Plant food allergens homologous to pathogenesis-related proteins

In general, pathogenesis-related (PR) proteins are expressed by plants in response to stress conditions like infection, exposure to certain chemicals, wounding and environmental conditions. In some plant tissues, however, PR proteins are constitutively expressed, e.g. in pollens or fruits, tissues that are more likely to be attacked (by insects or fungi) or exposed to atmospheric conditions (e.g. UV irradiation). PR proteins display multiple effects within the plant and possess antimicrobial activity, and can thus be regarded as a part of the plant's defense system. Analyzing known amino acid sequences and functions of characterized (cloned) food allergens, it is remarkable that many of these molecules can be classified as PR proteins. Many PR proteins are stable at low pH, and display considerable resistance to proteases, requirements to act as food allergens. According to sequence characteristics and their enzymatic or biologic activity, PR proteins can be divided into 14 groups. Seven of these 14 groups contain proteins with allergenic properties, six groups contain food allergens.     

Human hair is a potential source of cat allergen contamination of ambient air

BACKGROUND: We have previously shown that airborne cat allergen levels are significantly lower in school classes using special school clothing or in classes with no pet owners. However, cat allergen is present and the levels are in fact two- to threefold higher on cat owners' than noncat owners' school clothing which is used, washed and stored at school only. This suggests that allergen is transferred to schools by routes other than clothing. AIM: To analyse levels of cat allergen (Fel d 1) in hair from cat owners and noncat owners among children and adults. METHODS: Samples of unwashed hair (> or =1 day prior to sampling) from adults and children with (n = 22) or without (n = 22) cats at home were collected at a hairdresser. In addition, samples of newly washed hair (adults only, n = 11) were collected. The hair sample was extracted and analysed for Fel d 1 content with ELISA. RESULTS: The geometric mean levels were more than two orders of magnitude higher in unwashed hair from cat owners, compared with noncat owners (P < 0.0001) and more than 10-fold higher in newly washed hair from adults. The allergen contamination of unwashed hair among noncat owners appeared higher in children than in adults (P = 0.045). CONCLUSIONS: Hair may be an important source for transfer and deposition of cat allergen in schools and may explain why cat allergen is found in environments with strict allergen avoidance measures. Although it may be unrealistic to apply allergen avoidance strategies against this allergen source, it is important to be aware of it.     

In contrast to specific B cells, human basophils are unaffected by the toxic activity of an allergen toxin due to lack of internalization of immunoglobulin E-bound allergen

BACKGROUND: Specific immunotherapy is the only curative therapy for type I allergies and the alarming increase in allergy prevalence emphasizes the need for additional/alternative strategies for curative treatment. Allergen toxins (AT), fusion products of an allergen with an apoptosis inducing cytotoxin, are a new kind of immunotoxin. OBJECTIVE: AT should allow allergen-specific targeting and elimination of allergy-relevant cells, with B cells being the primary target. An important question is the fate of the effector cells, e.g. mast cells and basophils, which carry allergen-specific IgE: the immunotoxin might even prove to be harmful. METHODS: We established a reliable in vitro B cell model (using two mouse hybridoma cell lines) for testing specificity and toxicity of P5-ETA', a fusion protein of the major timothy grass pollen allergen Phl p 5b and truncated Pseudomonas Exotoxin A. In a second step, we investigated the impact of the AT on human basophils. RESULTS: P5-ETA' reliably eliminated Phl p 5-specific cells in the in vitro B cell model, leaving unspecific B cells unharmed. Human basophils of grass pollen allergic donors specifically bound P5-ETA', released IL-4 and up-regulated the activation marker CD203c, but were not subject to the toxic effect because of lack of internalization of IgE-bound allergen. CONCLUSION: According to our data, basophils are pure effector cells in the context of IgE-bound allergen and not involved in classical antigen presentation.     

