Arabia; 3Meakins-Christie Laboratories,
McGill University, Montreal, QC, Canada
Background: Asthma is a chronic inflam-
matory disorder of the lung airways that is
associated with airway remodeling and hy-
perresponsiveness. One of the most critical
structural changes that affect airway func-
tionality is fibrotic tissue deposition within
the airway wall. Eosinophils have been
proposed in different studies to contribute
to the production of several mediators and
cytokines, including the profibrotic cyto-
kines, TGF-b and IL-11. In this study, we
hypothesize that cytokines prevailing in
asthmatic tissue such as Th1, Th2, and
Th17 cytokines, may induce eosinophils to
produce pro-fibrotic cytokines.
Method: Eosinophils were isolated from
peripheral blood of six mild asthmatics
and six normal control subjects. Eosinoph-
ils were stimulated with Th1, Th2 and
Th17 cytokines and production of pro-
fibrotic cytokines, TGF-b and IL-11, were
determined using Intra-cellular cytokine
detection and FACS analysis, immunohis-
tochemistry, as well as real time PCR.
Result: The level of basal expression of
TGF-b and IL-11 was significantly upregu-
lated in asthmatic patients compared to
healthy individuals. Stimulating eosinophils
with Th1 and Th2 cytokines did not induce
expression of eosinophils derived pro-fibro-
tic cytokines. However, stimulating eosin-
ophils with IL-17 resulted in the
enhancement of the expression TGF-b and
IL-11 in asthmatic individuals.
Conclusion: The regulation of expression
of pro-fibrotic cytokines within eosinophils
is Th1/Th2 independent. However, IL-17
seems to regulate eosinophl profibrotic
cytokine release in asthmatic patients and
hence contributing to the accumulation of
fibrotic tissue in asthmatic airways.
340
Induction of epithelial to mesenchymal
transition in pulmonary epithelial cells by
gastric fluid
Shih, W; Chiu, C; Cheng, C
Biomedical Science and Environmental Biology,
Kaohsiung Medical University, Kaohsiung, Taiwan
Background: Inflammation in the context
of airway residential cells is now generally
recognized to be involved in the immune
modulation. Pulmonary epithelial lining
plays an important role in antigen defense.
Loss of control during damage repair in
epithelial lining upon antigen challenge
induces epithelial to mesenchymal transi-
tion (EMT) and subsequently leads to tis-
sue fibrosis and airway remodeling. It is
known that induction of EMT is affected
by combinatory effect of cytokines and
chemokines in vicinity. Epidemic studies
indicated that micro aspiration of gastric
fluid in gastro esophageal reflux disease
(GERD) patients is associated with the
prevalence of chronic respiratory diseases.
We had previously demonstrated that gas-
tric fluid acts as an inflammatory mediator
in macrophages and airway smooth muscle
cells. In the present study, we hypothesized
that, micro aspiration by gastric fluid
induces epithelial to mesenchymal transi-
tion in bronchial epithelial cell and initiates
the differentiation of epithelial to mesen-
chymal cells. Factors involved in epithelial
to mesenchymal differentiation were ana-
lyzed.
Method: To analyzed the effect of gastric
fluid in EMT, we had used human normal
bronchial epithelial cells (NL-20) to evalu-
ate the expression of EMT markers, a-SMA
protein, E-cadherin, and profibrotic factor,
TGF-b by western blot, RT-PCR and con-
focal microscope. Migration of the NL-20
stimulated by gastric fluid was analyzed by
boyden chamber assay. The expressions of
anti apoptotic marker, bcl-2 and the signal-
ing event of p38/pp38 were analyzed.
Poster Discussion Session 9 – Asthma: from bench to real life
146 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Result: Our data showed that gastric fluid
induced a-SMA protein expression after
48 h in both gastric fluid and condition
medium treated cells. The epithelial mar-
ker, E-cadherin was diminished by gastric
fluid after 24 h stimulation. The semi-
quantitative RT-PCR result showed
increased expression of m-RNA of TGF-b
by condition medium (P < 0.05). Migra-
tion of NL-20 cells were increased 4.5
times by gastric fluid (P < 0.05). The hall-
mark of anti apoptotic marker, bcl-2 was
increased at 24 and 48 h. The inhibition of
p38/pp38 signaling transduction was
observed in both gastric fluid and condi-
tion medium treated cells.
Conclusion: Proinflammatory mediators
induced by gastric fluid initiated epithelial
to mesenchymal transition in pulmonary
epithelial cells. The loss of epithelial cell
pattern is crucial in causative in pulmonary
fibrosis.
343
Effect of swimming on airway
inflammation: a 3-year prospective
follow-up study of competitive swimmers
Andrade, P1; Pinheiro, M1; Araujo, J1; Moreira, P2;
Padrao, P2; Fonseca, J3; Delgado, L4; Moreira, A4
1Immunology Department, Faculty of Medicine,
University of Porto, Porto, Portugal; 2Faculty of
Nutrition and Food Sciences, University of Porto, Porto,
Portugal; 3Immunoallergology, Hospital Sao Joao and
Department of Health Information and Decision
Sciences, University of Porto, Porto, Portugal;4Immunoallergology, Hospital Sao Joao and
Department of Immunology, Faculty of Medicine,
University of Porto, Porto, Portugal
Background: An increasing body of litera-
ture suggests an association between com-
petitive swimming and asthma. Recent data
seems to imply that high level competitive
swimming both increases the risk of incident
asthma, and changes prevalent asthma
towards a more difficult-to-control pheno-
type. In our 3 year follow up study, we
aimed to access changes in airway inflam-
mation, measured by exhaled nitric oxide in
high level competitive swimmers.
Methods: Swimmers from the two main
portuguese swimming teams were invited to
participate in this follow up study. From
120 athletes invited, informed consent was
obtained from 105 which were assessed at
the baseline visit. From these, 19 were lost
to follow up and 86 attended the 3 years fol-
low up visit and were included in the final
analysis. ‘Active swimmers’ (n = 47) were
defined as those remaining at high level
competitive swimming; if quited at least
6 months before the follow up visit they
were considered ‘past swimmers’ (n = 39).
Subjects completed a questionnaire report-
ing physician diagnosis of asthma and aller-
gic rhinitis, and use of asthma medication.
Physical activity (PA) levels and airway
inflammation were assessed using the Inter-
national Physical Activity Questionnaire
(IPAQ) and measuring exhaled nitric oxide
(NO) levels before a training session respec-
tively. Atopy was defined by positive skin
prick testing to common aeroallergens.
Differences in changes in exhaled NO after
the 3 year follow up were assessed by
general linear model adjusting on confound-
ing factors: gender, age, atopy, physician-
diagnosed asthma, and use of asthma
medication.
Results: A significant difference in changes
in exhaled NO between past and active
swimmers was observed [respectively, mean
difference -4.6 ppb (95%CI: -11.1 to 1.9)
vs 5.6 (95%CI: 0.9–10.3); P = 0.008]. All
subjects increased their overall physical
activity levels, however significant increases
in moderate and vigorous physical activity
level were only observed in active swim-
mers. After the 3 year follow up the preva-
lence of asthma, allergic rhinitis and use of
asthma increased significantly in each
group without differences among them.
Conclusion: This prospective study of com-
petitive swimmers showed those who
remained active at a 3 year of follow-up
significantly increased their levels of airway
inflammation, as accessed by exhaled nitric
oxide, independently of their gender, age,
atopy or asthma status.
344
Bronchial dilatation due to music in
asthmatics depends on the type of music
Cioca, I1; Popilean, F2; Iamandescu, I1
1Medical Psychology, University of Medicine and
Pharmacy ‘Carol Davila’, Bucharest, Romania;2University of Medicine and Pharmacy ‘Carol Davila’,
Bucharest, Romania
Background: Relaxation effects of music
have been demonstrated in a series of
medical fields, including bronchial asthma.
Method: In 40 asthmatics with mild or
moderate bronchial obstruction where
recorded values of FEV1 and MEF 50
before and after listening to symphonic
music pieces: four of them with joyful/exu-
berant and other four with meditative/
relaxing character
Result: Both FEV1 and MEF50 have
increased significantly after listening to both
kind of music but in a different manner:
meditative music increased more FEV1
(mean FEV1 before audition = 71.73,
mean FEV 1 after audition = 75.35), than
MEF 50 (t = 7.491, DF = 9, P < 0.001)
and conversely, joyful music increased more
MEF50 (mean MEF 50 before audi-
tion = 16.83 and mean MEF50 after audi-
tion = 21.10), than FEV1 (t = 6.32,
DF = 9, P < 0.001).
Conclusion: General bronchial dilatation
effects of music in asthmatics suggest inter-
vention of cathecolamines released by lis-
tening to music although other more
mediators may be involved. Receptive
music therapy may became an useful addi-
tional therapy in asthmatics.
345
A pictorial asthma action plan is effective
in achieving asthma control and can
improve quality of life of non-literate
women
Pur Ozyigit, L1; Ozcelik, B2; Ozcan Ciloglu, S3; Erkan, F2
1Allergy and Immunology, VKV American Hospital,
Istanbul, Turkey; 2Chest Department, Istanbul Medical
Faculty, Istanbul University, Istanbul, Turkey; 3Chest
Department, Mus State Hospital, Mus, Turkey
Background: Written asthma action plans,
recommended by all the guidelines, are
important parts of asthma management,
but cannot be applied to non-literate
people.
Method: In an under-developed city of
Turkey, 40 illiterate women diagnosed with
moderate-severe persistent asthma were
included in the study, demographic charac-
teristics, treatments were recorded, St
George’s Respiratory Questionnaire
(SGRQ) was performed. Divided into two
groups, asthma education was given to
both groups; the first group also received
pictorial asthma action plan formation.
Patients were questioned by telephone in
the first, second and sixth months and
non-scheduled hospital or emergency visits,
the needs for systemic corticosteroids and
their asthma control test (ACT) were also
evaluated. SGRQ was repeated at the sixth
month.
Result: Thirty-four of 40 cases completed
the study. The study group, to whom the
pictorial asthma action plan was given,
showed a significant change of ACT scores
in the first, second and the sixth months as
compared to the first evaluation (P =
0.0001). The control group, who received
only the asthma education, showed also a
significant improvement at ACT scores of
the first and the second month’s values
compared to the first evaluation
(P = 0.034, P = 0.01). The ACT scores of
the first and the second months were sig-
nificantly lower for the study group with
the pictorial asthma action plan. The sixth
month evaluation for SGRQ revealed sig-
nificantly higher results for the study group
(P = 0.033), which means better health
related quality of life. For both groups
there were statistically significant differ-
ences between mean emergency room visits
for the last year and during the follow-up
period of 6 months (P = 0.001). The study
group, over the same period, showed a sig-
Poster Discussion Session 9 – Asthma: from bench to real life
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 147
nificant change of mean emergency room
visit (P = 0.0001).
Conclusion: In our study; the achievement
of self-management skills through pictorial
asthma action plan in addition to educa-
tion resulted with greater improvement in
asthma control, better quality of life results
and lesser emergency room visit and hospi-
talization rates comparing to education
alone for illiterate asthma patients. This
study is the first implementation of the
pictorial asthma action plan at clinical
practice.
346
Evaluation of positive bronchial
responses to D. pteronyssinus in subjects
with confirmed local allergic rhinitis
Campo, P1; Mayorga, C2; Rondon, C1; Galindo, L1;
Garcia, R1; Garcia-Campos, J1; Melendez, L3;
Rodriguez-Bada, J3; Blanca, M1
1Allergy, Carlos Haya Hospital, Malaga, Spain; 2Allergy
Research Department, Carlos Haya Hospital, Malaga,
Spain; 3Allergy Research Laboratory, Carlos Haya
Hospital, Malaga, Spain
Background: Local allergic rhinitis (LAR)
is characterized by positive responses to
nasal challenge with allergens in absence of
systemic atopy. Subjects with LAR fre-
quently show other co-morbidities includ-
ing conjunctivitis and asthma. The possible
role of allergens in the bronchial symptoms
of subjects with LAR has not been
addressed.
Method: Twenty subjects with confirmed
LAR with D. pteronyssinus (DP) and asth-
matic symptoms (LARA), 20 subjects with
allergic rhinitis with DP and asthmatic
symptoms (ARA) and 10 healthy subjects as
control group (CG) were recruited. Bron-
chial challenge was performed with DP
extract at 4 lg/ml. Metacholine challenge
and induced sputum were performed prior
to and 24 h after challenge. Cell populations
and Th1/Th2 cytokines in sputum were
evaluated by flow cytometry, and ECP and
triptase in sputum by CAP method.
Result: Forty percent of LARA and all
ARA subjects had positive responses to
bronchial challenge with DP. PC20 values
decreased in both groups 24 h after chal-
lenge (P < 0.05). Sputum ECP values also
increased after challenge in both LARA and
ARA groups (16.8 ± 14.5 vs 20.1 ± 21
and 52 ± 69 vs 95 ± 98, respectively).
Also, an increase in sputum eosinophils was
observed (LARA: 6.9 ± 11 vs 14.2 ± 7.3,
ARA: 3.7 ± 3 vs 7.2 ± 6). LARA subjects
had higher values of IL5, IL8, IL10, IL12
and IFN-gamma at baseline compared to
ARA and controls, but no significant varia-
tions after challenge.
Conclusion: Positive responses to D. pter-
onyssinus were observed in the lower air-
ways of subjects with clear history of
asthmatic symptoms. Similar methacholine
responses and increases in ECP and eosin-
ophil levels were observed in both LARA
and ARA groups.
Poster Discussion Session 9 – Asthma: from bench to real life
148 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Poster Discussion Session 10
Hot topics in allergy diagnosis and molecular allergology
347
Recombinant protein assembling
epitopes from different allergens of
Dermatophagoides pteronyssinus
Puerta, L; Martinez, D; Munera, M; Cantillo, J;
Caraballo, L
Institute for Immunological Research, University of
Cartagena, Cartagena, Colombia
Background: The domestic mite Dermato-
phagoides pteronyssinus is an important
source of aero allergens affecting allergy
susceptible population around the world.
Recombinant allergens or their modifica-
tions have been proposed for replacing
advantageously natural allergenic extract
for diagnosis and immunotherapy. We
develop a fusion protein consistent of dif-
ferent segments of four allergens of this
species to explore its utility for diagnosis
and immunotherapy of mite allergy.
Method: We engineered a fusion protein
assembling several segments of different
allergens of D. pteronyssinus, the codon-
optimized nucleotide sequences of engineered
protein was cloned into an expression vector
and the protein expressed in Escherichia coli
with 6 · His tag. The purified protein was
tested for IgE-binding and IgG-binding
capacities by ELISA using sera from a
group of mite allergy patients and a group
of subjects without allergy, basophil activation
assays was determined by flow cytometric.
Result: A recombinant protein consistent
of different segments of the allergens Der p
1, Der p 2, Der p 7 and Der p 10, denomi-
nated DPx4, was obtained as inclusion
bodies, requiring solubilization with urea
followed by oxidative refolding. Frequency
of IgE reactivity in sera from mite allergy
patients sensitized to D. pteronyssinus was
37/90 (41%), specific IgE levels against the
recombinant were significant lower than
those against the allergenic extract of D.
pteronyssinus The IgG reactivity was
detected in all sera from both groups, with
mean optical density of 0.944 in the aller-
gic group vs 0.826 in the non-allergic
group (P = 0.0012). Basophil activation
induced by the recombinant as detected by
expression of CD203c ranged between 1%
and 13% in seven selected allergic patients
whereas that induced by the allergenic
extract ranged between 41.4% and 89%.
