Lebensmittelallergie — eine individuelle Gefahr: Wenn das Immunsystem falsch reagiert

Problems with food allergy and other adverse reactions to food are still a challenge for science. Both, in case of diagnostic and therapy and in characterization of allergens there is still a lot to do in research to help the continuously increasing number of allergic patients. But an exact elucidation of allergen structure, the development of hypoallergenic food by gentechnological processing of allergenic proteins, a better therapy, the knowledge of the pathogenetic effect or a complete declaration of ingredients in prepacked foods will never lead to absolutely sure food for allergic patients. As the pathogenesis, the symptoms, and sensibilization individually differ, at least for every allergic patient there will remain a risk.     

Purification,structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen,Phl p 4, with cross-reactive potential

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.     

义乌地区儿童过敏性结膜炎过敏原分析

目的了解过敏性结膜炎患儿常见过敏原及其分布特征,为预防和脱敏治疗提供依据。方法采用免疫印迹法对363例过敏性结膜炎患儿进行血清过敏原检测,并对结果进行分组比较。结果患儿中检出率居前3位的吸入性过敏原分别为尘螨(50.41%)、猫毛(3.03%)和霉菌(2.48%);检出率居前3位的食物性过敏原分别为淡水鱼(10.74%)、海鱼(7.44%)和鸡蛋白(6.34%)。吸入性过敏原(62.26%)和食物性过敏原(41.60%)总检出率比较差异有统计学意义(P0.05)。婴幼儿组、学龄前组和学龄组患儿均以尘螨为常见过敏原,其检出率分别为33.33%、52.76%和52.00%。不同年龄患儿牛奶过敏检出率比较差异有统计学意义(P0.05),婴幼儿组牛奶过敏检出率最高,为12.82%。男、女患儿过敏原阳性率比较差异无统计学意义(P0.05)。不同年龄患儿过敏原阳性率比较差异无统计学意义(P0.05)。婴幼儿组、学龄前组和学龄组患儿均以单纯吸入性过敏原和单一过敏原为常见。1月-3月为儿童过敏性结膜炎的低发季节。结论通过血清过敏原检测可明确过敏性结膜炎的过敏原。尘螨是本地区儿童过敏性结膜炎常见过敏原。     

Clinical study of peanut and nut allergy in 62 consecutive patients: new features and associations.

OBJECTIVE--To investigate clinical features of acute allergic reactions to peanuts and other nuts. DESIGN--Analysis of data from consecutive patients seen by one doctor over one year in an allergy clinic at a regional referral centre. SUBJECTS--62 patients aged 11 months to 53 years seen between October 1993 and September 1994. MAIN OUTCOME MEASURES--Type and severity of allergic reactions, age at onset of symptoms, type of nut causing allergy, results of skin prick tests, and incidence of other allergic diseases and associated allergies. RESULTS--Peanuts were the commonest cause of allergy (47) followed by Brazil nut (18), almond (14), and hazelnut (13). Onset of allergic symptoms occurred by the age of 2 years in 33/60 and by the age of 7 in 55/60. Peanuts accounted for all allergies in children sensitised in the first year of life and for 82% (27/33) of allergies in children sensitised by the third year of life. Multiple allergies appeared progressively with age. The commonest symptom was facial angioedema, and the major feature accounting for life threatening reactions was laryngeal oedema. Hypotension was uncommon. Of 55 patients, 53 were atopic--that is, had positive skin results of tests to common inhaled allergens--and all 53 had other allergic disorders (asthma, rhinitis, eczema) due to several inhaled allergens and other foods. CONCLUSIONS--Sensitisation, mainly to peanuts, is occurring in very young children, and multiple peanut/nut allergies appear progressively. Peanut and nut allergy is becoming common and can cause life threatening reactions. The main danger is laryngeal oedema. Young atopic children should avoid peanuts and nuts to prevent the development of this allergy.     

In vivo and in vitro techniques to determine the biological activity of food allergens

Methods for determination of the biological activity of food allergens comprise both determination of the allergenic potency, i.e. the capability to elicit an allergic reaction in an already sensitized individual, and the allergenic potential, i.e. the risk for sensitizing a hitherto non-allergic individual. Several methods are discussed for determination of potency including the double-blinded placebo-controlled food challenge, skin testing, in vitro effector cell assays such as basophil histamine release, and IgE-based techniques such as RAST and RAST inhibition. No reliable methods have yet been developed which can predict the allergenic potential of a food or a food allergen. The progress in the areas of stability studies and animal models for food allergy are discussed.     

