Allergen-specific oral immunotherapy is a disease-modifying treatment already established for respiratory allergies and tested for the treatment of several food allergies, with promising clinical and immunological outcomes. However, orally administered allergens must pass through the gastrointestinal tract, where they are exposed to proteolytic digestion. This study describes the design of multi-layered protein bodies (PBs) in Nicotiana benthamiana as a platform for allergen encapsulation, offering potential advantages for oral immunotherapy. By co-expression of three zein variants we generated multi-layered PBs with distinct core and shell structures containing derivatives of the major fish allergen parvalbumin. The specific layering and structural integrity of the PBs were confirmed by confocal microscopy. Correlative light and electron microscopy (CLEM), combined with immunolabelling, was then used to verify the exact position of the allergens in the different layers of the PBs. In vitro experiments simulating the gastrointestinal digestion process revealed a significantly increased, layer-specific resistance of PB-encapsulated allergens compared to soluble allergens. Additionally, the uptake of PBs by human intestinal epithelial cells was simulated using Caco-2 cells. Our work provides further insight into protein storage organelle formation and novel bioencapsulation strategies to produce customized delivery vehicles, whose compartments may offer increased protection against enzymatic degradation and support prolonged persistence upon oral administration.

Type I respiratory allergies to birch pollen and pollen from related trees of the order Fagales are increasing in industrialized countries, especially in the temperate zone of the Northern hemisphere, but the reasons for this increase are still debated and seem to be multifaceted. While the most important allergenic molecules of birch pollen have been identified and characterized, the contribution of other pollen components, such as lipids, non-allergenic immunomodulatory proteins, or the pollen microbiome, to the development of allergic reactions are sparsely known. Furthermore, what also needs to be considered is that pollen is exposed to external influences which can alter its allergenicity. These external influences include environmental factors such as gaseous pollutants like ozone or nitrogen oxides or particulate air pollutants, but also meteorological events like changes in temperature, humidity, or precipitation. In this review, we look at the birch pollen from different angles and summarize current knowledge on internal and external influences that have an impact on the allergenicity of birch pollen and its interactions with the epithelial barrier. We focus on epithelial cells since these cells are the first line of defense in respiratory disease and are increasingly considered to be a regulatory tissue for the protection against the development of respiratory allergies.

Scope Red meat, a staple food of Western diets, can also induce IgE‐mediated allergic reactions. Yet, apart from the heat‐labile protein serum albumin and the carbohydrate α‐Gal, the molecules causing allergic reactions to red meat remain unknown. Methods and results IgE reactivity profiles of beef‐sensitized individuals are analyzed by IgE‐immunoblotting with protein extracts from raw and cooked beef. Two IgE‐reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli . ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco–2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1. Conclusion MYLs are identified as novel heat‐stable bovine meat allergens.

The constant exposure to fungal spores, which constitute the largest proportion of aerobiological particles, can cause severe health problems, including allergic diseases.1, 2 Appropriate management of fungal allergies is hampered by the fact that our knowledge about fungal allergy is still limited to a small number of thoroughly investigated fungi, whereas for the majority of species, it is not yet known whether they play a role in allergic diseases.2 This lack of knowledge together with unreliable diagnostic results obtained with fungal allergen extracts of poor quality contributes to a general underdiagnosis of fungal allergy.3, 4 In the present study, we investigated the allergenic potential of Ulocladium chartarum, an opportunistic human pathogen5 that can grow on various substrates.6, 7 Exposure to Ulocladium species often occurs in the indoor environment as they are commonly detected inside damp buildings and are regarded as an indicator of water damages.6-9 The fact that U. chartarum belongs to the same family as the outdoor aeroallergen source Alternaria alternata suggests that U. chartarum might also represent an allergen source.6, 7 IgE immunoblots, performed with sera from patients sensitized to different mold species (Table S1), demonstrated the high allergenic potential of U. chartarum, since patients' IgE antibodies recognized several U. chartarum proteins (Figure 1A), whereas exposure of the blotted proteins to sera from non-allergic individuals or to immunodetection reagent did not lead to any unspecific binding (data not shown). Interestingly, despite their phylogenetic relationship, the IgE-binding profile of A. alternata (Figure S1) differed significantly from the one of U. chartarum. Mold-allergic patients not only recognized more proteins in U. chartarum than in A. alternata extract, but also showed an overall stronger reactivity to them. Peptide mass fingerprinting allowed to identify a 15 kDa U. chartarum protein (A in Figure 1A) as an Alt a 1-homologous protein, a 41 kDa protein (B) as formate dehydrogenase, a protein not yet known as an allergen, and a 48 kDa protein (C) as an Alt a 6-homologous enolase. The proteins were designated Ulo c 1aa NCBI Nucleotide Sequence Accession Number: MH716394. (A), Ulo c 2bb NCBI Nucleotide Sequence Accession Number: MN953052. (B), and Ulo c 6cc NCBI Nucleotide Sequence Accession Number: MH910061. (C). The IgE-reactive protein bands D, E, and F could not yet be identified by mass spectrometry. cDNAs coding for the identified allergens was generated and cloned into a bacterial expression vector, and recombinant proteins were produced in Escherichia coli. Circular dichroism spectroscopy revealed that the recombinant allergens contain considerable secondary structures. In case of rUlo c 1, mainly beta-sheet structures (minimum of far-UV spectrum at 213 nm) were found, whereas predominantly alpha-helical structures (minima at 207 nm and 220 nm) were detected for rUlo c 2 and rUlo c 6 (Figure S2). ELISAs performed with sera from mold-allergic patients showed the IgE-binding capacity of the three recombinant allergens, with rUlo c 1 displaying the highest levels of IgE reactivity (Figure 1B). Furthermore, IgE inhibition immunoblots, where nitrocellulose-blotted protein extracts from U. chartarum were exposed to serum pools that had been pre-incubated with the recombinant proteins, showed that the recombinant allergens were able to completely inhibit patients' IgE binding to their natural counterparts (Figure 1C). This indicates that the recombinant allergens represent well-folded proteins which contain all the IgE-binding epitopes present in their natural counterparts and suggests that rUlo c 1, rUlo c 