Specific T cell lines for ovalbumin, ovomucoid, lysozyme and two OA synthetic epitopes, generated from egg allergic patients' PBMC

Background Proteitis of hen egg white are common ingredients of food and difficult to eliminate. Allergens of egg while induce allergic symptoms among relatively high numbers of palients suffering from food allergy. B cell epitopes to hen egg white tnajor allergens have been reported. Considering that IgE antibody formation is mostly T cell dependent, the study of T cell epitopes is essential for both T cell dependent and independent IgE response. Objectives Little information on T cell epitopes recognizing food allergens has been reported. T cell responses to hen egg white allergens and two synthetic OA peptides located at amino acid residues No. 105–122 and 323–339 were investigated. Methods Peripheral blood mononuclear cells from hen egg allergic patients were investigated. Various allergens of hen egg white were used for stimulation. Primary proliferation responses were detected followed by the generation of long-term cultures which were examined for their specificity, phenotype, cytokine profile and IgE production. The allergen specific T cell lines were mapped using a panel of 13 synthetic peptides of ovalbumin. Results Human T cells recognizing ovomucoid, lysozyme and ovalbumin epitope 105–122 are reported for the first time. The cell lines were enriched CD4⁺/CD8⁺ T cells (CD2⁺ 95%). Ovomucoid and ovalbumin induced IgE synthesis by a small fraction of B cells (1%) present in the ovalbumin and ovomucoid specific T cell lines. Conclusions Human T cells recognized several egg white allergens and epitopes within the ovalbumin molecule. Specific IgE was produced in cultures stimulated with ovalbumin and ovomucoid. OA peptides 105–122 and 323–339 have no affinity to the specific IgE of the two patients; an observation which could be of particular interest regarding the mechanisms of peptide-based immunotherapy.     

The allure and peril of hematopoietic stem cell transplantation: overcoming immune challenges to improve success

Food allergy has become a major public health concern in westernized countries, and allergic reactions to peanuts are particularly common and severe. Allergens are defined as antigens that elicit an IgE response, and most allergenic materials (e.g., pollens, danders, and foods) contain multiple allergenic proteins. This has led to the concept that there are “major” allergens and allergens of less importance. “Major allergens” have been defined as allergens that bind a large amount of IgE from the majority of patients and have biologic activity. However, the ability of an allergen to cross-link complexes of IgE and its high-affinity receptor FcεRI (IgE/FcεRI), which we have termed its allergic effector activity, does not correlate well with assays of IgE binding. To identify the proteins that are the most active allergens in peanuts, we and others have employed in vitro model assays of allergen-mediated cross-linking of IgE/FcεRI complexes and have demonstrated that the most potent allergens are not necessarily those that bind the most IgE. The importance of a specific allergen can be determined by measuring the allergic effector activity of that allergen following purification under non-denaturing conditions and by specifically removing the allergen from a complex allergenic extract either by chromatography or by specific immunodepletion. In our studies of peanut allergens, our laboratory has found that two related allergens, Ara h 2 and Ara h 6, together account for the majority of the effector activity in a crude peanut extract. Furthermore, murine studies demonstrated that Ara h 2 and Ara h 6 are not only the major elicitors of anaphylaxis in this system, but also can effectively desensitize peanut-allergic mice. As a result of these observations, we propose that the definition of a major allergen should be based on the potency of that allergen in assays of allergic effector activity and demonstration that removal of that allergen from an extract results in loss of potency. Using these criteria, Ara h 2 and Ara h 6 are the major peanut allergens.     

过敏原蛋白组分sIgE检测及分析

目的 分析过敏性疾病患者血清中多种过敏原蛋白组分的sIgE,明确过敏原中的致敏蛋白组分,为临床过敏性疾病预防和治疗提供科学依据.方法 采用荧光酶联免疫法,用ImmunoCAP过敏原检测试剂检测受试者血液标本中过敏原蛋白组分及相关过敏原的特异性IgE(sIgE)水平,统计每种过敏原蛋白组分sIgE检出量和阳性率,并对主要致敏蛋白组分sIgE阳性率和相关过敏原的阳性率进行对比和分析.结果 40种过敏原蛋白组分的阳性率均小于过敏原的阳性率,致敏组分和交叉过敏组分共存于过敏原中.户尘螨、黄胡蜂毒、桃、猫毛、榛子、牛奶和鸡蛋的主要致敏蛋白组分阳性率高于过敏原阳性率的50%;百慕达草、意大利蜜蜂毒、桦树和花生的主要致敏蛋白组分阳性率介于过敏原阳性率的20%~50%之间;虾、狗毛、梯牧草和北艾草的主要致敏蛋白组分阳性率低于过敏原阳性率的20%.结论 过敏原蛋白组分检测能鉴别过敏原中真正致敏的蛋白组分,解决由于交叉反应所导致的过敏原特异性诊断难题,确保过敏性疾病特异性免疫治疗的精确性和有效性.     