Isolation of food allergens

The identification of food allergens is a priority in the management of food allergy, because of the need to obtain standardized extracts and pure allergens for diagnosis and therapy. It is thus important to develop methods for purification of allergenic molecules in order to study their biological and immunological characteristics. Protocols for protein extraction from foods and for allergen purification are reviewed in this paper. We report published methods for extraction of allergens from either animal and vegetable foods and detailed purification methodologies including ion-exchange, gel filtration and reversed-phase chromatography of well known allergens.     

Microarrayed allergens for IgE profiling

Diagnosis of type I allergy is based on anamnesis, provocation testing, and serological determination of total and specific IgE. Currently, in vivo and in vitro diagnostic tests employ allergen extracts prepared from various allergen sources (e.g., pollen, mites, animal dander, moulds, foods, venoms, etc.). The application of recombinant DNA technology to the field of allergen characterization has allowed to reveal the molecular nature of the most common allergens. To date a continuously increasing number of allergen sequences has become available and panels of recombinant allergens-assembling the epitope complexity of natural allergens sources-can be produced. The use of recombinant allergens instead of crude, natural extracts for allergy diagnosis allows us to determine the individual IgE reactivity profile of each patient. To enable a comprehensive analysis of the patient's IgE binding pattern to a large number of individual allergens, a new type of serological test is required. In this paper, we applied microarray technology to create a multi-allergen test system, based on microarrayed recombinant allergens.     

The Anisakis allergy debate: does an evolutionary approach help?

Allergic phenomena share common pathways with the immune response against helminth parasites. The definitions regarding allergens and their related concepts have their roots in the area of allergy research. The experience with the fish parasite Anisakis simplex-associated allergic features still nurtures an open debate on the necessity of larvae being alive to induce allergic reactions such as urticaria or anaphylaxis. Conceptual definitions of allergen, major allergen, as well as putatively crossreacting antibodies, as are used in food allergy, depend on the clinical relevance of specific IgE and deserve careful interpretation in the various forms of A. simplex-associated allergic features. Conversely, an evolutionary based interpretation of the presence of specific IgE depends on the viability of A. simplex.     

Proteomics and immunological analysis of a novel shrimp allergen,Pen m 2

Shellfish are a common cause of adverse food reactions in hypersensitive individuals and shrimp is one of the most frequently reported causes of allergic reactions. A novel allergen from Penaeus monodon, designated Pen m 2, was identified by two-dimensional immunoblotting using sera from subjects with shrimp allergy, followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the peptide digest. This novel allergen was then cloned and the amino acid sequence deduced from the cDNA sequence. The cloned cDNA encoded a 356-aa protein with an acetylated N terminus at Ala2, identified by postsource decay analysis. Comparison of the Pen m 2 sequence with known protein sequences revealed extensive similarity with arginine kinase (EC 2.7.3.3) from crustaceans. Pen m 2 was purified by anion exchange chromatography and shown to have arginine kinase activity and to react with serum IgE from shrimp allergic patients and induce immediate type skin reactions in sensitized patients. Using Pen m 2-specific antisera and polyclonal sera from shrimp-sensitive subjects in a competitive ELISA inhibition assay, Pen m 2 was identified as a novel cross-reactive Crustacea allergen. This novel allergen could be useful in allergy diagnosis and in the treatment of Crustacea-derived allergic disorders.     

糖蛋白过敏原N-糖链与过敏的相关性研究

糖蛋白是一种含有寡糖链的蛋白质,糖链与蛋白质之间以共价键相连。N-糖蛋白为常见过敏原之一,主要来源于食物、吸入物、昆虫毒素等,能够引起过敏反应。N-糖蛋白过敏原的N-糖链结构影响过敏原与IgE的结合,影响抗原提呈细胞（APC）对过敏原的识别和提呈。本文在介绍与过敏相关的N-糖蛋白、常见N-糖蛋白过敏原的N-糖链结构及与过敏相关的糖基化酶的基础上,进一步分析过敏原N-糖链影响过敏的机制,为临床预防与治疗过敏性疾病提供新的思路。     

Reproducibility of atopy patch tests with food and inhalant allergens

Although atopy patch tests (APT) seem a valuable additional tool in the diagnostic work-up for food allergy in children with atopic eczema/dermatitis syndrome, the immunopathology and some technical aspects of testing remain controversial. Few published data are available on the reproducibility of APT with inhalants and only two studies include fresh food allergens. In this study we therefore investigated the reproducibility of duplicate APT (left versus right side of the back) with native and commercially available food (cow s milk, hen s egg, tomato, wheat flour) and with inhalant allergens (Dermatophagoides pteronyssinus and mixed grasses) in a large unselected population of children. We tested a population of 277 Italian school children with three APT allergens: fresh food (cow s milk, hen s egg, tomato and wheat flour), standardised food allergens in petrolatum (the same four foods) and standardised inhalant allergens routinely used for skin prick testing. For the four food allergens (applied in the natural form or as the standardised commercial preparation) from one- to three quarters of the APT gave positive results on one side and negative reactions on the opposite side (Cohen s K coefficient between 0.38, fresh tomato and 0.81, fresh cow s milk). Conversely, APT with inhalant allergens were invariably reproducible (Cohen s K = 1.00). The possible technical and immunologic reasons explaining why reproducibility of APT differed for the two types of allergens await an answer from extensive controlled studies.     

