7th International Symposium on Molecular Allergology (ISMA 2017)

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Background:
The currently known cockroach allergen components do not account for the total IgE reactivity to cockroach extracts. The goal of this study was to assess the IgE reactivity to a wide panel of proteins from Blattella germanica that are known or potential allergens to cockroach sensitized patients from the United States (US). Methods: German cockroach allergens Bla g 1, Bla g 2, Bla g 4, Per a 7, Bla g 9 and Bla g 11 were expressed in Pichia pastoris. Bla g 1, Bla g 2 and Per a 7 were purified by specific-antibody affinity chromatography. Bla g 4 was purified by phenol Sepharose chromatography. Bla g 5 was expressed in Escherichia coli and purified by glutathione S-transferase affinity chromatography. Bla g 9 and Bla g 11 were purified by metal affinity chromatography. IgE antibody levels to these 7 purified allergens were measured by streptavidin ImmunoCAPs loaded with biotinylated purified allergens. Results: The prevalences of IgE antibody reactivity to cockroach allergens in a population of US cockroach allergic patients (n = 16) were: 31% (Bla g 1), 56% (Bla g 2), 31% (Bla g 4), 50% (Bla g 5), 31% (Per a 7), 50% (Bla g 9) and 63% (Bla g 11). Bla g 9 and Bla g 11 were identified as major allergens, in addition to the currently known major allergens 0.00 0.00 0.00 analyze the efficacy of AIT with Extract of Ambrosia (Diater Laboratories, Spain). Methods: 586 patients aged 5-58 were examined in Kyiv 327 (55.8%) and 259 (44.2%) in Lviv region. SPT was performed by Extract "Ambrosia". The patients were examined to undergo molecular diagnostics using ImunoCAP (Phadia) to identify major (n Amb a1) allergen. The SLIT was carried out with a mixture of Ambrosia. Results: The prevalence of Ambrosia sensitization diagnosed in 25 (9.6%) persons in the Lviv region. 3 (12.0%) children moved from the Crimea and 22 (88.0%) adults was born and have been living in Western Ukraine. Sensitization to Ambrosia major allergen (n Amb a1) was detected in 23 (92.0%) persons. Withal, sensitization to Ambrosia (SPT) in patients in the Kyiv region was determined 2.5 times higher: positive SPT were detected in 80 (24.5%) patients (28 (35.0%) children and 52 (65.0%) adults). The true sensitization has been confirmed in 88.0% of people. SLIT were prescribed in the patients from both regions. The efficacy of SLIT was assessed by a visual analogue scale (VAS-up). An assessment of SLIT showed that there was a significant decrease in the severity of symptoms in the study groups as after 2 years of treatment (94.1 and 94.5%, respectively).

Conclusions:
A high level of sensitization to major allergen (n Amb a1) Ambrosia in children and adults in Ukraine leads to a significant increase in allergic pathology. High efficacy of the SLIT provides the possibility of relative control of the prevalence of severe form of allergic diseases.

P10 Identification of IgE-binding epitopes on the surface of the non-specific lipid transfer protein Art V 3
Background: Pollen of Artemisia vulgaris (mugwort) are an important elicitor of allergic reactions in late summer and autumn. Art v 3 is an allergen of mugwort pollen which belongs to the non-specific lipid transfer protein (LTP) family. The aim of the study is to solve the structure of Art v 3 and to identify the structural epitopes of Art v 3 using murine monoclonal antibodies. Methods: Recombinant non-labeled and double-labeled (13C/15N) Art v 3.0201 were expressed in E. coli and purified using cation exchange chromatography. The three-dimensional structure of Art v 3 was solved by X-ray crystallography and resonance assignment was obtained by NMR spectroscopy. In addition, three Art v 3-specific murine monoclonal IgG antibodies (mAbs) were produced in hybridoma cells and purified using affinity chromatography. Binding affinities between Art v 3 and the mAbs were determined using the surface acoustic wave (SAW) technology. Cross-reactivity between the murine mAbs and the IgE from sera of mugwort allergic patients (n = 21) was investigated in an inhibition ELISA. Structural epitopes of Art v 3 were determined by NMR spectroscopy using the double-labeled Art v 3 and the murine mAbs. Results: Recombinant Art v 3 was produced as a non-tagged protein.
X-ray crystallography and NMR revealed a homodimeric assembly of Art v 3 containing four alpha-helices stabilized by four disulfide bonds per molecule. Binding affinities between Art v 3 and mAbs were in the nanomolar range. The binding to IgE from patients' serum was inhibited with a mean of 69-82% by the murine monoclonal antibodies indicating an overlap of the binding sites. Hydrogen/deuterium exchange detected by NMR spectroscopy with a resolution on the Clin Transl Allergy 2018, 8(Suppl 1):17 individual residues allowed the identification of epitope regions on the surface of Art v 3. Conclusions: Within this study we solved the 3-D structure of Art v 3 and identified potential IgE binding regions on the surface of Art v 3. These results will provide further insights into allergen cross-reactivity within the lipid transfer protein family.
Background: Seafood is one of the most common elicitors for foodallergic reactions while, among crustacean species, ingestion of prawn (Penaeus monodon) is considered as pre-dominant cause of adverse reactions. Tropomyosin, a muscle protein, is the major allergen in invertebrates such as crustaceans. Vertebrate tropomyosins are nonallergenic proteins, an observation which is not well understood. The aim of this study was first to isolate both allergenic (native, recombinant) and non-allergenic tropomyosins and following, to compare those proteins at the biomolecular levels and as to their allergenicity. Methods: Homologue tropomyosins from Black Tiger Prawn (P. monodon), chicken breast and leg muscle (Gallus gallus) were purified by column chromatography. Recombinant tropomyosins were expressed in E. coli, followed by protein purification. Purified proteins were compared by Edman degradation, mass spectrometry (MS), antibodybinding studies (immunoblot, ELISA) and circular dichroism analysis. Allergenicity was assessed by IgE-ELISA, basophil activation test (BAT) and skin testing using shrimp allergic patients. Results: Tropomyosins were purified to homogeneity by column chromatography in a milligram scale. MS and Edman analysis confirmed the identity of all proteins as muscle tropomyosins. Circular dichroism analysis revealed characteristic alpha-helical structures as well as high protein stability towards thermal treatment. Specific IgE sera titer were up to 9-times higher to shrimp than to chicken tropomyosin. BAT was positive with shrimp allergens at 100-times lower allergen concentrations than with chicken homologs. Biomolecular assays on allergen characterization as well as IgE-and BAT-assays gave similar results for both native and recombinant proteins. In addition, skin reactivity of shrimp-allergic patients was positive with both shrimp and chicken tropomyosins but at up to 100-times lower concentrations with the shrimp allergen. Conclusions: Tropomyosins from shrimp and chicken exhibit similar biomolecular characteristics while they vary by their allergenic potency. Both tropomyosins might be used as standard proteins, representing high and low allergenic molecules, in future experimental set-ups for the risk assessment of novel food sources. Background: Food processing, as well as digestibility and intestinal transport, are key factors to consider since they may affect the allergenic potential of food allergens. Typically, wheat based foods are always consumed after cooking which include some heating step. As regard to health aspects, wheat may trigger food allergy in some individuals. Numerous wheat allergens have been identified, and in particular the gliadins, that are among the main proteins responsible for food allergy to wheat. Complex foods such as bread or pasta are not easy to handle in 'in vitro' assays for allergenicity evaluation. We used total gliadins and the alpha-gliadin sub-fraction as simplified models to investigate the effect of heating on their capacity to maintain an allergenic potential. Successive steps of the "antigen transformation" were taken into account, from heating treatment to gastric digestion before considering the passage of the intestinal barrier. Methods: The heated and heated/digested total gliadins and alphagliadins were characterized for their size by laser light scattering. The chromatographic profiles of the soluble fractions were obtained by RP-HPLC chromatography. The IgE-binding capacity of the treated proteins was compared to that of the native forms with sera from wheat allergic patients. Furthermore their capacity to cross the intestinal barrier and to induce the mast cell degranulation was investigated by combining two in vitro cellular models, Caco-2 and RBL-SX38.

Results:
The heat treatment of total gliadins or of alpha-gliadins induced in both cases the production of large aggregates that were no more recognized by patients IgE. However, after limited pepsin hydrolysis, they recovered partial IgE-binding by unmasking epitopes in Dot Blot, but were not able to trigger RBL cells. After crossing the Caco2 cells, the treated proteins partially recovered their biological activity.

Conclusions:
The large aggregates of gliadins that can occur during bread-making displayed a decreased allergenicity in vitro compared to native gliadins. This may be related to the capacity of some patients to achieve hypo-responsiveness to wheat during oral immunotherapy protocols performed with bread or other heated wheat-based products.
Background: Prostaglandin (PG) E2 plays an important role in relation to mast cells (MCs) in different diseases. It mediates various and sometimes opposing effects on these cells through activation of four distinct receptors (EP1-4). Responses can be influenced by several factors such as variation among species or tissue sites. Differences in the genetic background within a species might likewise contribute to the reactivity and response pattern of MCs towards PGE2. Methods: In this study, we examined genetic variation as a factor influencing the responsiveness towards PGE2 in MCs from two mouse strains typically employed in studies of allergic diseases. We first analyzed serum levels of PGE2 in Balb/c and C57BL/6J mice. Then, the expression of EP1-4 receptors was determined using bone marrowderived cultured mast cells (BMcMCs). Subsequently, we assessed the impact of various concentrations of PGE2 and specific EP-agonists alone/in combination on IgE-mediated MC activation by detection of histamine release (HR). Results: Serum levels of PGE2 were significantly higher in Balb/c compared to C57BL/6 J mice. PGE2 receptors were likewise expressed to a greater extent in BMcMCs from Balb/c mice with the highest expression of EP3. PGE2 increased IgE-mediated HR in BMcMCs from Balb/c mice dose-dependently. In contrast, PGE2 led to an inhibition of HR in C57BL/6-derived MCs. EP receptor agonists achieved a comparable influence on HR in both strains. EP2-agonist decreased the IgEmediated response while the EP3-agonist elevated it in both strains. By contrast, EP4-agonist had no impact on MC activation. However, a combination of EP2 and EP4-agonists, decreased HR in BMcMCs taken from C57BL/6J mice only.