The antigenicity and allergenicity of microparticulated proteins: Simplesse®

New technologies are allowing the food industry to develop products from standard foods which may not be recognized in its modified form by food allergic patients. One such product, Simplesse, has been formulated by microparticulation of egg white and/or cows' milk proteins and is used as a fat substitute in many fat-laden foods. The purpose of this study was to determine whether the process of microparticulation altered the allergenicity/antigenicity of egg white and cows' milk proteins compared to the starting materials. Soluble protein fractions of Simplesse and its respective starting materials were compared to egg white, cows' milk protein, an ultra-filtered egg white/condensed milk mixture, and/or a whey concentrate by SDS-polyacrylamide gel electrophoresis. In addition, sera from 16 patients with documented egg and/or cows' milk hypersensitivity and two controls who were not allergic to egg or milk were used to assess potential allergenicity/antigenicity of these products by immunoblot (Western blot) analysis. There were heterogeneous IgE and IgG binding patterns to the food fractions among these food allergic patients suggesting differing sensitivity patterns among the individuals tested. However, utilizing both SDS-PAGE and immunoblot analyses, the major allergens in the microparticulated products were the same as those found in the starting materials, egg and cows' milk. In addition, there was no evidence of 'novel' protein fractions in the Simplesse test materials compared to the starting materials.     

Allergens causing bird fancier's asthma

Tauer-Reich I, Fruhmann G, Czuppon AB, Baur X. Allergens causing bird fancier's asthma.
The study investigates to what extent bird feathers contain relevant allergens/antigens involved in bird fancier's asthma. The study group consisted of two budgerigar fanciers, two parrot fanciers and one canary fancier. All subjects complained of asthmatic symptoms, caused by contact with their birds, and they showed a significant bronchial hyperreactivity to acetylcholinc. Positive IgE antibody reactions to bird sera as well as to extracts of feathers were observed in RAST. Well-defined major allergenic bands could be detected and identified in the IgE immunoblots with feather extracts as well as with serum proteins of budgerigar, parrot, pigeon, canary, and hen (mol. mass 20-30 kDa and 67 kDa). The most pronounced bands appeared with the extracts of species to which an exposure had taken place. Weaker IgG-binding patterns were also observed. Our results show that inhalable feather dust contains several allergenic components which cross-react with serum allergens/antigens of the same as well as of other bird species. This emphasizes the significance of bird feathers for immediate-type allergic reactions.     

Hidden food allergens

This review summarizes recent advances and findings in the area of 'hidden' food allergens, i.e. allergenic foods that can either contaminate other foods, or be 'disguised' as part of a food, and cause allergic reactions. Newly emerging allergenic foods of increasing importance, recently developed methods for the detection of allergenic residues, the potential allergenicity of genetically engineered foods, and some unexpected sources of food allergens are described.     

Classification of allergens by positive percentage agreement and cluster analysis based on specific IgE antibodies in asthmatic children]

Classification and characterization of allergens is important because allergic patients are sensitized by a variety of allergens. One hundred and sixty-one sera from asthmatic children were investigated for specific IgE antibodies against 35 allergens including 20 inhalants and 15 foods by means of the MAST method. We assessed the allergenic properties of the allergens based on positive percentage agreement and cluster analysis. There was a high positive percentage agreement of specific IgE antibodies between house dust and Dermatophagoides spp., a relatively high agreement between 5 molds, cat and dog epithelium, mugwort and wormwood and 5 grasses. Among the food allergens, the positive percentage agreements were relatively high, especially between cow's milk, casein, cheese, and between 3 cereal grains. In the cluster analysis, house dust and Dermatophagoides spp. made a big cluster; therefore 32 allergens except house dust and mites were analyzed. From the results of the cluster analysis, the major cluster consisted of (1) ragweed, (2) mugwort and wormwood, (3) timothy, sweet vernal, velvet and cultivated rye, (4) wheat, barley and rice, (5) molds, (6) cow's milk, casein, soybean and cheese, (7) shrimp and crab, (8) egg white, (9) Japanese cedar, (10) dog epithelium, (11) cat epithelium. The cluster of grass pollens and cereal grains made one cluster. These results tend to confirm the presence of species cross-reactivities within the major classes of allergens.     

