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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 1 Department of Medical Sciences: Clinical Physiology, Uppsala University, Uppsala, Sweden; 2Department of Medical Sciences: Respiratory Medicine and Allergology, Uppsala University, Uppsala, Sweden; 3 Immunodiagnostics, 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 airways inflammation or systemic inflammation in subjects with allergic asthma has been little studied. Method: Within the frame of an industryacademy collaboration on minimally-invasive 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 sensitisation to only aeroallergens (n = 59, median age 15 years) and atopic with IgE sensitisation to both aero- and food allergens (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 compared 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 sensitized to both aero- and food allergens had higher levels of FeNO and serum ECP 98

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 asymptomatic, 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 previous 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 previous 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 ovomucoid raised from a median of 3–8 mm (P < 0.001), whereas infants allergic to raw egg maintained a negative SPT to ovomucoid. 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 negative in 14 patients (52%). In the rest (13 patients, 48%) the trend was to maintain or decrease SPT. Eighteen patients had negative 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 component 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 allergenicity of different species of fish, Bangladeshi 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.

Allergy 67, Suppl. 96 (2012): 98–165 2012 John Wiley & Sons A/S

Poster Discussion Session 1 – Childhood and adolescent and asthma

Result: There was significant cross-reactivity 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%). Epipen (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 modified skin prick testing to cod, can be used as a guide for diagnosing allergy to Bangladeshi 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 1 Danone 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, characterized 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 distribution 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), thymus- 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 reference 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 IgfLC 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 difference in the biology of EoE to the already described non synonymous polymorphism 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 sensitisation 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 dermatitis were examined. Twenty-five agematched healthy infants were included in the control group. Total IgE, allergen-specific 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 accompanied 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 Pediatrics, Hanyang University Guri Hospital, Guri, Korea; 2Hanyang University Seoul Hospital, Seoul, Korea

1

Background: A number of studies has outlined mechanisms by which mycoplasma


infection may promote allergic lung inflammation and airway remodeling. In addition, there is increasing evidence from human studies suggesting that atypical bacterial infections contribute to asthma exacerbations, 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 nonatopic children with viral pneumonia (Group 4). The change of serum levels of interleukin (IL)-5, IL-13, vascular endothelial 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 concentration were significantly increased at clinical recovery compared to the serum concentration at admission. Conclusion: The outcomes of the present study implied the increased changes of VEGF and IL-5 during Mycoplasma infection may be associated with the mechanism by which the Mycoplasma pneumoniae contribute to the development of hypersensitivity during acute mycoplasma pneumonia. 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 particularly 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. 99


Aims: To identify risk factors associated with incidence of common childhood infections 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 prospective 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 session 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 traditional statistics and the data-driven Sparse Principal Component Analysis. Result: Three hundred and thirty-four children had complete follow-up for this analysis. 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 variant was associated with increased the risk of lower respiratory tract infections. Conclusion: Otherwise healthy children experience a mean frequency of five episodes 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 interindividual 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 1 Pediatric Allergy and Immunology, Gaziantep University Hospital, Gaziantep, Turkey; 2Pediatrics, Gaziantep University Hospital, Gaziantep, Turkey; 3 Pulmonology, Gaziantep University Hospital, Gaziantep, Turkey; 4Biostatistics, Gaziantep University Hospital, Gaziantep, Turkey

Background: Inflammation and oxidative stress are essential parts of asthma pathophysiology. A variety of methods are used to measure intense inflammation in the airways of asthmatics. Exhaled breath condensate (EBC) is a completely noninvasive method for the collection of airway secretions. Recently, it has been shown that asthma control test (ACT) is a good measure which may be used in the evaluation of asthmatic patients. It is not well known if ACT score and asthma control level 100

correlates with the airway inflammation in asthma. There is no study evaluating the relationship between exhaled cysteinyl leukotrienes (Cys-LTs) and 8-isoprostane levels 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 function tests and asthma severity and asthma control level were assesed according to GINA. EBC was collected and Cys-LTs and 8-isoprostane concentrations were determined using a specific immunoassay kit. Result: Exhaled 8-isoprostane levels in patients with moderate persistent asthma [114 (55–146) pg] was higher than mild persistent 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 CysLTs and serum IgE levels (P = 0.028, r = 0.467). In addition, exhaled 8-isoprostane levels correlated negatively with bronchodilator 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 exacerbation 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; Bjo¨rksteń, B2; Jenmalm, M1 1 Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, 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 lipopolysaccharide (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 probiotic 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 dependent 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. Probiotic supplementation may be associated with an increased immunoregulatory capacity during infancy, in line with our previous findings showing allergen hyporesponsiveness 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 1 NUTRIM School of Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands; 2Pediatric Pneumology and Immunology, Charite´ Universita¨tsmedizin, Berlin, Germany; 3Symbiopharm Herborn, Herborn, Germany; 4 Ruhr-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 composition, as a result of changed lifestyles in westernized countries, may have disrupted mechanisms involved in the development of immunological tolerance. The aim of the present 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 placebo-controlled trial (n = 606) on the primary 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 intervention) and of 7 months (n = 500, end of intervention) were subjected to real-time PCRs for the enumeration of bifidobacteria, 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 G were evaluated in AR patients and compared with controls. In a case-control study, 155 AR patients and 163 allergy-free controls were genotyped using polymerase chain reaction sequence-specific primer (PCR-SSP) technique. 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 polymorphism 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 hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation of adult Icelandic horses (1st generation) 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 indicate 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 function of equine FoxP3+CD4+CD25high cells and shown that upon stimulation with Culicoides extract, the proportion of FoxP3+CD4+CD25high cells was significantly 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 tolerance. Our aim was to characterize circulating 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 yearlings (1 year old) were examined for the presence of circulating FoxP3+CD4+CD25high cells using flowcytometry. 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 significantly 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 environmental 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 hypothesis’. Interestingly, this protection effect has in particular been observed in children born and raised in Alpine farming households who are protected from asthma. Animal 113

Poster Discussion Session 3 – Leukocyte activation in allergy

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 phenotype 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 cytokines 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 inflammation. 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+/FoxP3and 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 (gammaIFN), 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 Lo´pez, S1; Pozo, D1; Fernańdez, T2; Rodrı´guez-Bada, J2; Meleńdez, L2; Mayorga, C2; Blanca, M3 1 Department of Cell Therapy CABIMER-Andalusian Center for Molecular Biology and Regenerative Medicine, University of Seville, Sev, Seville, Spain; 2 Research Laboratory, Carlos Haya Hospital-Fundacioń IMABIS, Ma´laga, Spain; 3Allergy Service, Carlos Haya Hospital, Ma´laga, Spain

Background: Human invariant natural killer T cells (iNKT cells) are a unique population of T-cells that express a semiinvariantly rearranged T cell receptor (TCR) and play an important role in regulating the development of allergy. Objective: To characterize the iNKT population in peripheral blood from allergic patients to house dust mite compared with the iNKT population from tolerant controls. 114

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 phenotypes 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 determined the percentage of the CD3+ T cell, CD4+, CD8+ T cell subsets and the CD4/CD8 ratio in PBMC from both allergic patients and tolerant controls. The allergic patients to house dust mite population did not differ significantly from the investigated tolerant controls. No statistical relationships were found between the frequency 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 allergic 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, antigen 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, University of Zurich, Davos, Switzerland

Background: When using mouse models to study allergy and asthma it is important to quantitate allergen-specific IgE. Enzymelinked 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 experimental model. However, in common immunization procedures, large amounts of OVA specific IgG is also generated which could potentially interfere with IgE quantification. Method: There are currently two widespread, commercially available ELISAbased 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 detection using labelled OVA. Using monoclonal OVA-specific IgE we examined its recovery in both ELISA formats from serums of both immunized and non-immunized animals. As an improvement of ELISA, we have developed new instrumentation 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 similar 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 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.