2, and rUlo c 6 could be used as tools for in vitro diagnosis of U. chartarum sensitization. The prevalence of the molecules' IgE reactivity was analyzed by ELISA using sera from 85 individuals sensitized to different mold species (Figure S3). rUlo c 1 was recognized by 58% of the patients, indicating that this molecule represents a major mold allergen, whereas rUlo c 2 was recognized by 43% and rUlo c 6 by 40% of the patients, suggesting minor mold allergens. Sequence comparison of the three U. chartarum allergens with homologous A. alternata proteins revealed protein sequence identities of 89% between Ulo c 1 and Alt a 1, 99% between Ulo c 2 and its homologous A. alternata protein, and 100% between Ulo c 6 and Alt a 6. The high sequence homology between Ulo c 1 and Alt a 1 and the suggested presence of Alt a 1-homologous proteins in other species of the Pleosporaceae family10 prompted us to investigate the potential cross-reactivity between rUlo c 1 and rAlt a 1 in IgE inhibition immunoblots. As depicted in Figure 2A, pre-incubation of patients' sera with one allergen always significantly reduced or even abolished IgE binding to the other molecule, whereas IgE binding was not reduced by pre-incubation of the sera with the irrelevant respiratory allergen rBet v 1. This reduction in IgE binding obtained with rAlt a 1 and rUlo c 1 was comparable to the reduction obtained by self-inhibition and indicates that the two allergens share conserved IgE epitopes. Furthermore, both recombinant allergens, rUlo c 1 and rAlt a 1, induced a dose-dependent expression of CD63 in basophils from six of the eight analyzed mold sensitized individuals (Figure 2B), which provided evidence for the molecules' biological activity. Analysis of the release of allergens from allergen sources can provide useful information about the potential exposure to these allergens. Interestingly, our investigations on the release kinetics of Ulo c 1 and Ulo c 6 from U. chartarum and Alt a 1 and Alt a 6 from A. alternata showed that the allergens were all immediately released from the spores (Figure S4). These findings suggest that rapid allergen elution might also occur when spores reach mucosal surfaces, where they can then elicit allergic reactions. In conclusion, in this study we provide evidence that U. chartarum represents an important, so far underestimated, allergen source that shows at least partial cross-reactivity to the evolutionarily related species A. alternata. Owing to the fact that in contrast to the outdoor mold A. alternata, U. chartarum is an important component of the indoor environment, it is especially important to raise the awareness that U. chartarum represents a potential cause of respiratory allergic diseases and to include the species into routine allergy diagnosis. We identified the species' first allergens, Ulo c 1, Ulo c 2, and Ulo c 6, and produced them as IgE-reactive recombinant molecules. These recombinant allergens will expand the repertoire of fungal allergens available for fungal allergy diagnosis, thus improving diagnosis' specificity and sensitivity. The description of the used methods can be found in the supporting information. The authors would like to thank Merima Bublin and Stefan Kabasser from the Department of Pathophysiology and Allergy from the Medical University of Vienna for their valuable support. SP, PS, MR, MP, RMMC, CSB, KN, ERF, MFT, KS, and IS have nothing to disclose. SQ reports personal fees and non-financial support from GSK, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Sanofi, personal fees and non-financial support from Novartis, personal fees and non-financial support from Mundipharma, personal fees and non-financial support from Teva, and personal fees and non-financial support from Allergy Therapeutics, outside the submitted work. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Fungal allergy represents a worldwide public health burden.1 Owing to their ability to colonize and germinate in the respiratory mucosa, fungi can have a far greater impact on the patients' immune system than other respiratory allergen sources.1 However, the lack of knowledge about allergenic fungi is one of the factors that contribute to a general underdiagnosis of fungal allergy.2 In the present study, we investigated the allergenic potential of Paecilomyces variotii, a heat-resistant mold ubiquitously found in soil, decomposing organic material, food products, and clinical samples, that is regarded as an emerging cause of serious, sometimes life-threatening, infections, and is often detected in the indoor environment.3-6 Even though P. variotii has already been suggested as an aeroallergen source and occupational hazard, no allergens have yet been identified or characterized by this species.7 IgE immunoblots, performed with four different pools of sera from mold-sensitized patients (Table S1), showed the presence of several IgE-reactive proteins in P. variotii extract (Figure 1A), suggesting the species’ high allergenic potential (no reaction was observed in immunoblots incubated with the detection antibody only—data not shown). Mass spectrometry analysis led to the identification of the first three P. variotii allergens, which interestingly all represent enzymes. Protein A (~36 kDa) was identified as a transaldolase, an enzyme that is already known as an allergen in different fungal species.8 Protein B (~37 kDa) was identified as a glyceraldehyde 3-phosphate dehydrogenase, which represents an important allergen in various sources,9 and Protein C (~48 kDa) was identified as an enolase. Enolases have also already been identified as highly conserved and cross-reactive allergens in various allergen sources including different fungi.10, 11 The proteins were submitted to the WHO/IUIS Allergen Nomenclature Subcommittee and were tentatively assigned the allergen names Pae v 1aa NCBI Nucleotide Sequence Accession Number: MN953053 , Pae v 2bb NCBI Nucleotide Sequence Accession Number: MN953054 , and Pae v 6cc NCBI Nucleotide Sequence Accession Number: MN93795 . The IgE-reactive protein bands marked D to H still have to be identified by peptide mass fingerprinting. Full-length cDNAs coding for the P. variotii proteins Pae v 1, Pae v 2, and Pae v 6 were generated, cloned into a bacterial expression vector, and recombinant proteins were produced in Escherichia coli with a C-terminal hexa-histidine-tag. Reducing and non-reducing SDS-PAGEs (Figure S1) and immunoblots, performed with an anti-histidine-tag antibody, verified the purity of the recombinant proteins and showed that all proteins possess the capability to form disulfide bridges and to oligomerize under non-reducing conditions (Figure S1). Moreover, circular dichroism spectroscopy analysis of the recombinant allergens showed far-UV spectra with broad minima between 210 and 220 nm, which suggested the presence of both, alpha-helical structures, and beta-sheets, and indicated that the proteins represent properly folded molecules (Figure S2). ELISAs performed with sera from mold-allergic patients showed the IgE-binding capacity of the three recombinant proteins. With OD values of more than 3.0 (Figure 1B), the enolase rPae v 6 showed the highest levels of IgE reactivity, whereas the IgE reactivities of rPae v 1 (maximum OD value of 0.8) and rPae v 2 (maximum OD value of 0.390) were considerably