The investigation of asthmatic children by multiple factor analysis. II. The influence of 17 allergic factors on atopic dermatitis and allergic rhinitis in asthmatic children

We investigated possible influence of 17 allergy-associated factors on atopic dermatitis and allergic rhinitis using Multiple factor analysis in 150 asthmatic children. Atopic dermatitis was complicated in ninety-seven cases and allergic rhinitis in ninety-seven cases. 17 allergy-associated factors were as follows: 1) sex, 2) age, 3) onset age of asthma, 4) family history of allergy, 5) peripheral eosinophil counts, 6) IgE RIST, 7) IgE RAST score to egg white, 8) IgE RAST score to milk, 9) IgE RAST score to soybean, 10) IgG4 antibody titers to egg white, 11) IgG4 antibody titers to milk, 12) IgG4 antibody titers to soybean, 13) IgE RAST score to house dust, 14) IgE RAST score to Dermatophagoides farinae, 15) severity of asthma, 16) exercise-induced asthma, 17) atopic dermatitis or allergic rhinitis. We concluded as follows: 1) Factors which more strongly influenced both atopic dermatitis and allergic rhinitis were IgE RAST score to D.f., positive family history of allergy, IgE RIST and eosinophil counts. 2) Combination with high levels of IgG4 antibody to 3 food allergens such as egg-white, milk and soybean and IgE RAST to egg-white has a strong influence on atopic dermatitis, but high levels of IgG4 antibody to 3 food allergens except high level of IgG4 antibody to soybean have a weak influence on allergic rhinitis.     

A new in vitro model for testing of food allergens: Allergen‐specific mediator release of passively sensitized rat Basophil Leukaemia cells

An assay based on allergen‐specific mediator release of rat basophil leukaemia cells (RBL cells) was investigated as a possible new tool for the in vitro testing of food allergens. The RBL‐2H3 cells were sensitized passively with diluted murine pool sera containing IgE specific for food and pollen allergens. These sera were obtained from BALB/c mice after low‐dose intraperitoneal injection of birch pollen, celery and peanut extracts. Comparative immunoblotting with murine and human IgE demonstrated that the murine IgE response was directed predominantly against known major allergens. Subsequent to sensitization of the RBL cells with antiserum against birch pollen allergens, apple allergy was used as an immunological model of birch pollen related food hypersensitivities. The known cross‐reaction of the major allergens of birch pollen and apple was reproduced completely by the mediator release measured in the murine in vitro assay. The ranking of allergenic potency obtained for these allergens agreed closely with the results of histamine release analyses performed with blood samples of allergic patients. In addition, murine sera against celery tuber were used to study the influence of thermal and non‐thermal food processing on the allergenic potency of the food. Again, the results corresponded to previous data obtained in allergic humans and indicated a reduction of potency due to the application of heat, but a high residual biological activity in many mildly processed products. Finally, spurious contaminations of peanut protein in commercial foods could be detected specifically at a concentration of at least 0.01%. This assay is suitable for many applications in food allergen research.     

Identification of allergenic proteins in condoms by immunoenzymatic methods.

BACKGROUND: A large increase of allergy to latex proteins has been observed lately probably as a result of a great use of latex-containing goods. At present these untoward reactions have led to consideration of this problem as a health and occupational hazard. It is therefore, necessary to identify the allergens contained in latex-manufactured products and to develop effective diagnostic tools to detect sensitized individuals. OBJECTIVE: The objective of this study is to identify antigenic and allergenic components in latex condoms by using chemical, immunochemical, and immunoenzymatic methods. METHODS: The protein content of extracts obtained from several brands of condoms was determined and characterized by using a modified Lowry method, a quantitative ELISA assay and SDS-PAGE. The allergenic behavior of these proteins was studied by IgE immunoblotting, EAST and ELISA techniques, using sera from subjects allergic to latex products, particularly to latex condoms. RESULTS: Wide variations in the protein content (38 to 740 microg/g product) and composition were observed. The SDS-PAGE protein profiles showed components ranging from 7 to 94 kD of relative molecular weights; most of them were also detected in natural rubber latex. The most prominent bands were revealed in the 14 and 30 kD zones. A strong band of 69 kD in the SDS-PAGE profiles would correspond to a neoantigen, since it was not observed in natural latex. The immunoblotting analysis employing sera from 5 patients allergic to latex condoms showed the presence of 4 components with IgE binding capacity (14, 30, 69, and 94 kD). The EAST and ELISA methods showed the presence of allergens in all the condom brands studied. CONCLUSIONS: The presence of allergenic proteins in several condom brands was demonstrated by different immunoenzymatic methods.     