115

Poster Discussion Session 4 Exploring food allergy: bench to bedside

259 Dehydrated egg-white, a future treatment for egg allergy Ruiz-Garcia, M; Haroun Diaz, E; Landivar Encalada, M; Torres Hernańdez, J; Sastre Domıńguez, J Allergy Department, Fundacioń Jimeńez Dıáz, 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 desensitisation was 7.88 ± 2.55 years-old. Adult’s mean age 28.33 ± 9.50 years-old. OVODES 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 challenge with OVO-DES NM, 11/17 had oral challenge with liquid egg and two patients didn’t have oral challenge prior to desensitisation. 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 consisted 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, broncospasm, dyspnea and abdominal pain) but was able to complete desensitisation. 116

Conclusion: The main problem was updosing 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 desensitisation 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 proteins (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 possible 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 restrictive diet. The eligibility criteria were: 1) confirmed IgE-mediated CMPA via a positive 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 clinical 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 population 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-lactalbumin, 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 negative for immediate and late onset reactions. Notably 4 and 7 months after the negative pHF OFC, two out of seven children experienced an anaphylactic reaction due to accidental exposure to CM. Conclusion: The results of our study indicate that the use of pHF is safe in the management of selected cases of IgE-mediated CMP allergic patients. Advantages of the use of pHF include lower cost and better taste. Further studies should be held to examine the role of pHF in the diet of some infants allergic to cow’s milk proteins. Such studies should also include a cost/benefit analysis of the use of hydrolyzed 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 1 Department of Paediatric and Adolescence Science, Regina Margherita Children’s Hospital, Turin, Italy; 2 CNR-Institute of Science of Food Production, Turin, Italy; 3Neonatal Unit, Department of Paediatric and Adolescence Science, University of Turin, Turin, Italy; 4 Koelliker – Missionari della Consolata Hospital, Turin, Italy

Background: Children with cows’ milk protein allergy (CMPA) who are not exclusively breast-fed require a formula or alternative diet, but there is no agreement among the international scientific community 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,


Poster Discussion Session 4 – Exploring food allergy: bench to bedside

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 comprised subjects with mild-to-severe clinical forms of CMPA. Native electrophoresis and immunoblotting were used to identify CM and DM cross-reactive proteins. Zscores 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 transferrin, 1,25-OH2 vitamin D and serum prealbumin) 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 allergic 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 ‘hypoallergenic’ food for treatment of CMPA, according to American Accademy od Paediatrics 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 alternative 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 recommend 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 usually 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 lowdose 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 immediate 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 diagnosis, underwent a successful desensitization 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 specific-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 induction (SOTI) started at the onset of IgEmediated CM allergy seems to be a promising approach, able to modify the natural increasing pattern of sensitisation to CM during the 2nd-3rd half-year period. Furthermore it seems to be easier, faster and safer to perform in infant age in respect to other analogue experiences in older children.

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 cutaneous 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 sensitization 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 outpatient 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 treatment 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 tolerance 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 professionals and dieticians when evaluating food related problems in order to reach a correct 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 Schneider 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

1

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 calcium intake during the preceding 6 months and blood tests for calcium, phosphorus, alakaline phosphatase, parathyroid hormone (PTH) and vitamin D levels. Bone 117


strength was evaluated using quantitative ultrasound of the radius (Omnisense 7000P; Sunlight Medical, Petach Tikva, Israel); z- score value of 2SD and more below the mean according to age and sex was considered pathological. As control group served children with asthma without milk allergy. Results: The study group included 81 patients [38 (47%) of them with asthma, 35 (43.2%) girls]. The control group included 27 patients [8 (29.6%) girls]. Mean age of the study group was 7.01 ± 3.08 years, compared to 11.59 ± 4.17 years of the control group (P < 0.001). No significant differences in BMI percentiles were identified. The mean daily calcium intakes of the cow milk allergic 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 zscore 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 recommended 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 significance. 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 calcium 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; Plaćido, J2; Coimbra, A2; Moreira, P1; Barros, R1 1 Faculty 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. 118

Method: In a cross-sectional manner measurements of anthropometry (weight, height, skinfold thickness, waist circumference, and classification according to CDC Growth Chart percentiles and z-scores), dietary 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, demographic 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 evaluation and dietary counseling is essential as complementary approach to food allergy treatment, to ensure adequate nutritional 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 1 Utrecht 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 hydrolysed formulae. However, no validated animal models are currently available to assess the residual sensitising capacity, although guinea 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 formulae. 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 temperature 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 immunoglobulins and mMCP-1. All protocols, test substances and procedures were standardised. Result: All participating research laboratories detected elevated levels of whey-specific IgE/IgG1/IgG2a, serum mMCP-1 (reflection of mast cell degranulation) and acute allergic skin responses in whey sensitised 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 multicenter validation process, partially hydrolysates 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-Ibań˜ez, C1; Rodrigues, A2; Lobo, C3; Taborda-Barata, L4 1 Allergy Department, Unidade Local de Sau´de de Castelo Branco, EPE, CICS, Health Sciences Research Centre, University of Beira Interior, Castelo Branco, Portugal; 2EPE, Emergency Department, Unidade Local de Sau´de de Castelo Branco, Castelo Branco, Portugal; 3 Clinical Pathology Department, Unidade Local de Sau´de 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. Therefore, the aim of the present study was to determine the prevalence of FA in patients



attending an Allergy Outpatient Department, 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 analyzed. 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 children 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 children and 27% of adults were diagnosed with FA to more than one foodstuff. Both groups reported urticaria as the most frequent FA reaction (FAR) upon ingestion of seafood, fish, egg and dry fruits, whereas oral allergic syndrome (OAS) was most frequently 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 Hospital 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 prevalence of FA using the new SPAIC tool in Portugal, the prevalence of FA is similar to that observed in other allergy departments reported in the literature. Urticaria and OAS were the most frequent symptoms. Seafood, fresh fruits and latexrelated 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

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 Questionnaire – Parent Form FAQLQ-PF 0– 12 years, DunnGalvin et all) was administered to parents by a psychologist. FAQLQ-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 questionnaire 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) compared to 43 children 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 (CMIgE > 0.35 kU/l). Among them, 349 (74.1%) were sensitized to casein (CasIgE > 0.35 kU/l), 283 (60.1%) sensitized to ALA (ALA-IgE > 0.35 kU/l), and 245 (52.5%) were sensitized to BLG (BLGIgE > 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 positive predictive level to CM (CMIgE ‡ 15 kU/l). All eight were sensitized to ALA, BLG, and casein. In addition, 95 patients had at least two times of followup 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 sensitized 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 119


patients and follow-up studies indicate same results. Negative predictive value difference between patients whose CM-IgE, ALA-IgE, BLG-IgE, Cas-IgE 44 kDa. Determination of free lysines showed that the majority of the lysines were modified in both pollen allergoids (73% and 74%). Fluorescence indicated hydrophobic tertiary protein structures after modification of the pollen extracts. Conclusion: Applying a combination of physicochemical techniques was shown to be a suitable approach to characterise the pollen allergoids well: Identification of the relevant allergens, determination of the degree of polymerisation and cross-linking, and investigation of the protein structures were accomplished.


Poster Discussion Session 6 Common hypersensitivity reactions

288 Diagnosis in oxaliplatin hypersensitivity: a 2-year experience with skin tests and specific IgE Madrigal-Burgaleta, R1; Berges-Gimeno, M1; Angel-Pereira, D1; Sanchez-Moreno, V1; Venemalm, L2; Alvarez-Cuesta, E1 1 Allergy Division, Ramon y Cajal University Hospital, Madrid, Spain; 2Phadia AB Thermo Fisher, Uppsala, Sweden

Background: Diagnostic tests for oxaliplatin hypersensitivity have not been validated. 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 inclusion 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 allergological study. Gold Standard for conclusive 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. Sensitivity 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 sensitive 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.

reactors. Respiratory allergy was more frequent in immediate than in nonimmediate reactors (4/13 vs 0/8). The reasons for ICM use were CT scans in 16 patients, ourography 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 diagnosis of contrast medium allergy and may play an important role in selection of a safe product in previous reactors.