Conclusions:
In conclusion, BMcMCs from Balb/c and C57BL/6J mice exhibit heterogeneity in their responsiveness towards PGE2. PGE2 seems to increase MC degranulation via EP3 receptor, in Balb/c mice, while in C57BL/6 mice the inhibition of MC degranulation might be caused by simultaneous ligation of EP2 and EP4. Differences in the PGE2 network among genotypes may contribute to their differential susceptibility towards disease, as endogenous PGE2 has been implicated in the fine-tuning of allergic reactions. The current findings provide the basis to explore the modulation of MC signaling by the genetic background of the host.  [1]. More recently in Japan, a soap containing a-HWP elicited severe skin reactions and food allergy in more than 2000 people [2]. Gliadins and glutenins, the main components of wheat proteins, are characterized by homologous domains constituted of repeated sequences of 6-8 amino acids rich in glutamines. During acid-hydrolysis, the random process of deamidation results in heterogeneous deamidation in each repeated sequences [3]. This work investigated the effect of the deamidation rates of the repeated sequences of a-HWP on their triggering potency.

Intensity of deamidation in the epitopes of acid-hydrolyzed wheat proteins is a key parameter for their allergenicity
Methods: Three batches of deamidated gliadins were produced by increasing the acid-hydrolysis duration. These 3 samples and 5 industrial HWP samples involved in European or Japanese cases of allergy were characterized for their content in native, weakly deamidated and highly deamidated repeated sequences by competitive ELISA. Their triggering potency was determined using a basophils assay with HWPallergic patients' sera. Results: Competitive ELISAs showed that native sequences were progressively converted to deamidated sequences when acid-hydrolysis duration increased. Among the deamidated sequences the content in highly deamidated sequences progressively increased with the treatment duration while the content in weakly deamidated sequence remained constant. Industrial HWPs appeared extremely heterogeneous and displayed various levels of native, weakly and highly deamidated sequences. The ability to activate basophils sensitized with HWP-allergic patients appeared related to the content in highly deamidated sequences. Conclusions: Repeated domains of gliadins and glutenins in a-HWPs are a mix of native, weakly deamidated and highly deamidated sequences which proportions vary among the products released on the market. The content in highly deamidated sequences predominantly contributed to the triggering potency of a-HWP samples. Clin Transl Allergy 2018, 8(Suppl 1):17 context of food allergy to egg, how the degranulation ability changed during short-duration digestion process of native and aggregated OVA was studied. Methods: OVA solutions were heated to form nanometric linear or micrometric spherical-agglomerated aggregates. Native and aggregated OVAs were in vitro digested using a gastrointestinal digestion model based on the INFOGEST harmonized protocol with a final degradation with peptidases of the jejunal brush border membranes (BBM) enzymes. Degranulation abilities were studied for the three OVAs and their digests using a pool of eight sera from egg-allergic children and the RBL-SX38 cell line. Results: Undigested, both aggregates had similar degranulation abilities lower than the ability of native OVA. Native and aggregated OVAs exhibited a similar reduced ability at the end of the digestion but were differently affected during the digestion process. Heated aggregated OVAs were more and more rapidly digested than the native OVA and the small more than the large aggregates. The duodenal phase mostly participated to the digestion of the native OVA and no further digestion during the BBM phase was noticed. The degranulation abilities of the aggregates slightly changed during the digestion process. Although digestibility differed between the aggregates, they exhibited similar degranulation abilities at each step of the digestion process. The degranulation ability of native OVA was mostly decreased by the duodenal digestion; only a small decrease was noticed during the gastric phase and no further change with BBM digestion.
Conclusions: Compared to OVA aggregates, both the higher degranulation capacity of undigested native OVA and its late reduction during the duodenal phase of the digestion process could be responsible for the better tolerance of heated OVA by egg-allergic patients. Background: Peanut allergy belongs to one of the most severe food allergies in the westernized counties and has emerged as a problem in the German speaking counties, too. Whether breast feeding induces tolerance development or on the contrary, leads to sensitization to peanuts is still under discussion. In this study, we developed sensitive and specific diagnostic tools for the investigation of two clinically relevant peanut allergens, Ara h 2 and Ara h 6, in human breast milk in our German breast milk study. Methods: We recruited 40 lactating women without a history of peanut allergy, each consuming 100 g of dry roasted peanuts after which breast milk samples were retrieved at different time points. Two ELISA systems were developed and validated for the quantification of Ara h 2 and Ara h 6 in the low ng/mL range.

Results:
The Ara h 2 ELISA revealed a limit of detection (LOD) of 1.3 ng Ara h 2/mL breast milk and a quantification range of 2.3-250 ng/mL. The Ara h 6 ELISA showed an LOD of 0.7 ng/mL and a quantification range of 1.1-14.4 ng/mL. No relevant cross-reactivities against potentially relevant cross-reactive legume, tree nut and seed extracts were noted. By means of these assays, Ara h 2 could be measured in 14/40 (35%) lactating women in concentrations between 2.3 and 184 ng/ mL breast milk and Ara h 6 was detected in 9/40 (22.5%) of the participants between 1.1 and 9.7 ng/mL and one highly positive sample with 79 ng/mL. Notably, Ara h 2 and Ara h 6 were transferred at the same time courses of appearance after ingestion, but Ara h 6 in lower concentrations than Ara h 2.

Conclusions:
The Ara h 2 and Ara h 6 ELISA were developed as sensitive and specific diagnostic tools for the assessment of the allergen concentration in human breast milk. Evidently, Ara h 2 and Ara h 6 are transferred at the same time points after peanut exposition, however a difference in concentration was observed. By this means investigations on the allergens' sensitizing or tolerogenic properties in human breast milk become accessible on the molecular level. Background: Macadamia nuts (Macadamia integrifolia) are predominantly grown and consumed in Oceania, although they become more and more part of the diet in Europe primarily in bakery products and snacks. With rising consumption the number of cases of macadamia nut allergies increases. Today, they are commonly diagnosed by the detection of serum IgE antibodies against the nut extract. However, for a reliable diagnosis and for the differentiation between allergies to macadamia nuts and other tree nuts and seeds, it is necessary to analyse the subfractions of macadamia nut and to determine the immunoreactivity against defined partial allergens. The aim of this study was the identification of components of macadamia nut associated with allergy. Methods: Proteins from macadamia nut extract (MNE) were separated by 2D gel electrophoresis and subsequently blotted onto nitrocellulose membrane. Blots were incubated with sera of macadamia nut sensitized patients and non-sensitized controls. Specific immunoreactive spots were analysed by means of MALDI-TOF. Proteins were identified, cloned, expressed in E. coli, purified and coated on membranes to produce line blots. IgE reactivity was determined in the serum of patients and controls. Results: 2D Western blots of MNE with sera from macadamia nut sensitized patients showed major spots at a molecular mass around 53-67 kDa and in a pH interval of around 6-9. These spots corresponded to vicilin-like antimicrobial peptides of Macadamia integrifolia (Mac i-VLAP), which were apparently present in different modifications. 11 of 16 sera of macadamia nut sensitized patients reacted with recombinant Mac i-VLAP expressed in E. coli, but none of the controls. The cross reactivity of 24 rMac i-VLAP positive sera to 7 vicilins of other nuts and seeds was analysed in line blots. It was shown that 83% of the sera cross reacted with vicilins from peanut (rAra h 1) and cashew nut (rAna o 1). Furthermore, cross reactivity was observed to vicilins from pecan nut (rCar i 2, 67%), pistachio (rPis v 3, 63%), walnut (rJug r 2, 58%), sesame (rSes i 3, 54%) and hazelnut (rCor a 11, 46%). Conclusions: Mac i-VLAP was identified as a major allergen in macadamia nut. It is useful for detailed partial allergen diagnostics in allergologic laboratories. For a comprehensive diagnosis of nut allergies IgE detection using profiles including different nut allergens especially storage proteins like vicilins is crucial. Background: Peanut allergy is the most common cause of life-threatening anaphylaxis in adolescents and children in German speaking countries. Recently, we identified two novel peanut allergens, the defensins, denominated Ara h 12 and Ara h 13. Interestingly, in the course of purification we encountered a further IgE-reactive protein in the low molecular weight (LMW) range. In order to investigate the allergenic risk and improve diagnostic test systems, allergens that lead to clinical reactions need to be identified and characterized. Therefore, our aim was to isolate and characterize this potential novel allergen. Moreover, we wanted to compare the impact of thermal processing of this molecule with known LMW peanut allergens (Ara h 12/Ara h 13) on IgE reactivity as food processing (e.g. roasting) has been shown to increase the allergenicity of diverse peanut allergens. Clin Transl Allergy 2018, 8(Suppl 1):17

P26 Identification and characterization of IgE reactive low molecular weight peanut proteins
Methods: LMW peanut proteins of raw and in-shell roasted peanuts were isolated by lipophilic extraction and subsequent chromatographic separation techniques. Isolated proteins were identified by mass spectrometry and N-terminal sequencing. Sera of peanut-allergic patients with severe allergic symptoms, sensitized but peanut-tolerant patients and non-allergic individuals were screened by immunoblot analysis for IgE binding to these molecules. Additionally, the ability of the isolated proteins to trigger allergic reactions was assessed by basophil activation test.