Concentration of airborne mite allergens (Der I and Der II) during sleep

Using a low-noise air sampler and a sensitive radioimmunoassay, we measured the concentration of mite allergens in the air during sleeping with Japanese bedquilts (futon). The airborne allergen levels of Der I (Der p I plus Der f I) and Der II during sleep were 223 and 87.1 pg/m3 of air, respectively. These levels were about 10-fold higher than those during usual domestic life in the living room of the same houses. When the bedquilts were changed to new ones free of mite allergens, the airborne allergen levels of Der I and Der II were decreased to 11.5 and 12.0 pg/m3, respectively. This indicated that the mite airborne allergens during sleep were generated from the used bedding, not from the floor. We believe that exposure to airborne mite allergens during sleep might be an important factor in the development of mite allergies.     

The analysis of specific allergenicity of food allergens families

Classification of food allergens based on protein's structure and function contributes to the study of the relationship between bioinformatics and potential allergenicity of allergens, as well as the evaluation of novel proteins' allergenicity. Some researches were focused on classification within plant or animal food allergens respectively, but there is not any classification of the wholefood allergens. In this article, we classified all the food allergens included in the SDAP into different food allergen families and analysed their specific allergenicity. According to allergen families in AllFam Database, food allergens taken from SDAP (Structural Database of Allergenic Proteins) are classified into different allergen families. Moreover, Protean of DNAStar was applied to analyzing the allergenicity of food allergens. 60% of food allergens are included in the five allergen families: Prolamin superfamily, EF hand domain, Cupin superfamily Profilin and Bet v 1-related protein. Besides, three other cross-food allergen families are found: EF hand domain, Serpin serine protease inhibitor and Triose phosphate isomerase, which include food allergens from both plants and animals. Common characters of food allergens from the same family were easy to find. For instance, they share the same specific peptides and potential T lymphocyte antigen epitope. This classification and characterization of allergen allergenicity in different food allergen families provide foundation for studying the structure and function of novel food allergens as well as the data for the assessment of potential allergenicity.     

主要过敏原的物种分布及逐步聚类分析

目的 综合分析数据库中过敏原的总体状况，在剖析重组过敏原研究领域的共性问题后，对主要过敏原进行聚类分析，为重组过敏原的深入研究指明方向。方法 以国际免疫学联合会过敏原命名分会公布的正式过敏原名录表为原始数据，对过敏原的物种分布特点进行分析，并采用Kolmogorov-Smironov检验对过敏原类型分布与相应的物种分布的一致性进行检验；针对氨基酸序列数据，采用ClustalW 1．83及MEGA2等生物信息学软件，辅之以手动缩减，对来自公共数据库中的主要过敏原进行逐步聚类分析，以获得代表性过敏原。结果 过敏原物种分布分析显示，510个过敏原归属于9大类共179个物种，不同类型过敏原所涉及的物种数及过敏原例数产生的两类分布相同；用关键词在蛋白质数据库中搜索得到的60条主要过敏原序列，聚类成有着明显差异的7个簇后，逐步聚类缩减到21个氨基酸序列无任何相关的代表性过敏原。结论 过敏原的研究是依所涉及的物种不同而逐步平行地展开，没有明显的物种或过敏原的侧重点；依据氨基酸序列的相似性，可以将不同物种来源的主要过敏原缩减到少数代表性过敏原上，从而减少重组过敏原的工作量。     