289 Hypersensitivity reactions to non-ionic iodinated contrast media

290 Dual haptenic presentation in carrier molecules for the in vitro testing to detect IgE-antibodies in patients allergic to betalactams

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 immediate (1 h). Method: Twenty-one patients referred over a 4-year period for ICM hypersensitivity past reactions, either immediate or nonimmediate, 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 medical history was obtained in each case. Result: Positive skin tests were observed in seven of 13 patients with immediate reaction (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 moderate. In the skin positive group of immediate 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 previously. Cross reactivity was extended to 57% in skin positive group of immediate


Montanez, M1; Ruiz-Sanchez, A2; Ariza, A1; Mayorga, C1; Perez-Inestrosa, E2; Rodriguez-Bada, J1; Blanca, M3; Torres, M3 1 Research Laboratory, IMABIS Foundation-Carlos Haya Hospital, Malaga, Spain; 2Organic Chemistry, University of Malaga, Malaga, Spain; 3Allergy Service, Carlos Haya Hospital, Malaga, Spain

Background: Benzylpenicilloyl-dendrimer conjugates are recognized by IgE specific to benzylpenicillin. The use of dendrimers compared to the conventional poly-L-lysine carrier improves the reproducibility of in vitro tests to diagnose allergy. The aim of this study was to include other betalactam hapten, amoxicillin, and the concept of having two kinds of haptens in the same dendrimer 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 betalactams, for developing a universal in vitro allergy diagnostic test to betalactams. Method: Well-defined hapten-carrier conjugates were synthesized by haptenization of 4th generation PolyAMidoAMine dendrimers with either one kind of penicillin (benzylpenicillin and amoxicillin) or both penicillins. The chemical structures of the resulting conjugates were successfully characterized by means of nuclear magnetic resonance experiments. The different conjugates were immunologically tested by RAST (RadioAllergoSorbentTest) inhibition using 127

Poster Discussion Session 6 – Common hypersensitivity reactions

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 concentration, all sera allergic to benzylpenicillin showed 80% of RAST inhibition to both benzylpenicilloyl-dendrimer and mixed-dendrimer, and negative to amoxicilloyl-dendrimer. All sera allergic to amoxicillin showed 90% of inhibition to both amoxicilloyl-dendrimer and mixed-dendrimer, with no inhibition 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 similar values with monoantigenic and biantigenic conjugates. Thus, IgE recognition is specific and the sensitivity does not vary from biantigenic to monoantigenic conjugates. 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 1 Immunology – Allergology – Rheumatology, Antwerp University Hospital, Antwerp, Belgium; 2Anesthesiology, Antwerp University Hospital, Antwerp, Belgium

Background: Anaphylactic reactions occurring during anesthesia remain a major cause of concern for anesthesiologists. The authors report results of a 10-year survey of such reactions observed during anesthesia in Belgium. Method: Between January 1, 2001, and December 31, 2011, 344 patients who experienced 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%). 128

In 71 out of 93 NMBA-related cases, rocuronium was identified as the offending drug. Eighty-seven percent of the rocuroniuminduced 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 mastocytosis/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 reactions to local anaesthetics and dental materials is difficult, because of poor sensitivity of in vivo testing, which controlled administration provocative tests in order to confirm the diagnosis. Contrary, in vitro testing is a high sensitive and reliable diagnostic 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 symptoms of hypersensitivity (HS) to local anaesthetics (LA) – Group A and 264 outpatients with hypersensitivity to dental materials (DM) – Group B, were investigated for specific 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 positive history of multi hypersensitivity. In parallel 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, mepivacain, prilocain, lidocain; and dental materials 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 materials (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-testing 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 materials. 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 history 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 1 Immunoallergology 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 immunomediated 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 reactive 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 stimulation, in which peripheral blood mononuclear 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 IFXspecific 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 proliferative 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 oligoclonal expansion. An high proportion of



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 evidence of both effector and regulatory memory T cells specific for IFX and contributes to the understanding of pathogenic mechanisms of immunogenicity of biological 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 Luı´s, A Immunoallergology Department, Coimbra University Hospitals, Coimbra, Portugal

Background: Hypersensitivity reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) are currently responsible for 21– 25% of the reported adverse drug events. In this study we tried a novel approach in patients with no safe alternative NSAID. Method: We performed the retrospective study of the clinical charts of patients with history of cutaneous hypersensitivity reactions 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 symptoms. 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 paracetamol while the others had no known alternative. In four patients the reaction did not occurred in the first hour. The OCT performed by each patient are in table 1. The Patient 2 reacted to OCTs with cetirizine

and montelukate premedication so we proceed 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-histamine 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 Luı´s, A Immunoallergology Department, Coimbra University Hospitals, Coimbra, Portugal

Background: Hypersensitivity reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) are currently responsible for 21– 25% of the reported adverse drug events. This study aims to characterize the clinical profile of patients with suspected cutaneous hypersensitivity reactions to NSAIDs. Method: We performed the retrospective study of the clinical charts of patients followed in a Drug Outpatient Clinic in a 2 year period (2010–2011). The investigation included the analysis of involved drugs, symptoms, timing of reaction, underlying disease and challenge with culprit 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 implicated drugs were salicylates (41/28.5%) followed 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 urticaria 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 challenge 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 hypersensitivity reactions to multiple NSAIDs and in five patients the clinical history and the diagnostic work-up suggests hypersensitivity to a single NSAID, however it was not confirmed by means of OCT with culprit drug. Five patients had NSAIDs-exacerbated 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 majority 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 evaluate the diagnostic value of immediate and non immediate responses in skin tests (ST) with these drugs, to determine the frequency of allergic (specific, immediate and non immediate) and non allergic (intolerance/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 acetylsalicylic 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 Patient 2

Meloxicam+ (urticaria) Meloxicam+ (facial angioedema) Paracetamol+ Celecoxib+ (urticaria) Celecoxib+ (cutaneous pruritus) Nimesulide+ (urticaria)

Meloxicam Meloxicam+ (facial angioedema) ND Celecoxib Celecoxib ND

ND Meloxicam -

Patient Patient Patient Patient

Meloxicam+ (urticaria) Meloxicam+ (facial angioedema) Paracetamol ND ND Nimesulide -

3 4 5 6


ND ND ND ND

129


Table 1. Diagnosis (n) by means of

n

Non-allergic HS to all drugs Non-allergic HS with tolerance of paracetamol Immediate-type HS to ASA Delayed-type HS to ASA Immediate-type HS to ibuprofen Delayed-type HS to ibuprofen Immediate-type HS to paracetamol Delayed-type HS to paracetamol Total

4 2 2 3 2 3 9 2 27

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/worrying clinical history (CH) or positive CT with suspected drugs. Results: Most frequent reactions were urticaria 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 children 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 (immediate 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, diagnosis of NOA-AP-NSADS HS was primarily based on CT and on CH in highly suggestive 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.

often observed in children treated with betalactams. Many children are then labelled as ‘allergic’ without a reliable testing. In patients explored several years after a suspected 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 usefulness 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 maculopapular rash occurring within 12 h of a treatment with a betalactam antibiotic. Diagnosis was performed following International Guidelines by skin tests (ST), including Skin Prick Test (SPT) and Intradermal Test (ID) with (i) Major and (ii) Minor determinants of penicillin, (iii) benzylpenicillin, (iv) Amoxicillin and (v) the suspected drug. A drug provocation test (DPT) was performed in children with negative 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 reactions to betalactams, mainly amoxicillin. ST with betalactams (n = 1494) were negative in 1470 cases. DPTs with betalactams (n = 1475) resulted in negative response in 1470 cases. Betalactam allergy was diagnosed 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 accelerated mild skin reactions and exanthemas. However, these results should be confirmed by multicenter studies.