Conclusion: The fusion protein DPx4
shows a hypoallergenic profile, suggesting
that it could be useful for allergen immu-
notherapy of mite allergy.
348
Primary structure and recombinant
expression of Secale cerealemajor
allergen Sec c 5: immunologic comparison
with Phleum pratense Phl p 5 isoallergens
Nandy, A; Pump, L; Mitulski, L; Augustin, S; Klysner, S;
Reese, G
Allergopharma Joachim Ganzer KG, Reinbek, Germany
Background: Group 5 allergens are major
allergens of grasses and cereals. Whereas
primary structures of a number of group 5
allergens from different grass species are
known, the cDNA sequence coding for the
rye major allergen Sec c 5 has not been
identified so far. Rye pollen is believed to
be an important sensitizing and symptom
eliciting source for seasonal grass pollen
induced hayfever. Recombinant Sec c 5
can be used to obtain an in-depth knowl-
edge on crossreactivity and/or co-sensitiza-
tion of grasses and cereals.
Method: The cDNA coding for Sec c 5
was obtained using a PCR strategy and the
presence of natural Sec c 5 in rye pollen
extract was confirmed by mass spectrome-
try. Recombinant Sec c 5 was produced in
E. coli and subsequently purified to homo-
geneity. The IgE-reactivity of the produced
rSec c 5 was compared to the major
Phleum pratense allergens rPhl p 5a and
rPhl p 5b by immunoblot and human IgE
inhibition ELISA.
Result: The primary structure of Sec c 5
was solved and officially named Sec c 5.0101
by the IUIS Allergen Nomenclature Sub-
Committee. Sec c 5.0101 has approximately
65% and 60% sequence identity with Phl p
5a and Phl p 5b, respectively. In IgE-immu-
noblots, 41 of 46 grass pollen allergic indi-
viduals react with recombinant Sec c 5.
Seven of 46 allergic subjects show higher
IgE-reactivity towards Sec c 5 as compared
to Phl p 5. IgE-inhibition ELISA confirmed
the existence of crossreactive as well as spe-
cies specific IgE-epitopes. In addition a
higher IgE-reactivity to Phl p 5a as com-
pared to Phl p 5b was found.
Conclusion: The first Sec c 5 cDNA
sequence has been solved. Recombinant Sec
c 5 is an important tool for a more detailed
analysis of grass pollen and rye pollen
sensitization profiles in hay fever allergic
subjects. The high degree of primary
sequence identity of Phleum and rye group 5
pollen allergens explains their high IgE-
crossreactivity. The observed minor individ-
ual differences in IgE-binding strength sug-
gest different sources of sensitization or
different contributions of various species in
co-sensitized grass pollen allergic individuals.
349
b-lactoglobulin from buffalo’s milk is
responsible for anaphylactic shock
without cross-reactivity to other
mammalian milks
Borges, J1; Gironde, C1; Collin, F1; Grabowska, A2;
Rouge, P1; Barre, A1
1University of Toulouse, UMR 152 IRD-UPS, Toulouse,
France; 2IPBS, UMR 5089 CNRS-UPS, Toulouse, France
Background: In Italy, buffalo’s milk is used
for mozzarella cheese production. Buffalo’s
milk also accounts for more than 50% of
drinking milk in certain developing coun-
tries, such as India, Pakistan and Nepal.
Only few reports exist about allergy to buf-
falo’s milk or buffalo’s cheese. The goal of
our study was to investigate allergy to buf-
falo’s milk by identifying new allergens
and testing their cross-reactivity with milks
from phylogenetically closed related mam-
mals like cow, goat or sheep.
Method: Sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis and Western
blotting were used to reveal specific IgE
reactivity of patient’s allergic to buffalo’s
milk/cheese. Mass spectrometry was used
to identify IgE-binding proteins after in gel
digestion and detection of tryptic peptides.
Cross-reactivity with other mammalian
milks was investigated by Western-blot
inhibition experiments, using a range of
total milk protein concentrations.
Result: Western-blot analysis revealed only
one IgE-binding protein. The protein, with
an apparent mass between 10 and 15 kDa,
was identified as a b-lactoglobulin by mass
spectrometry analysis of tryptic digested
peptides. It can be considered as a new
allergen in buffalo’s milk. IgE-binding inhi-
bitions carried out with other milk protein
extracts from phylogenetically closed
related mammals like cow, goat and sheep
clearly demonstrated no cross-reactivity.
Conclusion: To our knowledge, our study
identified for the first time a b-lactoglobulin
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 149
as a new allergen in buffalo’s milk allergy.
Even if cross-reactivity between milk pro-
teins from different animal species (cow,
goat, ewe, buffalo) has been shown in vitro
in cow’s milk allergic patients, our study
clearly demonstrate, as it was already shown
for a single patient earlier, a difference of
IgE cross-reactivity for buffalo’s milk aller-
gic patients. Despite very high homology
with other b-lactoglobulins from mamma-
lian milks, the absence of cross-reactivity
suggest a bigger diversity of b-lactoglobu-
lin’s epitopes from this huge family. Taken
into account that buffalo’s milk is more and
more considered as an important source of
milk in developing countries, more investi-
gations has to be carried out to better
understand the IgE-reactivity of b-lacto-
globulins.
350
Analysis of the complement activating
ability of different types of allergens
Csuka, D1; Varga, L1; FA¼st, G1; Resch, Y2; Chen, K2;
Vrtala, S2; Valenta, R2
13rd Department of Internal Medicine, Semmelweis
University, Budapest, Hungary; 2Division of
Immunopathology, Department of Pathophysiology
and Allergy Research, Center for Pathophysiology,
Infectiology and Immunology, Medical University of
Vienna, Vienna, Austria
Background: The role of complement acti-
vation in both the sensitisation and effector
phases of allergic reactions is well known.
As shown previously, allergen extracts are
efficient activators of the complement sys-
tem. Our group previously reported that
complement activation and specific IgE
binding are distinct molecular properties of
ragweed pollen allergen. In current study,
our aim was to analyse the complement
activating effect of different recombinant
allergens.
Method: For determing complement acti-
vation in the fluid phase, serum pool of
healthy subjects (non-allergic to the aller-
gens tested) was incubated by recombinant
allergens (rPhlp1, rPhlp2, rPhlp5, rPhlp6,
rBetv1a, rBetv2, rDerp2, rDerp23). The
levels of the complement activation prod-
ucts, characteristic for the activation of the
classical, alternative and terminal comple-
ment pathways (C1rC1sC1-INH, C3bBbP,
SC5b-9) were measured by ELISA meth-
ods. For analysing complement activation
induced by recombinant allergens bound to
the solid phase, we detected complement
activation products (C4b, C3b, C5b-9)
bound to the allergens. Allergen-specific
antibodies (IgG1, IgG2, IgG3, IgG4 IgA,
IgM) were also determined in the serum
pool.
Result: In the fluid phase, no significant
activation of the complement pathways
was found, except of the mild activation
with rPhlp2 and rPhlp5. By contrast,
remarkably amounts of bound C3b, C4b
and C5b-9 were detected to allergens
attached to the solid phase. Allergen-spe-
cific IgM and IgA antibodies were detected
against all types of allergens. Allergen-spe-
cific IgG1 was present in the serum pool,
except against rPhlp2, rPhlp6 and rDerp23.
Conclusion: Recombinant allergens mostly
do not activate complement pathways in
the fluid phase, although specific antibodies
with complement activating capacity are
present. However, C3 and C4 were acti-
vated in case of bound allergens, which
indicates that complement may be acti-
vated through the classical pathway, in the
solid phase.
351
Development of a coeliac peptide
database to identify novel proteins of
potential risk for eliciting disease
Amnuaycheewa, P; Wise, J; Tetteh, A; Taylor, S;
Goodman, R
Food Allergy Research and Resource Program,
University of Nebraska-Lincoln, Lincoln, NE, United
States
Background: Celiac disease (CD) is an
autoimmune enteropathy of the small
intestine induced by some gluten proteins
of wheat, barley, rye, and possibly oats.
Approximately 1% of the global popula-
tion is affected, following the distribution
of specific isoforms of MHC Class II
genes, DQ2 and DQ8. While 95% of CD
patients have at least one copy of DQ2,
many DQ2 positive individuals are not
affected. Some native gluten peptides bind
and activate DQ2 or DQ8 restricted T
cells, while others require deamidation by
inducible endogenous human tissue trans-
glutaminase. The induced enzyme modifies
proteins in connective tissue as well, mak-
ing it a target for autoimmunity.
Rationale: Food safety regulators ask
developers of novel foods and genetically
modified organisms to evaluate proteins
developed from wheat-related grains to
evaluate the proteins for their potential
induction of CD. Our goal is to provide a
CD specific database and predictive pro-
gram to aid in the evaluation.
Method: CD active peptides were identified
from published studies identified from Pub-
Med using keywords ‘celiac’ and ‘coeliac’.
A total of 1016 native or deamidated pep-
tides with reported evidence of induction
of proliferation or secretion of inflamma-
tory cytokines were identified and included
in the peptide database. Representative
source proteins were identified for all 1016
peptides to construct a CD protein data-
base. The peptide database is searched by
an exact character matching program. The
protein database is searched by FASTA.
Selected gluten-like proteins from diverse
flowering plants and wheat gliadin
sequences modified by alanine substitutions
were used to test search efficacy.
Result: As expected, only prolamin and
glutelin proteins from wheat, spelt, barley,
rye and oats were found to contain CD
peptides. Homologous proteins from taxo-
nomically distantly related plants lacking
reports of CD induction (rice, millets, corn,
sorghum, sugarcane, and Job’s tears), did
not contain exact CD peptides and FAS-
TA alignments to CD proteins were of
moderate to low identity. Alanine substi-
tuted gliadins from wheat did not match
CD peptides, but FASTA alignments were
significant.
Conclusion: The CD peptide database with
exact matching algorithm is the most defin-
itive tool to identify proteins with a high
risk CD potential. The database is now
available (February 2012) as a link from
the AllergenOnline.org website, for use as
a tool for food safety evaluations.
352
Stromal interaction molecule 1
polymorphisms are associated with
coronary artery dilation only and not
with aneurysm formation in patients
with Kawasaki disease
Chang, W1; Kuo, H2
1Kaohsiung Medical University, Kaohsiung, Taiwan;2Pediatrics, Kaohsiung Chang Gung Memorial Hospital,
Kaohsiung, Taiwan
Background: Kawasaki disease (KD) is an
autoimmune disease that is associated with
systemic vasculitis and other cardiovascular
symptoms. Recent studies have shown that
the calcium sensor STIM1 is a key mole-
cule that modulates the functioning of the
immune system. In this study, we aimed to
investigate if Stromal interaction molecule
1 (STIM1) polymorphisms are associated
with KD.
Method: We analysed the Han Chinese in
Beijing (CHB) reference population sample
of the haplotype map (HapMap) database
and selected four tagging single nucleotide
polymorphisms (SNPs; rs2304891,
rs3750996, rs1561876, and rs3750994) with
more than 10% minor allele frequency in
the coding region of STIM1 gene from.
The genotyping of 381 KD patients was
performed using TaqMan allelic discrimi-
nation assay.
Result: By using a recessive model, we
showed that the SNP rs2304897 in the
STIM1 gene was significantly (P = 0.016)
associated with coronary artery dilation in
KD patients. Patients with the four STIM1
SNPs showed no significant differences
(P > 0.1) in resistance to intravenous
Poster Discussion Session 10 – Hot topics in allergy diagnosis and molecular allergology
150 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
immunoglobulin (IVIG) treatment and
incidences of aneurysm formation.
Conclusion: This study is the first to show
that SNP in the STIM1 gene (rs2304891) is
associated with coronary artery dilation,
but not with IVIG resistance or aneurysm
formation, in the Taiwanese population.
353
Basophil activation tests in childhood
food allergy
Caimmi, S1; De Amici, M2; Ciprandi, G3; Caimmi, D1;
Marchi, A1; Pieri, G1; Giunta, V2; Marseglia, G1
1Foundation IRCCS Policlinic San Matteo, University
Pediatric Department, Pavia, Italy; 2Foundation IRCCS
Policlinic San Matteo, Hospital Pediatric Clinic, Pavia,
Italy; 3Department of Internal Medicine, Azienda
Ospedaliera Universitaria San Martino – University of
Genoa, Genoa, Italy
Background: Oral Food Challenge (OFC)
is considered as the Gold Standard for the
diagnosis of Food Allergies (FA) in chil-
dren. Nevertheless, such a procedure is
time-consuming, expansive and sometimes
dangerous. Basophil Activation Test
(BAT) is a recent tool that has not been
extensively investigated yet for the diagno-
sis of food allergy. We aimed to investigate
if BAT may be a useful screening test that
could allow clinicians both to decide
whether or not to practice an OFC and to
evaluate the persistence of FA.
Method: We retrospectively collected data
from 55 pediatric patients (pt) who, on the
whole, underwent 59 OFCs. Before the
challenge, each pt underwent Prick By
Prick (PBP) testing, and blood samples
were collected to dose serum specific IgEs
(sIgEs) and BAT (kit Flow2 CAST-Buhl-
mann). The mean age of the pt was
4.8 years (±2.9 years); 47% of them had a
diagnosis of atopic dermatitis, 29% suf-
fered from recurrent bronchospasms and
24% had presented at least one episode of
urticaria. Children were tested for egg (43
pt), milk (12 pt), hazelnut (3 pt) and kiwi
(1 pt).
Result: Eight children who were tested for
egg had a positive OFC (18.6%): they all
presented mild reactions, with the excep-
tion of a pt who required adrenalin and
systemic steroids to control the presented
symptoms. As for the other food, no other
pt had a positive OFC. In our population,
BAT showed a sensibility of 63%, a speci-
ficity of 96%, an efficiency of 91%, with a
Positive Predictive Value (PPV) of 71%
and a Negative Predictive Value (NPV) of
94%. PBP and sIgE both had a 100% sen-
sibility and a 100% NPV, with 40% and
48% of specificity, 48% and 55% of effi-
cency, and a 21% and 24% PPV, respec-
tively. The percentage of activated
basophils in children tested for egg white
was significantly higher in children with
positive OFC (median value 17.75%) than
in those with negative OFC (median value
3.39%); a percentage of activated basophils
lower than 2.81% had a 100% NPV.
Conclusion: Although more data are
needed to strengthen our results, we believe
that BAT could be a useful test in deciding
whether or not to practice an OFC. For
example, BAT could be very effective in
low risk pt (sparing useless OFCs) or high
risk pt (sparing dangerous OFCs). The
decision is routinely based on PBP and
sIgE results. Adding BAT, which seems to
have good specificity, PPV and efficiency,
could simplify the allergic work-up in
many cases.
354
Association of centrosomal protein
68 single nucleotide polymorphisms
with hypersensitivity reactions to
non-steroidal antiinflammatory drugs
Cornejo-Garcia, J1; Plaza-Seron, M1; Flores, C2;
Dona, I3; Blanca-Lopez, N4; Jagemann, L1; Laguna, J5;
Fernandez, J6; Canto, G4; Blanca, M3
1Laboratorio de Investigacion, Fundacion
IMABIS-Hospital Carlos Haya, Malaga, Spain; 2Unidad
de Investigacion, Hospital Universitario N.S. de
Candelaria, Tenerife, Spain; 3Servicio de Alergologıa,
Hospital Carlos Haya, Malaga, Spain; 4Servicio de
Alergologıa, Hospital Infanta Leonor, Madrid, Spain;5Servicio de Alergologıa, Hospital Central de la Cruz
Roja, Madrid, Spain; 6Servicio de Alergologıa, Hospital
de Elche, Alicante, Spain
Background: Non-steroidal anti-inflamma-
tory drugs (NSAIDs) are the medicaments
most frequently involved in hypersensitivity
drug reactions (HRs), representing more
than 40% of all allergic reactions. HRs can
be classified as selective responses (IgE- or
T cells-mediated) (SR) and cross-intoler-
ance (non specific immunological mecha-
nism) (CI). Recent observations in
different populations have shown that CI
is the most relevant group. Genetic associ-
ation studies have been carried out only in
patients with CI and consist mainly in the
analysis of single nucleotide polymor-
phisms (SNPs) related with the arachidonic
acid pathway. However, other possibilities
should be considered. In this sense, the
centrosomal protein 68 (CP68) has recently
been proposed as a susceptible gen in aspi-
rin-exacerbated respiratory disease. In this
study, we analyzed CP68 polymorphisms
in a large group of patients with SR or CI
to NSAIDs.