Results:
In the course of Ara h 12/Ara h 13 purification, we encountered a novel LMW IgE reactive peanut protein which was able to stimulate basophils of peanut-allergic individuals in vitro. Mass spectrometric analysis and N-terminal sequencing revealed that the IgE reactive protein is a third novel peanut defensin with a homology of 32% to Ara h 12, 39% to Ara h 13.0101 and 41% to Ara h 13.0102, respectively. The majority of peanut-allergic patients sensitized to defensins displayed more severe allergic symptoms. Defensins from in-shell roasted peanuts showed a higher IgE binding capacity in western blot analysis and led to an increased basophil activation compared to peanut defensins from raw peanuts. Conclusions: Roasting enhances the IgE binding of the novel identified peanut defensin, as well as of Ara h 12 and Ara h 13. Furthermore, our data suggests that IgE binding to peanut defensins correlates with the severity of allergic symptoms. Background: Peach tree pollen has ben identified as relevant in areas of peach tree cultivar. After olive and grass, it is the third one inducing sensitisation in these areas. Our aim was to study if peach tree pollen contain other allergens that can induce sensitisation in addition to Pru p 3. Methods: Skin tests with peach pollen extracts were made in subjects with seasonal symptoms during the period of production of this pollen in an area of high exposure. Sera from positive skin tests cases were obtained and SDS-PAGE and immunoblotting analysis was made.

P27 IgE and allergenic activity against α-Gal containing proteins in the ticks ixodes Ricinus and Amblyomma americanum
Results: Using pool of sera of mono-sensitized cases negative to Pru p 3 and other pollens several bands were identified that corresponded to 45, 25 and 15 kD. We named the 15D band as Pru p X. This protein and Pru p 3 in 110 cases skin test positive to peach pollen. The 40% were prick positive to Pru p X and the 35% to Pru p 3. The 12% were positive to both and in the remaining cases with skin test positive to peach pollen both were negative. Conclusions: Peach pollen has several allergens that can be involved in the induction of sensitisation and allergy in highly exposed populations. From these we identify the Pru p X that has not been previously recognized. Because subjects were also positive to Pru p 3, the respiratory tract can be a pathway of sensitisation to this pan-allergen. The clinical relevance of these findings is under evaluation. Background: Pomegranate, Punica granatum, is a fruit whose consumption is growing as it is considered a health-promoting food. Mild to very severe allergic reactions to this fruit are also increasingly reported. Class III chitinase is one of the major protein components found in the extracts from pomegranate pulp, but its potential allergenicity has not been investigated so far.

P29
Methods: Aim of this study was the isolation of pomegranate class III chitinase and the investigation of its allergenicity. The protein was isolated from pomegranate pulp extract by RP-HPLC, identified by direct protein sequencing, purified by conventional chromatographic separations and the capacity to bind specific IgE in the sera of allergic subjects was investigated using the FABER ® test bearing five pomegranate's preparations in total: Pun g (seed extract), Pun g 1 (9k-LTP), Pun g 5 (Hevein like protein), Pun g 7 (Pommaclein), Pun g 14 (class III Chitinase). Allergome database shows a high similarity with the latex hevamine (Hev b 14) and with the raspberry homolog (Rub i Chitinase), sharing 69 and 62% sequence identity, respectively. A lower structural similarity has been recorded with other IgE binding class III chitinases, such as the one from Chinese-date (Ziz m 1) and from coffee (Cof a 1), sharing 42 and 38% identity, respectively. The purified Pun g 14 was spotted on the FABER ® biochip and the results obtained after testing a large population of allergic subjects were analyzed in comparison with those obtained for the other pomegranate allergenic preparations available on the FABER ® test. Out of 4537 tested patients 266 (6%) turned out to be sensitized to at least one of the 5 pomegranate allergenic preparations present on FABER ® , with the following prevalence calculated out of the 266 patients: Pun g seed extract and Pun g 1 54%, Pun g 14 23%, Pun g 7 13% and Pun g 5 6%.

Results
Conclusions: A new IgE binding protein, the class III chitinase, Pun g 14, has been identified in pomegranate. It displays high structural similarity with the homologous allergens from latex and raspberry. Pun g 14 can contribute to improve the allergy diagnosis to pomegranate and, in general, to plant allergenic sources.

P30 Evaluation and characterization of Ory c 3, a major rabbit allergen
Stephanie Background: Ory c 3 is a major rabbit allergen. It is a heterodimer composed of two peptide chains, lypophillins called CL2 and AL. Ory c 3 belongs to the secretoglobin family and it has high structural identity with Fel d 1, the major cat allergen. Objective: To produce Ory c 3 as recombinant protein, to compare it to the natural allergen and to set up a detection assay. Methods: cDNAs corresponding to Ory c 3.A.0101 (CL2) and Ory c 3.B.0101 (AL) were isolated from rabbit salivary gland by RACE PCR. Both cDNAs were cloned as head-to-tail construct with C-terminal His-tag. Recombinant Ory c 3 (rOry c 3) was expressed in E. coli and purified by affinity and ion exchange chromatography. Native Ory c 3 (nOry c 3) was purified from rabbit fur by gel filtration and ion exchange. Identity was assessed by by mass spectrometry. Secondary structure analysis was performed using circular dichroism. IgE-binding of rOry c 3 and nOry c 3 was analysed by ELISA using sera from 36 rabbit-allergic patients. Polyclonal anti-sera to rOry c 3 were raised in guinea-pigs and an Ory c 3 detection assay was established. Results: rOry c 3 was expressed as head-to-tail fusion protein. The recombinant protein showed a folding which was similar to nOry c 3. Thermal stability was very high and both proteins readily folded back to their initial structures. Mass spectrometry of purified nOry c 3 confirmed that the heterodimer is composed exclusively of CL and AL2. 81% of the rabbit-allergic patients were sensitized to nOry c 3 and IgEbinding to rOry c 3 and nOry c 3 was very similar (r = 0.9689). Ory c 3 could be detected in rabbit urine and dander. The allergen was also confirmed to be present in the New Zealand White rabbit, dwarf rabbit and two breeds raised for meat. Conclusions: The expression of rOry c 3 as fusion protein of two monomers yielded a recombinant protein of similar structure, stability and IgE-binding as the natural allergen. Ory c 3 is a specific marker of rabbit allergy and a valuable diagnostic tool for determining a primary sensitization. Background: Most fish-allergic patients are sensitized to muscle parvalbumin. Clinical cross-reactions are common, but a number of patients tolerate specific fishes. The knowledge on molecular and immunological properties of parvalbumins from different fishes is essential to understand this variable clinical reactivity. Angler fish (Lophius piscatorius) is a food fish which is popular as a delicacy but not yet characterized concerning its potency to induce allergic reactions. The aim of this project was to analyse angler fish parvalbumins regarding their properties as putative food allergens. Methods: Angler fish protein extracts were separated by gel electrophoresis, parvalbumins identified in immunoblots with specific antibodies and quantified in SDS-PAGE by densitometric analysis. cDNAs coding for parvalbumin isoforms were cloned and one isoform expressed in Escherichia coli. Natural, purified parvalbumins were analyzed regarding their IgE reactivity by ELISA, their stability towards in vitro gastrointestinal digestion and their structural properties by circular dichroism spectroscopy. The humoral immune response to angler fish parvalbumin was investigated in a BALB/c mouse model. Results: Angler fish contains 0.6-1.5 mg parvalbumins per gram muscle. We identified three parvalbumin isoforms which differed by their migration behavior in SDS-PAGE (6-14 kDa), their isoelectric points (pH 4-5) and in their N-termini. Protein sequence comparison of cloned parvalbumins gave an identity of 69%, confirming the presence of true isoforms. Purified natural angler fish parvalbumins and a recombinant parvalbumin were recognized by IgE antibodies from 70% of cod-allergic individuals. The natural parvalbumins showed thermally stable alpha-helical structures sensitive to calcium depletion. Analysis of the proteins' stability towards gastrointestinal digestion revealed that an angler fish parvalbumin isoform resisted partially to this treatment and was still detectable by specific antibodies. A mouse model substantiated that angler fish parvalbumins represent immunogenic molecules, although the humoral immune response to carp parvalbumin was stronger than to the angler fish homologs. Conclusions: Angler fish parvalbumins might be important food allergens as they are stable, highly abundant and recognized by fishallergic patients' IgE-antibodies. Recombinant angler fish parvalbumin could be an important reagent for a future diagnostic panel of standardized molecules.

