Cross-reactivity in plant food allergy. Clinical impact of Component Resolved Diagnostics (CRD)

Transcription

1 Cross-reactivity in plant food allergy Clinical impact of Component Resolved Diagnostics (CRD)

2 Author: Jan Hed, MD, PhD Ass. Prof., Clinical Immunology Karolinska Institute, Stockholm, Sweden All rights reserved. No part of this publication may be reproduced in any form without the written consent of Phadia AB. Phadia AB, 2009 Design: RAK Design AB, 2009 Printed by: Åtta.45 Tryckeri AB, Solna, Sweden

3 Contents Glossary...4 I. Component Resolved Diagnostics (CRD) a new trend...6 Panallergens important proteins in plant food allergy...8 Relations between some common plants...9 II. Factors important for the clinical expression of allergy...10 Allergen concentration...10 Allergen stability...10 IgE antibody concentration and cross-linkage...10 Polysensitization and multi-allergen exposure...11 IgE antibody specificities...11 III. Plant food allergy...12 PR-10 related plant food allergy...13 Sensitizing plant allergen...13 Clinical considerations...14 Diagnostic considerations...15 Summary PR-10 proteins (Bet v 1 homologues)...15 LTP-related plant food allergy...16 Sensitizing plant allergen...16 Clinical considerations...17 Diagnostic considerations...17 Summary nsltps (PR-14 proteins)...17 Profilin-related plant food allergy...18 Sensitizing plant allergen...18 Clinical and diagnostic considerations...19 Summary Profilins...19 Storage protein-related plant food allergy...20 Sensitizing plant allergen...20 Clinical considerations...21 Diagnostic considerations...21 Summary Storage proteins...21 CCD-related plant food allergy...22 Sensitizing plant allergen...22 Clinical considerations...23 Diagnostic considerations...23 Summary CCDs...23 IV. Identified food components of important plant protein families...24 References...27 Recent reviews related to Plant Food Allergy...27 References related to PR-10 proteins (Bet v 1 homologues)...28 References related to nsltps (PR-14 proteins)...30 References related to Profilins...32 References related to Storage Proteins...35 References related to CCDs...37 Allergen database references...37

4 Glossary Allergen vs. allergen components: allergen is here referring to an allergen source (e.g. birch pollen), while the allergen components are the single allergy provoking proteins. Apiaceae: Bet v 1: Bet v 1 homologues: Bet v 2: CCD: the Apiaceae or Umbelliferae (both names are allowed by the International Code of Botanical Nomenclature, ICBN) is a family of usually aromatic plants with hollow stems. It includes cumin, parsley, carrot, dill, caraway, fennel, and other relatives. the major allergen component of birch pollen (Betula verrucosa). also called PR-10 proteins; Bet v 1 related proteins in other allergens (pollens and foods) with similar structure as Bet v 1 in birch pollen. Birch pollen profilin, regarded as a minor allergen. Cross-reacting Carbohydrate Determinant; allergens are often glycoproteins, i.e. proteins with attached carbohydrate chains (glycans). IgE antibodies to plant CCDs are prone to extensive cross-reactivity, regarded as minor allergens and to have low clinical relevance. Component Resolved Diagnostics IgE antibodies to an allergen mixture of the source material are measured in (CRD) vs. traditional diagnostics: traditional testing, while in CRD IgE antibodies to the single allergen components can be detected. The allergen components have different features and thus may give rise to shifting symptoms and with varying severity. Cross-reaction: Determinant: Epitope: Extract-based test: Fabaceae: Glycan: Glycoprotein: Histamine: Homologous protein: IgE antibodies to one allergen may cross-react to other allergens; the clinical relevance of these antibodies varies due to the individual reactivity of the patient and due to the structural similarity of the allergens in question with the original sensitizing allergen. the amino acid sequence of a protein corresponding to the allergen binding part of the IgE antibody (Fab), epitope is another name. see determinant. a mixture of all allergens available in the source material (e.g. peanut) is coupled to the solid phase of the test, and the test will detect all antibodies in a patient sample directed to this allergen. the Fabaceae (or Leguminosae) family is a large and financially important family of flowering plants, which is commonly known as the legume family, pea family, bean family or pulse family. refers here to the carbohydrate chains of glycoproteins (allergens). MUXF3 is a common plant glycan structure (ImmunoCAP Ro214). allergens are most often glycoproteins, i.e. proteins that contain oligosaccharide chains (glycans) covalently attached to their polypeptide. a vasoactive amine causing some of the symptoms of an allergic reaction. related protein with similar structure. Immediate vs. late phase reaction: an IgE-mediated reaction is typically developing within minutes (immediate) after exposure, while a late phase reaction may occur after hours when cells such as eosinophils have been recruited into the tissue. ImmunoCAP : Invertebrate: In vitro test for measurement of IgE antibodies to allergens, from Phadia AB, Uppsala, Sweden. The most used test in this area and regarded as the gold standard. ImmunoCAP is also available for testing of other immunoglobulins (IgA/IgG) and other clinical areas (e.g. autoimmunity). also known as Protochordata, an animal lacking a vertebral column. Examples are insects, arachnids (e.g. mites) and crustaceans that consist of important allergy provoking glycoproteins with carbohydrate structures (glycans), which may cross-react with IgE antibodies to glycans on plant and food.

