Les CCD

[1] - Aalberse RC, Koshte V, Clemens JG. Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. J Allergy Clin Immunol 1981;68:356-364

IgE in some human sera reacted with an antigen present in a large number of unrelated foods: potato, spinach, wheat, buckwheat, peanut, honey and others. The antigen, which was periodate-sensitive and heat-stable, was also found in pollen. Even more surprisingly, these antibodies often reacted in vitro with bee and vespid venom and were sometimes apparently induced by Hymenoptera stings

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[2] - Pauli G, Bessot JC, Dietemann-Molard A, Braun PA, Thierry R. Celery sensitivity: clinical and immunological correlations with pollen allergy. Clin Allergy 1985;15:273-279

The authors studied twenty patients with celery allergy and concomitant hypersensitivity to certain pollens (mugwort, birch). The specific symptoms induced by eating celery were attacks of urticaria and angio oedema (seventeen out of twenty) respiratory complaints (eight out of twenty), systemic anaphylaxis with vascular collapse (three out of twenty). A strong association between clinical reactions to celery and mugwort sensitization, and to a lesser degree between celery allergy and birch pollen sensitization was established. Celery allergy is mediated by IgE antibodies and can be easily diagnosed by cutaneous tests using fresh material and/or by adequate RAST test. RAST inhibitions performed on individual sera suggest the existence of common antigens in celery and mugwort, and in celery and birch pollen. However, the exact nature of these common antigens has not yet been determined.

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[3] - Flinterman AE, Hoekstra MO, Meijer Y, van Ree R, Akkerdaas JH, Bruijnzeel-Koomen CA, et al. Clinical reactivity to hazelnut in children: Association with sensitization to birch pollen or nuts ? J Allergy Clin Immunol 2006;118:1186-1188

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[4] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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[5] - Lidholm J, Ballmer-Weber BK, Mari A, Vieths S. Component-resolved diagnostics in food allergy. Curr Opin Allergy Clin Immunol 2006;6:234-240

PURPOSE OF REVIEW: The purpose of this paper is to review and discuss recent studies on component-resolved diagnostics in food allergy, involving panels of pure allergen molecules or arrays of peptides derived from allergen sequences, and to summarize the reporting of new food allergens during the past 2 years. RECENT FINDINGS: Several component-resolved diagnostic studies in food allergy suggest that the use of panels of allergen molecules may allow refined clinical information to be obtained on the likelihood or severity of an allergic food reaction and regarding diagnostic specificity. Further, in some studies the use of pure allergen molecules has led to a clearly higher sensitivity of the immunoglobulin E immunoassay compared with conventional allergen extracts. SUMMARY: While common diagnostic methods in allergy assess the presence or absence of allergen-specific sensitization, to date, no in-vitro or in-vivo test exists which exhibits full correlation with clinical food allergy. A multitude of recently reported findings and observations indicate that molecular analysis of allergen sensitization pattern may serve to enhance the clinical utility of immunoglobulin E antibody-based allergy diagnostics. Pure natural and recombinant allergen molecules as well as panels of synthetic peptides have been used for this purpose.

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[6] - Ebo DG, Ahrazem O, Lopez-Torrejon G, Bridts CH, Salcedo G, Stevens WJ. Anaphylaxis from Mandarin (Citrus reticulata) : Identification of Potential Responsible Allergens. Int Arch Allergy Immunol 2007;144:39-43

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient's sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

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[7] - Hamilton RG, Williams PB. Human IgE antibody serology: a primer for the practicing North American allergist/immunologist. J Allergy Clin Immunol 2010;126:33-38

The allergist/immunologist judiciously diagnoses allergic disease by using confirmatory IgE antibody data from in vivo and in vitro assays after the collection of a clinical history. After an overview of historical events, clinically available allergen-specific IgE assays from Phadia, Siemens, and Hycor are contrasted by their design and performance characteristics. The assays share comparable working ranges, analytical sensitivities, and excellent precision, reproducibility, and linearity to a performance standard of <15% coefficients of variation. However, multiple interlaboratory studies have confirmed that the 3 IgE antibody assays either detect different populations of IgE antibody or do not measure the same antibodies with comparable efficiencies. The clinical consequence is that IgE antibody results from the 3 assays are not interchangeable or equivalent. Data generated with one assay cannot be directly extrapolated to published predictive outcomes based on IgE antibody levels from a different assay. The transition from allergen extract-based to allergenic components reagents is discussed, emphasizing the chip-based microarray's strength in identifying IgE antibody cross-reactivity. US Food and Drug Administration-cleared point-of-care IgE antibody lateral flow cassettes are overviewed. Finally, IgE antibody concentration, affinity, clonality (epitope specificity), and specific activity (specific/total IgE ratio) are examined as humoral immune response parameters measured by serologic assays that affect effector cell degranulation and ultimately allergic disease expression.

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[8] - Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR Hart GW and Etzler ME. Eds, Essentials of Glycobiology. Second Edition, CSHL Press, 2008 [http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=glyco2]

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[9] - Aalberse RC, van Ree R. Crossreactive carbohydrate determinants. Clin Rev Allergy Immunol 1997;15:375-387

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[10] - van Ree R. Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases. Int Arch Allergy Immunol 2002;129:189-197

Allergenicity of plant and invertebrate N-glycans has been shown to be caused by the presence of two typical nonmammalian substitutions: an (1,3)-fucose linked to the proximal N-acetylglucosamine and a (1,2)-xylose linked to the core mannose. IgE antibodies against these carbohydrate structures are induced upon exposure to pollen or after insect stings, and result in extensive cross-reactivity to plant and invertebrate foods. These cross-reactive IgE antibodies have been shown to possess variable degrees of biological activity, but have never been convincingly shown to induce clinical food allergy. The most likely explanation for this lack of clinical relevance has to be sought in a combination of epitope valency and antibody affinity. In diagnostic tests, these antibodies are at the basis of many false-positive test results for food allergy. Recombinant technologies offer the possibility to produce allergens that do not carry IgE-binding glycans. Whether their absence or presence is of importance for the application of recombinant allergens in immunotherapy is still largely unknown.

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[11] - Fötisch K, Vieths S. N- and O-linked oligosaccharides of allergenic glycoproteins. Glycoconj J 2001;18:373-390

Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions.

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[12] - Thomas WR, Smith W. How good are carbohydrates as allergens ? Clin Exp Allergy 2002;32:658-661

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[13] - Malandain H. IgE-reactive carbohydrate epitopes--classification, cross-reactivity, and clinical impact. Eur Ann Allergy Clin Immunol 2005;37:122-128

A glycan-related IgE-reactivity has been demonstrated in most allergen sources, especially in plant kingdom. Recent progress in glycobiology has allowed a clearer classification of these glyco-epitopes. Unlike classical peptide chain-based epitopes, glyco-epitopes can share significant structural homologies beyond the limits of protein families. These glycoepitopes are thus prone to extensive cross-reactivity. They have been called Cross-reactive Carbohydrate Determinants or CCD. Many of these glyco-epitopes behave as "panepitopes", leading to cross-reactivity between products as distant as pollens and hymenoptera venoms. But CCD are not universally cross-reactive and they rather cluster into subgroups such as plant CCD or fungal CCD. Because a monovalent IgE-binding is sufficient in serum-based assays, glyco-epitopes and CCD are classically considered as a potential source of positive in vitro results without clinical significance. But some authors recently demonstrated that glyco-epitopes could induce a response at the cell level and suggested that they might play a role in vivo. As long as in vitro assays include glycan- and CCD-related IgE responses, laboratory results should be carefully interpreted in the light of existing knowledge about the glycomes of natural products. IgE-reactivity of the patient's serum can also be tested towards a glycoprotein model such as bromelain.

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[14] - Malandain H. IgE-reactive carbohydrate epitopes--classification, cross-reactivity, and clinical impact (2nd part). Eur Ann Allergy Clin Immunol 2005;37:247-256

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[15] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[16] - Peter-Katalinic J. O-glycosylation of proteins. Methods Enzymol 2005;405:139-171

Cell surface and extracellular proteins are O-glycosylated, where the most abundant type of O-glycosylation in proteins is the GalNAc attachment to serine (Ser) or threonine (Thr) in the protein chain by an a-glycosidic linkage. Most eukaryotic nuclear and cytoplasmic proteins modified by a-linked O-GlcNAc to Ser or Thr exhibit reciprocal O-GlcNAc glycosylation and phosphorylation during the cell cycle, cell stimulation, and/or cell growth. Less-investigated types of O-glycosylation are O-fucosylation, O-mannosylation, and O-glucosylation, but they are functionally of high relevance for early stages of development and for vital physiological functions of proteins. Glycosaminoglycans are a-linked to proteoglycans via a xylose-containing tetrasaccharide, represented by linear chains of repetitive disaccharides modified by carboxylates and O- or/and N-linked sulfates. Analysis of O-glycosylation by mass spectrometry (MS) is a complex task due to the high structural diversity of glycan and protein factors. The parameters in structural analysis of O-glycans include determination of (i) O-glycosylation attachment sites in the protein sequence, (ii) the type of attached monosaccharide moiety, (iii) a core type in the case of GalNAc O-glycosylation, (iv) the type and size of the oligosaccharide portion, (v) carbohydrate branching patterns, (vi) the site of monosaccharide glycosidic linkages, (vii) the anomericity of glycosidic linkages, and (viii) covalent modifications of the sugar backbone chains by carbohydrate- and noncarbohydrate-type of substitutents. Classical and novel analytical strategies for identification and sequencing of O-glycans by MS are described. These include methods to analyze O-glycans after total or partial release from the parent protein by chemical or enzymatic approach or to analyze O-glycosylated peptides by mapping and sequencing from proteolytic mixtures. A recombination process of multiply charged glycopeptides with electrons by electron capture dissociation Fourier transform ion cyclotrone resonance (FTICR)-MS has been introduced and is instrumental for nonergodic polypeptide backbone cleavages without losses of labile glycan substituents. A method for O-glycoscreening under increased sensitivity and efficient sequencing as a combination of an on-line coupling of capillary electrophoresis separation, as well as an automated MS-tandem MS (MS/MS) switching under variable energy conditions collision-induced dissociation (CID) protocol, is beneficial for determination of O-acetylation and oversulfation (Bindila et al., 2004a; Zamfir et al., 2004a). O-glycomics by robotized chip-electrospray/ionization (ESI)-MS and MS/MS on the quadrupole time-of-flight (QTOF) and FTICR analyzers, accurate mass determination, and software for assignment of fragmentation spectra represent essentials for high-throughput (HTP) in serial screenings (Bindila et al., 2004b; Froesch et al., 2004; Vakhrushev et al., 2005). Dimerization of intact O-glycosylated proteins can be investigated by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF)-MS after blotting.

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[17] - Lerouge P, Cabanes-Macheteau M, Rayon C, Fitchette-Lainé AC, Gomord V, Faye L. N-glycosylation biosynthesis in plants: recent developments and future trends. Plant Mol Biol 1998;38:31-48

N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of beta(1,2)-xylose and alpha( 1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.

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[18] - Staudacher E, Altmann F, Wilson IBH, Marz L. Fucose in N-glycans: from plant to man. Biochim Biophys Acta 1999;1473:216-236

Fucosylated oligosaccharides occur throughout nature and many of them play a variety of roles in biology, especially in a number of recognition processes. As reviewed here, much of the recent emphasis in the study of the oligosaccharides in mammals has been on their potential medical importance, particularly in inflammation and cancer. Indeed, changes in fucosylation patterns due to different levels of expression of various fucosyltransferases can be used for diagnoses of some diseases and monitoring the success of therapies. In contrast, there are generally at present only limited data on fucosylation in non-mammalian organisms. Here, the state of current knowledge on the fucosylation abilities of plants, insects, snails, lower eukaryotes and prokaryotes will be summarised.

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[19] - Vidal C, Armisen M, Dominguez-Santalla MJ, Gude F, Lojo S, Gonzalez-Quintela A. Influence of alcohol consumption on serum immunoglobulin E levels in atopic and nonatopic adults. Alcohol Clin Exp Res 2002;26:59-64

BACKGROUND: Total and specific serum immunoglobulin E (IgE) are routinely used as diagnostic tools in allergy clinics. Several studies have demonstrated an increase of total serum IgE concentrations in alcoholics, but the possible influence of lower quantities of ethanol intake on serum IgE values has not been fully evaluated. This study was aimed at analyzing the influence of alcohol intake on both total and specific serum IgE concentrations in patients studied in an allergy clinic. METHODS: A total of 460 patients were included in the study. According to skin-prick tests to common aeroallergens, 325 were classified as atopics and 135 as nonatopics. Most atopic patients (253; 78%) were allergic to mites. Alcohol consumption was recorded as the number of standard (10-g) drinking units regularly consumed per week. Two hundred subjects (43%) were abstainers, and 260 (57%) were regular consumers of a median of 30 g of alcohol per week. Total serum IgE was measured in all patients by latex-enhanced nephelometry. Serum-specific IgE was assayed by fluoroenzymeimmunoassay. RESULTS: Total serum IgE increased along with ethanol consumption. On multivariate analysis, regular alcohol consumption greater than 70 g per week was associated with increased total serum IgE levels, even when adjusting for age, sex, atopy, and smoking. Among house-dust mite-allergic patients, specific serum IgE values against the house dust mite Dermatophagoides pteronyssinus were higher in regular alcohol consumers than in abstainers. This difference was not observed among patients allergic to grass pollen (Lolium perenne). CONCLUSIONS: Alcohol consumption, even in moderate quantities, is associated with increased total and specific IgE concentrations in subjects studied in an allergy clinic. Alcohol intake should be taken into account in epidemiological studies of total serum IgE levels.

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[20] - Friedrich N, Husemoen LL, Petersmann A, Nauck M, Völzke H, Linneberg A. The association between alcohol consumption and biomarkers of alcohol exposure with total serum immunoglobulin E levels. Alcohol Clin Exp Res 2008;32:983-990

BACKGROUND: A number of studies have shown that self-reported alcohol intake is positively associated with total serum immunoglobin E (IgE) levels. The purpose of the present study was to investigate the association of self-reported alcohol consumption and different biomarkers of alcohol exposure to total serum IgE levels in a general adult population. METHODS: A total of 3,443 subjects aged 20 to 79 years from the population-based cross-sectional Study of Health in Pomerania (SHIP) were included in the analyses. Information on alcohol consumption and serum carbohydrate-deficient transferrin (CDT), gamma-glutamyl transferase (GGT), aspartate-amino transferase (ASAT), andalanine-amino transferase (ALAT) levels were measured. Multivariable linear regression models were performed separately in atopic and nonatopic subjects. RESULTS: In nonatopic subjects positive associations between self-reported alcohol consumption as well as all considered biomarkers of alcohol exposure and total serum IgE levels were found. Further the results also suggested positive associations between alcohol consumption as well as alcohol biomarkers and total serum IgE level in atopic subjects, even though not all tests for linear trend reached statistical significance. CONCLUSIONS: In conclusion, biomarkers of alcohol exposure were positively associated with total serum IgE levels supporting that the positive association between self-reported alcohol intake and IgE levels observed in previous studies is real and not due to misclassification of alcohol intake or confounding by other factors that may be linked to both alcohol intake and total serum IgE levels.

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[21] - Serghini-Idrissi N, Ravier I, Aucouturier H, Ait Tahar H, Sonneville A. [Food allergy in the chronic alcoholic and alcohol in food allergy: apropos of 38 cases]. Allerg Immunol (Paris) 2001;33:378-382

Exacerbation or appearance of hypersensitivity of food origin in chronic alcoholics appears to be linked to a basis of catabolism of histamine, a facilitation of penetration of allergens under the impetus of the mucosa-irritating power of alcohol and its potential as a histamine-liberator; it results frequently in an elevated total IgE that predisposes to conflictual sub-cutaneous mechanisms, so explaining the easy appearance or exacerbation of food allergies. We have used skin tests of the principal food allergens to evaluate the influence of alcohol in the development of allergies, by determining the allergy profile of each patient and consequently, the titres of total IgE. Our results show the potentiating role in the physiopathology of allergy and food intolerance without which it helps as much by an evident inducing role in the appearance of true food allergy.

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[22] - Gonzalez-Quintela A, Gude F, Boquete O, Rey J, Meijide LM, Suarez F, et al. Association of alcohol consumption with total IgE levels and allergic sensitization in an adult population-based survey. Clin Exp Allergy 2003;33:199-205

BACKGROUND: Chronic alcoholism is associated with increased total serum IgE levels . OBJECTIVE: The study aimed to investigate the relationship between alcohol intake and both total serum IgE levels and allergic sensitization in a general adult population. MATERIALS AND METHODS: A total of 720 subjects was randomly selected (stratified by age) from the population older than 18 years of A-Estrada (Spain) and invited to participate in the study. From 697 eligible subjects, 469 (67%, median age 54 years, range 18 to 92 years, 44% males, 75% of cases from a rural environment) agreed to participate. A battery of 13 skin prick tests to common aeroallergens was performed in all subjects. Cases with at least one positive test (n = 121, 26%) were considered to have allergic sensitization. The most frequent sensitisers were mites and pollens (24% and 10% of subjects, respectively). Total serum IgE was measured in 465 subjects (99%). Alcohol consumption was registered as the number of standard (approximately 10 g) drinking units habitually consumed per week. A total of 244 subjects (52%) were alcohol consumers (median intake, 14 units/week, range 1 to 147 units/week). Abstainers (n = 225, 48%) constituted the reference category . RESULTS: Alcohol consumption of more than 14 units/week was associated with an increase in serum IgE levels after adjusting for age, gender, allergic sensitization and smoking (P = 0.02). Alcohol consumption was not significantly associated with either overall allergic sensitization or mite sensitization after adjusting for age, gender and smoking. However, alcohol consumption of more than 14 units/week was associated with an increased prevalence of pollen sensitization (adjusted OR 3.15, 95% CI 1.19 to 8.34, P = 0.02) . CONCLUSION: Alcohol consumption above a certain threshold is associated with an increase in total serum IgE levels. Alcohol consumption may also be associated with an increased prevalence of pollen sensitization.

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[23] - Bendtsen P, Grønbæk M, Kjærwz SK, Munkw C, Linneberg A, Tolstrup JS. Alcohol consumption and the risk of self-reported perennial and seasonal allergic rhinitis in young adult women in a population-based cohort study. Clin Exp Allergy 2008;38:1179-1185

BACKGROUND: Alcohol consumption has been suggested to be associated with the development of allergic rhinitis (AR), but there is limited data on the topic . OBJECTIVES: The objective of this study was to investigate the association between alcohol consumption and the risk of developing AR among young women . METHODS: Five thousand eight hundred and seventy Danish women aged 20-29 years participated in a prospective cohort study, and were free of seasonal and perennial AR at baseline (1991-1993). Alcohol consumption was assessed by a food frequency questionnaire. The main outcome measures were self-reported information on seasonal and perennial AR debuting during a mean follow-up period of 7.8 years . RESULTS: During follow-up, 831 women developed seasonal AR and 523 women developed perennial AR, corresponding to 14% and 9%. Alcohol consumption was positively associated with the risk of developing perennial AR. The adjusted odds ratio (OR) for perennial AR was 1.78 (95% CI, 1.13-2.80) among women drinking more than 14 drinks/week compared with women drinking <1 drink/week. There was no association between alcohol consumption and seasonal AR. Having one or two parents with asthma was, after adjustment, significantly associated with the risk of developing seasonal (OR, 2.01; 95% CI, 1.65-2.45) and perennial AR (OR, 2.28; 95% CI, 1.70-2.74). Smoking was not associated with an increased risk of developing AR . CONCLUSION: In this population of young adult women, alcohol consumption was associated with an increased risk of developing perennial AR.

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[24] - Linneberg A, Roursgaard M, Hersoug LG, Larsen ST. Effects of alcohol consumption on the allergen-specific immune response in mice. Alcohol Clin Exp Res 2008;32:553-556

BACKGROUND: There is evidence that chronic alcohol consumption impairs the T-helper 1 (Th1) lymphocyte-regulated cell-mediated immune response possibly favoring a Th2 deviation of the immune response. Moreover, a few epidemiological studies have linked alcohol consumption to allergen-specific IgE sensitization. OBJECTIVE: To investigate the effects of alcohol consumption on the allergen-specific immune response in mice. METHODS: BALB/cJBomTac mice were immunized intraperitoneally with ovalbumin (OVA) using a low dose sensitization protocol. Throughout the experiment, mice were kept on isocalorical liquid diets containing 0 to 6.2% ethanol. Evaluation of immunomodulatory effects of ethanol was based on measurements of total serum IgE, as well as OVA-specific IgE, IgG1, and IgG2a. Furthermore, levels of OVA-induced interleukin (IL)-4 and interferon-gamma were determined in ex vivo splenocyte cultures. RESULTS: Alcohol intake decreased the level of OVA-specific IgG2a in a dose-dependent manner, whereas high levels of alcohol markedly increased the level of total IgE, but not OVA-specific IgE. Th1 suppression was supported by the cytokine profile. CONCLUSIONS: Alcohol consumption induced a marked decrease in markers of the Th1-type allergen-specific immune response and an increase in total serum IgE. In this model, there was no effect of alcohol on OVA-specific IgE. Studies using other routes of immunization may be warranted.

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[25] - Domínguez-Santalla MJ, Vidal C, Viñuela J, Pérez LF, González-Quintela A. Increased serum IgE in alcoholics: relationship with Th1/Th2 cytokine production by stimulated blood mononuclear cells. Alcohol Clin Exp Res 2001;25:1198-1205

BACKGROUND: Increased serum immunoglobulin (Ig) E values are frequently found in alcoholics. Cytokines produced by T-helper-2 (Th2) lymphocytes are required for IgE synthesis. Chronic alcoholism is associated with altered cytokine balance. This study analyzed the relationship between Th1 and Th2 cytokine production by stimulated peripheral blood mononuclear cells (PBMCs) and serum IgE levels, both in atopic and nonatopic alcoholics. METHODS: Twenty-five patients admitted to the hospital with alcohol withdrawal syndrome were included in the study. Five were classified as atopic and 20 as nonatopic by means of skin-prick tests. Interleukin-4 (IL-4), IL-10, IL-12, IL-13, and interferon gamma were measured in the supernatants of 48-hr cultures of PBMCs stimulated with phytohemagglutinin. Total serum IgE was measured by chemiluminescent enzyme immunoassay. Results were compared with those of 15 healthy controls (seven atopics and eight nonatopics). RESULTS: Total serum IgE concentrations were higher in alcoholics than in controls, in both atopic and nonatopic subjects. The ratio of IL-4 to interferon gamma production by phytohemagglutinin-stimulated PBMCs (as an approach to Th2/Th1 balance) was significantly lower in alcoholics than in healthy controls, both in the atopic and in the nonatopic group. No difference was observed regarding IL-10, IL-12, and IL-13 production between alcoholics and controls. No correlation was demonstrated between cytokine production and total serum IgE levels in any group. CONCLUSIONS: Increased total serum IgE is observed in alcoholics together with a paradoxically low ratio of Th2 to Th1 cytokine production by phytohemagglutinin-stimulated PBMCs. These findings are independent of the atopic status of patients.

