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Background An imbalance in gut microbiota is implicated in several pathological conditions, including allergic diseases. This study investigates the association between gut microbiota composition and sensitization to two inhaled antigens. Methods The study comprised 1109 local residents who had participated in the Iwaki Health Promotion Project in 2016. Blood samples were analyzed for levels of antigen-specific IgE against Japanese cedar pollen (JCP) and house dust (HD1). Fecal samples were analyzed for bacterial 16S rRNA (ribosomal ribonucleic acid) using next generation sequencing. The percent composition of gut microbes was compared between patients sensitized and unsensitized group for JCP and HD1 to determine whether the rate of sensitization to inhaled antigens associates with specific bacterial orders composing the gut microbiota. Results In participants aged 20–49 years, the percent composition of Bacteroidales was significantly higher among participants sensitized to JCP than in those unsensitized. The percent composition of Lactobacillales was significantly higher in participants unsensitized to HD group than in those sensitized to that antigen. In addition, participants with low Bacteroidales and high Bifidobacteriales or Lactobacillales has low sensitization rates to HD compared with high Bacteroidales and low Bifidobacteriales or Lactobacillales. Conclusions The presence of bacteria of order Lactobacillales, Bifidobacteriales, and Bacteroidales in the gut microbiota may affect sensitization to inhaled allergens.

Food carries risks. Food allergies are risk that comes from certain food proteins (food allergens). These are natural constituents of food that can provokes life threatening conditions in significant part of population. With this chapter attempt is made to introduce basic data about food allergies and allergens. Focused to the question what makes a food protein an allergen, properties of certain allergens from dominant food allergen families are presented. Those parts of allergen molecule structure responsible for interactions with components of immune system are described. These information are important for understanding molecular mechanisms of allergy, assessment of risks, quality and safety control, and finding a best way for treatment and cure. Some of methods for prediction of allergenicity of food proteins emerged from the current knowledge are described. Food processing methods as a source of potential for increase or reduction of food allergenicity are listed in brief. Most promising strategies for treatment of food allergies are explained.

Foodborne diseases are becoming major scientific struggles for both developing and developed countries. Most foodborne infections are caused by microbial pathogens such as bacteria, viruses, and other food allergens. Hence early and accurate diagnosis of these foodborne pathogens is always preferable. To satisfy these concerns, plenty of isothermal amplification methodologies such as rolling circle amplification (RCA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA) have been developed. Among these, loop-mediated isothermal amplification (LAMP) is a widely usable, potential, and recognizable amplification technique achieved at a constant temperature around 60°C within 30–60 min by using only one kind of enzyme. As a robust gene amplification technique, it can be employed for the detection of bacteria, viruses, and other related food allergens. This technique has its own merits such as cost-effectiveness, facile manufacturing procedure, and consistency. In this chapter, we emphasize recent trends in designing the techniques, challenges, and the future prospects of LAMP in the detection of foodborne pathogens. These effective pathogen detection methods may offer potential benefits compared with existing conventional methods.

Food allergy (FA) is a relatively new food safety issue with an increasing prevalence worldwide and an increasing number of food allergens identified. It is due to an anomalous interaction between food components and immune system causing a broad spectrum of disorders extremely variable among individuals. Although FA-related diseases are restricted to a small category of individuals, the number of patients concerned is continuously growing in the last 10 years, making this field of a primary importance for clinicians as well as for food industry. Several foods are implicated in allergic reactions; however, a total of eight big ingredients have been identified accounting for most of the allergic reactions, that is, milk, egg, wheat, soy, peanuts, tree nuts (e.g., hazelnut, walnuts, pecans, almonds, and cashews), fish, and shellfish. This chapter illustrates the capabilities and potential of MS applied to the detection of allergens in food. Top-down and bottom-up approaches are presented. Finally, performances of high and low-resolution mass spectrometry to meet the desired allergen thresholds are presented.