Quantification of Ara h 1 in peanuts: why roasting makes a difference

BACKGROUND: Increased allergenicity of roasted vs. raw peanut has been reported by showing higher IgE binding to roasted peanut extracts. OBJECTIVE: To study the effect of roasting on Ara h 1 quantification in peanut using a specific monoclonal antibody-based ELISA, and to compare the Ara h 1 content from different kernel size peanuts from four runner cultivars. METHODS: Raw or oven-roasted (177 degrees C for 5-30 min) runner peanuts were crushed and extracted at 60 degrees C. Inhibition ELISA was used to study binding of Ara h 1 purified from raw or roasted peanut. Runner peanuts of four different cultivars were collected, shelled, sized and roasted for 15 min at 177 degrees C. Ara h 1 in the extracts was compared by ELISA. RESULTS: Ara h 1 levels were up to 22-fold higher in roasted than in raw peanuts (820 vs. 37 microg/mL, in a representative experiment) with an Ara h 1 peak at 10-15 min of roasting. Inhibition ELISA indicated that this increase was not due to conformational changes in the Ara h 1 monoclonal antibody epitopes. Ara h 1 was found at lower levels in number 1 than in jumbo- and medium-sized peanuts, and no differences were found among cultivars. CONCLUSION: These results suggest that roasting increases the efficiency of Ara h 1 extraction, and/or that the monoclonal antibody binding epitopes were more accessible in roasted peanut. Expression of Ara h 1 is associated with peanut maturity.     

Cloning of the American cockroach Cr-PII allergens: Evidence for the existence of cross-reactive allergens between species

Background: Previously, we have identified the 28 and 32 kd proteins as additional important allergens from the American cockroach (Periplaneta americana) Cr-PII allergenic fraction. Objective: The aim of this study was the cloning of P. americana Cr-PII allergens. Methods: A λgt22A cDNA library constructed from P. americana mRNA was packaged into Escherichia coli Y1090 (r^–), and clones recognized by murine anti-Cr-PII monoclonal antibodies and human IgE antibodies were isolated, sequenced, and subcloned into pET 21 and expressed in E. coli BL21(DE3). Results: Six Cr-PII–positive clones recognized by human IgE antibodies were isolated. Two clones, C6 and C17, were sequenced, and we found encoding proteins of 228 and 274 amino acids with no cysteine or any potential N-glycosylation site, with predicted masses of 25.8 and 31.14 kd, respectively. Both molecules contain internal repeated sequences with a 94% identity between them. C6 and C17 showed 59% and 77.3% skin reactivities, respectively, on 22 cockroach-sensitive atopic patients. Both clones were found to have 28.9% to 31.8% identities to ANG12 protein, a precursor of the African malaria mosquito (Anopheles gambiae) and 82.7% to 85.1% identity to a nucleotide sequence of the German cockroach (Blattella germanica) Bla g Bd90K allergen. The anti-C6 and anti-C17 antibodies were able to recognize Cr-PII, recombinant proteins, five commercial American extracts, and two German cockroach extracts. Moreover, the binding of anti-C6 and anti-C17 antibodies to recombinant protein can be inhibited by B. germanica crude extract. Furthermore, Northern blot analyses have shown that B. germanica mRNAs could be detected by both cDNA probes. Conclusion: Our findings provide the first evidence of antigenic cross-reactivity between P. americana and B. germanica allergens on molecular levels. The results will be a great aid in facilitating the epitope mapping and improving diagnostic and therapeutic reagents for both cockroach species. (J Allergy Clin Immunol 1998;101:832-40.)J Allergy Clin Immunol 1998;101:832-40.     

The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogeneously recognized among allergic subjects

Pollen allergens are multivalent proteins that cross-link IgE antibodies on mast or basophil cells, inducing secretion of biologic mediators, and resulting in various allergic symptoms. The IgE-binding regions of the Parietaria judaica (Pj) pollen major allergen rPar j 2 were investigated. Twenty-nine single sera from Pj-allergic subjects were tested by Western blot against five recombinant peptides. At least four putative IgE-binding epitopes were identified. The analysis of their diffusion suggested a heterogeneous IgE-binding response. In fact, 75% of the sera reacted with peptide 1-54, 48% with peptide 48-101, 24% with peptide 1-30, 7% with peptide 29-54, and none with peptide 48-76. These five peptides were analyzed with the histamine-release assay. Only peptide 48-101 was capable of inducing degranulation and release of histamine. These results suggest that the recombinant rPar j 2 allergen contains IgE epitopes that are heterogeneously recognized by sensitive patients, and that therefore the therapeutic approach based on the use of haptenic peptides needs a careful evaluation.     

