Birch Pollen Allergy: Molecular Characterization and Hypoallergenic Products
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Birch pollen allergy: molecular characterization and hypoallergenic products Martijn F. Schenk Promotoren: Prof. dr. ir. E. Jacobsen Hoogleraar Plantenveredeling, Wageningen Universiteit Prof. dr. L. J. Frewer Hoogleraar Voedselveiligheid en consumentengedrag, Wageningen Universiteit Co‐promotoren: Dr. M. J. M. Smulders Senior onderzoeker, Wageningen UR, Plant Research International Dr. L. J. W. J. Gilissen Senior onderzoeker, Wageningen UR, Plant Research International Promotiecommissie: Prof. dr. A. E. J. Dubois Universiteit van Groningen Prof. dr. ir. H. F. J. Savelkoul Wageningen Universiteit Prof. dr. R. Shepherd University of Surrey, United Kingdom Prof. dr. M. S. M. Sosef Wageningen Universiteit Dit onderzoek is uitgevoerd binnen de onderzoeksschool EPS (Experimental Plant Sciences) Birch pollen allergy: molecular characterization and hypoallergenic products Martijn F. Schenk Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. M. J. Kropff in het openbaar te verdedigen op dinsdag 19 februari 2008 des namiddags te vier uur in de Aula Birch pollen allergy: molecular characterization and hypoallergenic products Martijn F. Schenk PhD thesis, Wageningen University, The Netherlands, 2008 With references – with summaries in English and Dutch ISBN: 978‐90‐8504‐873‐2 Contents List of abbreviations Page 6 Chapter 1 General Introduction Page 7 Chapter 2 The influence of perceived benefits on acceptance of Page 23 GM applications for allergy prevention Chapter 3 Hypoallergenic food products as a novel approach Page 45 to alleviate mild food allergy Chapter 4 Seven different genes encode a diverse mixture of isoforms Page 63 Bet v 1, the major birch pollen allergen Chapter 5 Phylogenetic relationships in Betula (Betulaceae) based on Page 83 AFLP markers Chapter 6 Characterization of PR‐10 genes from eight Betula species Page 101 and detection of PR‐10 (Bet v 1) isoforms in birch pollen Chapter 7 Assessment of allergenicity of birch pollen from Page 125 several species of the genus Betula Chapter 8 General discussion Page 143 References Page 161 Summary Page 181 Samenvatting Page 185 Dankwoord Page 189 Curriculum Vitae Page 191 Education certificate Page 192 List of abbreviations bp. base pair(s) cDNA complementary DNA (based on mRNA template) gDNA genomic DNA ELISA Enzyme‐Linked ImmunoSorbent Assay EST Expressed Sequence Tag GM Genetic Modification GMO Genetically Modified Organism IgE Immunoglobulin E IL‐4, IL‐5, etc. InterLeukin‐4, InterLeukin‐5, etc. LC‐MS Liquid Chromatography‐Mass Spectrometry LC‐MS/MS Liquid Chromatography‐tandem MS LC‐MSE Alternate scanning LC‐MS LTP Lipid Transfer Protein mRNA messenger RNA NJ Neighbor Joining OAS Oral Allergy Syndrome pAB polyclonal AntiBody PR‐10 Pathogenesis‐Related class 10 RAST RadioAllergoSorbent Test RNAi RNA interference REML REsidual Maximum Likelihood SD Standard Deviation SDS‐PAGE Sodium Dodecyl Sulfate‐PolyAcrylamide Gel Electrphoresis SNP Single Nucleotide Polymorphism SPT Skin Prick Test SIT Specific ImmunoTherapy TH‐cells T helper‐cells Q‐TOF Quadrupole‐Time‐Of‐Flight 6 CHAPTER 1 General introduction General context: birch pollen allergy Birch trees grow in the temperate climate zone of the northern hemisphere, and release large amounts of pollen during spring. Birch pollen is a major cause of Type I allergies. The major birch allergen is a pollen protein from the European white birch (Betula pendula syn. B. verrucosa) termed Bet v 1 (Breiteneder et al. 1989; Jarolim et al. 1989). Bet v 1 and other Pathogenesis‐ Related class 10 (PR‐10) proteins constitute the largest group of aeroallergens (Breiteneder et al. 2000). Individuals who have been sensitized to birch pollen are particularly prone to develop Oral Allergy Syndrome (OAS) due to an IgE‐mediated cross‐reaction between Bet v 1 and PR‐10 food proteins (Ferreira et al. 2004; Wensing et al. 2002). As a result, PR‐10 proteins are also among the four most common food allergens (Breiteneder et al. 2000). Allergic diseases have a negative impact on the patient’s quality of life and are associated with high economic costs (Meltzer 2001; UCB 1997). Consequently, allergy to birch and OAS represent relevant targets for prevention. Genetic Modification (GM) can be applied to reduce the allergenicity of the plants involved (Bhalla et al. 2004; Le et al. 2006). However, developments which apply this technique should take societal concerns about GM into account (Frewer et al. 2004; Zechendorf 1994). Before describing the main aims of this thesis, an overview is presented which summarizes current knowledge regarding the prevalence of hay fever, the genetic background of the Bet v 1 allergens, OAS, potential prevention strategies and societal acceptance of different applications of GM. Hay Fever Allergic disorders affect a substantial number of people in westernized countries (Aberg et al. 1995; Wuthrich et al. 1995). Among the existing allergic disorders, hay fever (medical term: seasonal allergic rhinitis) is the most common disorder, affecting 11‐19% of the