lower. Furthermore, IgE inhibition immunoblots, where nitrocellulose-blotted protein extracts from P. variotii were exposed to serum pools that had been pre-incubated with either one of the recombinant proteins or, for control purposes, with buffer only, showed that the recombinant allergens rPae v 1, rPae v 2, and rPae v 6 were able to significantly reduce patients' IgE-binding to their natural counterparts (Figure 1C). This indicates that the recombinant molecules contain the IgE-binding epitopes of their natural counterparts and suggests that rPae v 1, rPae v 2, and rPae v 6 could be used as tools for in vitro diagnosis of P. variotii sensitization. Pre-incubation with rPae v 2, a GAPDH, caused the reduction of IgE-binding to several proteins with molecular weights of 25–45 kDa. This suggests the presence of different Pae v 2 variants with different posttranslational modification and Pae v 2 degradation products in the natural fungal extract. The presence of GAPDH variants, which can also influence the molecule's different functions, has already been suggested in the literature.12 Analysis of the prevalence of IgE recognition in a cohort of patients, sensitized to different mold species, by ELISA revealed different frequencies of IgE reactivity. In Figure S3, the presence (in green) or the absence (in red) of IgE-binding to the recombinant allergens is displayed for each patient. rPae v 1 was recognized by 22% (11 out of 49) of the tested patients, rPae v 2 by 6% (3 out of 50), and 68% of the tested patients (39 out of 57) reacted with rPae v 6. Among the patients tested with all three recombinant allergens, rPae v 1 was recognized by 22% (9 out of 41), rPae v 2 by 5% (3 out of 41), and rPae v 6 by 61% (25 out of 41) of the patients. Based on these results, rPae v 1 and rPae v 2 can be regarded as minor allergens of P. variotii, whereas rPae v 6 can be classified as a major mold allergen. Furthermore, evaluation of the allergenic activity of the recombinant major allergen rPae v 6 in basophil activation tests (BATs) showed a dose-dependent expression of CD63 in basophils from patient 4, suggesting the molecule's biological activity (Figure S4 in the supporting information). The availability of an anti-enolase antibody allowed us to further investigate the release kinetics of the enolase Pae v 6 from P. variotii spores. The experiments showed that the spores from P. variotii immediately released this major allergen upon exposure to a humid milieu (Figure S5 in the supporting information). It can thus be expected that Pae v 6 would be rapidly secreted when the spores get in direct contact with the mucosa, leading to immediate allergic symptoms on the accessible mucosa surfaces such as the conjunctiva and the nose. In conclusion, during this study the strong IgE reactivity of Paecilomyces variotii was demonstrated. Furthermore, the species first three allergens, tentatively designated Pae v 1, Pae v 2, and Pae v 6, were identified, recombinantly produced, and characterized regarding their biochemical and immunological features. It was shown that the three recombinant allergens represent IgE-reactive molecules, with Pae v 1 and Pae v 2 representing minor fungal allergens, whereas Pae v 6 can be classified as a major fungal allergen. We are aware that the use of sera from patients allergic to known fungal allergen sources is a limitation of our study, because, in this way, it was much more likely to identify highly conserved, potentially cross-reactive allergens rather than species-specific allergenic molecules. Indeed, one of the three identified allergens is the highly conserved fungal pan-allergen enolase. However, since P. variotii has not yet been thoroughly investigated as an allergen source, it was not possible to overcome this limitation. Nevertheless, the obtained data together with the facts that exposure to P. variotii represents an important component of the indoor mycobiota5 and is increasingly recognized as an important cause of infections13 suggest that this species should be considered as a potential allergen source and should be included in routine fungal allergy diagnosis. The description of the used methods can be found in the supporting information. The authors would like to thank Kay Holleis, MSc, from the Molecular Biotechnology Section, FH Campus Wien for his support during this study. All authors declared no conflict of interest. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The encapsulation of biopharmaceuticals into micro- or nanoparticles is a strategy frequently used to prevent degradation or to achieve the slow release of therapeutics and vaccines. Protein bodies (PBs), which occur naturally as storage organelles in seeds, can be used as such carrier vehicles. The fusion of the N-terminal sequence of the maize storage protein, γ-zein, to other proteins is sufficient to induce the formation of PBs, which can be used to bioencapsulate recombinant proteins directly in the plant production host. In addition, the immunostimulatory effects of zein have been reported, which are advantageous for vaccine delivery. However, little is known about the interaction between zein PBs and mammalian cells. To better understand this interaction, fluorescent PBs, resulting from the fusion of the N-terminal portion of zein to a green fluorescent protein, was produced in Nicotiana benthamiana leaves, recovered by a filtration-based downstream procedure, and used to investigate their internalization efficiency into mammalian cells. We show that fluorescent PBs were efficiently internalized into intestinal epithelial cells and antigen-presenting cells (APCs) at a higher rate than polystyrene beads of comparable size. Furthermore, we observed that PBs stimulated cytokine secretion by epithelial cells, a characteristic that may confer vaccine adjuvant activities through the recruitment of APCs. Taken together, these results support the use of zein fusion proteins in developing novel approaches for drug delivery based on controlled protein packaging into plant PBs.

Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen's calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patient's IgE binding to wildtype Phl p 7, indicating the mutant's potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule's surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.

SummaryBackgroundFish is a well‐recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under‐investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat‐processed fish from Vietnam.ObjectiveThe aim of this study was to identify the major heat‐stable allergens from frequently exported Asia‐Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta).MethodsHeated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV‐specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy.ResultsHeat‐stable IgE‐reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D‐gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species.Conclusions and Clinical RelevanceDifferent PVs were identified as the major heat‐stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world‐wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia‐Pacific species should be developed with focus on PV.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extractbased diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.