297 Drug provocation tests with betalactam in children presenting with rash Iglesias-Souto, J; Gonza´lez-Pe´rez, R; Sańchez-Machıń, I; Poza-Guedes, P; Matheu, V Allergy, Hospital del To´rax (Ofra), Santa Cruz de Tenerife, Spain

ST

CT

1 (confirmed by CT)

4 2 1 2 2 7

1 (confirmed by CH) 2

18

CH

1 3 2 1 7

298 Risk factors for developing immediate hypersensitivity to fluoroquinolones Torres, M1; Salas, M1; Donã, I1; Gomez, F1; Garcia, I1; Blanca-Lopez, N2; Ruiz, M1; Guerrero, M1; Blanca, M1 1 Allergy Service, Carlos Haya Hospital, Malaga, Spain; 2 Allergy 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 developing 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 fluoroquinolones administration. The diagnosis was confirmed by drug provocation tests or a clinical history of well-defined repeated episodes. The patients were classified as allergic 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 Chisquare 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 quinolone involved (being higher for moxifloxacin in the group of allergics, P = 0.021). There were no differences in age, gender, confirmed 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 culprit drug. Conclusion: In patients developing a reaction associated with quinolones, hypersensitivity is more frequently confirmed in those previously diagnosed as allergic to betalactams, those with immediate reactions and when moxifloxacin is the drug involved. Reasons for this association require further study. The study was funded by FIS-Thematic Networks (RIRAAF/RD07/0064), Junta de Andalucia (CTS 06603, PI-05452010) and FIS (09/01768).

Background: Immediate mild skin reactions, maculopapular rashes and exanthemas are 130



299 The prevalence of type 1 hypersensitivity drug reactions in school children – preliminary results Erkocoglu, M1; Kaya, A1; Civelek, E1; Banu, C2; Kocabas, C1 1 Department 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 selfreported 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 calculated 9096 out of a total of 210 000 students 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 hypersensitivity reaction prevelance (P) = 1% and a Delta value = 0.2 (a < 0.05,

b = 0.8). The participation in the questionnaire 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 questionnaires 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 1 Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore, Singapore; 2Department of Microbiology, National University of Singapore, Singapore, Singapore; 3 Department of Neurology, National Neuroscience Institute, Singapore, Singapore; 4Department of Neurology, Tan Tock Seng Hospital, Singapore, Singapore

cutaneous allergic reactions (SCARs), HLA-B*5801 with allopurinol-induced reactions and HLA-B*5701 with abacavirinduced 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-, phenytoinand allopurinol-induced SCARs and the drug-tolerant controls, recruited from Tan Tock Seng Hospital and the National Neuroscience 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 allopurinol 11 patients and nine control. For carbamazepine, 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 association between specific HLAs and SCAR does not appear to be as strong as suggested. Further studies are warranted as this has implications for wide implementation of pre-treatment genotyping.

Background: HLA-B*1502 is associated with carbamazepine- and phenytoin-induced severe


131

Poster Discussion Session 7 Immunotherapy: progress in clinical management

301 Preventing progression of allergic rhinitis into asthma in school children treated with Salsola kali immunotherapy Arifhodzic, N; Al-Ahmed, M; Al-Ahmed, N; Paniker, R; Ali, A Allergy Department, Al Rashed Allergy and Chest Centre, Kuwait, Kuwait

Background: Allergen immunotherapy (AIT) has the potential to reduce the progression 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 immunotherapy (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 rescue 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 medication 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 probability of superiority in the presence of a 132

large placebo effect particularly during early treatment phases, controlled studies in house dust mite (HDM) allergy commonly include clinical outcome measurements 2 or 3 years after initialization of SCIT. Patients’ expectations and compliance, however, warrant efficacy assessments 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 controlled 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 nonhyposensitized 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 during SCIT vs control patients (P < 0.0001). Conclusion: SCIT with the high-dose hypoallergenic 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 immunotherapy (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 minirhinoconjunctivitis quality of life questionnaire (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, demonstrated 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 improving symptoms and QoL in patients with HDM induced AR, the effects becoming


Poster Discussion Session 7 – Immunotherapy: progress in clinical management

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 University of Antioquia, Medellıń, Colombia; 2 Allergology Service, University of Antioquia, Medellıń, Colombia

1

Background: Subcutaneous allergen-specific immunotherapy is a proven, highly effective treatment for IgE–mediated diseases. However, rush immunotherapy is prescribed infrequently because of the perception 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 atopic dermatitis receiving HDM allergen-specific immunotherapy between January 2009 and august 2011. Subcutaneous Immunotherapy 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 subsequent monthly injections. A 30 min observation 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 reaction. Systemic reactions were graded using the World Allergy Organization Subcutaneous Immunotherapy Systemic Reaction Grading System. Results: Data from 102 patients with atopic 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 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 Department 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

1

Background: Life-threatening and even fatal bradykinin-induced angioedema (AE) attacks due to C1-inhibitor esterase deficiency may be triggered by oral trauma, including dental 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


Poster Discussion Session 8 – Cutaneous allergy

selected those treated with renin-angiotensin system (RAS) inhibitors, ACEI or angiotensin receptor blockers, needing dental care procedures provoking oral trauma, including tooth extraction, scaling and root planning, surgical debridement procedures, dental caries treatment, endodontic treatment, teeth preparations and dental impressions for prosthetic appliances like crowns, bridges, partial or complete dentures. Because an unmasking of C1-esterase inhibitor (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 regimens including ACEI, we detected two patients in treatment with ACEI presenting episodes of perioral AE and unilateral tongue AE, several hours after dental impressions 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 symptoms was obtained in maximum 24 h, without bradykinin B2 receptor antagonist icatibant use. Non-steroidal anti-inflammatory 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, prosthodontists, 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 cytokine 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 elobase cream for 14 days. Clinical efficacy of the treatment was assess on the following

features: SCORAD index, Investigators’ Global Assessment (IGA) score and subjective 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 immunological 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 transcription 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, IL12Aˆ, 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 cytokine 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, IL12Aˆ, 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 immunology, Skopje, Macedonia, Fyrom

Background: The balance between Tr1 and IL4 are crucial in the development of allergy. The aim of this study was to examine the association of IL-4, IL-10 and TGF beta cytokine gene polymorphism in the Macedonian population with atopic dermatitis (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 present 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 present 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 children 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 genotype 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 population. The frequency of alleles and genotypes of IL-4 are increased in patients with AD, while they are present in small frequency 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 Macedonian population.

321 The temporal and spatial dynamics of Treg cell-mediated suppression during contact hypersensitivity responses in a murine model Fyhrquist, N1; Lehtima¨ki, S1; Savinko, T1; Lahl, K2; Sparwasser, T3; Wolff, H1; Lauerma, A4; Alenius, H1 1 Finnish Institute of Occupational Health, Helsinki, Finland; 2Stanford University School of Medicine, Stanford, CA, United States; 3Centre for Experimental and Clinical Infection Research, Hannover, Germany; 4 University of Helsinki, Helsinki, Finland

Background: Regulatory T (Treg) cells suppress contact hypersensitivity (CHS) 139


responses, but the dynamics, mode and site of their action is not well characterised. We studied Foxp3+ Treg cells during the CHS response in conditional Foxp3 knock-out DEREG mice, where Foxp3+ cells can be transiently deleted by diphtheria toxin. Method: The mice were sensitised and challenged by oxazolone, and Foxp3+ cells were depleted by diphteria toxin treatment either during sensitisation or elicitation. Inflammatory parameters including ear swelling, the expression of cytokines and chemokines, and the recruitment of inflammatory cells, were analysed at different time points after challenge. Result: Treg cell depletion prior to sensitisation 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 similarly 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 demonstrate that endogenous Foxp3+ Treg cell function is important in the LNs during the sensitisation phase and in the skin during the resolution phase, but seem redundant 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 1 Department 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 measurement of inside-out skin barrier function. TEWL increases when skin barrier is damaged. 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 development 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 140

controlled room in the maternity hospital. Measurements were taken on the volar surface of the forearm after skin had been exposed and acclimatised. TEWL was measured using an open chamber device. Three measurements were taken and the average recorded. Parent’s completed a comprehensive 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 measurement 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 gestational 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 specific Maternal and Paternal history of Atopic 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 1 Faculty 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; 3 University Hospital Goethe-University Frankfurt, Pharmazentrum Frankfurt/ZAFES, Frankfurt, Germany