5 Linear peptide: Major allergen vs. minor allergen: Mast cell: Monovalent determinant: Multivalent determinant: Native allergen: nsltp: OAS: Panallergen: Peptide: Polysensitization: PR-10 protein: Profilin: Protease: Recombinant allergen: Rosaceae: Sensitization: Storage protein: Systemic reaction: an epitope that is recognized by antibodies by its linear sequence of amino acids, or primary structure. In contrast, most antibodies recognize an epitope that has a specific three-dimensional shape as its protein structure (conformational epitope). if the majority (> 50%) of the tested population reacts to an allergen or an allergen component it is described as a major allergen, while a minor allergen refers to less common reactions. a mast cell (or mastocyte) is a resident cell of several types of tissues and contains many granules rich in histamine and heparin, an effector cell in IgE mediated allergy. a single epitope. multiple epitopes. a native allergen is made from the original allergen source (e.g. peanut), i.e. extracted and/or purified (compare with recombinant allergen below). non-specific Lipid Transfer Protein; a protein family that plays a role in plant defense against fungi and bacteria, major allergen components of fruits from the Rosaceae family and clinically very important. Oral Allergy Syndrome; milder allergy reactions that most often only affect the mouth, the most well-known is the birch-fruit syndrome, caused by birch-related plant foods such as hazelnut, apple, peach and cherry, also called pollen-related food allergy. evolutionarily conserved and widely distributed allergen; ubiquitous component of several complex sources of allergens. IgE antibodies to a panallergen may cross-react with homologous allergens and thus also give rise to symptom in patients. is a short polymer formed from the linking, in a defined order, of a-amino acids. Proteins are polypeptide molecules (or consist of multiple polypeptide subunits). sensitization to several allergens; often related to more severe allergy. also called Bet v 1 homologue, belongs to the pathogenesis-related protein family PR-10, a subfamily of the Bet v 1-related superfamily, shows IgE antibody cross-reactivity between homologous allergens in pollens (Fagales) and various fruits and vegetables (Rosaceae). a protein family of actin-binding proteins involved in cytoskeleton dynamics found in most eukaryotic cells, regarded as a panallergen and showing great homology and cross-reactivity even between distantly related species, is described as a minor allergen with in general lower clinical relevance, but for some allergens (e.g. melon and tomato) profilins seem to be clinically important. enzymes present in the gastrointestinal tract that can destroy/denaturize proteins and thus the allergenicity in sensitive foods such as the Bet v 1-homologous proteins in the Rosaceae family. is a biotechnology produced allergen molecule originally identified from allergen extract. Often expressed in Escherichia coli (E. coli) and usually comparable with its natural templates in structural features and immunobiological properties, except for lacking carbohydrate chains (when produced in E. coli). the Rosaceae or rose family is a large family of plants including genera bearing fruits such as strawberry, raspberry, apple, pear, plum, almond and cherry. IgE antibody production as a result of allergen exposure in a sensitive individual. a heterogeneous group of proteins belonging to two superfamilies, Cupins (7/11 S globulins) and Prolamins (2S albumins), are dominating allergens in seeds, tree nuts and legumes, there seems to be limited cross-reactivity between storage proteins of different foods. a more general and severe allergy reaction that involves several organs and may in the worst scenario end up in an anaphylactic reaction.

6 I. Component Resolved Diagnostics (CRD) a new trend The result of an ImmunoCAP blood test to birch pollen is based on the detection of IgE antibodies to a number of different allergen components in the pollen extract (Figure 1a) coupled to the solid phase in the test. The serum profile of IgE antibodies to those allergen components (Figure 1b) differs, not only between patients but also geographically, due to local variation of allergen exposures. The inherent complexity of allergens and the variation between individual patient profiles of induced IgE antibody specificities makes it difficult to relate extract-based test results to certain clinical phenomenon such as cross-reactivities. However, the use of molecular biology techniques has lately dramatically increased the knowledge and understanding of the clinical relevance of IgE antibody responses to the variety of components in complex allergen mixtures such as pollen and plant food allergen extracts. IgE antibodies to specific single allergen components of native allergen extracts can be used as clinical markers in estimating the clinical risk for specified food allergies. The introduction of ImmunoCAP tests based on single allergen components of native allergen extracts makes it possible to describe a unique component-specific IgE antibody profile of the patient (Figure 2). This increased knowledge will change the diagnostic routines in laboratory medicine and improve the usefulness of blood tests also in the clinical routine and thus the value for the patients. This new diagnostic routine has been described as Component Resolved Diagnostics (CRD). a) Traditional diagnostics b) Component Resolved Diagnostics CRD Figure 1. Different allergen compositions in tests for traditional diagnostics (a) vs. tests for Component Resolved Diagnostics (b).

7 ku /l A Patient 1 ku /l A Patient Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 ku /l A Patient Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 ku /l A Timothy (g6) Patient 4 rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 ku /l A Patient Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 ku /l A Timothy (g6) Patient 6 rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 ku /l A Patient 7 ku A /l Patient Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p Timothy (g6) rphl p 1 rphl p 2 nphl p 4 rphl p 5 rphl p 6 rphl p 7 rphl p 11 rphl p 12 Figure 2. IgE antibody level to timothy pollen extract (Phleum pratense n) in 8 grass pollen allergic patients, compared with their IgE antibody profile to 8 different timothy components (Phl px n). Variation in the IgE antibody profiles influences the clinical expression and reactivity of the patients.