A Clinical and Immunological Study of Allergy to Hen's Egg White

Allergens in hen's egg white were studied in crossed radio-immunoelectrophoresis (CRIE). Specific IgE-antibodies against different proteins in the egg white were examined in sera from 70 atopic patients with clinical hypersensitivity, and RAST ≥2 to egg white. Ovalbumin, ovomucoid and an unidentified protein, antigen 22, were classified as major allergens. Specific IgE-antibodies against 10 more proteins in hen's egg white were detected. IgE-antibodies against lysozyme could not be detected.     

Relation between IgG antibodies to foods and IgE antibodies to milk, egg, cat, dog and/or mite in a cross-sectional study

BACKGROUND: Because IgG antibodies to foods can be detected before IgE antibodies to inhalants, increased levels of IgG antibodies to foods might be used as a predictor of IgE-mediated allergy in initially nonatopic children. OBJECTIVE: To examine the cross-sectional relation between IgG to foods (i.e. mixture of wheat and rice, mixture of soybean and peanut, egg white, cow's milk, meat, orange and potato) and specific IgE to cat, dog, mite, milk and egg white in 1-year-old children. METHODS: All atopic children (n = 120; 58 with and 62 without eczema) and a random sample of the nonatopic children (n = 144) of the Bokaal study were tested on their IgG response to foods. The IgG results of the food assays were dichotomized high or low using the 66th centile as a cut-off value. RESULTS: Atopic children more often had high IgG levels to foods than nonatopic children. IgG to egg white (OR = 7.50) and mixture of wheat and rice (OR = 4.79) were most strongly associated with positive specific IgE. In a stepwise logistic regression analysis egg white, mixture of wheat and rice, and orange were selected (OR = 3.76, OR = 2.43, and OR = 2.11, respectively). In children without eczema higher levels of IgG to foods were still significantly associated with atopy, which was most prominent for egg white, orange and cow's milk. CONCLUSION: An increased IgG antibody level to foods, especially to egg white, orange, and mixture of wheat and rice, indicates an increased risk of having IgE to cat, dog, mite, egg and/or milk allergens, even in the noneczematous group. Therefore, in another prospective study we are currently investigating the usefulness of IgG in early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing allergic diseases in the future.     

Allergenic activity of cat and dog skin fractions obtained by Sephadex gel filtration

Allergens derived from domestic mammals have been investigated by subjecting extracts of cat and dog skin to Sephadex gel filtration and assessing the activity of the eluted fractions by skin tests in appropriate allergic subjects. A certain heterogeneity of allergenic activity occurred and minor allergens, with an estimated molecular weight greater than 70,000, were found which showed activity in patients highly sensitive to cat and dog serum proteins. The major allergens of both mammalian skin extracts were eluted in similar positions between protein markers of 24,000 and 67,000 molecular weight.     

Surgical latex glove allergy: characterization of rubber protein allergens by immunoblotting.

Immunoblot analysis employing IgE antibodies derived from sera of 3 physicians and 2 nurses allergic to surgical latex gloves, disclosed 10 allergens in natural rubber sap. Nine of the 10 allergens were detected in ammoniated natural rubber latex, but only 4 allergens in a latex glove extract. The allergenic proteins had apparent molecular weights ranging from 14 to 70 kD. Allergens with molecular weights of 14 and 21 kD showed the most intense immunoblot reactions suggesting that these proteins could be the major allergens in the natural rubber. An 11-kD protein and a 26-kD protein were only seen in the glove extract, indicating that they could be modified rubber proteins formed during glove manufacture.     

Adverse reactions to foods.

