Serological IgE anti-Ascaris lumbricoides response is associated to a T1D diagnostic but not for...

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



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.



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.



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.




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).


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.



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


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



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



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?




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


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


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



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



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


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



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.




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,


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


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



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



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



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.




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,


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


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



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



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.




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


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


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


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


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


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



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



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.




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


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


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.



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



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



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.




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)


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


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



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



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.




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.


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


(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.



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.



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-



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


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.




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


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


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



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.



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.




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,


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


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.



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



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



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.




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-


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


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.



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-



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



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.



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