T. Cirkovic Velickovic

A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro

Background It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low‐pH conditions.

Immunoproteomics of processed beef proteins reveal novel galactose-α-1,3-galactose-containing allergens.

Red meat allergy presents a novel form of food allergy with severe delayed allergic reactions where IgE antibodies are directed against the carbohydrate α‐Gal epitope. Food preparation and processing can influence the allergenicity of proteins. The aim of this study was to characterize the proteomic profile of different beef preparations and to investigate their α‐Gal reactivity and potential allergenicity.

Red meat allergic patients have a selective IgE response to the α-Gal glycan

Galactose‐α‐1,3‐galactose (α‐Gal) is a mammalian carbohydrate with significance in a novel type of food allergy. Patients with IgE against α‐Gal report severe allergic symptoms 3–6 h after consumption of red meat. We investigated whether IgE from red meat allergic patients recognizes other mammalian glycans than α‐Gal or glycans from the plant kingdom and insects of importance in allergy. We found that none of the 24 red meat allergic patients investigated had an IgE antibody response against the other abundant mammalian glycan N‐glycolylneuraminic acid or against cross‐reactive carbohydrate determinants from plant or venom sources (nCup a 1, nArt v 1, and MUXF3). Deglycosylation of an α‐Gal‐containing protein, bovine thyroglobulin, significantly reduced the IgE response. In conclusion, we show that red meat allergic patients have a selective IgE response to the α‐Gal glycan found in red meat. Other common glycans reactive in allergic disease are not targets of red meat allergic patients’ IgE.

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1.

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation

Background Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies.

Influence of peanut matrix on stability of allergens in gastric-simulated digesta: 2S albumins are main contributors to the IgE reactivity of short digestion-resistant peptides

Most food allergens sensitizing via the gastrointestinal tract are stable proteins that are resistant to pepsin digestion, in particular major peanut allergens, Ara h 2 and Ara h 6. Survival of their large fragments is essential for sensitizing capacity. However, the immunoreactive proteins/peptides to which the immune system of the gastrointestinal tract is exposed during digestion of peanut proteins are unknown. Particularly, the IgE reactivity of short digestion‐resistant peptides (SDRPs; <10 kDa) released by gastric digestion under standardized and physiologically relevant in vitro conditions has not been investigated.

Peptidomics of an in vitro digested α-Gal carrying protein revealed IgE-reactive peptides

The mammalian carbohydrate galactose-α1,3-galactose (α-Gal) causes a novel form of food allergy, red meat allergy, where patients experience severe allergic reactions several hours after red meat consumption. Here we explored gastric digestion of α-Gal glycoproteins using an in vitro model. Bovine thyroglobulin (BTG), a typical α-Gal carrying glycoprotein, was digested with pepsin. The resulting peptides were characterised by SDS PAGE, immunoblot and ImmunoCAP using sera from 20 red meat allergic patients. During pepsinolysis of BTG, a wide range of peptide bands was observed of which 14 to 17 kDa peptides remained stable throughout the gastric phase. The presence of the α-Gal epitope on the obtained peptides was demonstrated by an anti-α-Gal antibody and IgE from red meat allergic patients. The α-Gal digests were able to inhibit up to 86% of IgE reactivity to BTG. Importantly, basophil activation test demonstrated that the allergenic activity of BTG was retained after digestion in all four tested patients. Mass spectrometry-based peptidomics revealed that these peptides represent mostly internal and C-terminal parts of the protein, where the most potent IgE-binding α-Gal residues were identified at Asn1756, Asn1850 and Asn2231. Thus allergenic α-Gal epitopes are stable to pepsinolysis, reinforcing their role as clinically relevant food allergens.

α-Gal on the protein surface affects uptake and degradation in immature monocyte derived dendritic cells

Red meat allergy is characterized by an IgE response against the carbohydrate galactose-α-1,3-galactose (α-Gal), which is abundantly expressed on glycoproteins from non-primate mammals. The mechanisms of how α-Gal is processed and presented to the immune system to initiate an allergic reaction are still unknown. The aim of this study was to reveal whether the presence of α-Gal epitopes on the protein surface influence antigen uptake and processing in immature monocyte-derived dendritic cells (iMDDCs). Immature MDDCs were prepared from healthy blood donors and red meat allergic patients. We found an increased internalization of α-Gal carrying proteins over time in iMDDCs by flow cytometric analysis, which was independent of the donor allergic status. The uptake of α-Gal carrying proteins was significantly higher than the uptake of non-α-Gal carrying proteins. Confocal microscopy revealed α-Gal carrying proteins scattered around the cytoplasm in most iMDDCs while detection of proteins not carrying α-Gal was negligible. Fluorescent detection of protein on SDS-PAGE showed that degradation of α-Gal carrying proteins was slower than degradation of non-α-Gal carrying proteins. Thus, the presence of α-Gal on the protein surface affects both uptake and degradation of the protein, and the results add new knowledge of α-Gal as a clinically relevant food allergen.

Authors reply to beta-lactam allergy in children.

Editor, We thank you for the opportunity to answer to the interesting remarks made by colleagues Perez-Rodrigues et al. (1) on our recent paper published in Pediatric Allergy and Immmunology on betalactam allergies in children. We found the comments very useful to clarify a few issues that a critical reading of our paper could actually raise. In our study, a group of 1170 children with already suspected immediate allergic reactions to penicillins and/or cephalosporins was evaluated. There were no self-reported allergies in the studied population. We agree that the percentage of betalactam (BL) allergic children found in our study is very high, particularly if compared with literature data. However, such percentage can be explained by the fact that penicillins, including benzyl penicillin, are the most frequently prescribed drugs for children in our country and by the characteristics of the sample assessed, which might be unique for genetic reasons. We used PPL and MDM produced by the Institute of Immunology and Virology of Torlak (Serbia). The highest concentrations for all tested drugs were given in the Methods section, and were the same proposed by Torres et al. (2) in the position paper of the European Network for Drug Allergy (ENDA). The testing was done in accordance with the ENDA position paper on diagnosis of immediate allergic reactions to BLs. As far as re-evaluation is concerned, we are aware of its importance in patients who suffered immediate reactions to BLs and display negative results in the first allergologic exam, including challenges. However, at the time we started this study, we did not include re-evaluations in our diagnostic protocol, as we have been doing since 2003. The skin testing results presented in the paper were obtained by both skin prick and intradermal testing (mentioned as skin tests at the beginning of Methods section). We did not show separately the results obtained by each of the two in vivo methods, as it was not the scope of the paper. We showed the separate data only for the challenge test, to emphasize the low percentage of positive responses to it, in case the skin test was negative. In parts of the Results section and in the Table 1, the in vivo tests were marked as skin prick tests, but they should have been marked as skin tests, as we did not show any separate data on the percentages of the results obtained by skin prick testing. We also want to underline that the Ethical Committee of the University Children’s Hospital in Belgrade approved the study. For the control group of 100 children without drug allergies the informed consent was obtained, and the testing was carried out according to a request on the part of parents because of a family history of drug hypersensitity. Only those children constituted the control group. We do agree that performing the testing without an informed consent would be unethical. With regard to the diagnosis of selective sensitizations, we performed provocation tests only with suspect BLs; therefore, such diagnosis actually was based only on responses to skin tests. However, in patients with immediate reactions to penicillins, the negative predictive value of skin tests with compounds other than the culprit ones is very high (3, 4).