Background: Der f 7 and Der p 7 are important house dust mite allergens. An IgE-binding inhibition monoclonal antibody WH9 reacts ten folds stronger against Der p 7 than to Der f 7. The purpose of this study is to identify the antigenic determinant(s) and the structural basis of Der f 7 recognize by WH9. Methods: WH9-reactive determinant(s) on Der f 7 was identified by immunoblot and immunoblot inhibition. The 3-D binary complex structures of WH9 and the group 7 allergens were simulated with homology modeling and docking methods. Results: WH9 reacted with the Der f 7 f9 fragment. Among the five site-directed Der f 7 mutants, WH9 showed reduced immunoblot reactivity against Der f 7 S156A, D159A and P160A mutants. Only the wild-type protein and the Der f 7 I157A and L158A mutants can inhibit significantly the WH9-binding against Der f 7. The structural model of the Der f 7-WH9 complex suggests residues S156 and D159 of Der f 7 can bind to WH9 via potential hydrogen bonds. Conclusion: The structure models of Der f 7-WH9 and Der p 7-WH9 complexes revealed that the differential modes of binding of Der p 7 and Der f 7 allergens on WH9 contribute to the differential reactivity of WH9 against the Der f 7 and the Der p 7 mite allergens. Keywords: Der f 7, Der p 7, Homology modeling and docking, MoAb WH9

Since 2–3 decades, food allergies seem to have increased in prevalence. They remain more common in children compared to adults, but some of them have a high rate of resolution in childhood. If peanut, tree nuts, fish, shellfish, egg, milk, wheat, soy and seeds account for the most serious allergic reaction and are acknowledged as being of public health importance; fish, shellfish, tree nuts, peanuts and seeds have a poor rate of rate of resolution in childhood. This article will discuss the prevalence, the symptoms, the eliciting dose, the characteristic allergenic proteins, the potential cross-reactivity of proteins and the potential treatment advances for three persistent food allergies: seafood, tree nuts and peanut. It will conclude with few words regarding VITAL (Voluntary Incidental Trace Allergen Labelling) risk assessment system, the development of reference material and the harmonization of suitable methodologies for the detection of these food allergens.

Alternaria alternata spores are considered a well-known biological contaminant and a very common potent aeroallergen source that is found in environmental samples. The most intense exposure to A. alternata allergens is likely to occur outdoors; however, Alternaria and other allergenic fungi can colonize in indoor environments and thereby increase the fungal aeroallergen exposure levels. A consequence of human exposure to fungal aeroallergens, sensitization to A. alternata, has been unequivocally associated with increased asthma severity. Among allergenic proteins described in this fungal specie, the major allergen, Alt a 1, has been reported as the main elicitor of airborne allergies in patients affected by a mold allergy and considered a marker of primary sensitization to A. alternata. Moreover, A. alternata sensitization seems to be a triggering factor in the development of poly-sensitization, most likely because of the capability of A. alternata to produce, in addition to Alt a 1, a broad and complex array of cross-reactive allergens that present homologs in several other allergenic sources. The study and understanding of A. alternata allergen information may be the key to explaining why sensitization to A. alternata is a risk factor for asthma and also why the severity of asthma is associated to this mold. Compared to other common environmental allergenic sources, such as pollens and dust mites, fungi are reported to be neglected and underestimated. The rise of the A. alternata allergy has enabled more research into the role of this fungal specie and its allergenic components in the induction of IgE-mediated respiratory diseases. Indeed, recent research on the identification and characterization of A. alternata allergens has allowed for the consideration of new perspectives in the categorization of allergenic molds, assessment of exposure and diagnosis of fungi-induced allergies. Keywords: Alternaria alternata allergens, Asthma, Cross-reactivity, Diagnosis of allergy, Exposure to fungal allergens, Poly-sensitization

The allergenic load of house dust mite allergy is largely constituted by a few proteins with a hierarchical pattern of allergenicity. The serodominant specificities are the group 1&2 and the group 23 faecal allergens. The collective IgE binding to the group 1&2 allergens can measure unequivocal HDM sensitisation better than HDM extracts although discrepancies have been found in regions with complex acarofauna suggesting a need to investigate the specificity with allergen components. The group 4, 5, 7&21 allergens that each induce responses in about 40% of subjects are mid-tier allergens accounting for most of the remaining IgE binding. Their titres are proportional to the concomitant responses to Der p1&2. Group 2 allergen variants have different antibody binding. Body proteins only occasionally induce sensitisation although a higher prevalence of binding by atopic dermatitis patients provides a new avenue of research. A broad spectrum of IgE binding has been associated with diverse symptoms but not with the severity of asthma which is associated with low IgG antibody. Some allergens such as the group 14 large lipid binding proteins and the recently described proteins Der f 24–33, need further investigation but with the cognoscence that other denominated allergens have been found to be minor sensitisers by comparative quantitative analyses. Scabies is a confounder for diagnosis with extracts, inducing cross-reactive antibodies with Der p 4&20 as is seafood allergy with cross reactivity to Der p 10 a minor HDM allergen. The HDM genome sequence can now be used to verify allelic and paralogous variations.