Method: Samples were obtained from
Allergy Services integrated into the Spanish
network for allergic diseases RIRAAF.
Patients who developed several episodes
with the same NSAID and good tolerance
to a strong COX inhibitor were considered
to have a SR, whereas patients with epi-
sodes with three or more different NSAIDs
were included in the CI group. We studied
6 tSNPs in CP68 by using TaqMan
probes.
Results: A total of 812 subjects with HRs to
NSAIDs were finally included, 636 with CI
and 176 with SR, and 350 age, sex-matched
controls. Statistically significant differences
were found in both groups of patients com-
pared with the control group in rs3732098
(P < 0.001 for both), and in rs7572857
(P = 0.002 and P = 0.025, respectively).
Conclusion: Our results suggest the impor-
tance of genetic variants in CP68 in HRs
to NSAIDs. Further studies are required
to analyze their functional role and the
involvement of other potential genes
nearby CP68.
355
Effects of glycoconjugates of ovalbumin
on alleviation of orally induced egg
allergy in a BALB/c mouse model
Rupa, P; Mine, Y
Food Science, University of Guelph, Guelph, ON, Canada
Background: Glycation of allergens (via
Maillard modifications) has been shown to
influence resistance or susceptibility to
food-induced allergies. Glycation structures
play a crucial role in the pathogenesis of
food allergy by acting as antigenic determi-
nants. It is hypothesized that mucosal
immune response bias can be favourably
altered by orally administering various
forms of glycated ovalbumin (Ova).
Objective: The research objectives seek to
investigate and describe the efficacy of var-
ious glycated forms of Ova as targets of
immune response polarizing candidates
and characterize immune mechanisms
underlying the influence of various glycated
forms of Ova in a Balb/c mouse model of
egg allergy.
Methods: Groups of Balb/c mice (n = 10)
were orally sensitized to Ova and subse-
quently administered various forms of
glycated-Ova (glucose, mannose, glucoman-
nan, galactomannan and mixture) followed
by oral challenge with Ova. Outcomes post
oral challenge were measured as clinical
signs, serum histamine, mast-cell protease
(MMCP-1), antibody activity (IgG; IgE;
IgG1, IgG2a, IgA), cytokines (IL-4, IFN-
gamma, IL-12p70, IL-10, TGF-beta, IL-17)
and T-regulatory cells (Tregs).
Results: Clinical signs were less frequent in
the Ova-glycated mannose and glucoman-
nan treated groups (P < 0.05). There was a
significant decrease in specific IgE antibody
activity and increase in the percentage of
Tregs (CD25+FOXP3+) cells in both the
groups; however the Ova-glycated mannose
treated group had less histamine, MMCP-1,
specific IgG, IL-4 and IL-17 (P < 0.05) and
more IL-12p70 (P < 0.001). Specific IgG1,
IgG2a, IgA-related antibody isotypes and
Poster Discussion Session 10 – Hot topics in allergy diagnosis and molecular allergology
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 151
cytokines IFN-gamma, IL-10 and TGF-
beta did not differ significantly among
groups. On the other hand, Ova-glycated
with glucose, galactomannan and the mix-
ture had no influence on the allergic status
of mice both in vivo and in vitro.
Conclusion: In summary, we demonstrated
that Ova-glycated mannose and glucoman-
nan have reduced susceptibility and subse-
quent frequency of allergy to Ova thus
biasing immune response into a balaced,
regulated, anti-allergy phenotype. This
study validates potential use of Ova-glycat-
ed mannose and glucomannan for feasible
health-enhancing therapeutic interventions
to model human food allergy via induction
of oral tolerance. cc
356
Mapping of the IgE sequential epitopes
of Ani s 1, the major Anisakis simplex
allergen, with a peptide microarray-based
immunoassay
Perez-Pinar, t1; Caballero, M1; Knaute, T2; Umpierrez,
A3; Rodriguez-Perez, R1
1Immunology, Carlos III Hospital, Madrid, Spain; 2JPT
Peptide Technologies GmbH, Berlin, Germany;3Allergy, Carlos III Hospital, Madrid, Spain
Background: Ani s1 is a major allergen
from Anisakis simplex with an IgE-binding
frequency higher than 80% among Anisa-
kis allergic patients. The diagnostic value
of Ani s 1 allergen has been extensively
demonstrated. Furthermore, it is also
expected to be a useful tool in future aller-
gen-specific immunotherapy. However, it is
desirable to use hypoallergenic mutants
devoid of IgE-binding epitopes to avoid
side-effects in immunotherapy. The aim of
this study was to determine the IgE-bind-
ing regions of Ani s 1 by using a peptide
microarray-based immunoassay.
Method: A library of 52 peptides, consisting
of 20 amino acids overlapping by 17 (3-off-
set), corresponding to the primary sequence
of Ani s 1 was printed on epoxy-coated
slides. A microarray immunoassay was per-
formed with sera from eight patients with
Anisakis allergy and positive Ani s 1 reac-
tion by western-blotting against a recombi-
nant Ani s 1 produced in Pichia pastoris.
Result: Three peptides were found to be
antigenically relevant for the majority of the
sera studied: peptide 9 (aminoacids from 25
to 44), peptide 13 (aminoacids from 37 to
56) and peptide 32 (aminoacids from 94 to
113).
Conclusion: A reaction with these peptides
has been observed for most of the samples
suggesting that these epitopes are part of
the main IgE interaction site. This data
can help to search for Ani s 1 hypoaller-
genic forms.
357
Pattern of sensitisation to nDer p 1 and
rDer p 2 in patients allergic to house dust
mite in Tarragona
Lopez-Patino, A1; Esteso, O1; Pastor, R2; Dalmau, G1;
Gazquez, V1; Gaig, P1
1Allergology Unit, Joan XXIII University Hospital, Tar-
ragona, Spain; 2Laboratory of Clinical Analysis, Joan
XXIII University Hospital, Tarragona, Spain
Background: nDer p1 and rDer p2 are the
specific markers of sensitisation to Derma-
tophagoides pteronyssinus (Dpt). There is
the possibility that the ratio of proportion
between them varies depending on the geo-
graphical area. The aim of this study was
to report the pattern of sensitisation to
nDerp1 and rDer p2 in our area of influ-
ence, a Mediterranean climate coastal city
100 km from Barcelona.
Method: Thirty-seven patients (13 men and
24 women) allergic to house dust mite were
included in the study. Mean age was
29.62 years (range: 13–54). Diagnosis was
made by clinical history related to house
dust mite, skin tests, and Dpt-specific IgE
determination. We collated information
about treatments (including immunother-
apy) and subjective response to immuno-
therapy. Serum specific IgE against nDer
p1 and rDer p2 was determined in all
patients using InmunoCAP Phadia (posi-
tive results were values >0.35 kU/l). The
immunotherapy response was measured
using the visual analogue scale.
Result: 78.4% (29) of patients lived in an
urban area, 83.7% (31) were living in an
apartment, 5.6% (2) of patients worked in
an outdoor environment and 27% of
patients had rhinitis without lung symp-
toms. Eighty-three percent (31) of patients
had positive IgE against nDer p1 and rDer
p2, 2.7% (1) only against nDer p1 and
another 2.7% (1) against rDer p2; 10.8%
(4) of patients had negative results. Aver-
age levels were 7.64 kU/l (range: 0–45.6)
for nDer p1 and 10.13 kU/l (range: 0–74.1)
for rDer p2. Serum IgE levels against rDer
p2 were higher than nDer p1 in 19
patients. Similar results were observed in
the ratio nDerp 1/rDer p2 in relation to
subjective response to immunotherapy.
Four patients who did not respond to
immunotherapy had a positive value for
Dpt and a negative value for nDer p1 and
rDer p2. Statistical power was lower than
80%.
Conclusion: The current study reveals that
most patients allergic to Dermatophagoides
pteronyssinus showed sensitisation to nDer
p1 and rDer p2. No statistically significant
differences were found on IgE levels
against nDer p1 and rDer p2 in terms of
subjective response to immunotherapy.
Negative results were always associated
with a poor response to this treatment.
358
Inhibition of anti-carbohydrate IgE
renders in vitro allergy diagnosis more
specific
Fellner, W1; Dalik, T2; Hemmer, W3; Holzweber, W4;
Altmann, F2
1Kwizda/Epignost, Vienna, Austria; 2Department of
Chemistry, University of Natural Resources and Life
Sciences (BOKU Vienna), Vienna, Austria; 3Floridsdorf
Allergy Center, Vienna, Austria; 4Medical Laboratory
Villach, Villach, Austria
Background: Allergen extracts from plants
and insects contain glycoproteins, whose
sugar moieties are bound by IgE from up
to 25% of allergic patients. While the bind-
ing to these cross-reactive carbohydrate
determinants (CCDs) is of high specificity
and affinity, experience indicates that it
does not cause clinical symptoms. Hence,
positive in-vitro results in such cases are
false-positives for most allergens, i.e. they
are just cross-reactive binding without clin-
ical significance. As most allergens of plant
and insect origin contain CCDs, doctors
and patients are confronted with an unin-
terpretable multitude of positive in-vitro
results. Wrong fears are nurtured and the
nature of the true allergen remains elusive.
Methods: Adding a glycoprotein with
CCD-type carbohydrate structures to the
patient’s serum could eliminate this inter-
ference. For this study, a plant glycopro-
tein was thoroughly digested with protease
to destroy protein epitopes. The CCDs
were coupled to an inert carrier to generate
a multi-valent inhibitor devoid of con-
cealed peptide epitopes. A number of
patients’ sera, most of them preselected for
CCD-reactivity, were tested both with the
ImmunoCAP (Thermo Scientific/Phadia)
as well as with the AllergyScreen (Medi-
wiss Analytic) system. Sera were incubated
with allergens in the absence or presence of
five volumes of inhibitor solution per 100
volumes of serum.
Results: CCD-inhibition had no effect on
recombinant allergens but exhibited drastic
reduction of cross-reactivity within natural
allergens and with the CCD-test allergens
ascorbate oxidase or horse radish peroxi-
dase. Remaining reactivities correlated well
with anamnestic data in all cases.
Conclusion: CCD-inhibition is a simple
strategy to improve the specificity of in vitro
allergy diagnosis in the routine laboratory.
Poster Discussion Session 10 – Hot topics in allergy diagnosis and molecular allergology
152 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
359
A human monoclonal IgE derived from
hybrid repertoire libraries defines an
epitope conserved in Bet v 1 and fagales
pathogenesis-related class 10 proteins
Diethers, A1; Hecker, J1; Schulz, D1; Sabri, A1; Plum, M1;
Michel, Y1; Mempel, M2; Jakob, T3; Blank, S1; Braren, I4;
Spillner, E1
1Institute of Biochemistry and Molecular Biology,
University of Hamburg, Hamburg, Germany;2Department of Dermatology, Venerology, and
Allergology, Georg-August-University, Gottingen,
Germany; 3Department of Dermatology, University
Medical Center Freiburg, Freiburg, Germany;4Hamburg Center for Experimental Therapy Research,
University Medical Center Hamburg, Hamburg, Germany
Background: Analyses of the molecular
basis underlying allergenicity and allergen
cross-reactivity, as well as improvement of
allergy diagnostics and therapeutics are
hampered by the lack of human monoclo-
nal IgE antibodies and knowledge about
their epitopes. Here we report the consecu-
tive generation and epitope delineation of
a human monoclonal IgE against the pro-
totype allergen Bet v 1 from birch pollen.
Method: Immune/synthetic hybrid libraries
were established from pollen allergic
donor-derived VH regions complemented
with synthetic VL regions. By selection,
antibody fragments with specificity for the
major birch pollen allergen Bet v 1 were
obtained, reconverted to human IgG, IgA
and IgE formats, and assessed for their
characteristics.
Result: The antibodies exhibited pro-
nounced reactivity with Bet v 1, but were
not reactive with the homologous PR10 pro-
tein Mal d 1. Based on a set of chimeric Bet
v 1 fusion proteins and fragments thereof,
the epitope as defined by the IgE paratope
could be assigned to a C-terminal helix-
structured motif comprised by the amino
acid residues 132–154, including the critical
residue E149. Grafting this motif re-estab-
lished reactivity of the per se non-reactive
Mal d 1 framework. Additionally, cross-
reactivities were evaluated by primary struc-
ture analyses of different isoforms and
PR10 proteins and verified by array based
analyses.
Conclusion: The obtained results demon-
strate that using hybrid IgE repertoires is a
suitable strategy for efficient establishment
of human authentic antibodies. The IgE-
derived information about the epitope of
Bet v 1 allows for detailed insights into
molecular aspects of allergenicity and cross-
reactivity within the PR10 protein family.
360
Targeting the extracellular membrane-
proximal domain of IgE memory B cell
Prati, M; Rhyner, C; Crameri, R
Swiss Institute for Allergy and Asthma Research (SIAF),
University of Zurich, Davos, Switzerland
Background: In industrialized countries
approximately 30% of the population is
affected by IgE-mediated allergies, including
asthma, atopic dermatitis or allergic rhino-
conjunctivitis. Allergen-specific IgE is the
key molecule in allergic diseases and it can
be expressed either as secreted IgE (sIgE) or
as membrane-bound form (mIgE), which
contains two additional domains termed
M1 and M2. The M1 region codes for an
extracellular membrane-proximal domain
(EMPD) and for the transmembrane
domain, whereas the M2 region codes for
the cytoplasmic domain expressed only in
memory B cells as integral part of the B cell
receptor (BCR).
Aims: We believe that specific targeting of
mIgE on memory B cells could be a useful
strategy for prophylactic therapeutic inter-
ventions. With the present study we aim
therefore to demonstrate that anti-EMPD
mAbs can be used to isolate IgE-switched
memory B cells from blood of allergic
patients.
Results: One anti-murine (mAbA9) and
three anti-human hybridomas (hAbC6,
hAbC33, hAbC20), were selected for fur-
ther characterization and for production
and purification of the corresponding
monoclonal antibodies. hAbC20, which
bind the human EMPD domain with
higher specificity and affinity (KD �10)10),
was used to sort IgE+ memory B cell from
allergic patients’ PBMC’s. Usually the fre-
quency of IgE+ B cells is very low. Preli-
minary results have shown that about
0.3% of the total B cell can be targeted
with the monoclonal hAbC20.
Conclusions: With this approach we want
to generate clones producing human aller-
gen-specific mAbs of the IgE isotype by
immortalization in order to elucidate IgE-
binding epitopes by co-crystallization of
allergens and allergen-specific human Fab
fragments. These initial studies and the epi-
tope mapping can aid in the discovery and
development of new therapeutics, vaccines,
and diagnostics, thus providing new
insights into allergy-related disorders.