Background: IL-18 is an important mediator involved in chronic inflammatory conditions such as chronic eczema. An imbalance between IL-18 and its endogenous antagonist IL-18 binding protein (BP) may account for increased IL-18 activity. IL-27 is a cytokine with dual function displaying pro- and anti-inflammatory properties. 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 (RTPCR) was used to investigate mRNA expression and enzyme-linked immunosorbent (ELISA) was used to investigate protein production. Western blotting and luciferase assays were used to determine levels and phosphorylation of signalling molecules. Result: Human keratinocytes and surprisingly also fibroblasts (which do not produce any IL-18) show a robust, dosedependent 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 sustained for up to 96 h. Investigation of the signalling pathway showed that IL-27 activates STAT1 in human keratinocytes and that a proximal GAS site at the IL-18BP promoter is of importance for the functional 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 tissue resident cells may prove an interesting treatment strategy for chronic inflammatory 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 1 Department 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, ChungAng University College of Medicine, Seoul, Korea; 5 Department 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 Lactobacillus plantarum CJLP133 improved clinical 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 SCORAD indexes, the use of topical corticosteroid (TCS), eosinophil count, total serum IgE, cytokines, and microbial colonization of the gut were evaluated. Result: Eighty-three patients (44 in probiotic 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 population 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 1 Nutrition and Health, Centre de Recherche Nestle´, Lausanne, Switzerland; 2Centre de Recherche Nestle´, Lausanne, Switzerland

Background: Probiotics have been suggested to play an important role in controlling 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 atopic dermatitis induced by two epicutaneous sensitizations with Aspergillus (ASP) followed 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 (prevention), the second skin exposure (management) or all along the experiments. Result: No difference in the total cell counts in the bronchoalveolar lavage (BAL) was observed across the different groups. Eosinophils were absent in the BAL of saline (SAL)-sensitized/ASP-challenged animals but were significantly increased in ASP-sensitized/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 ‘prevention’. 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 Pediatrics, Allergy & Respiratory Research Laboratory, Inje University, Seoul, Korea; 2Allergy & Respiratory Research Laboratory, Inje University, Seoul, Korea

1

Background: Vitamin D has also been inconsistently associated with atopic diseases, 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 significantly 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 compared 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 reduction. However, considering the significant decrease in SCORAD index andimprovement in vitamin D level, this may be a useful 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 internationally 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 consecutive patients (130 F; 45 M;) affected by non-physically induced CU underwent ASST. Antihistamines, Doxepin and corticosteroids 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 141


controls. A 30 min reading was performed. Antinuclear antibodies (ANA) with pattern and titer were also analized in all patients by IIF. Negative patients were treated with antihistamines. Positive ones with corticosteroids and/or immunosuppressants (Cyclosporine) Result: 133/175 patients showed positive reaction to ASST. Of this positive group in 59/133 patients was demonstrated the presence 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 Cyclosporine (3 mg/kg/day) were necessary to obtain a control of the itchy weals. Conclusion: Despite ASST can be considered 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 immunosuppressants are needed. A pathogenetic autoimmune mechanism is strongly suspected.

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328 Anxiety and depression are less common in patients with autoreactive chronic spontaneous urticaria Weller, K1; Koti, I2; Makris, M2 Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany; 2 Attikon University Hospital, Athens, Greece

1

Background: Patients with chronic spontaneous urticaria (csU) frequently exhibit psychiatric comorbidities such as anxiety and depression. Autoreactive csU is a common subform of csU that is caused by circulating 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 psychiatric comorbidities varies in different subforms of csU, particularly between ASST positive and negative patients. Method: For this joint project, data of 209 csU patients was collected in tertiary referral centers in Berlin and Athens. During their visits, most of the patients were subjected to the ASST (n = 164). In addition, all patients were asked to complete the Urticaria Activity Score for four consecutive 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 distribution, urticaria activity, quality of life impairment and frequency of ASST positive patients (36.4% vs 34.3%). Accordingly, both populations were pooled for the further comparison of autoreactive and nonautoreactive csU patients. In both groups, age and gender distribution was not different, 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 anxiety 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 depression although they tend to show a higher disease activity and quality of life impairment. These results suggest that autoreactive csU is different from other subtypes of csU and that psychiatric comorbidity plays a minor role in these patients.


Poster Discussion Session 9 Asthma: from bench to real life

329 Expression of CD206, CD124 and CD210 on peripheral blood monocyte subpopulations of house dust mite allergic patients during allergen challenge Kowal, K1; Moniuszko, M2; Dabrowska, M1; Bodzenta-Lukaszyk, A1 1 Allergology and Internal Medicine, Medical University of Bialystok, Bialystok, Poland; 2Allergology and Internal Medicine, Medcial University of Bialystok, Bialystok, Poland

Background: Alternatively activated mononuclear phagocytes participate in tissue remodeling. They are characterised by expression of the mannose receptor CD206. The aim of this study was to evaluate the expression of CD206, CD124 and CD210 on peripheral blood monocytes of house dust mite allergic patients (HDMAPs) before and after allergen challenge. Method: Blood samples were collected from 40 HDM-APs before (T0), 6 h (TLAR) and 24 h (T24) after intrabronchial 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 frequently expressed on CD14++CD16+ monocytes (25.3 ± 13.6%) than on CD14++CD16cells (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 predominantly 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 Department 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

1

Background: IL-33 is known to induce Th2type 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. Exposure to allergens through damaged skin is suspected to be a trigger for allergen sensitisation, resulting in development of such allergic disorders as asthma and atopic dermatitis. However, the role(s) of the IL-33/ ST2 pathway in allergic airway inflammation in individuals who had been epicutaneously 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 challenged 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, quantitative 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 chemoattractants (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 pulmonary 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 crucial for Th2-cytokine-mediated eosinophilic,


rather than Th17-cytokine-mediated neutrophilic, airway inflammation in mice that had been epicutaneously sensitised with antigens and then challenged with antigen. Our findings 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 1 Laboratory of Clinical Immunology, University of Leuven, Leuven, Belgium; 2Research Unit of Lung Toxicology, University of Leuven, Leuven, Belgium; 3 Department 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; 5 Laboratory 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 experiments, we showed that a single nasal instillation of low concentration of hypochlorite (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 pretreating animals with an antagonist of its specific receptor RP67580 and by immunofluorescent staining of large airways of treated 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 cinnamaldehyde 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 deficient KitW-sh/KitW-sh mice and by in vitro stimulation of bone marrow cultured mast cells (BMMCs) with incremental concentrations of OVA. 143

Poster Discussion Session 9 – Asthma: from bench to real life

Result: AHR induced by ClO- + OVA was reduced after pretreatment with RP67580. However, no increased SPexpression could be shown in the airways of treated mice. Low concentration of ClOinduces a Ca2+-influx in hTRPA1-transfected 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 stimulates BMMCs in vitro to produce and release TNFa, IL13 and IL6 in dosedependent way. Conclusion: Our findings show that nasal exposure to ClO- and OVA induces a noninflammatory AHR by a neuro-immune interaction mechanism.

higher in patients treated by inhaled corticosteroids 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 asthmatic airways. Whether the induction of these cells also play a role in the beneficial effects of corticosteroid treatment, has to be studied.

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 consequently the severity of OVA-induced lung inflammation.