Allergic reactions to foods represent a prominent, actual and increasing problem in clinical medicine. Symptoms of food allergy comprise skin reactions (urticaria, angioedema, eczema) respiratory (bronchoconstriction, rhinitis), gastrointestinal (cramping, diarrhea) and cardiovascular symptoms with the maximal manifestation of anaphylactic shock. They can be elicited by minute amounts of allergens. The diagnosis of food allergy is done by history, skin test, in vitro allergy diagnosis and--if necessary--oral provocation tests, if possible placebo-controlled. Avoidance of respective allergens for the allergic patient, however, is often complicated or impossible due to deficits in declaration regulations in many countries. Increasing numbers of cases including fatalities, due to inadvertent intake of food allergens are reported. It is therefore necessary to improve declaration laws and develop methods for allergen detection in foods. Allergens can be detected by serological methods (enzyme immunoassays, in vitro basophil histamine release or in vivo skin test procedures in sensitized individuals). The problem of diagnosis of food allergy is further complicated by cross-reactivity between allergens in foods and aeroallergens (pollen, animal epithelia, latex etc.). Elicitors of pseudo-allergic reactions with similar clinical symptomatology comprise low-molecular-mass chemicals (preservatives, colorings, flavor substances etc.). For some of them (e.g. sulfites) detection assays are available. In some patients classic allergic contact eczema can be elicited systemically after oral intake of low-molecular-mass contact allergens such as nickel sulfate or flavorings such as vanillin in foods. The role of xenobiotic components in foods (e.g. pesticides) is not known at the moment. In order to improve the situation of the food allergic patient, research programs to elucidate the pathophysiology and improve allergen detection strategies have to be implemented together with reinforced declaration regulations on a quantitative basis.     

Array-based profiling of ragweed and mugwort pollen allergens

Background: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. Objective: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme‐linked immunosorbent assay (ELISA). Methods: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. Results: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen‐allergic patients. Extensive cross‐reactivity was observed for both patient groups mainly involving the pan‐allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. Conclusions: Molecule‐based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.     

Allergens from fish and egg

Allergens from fish and egg belong to some of the most frequent causes of food allergic reactions reported in the literature. Egg allergens have been described in both white and yolk, and the egg white proteins ovomucoid, ovalbumin, ovotransferrin and lysozyme have been adopted in the allergen nomenclature as Gal d1–d4. The most reported allergen from egg yolk seems to be alpha-livitin. In fish, the dominating allergen is the homologues of Gad c1 from cod, formerly described as protein M. A close cross-reactivity exists within different species of fish between this calcium-binding protein family, denominated the parvalbumins. This cross-reactivity has been indicated to be of clinical relevance for several species, since patients with a positive double-blind, placebo-controlled food challenge to cod will also react with other fish species, such as herring, plaice and mackerel. In spite of the importance of these two allergen systems, only a few studies have been performed, and the scarcity of cloned allergens from both of the systems is emphasized.     

Studies of the allergens of Olea europea pollen

Allergens of Olea eurupea have been prepared by isoelectrofocusing and tested by RAST, RAST inhibition and skin tests. The major allergen was found to be an acidic protein of pI 6, consisting of two polypeptide chains of molecular weight 15000 and 17000 daltons, possibly non-covalently associated into a dimer. Most other allergens were acidic proteins, but basic proteins were also observed with allergenic activity. The RAST inhibition studies show a considerable degree of allergic cross-reactivity between the isoelectrofocusing fractions.     

IgE response to fur animal allergens and domestic animal allergens in fur farmers and fur garment workers

Objective The aim of this study was to compare the IgE response to the most commonly farmed fur animals with that to domestic animals. Methods IgE-immunoblotting and RAST-inhibition analyses were performed using RAST-positive sera from fur workers sensitized to fur allergens and sera from patients sensitized to domestic animal allergens. Results The urine extracts of mink, blue fox, silver fox, racoon dog and fitchew contained more protein bands than the fur extracts did. Allergens with the same molecular weight were found in all of the fur and urine extracts. The most prominent allergenic bands had molecular weights of 62–67 kDa, 23–25 kDa and 18–19 kDa. With crossreacting sera the reciprocal RAST inhibition with all five animal extracts indicated common IgE-binding epitopes, probably common allergens (especially the 62–67 kDa bands). Urine and fur contain common allergens, since urine allergens strongly inhibited the IgE-binding to fur allergens. The IgE binding to allergenic bands of fur animal extracts was also observed in immunoblotting when dog and cat RAST-positive sera were used, but not for cow RAST- positive sera. RAST inhibition of dog-positive sera with fur animal extracts and fur-positive sera with dog extract confirmed the crossreactivity of these IgE antibodies. No such inhibition was seen with cow extract. Conclusion The results of the RAST inhibition and immunoblotting suggest that fur animals have IgE binding epitopes or allergens in common with cat and dog – possibly albumin but not with cow.