Poster Discussion Session 10 – Hot topics in allergy diagnosis and molecular allergology
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 153
Poster Discussion Session 11
Food allergy: anaphylaxis and diagnosis
361
Network for online-registration of
anaphylaxis: towards a European registry
of severe allergic reactions – current
status
Worm, M1; Moneret-Vautrin, A2; Nikos G., P3;
Fernandez-Rivas, M4; Jutel, M5; Guilarte, M6; Kowalski,
M7; Mustakov, T8; Hompes, S1
1Department of Dermatology and Allergy, Charite –
Universitatsmedizin Berlin, Berlin, Germany; 2Reseau
Allergovigilance, Vandoeuvre Les Nancy, France;3Allergy Department, 2nd Pediatric Clinic, University of
Athens, Athen, Greece; 4Allergy Department, Hospital
Clinico San Carlos Madrid, Madrid, Spain; 5Department
of Clinical Immunology, Wroclaw Medical University,
Wroclaw, Poland; 6Allergy Section, Hospital Vall
d¢Hebron, Barcelona, Spain; 7Department of Clinical
Immunology and Allergy, University of Lodz, Lodz,
Poland; 8Allergology, Pediatrics, University Hospital
Alexandrovska, Sofia, Bulgaria
Background: Anaphylaxis is the most
severe manifestation of an allergic reaction
and can be life-threatening. Clinical data
from registries can support the identifica-
tion of risk factors and help to unravel
lacks e.g. concerning treatment but also
preventive measures. A first comparative
analysis of current registries in Europe
revealed that many countries within Eur-
ope do not acquire standardised data from
patients with anaphylaxis, or use different
methods of data acquisition.
Method: The aim is to harmonise the ana-
phylaxis questionnaire among different Euro-
pean countries, assess the quality of the data
and obtain first data at a European level.
Currently actively participating countries are
Bulgaria, France (Reseau Allergovigilance),
Germany, Greece, Poland and Spain.
Result: The questionnaire covers demo-
graphic data from the patient, detailed
information about the symptoms and the
elicitor(s), diagnostic tests and preventive
measures taken (e.g. consultation, prescrip-
tion of emergency medication). From 31
May 2011 to 11 January 2012, 281 cases
were obtained using the new consented
questionnaire. Fifty-three cases were from
Germany, 11 from Spain, 10 from Poland,
eight from Greece, and three from Bulgaria.
One hundred and ninety-nine cases from
France were entered into the system through
written information provided via e-mail. The
first data analysis indicates the major role of
food allergens in eliciting severe allergic reac-
tions. However as reported previously, drug-
and venom-induced anaphylaxis occur more
frequently in adult patients.
Conclusion: The data show that the system
used is suitable to acquire standardised data
from different allergy centres throughout
Europe. Currently the involvement of fur-
ther centres has started. Annual meetings
and distribution of the obtained data should
help not only to improve the understanding
of this complex disease, but also to enhance
awareness among the community. However,
right now only data covering clinical epide-
miology but not prevalence or incidence
rates can be gathered.
362
Anaphylaxis in children and adolescents:
a 1-year survey in an immunoallergy
department
Santos, N; Gaspar, A; Piedade, S; Santa-Marta, C;
Pires, G; Sampaio, G; Borrego, L; Arede, C;
Morais-Almeida, M
Immunoallergy Department, CUF Descobertas Hospital,
Lisbon, Portugal
Background: Anaphylaxis incidence is
increasing, especially in the pediatric age
group, but studies on characteristics of
anaphylaxis reactions are hampered due to
underreporting and underdiagnosis.
Aim: To determine the prevalence of ana-
phylaxis in an Immunoallergy outpatient
clinic, and to identify its main clinical man-
ifestations and triggers, in children and
adolescents.
Methods: From 3646 patients up to
18 years old observed in our Immunoaller-
gy department during 2011, we included
those with history of anaphylaxis voluntar-
ily reported by the clinical staff (‘at least
one episode of severe systemic reaction’).
Results: During the 1 year period, 64 chil-
dren had history of anaphylaxis (prevalence
of 1.76%). Mean age was 8.1 ± 5.5 years,
with 39 (61%) being male. The majority
(91%) had personal history of allergic dis-
ease, and 44% had asthma as co-morbidity.
Median age of the first anaphylactic episode
was 3 years (1 month–17 years old). In 14
children the first anaphylactic reaction
occurred in the first year of life. The major-
ity of patients had food-induced anaphy-
laxis (84%): cow’s milk (n = 21), egg
(n = 7), peanut (n = 6), tree nuts (n = 6),
fresh fruits (n = 6), crustaceans (n = 4),
fish (n = 4), wheat (n = 2) and goat milk
(n = 1). Food-associated exercise-induced
anaphylaxis was reported in two patients.
Drug-induced anaphylaxis occurred in 8%:
NSAID (n = 4), amoxicillin (n = 1). Three
children had cold-induced anaphylaxis, one
adolescent had anaphylaxis to latex and
latex-fruit syndrome, and one child had ana-
phylaxis to insect sting. The majority (73%)
of patients had no previous diagnosis of the
etiologic factor. Symptoms reported were
mainly cutaneous (94%) and respiratory
(84%), followed by gastrointestinal (42%)
and cardiovascular (25%); 86% beginning
in the first 30 min after exposure to trigger
agent. Forty-seven (73%) patients were
admitted to emergency department,
although only 21 (33%) were treated with
epinephrine. Recurrence of anaphylaxis
occurred in 25 patients (three or more epi-
sodes in 14 children).
Conclusions: In our pediatric population,
the main triggering agent of anaphylaxis is
IgE-mediated food allergy. Epinephrine is
clearly underused, as has been reported by
others. Often, children have several epi-
sodes before being assessed by an allergist.
We stress the importance of systematic
notification of anaphylaxis and improve-
ment of educational programmes in order
to achieve a better preventive and thera-
peutic management of this life-threatening
entity.
363
Anaphylaxis after gamba consumption
without clinical shrimp allergy: is it
thermal processing or a difference in
allergens?
de Jong, N1; Schreurs, M2; Hooijkaas, H2; Smolders, L3;
Savelkoul, H3; Gerth van Wijk, R1
1Internal Medicine, Allergology, ErasmusMC,
Rotterdam, The Netherlands; 2Immunology,
ErasmusMC, Rotterdam, The Netherlands; 3Cell
Biology and Immunology, Wageningen University,
Wageningen, The Netherlands
Background: Crustaceans are a common
food product in the western world; how-
ever they may cause severe allergic reac-
tions. In selective cases anaphylactic
reactions occur after consumption of
gamba, in the absence of similar reactions
to shrimp. The aim of this study was to
investigate whether the observed differ-
ences are caused by thermal food process-
ing or by differences in protein content.
Method: Five patients with severe allergic
reactions after consumption of gamba,
154 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
without similar reaction to shrimp were
included. Fresh gamba and shrimp were
prepared in the oven (separately at 100
and 180�C) ether extracted, freeze dried
and a 10% solution was made in PBS. Spe-
cific IgE against these gamba and shrimp
proteins was measured by SPT and visual-
ized by immunoblot analysis using SDS-
page.
Result: In Immunoblot, two novel gamba
specific allergens (18 and 80 kDa) were
detected in 4/5 patients that were not
detectable in shrimp. Furthermore, the
major shrimp allergen Tropomyosin (38
kDA) was only weak detected in two
patients in gamba and shrimp. Food pro-
cessing temperature had no influence on
the results.
Conclusion: This is the first study investi-
gating differences in gamba and shrimp
allergy. The selective anaphylactic reactions
observed in the five patients under study is
not simply due to differences in food pro-
cessing but most likely due to two novel
gamba specific proteins that apparently do
not exist in shrimp.
364
Basophil activation test role in food
allergy diagnosis: preliminary results
Aruanno, A; Mezzacappa, S; Buonomo, A; Pecora, V;
Colagiovanni, A; Rizzi, A; Pascolini, L; Ricci, A; Di
Rienzo, A; Liuzzo, M; Nucera, E; Schiavino, D
Allergy Department, Universita Cattolica del Sacro
Cuore – Policlinico A. Gemelli, Rome, Italy
Background: The ‘gold standard’ for diag-
nosis of IgE-mediated food allergy remains
at the moment a properly performed dou-
ble-blind, placebo-controlled food chal-
lenge (DBPCFC). However, there are
different practical (long duration, blinding)
and ethical (e.g. severe anaphylaxis) limita-
tions that hamper entrance of DBPCFC in
daily clinical practice. Basophil activation
test can constitute a sensitive and specific
instrument that can complement conven-
tional tests such as quantification of spe-
cific IgE (sIgE) and skin prick tests (SPTs).
The aim of the study was to evaluate the
relationship among sIgE, basophil activa-
tion test and oral food challenge in food
allergy.
Method: The study sample included 70
subjects who were evaluated using their
medical history, SPTs (with commercial
extracts of foods), quantification of sIgE
against foods, basophil activation test and
oral food challenge. The patients evaluated
were sensitized to eggs, milk, non-specific
Lipid Transfer Protein (nsLTP) or profilin.
Results: All the 70 enrolled patients pre-
sented positive SPTs, sIgE and DBPCFC
and 57 out of 70 patients (81.5%) resulted
contemporaneously positive to basophil
activation test. Only in 13 patients we
found a negative basophil activation test
with a positive oral food challenge.
Conclusion: Although further studies are
necessary, our work shows a high rate of
accordance among the quantification of spe-
cific IgE, basophil activation tests and oral
food challenge in the study population. On
the basis of these preliminary results, we
believe that in the future the basophil acti-
vation test could be a valid alternative to
oral food challenge in diagnosis of food
allergy in patients with severe reactions.
365
The basophil activation test confirms
cow’s milk allergy in non-sensitised
children with allergy symptoms
Grandne, V1; Agabriel, C2; Sainte-Laudy, J3; Fabre, A2;
Deneux, I2; Sarles, J2; Bongrand, P1; Vitte, J1
1Immunology Lab, APHM Conception, Marseille Univer-
sity Hospital, Marseille, France; 2Pediatrics Department,
APHM Timone, Marseille University Hospital, Marseille,
France; 3Immunology Lab, Limoges University
Hospital, Limoges, France
Background: The need for an oral food
challenge (OFC) surrogate is growing in
line with the continuous increase in the
prevalence and severity of pediatric food
allergy. The basophil activation test (BAT)
is currently used for the diagnosis of
venom and drug allergies.
<B<>We made the hypothesis that
BAT might improve the sensitivity of cow’s
milk (CM) allergy diagnosis in pediatric
patients with negative skin prick tests
(NSPT) and undetectable specific serum
immunoglobulin E (USSIgE).
Methods: BAT with CM extract (BAG-F2,
Flow and Flow2CAST, Buhlmann Labs,
Switzerland, final concentrations 25, 12.5, 5
and 1 ng/ml) was performed on freshly
drawn blood from 50 consecutive CM aller-
gic patients (0.5–14 years) with NSPT, USS-
IgE and positive CM oral challenge. For
each donor, three controls were assessed:
negative control (reaction buffer), positive
controls (anti-IgE receptor and fMLP).
BAT specificity was assessed with clinically
irrelevant allergens in the study group and
with CM extract BAG-F2 in non-allergic
donors. BAT was monitored through flow
cytometric membrane variations of CD63
expression and CCR3 downregulation. The
percentage of CD63+ basophils and the
mean fluorescence intensity of CD63 and
CCR3 were used to define the basophil stim-
ulation index. BAT was considered positive
if at least two consecutive concentrations of
CM extract induced a stimulation index of
two or more compared with the negative
control.
Results: Cow’s milk BAT was positive in
33 children and negative in 15. Two
patients’ results were not included in the
final statistical analysis (one IgE-receptor
non-responder and one rejection for techni-
cal reasons). BAT to irrelevant allergens in
patients, as well as BAT to CM extract in
nonallergic controls, were negative in 27
cases but positive for three BAT to CM in
CM-tolerant children. BAT was subse-
quently used for the follow-up of five posi-
tive children, two of them over a period of
4 years, with results closely matching those
of the oral food challenge.
Conclusion: BAT brought the biological
proof of food allergy in 33 out of 50 CM
allergic children with NSPT and USSIgE.
The sensitivity of the test was 69%, with
90% specificity, 90% positive predictive
value and 64% negative predictive value.
Clinical relevance: Cow’s milk BAT proved
to be a useful tool for the diagnosis and
follow-up of CM allergy in children with
NSPT and USSIgE. A positive CM BAT
in such patients might therefore avoid
OFC, but a negative test still needs OFC
confirmation.
366
Basophil reactivity to peanut
differentiates between mild and severe
peanut allergy and correlates with IgE to
peanut extract
Nentwich, I1; Herland, E2; Llohn, A3
1Allergy and Autoimmunity Unit, Department of
Immunology, Oslo University Hospital, Oslo, Norway;2Child and Youth Clinic, Akershus University Hospital,
Loerenskog, Norway; 3Department of Immunology and
Transfusion Medicine, Akershus University Hospital,
Loerenskog, Norway
Background: Basophil activation test is an
in vitro functional test in which peripheral
blood basophils are stimulated with aller-
gens and expression of activation markers
is measured. Various protocols are used
with varying diagnostic performance in
food allergy. The aim of the study is to
answer these questions: Can basophil acti-
vation test (Flow2Cast�, Buehlmann) on
stimulation with peanut extract distinguish
between patients with mild local form of
peanut allergy and the systemic form,
respectively? Does basophil sensitivity cor-
relate with specific IgE to peanut extract
and peanut components?
Patients and methods: We recruited 11
patients [six male, five female, mean age
25.8 (5–62) years.] with self-reported pea-
nut allergy. On the basis of questionary
data they were divided into a group with
mild local symptoms (oral allergic syn-
drome) and with severe systemic symptoms
(gastrointestinal, cutaneous, anaphylaxis),
respectively. We stimulated their full blood
samples with peanut extract (from Buehl-
mann) at concentrations ranging from
1000 to 0.1 ng/ml and measured the
expression of CD63 activation marker on
Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 155
basophils using flow cytometry. We calcu-
lated the CD-sens (after Nopp, Allergy
2009) as a measure of basophil reactivity.
In addition, we analyzed specific IgE to
peanut extract and peanut components
(Ara h 1 3, 8, 9).
Results: Basophil reactivity was undetect-
able (CD-sens 0.0) in all patients with mild
form of peanut allergy (n = 5) whilst the
CD-sens values in the group with severe
peanut allergy (n = 6) were as follows:
(min–median–max): 2.0–100.0–250.0. The
CD-sens values showed very strong posi-
tive correlation with IgE to Ara h 2
(Spearman r = 0.91; P = 0.0003) and
strong correlation with IgE to whole pea-
nut extract (r = 0.77), Ara h 1 (r = 0.77)
and Ara h 3 (r = 0.76) components,
respectively.
Conclusion: The basophil activation test
with single CD63 detection after stimula-
tion with peanut extract distinguishes well
between patients with the mild local form
of peanut allergy and the systemic one.
The basophil sensitivity shows a strong
positive correlation with specific IgE to
peanut storage proteins as well as to the
whole peanut extract. Our results support
the recently published data (Glaumann,
Allergy 2012).
367
Reproducibility of blinded, oral peanut
challenges and basophil allergen
threshold sensitivity, CD-sens, in
peanut-sensitised children
Glaumann, S1; Nopp, A2; Borres, M3; Johansson, S2;
Nilsson, C1
1Department of Clinical Science and Education,
Karolinska Institutet, Sodersjukhuset, Stockholm,
Sweden; 2Clinical Immunology and Allergy Unit,
Department of Medicine, Karolinska Institutet,
Stockholm, Sweden; 3Department of Paediatrics,
Sahlgrenska Academy of Gothenburg University,
Gothenburg, Sweden
Background: Double blind placebo con-
trolled food challenge (DBPCFC), the gold
standard for diagnosing food allergy, is
both time-consuming and potentially dan-
gerous. New diagnostic tools like the
basophil allergen threshold sensitivity test
(CD-sens) have been introduced in food
allergy. The aim of the present study was
to evaluate the reproducibility of oral food
challenge and CD-sens in peanut sensitized
children.