332 Airway regulatory T-cells: a feature of asthma severity or induced by daily treatment?

333 Interleukin-21 promotes T helper type 2 allergic airway responses by inhibition of Foxp3+ T regulatory cells

Seys, S1; Decraene, A2; Grabowski, M3; Adriaensen, W1; Dilissen, E1; Ceuppens, J1; Dupont, L4; Bullens, D5 1 Microbiology and Immunology, Lab of Clinical Immunology, University of Leuven, Leuven, Belgium; 2 Clinical and Experimental Medicine, Lab of Pneumology, University of Leuven, Leuven, Belgium; 3 Physiology, Wroclaw Medical University, Wroclaw, Poland; 4Pneumology, University Hospital Leuven, Leuven, Belgium; 5Microbiology and Immunology, Pediatric Immunology, University of Leuven, Leuven, Belgium

Pawelski, H1; Sonar, S1; Tortola, L2; Yadava, K2; Schneider, C2; Sparwasser, T3; Kopf, M2 1 ETH, Moleculare Biomedicine, Zu¨rich, Switzerland; 2 ETH, Zu¨rich, Switzerland; 3TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany

Background: Current evidence suggests that autoreactivity may underlie the pathogenesis of chronic allergic diseases. It still needs to be clarified whether detection of autoAbs (autoantibodies) can define a distinct 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 IgG4autoreactivity. Method: IgE and IgG4-autoAbs against some lung tissue Ags (antigens): keratin, III and VI collagen types, elastin and myosin 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 duration of asthma more than 5 years was attended with most strong elevation of IgEautoAbs 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

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 necessary 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, however 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 144

IL-21 plays an important role in the development of Th2-driven airway inflammation. 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 eosinophilia, airway hyperresponsiveness, blood basophils and type 2 granuloma in asthma and nematode infection. In this study we sought to identify IL-21 dependent mechanisms 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 population of Tregs in the lung of IL-21R-/- as compared to WT mice upon airway challenge 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 administration of diptheria toxin (DT) is possible. Depletion of Tregs led to an enhanced influx of Th2 cells into the lung and exaggerated features of asthma in both IL21R+/+xDEREG and IL21R-/-xDEREG mice; indicating that Tregs are potent suppressors of asthma. To identify the time point when Tregs are imperative for the outcome of asthma, administration of DT

334 IgE- and IgG4-autoantibodies in allergic asthma Konishcheva, A; Gervazieva, V Allergy Diagnosis Department, Mechnikov Research Institute for Vaccines and Sera, Moscow, Russian Federation



(2460; 4460) ng/ml]. Patients with BA, receiving IGS (inhaled corticosteroids) displayed significantly higher levels of IgG4Abs [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 durable clinical course of disease, whereas IgG4-autoAbs prevails during AR and intermittent BA. Besides, in patients with BA the levels of IgG4 – autoAbs can indicate therapeutic benefits of IGS therapy.

335 Pathological features of the upper respiratory 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 respiratory 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 difficulty in nasal breathing. Twenty-two (36.7%) of them were diagnosed with allergic rhinitis and 24 (40.0%) – vasomotor. Chronic rhinitis in 18 (30.0%) patients combined with the distortion of the nasal membrane. 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 testified 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 evidence of pronounced bronchonasal reflex.

Conclusion: Allergic and vasomotor rhinitis in patients with uncontrolled asthma is accompanied by pronounced vegetativevascular 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 Lundstro¨m, M1; Andersson, C2; Bjermer, L2; Erjefa¨lt, J1; Westergren-Thorsson, G1 1 Department of Experimental Medical Sciences, Lund University, Lund, Sweden; 2Department of Clinical Sciences, Lund University, Lund, Sweden

Background: Conventional asthma therapies, 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 asthmatics and how these are correlated to infiltrating leukocytes and clinical data. Method: Bronchial and transbronchial biopsies from controls, patients with mild to moderate uncontrolled asthma were processed for immunohistochemical analysis of and extracellular matrix molecules; versican, biglycan, decorin and collagen. Regularity of collagen fibrils were examined using transmission electron microscopy. Result: The density of collagen (trichrome staining) was significantly increased in distal lung tissue of patients with uncontrolled asthma compared to controls (P = 0.0095). In addition, there was a negative correlation between collagen in bronchial biopsies and FEV1 (rs = -0.66, P = 0.04) in asthmatic patients. Also, the expression of decorin was increased in distal lung of asthmatics compared to controls (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 versican and biglycan was not altered in central vs distal airways when comparing controls to asthmatic patients. The density of connective tissue mast cells (MCTC) correlated positively to the density of collagen (rs = 0.71 P = 0.027) in distal airways of asthmatic patients. Expression of a scavenger 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 localizations. 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 impairment. These findings strengthen the rational to target also the alveolar compartment in uncontrolled asthma.

337 Filarial immunomodulator alters grass allergic responses Danilowicz-Luebert, E1; Lucius, R2; Hamelmann, E3; Hartmann, S4 1 Department of Molecular Parasitology, Humboldt University Berlin and Free University Berlin, Berlin, Germany; 2Department of Molecular Parasitology, Humboldt University Berlin, Berlin, Germany; 3 University 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 industrialized countries was very successful, while the prevalence of allergic diseases is increasing dramatically. Possibly reduction of viral, bacterial or helminth infections, in return contribute to a disregulated immune system that leads to allergic and autoimmune disorders. It is known that parasitic helminths have developed complex mechanisms to evade and modulate host immune responses. As a bystander effect, wormderived immunomodulators temper responses to non-helminth antigens, like environmental allergens. Previous studies in our group characterized a single filarial molecule: a secreted cysteine protease inhibitor (AvCystatin), potent modulator of macrophages 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 hyperreactivity 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 challenged with timothy grass pollen. AvCystatin was applied during the sensitization phase. In the translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with AvCystatin prior stimulation with timothy grass pollen extract. 145


Result: Administration of AvCystatin suppressed allergen-specific Th2-related inflammation, 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 IL13. Moreover, ex vivo restimulation with AvCystatin significantly induced IL-10 production by spleen cells. In the human model, AvCystatin changed the allergy balance towards Th1 immune response by increasing production of IFN gamma. Conclusion: The results show that AvCystatin down-regulates allergic immune responses and improves murine airway disease after sensitization and challenges with a clinically relevant aeroallergen and provides a Th1-type shift in in vitro human model. The impact of AvCystatin might be exploited for the treatment of allergic diseases, 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; Sjo¨strand, M; Ra˚dinger, M; Malmha¨ll, C; Lo¨tvall, J; Bossios, A Sahlgrenska Academy, University, Krefting Research Centre, Gothenburg, Sweden

Background: Allergic asthma is characterised, among others, by eosinophilic-associated lung inflammation. We have previously shown that anti-IL-33 markedly attenuated the number of eosinophil-lineage-committed progenitors (CD45+CD34+IL-5Ra+CCR3+Sca1+SSClow) and immature eosinophils, including early (CD45+CD34+IL5Ra+CCR3+Sca-1+SSChigh), intermediate (CD45+CD34+IL-5Ra+CCR3+Sca1-SSChigh) and late (CD45+CD34-IL5Ra+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 Marrow (BM) cells were harvested 24 h after the last exposure. Lung CD45+ cells were labeled with CFSE and cultured with rmIL5, rmEotaxin-2 and rmIL-33 with or without anti-IL-33 for 60 h to assess the proliferation 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 146

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+ mononuclear cells and CD45+CCR3+ granulocytes. Blockage of IL-33 did not affect the first and second generations; however, it significantly decreased cells on the third generation 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 eotaxin-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 Pediatrics, 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

1

Background: Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. One of the most critical structural changes that affect airway functionality 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 cytokines, 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. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and production of profibrotic cytokines, TGF-b and IL-11, were determined using Intra-cellular cytokine detection and FACS analysis, immunohistochemistry, as well as real time PCR.

Result: The level of basal expression of TGF-b and IL-11 was significantly upregulated in asthmatic patients compared to healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of eosinophils derived pro-fibrotic cytokines. However, stimulating eosinophils 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 transition (EMT) and subsequently leads to tissue 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 gastric 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 transition in bronchial epithelial cell and initiates the differentiation of epithelial to mesenchymal cells. Factors involved in epithelial to mesenchymal differentiation were analyzed. Method: To analyzed the effect of gastric fluid in EMT, we had used human normal bronchial epithelial cells (NL-20) to evaluate the expression of EMT markers, a-SMA protein, E-cadherin, and profibrotic factor, TGF-b by western blot, RT-PCR and confocal 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 signaling 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 marker, E-cadherin was diminished by gastric fluid after 24 h stimulation. The semiquantitative RT-PCR result showed increased expression of m-RNA of TGF-b by condition medium (P < 0.05). Migration of NL-20 cells were increased 4.5 times by gastric fluid (P < 0.05). The hallmark 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 condition 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 1 Immunology 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; 4 Immunoallergology, Hospital Sao Joao and Department of Immunology, Faculty of Medicine, University of Porto, Porto, Portugal

Background: An increasing body of literature suggests an association between competitive 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 phenotype. In our 3 year follow up study, we aimed to access changes in airway inflammation, 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 follow 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 reporting physician diagnosis of asthma and allergic rhinitis, and use of asthma medication.