Table of contents ORAL ABSTRACTS Symposium 1: Biochemistry, structure and environment of the allergen: what makes a protein an allergen? O1 Two cell-membrane peptidases carrying galactose-alpha-1,3-galactose are implicated in delayed anaphylactic reactions upon pork kidney ingestion in patients with IgE-antibodies to alpha-Gal Christiane Hilger, Kyra Swiontek, Jörg Fischer, François Hentges, Christiane Lehners, Martine Morisset, Bernadette Eberlein, Tilo Biedermann, Markus Ollert O2 Structure solution of Pla l 1 suggests similar folding of Ole e 1-like family members but distinct immunological properties Sabrina Wildner, Teresa Stemeseder, Regina Freier, Peter Briza, Roland Lang, Eva Batanero, Mayte Villalba, Jonas Lidholm, Thomas Hawranek, Fatima Ferreira, Hans Brandstetter, Gabriele Gadermaier Symposium 2: New allergen molecules in the spotlight O3 Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed (Ambrosia artemisiifolia) Philippe Moingeon, Rachel Groeme, Julien Bouley, Véronique Bordas, Maxime Le Mignon, Laetitia Bussières, Aurélie Lautrette, Laurent Mascarell, Vincent Lombardi, Véronique Baron-Bodo, Henri Chabre, Thierry Batard, Emmanuel Nony O4 Production and characterization of polybia paulista recombinant antigen 5: a valuable diagnostic tool Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Daniele Danella Figo, José Roberto Aparecido Dos Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Therese Wohlschlager, Peter Briza, Sabrina Wildner, Fatima Ferreira-Briza, Gabriele Gadermaier, Keity Souza Santos Symposium 3: Progress in molecular and cellular diagnosis O5 Basophil activation test with recombinant Pru p 3; identifying genuine peach allergic patients Margaretha Faber, Athina Van Gasse, Vito Sabato, Margo M. Hagendorens, Chris H. Bridts, Luc S. De Clerck, Araceli Diaz Perales, Didier Ebo O6 Nanofluidic technology enables rapid, near-patient quantification of allergen-specific IgE Petra Zavadakova, Aurélie Buchwalder, Fabien Rebeaud, Iwan Märki Symposium 4: Relevance of molecular diagnostics for intervention and treatment O7 Longitudinal analysis of Bet v 1-specific epitope repertoires during birch pollen immunotherapy Barbara Gepp, Nina Lengger, Christian Möbs, Wolfgang Pfützner, Christian Radauer, Barbara Bohle O8 A natural CCD-free tool: is polistes sp. venom suitable for polybia paulista diagnosis and therapy? Karine Marafigo De Amicis, Alexandra Sayuri Watanabe, Clovis Eduardo Galvao, Daniele Danella Figo, Jose Roberto Aparecido Santos-Pinto, Mario Sergio Palma, Fabio Fernandes Morato Castro, Jorge Kalil, Fatima Ferreira, Gabriele Gadermaier, Keity Souza Santos Symposium 5: The advent of molecular allergology in epidemiology O9 Peanut oleosins: from identification to diagnostic testing Christian Schwager, Skadi Kull, Frauke Schocker, Jochen Behrends, Wolf-Meinhard Becker, Uta Jappe O10 Endotypes of oral allergy syndrome in childhood: a molecular diagnostic approach Carla Mastrorilli, Salvatore Tripodi, Carlo Caffarelli, Riccardo Asero, Arianna Dondi, Giampaolo Ricci, Carlotta Povesi Dascola, Elisabetta Calamelli, Andrea Di Rienzo Businco, Annamaria Bianchi, Tullio Frediani, Carmen Verga, Iride Dello Iacono, Diego Peroni, Giuseppe Pingitore, Roberto Bernardini, Paolo Maria Matricardi Symposium 6: Molecular AIT: which approaches will make it to market? O11 Mbc4: an innovative molecule to tackle birch pollen and concomitant food allergies Heidi Hofer, Claudia Asam, Michael Hauser, Peter Briza, Martin Himly, Christof Ebner, Fatima Ferreira O12 Challenges and solutions associated with the production of recombinant Bet v 1 allergen as a therapeutic protein Emmanuel Nony, Maxime Le Mignon, Pierrick Lemoine, Karine Jain, Kathy Abiteboul, Monica Arvidsson, Sabina Rak, Philippe Moingeon Clinical Cases: Breakthroughs and headaches from CRD: interactive session CC1 Anaphylaxis caused by lipid transfer proteins: a complex clinical pattern syndrome Inês Mota, Filipe Benito Garcia, Angela Gaspar, Cristina Arêde, Susana Piedade, Graça Sampaio, Graça Pires, Luís Miguel Borrego, Cristina Santa-Marta, Mário Morais-Almeida CC2 IgE sensitization profile in a patient with asteraceae pollen-exotic fruits association Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC3 Food-dependent: exercise induced anaphylaxis. Which component to blame? Rosa Anita Rodrigues Fernandes, Isabel Carrapatoso, Raquel Gomes, Celso Pereira, Ana Todo-Bom CC4 Anaphylaxis to intravenous iron preparations in a patient that tolerates oral administration María Cecilia Martín Fernández De Basoa, Javier Barrios Regio, Juan De Castro Cordova, Antón Fernández Ferreiro CC5 IgE sensitization pattern in an adult patient with oral allergy syndrome to peanuts and pollinosis from southern Romania Florin-Dan Popescu, Mariana Vieru, Florin-Adrian Secureanu CC6 Evidence of specific IgE to plant-derived cross-reactive carbohydrate determinant in a patient with delayed anaphylaxis to red meat Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu POSTER PRESENTATIONS Poster Session 1: Molecular allergology and epidemiology P1 Atopic children produce stronger and more frequent IgG responses than non-atopic children: longitudinal data from the German MAS birth cohort Olympia Tsilochristou, Serena Perna, Alina Schwarz, Alexander Rohrbach, Antonio Cappella, Laura Hatzler, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Raffael D’amelio, Ulrich Wahn, Thomas Keil, Susanne Lau, Paolo Maria Matricardi P2 The IgG sensitization profiles against 112 allergenic components support the absence of a protective role of IgG in allergic individuals, outside of the context of SIT Pol André Apoil, Claire Mailhol, Anne Broué-Chabbert, Agnès Juchet, Alain Didier, Elodie Carrer, Thomas Lanot, Antoine Blancher P3 The immune response against the timothy grass pollen allergen Phl p 5 in non-allergic humans Almedina Kurtaj, Christoph Hillebrand, Gerda Fichtinger, Martin Danzer, Christian Gabriel, Theresa Thalhamer, Sandra Scheiblhofer, Josef Thalhamer, Richard Weiss P4 Analyzing the cross-reactivity profile of the major ragweed allergen Amb a 1 Martin Wolf, Michael Hauser, Ulrike Pichler, Teresa Twaroch, Gabriele Gadermaier, Christof Ebner, Hidenori Yokoi, Toshiro Takai, Alain Didierlaurent, Adriano Mari, Peter Briza, Heidrun Behrendt, Angela Neubauer, Frank Stolz, Fátima Ferreira, Michael Wallner P5 LTP (Pru p 3) sensitisation in skin prick test: which means in clinical practice? Sara Carvalho, Tatiana Lourenço, Joana Cosme, Fátima Cabral Duarte, Amélia Spínola Santos, Ana Célia Costa, Manuel Pereira Barbosa P6 IgE profiles, allergen