P31
Background: Rationale: Many existing web technologies have made the jump to mobile devices. Scientific resources, however, have been slow to follow. Current allergen databases are a powerful source of bioinformatics knowledge, but their utility is diminished by a lack of accessibility. Most productive science occurs at the lab bench, away from desktop computers but accessible to mobile devices. Our aim was to develop an Android application that could provide up to date information about allergens and be immediately accessible. Methods: A C ++ program was written to download HTML content from Allergen.org. These HTML files were processed through the command-line tools grep and sed, as well as through a Python program. The entries were then validated and parsed into a SQLite database. Finally, a user interface was written in XML format with underlying logic written in Java. The source code is made freely available on github.com (https://github.com/ninjha01/Mast). Results: An Android application that will automatically update as new information is added to the WHO/ISIS allergen nomenclature database was successfully developed. This was made possible by constructing a web scraper that would periodically create a local, searchable database using the technologies outlined above. The app replicates functionality present in the WHO/IUIS website; allergens can be searched by name, taxonomy, source, or biochemical name. All information contained in the online database is stored in the application locally, so users are not required to maintain an internet connection-functionality that will never be present in the webpage-based implementation. Conclusions: With the rise of mobile computing, scientists should expect their tools to accompany them wherever they go, whether it be the desk or the bench. The App for Allergens updates and improves a valuable bioinformatics resource, the WHO/IUIS allergen database, for allergy/immunology research. In addition, it provides an upgradeable, extendible platform that can quickly absorb changes in the database, as well as provide new features (e.g. 3D structures and offline access) and research capabilities. Background: The alpha-gal syndrome is characterized by a delayed (3-6 h) allergic reaction to red meat and the presence of specific IgE antibodies directed at the carbohydrate epitope galactose-alpha-1,3-galactose (alpha-gal). In particular pork kidney and innards seem to be triggers that induce faster and more severe allergic reactions. Moreover, several drugs of mammalian origin have been reported to trigger allergic reactions in these patients, e.g. therapeutic antibodies (cetuximab), antivenoms and Gelofusine, a volume replacement. Objective: To determine if further drugs composed of proteins of porcine origin contain clinically relevant quantities of alpha-gal and induce positive skin tests and basophil activation in patients with alpha-gal syndrome. Methods: Creon ® and Enzynorm ® f were obtained from the hospital pharmacy. Creon is a pancreatic enzyme preparation, a combination of porcine-derived lipases, proteases, and amylases indicated for the treatment of exocrine pancreatic insufficiency. Enzynorm contains mainly the protease pepsin which is obtained from porcine gastric mucosa. Both drug preparations were analysed by immunoblot and ELISA using a monoclonal antibody directed against alpha-gal and sera of patients with alpha-gal syndrome. Different fresh meat sources as well as Creon and Enzynorm were used to perform skin tests in 10 patients. Furthermore basophil activation (Flow CAST ® ) with different concentrations of a pork kidney extract and these two drugs were assessed in 14 patients with alpha-gal syndrome and 4 controls. Results: The main ingredient of Enzynorm, pepsin, was shown to carry the alpha-gal epitope. Creon was shown to contain alpha-gal epitopes, but no protein carrying this epitope could be identified so far. Skin tests using Enzynorm produced systematically a stronger reaction than pork kidney. All patients were positive with Enzynorm and Creon, 9/10 with pork kidney, 5/10 with raw pork meat and 7/10 with raw beef meat. Creon showed a strong basophil activation in 13 out of 14 patients, Enzynorm in all 14 patients analysed. When compared to pork kidney extract, basophil sensitivity was found to be increased about tenfold with Creon and Enzynorm. No activation was detected in basophils of 4 control patients. Background: Allergies due to venoms of hymenoptera can cause severe anaphylaxis in untreated patients. In the last years, progress of component-resolution advanced the differential diagnosis of honeybee and wasp venom allergic patients. To date, the discrimination between Vespula and Polistes venom allergy is still challenging, as only few allergens have been identified for component-resolved diagnostics. Both species live side to side in Mediterranean regions and the US, but with Polistes dominula being an invasive species, Polistes venom allergy is likely to evolve in more moderate climate zones of Europe. In this study, Polistes venom was analyzed for the presence of additional allergens. Newly identified allergens were subsequently characterized in detail to broaden the available panel of important allergens. Methods: Polistes venom was analyzed by mass spectrometry. Identified components were cloned from venom gland mRNA and recombinantly produced in insect cells. The resulting purified proteins, together with their homologues of different hymenoptera species, were characterized by immunoblotting and assessed for IgE crossreactivity. Moreover, their capacity to activate basophils of either honeybee or wasp venom allergic patients was evaluated. Results: Several Polistes venom components were identified and two proteins (100 kDa and 41 kDa) were successfully produced in Sf9 insect cells together with the homologous allergens from Apis mellifera and Vespula vulgaris. The analysis of specific IgE in sera from honeybee, Vespula and Polistes venom allergic patients identified the novel components as major allergens. Additionally, basophil activation tests proved their clinical relevance. Cross-reactivity on IgE level and basophil activation indicates the presence of shared IgE epitopes, probably in conserved regions of venom proteins. Conclusions: The analysis of crude Polistes venom identified several, yet unknown components. The two novel recombinantly produced proteins proved to be allergens of Polistes venom and, therefore, might become key elements for molecular diagnostics in the future. Background: Epicutaneous immunotherapy (EPIT) is a promising treatment for food allergy under clinical investigation. In animal models, EPIT seems to confer sustained unresponsiveness and prevents further sensitization. In this study, we investigated the kinetics of miRNA expression patterns underlying the therapeutic effect of EPIT and its persistence compared to placebo or oral immunotherapy protocols (OIT). Methods: BALB/c mice were orally sensitized to peanut and then treated with EPIT or not treated (sham). Mice (n = 112) were sacrificed during treatment at 1, 2, 4, 6 and 8 weeks; and 8 weeks after the end of treatment. MiRNAs were analysed in sorted CD4+ cells from spleen using high-throughput sequencing on a HiSeq4000. Results: were validated in an independent experiment (n = 112) including also a group treated with OIT with mice sacrificed during treatment at 2, 4 and 8 weeks, and 8 weeks after the end of treatment by LNAenhanced qPCR assays targeting 40 miRNAs identified in the sequencing experiment. Results: Global miRNA profiles consisting of ~ 1000 miRNAs reproducibly distinguished EPIT-treated mice from controls as early as one week following initiation of treatment. Between 23 and 190 MiRNAs were found to be differentially expressed (padj < 0.05) with a large overlap of miRNAs between adjacent time points. Differentially expressed miRNAs include miRNAs controlling T cell stability and plasticity (e.g. Tregs, miR-10a) and Th2 cytokine production (e.g. miR-92a-3p and miR-423-5p). 34 miRNAs were differentially expressed eight weeks after the end of the treatment. Experiments in the second cohort confirmed large changes in miRNA early during treatment with 29 miRNAs differentially expressed at 2 weeks, and 12, 4 and 9 miRNAs at 4, 8, and 8 weeks after the end of the treatment. In contrast only a single of the selected miRNAs differed between sham and OIT treated animals. Conclusions: EPIT leads to early and reproducible changes in miRNA expression shortly after the initiation of treatment differentiating EPIT from sham or OIT-treated mice and expression changes are maintained after the termination of treatment. Differentially expressed miR-NAs include miRNAs in T cell plasticity and postulated targets include genes previously associated with allergy and asthma. Our study provides further evidence for the molecular alterations underlying sustained unresponsiveness in EPIT. Background: The pathological mechanism of allergic enteritis (AE) is not well known in comparison to other clinical phenotypes in food allergy. The aim of our study is to elucidate cellular and molecular mechanism of AE using a murine model. Our previous microarray analysis indicated that gene expressions of CC chemokine receptor Clin Transl Allergy 2018, 8(Suppl 1):17 8 (CCR8) and its ligand, CC chemokine ligand 1 (CCL1 or I-309) were up-regulated in the inflamed tissues of AE mice (unpublished data). In the present study, we investigated the role of CCR8 in induction of AE using CCR8 knock out (KO) mice. Methods: BALB/c wild type (WT) and CCR8 KO mice were sensitized by i.p. injection with ovalbumin (OVA, a major egg white allergen) plus ALUM, and challenged by feeding egg white diet. Morphological changes and granulocytes accumulation in the inflamed jejunum were assessed by histological analysis. The frequency of granulocytes in lamina propria of small intestines was assessed by FACS. Serum levels of OVA-specific IgE antibodies and concentrations of cytokines and CC chemokines in homogenates of small intestines were measured by ELISA. T cell responses in the mice were assessed by in vitro antigenrecall assay using CD4+ T-cells isolated from mesenteric lymph nodes. Results: CCR8 KO mice exhibited similar inflammatory features (e.g. disrupted villi, crypto elongation and goblet hyperplasia) but less accumulation of eosinophils in the inflamed tissues, when compared to WT mice. FACS analysis showed a decreased frequency of eosinophils (CD11b-SiglecF+ cells) and an increased frequency of neutrophils (Ly6G+ CD11b+ SiglecF-cells) in lamina propria leukocytes (CD45+ cells) of CCR8 KO mice. Interestingly, the concentrations of CCL11 (eotaxin-1), but not of IL-5, another eosinophil chemoattractant, were reduced in intestinal homogenates of CCR8 KO mice, compared to those of WT mice. Production of Th2 cytokines (IL-4 and IL-5) by CD4+ T-cells and the serum levels of OVA-specific IgE antibodies were similar in both mice, suggesting that deficiency of CCR8 does not influence T cell and antibody responses upon allergen challenge. Conclusions: Our results suggest that CCR8 is engaged in CCL11 production and thereby contribute to eosinophil migration to inflammatory sites in AE, whereas neutrophils migrate in a CCR8 independent mechanism. Through a better understanding of the AE mechanism, this study will provide the basis to establish a novel anti-inflammatory strategy for treatment of food allergy. Background: Eosinophilic esophagitis (EoE) is an inflammatory condition of the esophagus characterized by the presence of a large number of eosinophils. Currently, EoE diagnosis is based on invasive endoscopic procedures for histopathological examination in combination with the clinical history of the patient. Hence, the identification of noninvasive biomarkers would be valuable for diagnosis and monitoring of EoE. In this study, our aim was to select and validate short peptides with potential to be used as novel biomarkers for EoE detection. Methods: For the biomarkers selection, we performed a comparative proteomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of esophageal biopsies from pediatric patients with eosinophilic esophagitis, gastroesophageal reflux disease and healthy individuals. Phage display technology was used to select peptides against up-regulated proteins identified in patients with EoE. A total of twelve phage clones were selected after three biopanning rounds, while their reactivity was evaluated in a phage-ELISA assay using patient mucus samples. Furthermore, sequences of the peptides were determined by phage-DNA sequencing and the binding between peptide and protein target analyzed by in silico prediction tools. Results: Mass spectrometry results showed that eosinophil cationic protein (ECP) was up-regulated in EoE patients. ECP is an eosinophil granule protein that is deposited in tissues, indicating tissue damage. A high reactive ECP-binding peptide (E8) was able to distinguish mucus of eosinophilic esophagitis patients from gastroesophageal reflux disease and healthy individuals by ELISA, achieving sensitivity of 84.62, specificity of 82.72, a positive likelihood ratio of 4896, and an area under the curve of 0.84. Conclusions: This is the first study to demonstrate the detection of eosinophil cationic protein using peptides isolated from a phage display library. The peptide presented herein could be a useful diagnostic tool for the detection of EoE patients.