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[26] - Campos J, Gude F, Quinteiro C, Vidal C, Gonzalez-Quintela A. Gene by environment interaction: the -159C/T polymorphism in the promoter region of the CD14 gene modifies the effect of alcohol consumption on serum IgE levels. Alcohol Clin Exp Res 2006;30:7-14

BACKGROUND: Serum IgE is increased in heavy drinkers. Endotoxin mediates most of the immunological alterations associated with heavy drinking. The -159C/T polymorphism in the promoter region of the gene encoding CD14 (an endotoxin receptor) is associated with serum IgE levels in different populations. AIM: To investigate the possible interaction between alcohol intake and the -159C/T polymorphism in the promoter region of the CD14 gene for serum IgE levels. METHODS: A total of 415 individuals (51.6% males, median age 50 years, range 18-92 years) were studied. A total of 140 individuals were alcohol abstainers, 112 were moderate drinkers (1-280 g/week), and 163 were heavy drinkers (>280 g/week). Main determinations included the CD14/-159C/T genotype, a panel of skin prick tests, total serum IgE, and specific serum IgE against common aeroallergens (Phadiatop test). RESULTS: Heavy drinking was associated with increased total serum IgE values and with positive specific serum IgE to common aeroallergens, but the association was stronger in carriers of the CD14/-159C allele (either CC homozygotes or CT heterozygotes) than in CD14/-159TT homozygotes. Both additive and multiplicative interactions between heavy drinking and the CD14/-159C allele for total and specific serum IgE values was still present after adjusting for potential confounders. Neither alcohol consumption nor the CD14/-159 genotype was associated with skin prick test positivity. CONCLUSIONS: The CD14/-159C/T polymorphism modifies the effect of alcohol consumption on serum IgE levels.

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[27] - Linneberg A, Hertzum I, Husemoen LL, Johansen N, Jørgensen T. Association between alcohol consumption and aeroallergen sensitization in Danish adults. Clin Exp Allergy 2006;36:714-721

BACKGROUND: It has been proposed that alcohol consumption may be one of the lifestyle factors associated with a westernized, urban, and affluent lifestyle contributing to the rise in atopic disease. OBJECTIVE: The aim was to investigate the association between alcohol consumption and atopy (aeroallergen sensitization). METHODS: In 1982, a population-based cross-sectional study of 3608 Danes (79% of the invited), aged 30, 40, 50, and 60 years, was carried out. Information on alcohol consumption was obtained by a questionnaire. Aeroallergen sensitization was defined as a positive test for the detection of specific IgE against a panel of 19 common inhalant allergens in stored serum samples. A total of 3317 subjects with complete information on all variables were included in the analyses. RESULTS: We found a statistically significant association between alcohol consumption and aeroallergen sensitization (independent of the type of alcoholic drink consumed). This association appeared to relate only to those who consumed more than 8 drinks/week. After adjustment for confounders this association was only statistically significant for those who consumed 15-21 drinks/week (adjusted odds ratio 1.8, 95% confidence interval 1.2-2.8). CONCLUSION: In this adult general population, self-reported alcohol consumption was positively associated with aeroallergen sensitization.

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[28] - Coutinho V, Vidal C, Garrido M, Gude F, Lojo S, Linneberg A, et al. Interference of cross-reactive carbohydrates in the determination of specific IgE in alcohol drinkers and strategies to minimize it: the example of latex. Ann Allergy Asthma Immunol 2008;101:394-401

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are N-glycans in plant and invertebrate proteins that interfere with specific IgE determinations. The prevalence of IgE to Man2XylFucGlcNAc2 (MUXF), the CCD from bromelain, may be increased in heavy drinkers. OBJECTIVE: To further investigate the relationship of alcohol consumption to CCD specific IgE. Latex was used as an example for investigating CCD interference with in vitro allergy testing and how to minimize the interference by using nonglycosylated recombinant allergens and inhibition assays. METHODS: We determined the levels of IgE to CCD markers (MUXF and ascorbate oxidase) and natural rubber latex in 270 adults without a history of latex allergy (73 abstainers or occasional drinkers, 76 light drinkers, 47 moderate drinkers, and 74 heavy drinkers). In cases with latex reactivity, we performed inhibition assays with MUXF and screened for IgE to a panel of recombinant latex allergens. Fourteen-day serologic follow-up was available for a subset of individuals. RESULTS: Moderate to heavy drinkers displayed an increased prevalence of IgE to CCD markers. The presence of CCD specific IgE was closely associated with latex IgE reactivity. Inhibition studies and the absence of reactivity to nonglycosylated recombinant latex allergens indicated CCD interference in latex IgE determinations. Serum levels of specific IgE decreased with alcohol abstention. CONCLUSIONS: In this population, alcohol consumption is associated with an increased prevalence of IgE reactivity to natural rubber latex due to CCD interference. The use of nonglycosylated recombinant allergens and inhibition assays may help to minimize CCD interference in populations in which IgE to CCDs is common.

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[29] - Gonzalez-Quintela A, Garrido M, Gude F, Campos J, Linneberg A, Lojo S, et al. Sensitization to cross-reactive carbohydrate determinants in relation to alcohol consumption. Clin Exp Allergy 2008;38:152-160

BACKGROUND: Alcohol consumption is associated with increased serum IgE of unknown specificity . OBJECTIVE: To investigate the prevalence of specific IgE to cross-reactive carbohydrate determinants (CCDs) in adults, and its relation to alcohol consumption . METHODS: Population-based survey of 457 adults (218 abstainers, 195 light-to-moderate drinkers, 44 heavy drinkers). Specific IgE determinations included a CCD (MUXF(3), the N-glycan of bromelain), pollens (Lolium perenne and Olea europaea), Hymenoptera venoms (Apis mellifera and Vespula spp.), and a mite (Dermatophagoides pteronyssinus). We replicated these studies in an additional sample of alcoholics (n=138). Inhibition assays were performed in selected cases . RESULTS: In the general population, 5.6% of individuals (95% confidence interval 3.5-7.6%) showed positive (>/=0.35 kU/L) CCD-specific IgE. The levels of CCD-specific IgE were particularly high in heavy drinkers, who also showed a high prevalence of positive IgE to pollens and Hymenoptera venoms, doubling (at least) the prevalence found in alcohol abstainers and light-to-moderate drinkers. The presence of IgE to pollens and Hymenoptera venoms was closely correlated with the presence of CCD-specific IgE. These features were confirmed in the additional sample of alcoholics. Inhibition studies indicated a role of CCD interference in IgE positivity to pollen and Hymenoptera allergens in alcoholics . CONCLUSIONS: CCD-specific IgE is prevalent in heavy drinkers, and is associated with positive IgE to pollens and Hymenoptera venoms. Specific IgE results should be interpreted with caution in heavy drinkers.

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[30] - Vidal C, Vizcaino L, Díaz-Peromingo JA, Garrido M, Gomez-Rial J, Linneberg A et al. Immunoglobulin-E Reactivity to a Glycosylated Food Allergen (Peanuts) Due to Interference With Cross-Reactive Carbohydrate Determinants in Heavy Drinkers. Alcohol Clin Exp Res 2009;33:1322-1328

Background: N-glycans in plant and invertebrate glycoproteins can induce extensive IgE cross-reactivity therefore limiting the specificity of in vitro allergy tests. IgE sensitization to N-glycans (cross-reactive carbohydrate determinants, CCDs) may be increased in heavy drinkers, who therefore show IgE reactivity to aeroallergens, latex, and Hymenoptera venoms. The peanut, a CCD-bearing allergen, is the leading cause of severe food allergic reactions in many populations. Aim of the study: To investigate the potential interference of CCDs with determinations of IgE to peanuts in heavy drinkers. METHODS: We determined IgE to peanuts and IgE to a CCD marker (MUXF(3), the N-glycan from bromelain) in 41 heavy drinkers admitted to the hospital and 54 healthy controls. None of the participants reported symptoms of peanut allergy. In cases with positive (>/=0.35 kU/l) IgE to peanuts, we performed inhibition assays with a neoglycoprotein consisting of MUXF(3) molecules coupled to bovine serum albumin (MUXF(3)-BSA) and a similar neoglycoprotein lacking xylose and fucose (MM-BSA). In the same cases, we screened for IgE to a panel of recombinant nonglycosylated peanut allergens. SDS-PAGE immunoblotting and inhibition assays were performed in selected cases. RESULTS: The prevalence of positive IgE to peanuts was 22 and 3.7% in heavy drinkers and healthy controls, respectively (p < 0.001). Peanut-IgE positivity was closely related to the presence of IgE to CCDs. In most (8/9) heavy drinkers with positive IgE to peanuts, reactivity was inhibited by preincubation with MUXF(3)-BSA, but not with MM-BSA. IgE binding to multiple bands on immunoblotting studies was also inhibited by MUXF(3)-BSA preincubation. IgE to nonglycosylated recombinant peanut allergens was uniformly negative. CONCLUSION: Heavy drinking is associated with clinically asymptomatic IgE reactivity to peanuts, a relevant food allergen, in relation to CCD interference.

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[31] - Vidal C, Gude F, Boquete O, Fernandez-Merino MC, Meijide LM, Rey J, et al. Evaluation of the Phadiatop test in the diagnosis of allergic sensitization in a general adult population. J Investig Allergol Clin Immunol 2005;15:124-130

BACKGROUND: Phadiatop is a commercially available qualitative serological test employed for screening of allergic sensitization in patients with suspected allergic diseases. AIM: The study evaluated the diagnostic accuracy of Phadiatop for the diagnosis of allergic sensitization in a general adult population. METHODS: A total of 469 subjects from the population of A-Estrada (Spain) were selected by age-stratified random sampling (age range, 18-92 years). Phadiatop test (Uni-CAP method) was performed in serum samples from 465 of these subjects. Skin prick tests to a panel of 13 relevant aeroallergens in the studied area (including mites, pollens, moulds, and animal dander) were employed as the reference diagnostic procedure. Subjects with at least a positive skin prick test (> or =4 mm, n= 120) were considered to have allergic sensitization. RESULTS: Phadiatop sensitivity was 70.8% (95% CI 61.7-78.6%), specificity 90.7% (95% CI 87.0-93.5%), positive predictive value 72.6% (95% CI 63.5-80.3%), negative predictive value 89.9% (95% CI 86.2-92.8%), global accuracy 85.6% (95% CI 82.0-88.6%), negative likelihood ratio 0.3 (95% CI 0.2-0.4), and positive likelihood ratio 7.6 (95% CI 5.4-10.8). A high proportion of false-positive Phadiatop cases showed (a) increased total serum IgE levels, (b) significant alcohol consumption, and (c) small-sized (below the diagnostic cut-off) wheal reactions on SPT. A high proportion of false-negative Phadiatop cases showed exclusive storage mite sensitization. Sensitivity and positive predictive value of Phadiatop were somewhat higher among individuals with a history of nasal or bronchial symptoms. CONCLUSIONS: Phadiatop is a valuable tool for the diagnosis of allergic sensitization in a general adult population. However, limitations of the test should be taken into account in similar surveys.

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[32] - Gonzalez-Quintela A, Garrido M, Gude F, Campos J, Lojo S, Linneberg A et al. Discordant positive results of multiallergen immunoglobulin E tests in relation to crossreactive carbohydrate determinants and alcohol consumption. J Investig Allergol Clin Immunol 2009;19:70-71

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[33] - Merrett TG, Merrett J, Kekwick R. The prevalence of immunoglobulin E antibodies to the proteins of rubber (Hevea brasiliensis) latex and grass (Phleum pratense) pollen in sera of British blood donors. Clin Exp Allergy 1999;29:1572-1578

Although there have been many studies of the prevalence of latex allergy in populations deemed to be at risk, little is known of the potential allergic susceptibility to latex products prevailing in the general population. OBJECTIVE: To assess the possible prevalence of allergy to latex goods in a population of blood donors by measurement of specific antilatex immunoglobulin (Ig) E in blood, to relate this to prevalence of antigrass IgE in the blood donations, and to assess the prevalence of antibodies to grass, house dust mite and cat allergens in those donors having antilatex IgE antibodies. METHODS: Sera from two groups of donations obtained in the English West Midlands were assayed. A group of 2000 donations obtained in midwinter was assayed for antilatex and antigrass pollen IgE. A group of 5000 midsummer donations was assayed for total IgE, and antilatex IgE and the sera giving a positive reaction, assayed for antigrass pollen, antihouse dust mite and anticat IgE. The nature of the principal latex and grass pollen polypeptides reacting with IgE in the sera was assessed by immunoblotting. RESULTS: Anti-latex IgE was detected in approximately 4% of the winter and 7% of the summer donations. The prevalence of antigrass IgE in the winter donations was approximately 20% and amongst the latex-positive sera approximately 84% contained antigrass IgE. Of the summer donations of latex-positive sera, 96% contained antigrass, 48.6% antimite IgE and 34% anticat IgE. The prevalence of both antilatex and antigrass IgE was age and sex related. Inhibition studies indicated cross-reactivity of IgE with latex and grass pollen proteins. CONCLUSIONS: Whilst 4-7% of the population may have serum IgE reacting with latex, the levels are low compared with those reacting with the aeroallergens studied. The apparent cross-reactivity of some serum IgE with both latex and grass pollen taken with other evidence suggests that, in some individuals, allergy to latex may arise from an initial sensitization by grass pollen.

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[34] - Ebo DG, Lechkar B, Schuerwegh AJ, Bridts CH, De Clerck LS, Stevens WJ. Validation of a two-color flow cytometric assay detecting in vitro basophil activation for the diagnosis of IgE-mediated natural rubber latex allergy. Allergy 2002;57:706-712

BACKGROUND: IgE-dependent triggering of basophils not only elicits the release of different mediators but also the up-regulation of certain markers, e.g. CD63, which can be detected by flow cytometry. We intended to investigate if flow cytometric analysis of basophil activation could be a valuable tool in the diagnosis of latex allergy, and to evaluate if the basophil activation test (BAT) could be helpful in determining the clinical significance of a positive latex IgE in individuals with negative history and negative latex skin test. Additionally we aimed to determine the role of cross-reactive carbohydrate determinants (CCDs) in causing positive latex IgE without apparent clinical significance . METHODS: Twelve healthy controls without a history of latex hypersensitivity with a negative latex IgE and skin test (group 1), 24 individuals without a history of latex hypersensitivity with a negative latex IgE and skin test but with other inhalant allergies (group 2), and 29 latex allergic patients with a compelling history of latex allergy with a positive latex IgE and prick test (group 3) were enrolled. The diagnostic performances of the BAT were further evaluated in 13 individuals with a history of latex allergy but with negative specific IgE and/or skin test (group 4). Twenty-four individuals with positive latex IgE without apparent clinical relevance, i.e. without history of latex hypersensitivity and negative latex skin tests, were also analyzed (group 5). The putative role of CCDs causing positive latex IgE results without apparent clinical significance was evaluated by quantification of IgE for bromelain . RESULTS: According, to the receiver operating characteristics(ROC)-generated threshold value of 17% between latex allergic patients and the pooled group of nonlatex allergic individuals, the sensitivity and specificity of the basophil activation test was 93.1% and 91.7%, respectively. In healthy controls, allergic patients without latex hypersensitivity and latex allergic patients the number of positive BATs was 0/12, 3/24 and 27/29, respectively. In the individuals with an evocative history of latex allergy but with negative latex IgE and/or skin test the BAT was positive in all 13 cases. Twenty of 24 individuals without apparent latex allergy but with positive latex IgE had a negative BAT. IgE for bromelain was positive in 1/19 sera from group 2, 1/24 sera from group 3, none of the 8 sera from group 4, but in 16/18 sera from group 5, respectively . CONCLUSION: Flow cytometric analysis of activated basophils seems a highly sensitive and specific tool for diagnosing latex allergy. In addition, the technique might help to determine the clinical relevance of positive IgE quantification in the absence of overt latex allergy. CCDs of natural rubber latex allergens were confirmed to mimic latex sensitization.

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[35] - Bousquet J, Flahault A, Vandenplas O, Ameille J, Duron JJ, Pecquet C, et al. Natural rubber latex allergy among health care workers: A systematic review of the evidence. J Allergy Clin Immunol 2006;118:447-454

BACKGROUND: Natural rubber latex is a recognized allergen, but a recent meta-analysis failed to find any association between latex exposure and allergy in health care workers (HCWs) . OBJECTIVES: A meta-analysis was carried out under the auspices of the French National Regulatory Authority to assess the allergic risk induced by latex gloves in HCWs . METHODS: The risk of work-related exposure to latex for the development of latex allergy was assessed. Prevalence and incidence rates of latex sensitization or allergy were compared in HCWs and in the general population. Exposure-response relationships were assessed in HCWs . RESULTS: Latex allergy was found in 4.32% (range, 4.01% to 4.63%) of HCWs and in 1.37% (range, 0.43% to 2.31%) of the general population. Latex-positive skin prick test responses ranged from 2.1% to 3.7% in the general population and from 6.9% to 7.8% for the HCWs. HCWs exposed to latex showed an increased risk of hand dermatitis (odds ratio [OR], 2.46; 95% CI, 2.11-2.86), asthma or wheezing (OR, 1.55; 95% CI, 1.15-2.08), rhinoconjunctivitis (OR, 2.73; 95% CI, 1.97-3.81), and at least one generic symptom (OR, 1.27; 95% CI, 1.09-1.47). Sensitization to latex was significantly associated with asthma and rhinoconjunctivitis. By contrast, exposure to latex was not associated with a significantly increased risk of positive skin prick test responses to latex (OR, 1.47; 95% CI, 0.94-2.30) . CONCLUSION: HCWs have an increased risk of sensitization and allergic symptoms to latex. CLINICAL IMPLICATIONS: Prevention of latex allergy in HCWs is needed.

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[36] - Mahler V, Gutgesell C, Valenta R, Fuchs T. Natural rubber latex and hymenoptera venoms share Immunoglobin E-epitopes accounting for cross-reactive carbohydrate determinants. Clin Exp allergy 2006;36:1446-1456

BACKGROUND: Epidemiological data on the prevalence and risk factors of latex sensitization have suggested a significant association between latex sensitization and the presence of one or more positive skin prick test responses to aeroallergens, food allergens and to one or more insect venoms. Xylose and core 3-fucose are typical complex glycans in plants and are foreign to mammals. Plant N-glycans and insect N-glycans may cross-react in humans . OBJECTIVE: The aim of our study was to investigate whether there are cross-reactive IgE-binding structures in natural rubber latex (NRL) and hymenoptera venoms and to examine their nature . METHODS: Hundred and twenty-five consecutive patients with insect venom allergy were screened for coincidental latex-specific IgE. IgE-binding components in the venoms from Apis mellifera and/or vespula species and in NRL extracts were characterized by IgE-immunoblotting to the natural allergen sources and determination of specific IgE to recombinant allergens. Cross-reactive components were investigated by inhibition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE-epitopes was further examined by specific IgE-binding to cross-reactive carbohydrate determinants (CCD) in bromelain and horseradish peroxidase as well as by periodate treatment . RESULTS: NRL glove extracts inhibited patients' serum IgE-binding to venom allergens. Vice versa, the IgE-binding to latex glove extracts could be inhibited by pre-incubation with the insect venoms. Specific IgE-binding to recombinant latex allergens was absent, whereas the cross-reactive IgE-epitopes were sensitive to periodate treatment and specific IgE to CCD (MMXF and MUXF type) could be detected . CONCLUSION: Insect venoms and NRL share IgE-binding CCD that may be responsible for positive serological test results to NRL in patients with insect venom allergy. This copositivity occurs frequently (13.6%) among venom-allergic individuals and did not elicit clinical symptoms upon contact to latex in the patients examined. In contrast, true cosensitization to insect venoms and NRL allergens can occur and may not be missed.

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[37] - Palacin A, Quirce S, Sánchez-Monge R, Fernández-Nieto M, Varela J, Sastre J, et al. Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens. Ann Allergy Asthma Immunol 2008;101:200-205

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

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[38] - Sander I, Rozynek P, Rihs H, van Kampen V, Chew F, Lee W et al. Baker's asthma - determination of individual allergen sensitisation profiles to recombinant wheat flour proteins and crossreactive carbohydrate determinants (CCD). Allergy 2009;64(Suppl. 90):59

Background: Several wheat flour allergens relevant for baker's asthma were identified in the last 20 years. It was the aim of our study to determine the frequency of sensitization to the available identified single wheat flour allergens in a group of German bakers with workplace-related complaints. Methods: Using recombinant DNA-technology, the following wheat flour allergens were cloned, expressed in E.coli, purified by affinity chromatography, and finally bound to Streptavidin-ImmunoCAPs: five different subunits of the tetrameric and dimeric wheat a- amylase inhibitors (WTAI-CM1, WTAI-CM2, WTAI-CM3b, WDAI-0.19 and WDAI-0.28), thioredoxin, 1-cys-peroxiredoxin (1-Per), triosephosphat-isomerase (TPIS), aß-gliadin, serpin, glycerinaldehyde-3-phosphate dehydrogenase (GA3PD), a lipid transfer protein (LTP), profilin and peroxidase. In addition, ImmunoCAPs with the recombinant allergen .-5-gliadin and two CCD, horse radish peroxidase (HRP) and MUXF, were tested. Specific IgE was measured in sera from 40 German bakers with allergic symptoms at work, all with specific IgE to wheat flour >0.5 kU/L. Results: Thirty bakers (75%) had specific IgE to at least one of the tested single allergens. Most frequent was specific IgE to WDAI-0.19, HRP and MUXF (25% each), followed by WTAI-CM1 (20%), WTAI-CM3b (18%), WTAI-CM2, thioredoxin and 1-Per (13% each), WDAI-28, TPIS and aß-gliadin (10% each), serpin and GA3PD (5%), .-5-gliadin, LTP and profilin (3%). Fifteen bakers (38%) had specific IgE to any a-amylase inhibitor and twelve (30%) to at least one CCD. Conclusion: The single allergen sensitization profiles obtained with 15 recombinant wheat flour allergens and two CCD in 40 German bakers revealed no major allergens. The profiles differed individually with the highest frequencies found for a-amylase inhibitors and CCD.