exposure and lifestyle of 501 Austrian pupils: investigation of influences on the development of allergic sensitizations Teresa Stemeseder, Eva Klinglmayr, Bettina Schweidler, Lisa Lueftenegger, Stephanie Moser, Patrick Doppler, Roland Lang, Martin Himly, Gertie J. Oostingh, Arne Bathke, Joerg Zumbach, Thomas Hawranek, Gabriele Gadermaier P7 Molecular profiles of sensitization to perennial inhalant allergens in a middle European region Petr Panzner, Martina Vachova, Tomas Vlas, Marek Maly P8 Evolution of the IgE response to house dust mite allergen molecules in childhood Daniela Posa, Serena Perna, Stephanie Hofmaier, Laura Hatzler, Alexander Rohrbach, Carl-Peter Bauer, Ute Hoffmann, Johannes Forster, Fred Zepp, Antje Schuster, Philippe Stock, Ulrich Wahn, Linus Grabenhenrich, Thomas Keil, Susanne Lau, Kuan-Wei Chen, Yvonne Resch, Susanne Vrtala, Rudolf Valenta, Paolo Maria Matricardi P9 Tropomyosin (Pen a1): to include or not to include in skin prick testing? Joana Cosme, Sara Carvalho, Tatiana Lourenço, Amélia Spínola Santos, Manuel Pereira Barbosa Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal, Lisbon, Portugal; Immunoallergy Department - Hospital de Santa Maria – Centro Hospitalar Lisboa Norte, Lisbon, Portugal; Faculdade de Medicina de Lisboa, Lisbon, Portugal P10 Component-resolved IgE profiles in Georgian patients Tamar Abramidze, Nino Lomidze, Maia Gotua P11 Cross reactivity between food and pollen allergens in Lithuania according to spIgE evaluation Austeja Dapkeviciute, Ruta Einikyte, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene P12 Distribution of inhalant allergy in the population of Lithuania Ruta Einikyte, Austeja Dapkeviciute, Jolita Norkuniene, Laima Skrickiene, Asta Miskiniene, Violeta Kvedariene Poster Session 2: Allergen molecules: identification, characterization, structure and function P13 Interference of antigen 5-based cross-reactivity in the diagnosis of hymenoptera venom allergy Maximilian Schiener, Bernadette Eberlein, Carmen Moreno-Aguilar, Gunilla Pietsch, Mareike Mc Intyre, Lea Schwarze, Dennis Rußkamp, Tilo Biedermann, Edzard Spillner, Ulf Darsow, Carsten Schmidt-Weber, Markus Ollert, Simon Blank P14 IgE cross-reactivity between European Hymenoptera and Asian hornet (Vespa velutina) venom allergens Cyril Longé, Andrea Brazdova, Jean-Louis Brunet, Claire Schwartz, Bruno Girodet, François Lavaud, Joelle Birnbaum, Nhân Pham Thi, Magalie Duchateau, Julia Chamot-Rooke, Laurence Guilloux, Marie-Ange Selva, Rémy Couderc, Hélène Sénéchal, Jean-Pierre Sutra, Pascal Poncet P15 Carbohydrate composition of house dust mite extracts and major group 1 and group 2 allergens Steffen Augustin, Linda Pump, Martin Wald, Thomas Eichhorn, Frank Fischer, Christoph Willers P16 Specificity of monoclonal antibodies against cross-reactive carbohydrate determinants Michaela Miehe, Melanie Plum, Sara Wolf, Frederic Jabs, Tim Raiber, Frank Bantleon, Henning Seismann, Thilo Jakob, Edzard Spillner P17 Red meat allergic patients have a selective IgE response to the a-Gal glycan Danijela Apostolovic, Anh Thu Tran, Sara Sanchez-Vidaurre, Tanja Cirkovic Velickovic, Maria Starkhammar, Carl Hamsten, Marianne Van Hage P18 Specificity of non-specific lipid transfer proteins and influence of the ligands on their three-dimensional structure Pawel Dubiela, Piotr Humeniuk, Sabine Pfeifer, Merima Bublin, Tomasz Borowski, Karin Hoffmann-Sommergruber P19 Real-time PCR analysis of Pru av 1 and Pru av 3 allergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Defien Depoortere, Kay Foetisch, Stephan Scheurer, Harry J Wichers, Theo Noij P20 Specificity of anti-Pru av 1 antibodies for the detection of Pru av 1 isoallergens Martie C.M. Verschuren, Shanna Bastiaan-Net, Nikki M.E. Van Uden, Karel Vandenberghe, Kay Foetisch, Stephan Scheurer, Harry J. Wichers H.J., Theo H.M. Noij P21 Enhancing recombinant production yield of Bet v 1 through codon usage harmonization Anargyros Roulias, Maria Alejandra Parigiani, Heidi Hofer, Claudia Asam, Christof Ebner, Fátima Ferreira P22 Structural and dynamic insights into the world of PR-10 allergens Linda Ahammer, Sarina Grutsch, Martin Tollinger Poster Session 3: Allergen molecules: identification, characterization, structure and function P23 Purification of polcalcin from different pollen allergenic sources by antibody-affinity chromatography Raquel Moya, Mª Angeles López-Matas, Raquel Reyes, Jerónimo Carnés P24 Variations of wheat allergens in cultivars measured through a targeted quantitative mass spectrometry approach Colette Larré, Hélène Rogniaux, Roberta Lupi, Sandra Denery-Papini P25 Art v 1, Amb a 4 and Par h 1 defensin-like proteins share similar structural features but distinct immunological and allergenic properties Isabel Maria Pablos, Stephanie Eichhorn, Yoan Machado, Peter Briza, Christof Ebner, Jung-Won Park, Alain Didierlaurent, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P26 Homogeneity or diversity of IgE-binding proteins in wheat dependant exercise induced anaphylaxis? Sandra Denery-Papini, Charlene Tanaka, Florence Pineau, Roberta Lupi, Martine Drouet, Etienne Beaudouin, Martine Morisset, Susan Altenbach P27 Deciphering the role of disulfide bonds and of repetitive epitopes in immunoglobulin E binding to wheat gliadins Sandra Denery-Papini, Hamza Mameri, Chantal Brossard, Roberta Lupi, Florence Pineau, Jean Charles Gaudin, Denise Anne Moneret-Vautrin, Etienne Beaudouin, Evelyne Paty, Martine Drouet, Olivier Tranquet, Colette Larré P28 Assessment of the allergenicity of soluble fractions from bread and durum wheats genotypes Roberta Lupi, Stefania Masci, Olivier Tranquet, Denise-Anne Moneret-Vautrin, Sandra Denery-Papini, Colette Larré P29 Isolation and characterization of Ara h 12 and Ara h 13: defensins, a novel class of peanut allergens Skadi Kull, Arnd Petersen, Marisa Böttger, Sandra Rennert, Wolf-Meinhard Becker, Susanne Krause, Martin Ernst, Thomas Gutsmann, Johann Bauer, Buko Lindner, Uta Jappe P30 Allergenicity attributes of different peanut market types Stef Koppelman, Shyamali Jayasena, Dion Luykx, Erik Schepens, Danijela Apostolovic, Govardus De Jong, Tom Isleib, Julie Nordlee, Joe Baumert, Steve Taylor, Soheila Maleki P31 The impact of peanut lipids on Ara h 1-induced immune responses in monocytes-derived dendritic cells Chiara Palladino, Barbara Gepp, Sofía Sirvent, Alba Angelina, Merima Bublin, Christian Radauer, Nina Lengger, Thomas Eiwegger, Oscar Palomares, Heimo Breiteneder P32 Compared allergenicity of native and thermally aggregated ovalbumin as large agglomerated particles Mathilde Claude, Roberta