Background:
We have previously shown that reactivity and sensitivity of the early response of cultured human mast cells (MCs) increase with increasing IgE affinity. Similar results have been obtained with bone marrow-derived murine mast cells (BMMCs) activated with antigens with different relative affinities for binding to FcεRI-bound specific IgE. The late phase response was surprisingly different in BMMCs; the low affinity interaction gave rise to enhanced chemokine expression, whereas the high affinity interaction resulted in an enhanced cytokine expression. Here we explore whether differences in the affinity of IgE for allergen result in a similar pattern of mediator release from human mast cells. Methods: Human MCs generated from CD133+ stem cells were sensitized with pairs of recombinant human IgE clones with either high or low affinity for Dermatophagoides pteronyssinus antigen 2 (Der p 2). Activation of MCs was measured as upregulation of CD63 by flow cytometry. MC reactivity (fraction of MCs activated, %CD63+ MC) and sensitivity (allergen concentration triggering a half-maximal response, EC50) were estimated by non-parametric curve fitting. The release of cytokines and chemokines from activated MCs was measured using a multiplex immunoassay based on the Proximity Extension Assay (PEA) technology (Olink, Uppsala, Sweden). Results: The combination of two high affinity IgE clones significantly increased MC reactivity (p = 0.0286) and MC sensitivity (p = 0.0286) relative to a pair of low affinity IgE clones (n = 4). Interleukin (IL)-6 (p = 0.0187), IL-13 (p = 0.0018) and IL-8 (p = 0.003) secretion was significantly increased at high IgE affinity compared with baseline and with low affinity stimulation. Secretion of the chemokines CCL3 (p < 0.0001) and CCL4 (p < 0.0001), but not CCL2 (p; ns), was significantly increased at both high and low affinity stimulation compared with baseline. However, the response was not affected by IgE affinity. Conclusions: The differential chemokine response at low IgE affinity could not be reproduced. Increased IgE affinity for the allergen increased MC reactivity and sensitivity, and enhanced MC cytokine, but not chemokine, response. This suggests that affinity maturation of the IgE population is likely to substantially enhance the MC response in vivo and thus the extent and characteristics of the clinical response upon allergen encounter.
Background: Interleukin-10 (IL-10) is an anti-inflammatory cytokine secreted by many different cells, including antigen-presenting cells, mast cells, eosinophils, B cells, and T cells. The regulatory activity of IL-10 includes the inhibition of proinflammatory cytokines involved in Th1 and Th2 differentiation, chemokines, as well as antigen-presenting and costimulatory molecules in monocytes/macrophages, neutrophils, and T cells. Within the field of allergy, to investigate the immunosuppression of allergic reactions mediated by IL-10 produced by functional Tregs during the generation of immune tolerance to allergens is of high interest. In the present study, an IL-10-like peptide was investigated for its capability of suppressing a proinflammatory immune response. Methods: IL-10-like peptides were selected from a phage-displayed peptide library through their capabilities of binding to the IL-10 receptor on macrophages. Aiming to evaluate peptides action, structural analysis was carried out using in silico approaches, while immunoregulatory activity analyzes were carried out through ELISA, macrophage stimuli, mediator release and antigen-specific T cell proliferation. Results: A total of 46 different peptides were selected and based in the immunoreactivity obtained in a phage-ELISA assay performed, the mimIL-10-like peptide was chosen to be further investigated. However, the other IL-10-like peptides will be also investigated in future studies. In silico analysis showed that, the mim-IL-10-like peptide selected interacts with the IL-10 receptor in the same site as the IL-10 molecule. The synthetic peptide was able to decrease lL-6, MCP-1 and TNF-α secretion in macrophage, decrease basophil degranulation and antigen-specific T cell proliferation isolated from birch pollen allergic patients.

Conclusions:
The mimIL-10-like peptide described in this study was able to modulate the expression of proinflammatory cytokines and other events that are crucial for the development of an allergic or inflammatory response. Hence, these results suggest that the mimIL-10-like peptide has potential to be explored as an immunomodulatory compound.
Background: Most individuals with hazelnut allergy have milder allergic reactions to birch pollen when sensitized to the PR-10 protein Bet v 1 a homologue to the major hazelnut allergen Cor a 1. Severe symptoms to hazelnut have been shown in individuals primary sensitized to the hazelnut storage proteins Cor a 9 and Cor a 14. Th2 cells producing the cytokines IL-4, IL-5 and IL-13 constitute the majority of the allergen-specific Th cell responses in allergic diseases. However, other cytokines as IL-9 and IL-31 have recently been associated with a Th2 phenotype. To better understand the different clinical reactivity to PR-10 and storage proteins, we aimed to investigate if hazelnutspecific Th cells of primary PR-10 and storage protein sensitized individuals differ in their production of Th2 associated cytokines in a population of tree nut allergic subjects. Methods: Subjects (n = 36), with a clinical reactivity to tree nuts were included. PBMCs was stained with CFSE and stimulated (2 × 10 6 cells/ mL) with and without whole hazelnut extracts (100 µg/mL) for 7 days. Allergen-specific Th cell phenotypes was analyzed by flow cytometry. Results: The included individuals were first grouped by their sIgE levels in storage protein (Cor a 9+ Cor a 14 > Cor a 1), PR-10 protein (Cor a 1 > Cor a 9+ Cor a 14) and non-sensitized (< 0.35 kUA/L). As expected, the hazelnut-specific Th cells of the storage and PR-10 protein sensitized groups showed higher levels of highly differentiated IL-4+ IL-5+ Th2 cells than in the non-sensitized group. In contrast, only the hazelnut-specific Th cells of the PR-10 sensitized subjects had more IL-31+ Th2 cells compared with the non-sensitized. We next subdivided the subjects in three groups of sIgE (Cor a 1, Cor a 9 and Cor a 14) negative or positive with no birch pollen allergy as well as sIgE positive with birch pollen allergy. Interestingly, a higher frequency of IL-31+ IL-5-hazelnut-specific Th cells were found in the sIgE sensitized subjects with birch pollen allergy compared with both groups with no birch pollen allergy. Conclusions: A higher frequency of the Th2 cell associated itch cytokine IL-31 was found in the hazelnut-specific Th cells of PR-10 sensitized subjects compared to the non-sensitized. We furthermore found a larger fraction of IL-31+ IL-5-hazelnut-specific Th cells in the subjects having pollen allergy indicating a different allergen-specific Th2 response in PR-10 and storage sensitized subjects. Background: To investigate the role of CD28 signal on the steroid responsiveness in asthma, effects of CTLA4-Ig and glucocorticoid on T cell activation and asthma model was analyzed. Methods: Ovalbumin (OVA) specific murine helper T cell (Th) clones were derived from either Balb/c mice immunized with OVA/CFA or DO11.10 transgenic mice expressing T cell receptor specific for OVA/H-2d. To analyze steroid responsiveness in vitro, Th clones were cultured with antigen presenting cells and OVA in the presence of various concentration of dexamethasone (DEX). Proliferative responses of were measured by 3H-thymidine incorporation. For in vivo analysis, unprimed BALB/c mice were transferred with Th clones, challenged with OVA, and administered with DEX subcutaneously. Bronchoalveolar lavage fluid (BALF) was obtained 48 h after challenge, and the number of infiltrating cells was differentially counted. CTLA4-Ig was administered intravenously. Results: Steroid sensitive (SS) and steroid resistant (SR) clones were selected based on the effect of DEX on the proliferative responses of antigen-stimulated Th clones. Airway infiltration of eosinophils of mice transferred with SS clones were effectively inhibited by DEX administration. In contrast, those of mice transferred with SR clones were not significantly inhibited by DEX. Administration of CTLA4-Ig significantly suppressed the proliferation of DEX-treated SR clones in vitro, and the eosinophil infiltration of SR asthma model transferred with SR clones in vivo. In addition, CTLA4-Ig and DEX synergistically suppressed BALF eosinophilia of mice transferred with SS clones. Conclusions: CD28 signal is involved in steroid responsiveness both in vitro and in vivo, and a good therapeutic target.
Background: Atopy is a condition that predisposes a person to certain allergic responses. This pathology includes some diseases such as atopic dermatitis, bronchial asthma, hives, etc. Asthma particularly is one of the most prevalent chronic diseases, in which an innate immune component and epigenetic mechanisms take place (DNA methylation and regulation of gene expression by miRNA mainly). The aim of this study is to compare the level of methylation and expression of TLR2 and TLR4 genes in atopic diseases (bronchial asthma). Methods: Scrapings from the mucous membranes of the upper respiratory tract were taken from 43 children from the age of 2-7, who were treated for bronchial asthma in Scientific Center of Children`s Health. They also were divided in 3 groups: patients without any allergic diseases, autoimmune disorders or infections (16), children with moderate (13) and severe (14) asthma. During the research the following methods were used: DNA extraction, sodium bisulfite conversion, methylation-specific PCR, restriction and detection. Results: At the first stage of data analysis a strong correlation between the methylation degree and the severity of asthma was found out. It has been shown that healthy patients get methylated or partially methylated regions in 50% of cases. There is also a slight increase of incompletely methylated sites in children with moderate asthma. In contrast to the previous groups, a small amount of unmethylated gene promoters appears in patients who developed severe form of bronchial asthma. The same situation also holds for methylated promoter sites in TLR4. But this time the amount of unmethylated parts becomes bigger and occurs in all 3 experimental groups. It should be noted that the number of unmethylated sites in patients with severe asthma double that in healthy ones (from 25 to 50%). Based on data for methylation, the expression profile of targeted gene promoters was also estimated. Conclusions: The present study demonstrates a strong connection between methylation status and the incidence of bronchial asthma. TLR2 and TLR4 are significant markers of innate immunity. They might be used in early case detection and in further epigenetic discovery of asthma. Background: Mycoplasma pneumoniae is the etiological agent in about 60% of all cases of community-acquired pneumonia in children older than 7 years and teens, is associated with the development of asthma. The involvement of M. pneumoniae in the pathogenesis of respiratory diseases is because of the multiplicity of pathogenicity factors, the major of which is hydrogen peroxide, which is released in enzymatic reaction catalyzed by glycerol-3-phosphate oxidase (gene name MPN051). In the structure of the enzyme amino acid His in position 51 is significant, because His51 participates in proton transfer during the oxidase reaction and the rate of this process determines the rate of hydrogen peroxide formation. The aim of the study was to detect possible mutations in His51 codon containing fragment (location: from 50 to 260 in gene) of MPN051, capable to influence the rate of hydrogen peroxide formation in M. pneumoniae isolates. Methods: Mycoplasma pneumoniae was isolated from sputum and throat swabs of 54 children and teens (7-17 years old) with pneumonia, 18 of them had also asthma, with a preliminary positive result of M. pneumoniae DNA detection by PCR. MPN051 gene of all M. pneumoniae isolates was tested for mutations by sequencing. On the 10th day of culturing for all M. pneumoniae isolates the formation of hydrogen peroxide was studied in a semiquantitative peroxide test. Results: Mutations associated with reduced and enhanced levels of hydrogen peroxide production by M. pneumoniae were detected. Mutation A152T, leading to change His51Leu, was observed in 7 isolates of M. pneumoniae from children and teens with pneumonia without asthma and was associated with reduced production of hydrogen peroxide (about 4 mg/l) in comparison with M. pneumoniae isolates without mutations (about 10 mg/l). Mutation G163C, leading to change Asp55His, was associated with enhanced production of hydrogen peroxide (about 20 mg/l) and was prevalent in M. pneumoniae isolates from children and teens with pneumonia and asthma (61%). The newly appeared His55 near His51 might promote proton transfer during the oxidase reaction, thereby accelerating the formation of hydrogen peroxide and enhancing the pathogenic properties of M. pneumoniae. Conclusions: Missense mutation G163C in MPN051 is associated with enhanced M. pneumoniae pathogenic properties and is prevalent in children and teens with mycoplasma associated pneumonia and asthma. Background: The scope of ALLERT project is to provide a practical, portable, rapid, and effective diagnostic system to detect allergens in foods. The system includes a multiplex Lateral-Flow Immunochromatographic Assay and a handheld Reader providing a qualitative response ("yes/no") regarding the presence of targeted allergens. This diagnostic system answers a growing need in food safety management and mainly targets agro-alimentary industries and end-user affected by a severe risk in food allergy. The device is meant to be used in remote situations from the laboratory, must therefore be portable, easy to handle and to operate by unexperienced users, be impactresistant and withstand extreme conditions, works quickly (15 min maximum), have low production costs, and ensures a long shelf life. Furthermore, the device must provide clear and reproducible results at the cut-off level. Methods: Design and construction of the multiplex detection test. Multiplexing is achieved by spotting technology, which consists of printing small quantities of antibodies and proteins in the shape of spots on the nitrocellulose membranes. Multiplex conjugate pads were made by integrating the various antibodies of interest conjugated with the gold nanoparticles. Results: a panel of specific polyclonal antibodies directed against the allergens of interest (milk, egg, hazelnut, peanut, shrimp and mustard). Development of a device for the preparation of the food samples. This easy-to-use device allows the extraction of allergens from different food matrices by a standard collection, filtration and purification technique. Multiplex lateral flow method to detect simultanously six targeted allergens. Photonic innovation that consists in manufacturing a signal acquisition device compatible with multiplex detection and adapted to a mobile testing. To increase the quality and the sensibility of the reader, we use the potential of the multispectral imaging approach and therefore increase the dynamic range of signal detection. Signal analysis with a homemade software that provides an automatic qualitative interpretation of the test easily understandable by any untrained user.