The safety of sublingual-swallow immunotherapy: an analysis of published studies

BACKGROUND: As the main target of sublingual immunotherapy (SLIT) is to reduce at most the occurrence of adverse events (AE), safety represents a critical issue. This aspect deserves particular mention when a higher dose of allergen extract than traditional subcutaneous immunotherapy (SCIT) is required to be effective: that may be up to 500 times that employed for SCIT. OBJECTIVE: All published controlled studies concerning SLIT-swallow were analysed to evaluate AE rates. METHODS: Studies were subdivided in two groups: (i) studies using low allergen dose (LAD), i.e. ranging from 1 to 50 times the dose commonly administered with SCIT, and (ii) studies with high allergen dose (HAD), i.e. ranging from 50 to 500 times the dose administered with SCIT. RESULTS: Twenty-five studies were altogether analysed: 13 studies belonged to the low-dose group, 12 belonged to the high-dose group. We considered all patients with at least one AE. Local reactions were significantly more frequent in the LAD group than in the HAD group (P<0.0001), while there was no difference in the rate of systemic reactions. Severe systemic reactions were never reported. CONCLUSION: This study represents the first analysis of the safety of SLIT concerning the allergen dose employed in the treatment. There is evidence that AE occurrence is substantially not dose-dependent. This fact highlights two main clinical aspects: the elevated tolerability of SLIT in general and the safety of HAD regimen.     

High prevalence of sensitization to cat allergen among Japanese children with asthma, living without cats

BACKGROUND: Cat allergy is common among children with asthma. Many cat-allergic patients in Japan and elsewhere do not keep cats, but nonetheless become sensitized through environmental exposure to cat allergen. OBJECTIVE: To assess the frequency of cat allergy and cat-specific immunoglobulin E (IgE) and immunoglobulin G (IgG) antibody responses in young Japanese patients with asthma in relation to self-reported cat exposure and Fel d 1 levels in dust samples. METHODS: Cat dander-specific IgE antibody was measured in sera from asthma patients using the CAP system. IgE and IgG antibody to Fel d 1 was measured by antigen binding radioimmunoassay and by chimeric enzyme immunoassay. Fel d 1 levels in dust samples from a subset of patients' homes were measured by monoclonal antibody-based enzyme immunoassay. RESULTS: Cat-specific IgE (CAP class>/=2) was found in sera from 70% of 44 patients who kept cats and 34% of 394 patients who had never kept cats. The prevalence of sensitization increased progressively to age 6 years (40%: positive), and then increased gradually to age 16 years (approximately 60%: positive) in patients who had never kept cats. There was an excellent correlation between cat CAP values and IgE levels to Fel d 1. The absolute amount of IgE antibody to Fel d 1 ranged from 0.01 to 15.6% of total IgE. Most patients who did not keep cats were exposed to Fel d 1 levels ranging from 0.07-8 microg/g dust. CONCLUSIONS: Sensitization to cat allergen is common among young asthmatic patients in Japan, even among patients who do not keep cats. Use of CAP and the chimeric enzyme-linked immunosorbent assay allows accurate diagnosis of cat allergy and quantification of specific IgE antibody levels.     