population in Western European countries (UCB 1997). Symptoms are experienced periodically, during the flowering period of various pollen producing plants. Typical hay fever symptoms are sneezing, rhinorrhea and a blocked nose, while itchy eyes (conjunctivitis), throat and ears are other symptoms that may occur. The diagnosis of hay fever may be difficult, because of similarities with non‐atopic complaints. Hay fever can be defined on symptom criteria or pathophysiological characteristics, such as the detection of specific IgE by a positive RAST or Skin Prick Test (SPT). A combination of both symptom criteria and RAST/SPT leads to the most reliable diagnosis (UCB 1997). Allergic diseases have a genetic basis; a history of atopy in the family has been identified as an important risk factor (Jõgi et al. 1998; Rönmark et al. 2003). The occurrence of allergic diseases also depends on exposure to allergens and environmental or life‐style factors. 7 CHAPTER 1 These life‐style factors may explain the strong increase in hay fever which has been observed during the last decades (Wuthrich et al. 1995). To substantiate prevention strategies towards hay fever, knowledge on the basic mechanisms underlying allergic disorders is required. An allergic reaction is preceded by a sensitization phase, in which a T‐cell response is directed against a particular protein. Eventually, this leads to the production of IgE that is directed towards this protein, which is then considered an allergen. T‐ helper (TH) cells play a central role in this process. TH cells are subdivided according to the cytokines they produce. TH1 cells secrete, among others, interferon‐gamma when stimulated with viruses or intracellular bacteria. By contrast, TH2 cells secrete cytokines such as Interleukin‐4 (IL‐4), IL‐5 and IL‐13 when stimulated with allergens. These interleukins stimulate, among others, IgE production by B‐cells. IgE is then bound to receptors on the surface of mast cells. Mast cells are present in several tissue types, such as the nasal mucosa. Upon renewed allergen contact, the allergen binds to the specific IgE, causing the mast cells to release large amounts of histamine. Immediate clinical allergy symptoms are the result. The ‘hygiene hypothesis’ (Strachan 1989) proposes that increased affluence has lead to increased domestic hygiene and reduced incidence of infections. Infections in infants would stimulate the TH2‐directed immune system in newborns to mature and develop into a TH1‐directed immune system. A lack of infections causes an imbalance in the TH1/TH2 responses, which would induce an immune system that is skewed towards a TH2 response and consequently results in a higher risk for developing allergies (Yazdanbakhsh et al. 2002). Socio‐economic factors that relate to an increased affluence, such as lifestyle (Alm et al. 1999), increasing use of antibiotics and vaccination (Alm et al. 1997), and decreasing family size (Von Mutius et al. 1994) correlate with the increased prevalence of allergies. A childhood lived in a farm environment appears to protect against sensitization (Horak et al. 2002; Kilpeläinen et al. 2002; Riedler et al. 2000). It has recently been suggested that the lack of regulatory T‐cells, resulting from a low pathogen and parasite burden, causes a deficit in the regulatory network, and thereby underlies the imbalance in the immune system (Yazdanbakhsh et al. 2002). The hygiene hypothesis has undergone numerous modifications since it was first proposed. This has not lead to a unifying concept, although various pieces of the complex interplay between the human immune system, various types of infections and atopy have become apparent (Guarner et al. 2006; Prioult et al. 2005; Schaub et al. 2006). The occurrence of atopic diseases is age‐dependent. Atopic eczema and food allergies have a substantial prevalence shortly after birth. Sensitization to pollen allergens is relatively low among young children (Silvestri et al. 1996) and increases with age (Rönmark et al. 2003; Silvestri et al. 1996). Generally, the prevalence of respiratory allergies (allergic asthma and allergic rhinitis) peaks among young adults (Saarinen et al. 1995). Hay fever is sometimes considered to act as a ‘gate 8 General Introduction opener’ to asthma as 13‐38% of the allergic rhinitis patients suffer from allergic asthma (Meltzer 2001). Sensitization to pollen allergens is a risk factor for the development of asthma symptoms, although the size of the risk may depend on the source of the pollen (a higher risk for grass than for tree pollen) (Chinn et al. 1998; Jõgi et al. 1998; Plaschke et al. 2000; Raukas‐Kivioja et al. 2003; Soriano et al. 1999). However, others studies indicate that sensitization to pollen allergens is associated to sensitization to indoor allergens, which would represent the actual risk factor for developing asthma (Jaen et al. 2002; Kerkhof et al. 2003). The suspected induction of asthma involves the formation of small pollen‐derived particles that (unlike pollen) are able to reach lower airway tracts and induce asthmatic responses (Schäppi et al. 1997). a. b. Figure 1.1 – (a) Flowering birch tree (B.