Mycoplasma pneumoniae with enhanced pathogenic properties is prevalent in children and teens with mycoplasma associated pneumonia and asthma
Conclusions: This research has demonstrated the feasibility of multiplexing on a lateral flow chromatographic test. The future challenge will be to increase the level of multiplexing of the target allergens, to reduce the cross-reactions between the different molecules and to improve the quality of the signal obtained for each allergen. Background: Component-resolved diagnostics (CRD) has become of growing importance in the field of clinical allergology, providing information that cannot be obtained from extract-based tests. It utilizes purified native(n) or recombinant(r) allergens to detect IgE sensitivity to individual allergen molecules facilitating more precise diagnosis of allergic diseases and identifying sensitizations attributable to crossreactivity. The information may also aid the clinician in prescribing oral immunotherapy (OIT) in patients with severe symptoms, giving advice on allergen avoidance, or needing to perform food challenges. In this study, we evaluate the performance of allergen components on the new system assessing both diagnostic accuracy and intermethod comparison. Methods: The new system is a fully-automated immunoassay platform to quantify specific IgE concentrations in human serum. It utilizes magnetic microparticles to which allergens are coupled by a process called "on-board kitting, " allowing for individual or multiple allergens to be used in a single test at the discretion of the clinician. The assay then adds 4 µL of serum to the coated beads in order to quantify sIgE concentrations for the individual allergen or "custom mix". For this study, a total of 100 patients were used that were positive to 10 components found in either cow's milk, egg, peanut, hazelnut, or short ragweed pollen. A panel of 10 negative patients were also tested against each component. All patients were tested on the new system and on a commercially available allergy platform (Thermo Fisher Scientific, Uppsala, Sweden). Results: The overall agreement between the two systems was 95.5% To determine the degree of CCD-sIgE associated alteration of venomrelated molecular IgE diagnostic with focus on decision-relevant changes. Methods: The RIDA CCD-inhibitor (R-Biopharm AG, Darmstadt, Germany) was established in serological routine diagnostic of insect venom allergy. All sera were tested twice for sIgE against bee and wasp venom with extracted-based and recombinant allergens (Api m1, Ves v1, Ves v5) on an ImmunoCAP 250 automated platform (Thermo Fisher Scientific, Uppsala, Sweden) with and without pretreatment with CCD inhibitor. The effect of the CCD inhibitor was verified with an Immuno-CAP containing MUXF3 from Bromelin. Results: In total the CCD inhibition procedure was applied to 20 CCD-negative samples as controls and 68 CCD-positive samples, from which n = 60 showed sufficient CCD inhibition and were included in further analysis. For bee-related molecular diagnostic CCD-related effects were found in 26.7% of the samples and 20.0% of the results had to be classified as false positive. CCD-related effects at least in one of the wasp-related recombinant assays were found 18.4 and 11.7% of the results had to be classified as false positive. Translating these results on a routine diagnostic laboratory setting for molecular diagnostic, a rate of < 5% of false positive results can be assumed. Conclusions: ImmunoCAP assays with recombinant allergens are indeed partially biased by the CCD sIgE. The extent is substantially lower than the known phenomenon for extract-based allergens. CCD inhibition is a useful tool in special clinical situations but no prerequisite for a routine diagnostic laboratory. Background: Highly purified allergens are core components of in vitro molecular diagnostics. The absence of any allergenic impurities is a fundamental quality criterion for diagnostic allergen components. Manufacturing of high purity peanut allergens from peanut flour is known to be challenging. The aim of this study was to use mass spectrometry (LC-MS/MS) to aid the development of effective purification strategies, establish criteria of purity, and validate purified peanut allergens for use in molecular diagnostics. Methods: Natural peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6 were extracted from blanched or light roast peanut flour at neutral pH (7.4). Peanut allergens were purified by monoclonal antibody affinity chromatography, followed by gel-filtration-and/or hydrophobic interaction chromatography and analyzed by LC-MS/MS, ELISA, and FEIA or chimeric IgE ELISA. SDS-PAGE and Western Blots of peanut extracts and purified allergens were performed under non-reducing and reducing conditions using peanut allergen-specific monoclonal antibodies. Results: Monoclonal antibody chromatography for purification of peanut allergens results in co-purification of other un-wanted peanut allergens. Western Blots of peanut extracts suggest the formation of high molecular weight complexes, notably between Ara h 1 and 2S-albumins Ara h 2 and Ara h 6. After extensive chromatographic clean-up, allergen purity assessed by LC-MS/MS, was > 93%. Immunoreactivity of purified peanut allergens was confirmed in ELISA and by FEIA or chimeric IgE ELISA using sera from peanut-allergic patients. Conclusions: Optimized, ISO-9001 compliant bioprocessing pathways have been established to yield high purity natural peanut allergens. The sensitivity provided by mass spectrometry is critical to confirm allergen purity. Background: Fish is not only an important component in the Mediterranean diet, it is also a common elicitor of food-allergic reactions. The clinical work-up includes anamnesis, sera and skin reactivity analysis and, in some patients, oral provocations. Diagnostic algorithms allowing to predict the patients' clinical reactivity are missing representing an important medical need. The aim of this study was to analyse the correlation of clinical tests (in vitro, in vivo) in a well-characterized Spanish cohort. Methods: Fish-allergic patients (n = 34; mean age 13.1 years) were characterized by detailed clinical records, skin testing with commercial extracts (8 fishes) and ImmunoCAP sera IgE-testing (7 fishes, Gad c 1). IgE line blots were done with extracts from tuna, hake and sole. A total of 84 open food challenges was performed, in the order from tuna (canned, fresh), over hake to sole. Results: Reported clinical symptoms varied from mild to severe, with patients mostly (62%) knowing about the fish species. Skin tests were positive in almost all patients (94%). IgE tests were all positive with extracts, although variable for the different fishes. Specific IgE to parvalbumin (Gad c 1) was positive in 82% of the patients while line blots revealed IgE-reactivity to homologs at variable levels (tuna, 15%; hake 15%; sole 68%), indicating that conformational epitopes might be more common than linear epitopes. IgE-binding was also found for other allergens, such as enolases (tuna, hake, sole; each 9%), aldolases (tuna, 12%; hake 44%; sole 24%) and other 30 to 40 kDa-proteins (tuna, 18%; hake 15%; sole 24%). Food challenges to canned tuna were all negative (n = 29) whereas oral provocations were positive to fresh tuna in 14% (4/29), to hake in 40% (6/15) and to sole in 27% (3/11). Data from sera analysis as well as skin test were found to correlate poorly with results from diagnostic food challenges. Conclusions: The integration of large datasets, ranging from anamnesis, skin testing, allergen-based IgE-measurement, are an important challenge for clinicians in today's clinical practice. A significant number of children may tolerate specific fishes but this still needs to be confirmed by oral challenges as the golden standard. Clin Transl Allergy 2018, 8(Suppl 1):17 Methods: To detail the overall set up of the FABER ® test and some of its performances. FABER ® test bears 244 allergenic preparations, namely 122 molecules and 122 extracts, coupled to nanobeads. The particles are arrayed to a solid phase matrix and allow a one-step comprehensive array-based testing solution needing only 120 µL of serum per test. Each allergen particle population can be individually optimized to achieve the maximum testing performance. The Biorad Lyphocheck Allergen IgE (BL-IgE) is a standard polyclonal commercial preparation obtained by pooling human sera. Results: BL-IgE has been used for the evaluation of the specific IgE. By means of BL-IgE 174 out of 244 allergens gave positive IgE results. BL-IgE is supplied after being tested on 3 different IgE commercial testing systems (ImmunoCAP, Immulite, Hytech). IgE mean values and ranges are provided. Twelve allergen extract results out of 15 provided with the BL-IgE were used for comparison: Alt a, Ara h, Art v, Asp f, Bet v, Bos d, Can f, Der p, Equ c, Fel d, Gal d, Phl p. BL-IgE was tested on 22 consecutive FABER batches, and extract to extract comparison was performed when the same was available on FABER ® . FABER ® IgE, expressed as arbitrary units (FIU), gave the following results: Ara h, overlapping with CAP-IMM-HYT; Art v, slightly below CAP, overlapping with IMM, above the HYT; Bet v, slightly below CAP-IMM; Bos d, above CAP-IMM-HYT; Can f, slightly below CAP-IMM, overlapping with HYT; Fel d, reproducible but below the 3 systems; Gal d, below IMM, overlapping with CAP-HYT; Phl p, overlapping with CAP-IMM. Alt a 1 performed better than CAP-HYT, overlapping with IMM. The 6 Der p FABER allergens gave overlapping results with the 3. Moreover our study disclosed IgE binding to allergens not declared in the BL-IgE data sheet (e.g. Cup a 1, Pru p 3), mostly all the molecule detected specificities (e.g. mite allergens) and all the IgE co-recognized preparations (e.g. egg allergens). Conclusions: FABER ® IVD is a new lab test for multiplex specific IgE detection using allergenic molecules and extracts showing very good performances. All steps in assembling the test are verified and all allergens bind IgE. The 3 systems do not overlap each other as they have different reference standards. FABER ® IgE measurements performs very well with almost all allergens and it helps to disclose unknown sensitizations. Results: 80% of children whom participated OFC passed their challenges. All of 3 who failed OFC had other nut allergies. 7% (4/15) of children whom had the challenges did not have personal history of atopy. Further analysis showed that Ana O3 at threshold of < 0.35 kUA/L and < 1kUA/L provided satisfactory specificity (100%), positive predictive value (PPV at 100%). Cashew specific IgE at the cut-off of < 0.35 had a better sensitivity (100% as compared to 66.7%) and negative predictive value (NPV at 100% as compared to 92.3%). Sensitivity, specificity, PPV and NPV profile for cashew IgE threshold < 3.5 (Grade 2) was similar to those for Ana O3 threshold at < 1. Skin prick test were neither sensitive or specific in the diagnosis of cashew allergy (Table 1). Conclusions: Ana O3 might be used in addition to cashew IgE in the selection of children undergoing hospital based OFC while Cashew IgE < 0.35 may be a good guide for choosing patients for home OFC. Although the application of component resolved IgE testing for cashew allergy may have improved diagnostic characteristics than the use of cashew extract alone, it cannot yet replace clinical history and oral food challenge.  Results: HRV-16 capsid proteins VP1, VP2, VP3, and VP0 (VP2 plus VP4, as a single poly peptide chain) expressed mainly as insoluble proteins in inclusion bodies, while only small amounts expressed in the soluble fraction. Protein solubility was highly dependent on the presence of 0.5 M l-Arginine in most of the purification and storage buffers. The protein preparations were > 90% pure as assessed by silver-stained SDS-PAGE and western blot analysis using HIS-tag and HRV-16 VP2specific antibodies.