Lupi, Grégory Bouchaud, Marie Bodinier, Chantal Brossard, Sandra Denery-Papini P33 Simulation of the gastrointestinal digestion of the hazelnut allergens Cor a 9 and Cor a 11 by an in-vitro model and characterisation of peptidic products including epitopes by HPLC-MS/MS Robin Korte, Julia Bräcker, Jens Brockmeyer P34 Analysis of distribution of rice allergens in brown rice grain and allergenicity of the products containing rice bran Rie Satoh, Reiko Teshima Poster Session 4: Molecular approaches in AIT P35 Production of a recombinant hypoallergenic variant of the major peanut allergen Ara h 2 for allergen-specific immunotherapy Angelika Tscheppe, Dieter Palmberger, Merima Bublin, Christian Radauer, Chiara Palladino, Barbara Gepp, Nina Lengger, Reingard Grabherr, Heimo Breiteneder P36 Mutagenesis of amino acids critical for calcium-binding leads to the generation of a hypoallergenic Phl p 7 variant Marianne Raith, Linda Sonnleitner, Doris Zach, Konrad Woroszylo, Margit Focke-Tejkl, Herbert Wank, Thorsten Graf, Annette Kuehn, Ines Swoboda P37 Are birch pollen allergen immunotherapy induced blocking antibodies protective for cross-reactive allergens? Claudia Asam, Sara Huber, Heidi Hofer, Roland Lang, Thomas Hawranek, Fátima Ferreira, Michael Wallner P38 High success of 58 subcutaneous immunotherapy for pets allergy in a polyallergic cohort of patients: a component resolved individually adapted treatment (CRIAT) Fabienne Gay-Crosier P39 Neutrophils are potential antigen presenting cells in IgE- mediated allergy Dominika Polak, Birgit Nagl, Claudia Kitzmüller, Barbara Bohle P40 Characterization of allergen-specific CD8+ T cells in type I allergy Nazanin Samadi, Claudia Kitzmüller, Rene Geyeregger, Barbara Bohle, Beatrice Jahn-Schmid Poster Session 5: Molecular and cellular diagnostic tests P41 Nanofluidic-based biosensors allow quantification of total circulating IgE from a drop of blood in 5 minutes Aurélie Buchwalder, Ariel Gomez, Fabien Rebeaud, Iwan Märki P42 Allergen microarray for the analysis of serum IgE binding profile and allergenic activity Jaana Haka, Liisa Hattara, Marika Heikkinen, Merja H Niemi, Juha Rouvinen, Petri Saviranta, Pekka Mattila, Kristiina Takkinen, Marja-Leena Laukkanen P43 Generation of a well-characterized panel of periplaneta americana allergens for component resolved diagnosis Stephanie Eichhorn, Isabel Pablos, Bianca Kastner, Bettina Schweidler, Sabrina Wildner, Peter Briza, Jung-Won Park, Naveen Arora, Stefan Vieths, Gabriele Gadermaier, Fatima Ferreira P44 Improved diagnostic sensitivity of recombinant Api m 1 and Ves v 5 in diagnosis of Hymenoptera venom allergy Mira Silar, Julij Selb, Rok Kogovsek, Mitja Kosnik, Peter Korosec P45 Added value of biomarkers of primary sensitization and cross-reactivity in patients with hymenoptera venom allergy Leticia Pestana, Alcinda Campos Melo, Ana Mendes, Maria Elisa Pedro, Manuel Pereira Barbosa, Maria Conceição Pereira Santos P46 Cosensitization to Alt a 1 and Act d 2: more than a fortuitous association? Françoise Bienvenu, Claire Goursaud, Lorna Garnier, Sandrine Jacquenet, Michaël Degaud, Sébastien Viel, Annick Barre, Pierre Rougé, Jacques Bienvenu, Joana Vitte P47 Molecular diagnosis for peanut allergy: ALFA method performs as well as established methods for Ara h 1, Ara h 2, Ara h 6, Ara h 9 and CCD Amel Bensalah, Isabelle Cleach, Laurent Mousseau, Chantal Agabriel, Valérie Liabeuf, Joëlle Birnbaum, Jean-Louis Mège, Joana Vitte P48 Evaluation of a food challenge service in relation to specific IgE to molecular components in children with suspected peanut allergy James Gardner, Minal Gandhi, Harsha Kariyawasam, Giuseppina Rotiroti P49 Component resolved diagnosis in cereal allergy Isabel Carrapatoso, Celso Pereira, Frederico Regateiro, Emília Faria, Ana Todo-Bom Poster Session 6: Molecular diagnosis in prevention and therapy P50 Pretreatment molecular sensitizations determine the sIgG4 induction during the updosing of SCIT and may be useful to identify clinically relevant additional sensitizations Johannes Martin Schmid, Ronald Dahl, Hans Juergen Hoffmann P51 Usefulness of recombinant latex allergens in immunotherapy’s decision and follow-up Inês Mota, Filipe Benito Garcia, Angela Gaspar, Mário Morais-Almeida P52 Omega-5-gliadin in the diagnosis of wheat-dependent anaphylaxis induced by ibuprofen but not by exercise Joana Cosme, Letícia Pestana, Amélia Spínola Santos, Manuel Pereira Barbosa P53 Food dependent exercise-induced anaphylaxis: a component-resolved and in vitro depletion approach to access IgE cross-reactivity Diana Silva, Teresa Vieira, Ana Maria Pereira, André Moreira, Luís Delgado P54 Olive pollen allergens: what are we missing? Sara Prates, Cátia Alves, Elena Finelli, Paula Leiria Pinto P55 Purified Alt a 1 extract in Alternaria alternata allergy diagnosis Bárbara Kong Cardoso, Cíntia Cruz, Filipa Semedo, Elza Tomaz, Filipe Inácio P56 Use of specific IgE Bos d8 (casein) to aid early introduction of dietary baked milk in children with cows’ milk allergy James Gardner, Santanu Maity, Giuseppina Rotiroti, Minal Gandhi P57 Molecular characterisation and immunoreactivity of a peanut ingredient for use in oral food challenges Ivona Baricevic-Jones, Justin T. Marsh, Phil E. Johnson, Anuradha Balasundaram, Anya-May Hope, Aafke Taekema, Angela Simpson, Aida Semic-Jusufagic, E.N. Clare Mills P58 Specific IgE to recombinant allergens of hazelnut and oral food challenge in children Gourdon Dubois Nelly, Sellam Laetitia, Pereira Bruno, Michaud Elodie, Messaoudi Khaled, Evrard Bertrand, Fauquert Jean-Luc Poster session 7/8: miscellaneous P59 What defines a protein as an allergen? A discussion of sources and sufficiency Richard E. Goodman P60 Cat allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P61 Anaphylaxis to rabbit: the cat came in last Luis Amaral, Borja Bartolomé, Alice Coimbra, Jose L Placido P62 Dog allergy: relationship between clinical and molecular diagnostic María Cecilia Martín Fernández De Basoa, Antón Fernández Ferreiro, Elena Rodríguez Plata P63 Correlation of serum timothy grass-pollen specific IgE levels determined by two immunoblot test systems Mariana Vieru, Florin-Dan Popescu, Florin-Adrian Secureanu, Carmen Saviana Ganea P64 Development of oral food challenge formulations for diagnosis of fish allergy using powdered fish ingredients Carol Ann Costello, Ivona Baricevic-Jones, Martin Sorensen, Clare Mills, Adrian Rogers, Aage Otherhals P65 Fish and peanut allergens interact with plasma membranes of intestinal and bronchial epithelial cells and induce differential gene