8 Panallergens important proteins in plant food allergy Proteins with similar functions in different plant species may have more or less similar molecular structures. Structural similarities between proteins decline with decreasing degree of biological relation between species. Some of the most well-studied allergen components in plants belong to protein families listed in the table below. They are widely distributed among plants and IgE antibodies to these proteins often show cross-reactivity, i.e. they may detect similarities between allergen molecules from several different sources. The term panallergens is often used for these widely distributed proteins. Protein family Sensitivity to Examples of plant food allergen sources Clinical expression heat & proteases PR-10 proteins Sensitive* Betulaceae: hazelnut* Mainly OAS* (Bet v 1 homologues) Rosaceae: e.g. apple, cherry, peach Apiaceae: e.g. carrot, celery*, fennel, parsley Fabaceae: e.g. peanut*, soybean*, mungbean nsltps (non-specific Stable Betulaceae: hazelnut Systemic reactions Lipid Transfer Proteins) Rosaceae: e.g. apple, cherry, peach common Other: maize, peanut, barley, grape, cabbage Profilins Sensitive Profilins are widely distributed in plants Mainly OAS** (Bet v 2 homologues) Typically: citrus fruits, melon, banana, tomato Storage proteins Stable Kernels/nuts/seeds: e.g. peanut/soybean, tree nuts, Systemic reactions (2S albumins, seeds, cereals common 7S/11S globulins) CCDs (Cross-reactive Stable CCDs are widely distributed in plant food ** Carbohydrate CCD allergenicity has been proposed for celery, Determinants) tomato and zucchini * Bet v 1 homologues in hazelnut, celery, peanut & soybean have been found to be partially heat stable and systemic reactions exist. ** IgE antibodies to profilins and CCDs are in general considered to have less clinical relevance than to other allergen components, but should not be neglected. The probability of cross-reactivity between allergens is a function of both biology and chemistry. Peptide epitopes on native proteins can be very variable depending on the variation in amino acid sequences and complex conformations of the molecules. Therefore, it is unlikely to find cross-reactivity between proteins that are not related to each other (non-homologous), or between similar (homologous) proteins from very distantly related species. This is in great contrast to carbohydrate epitopes on glycoproteins. The carbohydrate epitopes do not show the same variability as the peptide epitopes. Therefore they commonly show IgE antibody cross-reactivity between nonrelated allergens and even very distantly related species such as between plants and stinging insects. The amino acid sequence of profilin is a conserved structure and the epitopes are very similar in a wide range of distantly related plants. This is in contrast to the amino acid sequence of Bet v 1, which shows a high degree of identity in birch-related tree pollens and plant foods, but not to homologous proteins in more distantly related species. Therefore, IgE antibodies to profilin show a much more extended cross-reactivity to different plant food species than IgE antibodies to Bet v 1 homologues.

9 Relations between some common plants Botanical relations Sequence identity between some homologous proteins (%) in comparison to the reference protein for each group (100%)

10 II. Factors important for the clinical expression of allergy Allergen concentration The allergen exposure is the obvious prerequisite for clinical allergic symptoms in a sensitized patient. However, the amount of inflammation induced in the respiratory tract is dependent on the allergen concentration and the duration of exposure. A high concentration of an allergen might induce symptoms immediately, whereas a low concentration can induce a subclinical inflammation without any obvious symptoms. This subclinical inflammation, however, may amplify in the tissue over time and could later on result in clinical symptoms, even on exposure to a very low concentration of the allergen (the iceberg phenomenon). Allergen stability The stability of the allergen molecules in the gastrointestinal tract is important for the clinical expression in plant food allergy. There is a great variability between allergen components with respect to stability to heat and protease digestion mimicking the gastric environment (see page 8). Stable allergens such as nsltps and storage proteins are still functional in the gastrointestinal tract and may result in systemic reactions (urticaria, dyspnea and anaphylaxis) in contrast to Bet v 1 homologues and profilins, which are easily destroyed and mainly give rise to the oral allergy syndrome (OAS). IgE antibody concentration and cross-linkage The concentration of IgE antibodies to an allergen is crucial for triggering clinical symptoms. Most of the IgE antibodies are, however, not circulating in the blood stream, but bound to the effector cells (mast cells) in the tissue. Equilibrium exists between free and cell bound IgE. Therefore, the concentration of IgE antibodies in patient sera reflects the amount of allergen-specific IgE antibodies bound to the cell surface of effector cells. Cross-linkage of cell surface IgE antibodies by allergens is a prerequisite to induce cell activation. The probability of cell activation increases with the number of cross-linked IgE molecules on the cell surface. This indicates that there must be at least two epitopes on the allergen component, which can bind to two different IgE molecules on the cell surface to achieve the activation. It could be two identical (A and C) or two different epitopes (B) on the allergen. In case B, IgE antibodies with different specificities (polysensitization) are needed to result in cross-linking and cell activation. If the patient only has IgE antibodies to one of the epitopes there will not be any cross-linking unless the epitopes are not repetitive. A Epitope 1 Epitope 2 Figure 3. Cross-linking of surface IgE antibodies on a mast cell by different types of allergens (A-C). B C 10

11 This complexity in cross-linking of cell bound IgE antibodies might explain some of the controversy over the clinical relevance of IgE antibodies to some allergens as described below (see page 22 about CCD-related plant food allergy). Polysensitization and multi-allergen exposure As described earlier, cell activation and inflammation are related to the amount of crosslinking of surface bound IgE. It is also known that the risk to develop asthma symptoms is related to the number of IgE antibody specificities in the sensitization profile of the allergic patient. Furthermore, a polysensitized patient has an increased risk for triggering effector cell activation when exposed to several allergens due to an increase in the total amount of cross-linking of IgE on the cell-surfaces. This stresses the importance of describing the sensitizing profile (different IgE antibody specificities) in a patient related to actual allergen exposure since each IgE specificity adds to the cell activation process if the allergen is present. Each IgE antibody specificity adds to the cell activation process if the allergen is present. IgE antibody specificities It is important to be aware of the fact that the concentration of allergen-specific IgE measured by extract-based tests is the sum of IgE-antibodies to different allergen components in the extracts and thus consists of different specificities. This difference in specificity profile is dependent on the individual sensitivity, but also the type of allergen that initially sensitized the patient. To gain more information, especially in food allergy, it is of great value to analyze the separate allergen specificities of the different IgE-antibodies since some components: u are widely distributed and may be involved in IgE antibody cross-reactivity and thus important to be aware of u have a high molecular stability to heat and proteases and will thus immunologically functional in the gastrointestinal tract, giving rise to anaphylaxis (e.g. nsltps and storage proteins) u will easily be destroyed by proteases in the gastric environment (Bet v 1 homologues and profilins) and thus immunologically functional only in the mouth, giving rise to the oral allergy syndrome (OAS) u will easily be destroyed by heat and thus not immunologically functional as cooked (e.g. Bet v 1 homologues and profilins) u are univalent (only one binding site for IgE) with respect to the IgE response of the patient and are thus inefficient to activate effector cells by cross-linking IgE-receptors (some CCD-containing allergens) 11