IgE antibodies to recombinant forms of Fel d I: Dichotomy between fluid-phase and solid-phase binding studies

Background: The major cat allergen Fel d I consists of two polypeptide chains linked by disulfide bonds, each of which has been expressed in bacteria. To investigate the antigenic structure of Fel d I, antibody binding to the native molecule and to each recombinant chain were compared. Methods: Polyclonal human IgE and IgG antibodies and monoclonal antibodies (mAbs) to Fel d I were compared for binding to Fel d I, chain 1, or chain 2 by fluid-phase inhibition radioimmunoassay, RAST, and immunoabsorption. Results: In the fluid-phase assay, neither recombinant chain significantly inhibited the binding of antibody to native Fel d I at concentrations of up to 10 μg/ml. Partial inhibition was observed when chain 1 was used, which inhibited the binding of two mAbs by 40% and 75%. In contrast, when the solid-phase RAST assay was used, IgE antibodies bound both chains with high specificity, and there was a good quantitative correlation between IgE antibody binding to Fel d I and both chain 1 (r = 0.58, p < 0.01) and chain 2 (r = 0.47, p < 0.01). Up to 70% of IgG or IgE anti-Fel d I antibodies could be absorbed by either chain 1 or chain 2, and both chains in combination produced similar absorption values in response to native Fel d I. Four mAbs were fully absorbed by chain 1, but not chain 2, and three mAbs were not absorbed by either chain. Conclusions: The results demonstrate a dichotomy between antibody binding to recombinant Fel d I chains, which may be explained by confirmational differences between the chains in the fluid phase or on solid supports. The results also suggest that chain 1 is an important site for mAb-defined B-cell epitopes on Fel d I. (J ALLERGY CLIN IMMUNOL 1995;95:1221-8.)     

Immunological and physical properties of allergen solutions

Lyophilised birch pollen allergen extracts, reconstituted with different diluents (H₂O, saline, Albumin diluent® (AD)) were investigated to determine whether the allergen activity and quality of the extracts deteriorated by nebulization with different nebulizers (Pari, Wright, and Samdoz). Allergen activity was measured by IgG₄ RAST inhibition technique and allergen quality was analysed by crossed immunoelectrophoresis (CIE). The distribution of particle sizes of aerosols different allergen solutions was determined by a TSI Aerodynamic Particle Sizer. A significant difference (P < 0.05) in allergen activity was found between the AD and H₂O diluents before and after using a Sandoz nebulizer and a Wright nebulizer equipped with a small chamber. This suggested greater allergen activity in AD-diluted solutions, and the pattern was repeated with the other two nebulizers, but was not statistically significant. The samples diluted with saline showed no significant differences in quality after nebulization except for the impacted aerosol in which one of the precipitates was slightly diminished. In the AD-diluted sample one of the precipitates disappeared from the impacted aerosol and from the nebulization chamber after 2 min nebulization. To CIE with rabbit anti-human albumin in an intermediate gel. By this procedure one precipitate was transformed to a precipitate in the intermediate gel, indicating that one or more proteins in the extracts may associate with albumin. No significant difference in output was observed between the nebulizers. The particle size distribution curves for each diluent (saline, AD) were identical for the Wright nebulizer with 99% of the dry particles distributed within 0.5–2.0 μm. Although allergen solutions reconstituted with different diluents were not deteriorated in different nebulizers, it may be that the addition of human albumin to an allergen solution may induce one or more proteins in the extracts to associate with it. This could reduce allergen adsorption over periods longer than this study. The use of different diluents did not change the distribution of particle size generated by the nebulizer tested.     