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[39] - Kespohl S, Schluenssen V, Jacobsen G, Sigsgaard T, Maryska S, Meurer U, et al. The impact of cross-reactive carbohydrates determinants (CCDs) on wood dust sensitisation. Allergy 2008;63(suppl. 88):39

Background: Occupational wood dust exposure can provoke allergic diseases like asthma and rhinitis. In this study the relation between allergic symptoms after wood dust exposure and wood dust-specific IgE (sIgE) were investigated. Methods: Seven hundred and two serum samples of persons employed in the Danish wood industry were tested for specific IgE against the occupationally relevant wood dust allergens pine and beech. Both woods were extracted under standardized conditions and coupled to streptavidin-Immuno- CAPs. If sIgE against one of the woods was measured additional testing on CCDs with horseradish peroxidase (HRP), bromelain and MUXF3 were performed. Cross-reactivity between CCDs and beech or pine wood was examined by inhibition tests. Results: In 26 serum samples (3.7%) sIgE against pine and/or beech wood was detected. Three sera recognized exclusively pine wood, seven sera bound solely to beech wood and 16 samples contained sIgE to pine and beech wood. All samples containing pine and beech sIgE had additionally sIgE against CCDs. Specific IgE to CCDs was independent of questionnaire-evaluated allergic symptoms (CCD-sensitisation rate in symptomatic wood workers: 2.6%; CCDsensitisation rate in non-symptomatic wood workers: 2.9%). The relevance of CCDs for sIgE-binding to wood allergens was proven by HRP-inhibition in 13 serum samples (10 symtomatic and 3 non-symptomatic workers) with sIgE to wood and CCDs. In the group of symptomatic workers three sera showed no reduction of IgE-binding by HRP-inhibition. In these cases the wood dust sensitisation was obviously based exclusively on wood dust proteins. In contrast, the residual seven samples of symptomatic workers and in all sera of the non-symptomatic workers the sIgE-binding to wood dust was completely inhibited by HRP. In these cases the sIgE-binding to wood allergens was probably conducted by CCDs. Conclusion: For assessment of clinical relevant wood dust sensitisation the additional application of CCD-ImmunoCAPs as well as CCD-inhibition tests in the in vitro diagnosis is highly recommended.

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[40] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[41] - Ebo DG, Hagendorens MM, Bridts CH, De Clerck LS, Stevens WJ. Specific IgE for bromelain: indicator for sensitisation against cross-reactive carbohydrate determinants (CCD) ? Evidence from skin tests and in vitro basophil activation with bromelain. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°109

Background: Non-mammalian glycoproteins can bear highly cross-reactive carbohydrate determinants that hamper serologic diagnosis of allergic disease. Bromelain is a protease isolated from pineapple that possesses a characteristic carbohydrate moiety but rarely induces an IgE-mediated allergy. Therefore, some authors have used quantification of bromelain-specific IgE as a marker for clinical irrelevant CCD sensitisation. Rationale: To assess the biologic activity of specific IgE for bromelain by 2 techniques that closely resemble the in vivo pathway leading to symptoms, i.e. skin prick test (SPT) and in vitro basophil activation (BAT). METHODS: IgE antibodies were quantified by Immuno-CAP. Skin prick tests for aeroallergens were performed with HAL extracts, except for bromelain (500 mg/mL, Sigma-Aldrich). Basophil activation with bromelain (10mg/mL) was measured flow cytometrically using CD63 up-regulation. Calculating the percentage of basophils that express CD63 relative to buffer allows assessment of basophil activation. RESULTS: In controls (n=9) no specific IgE and no SPT reactivity for bromelain was observed and bromelain-induced CD63 expression remained almost unaltered (<10%). Fourteen out of 62 (22.6%) pollen allergic patients without evidence for bromelain allergy, had a bromelain-IgE (median (range): 1.72 kUa/L (0.44->100). In patients with positive bromelain-IgE, SPT as well as BAT with bromelain was positive in 2 (14%) cases. In patients with a negative bromelain-IgE no SPT reactivity for bromelain was observed whereas a positive BAT for bromelain was found in 3 cases (6%). Only the patient with a bromelain-IgE > 100 kUa/L had a positive SPT and highly positive BAT for bromelain (CD63 expression 75%). The exact meaning of this finding remains unclear, but could suggest some biologic activity of CCD. Apart his severe grass pollinosis, he also had suffered from frequent (>5/y) large local reactions to yellow jacket and honeybee venom. CONCLUSION: Bromelain-specific IgE antibodies, in particular low titres (class 1 and 2), were observed in almost one quarter of our pollen allergic patients. According to SPT and in vitro basophil activation, these antibodies generally seemed to have no or little clinical relevance. These results suggest that specific IgE for bromelain can be used as an indicator for irrelevant sensitisation against CCD. However, additional more functional testing is mandatory.

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[42] - Vieths S, Mayer M, Baumgart M. Food allergy: specific binding of IgE antibodies from plant food sensitized individuals to carbohydrate epitopes. Food Agric Immunol 1995;6:453-463

The presence of anti-carbohydrate immunoglobulin E (IgE) antibodies was investigated in a group of 54 patients who were sensitive to at least 2 pollen species and at least 5 vegetable foods. ELISA and ELISA inhibition assays were performed with immobilized food-related oligosaccharides (e.g. stachyose from legumes) and extracts of plant gums (e.g. gum arabic), as well as with allergen extracts from apples, celery and other fruits and vegetables. Furthermore, a combined procedure of immunoblotting and periodate treatment was applied to identify the epitope nature of glycans from vegetable food glycoproteins. Results suggested that 12 of the 54 sera examined contained IgE antibodies against carbohydrate structures. The antibodies were highly cross-reactive. For 6 patients, galactose may be an important epitope. The clinical relevance of this phenomenon requires further investigation

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[43] - Petersen A, Schramm G, Schlaak M, Becker WM. Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen. Clin Exp Allergy 1998;28:315-321

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.

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[44] - Reuter A, Lidholm J, Andersson K, Ostling J, Lundberg M, Scheurer S, et al. A critical assessment of allergen component-based in vitro diagnosis in cherry allergy across Europe. Clin Exp Allergy 2006;36:815-823

BACKGROUND: Food allergy to cherry occurs throughout Europe, typically with restricted oral reactions in the central and northern parts but with frequent systemic reactions in the Mediterranean region. Previous studies have demonstrated insufficient sensitivity of commercially available cherry extract reagents in the diagnosis of cherry allergy . OBJECTIVE: To assess the diagnostic performance of specific IgE tests based on recombinant cherry allergens in comparison with an extract-based assay and to skin prick test (SPT). A secondary objective was to analyse the frequency of systemic reactions in cherry-allergic subjects across Europe, including the largest population of LTP-sensitized subjects from central Europe studied to date . METHODS: A total of 186 subjects from central Europe and Spain were studied. Serum IgE was analysed with ImmunoCAP tests carrying rPru av 1, 3 and 4, combined and separately, and cherry extract . RESULTS: Among the central European cherry allergics, the mix of rPru av 1, 3 and 4 had a sensitivity of 95%, compared with 65% for cherry extract, and the IgE binding capacity of the recombinant mix was considerably higher. The sensitivity of the two tests was more comparable in the Spanish population, 95% and 86%, respectively. The recombinant allergen ImmunoCAP equalled SPT in terms of sensitivity and specificity. Consistent with previous reports, major geographic differences in sensitization pattern and prevalence of systemic reactions were found. A significantly higher rate of systemic reactions was found in Spanish patients sensitized to Pru av 3 whereas German patients sensitized to LTP only had oral allergy syndrome . CONCLUSIONS: The recombinant cherry allergen ImmunoCAP is a highly sensitive diagnostic tool, clearly superior to any diagnostic method based on cherry extract. Three cherry allergens are sufficient for detecting sensitization in 95% of cherry-allergic subjects. Systemic reactions are common in LTP-sensitized individuals but seem to require at least one additional causative factor.

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[45] - Brandao A, Castro A, Gushken A, Soubihe L, Kibrit F, Pastorino A, et al. Is it Possible to Establish a Clinical and Laboratorial Profile in Persistent Cow's Milk Allergic Patients ? J Allergy Clin Immunol 2008;121:S241

RATIONALE: About 20% of IgE-mediated cow‚s milk (CM) allergic patients do not outgrow their allergy until five years-old. Family history of atopy, total IgE levels and multiple food intolerance are among the involved factors. Clinical and laboratorial findings were evaluated in CM persistently allergic children. METHODS: CM allergic patients were defined as clinical data and positive IgE specific associated to anaphylaxis or challenge test (open or double blind). These patients were divided in two groups: group 1 (cow‚s milk protein persistent allergic children older than five years) and group 2 (cow‚s milk protein children that had outgrown their allergy before five years old). The following factors were analyzed: age of the first exposition to CM and of the onset of symptoms, anaphylaxis, multiple food allergy and other allergic diseases, family history of atopy, IgE levels and blood eosinophils counts, skin prick-test (SPT) and RAST/ImmunoCAP to CM and fractions. Statistical analysis: Fisher‚s test, considering p < 0,05. RESULTS: Comparing the two groups (group 1 n 5 32; group 2 n 5 47), there was a significant difference favoring persistent patients regarding to: anaphylaxis presence higher levels of IgE, SPT to CM _8 mm, RAST/ ImmunoCAP to CM _15 kU/ml and casein _3,5 kU/ml . CONCLUSIONS: In this preliminary study, clinical and laboratorial findings could be related to persistentCMallergy. Further studies should be carried out to establish a profile of persistent CM allergic patients.

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[46] - Fötisch K, Vieths S. N- and O-linked oligosaccharides of allergenic glycoproteins. Glycoconj J 2001;18:373-390

Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions.

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[47] - Johansen N, Weber RW, Ipsen H, Barber D, Broge L, Hejl C. Extensive IgE Cross-Reactivity towards the Pooideae Grasses Substantiated for a Large Number of Grass-Pollen-Sensitized Subjects. Int Arch Allergy Immunol 2009;150:325-334

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[48] - Fötisch K, Son D, Altmann F, Aulepp H, Conti A, Haustein D, et al. Tomato (Lycopersicon esculentum) allergens in pollen-allergic patients. Eur Food Res Technol 2001;213:259-266

Abstract. This study was undertaken to investigate the relevance of tomato allergy in pollen-allergic patients, and to identify and characterize tomato allergens. Therefore, sera of a group of 32 pollen-allergic patients with a history of adverse reactions to tomato were investigated by means of immunoblotting and specific IgE measurements (CAP, EAST, EAST inhibition and CCD [cross-reactive carbohydrate determinants] - ELISA). IgE-reactive spots on 2D blots were analyzed by amino acid microsequencing. Tomato allergy was found to occur with a prevalence of about 9% in our group of birch pollen-allergic patients. Of the patients with adverse reactions to tomato, 44% presented IgE to tomato profilin and 35.5% specific IgE to CCD. Two patients were sensitized to a lipid transfer protein in tomato. Microsequencing of other IgE-reactive proteins, separated by 2D-gel electrophoresis and transferred to PVDF membranes, revealed beta-fructofuranosidase, polygalacturonase 2A, and pectinesterase as further tomato allergens. Our data show that tomato is a relevant allergenic food in pollen-allergic patients. It seems that the majority of pollen-associated allergies to tomato is caused by known ubiquitous allergenic structures such as profilin and cross-reactive IgE-binding N-glycans. Furthermore, lipid transfer protein, as well as polygalacturonase 2A, pectinesterase and beta-fructofuranosidase may represent birch pollen-independent tomato allergens for certain patients.

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[49] - Bencúrová M, Hemmer W, Focke-Tejkl M, Wilson IBH, Altmann F. Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 2004;14:457-466

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing 1,2-xylose and core 1,3-fucose, the latter being also found in insect glycoproteins. In order to determine the relative contributions of these two sugar residues to antibody binding we have prepared an array of glycomodified forms of human apo-transferrin. Using core-1,3-fucosyltransferase (EC 2.4.1.214) and 1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF) or none of them (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the 1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in Western blot, ELISA and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.

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[50] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[51] - Ebo DG, Hagendorens MM, Bridts CH, de Clerck LS, Stevens WJ. Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy. Clin Exp Allergy 2004;34:137-144

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[52] - Marknell DeWitt A, Andersson K, Peltre G, Lidholm J. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy 2006;36:77-86

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein . OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4 . METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies . RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner . CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

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[53] - Ballmer-Weber BK, Wüthrich B, Wangorsch A, Fötisch K, Altmann F, Vieths S. Carrot allergy: double-blind, placebo-controlled food challenge and identification of allergens. J Allergy Clin Immunol 2001;108:301-307

BACKGROUND: Allergic reactions to carrot affect up to 25% of food-allergic subjects. Clinical manifestations of carrot allergy and IgE responses to carrot proteins, however, have never been studied in subjects with carrot allergy confirmed by means of double-blinded, placebo-controlled food challenge (DBPCFC) . OBJECTIVE: The purposes of this investigation were to confirm clinically relevant sensitizations to carrot by means of DBPCFC, to validate current diagnostic methods, and to identify IgE-reactive carrot proteins in patients with true allergy . METHODS: DBPCFCs were performed in 26 subjects with histories of allergic reactions to carrot. Patients underwent skin prick tests with carrot extract, fresh carrot, and various pollen extracts. Specific IgE to carrot, celery, birch, and mugwort pollen and to rBet v 1, rBet v 2, and rBet v 6 were measured through use of the CAP method. Carrot allergens were identified by means of immunoblotting and blotting inhibition . RESULTS: Twenty of 26 patients had positive DBPCFC results. The sensitivity of the determination of carrot-specific IgE antibodies through use of the CAP method (> or =0.7 kU/L) was 90%, the sensitivity for skin prick testing with commercial extracts was 26%, and the sensitivity for prick-to-prick tests with raw carrot was 100%. The Bet v 1--related major carrot allergen Dau c 1 was recognized by IgE from 85% of patients; 45% were sensitized to cross-reactive carbohydrate determinants and 20% to carrot profilin. In 1 subject, a Bet v 6--related carrot allergen was recognized. In 4 patients, IgE binding to Dau c 1 was not inhibited or was weakly inhibited by rBet v 1 or birch pollen extract . CONCLUSION: This study confirmed the allergenicity of carrot by means of DBPCFC. DBPCFC-positive patients had exclusively specific IgE antibodies to birch pollen--related carrot allergens, Dau c 1 being the major allergen. The lack of inhibition of IgE binding to Dau c 1 by birch allergens in a subgroup of patients might indicate an secondary immune response to new epitopes on the food allergen that are not cross-reactive with Bet v 1.

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[54] - Lüttkopf D, Ballmer-Weber BK, Wüthrich B, Vieths S. Celery allergens in patients with positive double-blind placebo-controlled challenge. J Allergy Clin Immunol 2000;106:390-399

BACKGROUND: Recently, for the first time, allergy to celery was confirmed by double-blind placebo-controlled food challenge (DBPCFC). Api g 1, Api g 4, cross-reactive carbohydrate determinants (CCD), and a 60 kDa allergen have been described as celery allergens . OBJECTIVE: To get insights in IgE responses of patients with a positive DBPCFC to celery tuber (celeriac) compared with patients with a negative challenge test . METHODS: Specific IgE to native and heated celery tuber and to recombinant Api g 1, the major celery allergen, were determined by enzyme allergosorbent test and immunoblotting. IgE binding to Api g 1, Api g 4, and CCD was confirmed by inhibition experiments that used recombinant Api g 1, recombinant Api g 4, pure N-glycans, and extracts of celeriac, lychee fruit, and pollens of birch, mugwort, and timothy grass as inhibitors . RESULTS: Immunoblotting with sera from 22 patients with a positive DBPCFC to celeriac confirmed the presence of known allergenic structures: The major allergen Api g 1 (16 kDa) was recognized by IgE from 13 of 22 patients (59%). Another major allergen was CCD, determined by IgE reactivity in 12 of 22 patients (55%). Celery profilin, Api g 4, was recognized by IgE from 5 of 22 patients (23%) . CONCLUSION: Our DBPCFC-positive patients exclusively presented IgE to known celery allergens, although the prevalences were slightly different than were previously reported. No obvious differences were found in patients with positive IgE antibody but negative challenge test. IgE binding to all 3 structures in celeriac extract was inhibited by birch pollen extract, whereas mugwort pollen extract could only inhibit IgE reactivity to Api g 4 and CCD. Inhibition experiments with a purified carbohydrate moiety clearly showed that the IgE epitope mannose-xylose-fucose-glycan (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[ Fucalpha1-3]GlcNAc) or a closely related structure is present in celeriac extract and is important in patients with clinical allergy to celery.

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[55] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[56] - Sturm G, Jin C, Hemmer W, Kranzelbinder B, Focke M, Altmann F, et al. Double sensitisation in Hymenoptera venom allergy: comparison of western blot IgE binding patterns with results from skin testing, serum IgE determination, and basophil activation test. Allergy 2008;63(suppl. 88):12-13

Background: Double sensitisation (DS) is a frequent problem in the diagnosis of Hymenoptera allergy. Especially serum IgE determination by ImmunoCAP and intradermal testing at 1.0 mg/mL frequently shows double positive results for bee and wasp venom. As a consequence, it can be sophisticated to choose the relevant venom for specific immunotherapy. The aim of this study was to determine individual antibody patterns by immunoblot and to compare the outcome with usual diagnostic methods and the basophil activation test (BAT). Methods: Sixty-two patients allergic to bee or wasp venom with double positive ImmunoCAP were included. IgE-binding to honeybee and Vespula venom was studied by immunoblot, including inhibition by the synthetic neo-glycoprotein MUXF-BSA to clarify the role of carbohydrate epitopes. Additionally, individual sensitisation was also evaluated by specific IgE determination by Advia Centaur, intradermal testing (IDT) and the BAT. Results: The immunoblot was negative at least for one venom in 11/62 patients. Among the remaining patients, true DS was diagnosed in 20/51 patients (39.2%), putative cross-reactivity due to hyaluronidase (HYA) in 4/51 (7.8%), and double positive results caused by carbohydrate epitopes (CCD) in 27/51 (52.9%). The BAT was negative for the cross-reacting venom in all 4 patients with DS due to HYA, the IDT and the Advia Centaur revealed one positive and three negative results. In the 27 patients of the CCD group, BAT with the primary venom was positive in 76% compared to only 22% with the cross-reacting venom. IDT was positive in 100% with the primary venom and in 74% with the cross-reacting venom (although mostly only at 1 mg/mL). While the Advia system was slightly less sensitive than the ImmunoCAP concerning the primary venom (sensitivity 84%), much less falsepositive tests to the cross-reacting venom were obtained (4/27, 15%). Interestingly, specific IgE to CCDs was in 9 of these 27 patients not detectable by bromelain CAP, although double positivity was clearly associated to CCDs in the immunoblot. Conclusion: Our data confirm the important role of carbohydrate epitopes in double positivity to bee and wasp venom. The BAT and the IgE determination by Advia Centaur were excellent tools to determine the relevant insect venom. Importantly, detectable IgE for bromelain did not exclude true DS and conversely, lacking IgE antibodies for bromelain did not rule out crossreactivity due to CCDs.

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[57] - Mairesse M, Ledent C. Allergie aux venins d'hyménoptères: intérêt du dosage des IgE anti-broméline. Rev Fr Allergol Immunol Clin 2003;43:95

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[58] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[59] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[60] - Müller UR, Johansen N, Petersen AB, Fromberg-Nielsen J, Haeberli G. Hymenoptera venom allergy: analysis of double positivity to honey bee and Vespula venom by estimation of IgE antibodies to species-specific major allergens Api m 1 and Ves v 5. Allergy 2009;64:543-548

BACKGROUND: In patients with hymenoptera venom allergy diagnostic tests are often positive with honey bee and Vespula venom causing problems in selection of venoms for immunotherapy . METHODS: 100 patients each with allergic reactions to Vespula or honey bee stings and positive i.e. skin tests to the respective venom, were analysed for serum IgE to bee venom, Vespula venom and crossreacting carbohydrate determinants (CCDs) by UNICAP (CAP) and ADVIA Centaur (ADVIA). IgE-antibodies to species specific recombinant major allergens (SSMA) Api m1 for bee venom and Ves v5 for Vespula venom, were determined by ADVIA. 30 history and skin test negative patients served as controls . RESULTS: By CAP sensitivity was 1.0 for bee and 0.91 for Vespula venom, by ADVIA 0.99 for bee and 0.91 for Vespula venom. None of the controls were positive with either test. Double positivity was observed in 59% of allergic patients by CAP, in 32% by ADVIA. slgE to Api m1 was detected in 97% of bee and 17% of Vespula venom allergic patients, slgE to Ves v5 in 87% of Vespula and 17% of bee venom allergic patients. slgE to CCDs were present in 37% of all allergic patients and in 56% of those with double positivity and were more frequent in bee than in Vespula venom allergic patients . CONCLUSIONS: Double positivity of IgE to bee and Vespula venom is often caused by crossreactions, especially to CCDs. IgE to both Api m1 and Ves v5 indicates true double sensitization and immunotherapy with both venoms.

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[61] - Ebo DG, Hagendorens MM, Bridts CH, de Clerck LS, Stevens WJ. Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy. Clin Exp Allergy 2004;34:137-144

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[62] - Tretter V, Altmann F, Kubelka V, Marz L, Becker WM. Fucose alpha 1,3-linked to the core region of glycoprotein N-glycans creates an important epitope for IgE from honeybee venom allergic individuals. Int Arch Allergy Immunol 1993;102:259-266

The reactivity of sera from honeybee venom allergic patients with the N-glycan of phospholipase A2 was investigated using neoglycoproteins with an enzyme-linked immunosorbent assay. Of 122 sera with appreciable levels of IgE antibodies directed against bee venom as measured by radioallergosorbent test, 34 sera exhibited significant amounts of glycan-reactive IgE. These sera cross-reacted with the N-glycan from the plant glycoprotein bromelain. The interaction of IgE with the N-glycan from phospholipase could be inhibited with glycopeptides from bromelain which shares the alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine with bee venom phospholipase. Since defucosylated bromelain glycopeptides or glycopeptides containing a Man3GlcNAc2 oligosaccharide were not recognized by most of these sera, we conclude that alpha 1,3-fucosylation of the innermost N-acetylglucosamine residue of N-glycoproteins forms an IgE-reactive determinant. This structural element is frequent in glycoproteins from plants, and it occurs also in insects. It is suspected to be one of the major causes of the broad allergenic cross-reactivity among various allergens from insects and plants.

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[63] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[64] - Malandain H. Widening sensitization spectrum through carbohydrate panepitopes - A hypothesis. Eur Ann Allergy Clin Immunol 2004;36:297-299

Cross-reactive carbohydrate determinants (CCD) differ from classical peptide epitopes because their cross-reactivity is not restricted to a protein family or domain. They can be considered as panepitopes. Antigen presenting cells (APC) express membrane receptors for IgE. APC-bound IgE are able to widen the patient's sensitization spectrum through cross-binding of allergens belonging to the same protein family or domain. In the case of carbohydrate panepitopes the same mechanism could induce a much larger array of new sensitizations than peptide epitopes can do.