expression of cytokines and chemokines Tanja Kalic, Isabella Ellinger, Chiara Palladino, Barbara Gepp, Eva Waltl, Verena Niederberger-Leppin, Heimo Breiteneder P66 Interleukin 4 affects fat tissue metabolism and expression of pro-inflammatory factors in isolated rat adipocytes Dawid Szczepankiewicz, Ewa Pruszynska-Oszmalek, Marek Skrzypski, Krzysztof W. Nowak, Aleksandra Szczepankiewicz P67 Ozone induced airway hyperreactivity in PD-L2−/− mice model Gwang-Cheon Jang P68 Thymic stromal lymphopoietin (TSLP) and its receptor as targets for the development of anti-inflammatory inhibitory agents Iva Markovic, Andreas Borowski, Tina Vetter, Andreas Wohlmann, Michael Kuepper, Karlheinz Friedrich P69 The mononuclear phagocyte system in experimentally-induced allergic rhinitis Ibon Eguiluz Gracia, Anthony Bosco, Ralph Dollner, Guro Reinholt Melum, Anya C Jones, Maria Lexberg, Patrick G Holt, Espen Sønderaal Bækkevold, Frode Lars Jahnsen P70 Expression of histamine metabolizing enzymes is increased in allergic children Aleksandra Szczepankiewicz, Paulina Sobkowiak, Marta Rachel, Beata Narozna, Dorota Jenerowicz, Witold Swiatowy, Anna Breborowicz P71 Modifying the glycosylation of human IgE towards oligomannosidic structures does not affect its biological activity Melanie Plum, Sara Wolf, Frank Bantleon, Henning Seismann, Frederic Jabs, Michaela Miehe, Thilo Jakob, Edzard Spillner P72 Flying Labs: an educational initiative to transfer allergy research into high-school settings Michael Wallner, Heidi Hofer, Fatima Ferreira, Reinhard Nestelbacher P73 Clinical significance of antihistamines and Kujin, an anti-allergic Kampo medicine Hiroyuki Fukui

Lipid transfer protein (LTP) is a major allergen in peach often causing anaphylactic reactions with a high prevalence of sensitization in the Mediterranean area. The prevalence of LTP sensitization in Central Europe is currently unknown, where the diagnosis is complicated by a high background sensitization to the major birch pollen allergen, Bet v 1, and Bet v 1 cross-reactive food allergens, which cause only mild reactions. LTP and Bet v 1-related proteins are both distributed throughout the plant-kingdom. In this study, we aimed to characterize the sensitization-patterns of patients with severe reactions to plant-derived food in Austria. Thirteen patients reporting strong reactions after consumption of fruits, vegetables or herbs were invited to skin prick and prick to prick tests. For visualizing patients' IgE-reactivity patterns, immunoblots were performed, which allowed the determination of specific IgE to raw and boiled parsley, raspberry, apricot, and peach. These immunoblots showed that cooking did not influence IgE binding to LTPs, while cooking reduced IgE binding to all Bet v 1-related proteins. We further observed that LTP-sensitized patients showed variable IgE reactivity to different LTPs, however, the strongest reactions were seen to fruits from the Rosaceae family, especially to peach, suggesting that peach LTP (Pru p 3) contains the majority of IgE epitopes also for a Central European population. Analysis of IgE reactivity to single molecules (including Bet v 1 and Pru p 3) using an allergen micro-array (ISAC) and single ImmunoCAP measurements enabled to determine the following sensitization patterns: 1 patient was monosensitized to Bet v 1, 3 were monosensitized to LTP, whereas 8 showed a co-sensitization to Bet v 1 and LTP and 1 patient was monosensitized to profilin. Based on this study, we conclude that analysis of specific IgE to the LTP Pru p 3 from peach is also sufficient for diagnosis of reactions to Rosacea fruits even in regions where frequent co-sensitization to Bet v 1 makes the interpretation of extracts-based skin prick tests difficult. In addition, also other plant-related panallergens, such as profilin, should be included in such a differential diagnostic workup scheme.

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.

BACKGROUND:Duchenne muscular dystrophy (DMD) is one of the most frequent forms of muscular disorders. It is caused by the absence of dystrophin, a core component of the sarcolemma-associated junctional complex that links the cytoskeleton to the extracellular matrix. We showed previously that plectin 1f (P1f), one of the major muscle-expressed isoforms of the cytoskeletal linker protein plectin, accumulates at the sarcolemma of DMD patients as well as of mdx mice, a widely studied animal model for DMD.Based on plectin's dual role as structural protein and scaffolding platform for signaling molecules, we speculated that the dystrophic phenotype observed after loss of dystrophin was caused, at least to some extent, by excess plectin. Thus, we hypothesized that elimination of plectin expression in mdx skeletal muscle, while probably resulting in an overall more severe phenotype, may lead to a partial phenotype rescue. In particular, we wanted to assess whether excess sarcolemmal plectin contributes to the dysregulation of sugar metabolism in mdx myofibers. METHODS:We generated plectin/dystrophin double deficient (dKO) mice by breeding mdx with conditional striated muscle-restricted plectin knockout (cKO) mice. The phenotype of these mice was comparatively analyzed with that of mdx, cKO, and wild-type mice, focusing on structural integrity and dysregulation of glucose metabolism. RESULTS:We show that the accumulation of plectin at the sarcolemma of mdx muscle fibers hardly compensated for their loss of structural integrity. Instead, it led to an additional metabolic deficit by impairing glucose uptake. While dKO mice suffered from an overall more severe form of muscular dystrophy compared to mdx or plectin-deficient mice, sarcolemmal integrity as well as glucose uptake of their myofibers were restored to normal levels upon ablation of plectin. Furthermore, microtubule (MT) networks in intact dKO myofibers, including subsarcolemmal areas, were found to be more robust than those in mdx mice. Finally, myotubes differentiated from P1f-overexpressing myoblasts showed an impairment of glucose transporter 4 translocation and a destabilization of MT networks. CONCLUSIONS:Based on these results we propose that sarcolemma-associated plectin acts as an antagonist of MT network formation in myofibers, thereby hindering vesicle-mediated (MT-dependent) transport of glucose transporter 4. This novel role of plectin throws a bridge between extra-sarcomeric cytoarchitecture and metabolism of muscle fibers. Our study thus provides new insights into pathomechanisms of plectinopathies and muscular dystrophies in general.