Physical activity (PA) levels and airway inflammation were assessed using the International Physical Activity Questionnaire (IPAQ) and measuring exhaled nitric oxide (NO) levels before a training session respectively. 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 confounding factors: gender, age, atopy, physiciandiagnosed 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 swimmers. After the 3 year follow up the prevalence of asthma, allergic rhinitis and use of asthma increased significantly in each group without differences among them. Conclusion: This prospective study of competitive 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 Medical Psychology, University of Medicine and Pharmacy ‘Carol Davila’, Bucharest, Romania; 2 University of Medicine and Pharmacy ‘Carol Davila’, Bucharest, Romania 1

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/exuberant 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 audition = 16.83 and mean MEF50 after audition = 21.10), than FEV1 (t = 6.32, DF = 9, P < 0.001).


Conclusion: General bronchial dilatation effects of music in asthmatics suggest intervention of cathecolamines released by listening to music although other more mediators may be involved. Receptive music therapy may became an useful additional 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 Allergy and Immunology, VKV American Hospital, Istanbul, Turkey; 2Chest Department, Istanbul Medical Faculty, Istanbul University, Istanbul, Turkey; 3Chest Department, Mus State Hospital, Mus, Turkey

1

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 characteristics, 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 significantly lower for the study group with the pictorial asthma action plan. The sixth month evaluation for SGRQ revealed significantly higher results for the study group (P = 0.033), which means better health related quality of life. For both groups there were statistically significant differences 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 sig147


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 education resulted with greater improvement in asthma control, better quality of life results and lesser emergency room visit and hospitalization 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 1 Allergy, Carlos Haya Hospital, Malaga, Spain; 2Allergy Research Department, Carlos Haya Hospital, Malaga, Spain; 3Allergy Research Laboratory, Carlos Haya Hospital, Malaga, Spain

systemic atopy. Subjects with LAR frequently show other co-morbidities including 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 asthmatic symptoms (LARA), 20 subjects with allergic rhinitis with DP and asthmatic symptoms (ARA) and 10 healthy subjects as control group (CG) were recruited. Bronchial 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 challenge (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 variations after challenge. Conclusion: Positive responses to D. pteronyssinus were observed in the lower airways of subjects with clear history of asthmatic symptoms. Similar methacholine responses and increases in ECP and eosinophil levels were observed in both LARA and ARA groups.

Background: Local allergic rhinitis (LAR) is characterized by positive responses to nasal challenge with allergens in absence of

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Poster Discussion Session 10 Hot topics in allergy diagnosis and molecular allergology

347 Recombinant protein assembling epitopes from different allergens of Dermatophagoides pteronyssinus Puerta, L; Martinez, D; Munera, M; Cantillo, J; Caraballo, L Institute for Immunological Research, University of Cartagena, Cartagena, Colombia

Background: The domestic mite Dermatophagoides pteronyssinus is an important source of aero allergens affecting allergy susceptible population around the world. Recombinant allergens or their modifications have been proposed for replacing advantageously natural allergenic extract for diagnosis and immunotherapy. We develop a fusion protein consistent of different 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 codonoptimized 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, denominated 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 allergic 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 immunotherapy of mite allergy.

348 Primary structure and recombinant expression of Secale cereale major 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 knowledge on crossreactivity and/or co-sensitization 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 spectrometry. Recombinant Sec c 5 was produced in E. coli and subsequently purified to homogeneity. 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 SubCommittee. Sec c 5.0101 has approximately 65% and 60% sequence identity with Phl p 5a and Phl p 5b, respectively. In IgE-immunoblots, 41 of 46 grass pollen allergic individuals 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 species specific IgE-epitopes. In addition a higher IgE-reactivity to Phl p 5a as compared 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 IgEcrossreactivity. The observed minor individual differences in IgE-binding strength suggest 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 1 University 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 countries, such as India, Pakistan and Nepal. Only few reports exist about allergy to buffalo’s milk or buffalo’s cheese. The goal of our study was to investigate allergy to buffalo’s milk by identifying new allergens and testing their cross-reactivity with milks from phylogenetically closed related mammals like cow, goat or sheep. Method: Sodium dodecyl sulfate-polyacrylamide 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 inhibitions 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 149

Poster Discussion Session 10 – Hot topics in allergy diagnosis and molecular allergology

as a new allergen in buffalo’s milk allergy. Even if cross-reactivity between milk proteins 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 allergic patients. Despite very high homology with other b-lactoglobulins from mammalian milks, the absence of cross-reactivity suggest a bigger diversity of b-lactoglobulin’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 investigations has to be carried out to better understand the IgE-reactivity of b-lactoglobulins.

350 Analysis of the complement activating ability of different types of allergens ˜ ¼st, G1; Resch, Y2; Chen, K2; Csuka, D1; Varga, L1; FA Vrtala, S2; Valenta, R2 1 3rd 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 activation in both the sensitisation and effector phases of allergic reactions is well known. As shown previously, allergen extracts are efficient activators of the complement system. 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 activation in the fluid phase, serum pool of healthy subjects (non-allergic to the allergens tested) was incubated by recombinant allergens (rPhlp1, rPhlp2, rPhlp5, rPhlp6, rBetv1a, rBetv2, rDerp2, rDerp23). The levels of the complement activation products, characteristic for the activation of the classical, alternative and terminal complement pathways (C1rC1sC1-INH, C3bBbP, SC5b-9) were measured by ELISA methods. 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 150

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-specific IgM and IgA antibodies were detected against all types of allergens. Allergen-specific 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 activated in case of bound allergens, which indicates that complement may be activated 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 population 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 transglutaminase. The induced enzyme modifies proteins in connective tissue as well, making 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 program to aid in the evaluation. Method: CD active peptides were identified from published studies identified from PubMed using keywords ‘celiac’ and ‘coeliac’. A total of 1016 native or deamidated peptides with reported evidence of induction of proliferation or secretion of inflammatory cytokines were identified and included in the peptide database. Representative source proteins were identified for all 1016 peptides to construct a CD protein database. 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 taxonomically distantly related plants lacking reports of CD induction (rice, millets, corn, sorghum, sugarcane, and Job’s tears), did not contain exact CD peptides and FASTA alignments to CD proteins were of moderate to low identity. Alanine substituted 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 definitive 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 Kaohsiung Medical University, Kaohsiung, Taiwan; 2 Pediatrics, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan

1

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 molecule 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 discrimination 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



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 1 Foundation 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 children. 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 diagnosis 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-Buhlmann). The mean age of the pt was 4.8 years (±2.9 years); 47% of them had a diagnosis of atopic dermatitis, 29% suffered 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 exception 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 specificity 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% sensibility and a 100% NPV, with 40% and 48% of specificity, 48% and 55% of efficency, and a 21% and 24% PPV, respectively. 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.

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 compared 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 importance 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.