Method: Twenty-seven children, aged 4–
19 years, underwent a DBPCFC followed
by a single blind active oral food challenge
(SBAOFC). The peanut challenge was per-
formed using increasing amounts of peanut
(1 mg–5 g). Symptoms were scored accord-
ing to Astier et al. A venous blood sample
was drawn for CD-sens analyses at the two
first challenges. Basophils were stimulated
in vitro with whole peanut extract in
descending doses until the threshold sensi-
tivity, measured by flow cytometry, as the
lowest allergen concentration giving 50%
of maximum CD63% up-regulation, was
reached. The CD-sens value was defined as
the allergen dose giving 50% of maximal
basophil response expression of CD63.
Result: Fourteen children (52%) reacted at
both active peanut challenges but not on
placebo. Thirteen (48%) did not react at
any of the challenges. Of the children with
positive challenges two reacted at the same
allergen dose and with the same severity
score. All other children scored differently
or reacted at different doses. There were
no significant correlations between the
severity grades or the doses at the first and
second active challenge. However, there
was a positive correlation (r = 0.58
P = 0.03) between change in dose and
change in symptom score, at the two active
challenges, i.e. an increase in peanut dose
was associated with an increase in severity
score. Among the children positive in
active food challenge, 12 were positive in
CD-sens. Two children were low-respond-
ers and had a too low response to the posi-
tive control anti-FceRI (<16%) to be
further evaluated. All children negative in
CD-sens (n = 10) were negative at all
challenges. There were no significant differ-
ence between the first and second CD-sens
value (P = 0.79). The within patient CV
was estimated to 25%.
Conclusion: The reproducibility in qualita-
tive result (pos/neg) was 100% both for
‘active food challenge’ and CD-sens. A
comparison of the degree of sensitivity was
not possible since ‘active food challenge’
was not reproducible in contrast to CD-
sens.
368
Can allergen-specific immunological
markers predict severity and reaction
dose in oral food challenges in peanut
allergic children?
Blumchen, K1; Beder, A1; Beschorner, J1; Ahrens, F2;
Grubl, A3; Hamelmann, E4; Hansen, G5; Heinzmann, A6;
Nemat, K7; Niggemann, B1; Wahn, U1; Beyer, K1
1Department of Paediatric Pneumology and
Immunology, Charite University Medicine, Berlin,
Germany; 2Department of Paediatric Allergology,
Children’s Hospital Altona, Hamburg, Germany;3Department of Pediatrics, Technical University Munich,
Munich, Germany; 4Department of Pediatrics,
Ruhr-University Bochum, Bochum, Germany;5Department of Pediatic Pneumology, Allergology and
Neonatology, Hannover Medical School, Hannover,
Germany; 6Center for Pediatrics and Adolescent
medicine, University of Freiburg, Freiburg, Germany;7Department of Pediatrics, University Hospital Carl
Gustav Carus, Technical University of Dresden,
Dresden, Germany
Background: There is conflicting data
whether laboratory markers are predictive
for severity and threshold levels for posi-
tive reactions under oral provocation in
children with food allergy.
Objective: To correlate severity and reac-
tion dose under oral peanut provocation
with immunological markers for peanut
sensitization in a prospective study.
Method: Sixty-three peanut allergic chil-
dren (median age: 6 years, range: 3–
18 years) with median peanut-IgE of
73.5 kU/l (range: 0.6–624 kU/l) and posi-
tive oral peanut provocation were
recruited. They received a maximum of
eight, semi-log increasing titration steps of
roasted peanut (12–18 000 mg equal 3–
4500 mg peanut protein) every 2 h within
3 days until objective allergic reactions
were observed. Severity of symptoms was
graded in a scale of I-V. Peanut- and Ara
h2-specific IgE, wheal size of peanut-spe-
cific skin prick test (SPT), peanut-specific
activated basophils (CD63+/CD203c+/
CD123+/HLA-DR-/CD45+) and cytokine
production of PBMCs stimulated in vitro
with peanut extract were measured prior to
provocation.
Result: The reaction dose (median: 400 mg
peanut, range: 12–18 000 mg) was signifi-
cantly weakly to moderately and inversely
correlated with pre-challenge peanut-spe-
cific IgE levels (r = -0.35, P = 0.005), Ara
h2-IgE levels (r = -0.38, P = 0.002),
wheal size of SPT (r = -0.26, P = 0.039),
peanut-specific activation of basophils
(r = -0.46, P = 0.001), IL-4- (r = -0.36,
P = 0.004), IL-5- (r = -0.42, P = 0.001),
IL-2- (r = -0.35, P = 0.006) and IL-10-
(r = -0.37, P = 0.003) production by
PBMCs. The severity of symptoms (med-
ian: III, range: I-IV) did not correlate to
any of the markers nor to the reaction
dose (r = 0.03, P = 0.8).
Conclusion: Although the reaction dose
correlated to all assessed markers of aller-
gen-specific sensitization and immune reac-
tions, the severity of the allergic symptoms
did not correlate to any markers of sensiti-
zation. Thus, all peanut-sensitized children
undergoing oral provocation for proof of
diagnosis should be monitored closely and
precautionary safety measures have to
be applied during the whole process of
provocation.
Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis
156 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
370
Purified Pru p 3 (lipid transfer protein)
from peach for in vivo diagnosis
Santos, M1; Ibarrola, I1; Arilla, M1; Fernandez-Rivas, M2;
Cuesta-Herranz, J3; Brena, S1; Zamarreno, J1;
Martınez, A1; Asturias, J1
1Research and Development Department, Bial-Arıstegui,
Zamudio, Spain; 2Allergology Service, Hospital Clınico
San Carlos, Madrid, Spain; 3Allergology Service,
Fundacion Jimenez Dıaz, Madrid, Spain
Background: LTPs have been identified as
major allergens in fruits belonging to the
Rosaceae family (eg. peach). Peach is the
most frequently involved fresh fruit in
allergic reactions in Mediterranean coun-
tries and more than 60% of patients with
peach allergy are sensitized to Pru p 3 in
our area. Therefore, Pru p 3, the marker of
primary sensitization to LTPs, should be
tested in patients with allergic reactions to
plant foods especially in Mediterranean
countries. The availability of a prick test of
purified Pru p 3 means an easy, fast, and
low-cost component-resolved diagnosis for
plant food allergy for daily clinical prac-
tice. The aim of this work is to produce a
purified Pru p 3 allergen useful for this
purpose.
Method: Pru p 3 was purified from peach
peels with chromatographic procedures
(Capto S and Desalting HiTrap columns in
AKTA system). Quality control of the
purified protein was performed by size
exclusion chromatography (Superdex 75–
3.2/30 column), SDS-PAGE, Western-blot,
and ELISA. The production protocol was
standardized and specifications for product
releasing were established. Quantitative
skin prick tests (5–25–50–75 lg/ml) accord-
ing Nordic Council on Medicines guide-
lines were performed in two allergy centers
including patients which had a positive
case history with food allergy related to
peach.
Result: Purified Pru p 3 was characterized
by size exclusion chromatography (single
peak at 10.5 kDa), SDS-PAGE, Western-
blot and ELISA reactivity against human
IgE and experimental rabbit anti-serum
against Pru p 3. All the batches obtained fit
the specifications of purity (>95%), SDS-
PAGE (single band at 10 kDa), and IgE/
IgG reactivity. Biological activity determi-
nation of purified peach LTP was performed
on 19 peach-allergic patients and 10 control
patients with no adverse side-effects in all
the tested subjects. The concentration which
induced a wheal of similar size than that
produced by 10 mg/ml histamine (1 SPT)
was 15 lg/ml with a confidence interval of
8.7–21.3 lg/ml (P < 0.05). The expected
wheal area produced by Pru p 3 at 1 SPT
ranged from 14.6 to 66.1 mm2.
Conclusion: The biological activity of the
purified Pru p 3 has been assayed finding
that purified Pru p 3 is a cost-effective
diagnostic test for daily clinical practice.
371
Low sensitivity of a commercial
microarray in cow’s milk allergy
Vlaicu, C1; Rodriguez-Alvarez, M2; Cerecedo Carballo, I3;
Zayas, L2; Olano Rocha, M1; Terrados Cepeda, S1;
Robledo, T2; Dieguez, M3; de la Hoz Caballer, B1;
Fernandez-Rivas, M2
1Allergy Department, Hospital Universitario Ramon y
Cajal, Madrid, Spain; 2Allergy Department, Hospital
Clinico San Carlos, Madrid, Spain; 3Allergy Department,
Hospital del Sureste, Arganda del Rey, Spain
Background: The currently available micro-
array allows, in a single analytical step, the
determination of specific IgE against more
than 100 multiple recombinant or purified
natural allergens including five cow’s milk
proteins: nBos d 4 (a-lactalbumin, ALA),
nBos d 5 (b-lactoglobulin, BLG), nBos d 6
(bovine serum albumin, BSA), nBos d 8
(casein), and nBos d lactoferrin (bovine
lactoferrin). Currently, the diagnostic value
of multiplex IgE testing for cow’s milk
allergy remains unknown.
Method: In the multicenter study CoALE
(cohort of children allergic to cow’s milk)
we analysed the results of multiplex IgE
testing in 94 milk-allergic infants. All the
patients had a recent history of immediate
reactions to cow’s milk or a positive oral
challenge test, and had at least one positive
skin prick test and/or serum specific IgE
(CAP) to milk, ALA, BLG or casein. The
multiplex and singleplex (CAP) tests were
performed in the same serum samples.
Result: We performed 149 determinations
of specific IgE to milk and its proteins with
both systems, 94 measurements at the
initial visit and 55 measurements at follow-
up visits (53 at 6 months, and 2 at
18 months follow-up visits). Specific IgE
results by CAP were positive (‡0.35 kU/l)
to milk in 107 determinations (72%), to
ALA in 56 (38%), to BLG in 69 (47%)
and to casein in 62 (42%). By microarray
specific IgE results were positive to ALA
in 20 determinations (13%), to BLG in 14
(9%), to casein in 21 (14%), to BSA in 9
(6%) and to bovine lactoferrin in 2 (1%).
The frequency of positive specific IgE
antibodies by CAP is higher (P < 0.001)
than that found by multiplex, at both
initial evaluation and 6 months follow-up
visit.
Conclusion: The sensitivity of the multiplex
test to detect specific IgE to a-lactalbumin,
b-lactoglobulin and casein is significantly
lower than that of CAP to the same milk
allergens. The addition of BSA and bovine
lactoferrin, which are present in the micro-
array, does not improve the frequency of
positive results.
372
The Europrevall criteria for identifying
cases of food allergy are effective in an
unrelated Irish birth cohort study:
BASELINE
Kelleher, M1; Murray, D1; Irvine, A2; Hourihane, J1;
Baseline, T1
1Department of Paediatrics and Child Health, University
College Cork, Cork, Ireland; 2Clinical Medicine, Trinity
College Dublin, Dublin, Ireland
Background: Food allergy in childhood is
common, with reported prevalence of
between 6% and 9% in children under
3 years. The parental perception of food
allergy is much higher. The discrepancy
between perceived and true allergy results
in many infants having unnecessary dietary
restrictions imposed, at a time when nutri-
tion plays an integral role in growth and
development. The BASELINE study is Ire-
lands first Birth Cohort Study with 2185
infants enrolled.
Method: BASELINE Parents are advised
to report any incidence of suspected food
allergy to the Study Team and are specifi-
cally questioned about food allergy symp-
toms at each clinic visit. EuroPrevall
criteria are used to assess initial queries. If
the history of symptoms given is unlikely
causative for food allergen, parents are
advised to reintroduce the food at home
and follow up contact is made. If history is
likely causative for a suspected food aller-
gen, children are assessed and allergy test-
ing is carried out. Skin Prick Test (SPT) is
performed, and if negative parents advised
to reintroduce food. If SPT is positive,
Specific IgE is taken and an Oral Food
Challenge (OFC) is carried out. At
24 months all children on the study will
have SPT carried out to a panel of com-
mon food allergens and if positive an OFC
follows.
Result: To end of December 2011 there
were 176 reports of suspected food allergy
from the study. One hundred and fifty-
eight have been contacted. One hundred
and seven were deemed ‘Unlikely Causa-
tive’ and 86 of these reintroduced impli-
cated food at home safely (49% of total
queries). Of those not reintroduced, 6/21
parents refused reintroduction of food. In
15/21 cases a trial exclusion diet was insti-
gated with regular follow up. Fifty-one
queries were deemed ‘Likely Causative’
and all but one had positive SPT. Sensiti-
sation rate was 32% of reported queries
(50/158). Thirty-eight children have had
oral food challenges (some have had multi-
ple OFCs), 13 await challenge. There were
33 positive challenges (87% positive chal-
lenge rate). In the ‘Unlikely Causative’
group eczema was present in 32/107 (29%)
whereas in the ‘Likely Causative’ group
Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 157
eczema was present in 38/51 (74.5%)
P £ 0.0001.
Conclusion: The prevalence of true food
allergy is substantially lower than that of
reported allergy. We have shown how Eu-
roprevall diagnostic schedule for diagnos-
ing food allergy in children is successfully
employed in an Irish Healthcare setting.
373
History of oral symptoms and birch
pollinosis are best predictors for hazelnut
allergy in adults from a birch-endemic
area
Masthoff, L; de Reus, A; Bruijnzeel-Koomen, C;
van Hoffen, E; Pasmans, S; Knulst, A
Department of Dermatology/Allergology, University
Medical Center Utrecht, Utrecht, The Netherlands
Background: The double-blind placebo-
controlled food challenge (DBPCFC) is the
‘gold’ standard to diagnose food allergy,
but is time-consuming, expensive and limit-
edly available. The position paper from the
European Academy of Allergology and
Clinical Immunology (EAACI) 2004 stated
that patients with pollen related oral
allergy syndrome should only undergo a
DBPCFC in selected cases. However, sup-
portive data from clinical studies are lim-
ited. The aim of this study was to evaluate
the value of history, atopic features, spe-
cific IgE (sIgE) and skin prick test (SPT)
to diagnose hazelnut allergy in adults.
Method: One hundred and one adults who
underwent a DBPCFC for hazelnut were
selected for this study. Reported clinical
symptoms after accidental ingestion of
hazelnut and a history of birch pollinosis
were analyzed in relation to hazelnut
allergy. The positive and negative predic-
tive value (PPV/NPV) of level of sIgE and
SPT reactivity for hazelnut was deter-
mined.
Result: Hazelnut allergy was confirmed in
(79/101) 78% of the patients. Most adults
(89%) had sIgE for birch pollen
‡0.35 kUA/l. Birch pollinosis was more
common in adults with a hazelnut allergy
(P = 0.002) with a PPV of 86% and
accounting for 79% of the adults and a
NPV of 48%. Oral symptoms in the his-
tory to hazelnut were very suspect for a
hazelnut allergy (PPV 86%) and commonly
reported (91%) and the absence of oral
symptoms had a NPV of 71%. All patients
reported oral symptoms during the chal-
lenge. sIgE for hazelnut had an area under
the curve (AUC) of the ROC-curve of 0.65
(95% CI, 0.51–0.79). SPT had an AUC of
0.58 (95% CI, 0.39–0.77). sIgE for hazel-
nut had a PPV of 80% and NPV of 30%
for ‡0.35 kUA/l and SPT had a PPV of
88% and NPV of 14% for ‡3 mm. The
sIgE for hazelnut had additional value to
the size of SPT. The maximum reached
PPV was 96% for sIgE ‡ 5 kUA/l and
SPT ‡ 3 mm, accounting for 69% of the
adults.