Usefulness of component Ana O3 IgE in comparison with specific IgE and skin prick test in the diagnosis cashew allergy in children
Conclusions: Expression of individual HRV capsid proteins is feasible in E. coli and the purified proteins will provide useful tools to study the immune mechanisms involved in rhinovirus-induced asthma exacerbations, epitope mapping, and for diagnostic purposes. Background: Immunotherapy is the only causative treatment for type I allergies, however, it may cause severe side effects. Development of genetically engineered hypoallergenic molecules offers the possibility to improve the safety of immunotherapy. Methods: Previously, a hypoallergenic variant of the calcium-binding fish allergen parvalbumin was successfully engineered by mutating four calcium-coordinating amino acids. We aimed to analyse, whether mutating the same, highly conserved amino acids in the calcium-binding domains of the grass pollen allergen Phl p 7 would also lead to a hypoallergenic molecule. Recombinant wildtype and mutant Phl p 7 were expressed in Escherichia coli and purified to homogeneity. Results: Analysis of the allergenic activity using sera and blood from Phl p 7 sensitized patients in IgE dot blots and basophil activation tests revealed a drastically reduced IgE reactivity and a strongly reduced allergenicity of the mutant variant. To test whether the Phl p 7 mutant protein is an immunogenic molecule, we immunized rabbits with wildtype and mutant Phl p 7 and tested the sera for the presence of Phl p 7-specific IgG antibodies. We saw that rabbit IgG titers were increasing after immunization and that Phl p 7 mutant IgGs were able to block patients' IgE binding to the Phl p 7 wildtype protein. Both, the immunogenicity as well as the blocking potential are prerequisites for a potential applicability of the mutant molecule for immunotherapy of Phl p 7-sensitized individuals. Analysis of the protein structures using circular dichroism spectroscopy revealed that both variants were expressed as predominantly alpha-helical folded proteins. However, temperature scan experiments revealed a reduced thermal stability of the mutant. Size exclusion chromatography linked to inductively coupled mass spectrometry showed that the mutant protein has lost its calcium-binding capacity.

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Conclusions: By mutagenesis of specific amino acids involved in calcium-binding of the grass pollen allergen Phl p 7, we were able to produce an immunogenic molecule which showed diminished IgE reactivity and a highly reduced allergenic activity. We therefore suggest that mutating specific amino acids responsible for the coordination of calcium ions might represent a general strategy to generate hypoallergenic variants of calcium-binding allergens. . Three groups were analysed: allergic mice without SIT, allergic SIT treated mice and untreated control mice (n = 5/group). Results: The analysis of the three different organs showed significant results reflecting an overall tolerogenic environment in the SIT treated mice. T and B cells were less activated in the SIT group compared to allergic mice. NK cells showed a twofold higher production of TNFα in the treated mice with respect to the two other groups. We also found substantial changes in the myeloid compartment with dramatic fivefold decrease in Th2-type macrophage subpopulation and tenfold decrease in mast cells in SIT treated mice compared to the allergic group. This was accompanied by changes in eosinophils and others myeloid cells in the lung parenchyma. Conclusions: Using CyTOF 2, a high throughput and innovative immunophenotyping technology we analysed the immune cell specific changes in a CpG/Fel d 1 SIT model. Our promising results will help to further understand how CpG/allergen SIT treatment modulates the immune system towards tolerance. Our data will help to further develop novel SIT approaches using CpG as adjuvant for patients with perennial rhinitis/asthma.

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Inquiry about the association of cultivable human skin microbiota with asthma outcomes in a group of children and adolescents of Salvador-Bahia Talita  an environment with a high number of pathogens, protect themselves from allergic sensitization. Thus, to understanding the relationship between skin microbiote diversity with asthma and atopy, the present study aims to evaluate the skin microbiota of individuals in Salvador city, Bahia, belonging to a prospective study of respiratory allergies through metagenomics assays. Methods: The skin bacterial flora was collected from the forearm of 50 individuals, separated in five groups based in the phenotypes of asthma and atopy (Atopic Asthmatic-AA; Atopic Asthmatic Remission-AAR; Non-Atopic Asthmatic-NAA; Non-asthmatic Atopic-ANA; Healthy), grown in BHI medium, and bacterial DNA extracted using a commercial extraction kit. DNA from 16S rRNA region was sequencing in 454 GS-FLX Titanium platform by a specialized company. The identification of genders with modulatory action as Acinetobacter was did using specific primers through PCR, and the absence or presence of this bacteria was associated with the production of the regulatory cytokine IL-10 in blood culture stimulated with antigens of Ascaris lumbricoides, Blomia tropicalis, Dermatophagoides farinae and vitamin D.
Results: The result of the 16S rRNA sequencing of the cultivable skin bacteria resulted in the identification of 27 bacterial genera in the study population. The group of individuals classified as AAR and Healthy were found with the higher biodiversity of genders. The presence of Acinetobacter was confirming in 31 individuals and it was observed a higher production of IL-10 in the culture supernatant stimulated with the A. lumbricoides and B. tropicalis antigens in the AA group without presence of Acinetobacter. In all the supernatants analyzed it was possible to detect higher levels of IL-10 production in healthy individuals who presented Acinetobacter bacteria when compared to the Healthy group without the presence of this bacteria.