12 III. Plant food allergy A number of different plant food allergy syndromes have been described based on the plant related foods that trigger the symptoms. Examples of such syndromes are ragweedbanana-melon, birch-fruit-vegetable, birch-mugwort-hazelnut, latex-banana, mugwortcelery-spice syndrome, and so on. New knowledge about diverse sensitization profiles in different geographic areas and cross-reacting allergen components (panallergens) in pollens and plant foods indicates that the syndromes vary a lot and are overlapping. The appearance of polysensitization, when testing for IgE antibodies to different plant foods or pollen allergens, is often due to quite a few defined cross-reacting allergenic components (panallergens) rather than to many different pollen species and plant foods. In this presentation, we prefer to group different plant food allergies according to the allergen component triggering the symptoms: u PR-10 proteins (Bet v 1 homologues) u non-specific Lipid Transfer Proteins (nsltps) u Profilins u Storage proteins u Cross-reacting carbohydrate determinants (CCDs) The list is certainly not complete, but represents the most established and studied allergy triggering plant protein families of today. The number of clinically relevant panallergens will increase with increased knowledge of this complexity. The suggested type of classification used in this presentation also seems to be the current opinion in recent publications. Moreover, it is most likely a good and pedagogical starting point for evaluation and understanding in the clinical practice to predict plant food cross-reactivity and possible related clinical expressions. 12

13 PR-10 related plant food allergy Sensitizing plant allergen The major allergen component Bet v 1 from birch pollen (Betula verrucosa) belongs to a group of plant proteins termed pathogenesis-related protein family number 10 (PR-10). Homologous proteins to Bet v 1 are widely distributed in the plant kingdom. These types of far and wide distributed allergens are described as panallergens. The food reactions seen in birch pollen allergic patients are primarily explained by IgE antibodies to Bet v 1, induced by birch pollen, which cross-react with Bet v 1 homologous proteins in different plant foods. Bet v 1 homologues are primarily localized to the pulp of the fruit, in contrast to nsltps which are localized to the peel, and storage proteins which are localized to the fruit stone (seed/nut/kernel). In the birch-rich areas of northern and central Europe almost all birch pollen allergic patients are sensitized to Bet v 1 and most of them have Bet v 1-IgE antibodies as the only specificity. In southern Europe, where birch trees are uncommon or absent, a positive test to birch pollen often reflects sensitization to Bet v 1 homologues in other trees closely related to birch (e.g. alder, hazel, hornbeam, beech and chestnut) or sensitization to other pollen allergens such as profilins (Bet v 2 homologues) in grass and weed (e.g. mugwort and Parietaria). Table 1. Some identified PR-10 related plant food components (see extended list on page 24-26). [Family] Food allergen PR-10 protein (Bet v 1 superfamily) [Betulaceae] Hazelnut Cor a 1 [Rosaceae] Almond Pru du 1 Apple Mal d 1 Apricot Pru ar 1 Cherry Pru av 1 Peach Pru p 1 Pear Pyr c 1 Raspberry Rub i 1 Strawberry Fra a 1 [Apiaceae] Carrot Dau c 1 Celery Api g 1 Parsley Pet c 1 [Family] Food allergen PR-10 protein (Bet v 1 superfamily) [Fabaceae] Mung bean Vig r 1 Peanut Ara h 8 Soybean Gly m 4 [Asparagaceae] Asparagus [Solanaceae] Bell pepper Bonnet pepper Potato [Anacardiaceae] Mango Aspa o 17kD Cap a 17 kd Cap ch 17 kd STH-2/STH-21 Man i 14 kd [Cucurbitaceae] Melon (musk) Cuc m 3 13