The impact of plant biotechnology on food allergy

Concerns about food allergy and its societal growth are intertwined with the growing advances in plant biotechnology. The knowledge of plant genes and protein structures provides the key foundation to understanding biochemical processes that produce food allergy. Biotechnology offers the prospect of producing low-allergen or allergen null plants that could mitigate the allergic response. Modified low-IgE binding variants of allergens could be used as a vaccine to build immunotolerance in sensitive individuals. The potential to introduce new allergens into the food supply by biotechnology products is a regulatory concern.     

Improving diagnostic tests for food allergy with recombinant allergens

Food allergy is one important manifestation of atopic allergy. Primary food allergy mainly affects young children (class I), whereas adults frequently develop food allergy as a consequence of an inhalant sensitization (class II). At present, the diagnostic instrument for proving class II food allergy is not satisfactory. Skin tests as well as serological tests are in general neither very specific nor highly sensitive because they depend on food extracts, which differ in their content of individual allergens, vary between manufacturers, and even between different batches. Since the presence of food allergen-specific IgE antibodies does not always correlate with clinical symptoms against the respective food, oral provocation tests (ideally double-blind placebo-controlled food challenges) have to be performed to validate serological diagnosis or skin tests. However, oral provocation tests are connected to several practical problems and include a specific risk for the allergic patient. Applying DNA technology, up to 40 food allergens have been produced in recombinant form, which implies standardized quality and unlimited quantity of the respective proteins. Hence, such molecules might be used to solve problems of clinical and molecular allergology in diagnosis, research, and therapy of class II food allergies. First experiments with recombinant food allergens in this respect appear very promising.     

Immunological identification and characterization of individual food allergens

Food allergies of type-I-allergy are immunoglobulin E (IgE) mediated and caused by certain proteins or glycoproteins, which are called food allergens. An analytical marker of allergens is the IgE-reactivity to these substances. Normally food allergens are minor components in allergenic source material, which consist of a huge number of chemical different substances. Thus allergen extraction, separation and immunological detection methods are described which identify and characterize individual food allergens by a minimum of manipulation. Favoured separation methods of allergenic extracts are electrophoretic ones allowing the combination of highly resolved protein separations with immunological detection methods subsumed by the term immunoblotting. These techniques are a useful basis to characterize allergens by chemical methods. Once the primary protein structure of a food allergen is established, the way is cleared for the identification of epitopes. Epitopes are immunological detectable parts of a protein or glycoprotein generating the interface between chemical structure and immune-system. The nature of epitopes may differ, for instance, can be conformational, continuous, or built up by glycoconjugates, which determine the stability of food allergens, especially in the case of food processing. Progress in identification and characterization of food allergens will improve diagnostics and therapy of food allergy.     

Strategies for converting allergens into hypoallergenic vaccine candidates

Specific immunotherapy is based on the administration of increasing doses of allergens to allergic patients with the aim of inducing a state of antigen-specific unresponsiveness. Specific immunotherapy is one of the few causative treatment approaches for Type I allergy but may cause numerous side effects, including local inflammatory reactions, systemic manifestations (e.g., asthma attacks) and in the worst case, anaphylactic shock which may lead to death. Several attempts have been made in the past to reduce the rate of side effects. They included the chemical modification of allergen extracts to reduce their allergenic activity and the adsorption of allergen extracts to adjuvants to prevent the systemic release of allergens after administration. During the last decade, cDNAs coding for the most relevant allergens have been isolated and the corresponding allergens have been produced as recombinant molecules. Using allergen-encoding cDNAs, the amino acid sequence of allergens or purified recombinant allergens several strategies can now be applied to produce allergen derivatives with reduced allergenic activity for allergy vaccination in a controlled and reproducible manner. Currently, allergen-encoding cDNAs are used to engineer recombinant hypoallergenic allergen derivatives. According to the amino acid sequences and experimental epitope mapping data, synthetic peptides representing T- or B-cell epitopes are produced and purified recombinant allergens are coupled to novel adjuvants for vaccine formulation. In this article, strategies for the production and evaluation of allergen derivatives with reduced allergenic activity for allergy vaccination are described. These new vaccines hold great promise to improve the current practice of allergen-specific immunotherapy and maybe also used for prophylactic vaccination in the future.     