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[65] - Pramod SN, Ofori-Anti AO, Cerny R, Goodman RE. IgE Binding to Cross-Reactive Carbohydrate Determinants (CCD) on Proteins of Phaseolus vulgaris using Sera from Peanut Allergic Subjects Demonstrates the Potential for False Diagnosis. J Allergy Clin Immunol 2009;123:S24

RATIONALE: Sera from some peanut allergic subjects show in vitro IgE cross-reactivity to taxonomically diverse food legumes which they claim to tolerate. Specific N-glycans of plant glycoproteins are reported to act as pan-epitopes of little relevance for allergy. This study was performed to identify the IgE binding glycoproteins in commonly consumed navy and kidney bean and determine if glycan is the sole epitope. METHODS: Peanut sera with demonstrated IgE binding to HRP and bromelain were used to identify IgE binding proteins in navy and kidney beans using 2D-gel electrophoresis, immunoblotting and LC-MS/MS. The dominant IgE binding proteins were purified by anion exchange with Q sepharose. Immunoblotting following deglycosylation by periodate was used to differentiate IgE binding to protein versus glycan. RESULTS: Strong IgE binding was seen to 32 to 37 kDa proteins in kidney and navy beans using CCD-binding sera. The subjects had no history of reactions to beans. No IgE binding was measured to the major seed storage glycoprotein phaseolin. The dominant IgE binding glycoprotein in both navy and kidney bean were identified as phytohemaglutinin (PHA) by LC-MS/MS. Based on IgE binding to controls and publications, PHA glycans include a-(1,3) fucosyl residues while phaseolin does not. CONCLUSIONS: The presence of CCD on PHA and high abundance of PHA may lead to an incorrect diagnosis of allergy to common beans if serum IgE binding is used as a determining diagnostic tool, without inhibition to differentiate IgE that binds to protein from that binding only CCD.78 Characterizing Sesame Allergy: Role of Specific IgE and Skin

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[66] - Lee KL, Albee KL, Bernasconi RJ, Edmunds T. Complete amino acid sequence of ananain and a comparison with stem bromelain and other plant cysteine proteases. Biochem J 1997;327:199-202

The amino acid sequences of ananain (EC3.4.22.31) and stem bromelain (3.4.22.32), two cysteine proteases from pineapple stem, are similar yet ananain and stem bromelain possess distinct specificities towards synthetic peptide substrates and different reactivities towards the cysteine protease inhibitors E-64 and chicken egg white cystatin. We present here the complete amino acid sequence of ananain and compare it with the reported sequences of pineapple stem bromelain, papain and chymopapain from papaya and actinidin from kiwifruit. Ananain is comprised of 216 residues with a theoretical mass of 23464 Da. This primary structure includes a sequence insert between residues 170 and 174 not present in stem bromelain or papain and a hydrophobic series of amino acids adjacent to His-157. It is possible that these sequence differences contribute to the different substrate and inhibitor specificities exhibited by ananain and stem bromelain.

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[67] - Kolarich D, Altmann F, Sunderasan E. Structural analysis of the glycoprotein allergen Hev b 4 from natural rubber latex by mass spectrometry. Biochim Biophys Acta 2006;1760:715-720

The lecithinase homolog (Hev b 4) from Hevea brasiliensis (Q6T4P0_HEVBR) is an important natural rubber latex allergen. Hev b 4 is a highly glycosylated protein and its carbohydrate moiety has been implicated in the binding of IgE from natural rubber latex allergic patients. The cDNA for Hev b 4 has recently been cloned and sequenced. Here, we have analyzed the post-translational modifications of natural Hev b 4 by liquid chromatography/electrospray ionization-mass spectrometry of tryptic peptides. Seven of the eight potential glycosylation sites were found to be occupied. One site, however, was only partially glycosylated. Asn224 was substituted by complex type N-glycans with fucose and xylose, whereas all other sites carried either oligomannose glycans or a mixture of oligomannose and complex N-glycans. Glycosylation site Asn308, the most C-terminal one of the eight sites, was only found in the non-glycosylated form. The complex type N-glycans apparently form the molecular basis for the immune reaction with patients' sera. A large fraction of Hev b 4 molecules contains two or more complex N-glycans and thus a physiological reaction against these polyvalent allergens on the basis of the carbohydrate is in theory possible. Aside from allowing glycosylation analysis, the mass spectrometric data defined the N-terminal cleavage site of Hev b 4. This study once more demonstrates the outstanding analytical potential of electrospray ionization-mass spectrometry coupled with liquid chromatographic separation.

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[68] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[69] - Léonard R, Kolarich D, Paschinger K, Altmann F, Wilson IB. A genetic and structural analysis of the N-glycosylation capabilities. Plant Mol Biol 2004;55:631-644

The recent draft sequencing of the rice (Oryza sativa) genome has enabled a genetic analysis of the glycosylation capabilities of an agroeconomically important group of plants, the monocotyledons. In this study, we have not only identified genes putatively encoding enzymes involved in N-glycosylation, but have examined by MALDI-TOF MS the structures of the N-glycans of rice and other monocotyledons (maize, wheat and dates; Zea mays, Triticum aestivum and Phoenix dactylifera); these data show that within the plant kingdom the types of N-glycans found are very similar between monocotyledons, dicotyledons and gymnosperms. Subsequently, we constructed expression vectors for the key enzymes forming plant-typical structures in rice, N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), core alpha1,3-fucosyltransferase (FucTA; EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) and successfully expressed them in Pichia pastoris. Rice GlcNAc-TI, FucTA and xylosyltransferase are therefore the first monocotyledon glycosyltransferases involved in N-glycan biosynthesis to be characterised in a recombinant form.

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[70] - Wilson IBH, Zeleny R, Kolarich D, Staudacher E, Stroop CJM, Kamerling JP, et al. Analysis of Asn-linked glycans from vegetable foodstuffs: widespread occurrence of Lewis a, core alpha-1,3-linked fucose and xylose substitutions. Glycobiology 2001;11:261-274

The N-glycans from 27 "plant" foodstuffs, including one from a gymnospermic plant and one from a fungus, were prepared by a new procedure and examined by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). For several samples, glycan structures were additionally investigated by size-fractionation and reverse-phase high-performance liquid chromatography in conjunction with exoglycosidase digests and finally also (1)H-nuclear magnetic resonance spectroscopy. The glycans found ranged from the typical vacuolar "horseradish peroxidase" type and oligomannose to complex Le(a)-carrying structures. Though the common mushroom exclusively contained N-glycans of the oligomannosidic type, all plant foods contained mixtures of the above-mentioned types. Apple, asparagus, avocado, banana, carrot, celery, hazelnut, kiwi, onion, orange, pear, pignoli, strawberry, and walnut were particularly rich in Le(a)-carrying N-glycans. Although traces of Le(a)-containing structures were also present in almond, pistachio, potato, and tomato, no such glycans could be found in cauliflower. Coconut exhibited almost exclusively N-glycans containing only xylose but no fucose. Oligomannosidic N-glycans dominated in buckwheat and especially in the legume seeds mung bean, pea, peanut, and soybean. Papaya presented a unique set of hybrid type structures partially containing the Le(a) determinant. These results are not only compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens, but they also demonstrate that the Le(a)-type structure is very widespread among plants.

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[71] - Wilson IBH, Altmann F. Structural analysis of N-glycans from allergenic grass, ragweed and tree pollens: core alpha-1,3-linked fucose and xylose present in all pollens examined. Glycoconj J 1998;15:1055-1070

The N-glycans from soluble extracts of ten pollens were examined. The pyridylaminated oligosaccharides derived from these sources were subject to gel filtration and reverse-phase HPLC, in conjunction with exoglycosidase digests, and in some cases matrix-assisted laser desorption-ionisation mass spectrometry. In comparison to known structures, it was possible to determine the major structures of the N-glycans derived from Kentucky blue grass (Poa pratensis), rye (Secale cerale), ryegrass (Lolium perenne), short ragweed (Ambrosia elatior), giant ragweed (Ambrosia trifida), birch (Betula alba), hornbeam (Carpinus betulus), horse chestnut (Aesculus hippocastanum), olive (Olea europaea) and snake-skin pine (Pinus leucodermis) pollen extracts. For grass pollens the major glycans detected were identical in properties to: [structure in text] Grass pollens also contained some minor structures with one or two non-reducing terminal N-acetylglucosamine residues. In the ragweed pollens, the major structures carried core alpha1,3-linked fucose with or without the presence of xylose. In tree pollen extracts, the major structures were either xylosylated, with or without fucose and terminal N-acetylglucosamine residues, with also significant amounts of oligomannose structures. These results are compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens.

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[72] - Bencúrová M, Hemmer W, Focke-Tejkl M, Wilson IBH, Altmann F. Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 2004;14:457-466

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing 1,2-xylose and core 1,3-fucose, the latter being also found in insect glycoproteins. In order to determine the relative contributions of these two sugar residues to antibody binding we have prepared an array of glycomodified forms of human apo-transferrin. Using core-1,3-fucosyltransferase (EC 2.4.1.214) and 1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF) or none of them (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the 1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in Western blot, ELISA and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.

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[73] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[74] - Wilson IBH, Harthill JE, Mullin NP, Ashford DA, Altmann F. Core alpha-1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 1998;8:651-661

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.

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[75] - van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, et al. beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451-11458

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

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[76] - Fötisch K, Vieths S. N- and O-linked oligosaccharides of allergenic glycoproteins. Glycoconj J 2001;18:373-390

Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions Cross-linking of cell-bound IgE on mast cells or basophils by polyvalent antigens causes the release of histamine and other mediators of the allergic response which then lead to the development of allergic symptoms. In this event not only peptide epitopes, but also carbohydrates can act as cross-linking elements. Since peptide epitopes of allergens are subject of most published studies, this review is focused on glycosidic epitopes. The current knowledge of the structures and possible epitopes of oligosaccharides linked to allergenic glycoproteins is briefly reviewed, showing that complex plant N-glycans containing alpha1,3 fucose and beta1,2 xylose are most frequently involved in the structures of IgE epitopes. In own studies a prevalence of up to 29% anti-glycan IgE was determined among pollen-allergic patients. The clinical relevance of these carbohydrate specific IgE antibodies is still a matter of controversial discussions.

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[77] - Wilson IBH, Altmann F. Structural analysis of N-glycans from allergenic grass, ragweed and tree pollens: core alpha-1,3-linked fucose and xylose present in all pollens examined. Glycoconj J 1998;15:1055-1070

The N-glycans from soluble extracts of ten pollens were examined. The pyridylaminated oligosaccharides derived from these sources were subject to gel filtration and reverse-phase HPLC, in conjunction with exoglycosidase digests, and in some cases matrix-assisted laser desorption-ionisation mass spectrometry. In comparison to known structures, it was possible to determine the major structures of the N-glycans derived from Kentucky blue grass (Poa pratensis), rye (Secale cerale), ryegrass (Lolium perenne), short ragweed (Ambrosia elatior), giant ragweed (Ambrosia trifida), birch (Betula alba), hornbeam (Carpinus betulus), horse chestnut (Aesculus hippocastanum), olive (Olea europaea) and snake-skin pine (Pinus leucodermis) pollen extracts. For grass pollens the major glycans detected were identical in properties to: [structure in text] Grass pollens also contained some minor structures with one or two non-reducing terminal N-acetylglucosamine residues. In the ragweed pollens, the major structures carried core alpha1,3-linked fucose with or without the presence of xylose. In tree pollen extracts, the major structures were either xylosylated, with or without fucose and terminal N-acetylglucosamine residues, with also significant amounts of oligomannose structures. These results are compatible with the hypothesis that the carbohydrate structures are another potential source of immunological cross-reaction between different plant allergens.

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[78] - Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F. Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 2008;121:185-190

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance . OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies . METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance . RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG) . CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

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[79] - Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F. Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 2008;121:185-190

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance . OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies . METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance . RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG) . CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

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[80] - Cocco RR, Sole D, Mallozi MC, Naspitz CK, Jacob CMA, Wandalsen NF, et al. Characterization Of Soy Allergic Brazilian Patients Using A Microarray Technique For Detection Of Multiple IgE Reactivity. J Allergy Clin Immunol 2008;121:S241

RATIONALE: Since soy allergy is established as one of the most common food allergies, we decided to analyze the profile of Brazilian soy allergic patients, regarding their IgE recognition to different soy allergens, soy containing foods and allergens derived from peanut and relevant inhalants. METHODS: Nine patientswith immediate symptoms after ingestion of soy formula (failing oral food challenge) and IgE reactivity to soy extract as well as a control group (positive IgE to soy but no symptoms)were selected. Their IgE responses to a number of purified proteins and protein mixes were analysed in a multiplexed capillary-flow based microarray assay. RESULTS: b-conglycinin was the most recognized soy protein among the challenge positive patients although reactivity towards other soy proteins also was detected. For soy containing foods and food additives IgE recognition was directly correlated to the IgE reactivity to soy extract. The major peanut allergens also demonstrated correlation to IgE binding to the soy extract. IgE reactivity against pollen allergens was demonstrated in a majority of the patients and high CCD levels were shown in almost half of the patients. CONCLUSIONS: A major difference in IgE reactivity pattern was observed between the allergic patients and the control group. The allergic patients showed high reactivity to major allergens in peanut and soy and a lower response towards inhalants. In this group the major IgE reactivity to soy was directed to b-conglycinin. The microarray technology offers a convenient tool to simultaneously study of different IgE specificities in allergic and sensitized individuals.

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[81] - Kimura Y, Kamamoto M, Maeda M, Okano M, Yokoyama M, Kino K. Occurrence of Lewis a epitope in N-glycans of a glycoallergen, Jun a 1, from mountain cedar (Juniperus ashei) pollen. Biosci Biotechnol Biochem 2005;69:137-144

We have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo- and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-) occurs in the N-glycans of the pollen allergens.

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[82] - Kimura Y, Kuroki M, Maeda M, Okano M, Yokoyama M, Kino K. Glycoform Analysis of Japanese Cypress Pollen Allergen, Cha o 1: A Comparison of the Glycoforms of Cedar and Cypress Pollen Allergens. Biosci Biotechnol Biochem 2008;72:485-491

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen, the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).

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[83] - Maeda M, Kamamoto M, Hino K, Yamamoto S, Kimura M, Okano M, et al. Glycoform Analysis of Japanese Cedar Pollen Allergen, Cry j 1. Biosci Biotechnol Biochem 2005;69:1700-1705

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.

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[84] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[85] - Deshpande N, Wilkins MR, Packer N, Nevalainen H. Protein glycosylation pathways in filamentous fungi. Glycobiology 2008;18:626-637

Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.

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[86] - Haslam SM, Morris HR, Dell A. Mass spectrometric strategies: providing structural clues for helminth glycoproteins. Trends Parasitol 2001;17:231-235

Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as 'self' or 'foreign' by the immune system of the host

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[87] - Khoo KH, Dell A. Glycoconjugates from parasitic helminths: structure diversity and immunobiological implications. Adv Exp Med Biol 2001;491:185-205

We have provided an account of the progress we and others have made over the last decade on the structural characterization of glycans from parasitic helminths. We hope to have illustrated a few principles and patterns governing helminth glycosylation, as well as the experimental approaches adopted and their associated strengths and limitations. Schistosomes remain the best studied systems but are still punctuated with gaps of knowledge. An important theme developed here is the regulated developmental stage-specific expression of various glycan epitopes and their interplay with immediate host environments for successful parasitism. It is anticipated that more novel or unusual structures will continuously be uncovered in the future and that despite many difficulties, current analytical techniques should be well up to meet the challenge in at least elucidating the major or key glycoconjugates from each of the diverse range of worms. The bottle neck will in fact reside in finding suitable experimental models to test their putative immunobiological functions from which the intricate host-parasite interactions can be delineated and rational vaccine design be achieved. The glycobiology of parasitic helminths is an area waiting to be more fully explored and the rewards should be sweet

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[88] - Moneo I, Audicana MT, Alday E, Curiel G, del Pozo MD, Garcia M. Periodate treatment of Anisakis simplex allergens. Allergy 1997;52:565-569

Anaphylactic reactions after parasitized fish consumption are mediated by an IgE response. However, positive skin tests and specific IgE can also be found in many asymptomatic subjects who recognize a single medium-mol.-wt. antigen by IgE immunoblot. The study aimed to find out whether this unspecificity was due to the carbohydrate moieties of parasite antigens. Sixty-two patients with suspected parasite allergy, 51 blood donors, 18 bakers, and 38 atopic patients were studied by blotting. Parasite proteins were treated with periodate. Several selected sera were inhibited with a crude wheat extract and fungal amylase. Twelve patients (19%), eight donors (16%), six bakers (33%), and one atopic patient (3%) recognized a single medium-mol.-wt. band in blotting and should be considered false-positive. This band was periodate-sensitive, but specific IgE to this allergen could not be inhibited by a wheat extract nor by fungal amylase and was clinically irrelevant. Diagnosis of Anisakis simplex hypersensitivity by skin tests and/ or specific IgE values should always be confirmed by specific IgE immunoblotting in order to detect the presence of clinically unrelated antibodies directed to periodate-sensitive allergens. These allergens are probably not a carbohydrate moiety of a parasite glycoprotein.

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[89] - van Remoortere A, Bank CM, Nyame AK, Cummings RD, Deelder AM, van Die I. Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R. Glycobiology 2003;13:217-225

To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selected from a panel of anti-glycan mAbs generated from Schistosoma-infected or immunized mice because it recognized both a plant glycoprotein horseradish peroxidase and phospholipase A2 from honeybee venom. On further characterization, it was shown that mAb 100-4G11-A recognizes the truncated biantennary N-glycan Man3GlcNAc2-R. Immunocytochemical analysis and immunoblotting with this mAb demonstrated that Man3GlcNAc2-R structures occur on many glycoproteins of schistosomes and other invertebrates. Remarkably, Man3GlcNAc2-R is also expressed on a restricted number of vertebrate glycoproteins. Our data indicate that this truncated N-glycan is immunogenic in mice during the course of infection. Nevertheless, no elevated antibody levels against this glycan epitope could be detected in sera of individuals infected with Schistosoma mansoni.

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[90] - van Die I, Gomord V, Kooyman FN, van den Berg TK, Cummings RD, Vervelde L. Core alpha1-->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep. FEBS Lett 1999;463:189-193

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

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[91] - Faveeuw C, Mallevaey T, Paschinger K, Wilson IB, Fontaine J, Mollicone R, et al. Schistosome N-glycans containing core alpha 3-fucose and core beta 2-xylose epitopes are strong inducers of Th2 responses in mice. Eur J Immunol 2003;33:1271-1281

During murine schistosomiasis, egg-derived glycoconjugates play a key role in skewing the immune response towards a Th2 phenotype. Among the candidates responsible for this effect, complex-type N-glycans containing the core alpha 3-fucose and core beta 2-xylose determinants, two glycan epitopes found in some invertebrate- and plant-derived allergens, may be important. Here, we show that core alpha 3-fucose and core beta 2-xylose determinants are expressed in the different developmental stages of Schistosoma mansoni, particularly in the excretory-secretory systems of schistosomula and adult worms and in eggs deposited in the liver. Glycosyltransferase assays confirmed the presence of core alpha 3-fucosyltransferase and core beta 2-xylosyltransferase activities in egg extracts. Using a model of immunization with pulsed dendritic cells, we show that egg-derived glycoproteins containing the core alpha 3-fucose and core beta 2-xylose determinants generate a strong Th2-biased cellular response in mice and that the glycan moieties of this extract are important in this effect. During murine infection, these complex-type N-glycans induce a glycan-specific Th2 cellular response and elicit T-dependent anti-core alpha 3-fucose and anti-core beta 2-xylose IgG1 (a Th2-associated isotype), but not IgG2b (a Th1-associated isotype) Ab. Taken together, our results point out the importance of core fucosylated/xylosylated N-glycans in the Th2 immune response during murine schistosomiasis.

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[92] - Lorenzo S, Romaris F, Iglesias R, Audicana MT, Alonso JM, Leiro J, et al. O-glycans as a source of cross-reactivity in determinations of human serum antibodies to Anisakis simplex antigens. Clin Exp Allergy 2000;30:551-559

BACKGROUND: Anisakis simplex is a seafood-borne parasite that may both infect humans and cause allergy. Serodiagnosis of anisakiasis and allergy caused by this nematode is difficult since most Anisakis antigens show cross-reactivity problems . OBJECTIVE: To analyse the possible role of sugar epitopes contained in Anisakis simplex antigens as causes of false-positive results in serodiagnostic assays . METHODS: The antigens UA2R and UA3R recognized by two anti-Anisakis monoclonal antibodies were used in this study. Capture ELISA techniques were used to compare the reactivities with native or O-deglycosylated antigens of sera from Anisakis-free children (most of them infected by several other parasites) and from Anisakis allergy patients. O-deglycosylation was done by mild alkali treatment with NaOH. SDS-PAGE and immunoblotting were used to characterize the effects of NaOH or N-glycanase F treatment on UA3R . RESULTS: Native UA2R was recognized by IgG1 and IgM antibodies in the sera of both Anisakis-free subjects and allergy patients. Native UA3R was recognized by most sera from allergy patients (92% considering immunoglobulin (Ig) G1, 100% considering IgE), but also by a significant proportion of sera from Anisakis-free subjects (36% considering IgG1, 14% considering IgE). O-deglycosylation of UA3R greatly improved specificity: none of the sera from Anisakis-free patients showed either IgG1 or IgE reactivity with O-deglycosylated UA3R, while the proportion of sera from allergy patients showing IgE reactivity with this antigen was practically unaffected. O-deglycosylation of UA2R did not improve the specificity of assays using this antigen. Our results also show that the protein core of glycoproteins may be altered by even very mild alkali treatment, depending on the nature of the protein . CONCLUSION: Native glycoproteins of A. simplex should not be used for diagnostic purposes. O-deglycosylated UA3R seems to be an excellent candidate for use as target antigen in the serodiagnosis of anisakiasis and A. simplex allergy.

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[93] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[94] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[95] - Mertens M, Brehler R. The clinical relevance of CCDs in hymenoptera-venom allergy. Allergy 2008;63(suppl. 88):24-25

Background: About 20% of allergic patients have specific (s.)-IgE-antibodies against the carbohydrate-part of allergens (cross-reactive carbohydrate determinant5CCD). These glycans are found on a wide variety of allergens like pollens, hymenoptera-venoms and foods. In most cases patients with s.-IgE against CCDs show no clinical symptoms and skin prick tests (SPT) are negative. In sera-tests these antibodies are referred as ŒŒfalse-positive‚‚ results. However, for a small subset of allergens (e. g. a specific tomato-allergen; Lyc e 2) it is shown that CCDs alone have the property to induce histamine-release of basophils passively sensitised with serum of allergic patients. This leads to the assumption that CCDs can have the capability to induce allergic reactions in vivo. Methods: To investigate the role of CCDs in hymenoptera-venom allergy patients‚ serum was analysed for s.-IgE against insect venoms, CCD and horseradish-peroxidase (HRP) - hymenoptera venom allergy was also verified by skin testing and patients‚ history. Furthermore basophils of these patients were stimulated with different concentrations of periodate-treated venomextracts (lacking glycan-epitopes), proteinase K treated wasp venom (lacking proteins epitopes), their native counterparts and HRP. Afterwards activation of basophils was measured by FACS-analysis to get information whether CCDs elicit an activation in vitro or not. Results: Regarding the basophil activation test wasp venom allergic patients with s.-IgE against CCD and HRP can be divided into two subgroups. Subgroup one shows only an activation with wasp venom (native and without glycan-epitopes) and no activation with any honeybee venom, whereas subgroup two shows an additional activation with native honeybee venom (at least in the highest concentration) but not with the glycan-lacking venom. Basophils stimulated with HRP show the same deviation-pattern: basophils of subgroup one show only an activation with high concentrations of HRP, whereas basophils of subgroup two show a stimulation with already about a 100 times less HRP. The same results were found for honeybee venom-allergic patients. Conclusion: We assume that CCDs are relevant in vitro in a subgroup of hymenoptera- venom allergic patients. Currently we analyse the clinical relevance of anti-CCDantibodies in patients with a positive basophil activation test by further skin tests with other glycan-rich allergens and a clinical follow-up.

