P. Rozynek

IgE binding of the recombinant allergen soybean profilin (rGly m 3) is mediated by conformational epitopes

BACKGROUND Soybean proteins are constituents of a number of food products and represent a panel of potential allergens. Thus far, little is known about the molecular characteristics of soybean allergens. OBJECTIVE The aim of this study was to identify the soybean profilin by PCR-based complementary (c)DNA cloning and to elucidate its allergenic characteristics. METHODS Highly degenerate profilin-specific primers were used to identify, by means of PCR, 2 soybean profilin isoforms (GmPRO1 and GmPRO2) by using soybean cDNA as a target. One isoform (GmPRO1) with a length of 394 bp corresponding to 131 amino acid residues was subcloned and expressed in fusion with the maltose-binding protein. Moreover, 3 overlapping recombinant soybean profilin fragments comprising amino acid residues 1-65, 38-88, and 50-131 were also prepared as maltose-binding protein fusion proteins. IgE-binding reactivity of the recombinant proteins and the cross-reactivity of soybean profilin with birch profilin was studied by immunoblotting, enzyme-linked allergosorbent assays (EASTs), and competitive inhibition experiments by using serum samples from 13 soybean-sensitized subjects. RESULTS Results of immunoblot analysis, EAST, and EAST-inhibition experiments indicate the presence of profilin in soybean extract. The recombinant soybean profilin (rGly m 3) was recognized by IgE in 9 (69%) of the 13 sera tested. Only the full-length rGly m 3 was able to bind with IgE antibodies, whereas the 3 soybean profilin fragments did not show significant binding reactivity, indicating that the IgE binding to rGly m 3 depends on the integrity of a conformational structure, which was not present in the overlapping profilin fragments. The rGly m 3 cross-reacted with birch pollen profilin (Bet v 2), and the IgE binding to Bet v 2 could be inhibited by rGly m 3. CONCLUSIONS rGly m 3 represents a new soybean allergen with well-characterized primary sequence, and its IgE-binding reactivity is mediated by conformational epitopes.

Update of the WHO/IUIS Allergen Nomenclature Database based on analysis of allergen sequences

The IUIS Allergen Nomenclature Sub‐Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, www.allergen.org. In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cows milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub‐Committee encourages researchers to use these updated allergen names in future publications.

Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis)

Background Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles.

Multiple wheat flour allergens and cross-reactive carbohydrate determinants bind IgE in baker’s asthma

To cite this article: Sander I, Rozynek P, Rihs H‐P, van Kampen V, Chew FT, Lee WS, Kotschy‐Lang N, Merget R, Brüning T, Raulf‐Heimsoth M. Multiple wheat flour allergens and cross‐reactive carbohydrate determinants bind IgE in baker’s asthma. Allergy 2011; 66: 1208–1215.

Overview on denominated allergens.

Type I allergy is a heightened or altered reactivity ofthe immune system in response to external substances involving immunoglobulins of the IgE class. More than 15% of the population in industrial countries suffer from immediate-type allergic symptoms [1]. Tn recent years, allergy research into the immunological, biochemical and structural characterization of allergens has led to an enormous progress alTecting diagnostics and therapeutics. This article summarizes important aspects of allergy research work on purified allergens. Characterized and denominated allergens have been listed. These data include biochemical and structural allergen characteristics as well as research results on human histocompatibility leucocyte antigens (HLA) restriction of ihe immune response, Tand B-cell epitopes and recombinant allergen expression. Allergens were isolated from a variety of dilTerent species. The taxonomy and relationship of dilTerent vertebrate, invertebrate as well as plant species from which allergens are isolated are also shown.