锯缘青蟹主要过敏原的纯化与鉴定

以锯缘青蟹为研究对象,从免疫鉴定、分离纯化、抗体制备和免疫学分析等方面对其主要过敏原进行研究。首先利用过敏者血清的免疫印迹法,确定锯缘青蟹的主要过敏原为分子量约38kD的蛋白。然后通过制备丙酮粉、等电点沉淀、硫酸铵沉淀及加热处理对分子量为38kD的主要过敏蛋白进行了高度纯化。该蛋白的pI约为4.5,与虾的原肌球蛋白Pena1性质相近,证实了锯缘青蟹的主要过敏原为原肌球蛋白。通过免疫新西兰大白兔,制备了原肌球蛋白的抗血清,采用Protein A Sepharose亲和层析柱对动物抗体进行了纯化。该抗血清效价高,经4×105倍稀释后仍能与抗原进行反应。该抗体与甲壳类动物及软体动物的原肌球蛋白具有较强的免疫交叉反应,可用于食品过敏原检测。       

Oxidized cellulose binding to allergens with a carbohydrate-binding module attenuates allergic reactions

Grass and mite allergens are of the main causes of allergy and asthma. A carbohydrate-binding module (CBM) represents a common motif to groups I (β-expansin) and II/III (expansin-like) grass allergens and is suggested to mediate allergen-IgE binding. House dust mite group II allergen (Der p 2 and Der f 2) structures bear strong similarity to expansin's CBM, suggesting their ability to bind carbohydrates. Thus, this study proposes the design of a carbohydrate-based treatment in which allergen binding to carbohydrate particles will promote allergen airway clearance and prevent allergic reactions. The aim of the study was to identify a polysaccharide with high allergen-binding capacities and to explore its ability to prevent allergy. Oxidized cellulose (OC) demonstrated allergen-binding capacities toward grass and mite allergens that surpassed those of any other polysaccharide examined in this study. Furthermore, inhalant preparations of OC microparticles attenuated allergic lung inflammation in rye grass-sensitized Brown Norway rats and OVA-sensitized BALB/c mice. Fluorescently labeled OC efficiently cleared from the mouse airways and body organs. Moreover, long-term administration of OC inhalant to Wistar rats did not result in toxicity. In conclusion, many allergens, such as grass and dust mite, contain a common CBM motif. OC demonstrates a strong and relatively specific allergen-binding capacity to CBM-containing allergens. OC's ability to attenuate allergic inflammation, together with its documented safety record, forms a firm basis for its application as an alternative treatment for prevention and relief of allergy and asthma.     

Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7.

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.     

Structure and Function of the Peanut Panallergen Ara h 8

The incidence of peanut allergy continues to rise in the United States and Europe. Whereas exposure to the major allergens Ara h 1, 2, 3, and 6 can cause fatal anaphylaxis, exposure to the minor allergens usually does not. Ara h 8 is a minor allergen. Importantly, it is the minor food allergens that are thought to be responsible for oral allergy syndrome (OAS), in which sensitization to airborne allergens causes a Type 2 allergic reaction to ingested foods. Furthermore, it is believed that similar protein structure rather than a similar linear sequence is the cause of OAS. Bet v 1 from birch pollen is a common sensitizing agent, and OAS results when patients consume certain fruits, vegetables, tree nuts, and peanuts. Here, we report the three-dimensional structure of Ara h 8, a Bet v 1 homolog. The overall fold is very similar to that of Bet v 1, Api g 1 (celery), Gly m 4 (soy), and Pru av 1 (cherry). Ara h 8 binds the isoflavones quercetin and apigenin as well as resveratrol avidly.     

Immunoglobulin E mediated food allergy.Modelling and application of diagnostic and predictive tests for existing and novel foods

It is known that some foods cause an allergenic response in certain individuals. Clinical and immunological tests are available for the diagnosis of food allergy and identification of food allergens. However, there are no valid tests for the prediction of the allergenic potential of foods not normally recognized as allergenic. Such foods include: food products developed from foods which may not be recognizable as allergenic in their modified forms; foods produced using novel processes (novel foods), for example genetically modified foods; and foods not normally consumed but that are being used increasingly as alternatives to more traditional foods. Both the risks associated with food allergy and the fact that foods such as the ones described above will become available to the consumer, highlight the need for methods to screen for potential food allergens. This review provides a general overview of food allergy including mechanism, development and prevalence, but focuses on and discusses: 1) the possible risks (with specific reference to food allergy) associated with new and novel foods; and 2) the development/use of food allergy models (in vivo and in vitro) to assess the allergenic potential of new and novel foods.