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[96] - Bublin M, Radauer C, Wilson IB, Kraft D, Scheiner O, Breiteneder H, et al. Cross-reactive N-glycans of Api g 5, a high molecular weight glycoprotein allergen from celery, are required for immunoglobulin E binding and activation of effector cells from allergic patients. FASEB J 2003;17:1697-1699

Allergy diagnosis relying on the determination of specific IgE is frequently complicated by the presence of cross-reacting IgE of unclear clinical relevance. Particularly, the anaphylactogenic activity of IgE directed to cross-reactive carbohydrate moieties of glycoproteins from plants and invertebrates has been a matter of debate. In this study, we present the biochemical and immunological characterization of Api g 5, a glycoprotein allergen from celery with homology to FAD containing oxidases. Carbohydrate analysis of the allergen revealed the presence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. Chemical deglycosylation of the protein completely abolished binding of serum IgE from all 14 patients tested. Likewise, basophils from a patient allergic to mugwort pollen and celery were stimulated only by native Api g 5, whereas the deglycosylated allergen did not trigger release of histamine. IgE inhibition immunoblots showed that native Api g 5 other than the deglycosylated protein completely inhibited IgE binding to high molecular weight allergens in protein extracts from birch pollen, mugwort pollen, and celery. A similar inhibition was accomplished using the IgE binding oligosaccharide, MUXF, coupled to bovine serum albumin. All these observations taken together confer convincing evidence that IgE directed to cross-reactive carbohydrates is capable of eliciting allergic reactions in vivo.

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[97] - Wicklein D, Lindner B, Moll H, Kolarich D, Altmann F, Becker WM, et al. Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens. Biol Chem 2004;385:397-407

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.

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[98] - Batanero E, Crespo JF, Monsalve RI, Martin-Esteban M, Villalba M, Rodriguez R. IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e1. J Allergy Clin Immunol 1999;103:147-153

BACKGROUND: Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE: The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS: Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS: The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION: The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.

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[99] - Seifert U, Wagner S, Ebner C, Hafner C, Mari A, Arija M Arif S, et al. To what extent does glycosylation of nHev b 2, the b-1,3-glucanase from Hevea brasiliensis, contribute to IgE recognition of sera of latex sensitized patients ? EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1058

Background The b-1,3-glucanase of Hevea brasiliensis, Hev b 2, is regarded as a major latex allergen. Most isoforms of this protein are glycosylated with the MMXF structure, a common sugar motif of plant glycoproteins. The aim of this study was to elucidate the contribution of the carbohydrate moiety to IgE recognition. Methods Hev b 2 was purified from fresh Hevea latex. An aliquot was treated with periodate to destroy carbohydrate epitopes. An activity assay and ELISA experiments with antibodies to the carbohydrate moiety and to protein epitopes were performed for both samples. Sera from 105 latex sensitized patients were tested in IgE-ELISA experiments on nHev b 2 and periodate treated Hev b 2. Results A positive activity assay confirmed that the secondary structure of the periodate treated sample was not significantly altered. Of 105 sera from latex sensitized patients, 65 (62%) showed IgE binding to nHev b 2. 65% (42/65) of these patients showed allergic symptoms, the remaining patients (23/65) had no symptoms, but IgE to nHev b 2. 48% (20/42) of the allergic patients´sera lost IgE reactivity to the periodate treated Hev b 2.Of the sera from patients without symptoms, 70% (16/23) did not react with the periodate treated sample. Of all patients with IgE to Hev b 2 protein epitopes (29), 76% (22/29) show allergic symptoms to latex. Whereas only 56 % (20/36) of the patients with IgE to a glycan epitope suffer from symptoms. Conclusions Our data show that protein epitopes are more frequently recognized by allergic subjects´ IgE than by IgE from sensitized subjects. Furthermore, the data indicate that the carbohydrate moiety of this glycosylated allergen is of minor clinical importance. Experiments to elucidate the clinical relevance of the cross-reactive carbohydrate structure of Hev b 2 are in progress.

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[100] - Seifert U, Wagner S, Bublin M, Ebner C, Hafner C, Mari A, et al. The Role of Glycosylation of nHev b 2, The ß-1,3-Glucanase from Hevea Brasiliensis Latex, in IgE Recognition. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°515

RATIONALE: The aim of this study was to determine the contribution of the carbohydrate moiety of nHev b 2 for IgE binding. nHev b 2 is glycosylated with the MMXF structure, a common sugar-motif of plant glycoproteins METHODS: nHev b 2 was purified from fresh Hevea latex. A portion was treated with periodate to destroy carbohydrate epitopes. CD-spectroscopy and a glucanase activity assay were performed for both samples Out of 107 latex sensitized patients, 47 (44%) sensitized to nHev b 2 were tested by IgE-ELISA experiments on nHev b 2 and periodate treated nHev b 2 RESULTS: No significant differences in the secondary structure between native and periodate treated nHev b 2 were observed. 55% (26/47) of patients with IgE to nHev b 2 showed latex allergic symptoms, 45% (20/46) did not. 39% (10/26) of latex allergic patients did not react to nHev b 2 treated with periodate. However, 61% (16/26) of sera contained IgE reactive only to protein epitopes. From patients without symptoms, 67% (13/21) reacted only with the carbohydrate chain, whereas 33 % (7/21) of the sera showed a positive reaction to nHev b 2 and periodatetreated nHev b 2 CONCLUSIONS: In general, IgE of individual patients is directed either to protein or carbohydrate epitopes. Protein epitopes are more frequently recognized by allergic subjects' IgE (61%) than by sensitized subjects (33%). The clinical relevance of the cross-reactive carbohydrate structure of nHev b 2 remains to be investigated This work was supported by a research grant from BIOMAY Funding: BIOMAY

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[101] - van Ree R, Vieths S, Poulsen LK. Allergen-specific IgE testing in the diagnosis of food allergy and the event of a positive match in the bioinformatics search. Mol Nutr Food Res 2006;50:645-654

Current documents on risk assessment of genetically modified foods recommend including IgE-binding tests on sera from allergic patients. However, there is no generally accepted recommendation on technical aspects of the testing procedures or on the interpretation of the results, despite that fact that both false positive and false-negative results may be caused by variability of the test procedures. The present article discusses the state-of-the-art of serological test procedures for qualitative and quantitative determination of specific IgE and interpretation of test results. It is emphasized that the use of sera from clinically well-characterized subjects is of high importance. In the case of a positive test result, the biological activity of the detected IgE antibodies, i. e., the potential to trigger mediator release from basophils or mast cells in an allergen-specific manner, should be taken into account. However, present data also indicate that validation of such mediator release tests is required, both in terms of experimental protocols and with respect to correlation of the test results with the clinical situation. Further studies are also required to prove the usefulness of targeted serum screening, i. e., the testing of gene products from organisms not known to be allergenic with sera from subjects allergic to related species.

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[102] - van der Veen MJ, van Ree R, Aalberse RC, Akkerdaas J, Koppelman SJ, Jansen HM, et al. Poor biologic activity of cross-reactive IgE directed to carbohydrate determinants of glycoproteins. J Allergy Clin Immunol 1997;100:327-334

BACKGROUND: In our outpatient population, approximately one third of patients sensitized to grass pollen were found to have significant serum levels of anti-peanut IgE in the RAST, without positive peanut skin prick test (SPT) response and without peanut-related allergic symptoms. It was suggested earlier that poor biologic activity of IgE antibodies directed to cross-reactive carbohydrate determinants (CCD) of glycoproteins might explain these discrepancies . OBJECTIVE: In this study we investigated the biologic activity of IgE directed to CCD . METHODS: Sera of 32 patients allergic to grass pollen with significant levels of anti-peanut IgE, a negative response on peanut SPT, and no symptoms of peanut allergy were tested for the presence of anti-CCD IgE. Eleven of these patients with greater than 3.0 IU/ml anti-peanut IgE (patients 1 to 11) were selected together with four control patients allergic to peanut, on the basis of a positive response on peanut SPT and a history of peanut allergy (patients 12 to 15). Inhibition of the peanut RAST was performed by using proteinase K-treated grass pollen extract as a CCD source. Basophil histamine release assays (BHRAs) were performed with peanut extract and the isolated peanut major allergens Ara h 1 and Ara h 2. In addition, intracutaneous tests with peanut extract were performed . RESULTS: In 29 (91%) of 32 patients with discrepant peanut RAST and SPT responses, anti-CCD IgE (> or =0.1 IU/ml) was detected. In patients 1 to 11 almost complete inhibition of the peanut RAST with CCD was found (94.3% +/- 5.5%; mean +/- SD). In contrast, in the patients allergic to peanut only partial inhibition (59%) was found in one subject (p = 0.002, Mann-Whitney test). In the BHRAs and the intracutaneous tests of patients with discrepant peanut RAST and SPT results, reactivity was found only at high concentrations of peanut allergens. When related to specific IgE levels, reactivity to peanut allergens in the BHRAs of these patients was found to be at least a factor of 1000 less when compared with reactivity to control inhalant allergens . CONCLUSION: We conclude that cross-reactive IgE directed to carbohydrate determinants of glycoproteins, as found in grass pollen-sensitized patients, has poor biologic activity. It can therefore cause positive RAST results without apparent clinical significance.

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[103] - Fötisch K, Altmann F, Haustein D, Vieths S. Involvement of carbohydrate epitopes in the IgE response of celery-allergic patients. Int Arch Allergy Immunol 1999;120:30-42

This study was performed to get further insights into antibody responses to cross-reactive carbohydrate determinants (CCD), including initial experiments to prove the biological activity of anti-CCD IgE. Earlier studies have shown that IgE specific for CCD occurs in about 25% of celery-allergic patients. The clinical significance of these antibody specificities is doubtful. METHODS: Patient sera were selected on the basis of a positive case history of celery allergy and multiple binding to high molecular weight celery allergens on immunoblots. Specific IgE to native and heated celery tuber was determined by the enzyme allergosorbent test (EAST). N-glycans were purified after extensive digestion of specific glycoproteins, such as pineapple stem bromelain, bovine fibrin, and human IgG, and used as antigens in an IgE ELISA as well as in EAST and immunoblotting inhibition experiments. Dose-related histamine release was performed with BSA neoglycoproteins containing 3-4 units of the purified glycopeptides. RESULTS: Seven celery-allergic patients were identified who clearly presented IgE against the N-glycan purified from bromelain which is a common structure within the plant kingdom. Chemical defucosylation showed that alpha1, 3-fucose is a key structure for IgE binding. In patients with anti-CCD IgE, the maximal inhibition of celery EAST by the bromelain glycan ranged from 22 to 100%. Inhibition of celery immunoblots by preincubation of patient serum with this glycan led to a quenching of multiple bands at masses >40 kD. After linking the bromelain glycopeptide to BSA, a strong dose-related histamine release was obtained in a celery-allergic patient, occurring at lower concentrations than with the recombinant major protein allergen from celery, Api g 1. CONCLUSIONS: Our results demonstrate that IgE specific for CCD is common in celery-allergic patients, and can represent the major proportion of IgE against this food. alpha1, 3-fucose was confirmed to be an essential part of the IgE epitope. Immunoblotting inhibition indicated the presence of this carbohydrate determinant on multiple glycoproteins in celery extract. Although histamine release was only performed in 1 patient, our data show that proteins carrying multiple glycan units can be biologically active in patients sensitized to CCD.

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[104] - van Ree R. Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases. Int Arch Allergy Immunol 2002;129:189-197

Allergenicity of plant and invertebrate N-glycans has been shown to be caused by the presence of two typical nonmammalian substitutions: an (1,3)-fucose linked to the proximal N-acetylglucosamine and a (1,2)-xylose linked to the core mannose. IgE antibodies against these carbohydrate structures are induced upon exposure to pollen or after insect stings, and result in extensive cross-reactivity to plant and invertebrate foods. These cross-reactive IgE antibodies have been shown to possess variable degrees of biological activity, but have never been convincingly shown to induce clinical food allergy. The most likely explanation for this lack of clinical relevance has to be sought in a combination of epitope valency and antibody affinity. In diagnostic tests, these antibodies are at the basis of many false-positive test results for food allergy. Recombinant technologies offer the possibility to produce allergens that do not carry IgE-binding glycans. Whether their absence or presence is of importance for the application of recombinant allergens in immunotherapy is still largely unknown.

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[105] - Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, et al. Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 2003;270:1327-1337

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manalpha1-6(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

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[106] - Iacovacci P, Afferni C, Butteroni C, Pironi L, Puggioni EMR, Orlandi A, et al. Comparison between the native glycosylated and the recombinant Cup a1 allergen: role of carbohydrates in the histamine release from basophils. Clin Exp Allergy 2002;32:1620-1627

Background Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. Objective : To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. Method s : Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. Result s : Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. Conclusion : A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy

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[107] - Mari A, Ooievaar-de Heer P, Scala E, Giani M, Pirrotta L, Zuidmeer L, et al. Evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of IgE antibodies against plant glycans. Allergy 2008;63:891-896

BACKGROUND: The clinical relevance of immunoglobulin E (IgE) to plant glycans is a longstanding debate. We sought to evaluate their clinical reactivity using the human glycoprotein lactoferrin expressed in rice . METHODS: Allergic patients with IgE antibodies against plant glycans were analyzed for the presence of IgE against rice-produced lactoferrin. The potency of IgE to induce mediator release was assessed by basophil histamine release and skin prick tests (SPTs). Clinical relevance was evaluated by double-blind placebo-controlled oral challenge (DBPCOC) . RESULTS: Twenty-four of 29 sera (82.7%) with IgE antibodies against plant glycans demonstrated IgE binding to transgenic lactoferrin. In three of five cases transgenic lactoferrin induced histamine release. Compared to a control major grass pollen allergen lactoferrin concentrations needed for biological activity of IgE were 5-6 orders of magnitude higher. Skin prick test and DBPCOC were negative in five patients with potential clinical reactivity that volunteered to undergo these in vivo challenges . CONCLUSIONS: Poor or no biological activity and lack of clinical relevance of IgE-binding plant glycans (five out of five) was demonstrated using human lactoferrin expressed in rice as a model.

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[108] - Ebo DG, Hagendorens MM, Bridts CH, De Clerck LS, Stevens WJ. Specific IgE for bromelain: indicator for sensitisation against cross-reactive carbohydrate determinants (CCD) ? Evidence from skin tests and in vitro basophil activation with bromelain. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°109

Background: Non-mammalian glycoproteins can bear highly cross-reactive carbohydrate determinants that hamper serologic diagnosis of allergic disease. Bromelain is a protease isolated from pineapple that possesses a characteristic carbohydrate moiety but rarely induces an IgE-mediated allergy. Therefore, some authors have used quantification of bromelain-specific IgE as a marker for clinical irrelevant CCD sensitisation. Rationale: To assess the biologic activity of specific IgE for bromelain by 2 techniques that closely resemble the in vivo pathway leading to symptoms, i.e. skin prick test (SPT) and in vitro basophil activation (BAT). METHODS: IgE antibodies were quantified by Immuno-CAP. Skin prick tests for aeroallergens were performed with HAL extracts, except for bromelain (500 mg/mL, Sigma-Aldrich). Basophil activation with bromelain (10mg/mL) was measured flow cytometrically using CD63 up-regulation. Calculating the percentage of basophils that express CD63 relative to buffer allows assessment of basophil activation. RESULTS: In controls (n=9) no specific IgE and no SPT reactivity for bromelain was observed and bromelain-induced CD63 expression remained almost unaltered (<10%). Fourteen out of 62 (22.6%) pollen allergic patients without evidence for bromelain allergy, had a bromelain-IgE (median (range): 1.72 kUa/L (0.44->100). In patients with positive bromelain-IgE, SPT as well as BAT with bromelain was positive in 2 (14%) cases. In patients with a negative bromelain-IgE no SPT reactivity for bromelain was observed whereas a positive BAT for bromelain was found in 3 cases (6%). Only the patient with a bromelain-IgE > 100 kUa/L had a positive SPT and highly positive BAT for bromelain (CD63 expression 75%). The exact meaning of this finding remains unclear, but could suggest some biologic activity of CCD. Apart his severe grass pollinosis, he also had suffered from frequent (>5/y) large local reactions to yellow jacket and honeybee venom. CONCLUSION: Bromelain-specific IgE antibodies, in particular low titres (class 1 and 2), were observed in almost one quarter of our pollen allergic patients. According to SPT and in vitro basophil activation, these antibodies generally seemed to have no or little clinical relevance. These results suggest that specific IgE for bromelain can be used as an indicator for irrelevant sensitisation against CCD. However, additional more functional testing is mandatory.

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[109] - Vassilopoulou E, Zuidmeer L, Akkerdaas J, Tassios I, Rigby NR, Mills ENC, et al. Severe Immediate Allergic Reactions to Grapes: Part of a Lipid Transfer Protein-Associated Clinical Syndrome. Int Arch Allergy Immunol 2007;143:92-102

BACKGROUND: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected . OBJECTIVES: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur . METHODS: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens . RESULTS: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa beta-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity . CONCLUSIONS: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The 'LTP syndrome' is the appropriate term to describe this condition.

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[110] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[111] - Schmid-Grendelmeier P, Holzmann D, Himly M, Weichel M, Tresch S, Rückert B, et al. Native Art v 1 and recombinant Art v 1 are able to induce humoral and T cell–mediated in vitro and in vivo responses in mugwort allergy. J Allergy Clin Immunol 2003;111:1328-1336

BACKGROUND: Mugwort pollen is an important allergen source in hay fever and pollen-related food allergy. Little is known about the clinical relevance of the major mugwort allergen Art v 1 and its importance in allergy . OBJECTIVE: In this study we aimed to investigate the allergenicity of mugwort extract compared with the allergenicity of native (n)Art v 1 and recombinant (r)Art v 1, one major allergen of mugwort, in vivo and in vitro . METHODS: Thirty-two patients allergic to mugwort and 10 control subjects were investigated by means of skin prick and nasal provocation testing with different concentrations of mugwort extract, nArt v 1, and rArt v 1. nArt v 1 was purified from aqueous mugwort extract, and rArt v 1 was cloned, expressed in Escherichia coli, and then purified. The in vitro allergenicity was measured by means of ImmunoCAP, ELISA, ELISA-inhibition experiments, and T-cell proliferation assays . RESULTS: nArt v 1 and rArt v 1 were able to elicit positive in vivo and in vitro reactions. The IgE-binding capacity, as determined by means of ELISA, was slightly higher for nArt v 1 than for rArt v 1, and both allergens were able to induce T-cell proliferation in sensitized patients. However, rArt v 1 elicited a reduced response in skin and nasal provocation tests compared with nArt v 1. Compared with mugwort extract, both nArt v 1 and rArt v 1 showed lower sensitivity in patients with mugwort allergy in vivo . CONCLUSIONS: Art v 1, either in its native or recombinant form, is able to induce allergic reactions in patients with mugwort allergy. rArt v 1 induced comparable humoral and cell-mediated responses in vitro but showed reduced in vivo allergenicity compared with biochemically purified nArt v 1.

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[112] - Weber A, Schroder H, Thalberg K, Marz L. Specific interaction of IgE antibodies with a carbohydrate epitope of honey bee venom phospholipase A2. Allergy 1987;42:464-470

Phospholipase A2 (E.C. 3.1.1.4.) is a major allergen of honey bee venom. It exists in a glycosylated and an unglycosylated variant. Both forms and the glycopeptide isolated after exhaustive proteolytic digestion were tested in RAST and RAST inhibition studies. IgE from 11 of 14 bee venom allergy sera exhibited significantly higher, and in two cases exclusive, affinity towards glycosylated phospholipase. In RAST inhibition experiments using phospholipase coupled to discs five of the sera were completely inhibited by glycopeptide at 0.1 mg/ml four sera were partially inhibited and two sera could not be inhibited. Glycoasparagine, lacking all amino acids except the carbohydrate-linking asparagine, inhibits IgE-binding to glycopeptide discs up to 100%. These data clearly demonstrate that an oligosaccharide of a structural type frequently found in glycoproteins can represent an epitope which is recognized by IgE antibodies from allergic patients, which are specifically directed against the parent glycoprotein.

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[113] - Lüttkopf D, Ballmer-Weber BK, Wüthrich B, Vieths S. Celery allergens in patients with positive double-blind placebo-controlled challenge. J Allergy Clin Immunol 2000;106:390-399

BACKGROUND: Recently, for the first time, allergy to celery was confirmed by double-blind placebo-controlled food challenge (DBPCFC). Api g 1, Api g 4, cross-reactive carbohydrate determinants (CCD), and a 60 kDa allergen have been described as celery allergens . OBJECTIVE: To get insights in IgE responses of patients with a positive DBPCFC to celery tuber (celeriac) compared with patients with a negative challenge test . METHODS: Specific IgE to native and heated celery tuber and to recombinant Api g 1, the major celery allergen, were determined by enzyme allergosorbent test and immunoblotting. IgE binding to Api g 1, Api g 4, and CCD was confirmed by inhibition experiments that used recombinant Api g 1, recombinant Api g 4, pure N-glycans, and extracts of celeriac, lychee fruit, and pollens of birch, mugwort, and timothy grass as inhibitors . RESULTS: Immunoblotting with sera from 22 patients with a positive DBPCFC to celeriac confirmed the presence of known allergenic structures: The major allergen Api g 1 (16 kDa) was recognized by IgE from 13 of 22 patients (59%). Another major allergen was CCD, determined by IgE reactivity in 12 of 22 patients (55%). Celery profilin, Api g 4, was recognized by IgE from 5 of 22 patients (23%) . CONCLUSION: Our DBPCFC-positive patients exclusively presented IgE to known celery allergens, although the prevalences were slightly different than were previously reported. No obvious differences were found in patients with positive IgE antibody but negative challenge test. IgE binding to all 3 structures in celeriac extract was inhibited by birch pollen extract, whereas mugwort pollen extract could only inhibit IgE reactivity to Api g 4 and CCD. Inhibition experiments with a purified carbohydrate moiety clearly showed that the IgE epitope mannose-xylose-fucose-glycan (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[ Fucalpha1-3]GlcNAc) or a closely related structure is present in celeriac extract and is important in patients with clinical allergy to celery.