14 Clinical considerations Oral Allergy Syndrome (OAS) 50 to 90% of birch pollen allergic patients have been reported to have some pollen related food allergy. Bet v 1-related food allergy is closely associated with OAS to hazelnut and different plant foods of the Rosaceae family such as apple, peach and cherry. The clinical sensitivity can be expanded to other plant foods, but not without having clinical reactions to hazelnut and Rosaceae fruits as well. Other clinically important food allergies commonly associated with Bet v 1 are reactions to foods from the Apiaceae family (e.g. celery and carrot) and/or to the Fabaceae family (e.g. peanut and soybean). The risk to develop clinical symptom such as OAS is related to the concentration of IgE antibodies to Bet v 1. Birch pollen sensitized patients who remain OAS-free often have low concentrations of IgE antibodies to birch. The Bet v 1 homologous proteins in the Rosaceae family are very sensitive to heat and proteases. Therefore, allergic reactions to Rosaceae fruits in Bet v 1 homologous food allergy are mainly triggered by fresh fruits and primarily restricted to the mouth (OAS) since Bet v 1 will be destroyed in the stomach. This is in great contrast to Rosaceae fruit reactions provoked by IgE antibodies to nsltps, which often give more serious and sometimes life threatening reactions. However, OAS may also occur due to nsltp and allergic reactions from Bet v 1 homologous foods may occasionally be more severe. Patients with Bet v 1 homologous food allergy will neither get symptoms when drinking processed fruit juice or eating heated fruits, also in contrast to the serious Rosacea fruit reactions based on nsltp food allergy. Furthermore, since the nsltps, but not Bet v 1 homologues, are primarily concentrated to the peel of the apple or the fuzz and peel of the peach, peeling will not decrease the symptoms of OAS in Bet v 1-related food allergy. Systemic reactions The Bet v 1 homologous proteins in hazelnut, celery and peanut/soybean have been shown in some studies to be more heat stable than fruits from the Rosaceae family. This may explain why those plant foods sometimes trigger more serious systemic reactions in Bet v 1-related food allergy. During recent years this has been clearly shown with some products based on soybean. Allergic reactions to celery/carrot (Apiaceae) and soybean/peanut (Fabaceae) are also associated with higher concentrations of IgE antibodies to Bet v 1. Difference in triggering immediate and late phase allergic reaction Most of the plant-related allergen determinants described in IgE-mediated food allergy are exposed on the native allergen molecule. Recently it has been shown that birch pollen-related foods may induce worsening of atopic dermatitis two days after food exposure to fresh food. Furthermore, heat treatment of Bet v 1-related food allergens destroys the ability to trigger an immediate symptom such as OAS, whereas the denatured allergen still can trigger the lymphocytes relevant for late clinical reactions. This has been shown to be the case in some patients with atopic dermatitis and might be relevant for other clinical conditions as well. 14

15 The mechanistic background is that heat treatment destroys the native three-dimensional molecular structure, which is important for binding to the IgE molecule and the immediate reaction, but does not affect the linear peptides important for the late phase cellular reaction. This phenomenon has also been proposed as a possible mechanism to explain why serum IgE antibody levels to pollen allergens remain high outside the pollen season. Heat treatment destroys the native three-dimensional molecular structure of PR-10 proteins, which is important for binding to the IgE molecule and the immediate reaction, but does not affect linear peptides important for the late phase cellular reaction. Diagnostic considerations The most important clinical question in food allergy is to identify those patients with a high risk for systemic reactions. As described above, apple allergy (Rosaceae allergy) in northern Europe is in most cases a rather mild food allergic reaction in Bet v 1 sensitized patients and most often restricted to the mouth (OAS). This is in great contrast to apple allergy in southern Europe where it more often triggers a severe systemic reaction based on IgE antibodies to nsltp. This serious form of apple allergy is often seen without any sensitization to common pollen allergens and without clinical symptom of pollen allergy. Such cases of IgE sensitization to nsltp do also exist in the birch-rich areas of northern Europe, but might be disguised by a concomitant positive test to birch pollen and the presence of IgE antibodies to Bet v 1. In birch pollen sensitized patients, with low sensitization to Bet v 1 and high sensitization to species of the Rosaceae family and/or hazelnut, the possibility of sensitization to nsltp (and/or storage proteins) should be suspected with an obvious risk for more severe clinical reactions. Furthermore only about 50% of hazelnut sensitized children in birch-rich areas have a clinical sensitivity to hazelnut and those with systemic reactions often have lower concentration of antibodies to Bet v 1 and are often sensitized to other nuts/peas (tree nuts and peanuts) as well. u It is important to investigate the presence of IgE antibodies to Bet v 1 and nsltp in patients sensitized to Rosaceae food allergens or having clinical symptoms to Rosaceae foods to evaluate the risk for serious systemic reactions u Sensitization or clinical symptoms to Rosaceae plant foods and hazelnut, without broad sensitization to other fruits and vegetables, are typical markers of Bet v 1(or nsltp) but not profilin sensitization u High concentration of IgE antibodies to Bet v 1 might be a risk factor for serious reactions to hazelnut, celery, soybean and peanut, even if these food products are processed to a certain degree Summary PR-10 proteins (Bet v 1 homologues) l Heat labile proteins, primarily localized to the pulp of the fruit l Cooked and processed foods are often tolerated l Often associated with local symptoms such as oral allergy syndrome (OAS) l Commonly related to allergic reactions to fruit and vegetables in northern Europe 15

16 LTP-related plant food allergy Sensitizing plant allergen Non-specific lipid transfer proteins (nsltp) are very stable small molecules widespread in plants. IgE sensitization to nsltp has primarily been described from southern Europe in patients with severe reactions to peach and other fruits belonging to the Rosaceae family and not associated with pollen allergy. The sensitization pathway is not yet completely understood, but an association with mugwort and plane tree has been described. Since nsltp is very concentrated in the fuzz of peach this has also been proposed. The presence of nsltp sensitization in food allergic patients in central and northern Europe and the US is not well investigated but has been described. Table 2. Some identified LTP-related plant food components (see extended list on page 24-26). [Family] Food allergen nsltp [Betulaceae] Hazelnut Cor a 8 [Rosaceae] Almond Pru du 8 Apple Mal d 3 Apricot Pru ar 3 Cherry Pru av 3 Peach Pru p 3 Pear Pyr c 3 Plum Pru d 3 Raspberry Rub i 3 Strawberry Fra a 3 [Asparagaceae] Asparagus Aspa o 1 [Brassicaceae] Cabbage Bra o 3 Turnip Bra r 3 [Apiaceae] Carrot Dau c 3 Parsley Pet c 3 [Euphorbiaceae] Castor bean Ric c LTP [Family] Food allergen [Lamiaceae] Rape seed nsltp Bra n LTP [Vitaceae] Grape Vit v 1 [Asteraceae (Compositae)] Lettuce Lac s 1 Sunflower Hel a 3 [Poaceae (Gramineae)] Barley Hor v 14 Maize Zea m 14 Rice Ory s 14 Spelt Tri s 14 Wheat Tri a 14 [Rutaceae] Lemon Cit l 3 [Fabaceae] Peanut Ara h 9 [Solanaceae] Tomato Lyc e 3 [Juglandaceae] Walnut Jug r 3 16