Conclusions:
The identification of species with modulatory action as Acinetobacter in AAR and Healthy group corroborates the hypothesis that the individual microbiota can influence the development or remission of asthma symptoms. And further studies with a higher number of individuals should be performed to confirm the correlation between the presence of Acinetobacter and the asthma and atopy outcomes. Background: Transforming growth factor-β1 (TGF-β1) has been shown to exert immunosuppressive functions, as reflected by inhibition of immune cell differentiation (Th1 and Th2 responses) and induction of regulatory T cells (Treg), thus playing an important role against the development of immunological disorders. Hence, peptides that mimic the active core of TGF-β1 could be highly promising candidates for immune modulation. In the present study, a TGF-β1-like peptide was evaluated for its capability of modulating the immune response. Methods: TGF-β1-like peptide was selected by phage display technology through competitive elution with the recombinant TGF-β1. Flow cytometry, ELISA, ELISpot, reporter gene, mediator release, intravital microscopy and peritonitis assays were conducted to evaluate the capacity of the peptide to modulate the in vitro or in vivo immune response under inflammatory or allergic conditions. Results: The synthetic TGF-β1-like peptide was able to decrease TNF-α and increase IL-10 production in human PBMCs, and to decrease IL-8 gene expression and cytokine production in Jurkat cells. In vivo experiments showed that in mice sensitized with Phl p 5, the major allergen from timothy grass pollen, the TGF-β1-like peptide was able to decrease IL-4 and IFN-γ, and increase IL-10 production in murine splenocytes. In the same model, the peptide was also able to decrease basophil degranulation and induce Treg cell differentiation. In another mouse model, the TGF-β1-like peptide was able to decrease leukocyte rolling and neutrophil migration under an inflammatory condition. The TGF-β1-like peptide presented herein was able to  induce Treg cell differentiation, modulate Th1 and Th2 responses, as well as other important events that promote the exacerbation of an inflammatory or allergic microenvironment. These findings strongly imply a potential use of the TGF-β1-like peptide as immunomodulatory compound for therapeutic approaches. Background: The chronicle, progressive character of bronchial asthma, the repeated episodes of hypoxemia and hypoxia in bronchial asthma (BA), worsen functional condition of myocardium, which is accompanied by development of pulmonary hypertension. The mentioned thing determines the early diagnostic necessity of transit changes of cardiovascular system in children with BA.

Methods:
The integral estimation of the cardio-respiratory changes in the children with BA. The research covered 52 children with BA aged 6-15. In the first group, 22 children aged 6-10 years, the maximum asthma duration was ≥ 6 years. Whereas in the second group 30 children aged 11-15 years, the maximum asthma duration was < 6 years. All subjects were examined by conventional Doppler echocardiography and they had pulmonary function tests on spirometry. The statistic procession of the received data was performed on the bases of the program package SPSS/V.12. Results: Haemodynamic changes characteristics of BA was dependent on duration of the disease. The patients, who had the Doppler echocardiography normal pressure gradient (9-13 mmHg) in the pulmonary artery, had moderate decrease of Spirometric indices (VC, FEV1, IT). The mentioned data were detected in the first group of BA, where the duration of disease was ≥ 6 years. In the second groups of the patients, with < 6 years BA period, Spirometric datum (VC, FEV1, IT) decrease is detected and pressure gradient increase (21-35 mmHg) in pulmonary artery. The reliable negative correlation connection was revealed in the pulmonary artery between the pressure gradient and the most indexes of external respiration. It is worth mentioning, that there is the moderate connection for IT (r = 0.45, p = 0.01). There was no connection revealed in the pulmonary artery between the pressure gradient VC and FEF75. The sensitivity of the mentioned connections turned out low (p = 0.05): from 1.1% for FEV1, to 21% for FEF25. Conclusions: These findings signify the diagnostic value of Doppler echocardiography in the early detection and monitoring of such deleterious effects among asthmatic patients.

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Oropharyngeal symptoms on first exposure to persimmon Diospyros kaki in an adult patient with Bet V 1-related allergy Background: Allergy to Diospyros kaki, commonly called Oriental persimmon, kaki or Sharon fruit (Ebenaceae family), has been very rarely reported. Cross-reactivity with pollen was involved in some cases, being discussed Bet v 1-like Dio k 1, profilin Dio k 4 and Bet v 6-like isoflavone reductase.

Methods:
We selected a 46-year-old male patient with seasonal allergic rhinoconjunctivitis and convincing history of oral allergy syndrome to hazelnuts, apple and kiwifruit, reporting immediate oropharyngeal allergy symptoms after first eating exposure to raw ripe nonastringent variety of kaki. He never ate other Diospyros fruits, such as American persimmon (D. virginiana) or date plum (D. lotus). Skin prick testing was performed with commercial allergen extracts and soymilk, while prick-prick testing was done with Bet v 1-containing plant foods: apple, hazelnut, kiwifruit, and persimmon D. kaki from South Africa. Multi-parameter line blot immunoassays with native extracts and recombinant molecular allergen components were used for in vitro allergy diagnosis. Results: Regarding Bet v 1-related allergy, the patient presented positive skin prick tests to birch and hazel pollen commercial extracts (each 18 mm diameter wheal), positive prick-prick tests with fresh apple and kaki persimmon (each 4 mm wheal), kiwifruit (3 mm wheal), and hazelnut (6 mm wheal), and negative skin test to soymilk, while serum specific IgE levels were found increased for birch (3.5 kU/L), hazel (0.43 kU/L) and alder (13.4 kU/L) pollen. Serum specific IgE antibodies against Rosaceae (apple, peach) and Actinidiaceae (kiwi) fruits, Betulaceae (hazelnut) and Rosaceae (almond) nuts, Apiaceae vegetables (celery, carrot), Fabaceae legumes (peanut, soybean) and tomato, were not found (< 0.35 kU/L). Specific IgE profile to recombinant components revealed sensitization to rBet v 1 (0.51 kU/L), while serum IgE antibodies to profilin (rBet v 2, rPhl p 12) and polcalcin (rBet v 4, rPhl p 7) biomarkers, and to isoflavone reductase rBet v 6, were not detected (< 0.35 kU/L). Serum IgE level to cross-reactive carbohydrate determinant marker was also below detection (< 0.35 kU/L). 1-cross-reactive plant foods, not only apples and hazelnuts, but also persimmon, a relatively new introduced edible fruit in Europe.

Consent to publish:
Written informed consent to publish was obtained from the patient involved in this study. Background: Anaphylaxis is a life threatening allergic reaction, can cause also severe anxiety disorder and bad quality of life for the patients ever experienced it. Recurrent anaphylaxis is very rare in the literature and due to lack of known triggering factors most of them considered idiopathic. During the 3rd trimester of her 2nd pregnancy a 33 year old women experienced allergic rhinitis symptoms in the BIRCH pollen season. She used no medication. She was otherwise a healthy female and had no atopic disease in her medical history. Case Report: The next birch season while eating RAW FRUITS, like cherry, apple, carrot, plum, celery or peach she experienced swallowing difficulty, and was diagnosed with supraglottic oedema. She had no problem with cooked carrot or celery.

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In the 3rd birch season she experienced bad cough while consuming PEANUT. That time she also had an early miscarriage. The 4th birch season: 1st ANAPHYLAXIS: 2 min after eating HASELNUT for breakfast in a ski resort she felt itchy and swollen throat, then dizziness and collapsed. No adrenalin was administered by the ambulance staff (Poland). 2nd ANAPHYLAXIS: 1 month later they went to another ski resort in Austria. 2 min after drinking SOY extract another anaphylactic episode occured. Her husband gave her adrenalin shot im. She was hospitalized. 3rd ANAPHYLAXIS: Next day in the hotel she ate only 1 bite of a TUNA salad and immediately felt low blood pressure, dizziness and fainted. 4th ANAPHYLAXIS attacked her next morning with having no meal. 5th ANAPHYLAXIS: 3 month later at home 20 min after eating a BANANA she felt discomfort, light headedness and dizziness. She promptly laid down waiting for the ambulance car. 6th ANAPHYLAXIS: 2 weeks later having OATFLAKES in coconut milk with cacao powder caused the same symptoms as happened before. She has been undergone many examinations, blood tests, CRD, a bone marrow sample was also taken. We will unveil her results and her recent treatment. We would appreciate if you could share your opinion with us in order to find the best medical solution.
Conclusions: How this report contributes to current knowledge: No such case has been reported to date.

Consent to publish:
Written informed consent to publish was obtained from the patient involved in this study. Background: Nanotechnology is revolutionizing many aspects of modern medicine, including diagnostics and therapeutics. It is now well established that an antigen presented in soluble monomeric form is a relatively weak immunogen, whereas the same antigen presented on the surface of an organized 3D structure will trigger a broader and stronger immune response. From the strong immunogenicity of these 3D structures, it is hypothesized that bioparticles presenting allergens at their surface should enable protective immunization against major allergens with a low number of injections. In this context, we have investigated the efficiency of a new plant-based platform for the production of allergen presenting bioparticles. Methods: A plant host has been devised for the spontaneous expression of allergen presenting bioparticles. This proceeds through transient expression of the fusion of a synthetic non-immunogenic carrier with an allergen component presented in a geometrically repetitive pattern. Bioparticles harboring spikes of oligomerized major dust mite allergen Der p 2 were produced. These bioparticles were purified, characterized and used for a head-to-head mouse immunogenicity and efficacy study in comparison with the same allergen in a soluble form and whole dust mite commercial extracts. Results: Among the great advantages of this new approach to allergen immunotherapy through allergen presenting bioparticles are its features of simplicity and efficiency. These plant produced bioparticles have been designed to elicit an immune response by direct immunostimulation of antigen-presenting cells. They have at their surface a constant and high density of organized allergens. Product quality is easily standardizable and its composition is perfectly reproducible from batch to batch. The production technology is GMP compliant, low cost and has unparalleled scalability. Its functionality has now been tested with several major allergens. These allergen bioparticles contain several hundreds of allergen molecules on their surface. Current trials in murine allergy models suggest their high potential for fast and efficient desensitization. Conclusions: Our current results suggest that plant-made allergen bioparticles have a real potential to create a strong protective immune response against respiratory allergens.

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Distinct parameters of the basophil activation test are useful as biomarkers for the clinical outcome of patients with Alpha-Gal sensitization