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[114] - Anliker MD, Reindl J, Vieths S, Wüthrich B. Allergy caused by ingestion of persimmon (Diospyros kaki): detection of specific IgE and cross-reactivity to profilin and carbohydrate determinants. J Allergy Clin Immunol 2001;107:718-723

BACKGROUND: Allergy to persimmon (Diospyros kaki) is very rare and not yet confirmed by means of double-blind, placebo-controlled, food-challenge (DBPCFC). Thus far, specific IgE to this fruit and cross-reactivity to pollen and other foods has not been determined. OBJECTIVE: The objective was to confirm allergy to persimmon in 3 patients with an according personal history and to characterize allergens and cross-reactivity of specific IgE antibodies to pollen and food allergens. One patient reacted with pruritus, penis edema, urticaria, and asthma; the second reacted with nausea and vomitus; and the third reacted with rhinoconjunctivitis, asthma, and stomachache after ingestion of persimmon. METHODS: Patients underwent skin prick testing with routine allergens, latex, persimmon, and other foods. Allergy to persimmon was confirmed by means of a DBPCFC. Specific serum IgE levels were measured with CAP-FEIA and the enzyme allergosorbent test (EAST) method. EAST and immunoblot inhibitions were carried out with persimmon; birch, grass, and ragweed pollen; latex; and N-glycans as inhibitors. RESULTS: All patients had positive skin test responses, DBPCFC and specific IgE assays to persimmon. Blot and EAST inhibition assays revealed IgE to cross-reactive profilin in one patient and IgE to cross-reacting carbohydrate determinants in all patients. CONCLUSIONS: This is the first report on 3 cases of allergy to persimmon verified by means of DBPCFC and detection of specific IgE. The sensitization is due to cross-reactive profilin and carbohydrate

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[115] - Pizzano R, Nicolai MA, Manzo C, Giannattasio M, Addeo F. Human IgE Binding to the Glycosidic Moiety of Bovine kappa-Casein. J Agric Food Chem 2005;53:7971-7975

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.

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[116] - Wicklein D, Lindner B, Moll H, Kolarich D, Altmann F, Becker WM, et al. Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens. Biol Chem 2004;385:397-407

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.

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[117] - Vieths S, Lüttkopf D, Reindl J, Anliker MD, Wüthrich B, Ballmer-Weber BK. Allergens in celery and zucchini. Allergy 2002;57(Suppl. 72):100-105

The aim of this study was to confirm allergy to celery tuber and to zucchini, for the first time, by DBPCFC, and to identify the allergens recognized by IgE from DBPCFC-positive patients. Therefore, raw vegetables were hidden in a broccoli drink, and a DBPCFC-procedure was developed that consisted of a spit and swallow protocol, making sure that the procedure was safe for the patients and that reactions strictly localized to the oral cavity as well as systemic reactions could be reproduced by DBPCFC. The allergens in celery and zucchini extract were identified by immunoblot inhibition using allergen extracts, recombinant allergens and purified N-glycans as inhibitors. Celery allergy was confirmed in 69% (22/32) of subjects with a positive case history. Four subjects with a history of allergic reactions to zucchini had a positive DBPCFC to this vegetable. During DBPCFC, systemic reactions were provoked in 50% (11/22) of the patients to celery, and in 3/4 of the zucchini-allergic patients. The Bet v 1-related major celery allergen was detected by IgE of 59% (13/22) of the patients. Cross-reactive carbohydrate epitopes (CCD) bound IgE of 55% (12/22) of the celery-allergic patients and in 2/4 of the subjects with zucchini allergy. Profilin was a food allergen in celery in 23% (5/22) and in zucchini in 2/4 of the cases. A zucchini-specific allergen was detected by IgE from one patient. We conclude that ubiquitous cross-reactive structures are important in allergy to both, celery and zucchini, and that a specific association to birch pollen allergy exists in allergy to celery (mediated by Api g 1), but not in zucchini allergy.

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[118] - van Ree R, Aalberse RC. Specific IgE without clinical allergy. J Allergy Clin Immunol 1999;103:1000-1001

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[119] - Vieths S, Scheurer S, Ballmer-Weber B. Current Understanding of Cross-Reactivity of Food Allergens and Pollen. Ann N Y Acad Sci 2002;964:47-68

-Pollen-allergic patients frequently present allergic symptoms after ingestion of several kinds of plant-derived foods. The majority of these reactions is caused by four distinct cross-reactive structures that are present in birch pollen. Proteins that share common epitopes with Bet v 1, the major birch pollen allergen, occur in pollens of several tree species: apples, stone fruits, celery, carrot, nuts, and soybeans. Approximately 70% of our patients who are allergic to birch pollen may experience symptoms after consumption of foods from these groups. In contrast, two minor allergenic structures-profilins and cross-reactive carbohydrate determinants (CCD)-that sensitize approximately 10-20% of all pollen-allergic patients are also present in grass pollen and weed pollen. Moreover, IgE-binding proteins related to the birch pollen minor allergen Bet v 6 have been found in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini, and carrot. Frequently, the occurrence of cross-reactive IgE antibodies is not correlated with the development of clinical food allergy. In particular, the clinical relevance of sensitization to CCD is doubtful. Generally, pollen-related allergens tend to be more labile during heating procedures and in the digestive tract compared to allergens from classical allergenic foods such as peanut. However, recent DBPCFC studies have shown that both cooked celery and roasted hazelnuts still pose an allergenic risk for pollen-sensitized subjects. Since pathogenesis-related proteins share several common features with allergens and both the Bet v 1 and the Bet v 6-related food allergens are defense-related proteins, approaches to introduce such proteins as a measure to protect plants against diseases should be performed with caution as they may increase the allergenicity of these crops

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[120] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[121] - Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. The Protein Data Bank. Nucleic Acids Res 2002;28:235-242

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[122] - van Ree R. Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases. Int Arch Allergy Immunol 2002;129:189-197

Allergenicity of plant and invertebrate N-glycans has been shown to be caused by the presence of two typical nonmammalian substitutions: an (1,3)-fucose linked to the proximal N-acetylglucosamine and a (1,2)-xylose linked to the core mannose. IgE antibodies against these carbohydrate structures are induced upon exposure to pollen or after insect stings, and result in extensive cross-reactivity to plant and invertebrate foods. These cross-reactive IgE antibodies have been shown to possess variable degrees of biological activity, but have never been convincingly shown to induce clinical food allergy. The most likely explanation for this lack of clinical relevance has to be sought in a combination of epitope valency and antibody affinity. In diagnostic tests, these antibodies are at the basis of many false-positive test results for food allergy. Recombinant technologies offer the possibility to produce allergens that do not carry IgE-binding glycans. Whether their absence or presence is of importance for the application of recombinant allergens in immunotherapy is still largely unknown.

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[123] - Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F. Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 2008;121:185-190

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance . OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies . METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance . RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG) . CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

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[124] - Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, et al. Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 2003;13:427-434

The presence of nonmammalian core alpha(1,3)-fucose and core xylose glyco-epitopes on glycans N-linked to therapeutic glycoproteins produced in plants has raised the question of their immunogenicity in human therapy. We address this question by studying the distribution of these N-glycans in pea, rice, and maize (which are the crops intended for the production of therapeutic proteins) and by reinvestigating their immunogenicity in rodents. We found that immunization with a model glycoprotein, horseradish peroxidase, elicits in C57BL/6 mice and rats the production of antibodies (Abs) specific for core alpha(1,3)-fucose and core xylose epitopes. Furthermore, we demonstrated that about 50% of nonallergic blood donors contains in their sera Abs specific for core xylose, whereas 25% have Abs against core alpha(1,3)-fucose. These Abs probably result from sensitization to environmental antigens. Although the immunological significance of these data is too speculative at the moment, the presence of such Abs might introduce some limitations to the use of plant-derived biopharmaceutical glycoproteins, such as an accelerated clearance during human therapy.

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[125] - Jin C, Nitsch S, Hemmer W, Altmann F. Improving allergy diagnosis by removal of CCD-specific IgE from patients' sera. Allergy 2009;64(Suppl. 90):30

Background: Carbohydrate cross-reactive determinants (CCDs) are sugar moieties of glycoprotein which cause cross-reactivity by specific IgE. Many allergens isolated from plants, insects and molluscs are glycoproteins containing core a1,3-fucose, which is the major CCD of non-mammalian glycoproteins. CCD-specific IgE can cause false positive results in in vitro test and leads to misdiagnosis, which further causes unnecessary avoidance and/or inappropriate immunotherapy that may even induce new allergy. CCD-specific IgE has a high prevalence in polysensitized allergic patients. In order to improve the specificity of in vitro diagnosis, we designed and evaluated an adsorption matrix for anti-CCD IgE. Methods: CCD-specific IgE was determined in a large cohort of subjects (n=174) by ELISA using horse radish peroxidase (HRP) as indicator. Allergen-specific IgE was determined by CAP system before and after CCD-adsorber. Results: From 174 sera, 35% contained measurable CCD-specific IgE and 53% contained CCD-specific IgG. In the mono-sensitized group, 24% (17/70) contained CCD-specific IgE, while in the poly-sensitized group this was the case in 41% (43/104) and the amounts of CCD-specific IgE were mostly higher. The CCD-specific IgE was removed by the CCD-adsorber in selected polysensitized sera. Conclusion: As anti-CCD IgE is considered clinically irrelevant, removal of this cross-reactive IgE can be expected to increase the specificity of allergy in vitro diagnosis.

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[126] - Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F. Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 2008;121:185-190

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance . OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies . METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance . RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG) . CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

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[127] - Faveeuw C, Mallevaey T, Paschinger K, Wilson IB, Fontaine J, Mollicone R, et al. Schistosome N-glycans containing core alpha 3-fucose and core beta 2-xylose epitopes are strong inducers of Th2 responses in mice. Eur J Immunol 2003;33:1271-1281

During murine schistosomiasis, egg-derived glycoconjugates play a key role in skewing the immune response towards a Th2 phenotype. Among the candidates responsible for this effect, complex-type N-glycans containing the core alpha 3-fucose and core beta 2-xylose determinants, two glycan epitopes found in some invertebrate- and plant-derived allergens, may be important. Here, we show that core alpha 3-fucose and core beta 2-xylose determinants are expressed in the different developmental stages of Schistosoma mansoni, particularly in the excretory-secretory systems of schistosomula and adult worms and in eggs deposited in the liver. Glycosyltransferase assays confirmed the presence of core alpha 3-fucosyltransferase and core beta 2-xylosyltransferase activities in egg extracts. Using a model of immunization with pulsed dendritic cells, we show that egg-derived glycoproteins containing the core alpha 3-fucose and core beta 2-xylose determinants generate a strong Th2-biased cellular response in mice and that the glycan moieties of this extract are important in this effect. During murine infection, these complex-type N-glycans induce a glycan-specific Th2 cellular response and elicit T-dependent anti-core alpha 3-fucose and anti-core beta 2-xylose IgG1 (a Th2-associated isotype), but not IgG2b (a Th1-associated isotype) Ab. Taken together, our results point out the importance of core fucosylated/xylosylated N-glycans in the Th2 immune response during murine schistosomiasis.

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[128] - van Ree R, Aalberse RC. Demonstration of carbohydrate-specific immunoglobulin G4 antibodies in sera of patients receiving grass pollen immunotherapy. Int Arch Allergy Immunol 1995;106:146-148

From a group of 92 patients receiving grass pollen immunotherapy, and selected on grounds of high IgG4 titers against Lol p I, sera were tested for IgG4 antibodies against the glycosylated grass pollen allergen Lol p XI. In 72 of 92 cases IgG4 antibodies were demonstrated. The N-glycan of Lol p XI was earlier shown to be an epitope for IgE antibodies. In the present study it was demonstrated that the carbohydrate structure is also recognized by IgG4 antibodies, illustrating that IgG4 responses are not immuno-restricted to peptide epitopes, as was suggested in several reports

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[129] - Chung CH, Mirakhur B, Chan E, Le QT, Berlin J, Morse M, et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 2008;358:1109-1117

BACKGROUND: Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, is approved for use in colorectal cancer and squamous-cell carcinoma of the head and neck. A high prevalence of hypersensitivity reactions to cetuximab has been reported in some areas of the United States . METHODS: We analyzed serum samples from four groups of subjects for IgE antibodies against cetuximab: pretreatment samples from 76 case subjects who had been treated with cetuximab at multiple centers, predominantly in Tennessee, Arkansas, and North Carolina; samples from 72 control subjects in Tennessee; samples from 49 control subjects with cancer in northern California; and samples from 341 female control subjects in Boston . RESULTS: Among 76 cetuximab-treated subjects, 25 had a hypersensitivity reaction to the drug. IgE antibodies against cetuximab were found in pretreatment samples from 17 of these subjects; only 1 of 51 subjects who did not have a hypersensitivity reaction had such antibodies (P<0.001). IgE antibodies against cetuximab were found in 15 of 72 samples (20.8%) from control subjects in Tennessee, in 3 of 49 samples (6.1%) from northern California, and in 2 of 341 samples (0.6%) from Boston. The IgE antibodies were shown to be specific for an oligosaccharide, galactose-alpha-1,3-galactose, which is present on the Fab portion of the cetuximab heavy chain . CONCLUSIONS: In most subjects who had a hypersensitivity reaction to cetuximab, IgE antibodies against cetuximab were present in serum before therapy. The antibodies were specific for galactose-alpha-1,3-galactose

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[130] - Chung CH, Mirakhur B, Chan E, Le QT, Berlin J, Morse M, et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 2008;358:1109-1117

BACKGROUND: Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, is approved for use in colorectal cancer and squamous-cell carcinoma of the head and neck. A high prevalence of hypersensitivity reactions to cetuximab has been reported in some areas of the United States . METHODS: We analyzed serum samples from four groups of subjects for IgE antibodies against cetuximab: pretreatment samples from 76 case subjects who had been treated with cetuximab at multiple centers, predominantly in Tennessee, Arkansas, and North Carolina; samples from 72 control subjects in Tennessee; samples from 49 control subjects with cancer in northern California; and samples from 341 female control subjects in Boston . RESULTS: Among 76 cetuximab-treated subjects, 25 had a hypersensitivity reaction to the drug. IgE antibodies against cetuximab were found in pretreatment samples from 17 of these subjects; only 1 of 51 subjects who did not have a hypersensitivity reaction had such antibodies (P<0.001). IgE antibodies against cetuximab were found in 15 of 72 samples (20.8%) from control subjects in Tennessee, in 3 of 49 samples (6.1%) from northern California, and in 2 of 341 samples (0.6%) from Boston. The IgE antibodies were shown to be specific for an oligosaccharide, galactose-alpha-1,3-galactose, which is present on the Fab portion of the cetuximab heavy chain . CONCLUSIONS: In most subjects who had a hypersensitivity reaction to cetuximab, IgE antibodies against cetuximab were present in serum before therapy. The antibodies were specific for galactose-alpha-1,3-galactose

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[131] - Chung CH, Mirakhur B, Chan E, Le QT, Berlin J, Morse M, et al. Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N Engl J Med 2008;358:1109-1117

BACKGROUND: Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, is approved for use in colorectal cancer and squamous-cell carcinoma of the head and neck. A high prevalence of hypersensitivity reactions to cetuximab has been reported in some areas of the United States . METHODS: We analyzed serum samples from four groups of subjects for IgE antibodies against cetuximab: pretreatment samples from 76 case subjects who had been treated with cetuximab at multiple centers, predominantly in Tennessee, Arkansas, and North Carolina; samples from 72 control subjects in Tennessee; samples from 49 control subjects with cancer in northern California; and samples from 341 female control subjects in Boston . RESULTS: Among 76 cetuximab-treated subjects, 25 had a hypersensitivity reaction to the drug. IgE antibodies against cetuximab were found in pretreatment samples from 17 of these subjects; only 1 of 51 subjects who did not have a hypersensitivity reaction had such antibodies (P<0.001). IgE antibodies against cetuximab were found in 15 of 72 samples (20.8%) from control subjects in Tennessee, in 3 of 49 samples (6.1%) from northern California, and in 2 of 341 samples (0.6%) from Boston. The IgE antibodies were shown to be specific for an oligosaccharide, galactose-alpha-1,3-galactose, which is present on the Fab portion of the cetuximab heavy chain . CONCLUSIONS: In most subjects who had a hypersensitivity reaction to cetuximab, IgE antibodies against cetuximab were present in serum before therapy. The antibodies were specific for galactose-alpha-1,3-galactose

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[132] - Sampson HA, Muñoz-Furlong A, Bock A, Schmitt C, Bass R, Chowdhury BA, et al. Symposium on the Definition and Management of Anaphylaxis: Summary report. J Allergy Clin Immunol 2005;115:584-591

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[133] - Commins SP, Satinover SM, Hosen J, Mozena J, Borish L, Lewis BD et al. Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-alpha-1,3-galactose. J Allergy Clin Immunol 2009;123:426-433

Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions. This study sought to determine whether IgE antibodies to alpha-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb. Twenty-four patients with IgE antibodies to alpha-gal were identified. These patients described a similar history of anaphylaxis, angiodema or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow's milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for alpha-gal.

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[134] - Commins SP, Satinover SM, Hosen J, Mozena J, Borish L, Lewis BD et al. Delayed anaphylaxis, angioedema, or urticaria after consumption of red meat in patients with IgE antibodies specific for galactose-alpha-1,3-galactose. J Allergy Clin Immunol 2009;123:426-433

Carbohydrate moieties are frequently encountered in food and can elicit IgE responses, the clinical significance of which has been unclear. Recent work, however, has shown that IgE antibodies to galactose-alpha-1,3-galactose (alpha-gal), a carbohydrate commonly expressed on nonprimate mammalian proteins, are capable of eliciting serious, even fatal, reactions. This study sought to determine whether IgE antibodies to alpha-gal are present in sera from patients who report anaphylaxis or urticaria after eating beef, pork, or lamb. Twenty-four patients with IgE antibodies to alpha-gal were identified. These patients described a similar history of anaphylaxis, angiodema or urticaria 3 to 6 hours after the ingestion of meat and reported fewer or no episodes when following an avoidance diet. SPTs to mammalian meat produced wheals of usually less than 4 mm, whereas intradermal or fresh-food SPTs provided larger and more consistent wheal responses. CAP-RAST testing revealed specific IgE antibodies to beef, pork, lamb, cow's milk, cat, and dog but not turkey, chicken, or fish. Absorption experiments indicated that this pattern of sensitivity was explained by an IgE antibody specific for alpha-gal.

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[135] - Adédoyin J, Johansson SG, Gronlund H, van Hage M. Interference in immunoassays by human IgM with specificity for the carbohydrate moiety of animal proteins. J Immunol Methods 2006;310:117-125

In allergen characterization common immunoassays such as ELISA and immunoblotting are often used to evaluate human IgE binding of sera from allergic individuals. In an attempt to find and characterize new cat allergens, heavily stained bands were found at 67 and 90 kDa on blotted cat dander extract. Further characterization confirmed these bands to be cat IgA and IgM heavy chains. Analyses in ELISA of purified cat immunoglobulins linked the human IgE binding to cat IgA and IgM and also revealed complete mutual cross-reactivity between the two isotypes. IgE reactivity was found to be directed to carbohydrates of the immunoglobulins and to IgM from 7 out of 9 animal species, but not to human immunoglobulins. Further investigations revealed that the reaction was not mediated by human IgE but by a factor in the serum cross-linking animal immunoglobulins and alkaline phosphatase. The factor was further studied and shown to be human IgM. This IgM fits into the heterophile antibody classification and is directed against carbohydrates on animal immunoglobulins and on calf intestine alkaline phosphatase.

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[136] - Van Nunen SA, O'Connor KS, Clarke LR, Boyle RX, Fernando SL. An association between tick bite reactions and red meat allergy in humans. Med J Aust 2009;190:510-511

Twenty-five patients living in a tick-endemic region of Sydney, New South Wales developed red meat allergy after experiencing large local reactions to tick bites. This represents a potentially novel cross-reaction between an arthropod and a food protein. (MJA 2009; 190: 510-511).

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[137] - Rowan AD, Buttle DJ, Barrett AJ. The cysteine proteinases of the pineapple plant. Biochem J 1990;266:869-875

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

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[138] - Maurer HR. Bromelain: biochemistry, pharmacology and medical use. Cell Mol Life Sci 2001;58:1234-1245

Bromelain is a crude extract from the pineapple that contains, among other components, various closely related proteinases, demonstrating, in vitro and in vivo, antiedematous, antiinflammatory, antithrombotic and fibrinolytic activities. The active factors involved are biochemically characterized only in part. Due to its efficacy after oral administration, its safety and lack of undesired side effects, bromelain has earned growing acceptance and compliance among patients as a phytotherapeutical drug. A wide range of therapeutic benefits has been claimed for bromelain, such as reversible inhibition of platelet aggregation, angina pectoris, bronchitis, sinusitis, surgical traumas, thrombophlebitis, pyelonephritis and enhanced absorption of drugs, particularly of antibiotics. Biochemical experiments indicate that these pharmacological properties depend on the proteolytic activity only partly, suggesting the presence of nonprotein factors in bromelain. Recent results from preclinical and pharmacological studies recommend bromelain as an orally given drug for complementary tumor therapy: bromelain acts as an immunomodulator by raising the impaired immunocytotoxicity of monocytes against tumor cells from patients and by inducing the production of distinct cytokines such as tumor necrosis factor-a, interleukin (Il)-1beta, Il-6, and Il-8. In a recent clinical study with mammary tumor patients, these findings could be partially confirmed. Especially promising are reports on animal experiments claiming an antimetastatic efficacy and inhibition of metastasis-associated platelet aggregation as well as inhibition of growth and invasiveness of tumor cells. Apparently, the antiinvasive activity does not depend on the proteolytic activity. This is also true for bromelain effects on the modulation of immune functions, its potential to eliminate burn debris and to accelerate wound healing. Whether bromelain will gain wide acceptance as a drug that inhibits platelet aggregation, is antimetastatic and facilitates skin debridement, among other indications, will be determined by further clinical trials. The claim that bromelain cannot be effective after oral administration is definitely refuted at this time.

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[139] - Erwin EA, Hosen J, Pollart SM, Reid MJ, Platts-Mills TAE. High-titer IgE antibody specific for pollen allergens in northern California is associated with both wheezing and total serum IgE. J Allergy Clin Immunol 2009;123:706-708

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[140] - Raison-Peyron N, Roulet A, Guillot B, Guilhou JJ. Bromelain: an unusual cause of allergic contact cheilitis. Contact Dermatitis 2003;49:218-219

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[141] - van Kampen V, Merget R, Brüning T. [Occupational allergies to bromelain]. Pneumologie 2007;61:159-161

The protease bromelain originating from the pineapple fruit (Ananas comosus) finds frequent use in industry. Exposure to enzyme dusts has long been known to cause occupational allergies. The present paper reviews the results of the evaluation of literature data concerning occupational airway sensitization due to bromelain. Cases of specific airway sensitization caused by bromelain could be shown clearly by the presented studies. Since the symptoms, results of skin prick tests, detection of specific IgE antibodies and results of specific bronchoprovocation tests are consistent, an immunological mechanism can be assumed.