17 Clinical considerations LTP-related plant food allergy has primarily been described to peach, cherry and apple, plants belonging to the Rosaceae family, but also to hazelnut (Betulaceae). Severe clinical symptoms based on IgE antibodies to nsltp have furthermore been shown for less related food allergens such as maize, peanut, barley, grape, cabbage and more (see page 24-25). In Rosacea allergy, based on nsltp, it is therefore important to be aware of the possibility of severe reactions also to unrelated vegetables and fruits. However, it has recently been shown that nsltp-allergic patients seem to tolerate typical Bet v 1-related plant foods such as potato, and carrot but also banana and melon (profilin-related). The important characteristic of nsltp, which explains its clinical relevance, is the high resistance to heat and proteases. The molecule will not be destroyed by the gastric fluid and will thus be immunologically functional in the gastrointestinal tract with the possibility to trigger systemic reactions such as anaphylaxis, urticaria/angioedema and asthma. The chemical stability of nsltp, in contrast to Bet v1 and profilin, is the explanation why these patients will get symptoms after drinking processed juice or heated food. Since the allergen component is primarily localized to the peel of the fruit patients get less symptoms after eating peeled fruit than patients with allergy based on Bet v 1 homologues or profilins. The prevalence of systemic reactions in apple allergic patient in northern Italy has been described to be 35% compared to less than 10% in apple allergic patients in the birch-rich areas of Europe. However, how common nsltp-sensitization is in northern Europe and the US has not been thoroughly investigated. In two very recent studies from birch endemic areas, IgE-antibodies to nsltp were common in children with provocation verified apple allergy, and in children with objective symptoms to hazelnut challenge. The risk of clinical symptoms is related to the concentration of IgE antibodies to nsltp. Diagnostic considerations The typical nsltp allergic case is a patient from southern Europe with clinical, often severe, symptoms of Rosaceae allergy, but without concomitant pollen allergy. A high degree of sensitization to mugwort or plane tree and a low sensitization to Bet v1 have been described as risk markers for nsltp sensitization in apple allergic patients in northern Italy. In a Spanish population cherry allergy based on IgE sensitization to nsltp was associated with plane tree and mugwort, but not with Parietaria. High homology is found between nsltps from different fruits and suggests that one nsltp such as Pru p 3 from peach can be a marker for genuine Rosaceae allergy. Summary nsltps (PR-14 proteins) l Proteins stable to heat and digestion, primarily localized to the peel of the fruit/vegetable l Reactions also to cooked and processed foods l Often associated with systemic and more severe reactions in addition to Oral Allergy Syndrome (OAS) l Commonly related to allergic reactions to fruit and vegetables in southern Europe 17

18 Profilin-related plant food allergy Sensitizing plant allergen Profilins are small proteins in the cytoplasm of nucleated cells and they are involved in the function of the intracellular fibrils of the cells. Plant profilins are described as minor allergens in most pollen species and plant food allergens and show great homology and cross-reactivity even between distantly related species. The prevalence of profilin sensitization in pollen allergic patients in central and southern Europe has been estimated to 10 to 35%, but seems to be rarer in northern Europe. The prevalence increased to 55% in patient populations with multi-pollen sensitization where grass pollen sensitization is dominating. Table 3. Some identified profilin-related plant food components (see extended list on page 24-26). [Family] Food allergen Profilin [Betulaceae] Hazelnut Cor a 2 [Rosaceae] Almond Pru du 4 Apple Mal d 4 Cherry Pru av 4 Peach Pru p 4 Pear Pyr c 4 Plum Pru d 4 Strawberry Fra a 4 [Asparagaceae] Asparagus Aspa o 4 [Musaceae] Banana Mus xp 1 [Poaceae (Gramineae)] Rice Ory s 12 Wheat Tri a 12 [Solanaceae] Bell pepper Cap a 2 Potato Sola t 8n Tomato Lyc e 1 [Apiaceae] Carrot Dau c 4 Celery Api g 4 Parsley Pet c 2 [Family] Food allergen Profilin [Sapindaceae] Litchi Lit c 1 [Anacardiaceae] Mango Man i 3 [Cucurbitaceae] Melon (musk) Cuc m 2 Watermelon Cit la profilin [Fabaceae] Garden pea Pis s 5 Peanut Ara h 5 Soybean Gly m 3 [Bromeliaceae] Pineapple Ana c 1 [Juglandaceae] Walnut Jug r 5 [Lamiaceae] Rape seed Bra n 8 [Pedaliaceae] Sesame seed Ses i 8 [Asteraceae (Compositae)] Sunflower Hel a 2 18