354 Association of centrosomal protein 68 single nucleotide polymorphisms with hypersensitivity reactions to non-steroidal antiinflammatory drugs

355 Effects of glycoconjugates of ovalbumin on alleviation of orally induced egg allergy in a BALB/c mouse model

Cornejo-Garcia, J1; Plaza-Seroń, M1; Flores, C2; Donã, I3; Blanca-Lo´pez, N4; Jagemann, L1; Laguna, J5; Fernańdez, J6; Canto, G4; Blanca, M3 1 Laboratorio de Investigacioń, Fundacioń IMABIS-Hospital Carlos Haya, Ma´laga, Spain; 2Unidad de Investigacioń, Hospital Universitario N.S. de Candelaria, Tenerife, Spain; 3Servicio de Alergologıá, Hospital Carlos Haya, Ma´laga, Spain; 4Servicio de Alergologıá, Hospital Infanta Leonor, Madrid, Spain; 5 Servicio de Alergologıá, Hospital Central de la Cruz Roja, Madrid, Spain; 6Servicio de Alergologıá, Hospital de Elche, Alicante, Spain

Background: Non-steroidal anti-inflammatory 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-intolerance (non specific immunological mechanism) (CI). Recent observations in different populations have shown that CI is the most relevant group. Genetic association studies have been carried out only in patients with CI and consist mainly in the analysis of single nucleotide polymorphisms (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 aspirin-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 episodes with three or more different NSAIDs were included in the CI group. We studied


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 determinants. 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 various 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 subsequently administered various forms of glycated-Ova (glucose, mannose, glucomannan, 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, IFNgamma, 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 glucomannan 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 151


cytokines IFN-gamma, IL-10 and TGFbeta did not differ significantly among groups. On the other hand, Ova-glycated with glucose, galactomannan and the mixture 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 glucomannan have reduced susceptibility and subsequent frequency of allergy to Ova thus biasing immune response into a balaced, regulated, anti-allergy phenotype. This study validates potential use of Ova-glycated 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-Pinãr, t1; Caballero, M1; Knaute, T2; Umpierrez, A3; Rodriguez-Perez, R1 1 Immunology, Carlos III Hospital, Madrid, Spain; 2JPT Peptide Technologies GmbH, Berlin, Germany; 3 Allergy, Carlos III Hospital, Madrid, Spain

Background: Ani s1 is a major allergen from Anisakis simplex with an IgE-binding frequency higher than 80% among Anisakis 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 allergen-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-binding 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-offset), corresponding to the primary sequence of Ani s 1 was printed on epoxy-coated slides. A microarray immunoassay was performed with sera from eight patients with Anisakis allergy and positive Ani s 1 reaction by western-blotting against a recombinant 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 hypoallergenic forms. 152

357 Pattern of sensitisation to nDer p 1 and rDer p 2 in patients allergic to house dust mite in Tarragona Lopez-Patinõ, A1; Esteso, O1; Pastor, R2; Dalmau, G1; Gazquez, V1; Gaig, P1 1 Allergology Unit, Joan XXIII University Hospital, Tarragona, Spain; 2Laboratory of Clinical Analysis, Joan XXIII University Hospital, Tarragona, Spain

Background: nDer p1 and rDer p2 are the specific markers of sensitisation to Dermatophagoides pteronyssinus (Dpt). There is the possibility that the ratio of proportion between them varies depending on the geographical area. The aim of this study was to report the pattern of sensitisation to nDerp1 and rDer p2 in our area of influence, 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 immunotherapy) and subjective response to immunotherapy. Serum specific IgE against nDer p1 and rDer p2 was determined in all patients using InmunoCAP Phadia (positive 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 symptoms. 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. Average 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 1 Kwizda/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 binding 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 clinical significance. As most allergens of plant and insect origin contain CCDs, doctors and patients are confronted with an uninterpretable 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 interference. For this study, a plant glycoprotein 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 concealed 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 (Mediwiss 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 peroxidase. 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 1 Institute of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany; 2 Department of Dermatology, Venerology, and Allergology, Georg-August-University, Go¨ttingen, Germany; 3Department of Dermatology, University Medical Center Freiburg, Freiburg, Germany; 4 Hamburg 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 monoclonal IgE antibodies and knowledge about their epitopes. Here we report the consecutive generation and epitope delineation of a human monoclonal IgE against the prototype 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 pronounced reactivity with Bet v 1, but were not reactive with the homologous PR10 protein 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 helixstructured motif comprised by the amino acid residues 132–154, including the critical residue E149. Grafting this motif re-established reactivity of the per se non-reactive Mal d 1 framework. Additionally, crossreactivities were evaluated by primary structure analyses of different isoforms and PR10 proteins and verified by array based analyses. Conclusion: The obtained results demonstrate that using hybrid IgE repertoires is a suitable strategy for efficient establishment of human authentic antibodies. The IgEderived information about the epitope of Bet v 1 allows for detailed insights into molecular aspects of allergenicity and crossreactivity within the PR10 protein family.

360 Targeting the extracellular membraneproximal 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 rhinoconjunctivitis. 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 interventions. 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 further 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 frequency of IgE+ B cells is very low. Preliminary 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 allergen-specific mAbs of the IgE isotype by immortalization in order to elucidate IgEbinding epitopes by co-crystallization of allergens and allergen-specific human Fab fragments. These initial studies and the epitope mapping can aid in the discovery and development of new therapeutics, vaccines, and diagnostics, thus providing new insights into allergy-related disorders.

153

Poster Discussion Session 11 Food allergy: anaphylaxis and diagnosis

361 Network for online-registration of anaphylaxis: towards a European registry of severe allergic reactions – current status Worm, M1; Moneret-Vautrin, A2; Nikos G., P3; Fernandez-Rivas, M4; Jutel, M5; Guilarte, M6; Kowalski, M7; Mustakov, T8; Hompes, S1 1 Department of Dermatology and Allergy, Charite´ – Universita¨tsmedizin Berlin, Berlin, Germany; 2Re´seau Allergovigilance, Vandoeuvre Les Nancy, France; 3 Allergy 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 identification 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 Europe do not acquire standardised data from patients with anaphylaxis, or use different methods of data acquisition. Method: The aim is to harmonise the anaphylaxis questionnaire among different European countries, assess the quality of the data and obtain first data at a European level. Currently actively participating countries are Bulgaria, France (Re´seau Allergovigilance), Germany, Greece, Poland and Spain. Result: The questionnaire covers demographic data from the patient, detailed information about the symptoms and the elicitor(s), diagnostic tests and preventive measures taken (e.g. consultation, prescription 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 reactions. However as reported previously, drugand venom-induced anaphylaxis occur more frequently in adult patients. 154

Conclusion: The data show that the system used is suitable to acquire standardised data from different allergy centres throughout Europe. Currently the involvement of further 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 epidemiology but not prevalence or incidence rates can be gathered.

362 Anaphylaxis in children and adolescents: a 1-year survey in an immunoallergy department ˆ ; Piedade, S; Santa-Marta, C; Santos, N; Gaspar, A Pires, G; Sampaio, G; Borrego, L; Areˆde, 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 anaphylaxis in an Immunoallergy outpatient clinic, and to identify its main clinical manifestations and triggers, in children and adolescents. Methods: From 3646 patients up to 18 years old observed in our Immunoallergy department during 2011, we included those with history of anaphylaxis voluntarily reported by the clinical staff (‘at least one episode of severe systemic reaction’). Results: During the 1 year period, 64 children 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 disease, 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 majority of patients had food-induced anaphylaxis (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 anaphylaxis 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 episodes 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 episodes before being assessed by an allergist. We stress the importance of systematic notification of anaphylaxis and improvement of educational programmes in order to achieve a better preventive and therapeutic 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 1 Internal 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; however they may cause severe allergic reactions. 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 differences are caused by thermal food processing or by differences in protein content. Method: Five patients with severe allergic reactions after consumption of gamba,


Poster Discussion Session 11 – Food allergy: anaphylaxis and diagnosis

without similar reaction to shrimp were included. Fresh gamba and shrimp were prepared in the oven (separately at 100 and 180C) ether extracted, freeze dried and a 10% solution was made in PBS. Specific IgE against these gamba and shrimp proteins was measured by SPT and visualized by immunoblot analysis using SDSpage. 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 processing temperature had no influence on the results. Conclusion: This is the first study investigating 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 processing 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 diagnosis of IgE-mediated food allergy remains at the moment a properly performed double-blind, placebo-controlled food challenge (DBPCFC). However, there are different practical (long duration, blinding) and ethical (e.g. severe anaphylaxis) limitations that hamper entrance of DBPCFC in daily clinical practice. Basophil activation test can constitute a sensitive and specific instrument that can complement conventional tests such as quantification of specific IgE (sIgE) and skin prick tests (SPTs). The aim of the study was to evaluate the relationship among sIgE, basophil activation 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 presented 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 specific 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 activation 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 1 Immunology Lab, APHM Conception, Marseille University 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.