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[142] - Galleguillos F, Rodriguez JC. Asthma caused by bromelin inhalation. Clin Allergy 1978;8:21-24

Two cases are reported of bronchial asthma caused by inhalation of bromelin, a proteolytic enzyme utilized in some anti-inflammatory medicine. Prick test results with bromelin extract were positive. Inhalation tests gave immediate reactions which were blocked by sodium cromoglycate, but not corticosteroids

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[143] - Baur X, Fruhmann G. Allergic reactions, including asthma, to the pineapple protease bromelain following occupational exposure. Clin Allergy 1979;9:443-450

A 58-year-old pharmaceutical worker regularly developed asthma and rhinitis when handling bromelain, a purified protease of pineapple (Ananas comosus), at her work-place, where she had been employed for about 10 years. RAST and prick test showed strong positive reactions to bromelain. Both inhalation test with 0.03 mg bromelain and peroral challenge by ingestion of 190 g pineapple resulted in asthmatic reactions; the latter challenge was accompanied by gastrointestinal symptoms. Five of six workers sensitized to papain, showed positive RAST and skin test results to bromelain, two of them also showed immediate asthmatic reactions after bronchial challenge with bromelain. Out of sixty asthmatics not exposed to airborne proteases but probably to these as constituents of foods, two had positive skin test results and eight had positive RAST results to bromelain; but in no case was there clear evidence for clinical sensitization. The presented data prove conclusively that bromelain is capable of inducing IgE mediated respiratory and gastrointestinal allergic reactions. Furthermore, there is evidence for immunological cross-reaction between the two plant proteases bromelain and papain in human subjects.

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[144] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[145] - Ishihara H, Takahashi N, Oguri S, Tejima S. Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides. J Biol Chem 1979;254:10715-10719

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2- (Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.

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[146] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[147] - Bencúrová M, Hemmer W, Focke-Tejkl M, Wilson IBH, Altmann F. Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 2004;14:457-466

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing 1,2-xylose and core 1,3-fucose, the latter being also found in insect glycoproteins. In order to determine the relative contributions of these two sugar residues to antibody binding we have prepared an array of glycomodified forms of human apo-transferrin. Using core-1,3-fucosyltransferase (EC 2.4.1.214) and 1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF) or none of them (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the 1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in Western blot, ELISA and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.

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[148] - van Huystee RB, McManus MT. Glycans of higher plant peroxidases: recent observations and future speculations. Glycoconj J 1998;15:101-106

Plant peroxidases are composed of a peptide and associated heme, calcium and glycans. The 3D structure of the major cationic peanut peroxidase has revealed the sites of the heme and calcium. But the diffraction of the glycans was not sufficient to show their structure. This review presents research that has been executed to obtain putative glycans and their binding sites, and to gain an indirect insight into these glycans. It also offers approaches that will be used to determine the function of the glycans on the peanut peroxidase. Some comparisons are made with other plant glycoproteins including peroxidases from plants other than peanut.

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[149] - Yang BY, Gray JSS, Montgomery R. The glycans of horseradish peroxidase. Carbohydr Res 1996;287:203-212

Horseradish peroxidase (E.C. 1.11.1.7) isozyme c (HRPc) is a glycoprotein found to contain 21.8% carbohydrate with the average composition: 2 mol GlcNAc, 2.6 mol Man, and 0.8 mol each of Fuc and Xyl. The oligosaccharides of HRPc were investigated by a combination of High pH Anion-Exchange Chromatography with Pulsed Amperometric Detection, methylation analysis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. The structure of the major oligosaccharide released by digestion with glycopeptidase A, accounting for between 75 and 80% of the total, was confirmed to be [sequence: see text]. Most of the remaining oligosaccharides were found to belong to the (Xyl)xManm(Fuc)fGlcNAc2 (m = 2, 4, 5, 6; f = 0 or 1; x = 0 or 1) family. Less than 5% of the oligosaccharides were of the ManmGlcNAc2 (m = 4 to 7) type. Methylation analysis of holo- and apo-HRPc and its tryptic glycopeptides support the structures proposed for the oligosaccharides. Furthermore, methylation analysis of the tryptic glycopeptides provides evidence for the heterogeneity of the oligosaccharides occurring at each of the N-linked sites

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[150] - Jin C, Hantusch B, Hemmer W, Stadlmann J, Altmann F. Affinity of IgE and IgG against cross-reactive carbohydrate determinants on plant and insect glycoproteins. J Allergy Clin Immunol 2008;121:185-190

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance . OBJECTIVE: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies . METHODS: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance . RESULTS: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG) . CONCLUSION: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

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[151] - Altmann F. Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini. Glycoconj J 1998;15:79-82

The N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.

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[152] - Coutinho V, Vidal C, Garrido M, Gude F, Lojo S, Linneberg A, et al. Interference of cross-reactive carbohydrates in the determination of specific IgE in alcohol drinkers and strategies to minimize it: the example of latex. Ann Allergy Asthma Immunol 2008;101:394-401

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are N-glycans in plant and invertebrate proteins that interfere with specific IgE determinations. The prevalence of IgE to Man2XylFucGlcNAc2 (MUXF), the CCD from bromelain, may be increased in heavy drinkers. OBJECTIVE: To further investigate the relationship of alcohol consumption to CCD specific IgE. Latex was used as an example for investigating CCD interference with in vitro allergy testing and how to minimize the interference by using nonglycosylated recombinant allergens and inhibition assays. METHODS: We determined the levels of IgE to CCD markers (MUXF and ascorbate oxidase) and natural rubber latex in 270 adults without a history of latex allergy (73 abstainers or occasional drinkers, 76 light drinkers, 47 moderate drinkers, and 74 heavy drinkers). In cases with latex reactivity, we performed inhibition assays with MUXF and screened for IgE to a panel of recombinant latex allergens. Fourteen-day serologic follow-up was available for a subset of individuals. RESULTS: Moderate to heavy drinkers displayed an increased prevalence of IgE to CCD markers. The presence of CCD specific IgE was closely associated with latex IgE reactivity. Inhibition studies and the absence of reactivity to nonglycosylated recombinant latex allergens indicated CCD interference in latex IgE determinations. Serum levels of specific IgE decreased with alcohol abstention. CONCLUSIONS: In this population, alcohol consumption is associated with an increased prevalence of IgE reactivity to natural rubber latex due to CCD interference. The use of nonglycosylated recombinant allergens and inhibition assays may help to minimize CCD interference in populations in which IgE to CCDs is common.

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[153] - Kubelka V, Altmann F, Staudacher E, Tretter V, Marz L, Hard K, et al. Primary structures of the N-linked carbohydrate chains from honeybee venom phospholipase A2. Eur J Biochem 1993;213:1193-1204

The N-linked carbohydrate chains of phospholipase A2 from honeybee (Apis mellifera) were released from glycopeptides with peptide-N-glycanase A and reductively aminated with 2-aminopyridine. The fluorescent derivatives were separated by size-fractionation and reverse-phase HPLC, yielding 14 fractions. Structural analysis was accomplished by compositional and methylation analyses, by comparison of the HPLC elution patterns with reference oligosaccharides, by stepwise exoglycosidase digestions which were monitored by HPLC, and, where necessary, by 500-MHz 1H-NMR spectroscopy. Ten oligosaccharides consisted of mannose, N-acetylglucosamine and fucose alpha 1-6 and/or alpha 1-3 linked to the innermost N-acetylglucosamine. Four compounds, which comprised 10% of the oligosaccharide pool from phospholipase A2, contained a rarely found terminal element with N-acetylgalactosamine. The structures of the 14 N-glycans from honeybee phospholipase A2 can be arranged into the following three series: [formula: see text]

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[154] - Godfrin D, Sénéchal H, Haddad I, Sutra JP, Chardin H, Desvaux FX, et al. Caractérisation par électrophorèse 2D et spectrométrie de masse des allergènes de pollen et de graines de colza (Brassica napus). Rev Fr Allergol Immunol Clin 2007;47:270

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[155] - Chardin H, Sénéchal H, Wal JM, Desvaux FX, Godfrin D, Peltre G. Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization. Clin Exp Allergy 2008;38:680-685

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues . RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

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[156] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[157] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[158] - Sturm GJ, Schuster C, Kranzelbinder B, Wiednig M, Groselj-Strele A, Aberer W. Asymptomatic Sensitization to Hymenoptera Venom Is Related to Total Immunoglobulin E Levels. Int Arch Allergy Immunol 2009;148:261-264

BACKGROUND: The detection of specific serum immunoglobulin E (sIgE) to Hymenoptera venoms is an established diagnostic tool to diagnose insect venom hypersensitivity. However, the specificity of sIgE detection is a debated issue . METHODS: In 145 subjects, total IgE (tIgE) and sIgE to Hymenoptera venoms as well as sIgE to rapeseed as a marker of cross-reactive carbohydrate determinants were measured. In addition, an atopy score was determined for each patient. We looked for a possible association between tIgE and the presence of sIgE in subjects with a negative history of large local or systemic reactions to insect stings . RESULTS: Fifteen of 65 subjects (23.1%) with low levels of tIgE (<50 kU/l) had sIgE for bee or wasp venom, and 23 of 47 subjects (48.9%) with a tIgE from 50 to 250 kU/l showed sIgE. The highest rate of asymptomatic sensitization (22 of 33; 66.7%) was found in patients with tIgE levels higher than 250 kU/l. Median sIgE was approximately 4.8 times higher in subjects with tIgE levels above 250 kU/l than in those with tIgE levels <50 kU/l. Interestingly, a significant difference in median tIgE was recorded between individuals with and without sIgE to rapeseed [776.5 kU/l (25, 75% percentiles: 252.5, 2,000.0) vs. 50.5 kU/l (20.1, 172.0), respectively; p < 0001] . CONCLUSION: Specific antibodies are frequently seen in individuals with high tIgE, but appear to be largely irrelevant in clinical terms. This might lead to misdiagnosis in persons with an inconclusive sting history.

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[159] - Focke M, Hemmer W, Hayek B, Gotz M, Jarisch R. Identification of allergens in oilseed rape (Brassica napus) pollen. Int Arch Allergy Immunol 1998;117:105-112

BACKGROUND: Pollen from oilseed rape (OSR), Brassica napus, an increasingly cultivated oilplant from the Brassicaceae, has been recognized as a potential cause of allergic sensitization. Allergens have been hardly investigated. METHODS: We characterized IgE binding proteins in OSR pollen by immunoblot, immunoblot inhibition and specific monoclonal antibodies using sera from 89 patients sensitized to OSR. RESULTS: Two low-molecular-weight allergens of 6/8 kD and 14 kD as well as a high molecular-weight cluster (27-69 kD) comprising six cross-reactive peptides could be identified. The three allergens were recognized by 50, 34 and 80% of patients, respectively. Immunoblot IgE binding to OSR could be totally inhibited by rye pollen and moderately by birch pollen (6/8 and 14 kD) while mugwort had little effect. An anti-profilin-specific monoclonal antibody bound specifically to a 14-kD protein in OSR. Binding to the 6/8-kD rape allergen could be effectively inhibited by rAln g 2, a calcium-binding protein from alder. Periodate treatment led to a significant reduction in IgE binding to the 27 to 69-kD OSR allergens indicating that carbohydrate determinants are involved in IgE binding. OSR proteins were capable to quench IgE binding to timothy grass pollen proteins of >/=60 kD suggesting that grass pollen group 4 allergens cross-react with the 27 to 69-kD cluster in OSR. CONCLUSIONS: The data demonstrate that OSR pollen is allergenic and indicate that the identified allergens represent cross-reacting homologues of well-known pollen allergens, i.e. calcium-binding proteins, profilins, and high-molecular-weight glycoproteins. Via cross-reactivity, exposure to OSR pollen may be a prolonging and aggravating factor in underlying birch and grass pollen allergy.

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[160] - Flinterman AE, Hoekstra MO, Meijer Y, van Ree R, Akkerdaas JH, Bruijnzeel-Koomen CA, et al. Clinical reactivity to hazelnut in children: Association with sensitization to birch pollen or nuts ? J Allergy Clin Immunol 2006;118:1186-1188

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[161] - van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, et al. beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451-11458

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

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[162] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[163] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[164] - Jappe U, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Specific IgE-antibody reaction to cross-reactive carbohydrate determinants (CCD) in hymenoptera venoms: comparison of two different in vitro-tests. Allergy 2007;62(suppl. 83):50

Background: IgE-positivity for both hymenoptera venoms is mostly due to cross-reactive carbohydrate determinants (CCD). This study evaluated CCD-IgE-binding intensity in two in vitro-IgE-detection-methods. Patients and methods: 56 patients with stinging insect allergy and anti-CCD-sIgE were investigated with further CCD-allergens: timothy grass (Phl p), natural rubber latex (NRL), bromelain (BRO), and horse radish peroxidase (HRP) in 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (DPC Biermann, Germany), based on liquid phase technology. The new CCD-ImmunoCAP, a MUXF3-type carbohydrate and ascorbate oxidase (DPC), also a glykan-containing allergen, were used as well. The sera were further investigated via hymenoptera venom- immunoblot (DPC). Results: All sera had sIgE to both insect venoms. In CAP FEIA, 47/56 (83.9%) had sIgE to NRL, 52/56 (92.9%) to Phl p, 50/56 (89.2%) to BRO, 50/56 (89.2%) to HRP and 48/56 (85.7%) to CCD-CAP. In Immulite, 30/56 (53.6%) had sIgE to NRL, 40/56 (71.4%) to BRO, 46/56 (82.1%) to HRP, 40/56 (71.4%) to Phl p, and 45/56 (80.4%) to ascorbate oxidase. In CAP FEIA sIgE-concentrations to BRO and HRP, showed a strong Pearson correlation with 0.9994 (p<0.0001). Although they differed individually in Immulite, the Pearson correlation was still good (0.6953; p=0.01). However, via Immulite, HRP-sIgE could be measured in 8 subjects, where no BRO-sIgE were detected, whereas only 2 individuals had sIgE to BRO and none to HRP. Regarding the class-grading, the differences between BRO and HRP in Immulite measured were 3 classes in 3, 2 classes in 20, and 1 class in 16 subjects. This phenomenon is dependent on BRO-DPC and is mirrored by a non-significant Pearson correlation for BRO-DPC/BRO-CAP (0.488; p=0.56), BRO-DPC/HRP-CAP (0.497; p=0.51) and a significant Pearson correlation for HRP-DPC/BRO-CAP (0.8905; p<0.0001). In immunoblot, all sera reacted with the glycosylated allergens. As in CAP FEIA the IgE-binding to the CCD structures was detected in more sera and was comparable with all the tested CCD allergens, it might be assumed that these epitopes are equally presented on the different ImmunoCAPs. The differences in the IgE-binding to BRO and HRP in the Immulite E 2000 system might be due to their presentation in these allergens in liquid phase. However, HRP seems to be the favourable screening allergen in cases of IgE-double-positivity in insect venom allergic patients.

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[165] - Unger E, Holtz A, Andersson K, Sjolander S, Holmquist I, Poorafshar M. Tools for studying CCD (Cross-reactive carbohydrate determinants). Allergy Clin Immunol Int 2005;17(Suppl. 1):192

Background: In diagnosis of allergy, there is a growing interest concerning the occurrence and relevance of IgE antibodies for a limited set of glycan structures, cross-reactive carbohydrate determinants (CCD). In order to study the complexities of CCD-reactivity in patients, well-defined tools, such as standardised inhibitory reagents and a CCD specific ImmunoCAP˙ should be helpful. We present here some preliminary results with such reagents. It has been demonstrated that specific carbohydrate structures, restricted to plants, insects and some molluscs, give rise to IgE antibodies. There are two main problems with this IgE-response to CCD-structures, relating to prevalence and clinical relevance. The widespread occurrence of CCD in plants and invertebrates is a basis for a high degree of cross-reactivity. Furthermore, the CCD-specific IgE is readily detected in vitro but its clinical importance is often uncertain. Based on the literature a protein in pineapple stem, bromelain, carries the most general CCD-glycan structure: Mana1-6(Xylb1-2)Manb1-4GlcNAcb1-4(Fuca1-3)GlcNAc or in short: MUXF3 Methods: CCD-reagents of MUXF3-type and of MMXF3-type (a glycan found in horseradish peroxidase, with one extra Man compared to MUXF3) were prepared. The MUXF3-reagent was coupled to an ImmunoCAP matrix. Potentially CCD-reactive human sera were selected by concurrent IgE responses to a number of CCD containing allergens, using the ImmunoCAP system. We made inhibition studies by mixing sera with CCD-reagent or buffer, comparing the resulting responses for different allergen ImmunoCAP. Results: For many of the selected sera the IgE response to a number of allergen extracts containing CCD was completely or partly inhibited by the CCDreagents. The two inhibitory reagents differed in efficiency, while for some sera they were equally inhibitory, for other sera and allergens the MUXF3-type was far more efficient. The experimental CCD ImmunoCAP was tested using a number of sera with or without confirmed CCD-reactivity, responses were reproducible, completely inhibitable by the MUXF3-type reagent, and consistent with the inhibition patterns for other allergens. Conclusion: Preliminary studies with inhibitory solutions and an experimental CCD ImmunoCAP based on the MUXF3-type carbohydrate support the suggested generality of this carbohydrate, and the usefulness of these reagents for studying the complexities of CCD.

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[166] - Mahler V, Gutgesell C, Valenta R, Fuchs T. Natural rubber latex and hymenoptera venoms share Immunoglobin E-epitopes accounting for cross-reactive carbohydrate determinants. Clin Exp allergy 2006;36:1446-1456

BACKGROUND: Epidemiological data on the prevalence and risk factors of latex sensitization have suggested a significant association between latex sensitization and the presence of one or more positive skin prick test responses to aeroallergens, food allergens and to one or more insect venoms. Xylose and core 3-fucose are typical complex glycans in plants and are foreign to mammals. Plant N-glycans and insect N-glycans may cross-react in humans . OBJECTIVE: The aim of our study was to investigate whether there are cross-reactive IgE-binding structures in natural rubber latex (NRL) and hymenoptera venoms and to examine their nature . METHODS: Hundred and twenty-five consecutive patients with insect venom allergy were screened for coincidental latex-specific IgE. IgE-binding components in the venoms from Apis mellifera and/or vespula species and in NRL extracts were characterized by IgE-immunoblotting to the natural allergen sources and determination of specific IgE to recombinant allergens. Cross-reactive components were investigated by inhibition experiments. The involvement of carbohydrates in the constitution of cross-reactive IgE-epitopes was further examined by specific IgE-binding to cross-reactive carbohydrate determinants (CCD) in bromelain and horseradish peroxidase as well as by periodate treatment . RESULTS: NRL glove extracts inhibited patients' serum IgE-binding to venom allergens. Vice versa, the IgE-binding to latex glove extracts could be inhibited by pre-incubation with the insect venoms. Specific IgE-binding to recombinant latex allergens was absent, whereas the cross-reactive IgE-epitopes were sensitive to periodate treatment and specific IgE to CCD (MMXF and MUXF type) could be detected . CONCLUSION: Insect venoms and NRL share IgE-binding CCD that may be responsible for positive serological test results to NRL in patients with insect venom allergy. This copositivity occurs frequently (13.6%) among venom-allergic individuals and did not elicit clinical symptoms upon contact to latex in the patients examined. In contrast, true cosensitization to insect venoms and NRL allergens can occur and may not be missed.

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[167] - Raulf-Heimsoth M, Rihs HP, Rozynek P, Cremer R, Gaspar A, Pires G, et al. Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis). Clin Exp Allergy 2007;37:1657-1667

BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles . OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis . METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology . RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs . CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.

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[168] - Mari A. IgE to Cross-Reactive Carbohydrate Determinants: Analysis of the Distribution and Appraisal of the in vivo and in vitro Reactivity. Int Arch Allergy Immunol 2002;129:286-295

Background: IgE to cross-reacting carbohydrate determinants has already been described by several authors, but their function and distribution are still a matter of debate. In previous studies we showed how the presence of IgE to bromelain could be a useful and simple marker of the presence of IgE to carbohydrate epitopes. Methods: A survey of 1,831 subjects with a suspected allergic respiratory disease has been carried out by detecting IgE to bromelain. Data were analysed on the basis of demographical and allergological parameters. To find out whether a glycoprotein is capable of triggering an allergic reaction, 1,076 subjects were also skin tested with several purified molecules bearing carbohydrate side chains differing in number, composition and complexity. Results: An overall prevalence of 23% of positive IgE to cross-reacting carbohydrate determinants was recorded. Prevalence varied when subsets of non-allergic (5%), non-pollen-allergic (10%), and pollen-allergic (31%) subjects were considered. Prevalence further increased in subsets with multiple pollen sensitization (71%), and with a previous pollen immunotherapy course (46%), whereas minor differences were found in gender and age distribution. Almost all the allergenic extracts recorded negative in the skin test gave a positive IgE test in vitro. A higher correlation was found mainly with plant-derived allergenic extracts, whereas a lower one was recorded with mites and fungi. Horseradish peroxidase was the only glycoprotein capable of exerting a positive skin test in 21% of the subjects with IgE to cross-reacting carbohydrate determinants, 80% of them having IgE to the HRP molecule. Conclusions: IgE to cross-reacting carbohydrate determinants are common among the allergic population and the binding to skin test negative allergenic extracts further confirms their poor biological activity. Further studies on horseradish peroxidase should be carried out to define the role of the glycan side chains in its allergenic activity.

