Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE‐mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE‐mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well‐defined, highly pure molecules for component‐resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the “EAACI Molecular Allergology User's Guide” (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state‐of‐the‐art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

The number of foreign protein molecules expressed on the cell surface of the budding yeast Saccharomyces cerevisiae by cell surface engineering was quantitatively evaluated using enhanced green fluorescent protein (EGFP). The emission from EGFP on the cell surface was affected by changes in pH. The amount of EGFP on the cell surface, displayed as alpha-agglutinin-fusion protein under control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, was determined at the optimum pH of 7.0. The fluorometric analysis and the image analysis by confocal laser scanning microscopy (CLSM) showed a similar number of molecules displayed on the cell surface, demonstrating that 10(4)-10(5) molecules of alpha-agglutinin-fused molecules per cell were expressed. Furthermore, the amount of fluorescent protein expressed on cells harboring a multicopy plasmid was three to four times higher than that on cells harboring the gene integrated into the genome.

To the Editor, Pollen-food allergy syndromes (PFAS) have been clinically reported and are based on structurally similar sensitizing allergens in pollen and plant food. Besides PR-10 or LTP allergens, the recently described Gibberellin-regulated proteins (GRP) are also allergens found in pollen and plant derived food, therefore are allergens involved in PFAS. 1,2 They are found in Cupressaceae pollen and some fruits such as peach/apricot, citrus, pomegranate, and cherry. 3 The aim of the study was to compare GRP sensitization in two cohorts of Japanese children allergic to Japanese cedar (Cryptomeria japonica, Cryj) pollen: group 1 allergic to pollen-only (n = 21), group 2 allergic to pollen and fruit (n = 22) suffering from systemic reaction (Table 1). Clinical data of recruited allergic patients were recorded at the department of pediatrics, Sagamihara National Hospital. Allergomics analysis was performed using direct or competitive IgE immunoblots after separation of the Japanese cedar pollen extracts in electrophoresis in 1 dimension (1-DE) or 2-DE followed by identification of allergens by mass spectrometry. Recombinant pollen and fruit GRPs were produced.Associated fruit IgE-dependent allergy in group 2 mainly involved peach, citrus, and apple but also cherry, Japanese apricot, strawberry, and grape. In a cohort of fruit-only allergic children,

Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of D-mannose to D-glucose, whereas they did not act on β-(1→4)-mannobiose, N-acetyl-D-glucosamine, and D-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for D-mannose epimerization were 3.89 and 3.51 min −1 mM −1 , respectively. Monitoring the Runsl_4512 reaction through 1 H-NMR showed the formation of β-D-glucose and β-D-mannose from D-mannose and D-glucose, respectively. In the reaction with β-D-glucose, β-D-mannose was produced at the initial stage of the reaction, but not in the reaction with α-D-glucose. These results indicate that Runsl_4512 catalyzed the 2epimerization of the β-anomer substrate with a net retention of the anomeric configuration. Since 2 H was obviously detected at the 2-C position of D-mannose and D-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2 H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L D-glucose at 50°C and pH 8.0, Runsl_4512 and Dfer_5652 produced D-mannose with a 24.4 and 22.8% yield, respectively. These D-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing D-mannose.

Megalo-type isomaltosaccharides are an enzymatically synthesized foodstuff produced by transglucosylation from maltodextrin, and they contain a mid-chain length polymer of D-glucose with α-1,6-glycoside linkages. The injection of a solution of megalo-type isomaltosaccharides (1-4%(w/v), average DP = 12.6), but not oligo-type isomaltosaccharides (average DP = 3.3), into the intestinal lumen dose-dependently reduced the transport rates of tight junction permeable markers in a ligated loop of the anesthetized rat jejunum. Application of the megalosaccharide also suppressed the transport of tight junction markers and enhanced transepithelial electrical resistance (TEER) in Caco-2 cell monolayers. Cholesterol sequestration by methyl-β-cyclodextrin in the Caco-2 monolayers abolished the effect of megalosaccharide. Treatment with anti-caveolin-1 and a caveolae inhibitor, but not clathrin-dependent endocytosis and macropinocytosis inhibitors, suppressed the increase in TEER. These results indicate that isomaltosaccharides promote the barrier function of tight junctions in the intestinal epithelium in a chain-length dependent manner and that caveolae play a role in the effect.