19 This might reflect geographical differences in pollen exposure where birch pollen sensitization is dominating in northern Europe and grass pollen sensitization in southern Europe. Profilins are also important allergens in mugwort and Parietaria. Olive, plane tree and cypress are other important profilin sources in the Mediterranean areas and so is ragweed in US. Clinical and diagnostic considerations It has been proposed that the use of profilin from just one plant species is enough for testing IgE-sensitization to profilin, due to the large homology and IgE antibody crossreactivity between plant food profilins. Profilins from birch (Bet v 2) and/or timothy grass (Phl p 12) are most widely used. This extended cross-reactivity is not only seen among botanically unrelated pollens but also between pollen and food as well as between pollen and latex. The serum levels of IgE antibodies to profilins have been shown to be related to the number of positive food tests, but also to the expression of clinical symptoms. In patients with reported plant food symptoms, IgE antibodies to profilin have shown highest correlation to IgE sensitization to potato, carrot, celery, buckwheat, paprika and tomato. In some types of food allergy the prevalence of IgE-sensitizations to profilins is very high. Examples of this are tomato allergy with 44% profilin sensitization and citrus fruit allergy with up to 95% profilin sensitization. Symptoms to citrus fruits, melon, banana and/or tomato have been described as clinical markers of profilin sensitization. Profilins are, like Bet v 1 homologues, sensitive to heat and proteases and will thus primarily give rise to OAS as the clinical manifestation of food allergy. However, the most accepted opinion today is that IgE-sensitization to profilin seems to have less clinical relevance than sensitization to Bet v 1, although in some cases profilin sensitization may cause severe reactions. Summary Profilins l Panallergens that show great homology and cross-reactivity even between distantly related plant species l Sensitization seldom associated with clinical symptoms, but may cause demonstrable or even severe reactions in a small minority of patients (e.g. citrus fruits, melon, banana and/or tomato) l Broad plant and plant food sensitization profiles may be explained by profilin-specific IgE antibodies 19

20 Storage protein-related plant food allergy Sensitizing plant allergen The storage protein family consists of a heterogeneous group of proteins belonging to two different superfamilies; Cupins and Prolamins. They are often designated according to their sedimentation rate, 7/8S and 11S globulins belonging to Cupins and 2S albumins belonging to Prolamins. Storage proteins are the dominating allergens in seeds, fruit stones and kernels. The molecule structures are complex and the relation to allergy is not fully understood. IgE sensitization to storage proteins is important in peanut/soybean, tree nut, seed and cereal allergy. The chemical structure of storage proteins is commonly regarded as much more stable to heat and proteases compared to Bet v1 homologues and profilins. There are indications that the 2S albumin is the most stable molecule of the storage proteins and therefore believed to be most clinically important. Bet v 1 homologous proteins, profilins and nsltps are also present in nuts, but in much lower concentrations. Table 4. Some identified storage protein-related plant food components (see extended list on page 24-26). Storage proteins 2S Albumin Vicilin-like Legumin-like [Family] protein/ protein/ Food allergen 7/8S Globulin 11S Globulin [Betulaceae] Hazelnut Cor a 14 Cor a 11 Cor a 9 [Rosaceae] Almond Pru du 2S Pru du 11S Albumin Globulin [Lecythidaceae] Brazil nut Ber e 1 Ber e 2 [Anacardiaceae] Cashew nut Ana o 3 Ana o 1 Ana o 2 Pistachio Pis v 1 Pis v 3 Pis v 2, 5 [Euphorbiaceae] Castor bean Ric c 1, 3 Ric c 2 [Fabaceae] Chick pea Cic a 2S Cic a 11S Albumin Globulin Garden pea Pis s 1, 2 Lentil Len c 1 Lupine Lup a Vicilin Lup a 11S Globulin Peanut Ara h 2, 6, 7 Ara h 1 Ara h 3, 4 Soybean Gly m 2S Gly m 5, Gly m 6 Albumin Gly m BD28K/60K Storage proteins 2S Albumin Vicilin-like Legumin-like [Family] protein/ protein/ Food allergen 7/8S Globulin 11S Globulin [Pedaliaceae] Sesame seed Ses i 1, 2 Ses i 3 Ses i 6, 7 [Asteraceae (Compositae)] Sunflower Hel a 2S Albumin [Brassicaceae] Mustard (white) Sin a 1 Sin a 2 Turnip Bra r 1 [Juglandaceae] Pecan Car i 1 Walnut Jug n 1; Jug n 2; Jug r 4 Jug r 1 Jug r 2 [Lamiaceae] Rape seed Bra n 1 [Polygonaceae] Buckwheat Fag e 10/16 kd Fag e 19 kd Fag e 1 20