Plectin, ein ausergewohnlich groses Cytolinkerprotein (>500kDa), weist eine besonders hohe Vielseitigkeit basierend auf der Expression von verschiedenen Isoformen auf. Diese, durch alternatives Splicing N-terminaler Sequenzen generierten Isoformen weisen im Gewebe sowohl unterschiedliche Lokalisation als auch Funktion auf. Plectin 1f (P1f), eine im Muskel exprimierte und mit dem Sarkolemma assoziierte Isoform, wird in mdx-Mausen, einem Tiermodell fur Duchenne-Muskeldystrophie, uberexprimiert. In Teil I der vorliegenden Arbeit wird gezeigt, dass die Akkumulation von Plectin am Sarkolemma von mdx-Mausen einerseits eine Schutzfunktion fur die strukturelle Integritat des Dystrophin-defizienten Muskels hat, andererseits aber Mikrotubuli-abhangige Prozesse wie die Translokation des Glukosetrans¬porters 4 (GLUT4) beeinflusst. Dadurch kommt es zu einer verlangsamten Aufnahme von Glukose aus dem Blut. Um die diesen Phanomenen zugrundeliegenden Mechanismen zu analysieren, wurden Mause generiert, welchen sowohl Dystrophin als auch Plectin fehlen. Diese Tiere wiesen eine schwerwiegendere Muskeldystrophie im Ver¬gleich zu mdx-Mausen auf, allerdings wurde eine weitgehend wiederhergestellte Integritat des Sarkolemmas, sowie eine ungestorte GLUT4 Translokation beobachtet. Diese Beobachtungen geben Anlass zu einem Modellvorschlag, in dem P1f als lokaler Gegenspieler von MT-Netzwerksbildung fungiert, wobei es bei einer Uberexpression am Sarkolemma, zu einer Reduktion des MT-abhangigen Vesikeltransportes kommt. Um die Rolle Plectins in Entwicklungs- und Regenerationsprozessen aufzuklaren, untersuchte ich in Teil II der vorliegenden Arbeit konditionelle Plectin-Knockout-Mause, in denen die Ausschaltung von Plectin in Vorlauferzellen der Skelettmuskulatur wahrend der fruhen Embryonalentwicklung (Pax7-Cre/cKO) erfolgte. Durch die Abwesenheit von Plectin wahrend der Skelettmuskelentwicklung sowie in Satellitenzellen zeigten diese Mause Entwicklungsstorungen, die sich als reduzierte Anzahl von Muskel¬fasern manifestierte, was schlieslich zu einer verminderten Korpergrose der Tiere fuhrte. Strukturell waren starke Anzeichen von Muskeldystrophie zu erkennen, einschlieslich des Ersatzes von Muskelfasern durch Bindegewebe, Kyphose und reduzierte Lebensdauer. Die gravierende Muskeldystrophie konnte auf eine fruhe Erschopfung des Pools an Satellitenzellen, aufgrund des schnelleren Ablaufs von deren Zellzyklus zuruckgefuhrt werden. Zusatzlich konnte in diesen Mausen eine Uberakti¬vierung des AMPK-Signalweges, einem Insulin-unabhangigen Ubertragungsweg, beobachtet werden. Im Pax7-Cre/cKO Muskel zeigte sich die AMPK Untereinheit α leichter fur akti¬vierende Kinasen zuganglich. Normalerweise ist die Aktivierungs¬stelle der katalytischen AMPK-Untereinheit α (Threonin 172) durch die Untereinheit γ blockiert, welche als Energie¬sensor dient und durch AMP-Bindung eine Konformationsanderung erfahrt. Aufgrund der Ergebnisse dieser Studie wird ein Arbeitsmodell vorgeschlagen, in dem Plectin im Normalfall die γ-Untereinheit des AMP Komplexes strukturell stabilisiert und es bei seinem Fehlen zur Uberaktivierung des Komplexes kommt.

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein α-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like α-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and α-actinin-2, and its NH2-terminus, which contains an actin-binding domain, directly interacted with that of plectin and α-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Chemokines are involved in the recruitment of inflammatory cells to vascularized allografts. The chemokine CCL2/MCP-1 is expressed during allograft dysfunction, which is associated with the recruitment of inflammatory cells. Both intrinsic renal cells (donor origin) as well as infiltrating inflammatory cells (recipient origin) can be a source of CCL2/MCP-1. We previously demonstrated that the recipient MCP-1-2518G polymorphism is associated with increased CCL2/MCP-1 production by inflammatory cells and decreased renal allograft survival. We evaluated the impact of the MCP-1-2518G polymorphism in donor cells on renal allograft outcomes. We enrolled 252 recipients of kidney allografts in this retrospective study who had received grafts from 152 cadaveric donors. The CCL2/MCP-1 genotype was assessed using genomic DNA isolated from cryopreserved donor splenocytes. Outcome parameters studied were acute biopsy proven rejection (Banff criteria), serum creatinine, and glomerular filtration rate (GFR) at 1 year after transplantation, allograft loss, and death. MCP-1-2518 genotypes were in HW equilibrium. A/A was present in 125 (49.6%), A/G in 107 (42.5%), and G/G in 20 (7.9%) donor kidneys. There were no significant differences in the number of rejection episodes, the number of allograft losses, serum creatinine, GFR, or overall survival 1 year after transplantation. In contrast with the detrimental effect of the CCL2/MCP-1 polymorphism of the recipient, the CCL2/MCP-1 polymorphism of the donor has no impact on the allograft outcome during the first year after transplantation. The impact on the long-term outcomes needs further evaluation.