Conclusion: In adults from a birch-endemic
area, a history of oral symptoms to hazel-
nut and birch pollinosis are most predictive
for a hazelnut allergy, independent of sIgE
and SPT reactivity for hazelnut. The com-
bination of sIgE and SPT is diagnostic for
a hazelnut allergy for specific cutoff values.
This study supports the expert opinion
from the EAACI 2004 position paper that
a DBPCFC needs not to be routinely per-
formed in patients with a history of only
oral symptoms to hazelnut from a birch
endemic area.
374
Safety and clinical value of oral
challenges with fish: a case series
Pereira, A; Coimbra, A; Cruz, L; Placido, J
E.P.E., Servico de Imunoalergologia, Centro Hospitalar
Sao Joao, Porto, Portugal
Background: Oral challenges (OC) are
important in the management of patients
with suspicion of fish food allergy. The
aim of this study was the characterization
of a series of patients who underwent OC
with fish as well as the comparison of skin
prick to prick tests (SPPT) and specific IgE
(sIgE) to the OC results. In our region fish
is an important part of our diet.
Method: A review of the food allergy med-
ical files of our department was made and
all the patients who underwent OC with
fish between June 2009 and August 2011
were selected. All the OC were open; the
fishes were selected according to the reac-
tion, SPPT, sIgE and the patient’s prefer-
ences. Diagnostic OC were not performed
in any cases of severe anaphylaxis.
Result: A total of 61 OC were performed
in 24 patients (1–7 per patient). Fifteen
(63%) were female with a median age (in-
terquartile variation) of 19 (6–38) years;
45% were atopic; 67% had rhinitis, 42%
asthma, 17% dermatitis and 17% allergy
to other foods. Seven of the 24 had ana-
phylactic reactions possibly related to fish.
Fifty-one (84%) of the OC were negative
with a median (interquartile variation) tol-
erated dose of 87.6 (63.1–127.3) g. Four
were doubtful due to the unspecific symp-
toms that led to the interruption of the
OC; one was later repeated and was nega-
tive. Six (10%) OC were positive in five
patients; two reactions occurred at the ini-
tial contact dose and two in the first
ingested dose (1 g). Adrenaline was not
required. Five of the six positive OC were
in four patients with a history of anaphy-
laxis and only one of these OC was with
the culprit fish. Three patients with positive
OC underwent OC with alternative fishes
and these were negative. The proportions
of disagreement between SPPT and OC
were high (29% for tuna, 60% cod fish,
29% salmon and 33% hake) as well as
between sIgE and OC (54% for tuna and
20% for codfish).
Conclusion: Oral challenges with fish are
safe when performed in specialized centers.
In this study, we were able to provide one
fish for the vast majority (92%) of these
patients (22 of the 24). A multicentric study
may allow a better evaluation of the concor-
dance between the different diagnostic
methods and better knowledge of allergens
and cross reactivity mechanisms between
fishes.
375
Extraintestinal manifestations in children
with non-IgE-mediated food allergies
may aid the clinical diagnosis
Dominguez Ortega, G1; Meyer, R1; Lindley, K1;
Thapar, N1; Chakravati, V2; Fox, A3; Shah, N1
1Great Ormond Street Hospital, London, United
Kingdom; 2Princess Alexandra Hospital, Harlow,
United Kingdom; 3St Thomas’ Hospital, London,
United Kingdom
Background: Non-Ig-E/mixed mediated
allergic reactions frequently present with
very non-especific symptoms. The lack of a
diagnostic test and the reliance on elimina-
tion and challenge of dietary antigens
reflects the dependence on a good clinical
history. We set out to investigate whether
there are extraintestinal features/symptoms
that may aid the diagnosis of non-IgE FA
(food allergy).
Method: We did an observational analysis
at a Gastroenterology department in a ter-
tiary referral centre. Records from 2002 to
2009 that included the following search
terms: allergy, reflux, eosinophilic oesopha-
gitis, enteropathy, colitis, proctocolitis,
constipation, diarrhoea, abdominal discom-
fort together with cow’s milk, soy, egg,
diary and wheat exclusion/elimination were
considered for inclusion. All families were
contacted to ensure accuracy of the clinical
record. The FA diagnosis had to be con-
firmed by at least two elimination diets for
4–6 weeks, followed by a home challenge
resulting in reproducible deterioration and
then recovery on re-elimination. The next
data was recorded from the medical notes:
symptoms at time of diagnosis, age of
onset, age of diagnosis, comorbid allergic
disease and dietary management. Blood
markers were documented when available.
We used a control group of 74 patients
diagnosed with inflammatory bowel dis-
ease.
Result: We collected 615 patient records
which fulfilled the criteria for inclusion.
Data from 437 children (203 female) was
Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis
158 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
included in the analysis. The median age of
onset of symptoms was 5 months. The med-
ian medical diagnosis was at 63 months of
age. The most frequent gastrointestinal
symptoms were: abdominal pain (90%),
diarrhoea (81%), abdominal distension/
bloating (74%), vomiting (57%), back arch-
ing and screaming (50%), constipation
(44%) and rectal bleeding (38%). Three
hundred and thirteen (71%) had other sys-
temic manifestations: mouth ulceration
(39%), poor sleep (34%), headaches (22%),
fatigue (53%), nights sweats (34%), bed
wetting (18%), joint pain/hypermobility
(35%). All extraintestinal manifestations
were more frequent in the allergic group
than in the control group, with a significant
difference (P < 0.05) in all except for head-
ache. Median total IgE was 28.5 KU/l.
Conclusion: This study highlights the sys-
temic manifestations commonly seen in
food allergic children and may impact on
the better recognition and management of
these patients. They support the diagnosis
of Non-IgE/mixed mediated food allergy.
Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 159
Poster Discussion Session 12
Allergic and non-allergic inflammatory mechanisms
376
Gut T-cell receptor gamma-delta positive
(TCRcd+) intraepithelial lymphocytes are
selectively activated by cholera toxin to
break oral tolerance in mice
Frossard, C1; Eigenmann, P1; Asigbetse, K1; Burger, D2
1Departments of Pediatrics and Internal Medicine,
University Hospitals of Geneva and University of
Geneva, Geneva, Switzerland; 2Faculty of Medicine,
University of Geneva, Geneva, Switzerland
Background: The gut immune system is
usually tolerant to harmless foreign anti-
gens such as food proteins. However, toler-
ance breakdown may occur and lead to
food allergy. To study mechanisms under-
lying food allergy, animal models have
been developed in mice by using cholera
toxin to break tolerance.
Objective: To study the role of cholera
toxin-activated TCRcd+ intraepithelial
lymphocytes in modulation of tolerance to
food allergens.
Methods: After cholera toxin gavage of
naıve mice, intraepithelial lymphocytes
were characterized for cholera toxin endo-
cytosis, expression of co-stimulatory mole-
cules, and cytokine production. In
addition, activated intraepithelial lympho-
cytes were transferred to naıve mice in
order to measure antibody production.
Results: TCRcd+ intraepithelial lympho-
cytes isolated from mice fed with cholera
toxin and transferred to naıve mice hamper
tolerization to the food allergen b-lacto-
globulin in recipient mice which produce
anti-b-lactoglobulin IgG1 antibodies. Fur-
thermore, adoptive transfer of TCRcd+
cells from cholera toxin-fed mice triggers
the production of anti-cholera toxin IgG1
antibodies in recipient mice that were never
exposed to cholera toxin, suggesting anti-
gen presenting functions of TCRcd+ in-
traepithelial lymphocytes. In contrast with
TCRab+ cells, TCRcd+ intraepithelial
lymphocytes bind and internalize cholera
toxin both in vitro and in vivo. cholera
toxin-activated TCRcd+ intraepithelial
lymphocytes express MHC class II mole-
cules, CD80, and CD86 demonstrating an
APC phenotype. Cholera toxin-activated
TCRcd+ intraepithelial lymphocytes
migrate to the lamina propria where they
produce IL-10 and IL-17.
Conclusion: These results provide in vivo
evidence for a major role of TCRcd+
intraepithelial lymphocytes in the modula-
tion of oral tolerance in the pathogenesis of
food.
377
CD8aa TCRab T-cells appear at extra-
intestinal sites in mice with experimental
colitis and change their gene expression
profile
Dickgreber, N; Zufferey, C; Bunter, A; Rihs, S; Saurer, L;
Mueller, C
Experimental Pathology, Institute of Pathology,
University of Bern, Bern, Switzerland
Background: Intestinal intraepithelial lym-
phocytes (IEL) contain a large proportion
of T cells, which differ in their phenotypic
composition from circulating T cells. One
predominant, unconventional T cell subset
with proposed regulatory functions are
CD8aa+CD8b-TCRab+ IEL, which were
shown to be resident in the small intestinal
epithelium under homeostatic conditions.
Method: We aimed to study the behaviour
of this cell type during inflammatory con-
ditions. To this end, we used transgenic
mice that contain self-specific CD8aa+T-
CRab+ IEL, but no conventional CD4+
and CD8+ T cells.
Result: The presence of resident
CD8aa+TCRab+ IEL did not protect
mice from colitis development after transfer
of naıve CD4+CD45RBhi T cells. Intrigu-
ingly, we observed a substantial increase in
CD8aa+TCRab+ T cells in peripheral
blood, lymph nodes, spleen and colon after
colitis induction. The appearance of
CD8aa+TCRab+ T cells at extra-intesti-
nal sites in colitic mice was accompanied by
increased proliferation and changes in hom-
ing marker expression. Under homeostatic
conditions, resident CD8aa+TCRab+ IEL
express genes that are associated with
immune regulation such as TGF-b1, TGF-
b3, fgl2 and LAG3 and lack expression of
genes associated with a pro-inflammatory
phenotype like IFN-g. During the course of
colitis, expression of TGF-b3, fgl2 and
LAG3 was reduced, while CD8aa+T-
CRab+ gained the ability to produce IFN-
g and TNF-a after restimulation in vitro.
These changes were most prominent in cells
obtained from extra-intestinal sites.
Conclusion: While the exact functional role
of intestinal and extra-intestinal CD8aa+
TCRab+ T cells or CD8aa+TCRab+ T
cell subsets still need further investigation,
our results indicate a so far unanticipated
plasticity of the CD8aa+TCRab+ T cell
population.
379
Impact of the exposure period to
galactooligosaccharides/inulin prebiotics
on immune system orientation
Gourbeyre, P1; Desbuards, N2; Gremy, G1; Tranquet,
O1; Champ, M2; Denery-Papini, S1; Bodinier, M1
1French National Institute of Agronomic Research
(INRA), Research Unit 1268 Biopolymers Interplays
Assemblies (BIA), Allergy Team, Nantes, France;2French National Institute of Agronomic Research
(INRA), Research Unit Physiology of Nutritional
Aptitudes (UMR PhAN), Nantes, France
Aim: To define the best exposure period
(perinatal, postnatal) to a prebiotic mix
able to induce immune pathways related to
tolerance mechanisms.
Background: Prebiotics are digestion resis-
tant molecules able to stimulate intestinal
microbiota and immune system. They con-
stitute emerging tools to alleviate some
pathologies including allergies. However,
there are not enough data proving their
efficacy and their impact on immune sys-
tem is yet not fully understood.
Method: Balb/c mice were fed with a ga-
lactooligosaccharides/inulin prebiotic mix
enriched diet along perinatal period (gesta-
tion, parturition, lactation) and/or postna-
tal period (from weaning to 12 weeks of
age). Some markers of immune pathways
related to tolerance mechanisms [T regula-
tory (Treg) response: IgA, interleukin-10
(IL-10) and transforming growth factor-b
(TGF-b); Th1 response: IgG2a and inter-
feron-c (IFN-c)] and to allergy (Th2
response: IgE, IgG1 and IL-4) were analy-
sed.
Result: When prebiotic enriched diet was
introduced only during postnatal period,
the IgG2a levels remained unchanged while
IgA levels were decreased. When prebiotics
were introduced during both perinatal and
postnatal period, IL-10, IgA and IgG2a
levels were increased.
Conclusion: Prebiotic exposure period exert
a major effect on cytokine and immuno-
globulin secretion. This study proves that
the combined exposure period (perinatal
+ postnatal) to prebiotic mix must be benefi-
160 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
cial to induce Th1 and Treg related immuno-
globulin production. Thus, these experimental
conditions seem interesting to prevent aller-
gies which are related to Th2 response.
380
Immunomodulatory effect of beta-glucan
isolated from Pleurotus ostreatus on
adaptive immunity in children with
recurrent respiratory tract infections
Jesenak, M1; Banovcin, P2; Rennerova, Z3; Majtan, J4;
Babusikova, E5; Kuniakova, R6; Hrubisko, M7
1Department of Paediatrics, Center for
Immunopathological Conditions, Jessenius Faculty of
Medicine of Comenius University, Martin, Slovakia;2Department of Paediatrics, Center for
Immunopathological Conditions, Jessenius Faculty of
Medicine of Comenius University in Bratislava, Martin,
Slovakia; 3Pneumo-Alergo Centrum, Bratislava,
Slovakia; 4Slovak Academy of Sciences, Institute of
Zoology, Bratislava, Slovakia; 5Department of Medical
Biochemistry, Jessenius Faculty of Medicine of
Comenius University in Bratislava, Martin, Slovakia;6Pleuran, Bratislava, Slovakia; 7Department of Clinical
Immunology, St. Elizabeth’s Oncology Institute,
Bratislava, Slovakia
Background: Recurrent respiratory tract
infections (RRTI) presents very frequent
and important problem in paediatric
immunology. In these children, usually
only mild deviations in the immune system
parameters could be detected, as true im-
munodeficiencies are only rare reasons for
RRTI. Several preparations of natural ori-
gin have been used for prevention of
RRTI, but only few of them have scientific
evidence for real influence on the immune
system functions.
Method: In double-blind, placebo-con-
trolled, multicenter study we studied a
group of 175 children (aged 5.65 ±
2.39 years) with more than five respiratory
infections in previous 12 months before
enrolling into the study. The children suffer-
ing from severe conditions (e.g. cystic fibro-
sis, ciliary dyskinesia, immunodeficiencies)
were excluded. Children were randomized
into active group (treated with imunoglukan
(pleuran) – beta-glucan from Pleurotus os-
treatus – in the form of syrup for 6 months)
and placebo group. During three visits (V0
– at the beginning, V1 – after 6 months, V3
– after 12 months) questionnaires were ful-
filled and blood sampling for the examina-
tion of the parameters of adaptive and
innate immunity was performed.
Result: Besides clinically proven effect on
the frequency of respiratory infections and
general morbidity, treatment with beta-glu-
can caused significant changes in immune
parameters compared to placebo. Imuno-
glukan significantly increased the plasmatic
concentration of all the three isotypes of
immunoglobulins (IgG, IgA, IgM), espe-
cially during the treatment period. In
active group, during the treatment period
the total number of CD3+, CD4+ and
CD8+ T-lymphocytes remained stable
whereas in placebo group they decreased
slowly. At the end of the study, total num-
ber of CD8+ T-lymphocytes was signifi-
cantly higher compared to placebo group
(V3: 0.85 ± 0.39.109/l vs 0.75 ± 0.28.109/
l, P < 0.05). Imunoglukan also increased
the number of NK-cells (V1 vs V2:
0.33 ± 0.21.109/l vs 0.39 ± 0.31.109/l,
P < 0.05).