Assessment of mRnA and microRnA stabilization in peripheral Human Blood for Multicenter studies and Biobanks

In this study we evaluate the suitability of two methods of RNA conservation in blood samples, PAXgene and RNAlater, in combination with variable shipping conditions for their application in multicenter studies and biobanking. RNA yield, integrity, and purity as well as levels of selected mRNA and microRNA species were analyzed in peripheral human blood samples stabilized by PAXgene or RNAlater and shipped on dry ice or at ambient temperatures from the study centers to the central analysis laboratory. Both examined systems were clearly appropriate for RNA stabilization in human blood independently of the shipping conditions. The isolated RNA is characterized by good quantity and quality and well suited for downstream applications like quantitative RT-PCR analysis of mRNA and microRNA. Superior yield and integrity values were received using RNAlater. It would be reasonable to consider the production and approval of blood collection tubes prefilled with RNAlater to facilitate the use of this excellent RNA stabilization system in large studies.

Cross‐reacting IgE antibodies recognizing latex allergens, including Hev b 1, as well as papain

The cross‐reactivity of IgE antibodies recognizing epitopes of latex allergens and papain was studied in sera of 36 latex‐exposed subjects and 22 papain workers. Eight out of 24 latex‐sensitized persons also showed positive reaction to papain in the CAP assay (mostly of low or moderate degree). On the other hand, six out of the 12 sensitized papain workers also revealed IgE binding to latex allergen(s). Reciprocal inhibition experiments confirmed that groups monosensitized to one of the two allergens can be separated from a group showing partial or nearly complete immunologic cross‐reactivity. Papain inhibited IgE binding by 20–33% in these subjects, whereas IgE binding to papain was strongly blocked in most cases. Comparison between the primary sequences of Hev b 1, a major latex allergen, and papain suggests that the cross‐reactivity may be due to several identical trimers and tetramers.

Polymerase Chain Reaction Based cDNA Cloning of Wheat Profilin: A Potential Plant Allergen

Highly degenerate profilin-specific primers were used to amplify by polymerase chain reaction the profilin-coding sequence of wheat cDNA. Analyzing the sequencing data after subcloning revealed the existence of three isoforms. The high amino acid sequence identity with the birch pollen allergen Betv II suggests that wheat profilin may be a potential plant allergen, relevant in food allergy.

PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8).

Background: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14‐kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE‐binding reactivity.

Relevance of the recombinant lipid transfer protein of Hevea brasiliensis: IgE-binding reactivity in fruit-allergic adults.

BACKGROUND Lipid transfer proteins (LTPs) are relevant allergens in certain plants. The role of the LTP of Hevea brasiliensis in the latex-fruit syndrome is widely unknown. OBJECTIVE To study IgE reactivity with recombinant Hevea LTP in sera of fruit-allergic adults with and without natural rubber latex (NRL) allergy. METHODS An LTP-specific complementary DNA of H brasiliensis leaves was amplified, subcloned into the pMAL expression system, and analyzed. The recombinant protein was coupled to ImmunoCAP, and the IgE-binding properties were studied in sera of 10 NRL-allergic patients without symptoms to fruit and 48 atopic patients with fruit allergy. Eleven of these 48 patients were also allergic to NRL, 14 displayed sensitization to NRL without symptoms on NRL exposure so far, and 23 had neither symptoms nor IgE antibodies to NRL. RESULTS After expression in Escherichia coli, a soluble maltose-binding protein-rHev b 12 fusion protein was isolated and coupled to ImmunoCAP to determine rHev b 12 specific IgE reactivity. rHev b 12 specific IgE binding was found in 3 fruit-allergic patients with NRL sensitization (0.68, 0.88, and 0.96 kU/L) and in 3 fruit-allergic patients without NRL sensitization (1.58, 2.25, and 2.27 kU/L). The remaining 52 serum samples and all maltose-binding protein control test results were negative (< 0.35 kU/L). CONCLUSIONS In these patients, rHev b 12 specific IgE reactivity seems to result from common cross-reactive epitopes with some of the fruit LTPs tested and underscores only an involvement in co-recognition. No clinical relevance of IgE binding to the LTP of H brasiliensis in association with NRL allergy was detected.