Mammalian lipocalin allergens--insights into their enigmatic allergenicity

Most of the important mammal-derived respiratory allergens, as well as a milk allergen and a few insect allergens, belong to the lipocalin protein family. As mammalian lipocalin allergens are found in dander, saliva and urine, they disperse effectively and are widely present in the indoor environments. Initially, lipocalins were characterized as transport proteins for small, principally hydrophobic molecules, but now they are known to be involved in many other biological functions. Although the amino acid identity between lipocalins is generally at the level of 20-30%, it can be considerably higher. Lipocalin allergens do not exhibit any known physicochemical, functional or structural property that would account for their allergenicity, that is, the capacity to induce T-helper type 2 immunity against them. A distinctive feature of mammalian lipocalin allergens is their poor capacity to stimulate the cellular arm of the human or murine immune system. Nevertheless, they induce IgE production in a large proportion of atopic individuals exposed to the allergen source. The poor capacity of mammalian lipocalin allergens to stimulate the cellular immune system does not appear to result from the function of regulatory T cells. Instead, the T cell epitopes of mammalian lipocalin allergens are few and those examined have proved to be suboptimal. Moreover, the frequency of mammalian lipocalin allergen-specific CD4(+) T cells is very low in the peripheral blood. Importantly, recent research suggests that the lipocalin allergen-specific T cell repertoires differ considerably between allergic and healthy subjects. These observations are compatible with our hypothesis that the way CD4(+) T-helper cells recognize the epitopes of mammalian lipocalin allergens may be implicated in their allergenicity. Indeed, as several lipocalins exhibit homologies of 40-60% over species, mammalian lipocalin allergens may be immunologically at the borderline of self and non-self, which would not allow a strong anti-allergenic immune response against them.     

Etiologic role of unapparent exposure in cat allergy

To determine the importance of unnoticed exposure to cat, we studied 20 patients with a history of respiratory allergy. All the patients had a positive prick test to cat dander extract, and none of them kept cats as pets. The prick test was carried out with a dander extract from cat at a concentration of 100 BU/ml. The specific IgE was determined by the commercially available Pharmacia CAP System. We carried out a conjunctival challenge test. The concentration of Fel d I was quantified in dust samples from the patients' homes by a commercially available method. The patients were reassessed in order to establish a relation between exposure and symptoms, and concealed allergen sources. Sixteen patients, showed significant levels of Fel d I in their homes (mean of 3.35 μg g of dust). The conjunctival challenge test was positive in 15 patients. These patients showed an exposure mean of 0.4 μg/g of dust. The mean levels of specific serum IgE were higher in those patients with a positive challenge than in those with a negative challenge ( P = 0.0145). In nine reassessed patients, a relation was established between natural exposure and the onset of the symptoms. A possible hidden allergen source was established in 11 patients. Hidden exposure to cat allergen may play a role in the symptomatology of many atopic patients, and investigation of sensitization to Fel d I should be included in the routine allergologic evaluation of all patients with asthma or perennial rhinitis.     

Mouse allergen-specific immunoglobulin G4 and risk of mouse skin test sensitivity

BACKGROUND: High serum levels of cat-specific IgG and IgG4 are associated with protection against allergic sensitization to cat, but whether this association applies to other animal allergens remains unclear. OBJECTIVE: To determine if high levels of mouse-specific IgG and IgG4 are associated with a decreased risk of mouse skin test sensitivity. METHODS: Two hundred and sixty workers of a mouse facility underwent skin prick testing and completed a questionnaire. Serum levels of mouse-specific IgG and IgG4 were quantified by solid-phase antigen binding assays. Room air samples were collected and airborne Mus m 1 was quantified by ELISA. RESULTS: Forty-nine participants had a positive skin prick test to mouse. Mouse-specific IgG was detected in 219 (84%) participants and IgG4 was detected in 72 (28%) participants. A detectable mouse-specific IgG4 level was associated with an increased risk of mouse skin test sensitivity (odds ratios (OR) 6.4, 95% confidence intervals (CI) 3.3-12.4). Mouse-specific IgG and IgG4 were both positively correlated with mouse allergen exposure (r(s)=0.31, P=0.0001, and r(s)=0.27, P=0.0006, respectively). The odds of skin test sensitivity peaked at moderate levels of IgG4, but decreased at the highest levels of mouse-specific IgG4. In contrast, the odds of skin test sensitivity increased monotonically with IgG levels. CONCLUSIONS: A detectable level of mouse-specific IgG4 is associated with an increased risk of skin test sensitivity to mouse. However, the highest IgG4 levels appear to be associated with an attenuated risk of mouse skin test sensitivity, suggesting that induction of high levels of IgG4 through natural exposure may protect against the development of allergic sensitization.