Conclusion: Our study provided relevant
evidence for the immunomodulatory activ-
ity of imunoglukan (beta-glucan) from
Pleurotus ostreatus in children with RRTI.
Such complex immunomodulatory activity
of none beta-glucan has not been showed
and studied before. This could be basis for
its clinical efficacy in children with RRTI.
This is the first double-blind, placebo-con-
trolled study in children with RRTI aimed
on the changes of immune parameters dur-
ing the treatment with beta-glucans.
381
Effects of atorvastatin on relevant
functions of human T lymphocytes
in vitro
Turuntas, V1; Colic, M2; Vasilic, S2
1Pediatrics/Allergology, Clinical center East Sarajevo-
Foca, Foca, Bosnia and Herzegovina; 2Institute for
Medical Research, MMA Belgrade, Belgrade, Serbia
Statins, which are used as cholesterol-lower-
ing agents, have anti-inflammatory and
immune-regulating properties. Little com-
prehensive analysis has been made to inves-
tigate the impact of statins on human T
lymphocytes function, their proliferation
and secretory function. We also investigated
if the immunossupresive effects of statins
are due to their impact on human T lym-
phocytes or proffesional antigen-presenting
dendritic cells (DC) alone, or both. Ator-
vastatin reduced phytohemagglutinin-stimu-
lated lymphocyte proliferation and cytokine
release, dose dependently. The results
showed that atorvastatin significantly
decreased the expression of cytokines IL-2,
IL-6, IFN-gama and TNF-alfa, and signifi-
cantly increased levels of antiinflammatory
cytokine IL-10. Thus the balance between
pro-inflammatory and anti-inflammatory
cytokines was shifted in the anti-inflamma-
tory direction, as shown by a significantly
decreased TNF-alfa/IL-10 ratio. Atorvasta-
tin promoted the generation of Treg from
primary T cells and enhanced preexisting
Treg function. Atorvastatin decreased the
CD80, CD86, CD40, CD54 and HLA-DR
expression, and significantly stimulated
CD1a, CD14 and CD83 expression in
immature DC. In LPS timulated DC,
expression of CD25 and mean fluorescence
intensity of HLA-DR, CD80, CD86, CD40,
CD25 were significantly reduced by ator-
vastatin, while the expression of HLA-DR
and CD40 was similar to that in control
cells. Atorvastatin significantly inhibited the
basal secretion of TNF-alfa, but not
affected IL-10, IL-12 and IL-18 secretion.
Release of IL-18 from LPS-stimulated DC
was inhibited, but inhibition of IL-12 pro-
duction and increase in IL-10 production
were not statistically significant. Atorvasta-
tin decreased the level of IFN-gama,
increase the level of IL-10, and not affected
the level of IL-4 in DC culture supernatants
when the atorvastatin was added during
maturation. If the atorvastatin was added in
monocite culture, no significant change in
cytokine level was observed. Our data pro-
vide strong evidence that atorvastatin can
act as an immunomodulator by reducing T
lymphocite proliferation and cytokine secre-
tion, inhibiting the immune response of
Th1, decreasing the expression of co-stimu-
latory molecules, and up-regulating the
number of T regulatory cells, which can help
in better understanding how to take advan-
tage of these new mechanistic insights in
increased use of statins in therapeutic strat-
egy in different immune/inflamatory disor-
ders in future.
382
Effect of CD180 ligation or CD180/IgM
sequential ligation on signalling
pathways in chronic lymphocytic
leukaemia cells
Kulikova, N1; Lydyard, P2; Vispute, K2; Steele, A3;
Tsertsvadze, T1; Botchorishvili, E1; Tsagareishvili, P1;
Mitskevich, N1; Nathwani, A4; Clark, E5; Porakishvili, N2
1Immunology&Microbiology, Iv. Javakhishvili Tbilisi
State University, Tbilisi, Georgia; 2School of Life
Sciences, University of Westminster, London, United
Kingdom; 3University of Southampton, Southampton,
United Kingdom; 4UCL Cancer Institute, London, United
Kingdom; 5University of Washington, Seattle, WA,
United States
Background: CD180/RP105 is a membrane-
associated orphan receptor that belongs to
the TLR family, is expressed by professional
antigen-presenting cells, and drives normal
B-cell activation and proliferation. We have
previously shown that approximately 60%
of CLL clones expressed surface CD180,
but only half of these clones responded
to ligation with anti-CD180 monoclonal
antibody (mAb) by activation, cycling, and
reduced basal apoptosis. These CLL clones
upregulated CD86 and Ki-67 upon stimula-
tion with anti-CD180 mAb and were termed
responders (R). In contrast, CD180+CLL
samples that failed to respond to anti-
CD180 mAb, despite expressing a high den-
sity of CD180 receptors, were termed non-
responders (NR).
Method: R-CLL clones from 22 CLL
patients were stimulated with anti-CD180
Poster Discussion Session 12 – Allergic and non-allergic inflammatory mechanisms
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 161
mAb for 30 min, and then with F(ab¢)2
fragments of goat anti-human IgM anti-
bodies for another 30 min. Fourteen R-
CLL clones were stimulated with anti-
CD180 mAb for various periods of time:
30 min–24 h. Stimulated cells were stained
with anti-CD19~PE/Cy5 mAb, fixed, per-
meabilized, counterstained with mAb
(FITC) to phosphorylated-Akt (pAkt), and
analysed by flow cytometry.
Result: Ligation of CD180 and sIgM mea-
sured by activation of Akt was heteroge-
neous, but the percentages of pAkt+ cells
were significantly above that of unstimu-
lated cultures: 25.3 ± 9.3% (CD180:
40.2 ± 22.5%, P = 0.0034 IgM: 41.8 ±
20.4%, P = 0.001). However, the percent-
ages of pAkt+ cells following sequential
ligation of CD180 and sIgM, dropped sig-
nificantly down to basal levels, compared to
the ligation of CD180 or sIgM alone
(27.7 ± 18.6%, P = 0.048). We showed
that R-CLL clones differed in the speed of
signal transduction downstream to Akt thus
identifying early responders (30 min stimu-
lation, ER) and late responders (24 h stimu-
lation, LR). Interestingly, ER-CLL cells
expressed significantly higher density of
CD79b compared to LR: 2273 ± 1664 vs
760 ± 532 antibody relative binding sites
(RBS)/cell, P = 0.039. ER were also
enriched for IgM+ cells, compared to LR:
66.2 ± 13.1% vs 32.3 ± 31.1%, P = 0.022,
indicating involvement of IgM/CD79b in
CD180 signal transduction.
Conclusion: Irrespective of the category, an
increase in pAkt was seen in all R-CLL cells,
and we now consider activation of Akt to be
a more precise indicator of R-CLL type,
compared to CD86 upregulation. We hy-
pothesise that CD180 and sIgM operate via
joint pathways which become anergic after
one of the pathways is activated.
383
Omalizumab in urticarial vasculitis
associated with Churg-Strauss syndrome:
a 12 month follow-up
Varricchi, G; Detoraki, A; Liccardo, B; Spadaro, G; de
Paulis, A; Marone, G; Triggiani, M
Division of Allergy and Clinical Immunology and Center
for Basic and Clinical Immunology Research (CISI),
University of Federico II, Naples, Italy
Background: Churg-Strauss Syndrome (CCS)
is a rare systemic necrotizing small vessel
vasculitis associated with bronchial asthma,
peripheral blood eosinophilia and eosino-
philic lung infiltration. Skin changes com-
patible with vasculitis are present in about
75% of patients. Previous reports suggest
that patients with CSS can be treated with
anti-IgE (omalizumab) in addition to con-
ventional therapy to achieve asthma con-
trol. Here we report the efficacy of a 12-
month treatment with omalizumab in a
patient with CSS characterised by severe
asthma, rhinosinusitis and urticarial vasculi-
tis.
Method: A 44 year old Caucasian female
with a 5 year history of severe asthma,
chronic urticaria and mild eosinophilia
(1100/ml) was evaluated for possible CSS.
Total serum IgE was 662 KU/l with posi-
tive skin prick tests for dust mites. Bron-
chial asthma was not controlled and FEV1
was 60% despite treatment with budeso-
nide (640 lg/die) and formoterol (18 lg/
die). Diffuse and confluent urticarial rash
occurred in the last 6 months before evalu-
ation and skin biopsy revealed a leukocyto-
clastic vasculitis. The urticarial rash
responded neither to prednisone (10 mg/
die) and rupatadin (10 mg/die) nor to
immunosuppressive agents (cyclosporin
200 mg/die or azathioprin 100 mg/die).
The patient was treated, as add-on ther-
apy, with omalizumab (300 mg s.c. every
2 weeks) accordingly to total IgE and
weight parameters reported in the drug
information leaflet.
Result: After 12 months of treatment the
patient reported a significant improvement
in asthma control with 50% reduction of
nocturnal awakenings and asthma exacer-
bations and a major FEV1 improvement
(101% at 16 weeks, 103% at 24 weeks,
105% at 48 weeks). Eosinophil count was
reduced to 600/ll after 24 weeks and never
reincreased until the 48 week evaluation. A
75% reduction of oral prednisone was reg-
istered after 8 weeks of treatment; predni-
sone was completely withdrawn after
24 weeks of treatment. Importantly, urti-
carial lesions disappeared after the first
injection of omalizumab. Omalizumab
injections were well tolerated and no
adverse event was recorded.
Conclusion: This case suggests that oma-
lizumab can be beneficial and safe in
patients affected by CSS with moderate to
severe asthma and urticarial vasculitis. In
addition to its effect on serum IgE, efficacy
of omalizumab in CSS may be related to
an inhibitory effect on blood eosinophilia.
384
A 1-year-old girl with STAT-1
gain-of-function mutation treated with
haematopoietic stem cell transplantation:
a 2 month follow-up
Aldave, J
Centro de Alergias y Asma ‘Themme Afan’, Primary
Immunodeficiencies, Lima, Peru
Background: Primary Immunodeficiencies
(PID) are inherited disorders of immune
system function that predispose affected
individuals to increased rate and severity of
infection, immune dysregulation with auto-
immune disease, and malignancy. In Peru
there is a sub diagnosis and report of these
diseases.
Method: I present a case of a 1-year-old girl
with recurrent mucocutaneous candidiasis
and recurrent diarrhea by gram negative
bacteria since 3 days old. The patient
needed antifungal therapy all the time.
Without antifungals the patient developed
dysphonia and persistent cough, suggestive
of laryngeal and lower respiratory tract
involvement. Sepsis in one oportunity. We
sent peripheral blood sample to the Labora-
toire de Genetique Humaine des Maladies
Infectieuses (Paris, France), where a diagno-
sis of STAT1 gain-of-function mutation was
made.
Result: The patient had progressive resis-
tance to antifungal therapy and failure to
thrive. At 1 year old, due to her poor prog-
nosis, we performed a full-matched hemato-
poietic stem cell transplantation (HSCT).
The donor was her 9-year-old brother. Two
months later the patients looks notably
healthier, has gained two kilograms, and
doesn’t receive antifungals for about
1 month, without Candida recurrence. We
are following the patient carefully.
Conclusion: HSCT may be an appropriate
therapy for patients with STAT1 gain-of-
function mutation, especially in the context
of antifungal resistance and poor progno-
sis. There is a huge work to do in the field
of PI in Peru. There has been until now a
subdiagnosis and subreport of PI, but we
have recently started working hard in pur-
pose of giving affected patients a specific
diagnosis and appropriate treatment.
385
Serological IgE anti-Ascaris lumbricoides
response is associated to a T1D
diagnostic but not for juvenile idiopathic
arthritis in Colombian patients
Navarro, E1; Mendoza, D2; Del Toro, K3; Revolledo, E3;
Iglesias, A4; Egea, E1; Garavito, G1
1Division ciencias de la Salud, Universidad del Norte,
Barranquilla, Colombia; 2Facultad de Medicina,
Universidad del Magdalena, Santa Marta, Colombia;3Ambulatories Pediatric Centers, Barranquilla,
Colombia; 4Universidad Nacional de Colombia,
Bogota, Colombia
Background: Type 1 Diabetes (T1D) and
Juvenile Idiopathic Arthritis (JIA) are
influenced by both genetic and environ-
mental factors. The increased prevalence of
T1D is not explained in an expeditious
manner by the genetic load alone, high-
lighting the influence of ecological factors,
such as Ascaris lumbricoides infection (Al).
The aim of our study was to examine the
total IgE response and IgE-anti-Al in
patients with T1D and JIA and compared
to a control group.
Poster Discussion Session 12 – Allergic and non-allergic inflammatory mechanisms
162 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
Method: This is an analytical, descriptive,
case-control sample with two group of
patients: 53 diagnosed with T1D, 54
patients with JIA. One hundred and three
healthy individuals was enrolled as a con-
trols. Total IgE levels were measured with
a commercial kit. Levels of specific IgE
againts Df and Al were measured by a in
house ELISA Indirect test. Specific IgE
against Df was determined in all of sub-
jects as an internal control of this test.
Result: In the T1D group of patients 28/53
(53%) showed elevated levels of total IgE.
32/53 (60%) T1D patients were positive
for IgE anti-Al and 9/53 (17%) of them
were positive for IgE anti Df. In the JIA
group of patients 22/54 (40.7%) showed
elevated levels of total IgE, and 4/54
(7.3%) were positive for IgE antiAL and
15/54 (27.4%) were positive for IgE anti
Df. In the control group 29/103 (28%)
showed elevation of total IgE, 30/103
(29%) had high level of IgE antiDf and
15/103 (14%) had a positive IgE anti-Al.
There was a Significant differences between
the concentrations of total IgE in the
groups of JIA and T1D patients comparat-
ed with controls (P = 0.0038).
Conclusion: In This work a greater number
of subjects suffering from these two dis-
eases had elevated total IgE levels when
compared with the control group. The
results also suggest an association between
a positive response of IgE anti Al and the
clinical diagnosis of T1D, but not by JIA
T1D and JIA are autoimmune diseases
characterized by a Th1 profile, however
infection with helmints as Al could polar-
ize LT toward a Th2 profile in the T1D
that could explains these results. More
studies should be performed to validate
our results.
386
Autosomal recessive hyper-IgE syndrome
caused by DOCK8 mutation presenting
predominantly with eczema, asthma and
food allergy
Cavkaytar, O1; Cagdas Ayvaz, D2; Keskin, O3;
Arik Yilmaz, E1; Buyuktiryaki, B1; Sahiner, U1; Yavuz, S1;
Tuncer, A1; Sackesen, C1; Sanal, O1
1Pediatric Allergy and Asthma Unit, Hacettepe
University Ihsan Dogramaci Childrens’ Hospital, Ankara,
Turkey; 2Pediatric Immunology Unit, Hacettepe
University Ihsan Dogramaci Childrens’ Hospital, Ankara,
Turkey; 3Department of Pediatric Immunology and
Allergic Diseases, Gaziantep University Faculty of
Medicine,Gaziantep, Turkey
Background: Autosomal recessive Hyper-
IgE syndrome is characterized with
eczema, recurrent pneumonia, viral skin
infections and high IgE levels. Atopic der-
matitis may begin at as early as the neona-
tal period and then clinical features of
asthma and anaphylaxis and may super-
vene. Herein a DOCK8 deficient patient
whose parents’ complaints were predomi-
nantly asthma and food allergy is reported
Case presentation: A 7-year old girl was
referred to our hospitaldue to refractory
eosinophilia and eczematous lesions ongo-
ing from 15th day of life. She hadangioe-
dema of the lips and increase in the severity
of eczema after milk and egg ingestion dur-
ing infancy. Shehad similar symptoms after
ingestion of fish, kiwi hazelnut and wheat
containing foods in the following years. She
had episodic wheezing attacks since she was
1 year old and had been taking inhaled cor-
ticosteroid, montelukaston a daily basis and
inhaled salbutamol treatment on demand.