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[169] - Jappe U, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Specific IgE-antibody reaction to cross-reactive carbohydrate determinants (CCD) in hymenoptera venoms: comparison of two different in vitro-tests. Allergy 2007;62(suppl. 83):50

Background: IgE-positivity for both hymenoptera venoms is mostly due to cross-reactive carbohydrate determinants (CCD). This study evaluated CCD-IgE-binding intensity in two in vitro-IgE-detection-methods. Patients and methods: 56 patients with stinging insect allergy and anti-CCD-sIgE were investigated with further CCD-allergens: timothy grass (Phl p), natural rubber latex (NRL), bromelain (BRO), and horse radish peroxidase (HRP) in 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (DPC Biermann, Germany), based on liquid phase technology. The new CCD-ImmunoCAP, a MUXF3-type carbohydrate and ascorbate oxidase (DPC), also a glykan-containing allergen, were used as well. The sera were further investigated via hymenoptera venom- immunoblot (DPC). Results: All sera had sIgE to both insect venoms. In CAP FEIA, 47/56 (83.9%) had sIgE to NRL, 52/56 (92.9%) to Phl p, 50/56 (89.2%) to BRO, 50/56 (89.2%) to HRP and 48/56 (85.7%) to CCD-CAP. In Immulite, 30/56 (53.6%) had sIgE to NRL, 40/56 (71.4%) to BRO, 46/56 (82.1%) to HRP, 40/56 (71.4%) to Phl p, and 45/56 (80.4%) to ascorbate oxidase. In CAP FEIA sIgE-concentrations to BRO and HRP, showed a strong Pearson correlation with 0.9994 (p<0.0001). Although they differed individually in Immulite, the Pearson correlation was still good (0.6953; p=0.01). However, via Immulite, HRP-sIgE could be measured in 8 subjects, where no BRO-sIgE were detected, whereas only 2 individuals had sIgE to BRO and none to HRP. Regarding the class-grading, the differences between BRO and HRP in Immulite measured were 3 classes in 3, 2 classes in 20, and 1 class in 16 subjects. This phenomenon is dependent on BRO-DPC and is mirrored by a non-significant Pearson correlation for BRO-DPC/BRO-CAP (0.488; p=0.56), BRO-DPC/HRP-CAP (0.497; p=0.51) and a significant Pearson correlation for HRP-DPC/BRO-CAP (0.8905; p<0.0001). In immunoblot, all sera reacted with the glycosylated allergens. As in CAP FEIA the IgE-binding to the CCD structures was detected in more sera and was comparable with all the tested CCD allergens, it might be assumed that these epitopes are equally presented on the different ImmunoCAPs. The differences in the IgE-binding to BRO and HRP in the Immulite E 2000 system might be due to their presentation in these allergens in liquid phase. However, HRP seems to be the favourable screening allergen in cases of IgE-double-positivity in insect venom allergic patients.

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[170] - Magne H, Boyer-Hoenner C, Ughetto S, Goutte B, Fauquert JL, Tridon A. IgE anti CCD chez des enfants polliniques ayant des IgE positives au latex. Rev Fr Allergol Immunol Clin 2007;47:265

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[171] - Jappe U, Hoffmann M, Huebsch-Mueller C, Enk A, Raulf-Heimsoth M. Specific IgE-antibody reaction to cross-reactive carbohydrate determinants (CCD) in hymenoptera venoms: comparison of two different in vitro-tests. Allergy 2007;62(suppl. 83):50

Background: IgE-positivity for both hymenoptera venoms is mostly due to cross-reactive carbohydrate determinants (CCD). This study evaluated CCD-IgE-binding intensity in two in vitro-IgE-detection-methods. Patients and methods: 56 patients with stinging insect allergy and anti-CCD-sIgE were investigated with further CCD-allergens: timothy grass (Phl p), natural rubber latex (NRL), bromelain (BRO), and horse radish peroxidase (HRP) in 1. CAP FEIA, based on allergens coupled to a solid ImmunoCAP matrix (Phadia, Sweden) and 2. Immulite E 2000 (DPC Biermann, Germany), based on liquid phase technology. The new CCD-ImmunoCAP, a MUXF3-type carbohydrate and ascorbate oxidase (DPC), also a glykan-containing allergen, were used as well. The sera were further investigated via hymenoptera venom- immunoblot (DPC). Results: All sera had sIgE to both insect venoms. In CAP FEIA, 47/56 (83.9%) had sIgE to NRL, 52/56 (92.9%) to Phl p, 50/56 (89.2%) to BRO, 50/56 (89.2%) to HRP and 48/56 (85.7%) to CCD-CAP. In Immulite, 30/56 (53.6%) had sIgE to NRL, 40/56 (71.4%) to BRO, 46/56 (82.1%) to HRP, 40/56 (71.4%) to Phl p, and 45/56 (80.4%) to ascorbate oxidase. In CAP FEIA sIgE-concentrations to BRO and HRP, showed a strong Pearson correlation with 0.9994 (p<0.0001). Although they differed individually in Immulite, the Pearson correlation was still good (0.6953; p=0.01). However, via Immulite, HRP-sIgE could be measured in 8 subjects, where no BRO-sIgE were detected, whereas only 2 individuals had sIgE to BRO and none to HRP. Regarding the class-grading, the differences between BRO and HRP in Immulite measured were 3 classes in 3, 2 classes in 20, and 1 class in 16 subjects. This phenomenon is dependent on BRO-DPC and is mirrored by a non-significant Pearson correlation for BRO-DPC/BRO-CAP (0.488; p=0.56), BRO-DPC/HRP-CAP (0.497; p=0.51) and a significant Pearson correlation for HRP-DPC/BRO-CAP (0.8905; p<0.0001). In immunoblot, all sera reacted with the glycosylated allergens. As in CAP FEIA the IgE-binding to the CCD structures was detected in more sera and was comparable with all the tested CCD allergens, it might be assumed that these epitopes are equally presented on the different ImmunoCAPs. The differences in the IgE-binding to BRO and HRP in the Immulite E 2000 system might be due to their presentation in these allergens in liquid phase. However, HRP seems to be the favourable screening allergen in cases of IgE-double-positivity in insect venom allergic patients.

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[172] - Guilloux L, Morisset M, Codreanu F, Parisot L, Moneret-Vautrin DA. Peanut Allergy Diagnosis in the Context of Grass Pollen Sensitization for 125 Patients: Roles of Peanut and Cross-Reactive Carbohydrate Determinants Specific IgE. Int Arch Allergy Immunol 2009;149:91-97

BACKGROUND: In vitro testing for food allergy may yield clinically irrelevant results due to cross-reactive carbohydrate determinants (CCD) specific immunoglobulin E (sIgE) induced by pollen exposure. The performances of 2 in vitro methods were evaluated for peanut sIgE measurement in patients allergic to grass pollen with or without subsequent allergy to peanuts. The correlation between clinically irrelevant peanut sIgE and the presence of CCD sIgE was investigated . METHODS: In vitro measurement of peanut sIgE was performed using the Pharmacia ImmunoCap system Radio Immuno Assay (RIA) and the Immulite 2000 3gAllergy system. Discrepancies between in vitro results and peanut allergy diagnosis were evaluated by measurement of CCD sIgE using bromelain and ascorbic acid oxydase (AAO) . RESULTS: The sensitivity was 100% with both systems for the diagnosis of allergy to peanut (58 patients), nevertheless the specificity obtained with Immulite (73%) was better than that obtained using ImmunoCap (46%) in patients who were not allergic to peanuts, but who had a grass pollen allergy (n = 41). In 22 out of 41 patients who presented clinically irrelevant peanut sIgE results using ImmunoCAP, CCD sIgE was detected in 72% of the cases by bromelain and in 86% by AAO. In 11 patients out of 41 who presented irrelevant peanut sIgE results using Immulite, CCD sIgE was detected in 81% of the cases by bromelain and in 100% by AAO . CONCLUSION: The Immulite 2000 system had better specificity than the ImmunoCap system for accurate diagnosis of peanut allergy in patients allergic to grass pollen. CCD sIgE was identified in most of the false-positive peanut sIgE results.

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[173] - Coutinho V, Vidal C, Garrido M, Gude F, Lojo S, Linneberg A, et al. Interference of cross-reactive carbohydrates in the determination of specific IgE in alcohol drinkers and strategies to minimize it: the example of latex. Ann Allergy Asthma Immunol 2008;101:394-401

BACKGROUND: Cross-reactive carbohydrate determinants (CCDs) are N-glycans in plant and invertebrate proteins that interfere with specific IgE determinations. The prevalence of IgE to Man2XylFucGlcNAc2 (MUXF), the CCD from bromelain, may be increased in heavy drinkers. OBJECTIVE: To further investigate the relationship of alcohol consumption to CCD specific IgE. Latex was used as an example for investigating CCD interference with in vitro allergy testing and how to minimize the interference by using nonglycosylated recombinant allergens and inhibition assays. METHODS: We determined the levels of IgE to CCD markers (MUXF and ascorbate oxidase) and natural rubber latex in 270 adults without a history of latex allergy (73 abstainers or occasional drinkers, 76 light drinkers, 47 moderate drinkers, and 74 heavy drinkers). In cases with latex reactivity, we performed inhibition assays with MUXF and screened for IgE to a panel of recombinant latex allergens. Fourteen-day serologic follow-up was available for a subset of individuals. RESULTS: Moderate to heavy drinkers displayed an increased prevalence of IgE to CCD markers. The presence of CCD specific IgE was closely associated with latex IgE reactivity. Inhibition studies and the absence of reactivity to nonglycosylated recombinant latex allergens indicated CCD interference in latex IgE determinations. Serum levels of specific IgE decreased with alcohol abstention. CONCLUSIONS: In this population, alcohol consumption is associated with an increased prevalence of IgE reactivity to natural rubber latex due to CCD interference. The use of nonglycosylated recombinant allergens and inhibition assays may help to minimize CCD interference in populations in which IgE to CCDs is common.

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[174] - Jappe U, Raulf-Heimsoth M, Hoffmann M, Burow G, Hübsch-Müller C, Enk A. In vitro hymenoptera venom allergy diagnosis: improved by screening for cross-reactive carbohydrate determinants and reciprocal inhibition. Allergy 2006;61:1220-1229

BACKGROUND: Immunoglobulin (Ig) E-double positivity for honeybee (HB) and yellow jacket (YJ) venom causes diagnostic difficulties concerning therapeutical strategies. The aim of this study was to clarify the cause and relation of the cross-reactivity in patients with insect venom allergy . METHODS: For this purpose, 147 patients with suspected stinging insect allergy and CAP-FEIA-double positivity were investigated for specific sIgE to additional cross-reactive carbohydrate determinant (CCD)-containing allergens: timothy grass pollen, rape pollen, natural rubber latex (NRL), bromelain, and horseradish peroxidase (HRP). Sera with sIgE to NRL were further investigated with the commercially available recombinant latex allergens. Reciprocal inhibition assays with both venoms and HRP were performed . RESULTS: About 36 of 147 (24.5%) patients had sIgE to both venoms only. However, 111 of 147 (75.5%) additionally reacted to CCD-carrying allergens. 89 of 111 CCD-reactive sera had NRL-sIgE. In cases where inhibition experiments were performed, the NRL-sIgE binding was completely abolished in the presence of HRP. Only nine of 61 sera were positive for at least one recombinant latex allergen; all of them were negative in history and NRL-skin prick test. In 43 sera containing sIgE to CCD, HRP inhibition revealed unequivocal results: In 28 of 43 (65%) an HRP-inhibition >70% of sIgE to one venom occurred, pointing out the relevant venom. In three of 43 sIgE proved to be entirely CCD-specific . CONCLUSIONS: Our data indicate that in cases of IgE positivity to both insect venoms supplementary screening tests with at least one CCD-containing allergen should be performed; HRP being a suitable tool for this test. In addition, subsequent reciprocal inhibition is an essential diagnostic method to specify cross-reacting sIgE results.

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[175] - van Ree R, Aalberse RC. Specific IgE without clinical allergy. J Allergy Clin Immunol 1999;103:1000-1001

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[176] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[177] - Guilloux L, Morisset M, Codreanu F, Parisot L, Moneret-Vautrin DA. Peanut Allergy Diagnosis in the Context of Grass Pollen Sensitization for 125 Patients: Roles of Peanut and Cross-Reactive Carbohydrate Determinants Specific IgE. Int Arch Allergy Immunol 2009;149:91-97

BACKGROUND: In vitro testing for food allergy may yield clinically irrelevant results due to cross-reactive carbohydrate determinants (CCD) specific immunoglobulin E (sIgE) induced by pollen exposure. The performances of 2 in vitro methods were evaluated for peanut sIgE measurement in patients allergic to grass pollen with or without subsequent allergy to peanuts. The correlation between clinically irrelevant peanut sIgE and the presence of CCD sIgE was investigated . METHODS: In vitro measurement of peanut sIgE was performed using the Pharmacia ImmunoCap system Radio Immuno Assay (RIA) and the Immulite 2000 3gAllergy system. Discrepancies between in vitro results and peanut allergy diagnosis were evaluated by measurement of CCD sIgE using bromelain and ascorbic acid oxydase (AAO) . RESULTS: The sensitivity was 100% with both systems for the diagnosis of allergy to peanut (58 patients), nevertheless the specificity obtained with Immulite (73%) was better than that obtained using ImmunoCap (46%) in patients who were not allergic to peanuts, but who had a grass pollen allergy (n = 41). In 22 out of 41 patients who presented clinically irrelevant peanut sIgE results using ImmunoCAP, CCD sIgE was detected in 72% of the cases by bromelain and in 86% by AAO. In 11 patients out of 41 who presented irrelevant peanut sIgE results using Immulite, CCD sIgE was detected in 81% of the cases by bromelain and in 100% by AAO . CONCLUSION: The Immulite 2000 system had better specificity than the ImmunoCap system for accurate diagnosis of peanut allergy in patients allergic to grass pollen. CCD sIgE was identified in most of the false-positive peanut sIgE results.

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[178] - Palomares O, Swoboda I, Villalba M, Balic N, Spitzauer S, Rodríguez R, et al. The Major Allergen of Olive Pollen Ole e 1 Is a Diagnostic Marker for Sensitization to Oleaceae. Int Arch Allergy Immunol 2006;141:110-118

BACKGROUND: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen . METHODS: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1 . RESULTS: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae . CONCLUSIONS: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

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[179] - Ebo DG, Hagendorens MM, Bridts CH, de Clerck LS, Stevens WJ. Sensitization to cross-reactive carbohydrate determinants and the ubiquitous protein profilin: mimickers of allergy. Clin Exp Allergy 2004;34:137-144

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[180] - Mari A, Iacovacci P, Afferni C, Barletta B, Tinghino R, Di Felice G, et al. Specific IgE to cross-reactive carbohydrate determinants strongly affects the in vitro diagnosis of allergic diseases. J Allergy Clin Immunol 1999;103:1005-1011

BACKGROUND: Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population. OBJECTIVE: We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests. METHODS: The involvement of CCDs was studied by specific IgE inhibition by using glycoproteins with a known carbohydrate structure. Direct and inhibition assays were performed by commercially available systems, in-house ELISA, and the immunoblotting technique. The binding to the periodate-oxidated carbohydrate structure of glycoproteins and allergenic extracts was also evaluated. A comparative study between skin test and specific IgE responses to the antigens studied was carried out in 428 consecutive allergic subjects. RESULTS: All the tests performed suggested that cross-reacting carbohydrate epitopes were the cause of false-positive specific IgE results in one of the commercial systems and the high reactivity in all the solid-phase in vitro tests. None of the cross-reacting carbohydrate allergens yielded a positive skin test response. Periodate treatment caused variable degrees of reduction of IgE binding to the different antigens studied, indicating that CCDs played a different role in each of them. About 41% of patients allergic to pollen had specific IgE for a glycoprotein, without a positive skin test response to the same molecule. CONCLUSIONS: CCDs must be taken into account when evaluating the clinical relevance of positive results in in vitro specific IgE assays, at least in the diagnosis of patients with pollen allergy. Commercial systems should be carefully assessed for the ability to detect specific IgE for carbohydrate determinants to avoid false-positive or clinically irrelevant results.

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[181] - Holmquist I, Jortso L, Kober A, Sjolander S. Investigation of cross-reactions and co-sensitisations patterns in cockroach sensitized individuals. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°318

Background: IgE sensitisation to cockroach is often associated with multi-sensitisation to a number of allergens, which may complicate the evaluation of IgE antibody (ab) responses to cockroach. Concurring responses to cockroach, house dust mite, shrimp and bromelain (BR) are often seen. Mite, shrimp and cockroach are known to share the allergen tropomyosin (TM) and cross-reactions have been demonstrated. BR has been used as an indicator of IgE ab to cross-reacting carbohydrate determinants (CCD) and indications of their presence on cockroach allergens has been demonstrated. METHODS: 730 human sera with known IgE ab responses to cockroach were analysed for responses to mite and shrimp, using the ImmunoCAP System. 84 of the sera were analysed for responses to BR and ImmunoCAP RAST-inhibition in 20 sera by the use of a BR based CCD inhibitor was performed. RESULTS: Simultaneous responses to cockroach and mite/shrimp were found in 83% and 67% of the sera, respectively. IgE ab responses to mite were significantly higher than responses to cockroach (p<0.001) although no significant correlation was seen. No significant difference was demonstrated between response levels to shrimp and cockroach, but a strong correlation (p<0.05) was shown. The response patterns indicate that simultaneous responses to cockroach and mite, beside the known cross-reaction to TM, may comprise additional co- or cross-mechanisms. Co-exposure of mite and cockroach has been demonstrated earlier and co-sensitisation therefore appears likely. The response pattern also indicates that simultaneous responses to cockroach and shrimp to a high degree are represented by cross-reactions to, for example, TM. Finally, a significant response to BR was found in 74% of the sera. Inhibition using a CCD inhibitor showed inhibition in 80% of the sera. The results strongly indicate the presence of CCD on cockroach allergens and that cross-reactions of this type seem to occur frequently. CONCLUSION: In this study we describe concurrent IgE responses to cockroach, mite and shrimp, and suggest that they either are due to co-sensitisation (mite) and/or cross-reactions specific for TM (mite and shrimp). We also propose the presence of recurrent cross-reactions to CCD. Awareness of these patterns will facilitate the evaluation of IgE responses to cockroach, and concomitant testing for several allergens and/or components may therefore be a useful tool in the subsequent diagnosis of cockroach allergy.

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[182] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[183] - Pevec B, Pevec MR, Markovic AS, Batista I. IgE cross-reactivity between cockroach, birch pollen, and insect venom allergens. 3rd International Symposium on Molecular Allergology, Salzburg, April 18-20, 2008, Poster n°35

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[184] - Aalberse RC, Koshte V, Clemens JG. Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. J Allergy Clin Immunol 1981;68:356-364

IgE in some human sera reacted with an antigen present in a large number of unrelated foods: potato, spinach, wheat, buckwheat, peanut, honey and others. The antigen, which was periodate-sensitive and heat-stable, was also found in pollen. Even more surprisingly, these antibodies often reacted in vitro with bee and vespid venom and were sometimes apparently induced by Hymenoptera stings

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[185] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[186] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[187] - Jin C, Nitsch S, Hemmer W, Altmann F. Improving allergy diagnosis by removal of CCD-specific IgE from patients' sera. Allergy 2009;64(Suppl. 90):30

Background: Carbohydrate cross-reactive determinants (CCDs) are sugar moieties of glycoprotein which cause cross-reactivity by specific IgE. Many allergens isolated from plants, insects and molluscs are glycoproteins containing core a1,3-fucose, which is the major CCD of non-mammalian glycoproteins. CCD-specific IgE can cause false positive results in in vitro test and leads to misdiagnosis, which further causes unnecessary avoidance and/or inappropriate immunotherapy that may even induce new allergy. CCD-specific IgE has a high prevalence in polysensitized allergic patients. In order to improve the specificity of in vitro diagnosis, we designed and evaluated an adsorption matrix for anti-CCD IgE. Methods: CCD-specific IgE was determined in a large cohort of subjects (n=174) by ELISA using horse radish peroxidase (HRP) as indicator. Allergen-specific IgE was determined by CAP system before and after CCD-adsorber. Results: From 174 sera, 35% contained measurable CCD-specific IgE and 53% contained CCD-specific IgG. In the mono-sensitized group, 24% (17/70) contained CCD-specific IgE, while in the poly-sensitized group this was the case in 41% (43/104) and the amounts of CCD-specific IgE were mostly higher. The CCD-specific IgE was removed by the CCD-adsorber in selected polysensitized sera. Conclusion: As anti-CCD IgE is considered clinically irrelevant, removal of this cross-reactive IgE can be expected to increase the specificity of allergy in vitro diagnosis.

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[188] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[189] - Giroux F, Cano Y, Malandain H. Validation of a simple method to overcome the interference of cross-reactive carbohydrate determinants (CCD) in specific IgE assays. Allergy 2009;64(Suppl. 90):8-9

Background: Some patients develop IgE reactivity to glyco-epitopes, ie. carbohydrate chains present on glycoproteins. This is especially the case for patients allergic to pollens or to Hymenoptera venoms. These glyco-epitopes are often cross-reactive, leading to so-called IgE anti-CCD (Cross-reactive Carbohydrate Determinants). Several studies failed to find clinical relevance for these IgE. But in vitro diagnostic tests do not differenciate between peptide- and glycan-specific IgE (sIgE) and their results can be interfered by the presence of IgE anti-CCD in the patient's serum. We sought to design a serum pretreatment to overcome this drawback. We especially aimed at simplifying the process and minimalizing cost and serum dilution so that the method could be suited to most daily routine situations. METHODS: Serum pretreatment: a reagent made of heat-inactivated bromelain and horseradish peroxidase (HRP) was added to the serum. After a waiting time, sIgE reactivity was directly measured on UniCAP (Phadia). To set up the method we used 2 sera positive to IgE anti- CCD (CAP bromelain >0,35 kU/l) (Eur Ann Allergy Clin Immunol 2007;39:216) and a pool of negative sera (total IgE <2 UI/ml). For validating the method, we selected 40 sera from our serum bank, 13 being positive to IgE anti-CCD. We studied 7 allergen sources (peanuts, white of egg, milk, cat ,dog, mite, cod) and 6 non glycosylated recombinant allergens (rBet v 1, rBet v 2, rPhl p 1, rPhl p 7, rPhl p 12, rFel d 1). RESULTS: The best compromise between the highest capture of IgE anti-CCD and the lowest serum dilution was to mix 4 parts of serum with 1 part of reagent (bromelain and HRP 25 g/l, a.a.). Serum pretreatment did not generate false positive IgE-reactivity in negative sera, nor significantly modified positive results for milk, cat, dog, mite, cod, white of egg, and non glycosylated allergens (n = 54 comparisons). It did not modify peanut results for 7 sera with bromelain <0,5 kU/l, and corrected peanut diagnostic for 7 patients with falsely high peanut sIgE (bromelain 1.5-28 kU/l). CONCLUSION: We think our method could be applied to daily lab routine. Components of the pretreatment reagent are ready-to-use, stable (>1 month, 4°C), and cheap (<0.1 •/test). Serum dilution is minimal so that CCD capture can be performed with IgE reactivity as low as 0,5 kU/ l. The method could prove especially useful to CCD-prone sIgE tests such as peanuts, latex and Hymenoptera venoms.

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Nature des CCD

Quels patients sont susceptibles de posséder des IgE anti-CCD ?

A quoi ressemblent les CCD ?

Existe-t-il des glycannes plus CCD que d’autres ?

Quels glycannes dans quels produits ?

Est-on sûr que les CCD n’ont pas de relevance clinique ?

Les « Alpha-Gal »

Quels outils pour détecter la présence d’IgE anti-CCD ?

Des tests in vitro exempts de réactivité CCD ?

Rubriques

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Origine des réactions croisées