21 Clinical considerations IgE sensitization to storage proteins in peanut/soybean, tree nuts or seeds is regarded as an important risk marker for severe systemic reactions. However, there seems to be great geographical differences. In southern Europe IgE sensitization to nsltp is shown to be the most important allergen component for systemic reaction in hazelnut allergy, whereas 11S globulin is described as the most important in the US. LTP is not a storage protein per se, but belongs to the same superfamily (Prolamins) as 2S albumins. The 2S albumin seems to be the dominating allergen in other tree nut, seed and peanut allergies. Polysensitization to different nuts and seeds is common and is a clinical phenomenon to be aware of. The IgE sensitization to storage proteins, and the related cross-reactivity, is complex since sensitization to unrelated nuts and seeds are often seen and increases with age. IgE antibody cross-reactivity can also be seen between tree nuts and peanut, this is a new finding. In a recent study 66% of peanut allergic patients showed sensitization to tree nuts as well. The risk for clinical symptoms is related to the level of IgE sensitization and has been well documented in childhood peanut allergy. In a recent European study it was shown that all patients with proven peanut allergy were sensitized to 2S albumin (Ara h 2) and less frequently to 7/8S globulin (Ara h 1) and 11S globulins (Ara h 3). However, patients polysensitized to the different storage proteins had a more severe disease than those monosensitized to the 2S albumin component and they also had higher levels of peanutspecific serum IgE antibodies. The immunological and biological background for the sensitization to storage proteins is not completely understood. There is no obvious relation between sensitization to pollens and sensitization to storage proteins. In pollen sensitized patients a positive peanut test could be due to IgE antibodies to profilin, Bet v 1 or CCD, but are then often of less clinical relevance in the majority of all cases. Diagnostic considerations Cross-reactivity between peanuts, tree nuts and seeds is common even if they are botanically unrelated. Therefore it is often recommended that IgE antibodies to several nuts should be tested if there is a risk for severe systemic reactions. High levels of IgE antibodies to peanuts have been shown to have a high positive predictive value for clinical relevant peanut allergy in children. Food challenge tests with their clinical risks are omitted in those cases. The important clinical question is to identify patients with increased risk for systemic reaction. A history of anaphylaxis following the ingestion of peanut, sesame seed, sunflower seed, mustard or tree nuts in a patient not sensitized to peach and/or other Rosaceae foods (except almond) suggests hypersensitivity to seed storage proteins. Tests based on nsltp and different storage proteins are crucial to identify possible provoking foods for best treatment/avoidance recommendations. In peanut allergy IgE antibodies to 2S albumin (Ara h 2) seem to have a very high sensitivity and specificity to identify a clinically relevant peanut sensitization. Summary Storage proteins l Proteins stable to heat and digestion, primarily localized to the seed/nut/kernel l Reactions also to cooked and processed foods l Sensitization is regarded as an important risk marker for severe systemic reactions l Cross-reactivity is often seen to unrelated nuts and seeds and seems to increase with age 21

22 CCD-related plant food allergy Sensitizing plant allergen Glycoproteins in plants and in invertebrate animals carry glycans with carbohydrate determinants, which do not exist in mammals. Since these determinants function as foreign epitopes in humans they are highly immunogenic and will give rise to antibodies such as IgE. Glycoproteins can carry two basic types of glycans, i.e. N-linked and O-linked glycans. The two most studied carbohydrate determinants in allergic reactions in humans are based on a fucose or a xylose bound to N-linked glycans. This linkage is not found in mammals. The widespread presence of fucose and xylose on N-linked glycans of plants, but also of invertebrates (to less extent), explains the high degree of cross-reactivity that has been reported for carbohydrate-specific IgE antibodies. These carbohydrate determinants have been termed Cross-reactive Carbohydrate Determinants (CCDs) and the IgE antibodies are termed anti-ccd IgE. Approximately 20% of patients with allergy to pollen have IgE antibodies to pollen allergens with molecular masses higher than 30kDa. A great part of their IgE-binding is dependent on carbohydrate determinants. Allergen A Allergen B Allergen C Carbohydrate epitope Peptide epitope Active Active Inactive Figure 4. Degranulation of mast cells require the binding of at least two epitopes to two adjacent IgE antibody molecules. This cross-linking may be achieved by two peptide epitopes, but also by one glycan and one peptide epitope (A) as well as by two glycan epitopes (B) if IgE antibodies with these specificities co-exist. However, if there is only one epitope (C), or only one IgE antibody specificity in case A, no cross-linking and thus no degranulation will occur. 22

23 Clinical considerations There is an ongoing discussion about the clinical relevance of these CCD-specific IgE antibodies. At the one extreme these antibodies are claimed to be without any clinical relevance and at the other it is suggested that they can induce anaphylactic reactions. One important factor for the clinical relevance of IgE antibodies to CCD is if the allergen components are monovalent or multivalent with respect to the carbohydrate determinant. Monovalent allergen components, such as the major peanut allergen (Ara h 1), cannot cross-link anti-ccd IgE bound on mast cells and thus do not induce histamine release and clinical symptoms. However, if there also exists IgE response to other protein determinants of the CCD-monovalent component a cross-linking is possible and can give rise to histamine release (Figure 4). Therefore, when evaluating the clinical relevance of anti-ccd IgE it is important to identify the sensitizing allergen and thus the risk of concomitant IgE to protein epitopes. It has been shown that patients sensitized to grass pollen develop anti-ccd IgE that also binds to CCD monovalent peanut allergens, but does not give rise to any clinical symptoms. However, anti-ccd IgE is of clinical relevance in proteins with multiple CCD epitopes, as have been shown in allergy to tomato and celery. In conclusion, even if the clinical relevance of anti-ccd IgE has been shown in some cases most researchers agree that it has less clinical relevance than many other allergen components. Diagnostic considerations Since these CCD epitopes are widely distributed in plants and invertebrate animals the corresponding IgE antibodies will give rise to positive in vitro tests (cross-reactivity) to many different and unrelated plant allergens including latex, but also invertebrate animals such as bee/wasp, cockroach, mite and shellfish. When very broad allergen profiles are obtained in allergy testing it can be due to anti-ccd IgE or anti-profilin IgE. Therefore it is important to investigate if CCD antibodies are present. This can be done routinely by IgE antibody testing with bromelain or horseradish peroxidase, but also by the use of a test specific to MUXF3, a common plant glycan structure. A positive in vitro test and a negative skin prick test to the same plant food allergen may indicate presence of non-cross linking CCD-specific IgE antibodies to that allergen. However, this does not exclude cross-linking to other allergens with multivalent CCD epitopes or the presence of concomitant IgE antibodies to peptide epitopes. Summary CCDs l CCD epitopes are widely distributed in plants and invertebrate animals l Sensitization is rarely associated with clinical symptoms, but may cause demonstrable or even severe reactions in a small minority of patients (has been proposed for celery, tomato and zucchini) l Broad allergen sensitization profiles may be explained by CCD-specific IgE antibodies 23