On her past medical history she had recur-
rent pneumonia, acute otitis media, herpes
labialis infections and had been hospitalized
for pyoderma after a zona zoster infection
recently. On physical examination her
height and weight were below 3rd percentile,
she had diffuse and severe atopic dermatitis,
lichenification on both antecubital fossae,
vesicles and brownish crusts forming a lin-
ear pattern on both sides of anterior and
posterior thoracic wall. Laboratory findings
showed low IgM, very high IgE levels,
eosinophilia, low antibody titers against
Hepatitis B vaccine, low isohemagglutinin
levels and low percentages of CD3+ and
CD4+lymphocytes. Lymphocyte prolifera-
tion test showed half of the control’s levels
after stimulation with conconavalin A and
phytohemaglutinin. With these findings
DOCK8 deficiency was suspected and the
homozygous mutation was established by
sequence analysis. Matched unrelated donor
has been found recently andstem cell
transplantation is planned.
Conclusion: In the presence of recurrent in-
fectionsprimary immunodeficiency must be
suspected in patiens with asthma, food
allergy and eczematous findings. Autoso-
mal recessive Hyper-IgE syndrome caused
by DOCK8 mutation is differentiated from
the other immunodeficiencies with compan-
ion of recurrent viral (HPV, HSV) cutane-
ous infections and food allergy. Stem cell
transplantation is the only treatment
option and must be done as soon as possi-
ble in these patients.
387
DOCK8 deficiency and diagnostic
guidelines for hyper-IgE syndromes
Engelhardt, K1; Gertz, E2; Keles, S3; Schaffer, A2; Ceja,
R3; Sassi, A4; Graham, L1; Massaad, M5; Mellouli, F6;
Benmustapha, I4; Khemiri, M7; Kilic, S8; Etzioni, A9;
Freeman, A10; Thiel, J11; Schulze, I11; Al-Herz, W12;
Metin, A13; Sanal, O14; Yeganeh, M15; Niehues, T16;
Siepermann, K16; Weinspach, S17; Unal, E18; Patiroglu,
T18; Dasouki, M19; Yilmaz, M20; Genel, F21; Aytekin, C22;
Kutukculer, N23; Somer, A24; Kilic, M25; Reisli, I26;
Camcioglu, Y27; Gennery, A28; Cant, A28; Jones, A29;
Gaspar, H29; Pietrogrande, M30; Baz, Z31; Al-Tamemi,
S32; Lougaris, V33; Lefranc, G34; Megarbane, A35;
Boutros, J36; Galal, N36; Bejaoui, M6; Barbouche, M4;
Geha, R37; Chatila, T38; Grimbacher, B39
1Department of Immunology and Molecular Pathology,
University College London, London, United Kingdom;2National Institutes of Health, National Center for
Biotechnology Information, Bethesda, United States;3Division of Immunology, Allergy and Rheumatology,
Department of Pediatrics, David Geffen School of
Medicine, University of California at Los Angeles, Los
Angeles, CA, United States; 4Department of
Immunology, Institut Pasteur de Tunis, Tunis, Tunisia;5Immunology Division, Children’s Hospital Boston,
Boston, MA, United States; 6Department of Pediatrics,
Bone Marrow Transplantation Center, Tunis, Tunisia;7Department of Pediatrics, Children’s Hospital, Tunis,
Tunisia; 8Department of Pediatric Immunology, Faculty
of Medicine, Uludag University, Bursa, Turkey;9Rambam Health Care Campus and Rappaport Faculty
of Medicine, Technion-Israel Institute of Technology,
Meyer’s Children Hospital, Haifa, Israel; 10National
Institutes of Health, Laboratory of Clinical Infectious
Diseases, Bethesda, MD. United States; 11University
Medical Center Freiburg, Centre of Chronic
Immunodeficiency, Freiburg, Germany; 12Department
of Pediatrics, Al-Sabah Hospital, Kuwait, Kuwait;13Pediatric Immunology Unit, SB Ankara Diskapi
Children’s Hospital, Ankara, Turkey; 14Immunology
Division, Hacettepe University Children’s Hospital,
Ankara, Turkey; 15Immunology, Asthma and Allergy
Research Institute, Children Medical Centre, Tehran
University of Medical Sciences, Tehran, Islamic
Republic of Iran; 16HELIOS Klinikum Krefeld, Zentrum
fuer Kinder-und Jugendmedizin, Krefeld, Germany;1718Department of Pediatric Oncology, Hematology and
Clinical Immunology, Center of Child and Adolescent
Medicine, Heinrich-Heine-University Dusseldorf,
Dusseldorf, Germany; 18Division of Pediatric
Immunology, Department of Pediatrics, Faculty of
Medicine, Erciyes University, Kayseri, Turkey;19Department of Pediatrics, University of Kansas
Medical Center, Kansas City, KS, United States;20Cukurova University, Adana, Turkey; 21Division of
Pediatric Immunology, Behcet Uz State Hospital, Izmir,
Turkey; 22Dr. Sami Ulus State Hospital, Ankara, Turkey;23Department of Pediatrics, Ege University Faculty of
Medicine, Izmir, Turkey; 24Division of Infectious
Diseases and Immunology, Istanbul Medical Faculty,
Istanbul University, Istanbul, Turkey; 25Firat University,
Elazig, Turkey; 26Division of Pediatric Allergy and
Immunology, Selcuk University, Konya, Turkey;27Division of Pediatric Allergy-Immunology and
Infectious Diseases, Cerrahpasa Medical Faculty,
Istanbul University, Istanbul, Turkey; 28Children’s Bone
Marrow Transplant Unit, University of Newcastle upon
Tyne, Newcastle upon Tyne, United Kingdom; 29Great
Ormond Street Hospital for Children, London, United
Kingdom; 30Department of Maternal and Pediatric
Sciences, University of Milan, Fondazione IRCCS
Policlinico Milano, Milan, Italy; 31Department of
Pediatrics, St George Hospital University Medical
Center, Beirut, Lebanon; 32Department of Pediatrics,
Sultan Qaboos University, Muscat, Oman;33Department of Pediatrics and Institute of Molecular
Medicine, University of Brescia, Brescia, Italy;34University Montpellier 2 and CNRS Institute of Human
Genetics, Montpellier, France; 35Medical Genetics Unit,
Saint Joseph University, Beirut, Lebanon; 36Primary
Immunodeficiency Clinic, Cairo University, Specialized
Pediatric Hospital, Cairo, Egypt; 37Division of
Immunology, Children’s Hospital Boston, Boston, MA,
United States; 38Division of Immunology, Allergy and
Rheumatology, Department of Pediatrics, David Geffen
School of Medicine, University of California at Los
Angeles, Los Angeles, CA, United States; 39Center of
Chronic Immunodeficiency, University Medical Center
Freiburg, Freiburg, Germany
Background: Hyper-IgE syndromes (HIES)
are rare primary immunodeficiencies.
Autosomal-dominant (AD) HIES due to
STAT3 defects and autosomal-recessive
(AR) HIES due to DOCK8 defects share
most but not all clinical features. Distin-
Poster Discussion Session 12 – Allergic and non-allergic inflammatory mechanisms
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 163
guishing AR- from AD-HIES is of clinical
importance (e.g. indication for transplanta-
tion).
Method: DOCK8 was analysed in 74 AR-
HIES patients. Of those, clinical data were
collected and compared between 36
patients with and 10 without DOCK8
mutation using regression analysis. The 36
patients with DOCK8 mutation were also
compared to 58 AD-HIES patients with
STAT3 mutation. A machine-learning
approach was used to identify features that
better predict a DOCK8 or STAT3 muta-
tion, respectively.
Result: We found mutations in DOCK8 in
55 patients. In 41 families, mainly from the
Middle East, we found 27 deletions, one 2-
bp insertion, 12 splice site or nonsense
mutations, and one gene transcription fail-
ure. All but one DOCK8-deficient patient
had high serum levels of IgE (mean
14 793 IU/ml), frequent respiratory tract
infections and eczema. Most had hypereo-
sinophilia (98%), skin abscesses (65%),
candidiasis (69%), multiple allergies (73%),
and severe viral infections (77%), mainly
with herpes viruses, molluscum contagio-
sum, and HPV. Sixteen patients had
asthma, 16 bronchiectasis, and one pneu-
matoceles. Failure to thrive (57%) and
neurological complications (35%) were also
present. Extra-immune features were less
prevalent: 13 patients had facial abnormal-
ities (mainly mild), seven retained primary
teeth, five hyperflexibility, and two frac-
tures after minor trauma. Several features
were significant in distinguishing DOCK8
from STAT3 patients. Using features with
uncorrected P-value of at most 0.1, we
generated a DOCK8 scoring sheet includ-
ing eosinophilia, upper respiratory infec-
tions (more present in DOCK8 patients),
lung abnormalities, retained primary teeth,
and fractures with minimal trauma (more
present in STAT3 patients). The error rate
was 12.7% with sensitivity for predicting a
DOCK8 mutation of 88.9% and specificity
of 86.2%. No significant difference could
be found between the phenotype of AR-
HIES patients with or without DOCK8
mutation.
Conclusion: After the diagnosis of HIES
has been established, looking at further
specific features such as consanguinity of
parents, severe viral infections and aller-
gies, as well as determining the DOCK8
and STAT3 scores, helps distinguish
STAT3 from DOCK8 deficiency.
388
Dedicator of cytokinesis 8 deficiency in
two brothers with hyper-IgE syndrome at
the two ends of the disease spectrum
AL-Zahrani, D1; AL-Raddadi, A2; Massaad, M3; Geha, R3
1Immunology/Allergy, Pediatric Department, King
Abdulaziz Medical City-WR, Jeddah, Saudi Arabia;2Dermatology Department, King Abdulaziz Medical
City-WR, Jeddah, Saudi Arabia; 3Allergy/Immunology/
Rheumatology/Dermatology, Harvard Medical School,
Boston, MA, United States
Background: Dedicator of cytokinesis 8
(DOCK8) is a type of Hyper-IgE syn-
drome that manifests recurrent sinopulmo-
nary and skin infections, mucocutaneous
candidiasis, anaphylaxis, eczema, asthma
and increased risk for malignancy.
Method: Two brothers with DOCK8 defi-
ciency are described. From the 1st year of
life the 10-year-old manifested eczema,
asthma, molluscum contagiosum, recurrent
skin abscesses, one episode of sepsis and
severe chickenpox that required hospital-
ization for 2-weeks. He presented to us
with severe generalized warts and recurrent
purulent otitis media. The 6-year-old
brother has only four small warts. Immu-
nologic assessment was undertaken.
Result: The investigations for the 10-year-
old showed; leucocytes 5100 cells/ll, neu-
trophils 2550 cells/ll, monocytes
360 · cells/ll, lymphocytes1390 · cells/ll
and eosinophils 740 · cells/ll. Lymphocyte
subset (cells/ll); CD3+ 960, CD4+ 650,
CD8+ 290, CD19+ 330 and CD16+ 70.
Lymphocytes expression of CD69 was 40%
of control in response to PHA. IgG 24.6 g/l,
IgA 3.8 g/l, IgM 0.53 and IgE 28 375 IU/
ml. VZV and vaccines serology were nega-
tive. The investigation for the 6-year-old
showed; leucocytes 6000 · cells/ll, neu-
trophils 1260 · cells/ll, monocytes 630 ·
cells/ll, lymphocytes 3660 · cells/ll and eo-
sinophils 410 · cells/ll. Lymphocyte subset
(cells/ll); CD3+ 2670, CD4+ 1460, CD8+
1060, CD19+ 440 and CD16+ 510. Lym-
phocytes expression of CD69 was 36% of
control in response to PHA. IgG 9.76 g/l,
IgA 0.75 g/l, IgM 0.78 and IgE 223 IU/ml.
Vaccines serology were strongly positive.
Both patients have absent DOCK8 expres-
sion on PBMCs and EBV-B-cell line. Dis-
cussion: the 10-year-old has significant
severe infections and his investigations con-
firmed combined primary immunodeficiency
with high IgE level. While his brother has
normal lymphocyte subset, strong response
to vaccines and no infection history of sig-
nificance apart from what might be consid-
ered as a transient warts with slightly
elevated IgE. His low CD69 expression
could be due to recent viral throat infection.
Conclusion: DOCK8 deficiency is an auto-
somal recessive form of hyper-IgE syn-
drome that can cause significant
immunodeficiency making the patient sus-
ceptible to frequent life-threatening infec-
tions. Even though both brothers have
DOCK8 deficiency, but its clinical manifes-
tations are variable. Thus, the disease man-
ifestations range from mild to severe
category both at cellular and clinical levels
even when it affects members from the
same family.
389
H2R knockout mice develop more severe
allergic airway inflammation
independently of decreased numbers of
regulatory T-cells
Ferstl, R; Ziegler, M; Frei, R; Konieczna, P; Akdis, C;
O’Mahony, L
Swiss Institute of Allergy and Asthma Research, Davos,
Switzerland
Background: In the last decades the number
of persons suffering from Allergy and
Asthma has increased dramatically to over
300 million people in the western popula-
tion. In order to develop new and more
effective therapies, a better understanding
of the molecular mechanisms underpinning
this chronic disease is required. Histamine,
released by activated mast cells and immune
cells, causes many of the symptoms associ-
ated with allergy and asthma. Histamine is
recognised by four different Histamine
receptors (H1R-H4R), each inducing its
own signaling cascade. H2R is recognised as
an immune regulatory receptor but its role
in allergy is not well described.
Aim: To investigate the role of H2R in a
murine model of allergic airway inflamma-
tion.
Method: Female H2R-/- mice and BALB/c
wild-type mice were sensitized i.p. and
OVA-aerosol challenged to induce an aller-
gic airway inflammation similar to asthma.
Result: Mice lacking H2R showed signifi-
cant increased cell numbers in bronchoalve-
olar lavages, mainly due to elevated
eosinophil numbers. Lung histology con-
firmed increased inflammatory scores in
knockout animals. Furthermore, in vitro re-
stimulation with OVA induced higher Th1
and Th2 cytokine release from single cell
suspensions from lungs, spleen and lymph-
nodes. All together, H2R-/- mice develop
more severe allergic airway inflammation.
CD4+CD25+Foxp3+ Treg numbers were
Poster Discussion Session 12 – Allergic and non-allergic inflammatory mechanisms
164 Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S
evaluated by flow cytometry in Peyer’s
patch, mesenteric lymph nodes, spleen,
lung-draining lymph nodes, lung tissue and
bronchoalveolar lavages. Treg numbers
were similar in wildtype and knockout ani-
mals at all sites, with a tendency towards
increased Treg numbers in H2R-/- inflam-
med lung, perhaps related to altered anti-
inflammatory compensatory mechanisms in
the H2R-/- animals.
Conclusion: H2R is an important immuno-
regulatory receptor that influences the
severity of allergic airway inflammation in
murine models. In addition, the increased
severity of disease was not associated with
decreased Treg numbers suggesting that
other immune cell populations may be
directly influenced by histamine signaling
through the H2R.
Poster Discussion Session 12 – Allergic and non-allergic inflammatory mechanisms
Allergy 67, Suppl. 96 (2012): 98–165 � 2012 John Wiley & Sons A/S 165