PURPOSE OF REVIEW: Molecular allergology uses pure, mainly recombinant and structurally defined allergen molecules and allergen-derived epitopes to study mechanisms of IgE-associated allergy, to diagnose, and even predict the development of allergic manifestations and to treat and prevent IgE-associated allergies. Atopic dermatitis, a chronic inflammatory skin disease is almost always associated with IgE sensitization to allergens. However, also non-IgE-mediated pathomechanisms seem to be operative in atopic dermatitis and it is often difficult to identify the disease-causing allergens. Here we review recent work showing the usefulness of molecular allergology to study mechanisms of atopic dermatitis, for diagnosis and eventually for treatment and prevention of atopic dermatitis. RECENT FINDINGS: IgE sensitization to airborne, food-derived, microbial allergens, and autoallergens has been found to be associated with atopic dermatitis. Using defined allergen molecules and non-IgE-reactive allergen derivatives, evidence could be provided for the existence of IgE- and non-IgE-mediated mechanisms of inflammation in atopic dermatitis. Furthermore, effects of epicutaneous allergen administration on systemic allergen-specific immune responses have been studied. Multi-allergen tests containing micro-arrayed allergen molecules have been shown to be useful for the identification of culprit allergens in atopic dermatitis and may improve the management of atopic dermatitis by allergen-specific immunotherapy, allergen avoidance, and IgE-targeting therapies in a personalized medicine approach. SUMMARY: Molecular allergology allows for dissection of the pathomechanisms of atopic dermatitis, provides new forms of allergy diagnosis for identification of disease-causing allergens, and opens the door to new forms of management by allergen-specific and T cells-targeting or IgE-targeting interventions in a personalized medicine approach.

The proteins in the food are the source of common allergic components to certain patients. Current lists of plant endogenous allergens were based on the medical/clinical reports as well as laboratory results. Plant genome sequences made it possible to predict and characterize the genome-wide of putative endogenous allergens in rice (Oryza sativa L.). In this work, we identified and characterized 122 candidate rice allergens including the 22 allergens in present databases. Conserved domain analysis also revealed 37 domains among rice allergens including one novel domain (histidine kinase-, DNA gyrase B-, and HSP90-like ATPase, PF13589) adding to the allergen protein database. Phylogenetic analysis of the allergens revealed the diversity among the Prolamin superfamily and DnaK protein family, respectively. Additionally, some allergens proteins clustered on the rice chromosome might suggest the molecular function during the evolution.

Background Peanut allergy is increasing at an alarming pace in developed countries. Peanut (Arachis hypogaea) is a common food in Cuba. Nevertheless, reported values of sensitization and symptom severity are usually low. As our objective, we carried out an evaluation of allergic sensitivity to perform an assessment of allergic sensitization and IgE specificity profile to peanut allergens in Cuban allergic patients.Methods The Skin Prick Test (SPT) was performed for each patient, using two glycerinated allergenic extracts, prepared from raw or roasted peanuts. Overall, 316 food allergic patients (159 adults and 157 children) attending allergy services at four hospitals in Havana were included, as well as 303 adult non- allergic volunteers. The IgE binding profile of 26 selected SPT positive patients was further analyzed by immunoblotting.Results The prevalence of sensitization to peanut was 4.6% in general adult population, whereas in adult food-allergic patients it was 18.6%. Prevalence rates were even greater in food allergic children achieving 25.8%. Sensitization frequencies were apparently greater for roasted, as compared to raw peanuts, although the difference was not significant (p> 0.05, Mc Nemar's). IgE binding was shown mostly by the 15 and 17 kDa bands, tentatively identified as the major allergens Ara h 2 and Ara h 6. The IgG4 binding profile was similar to IgE, although with more prominence of the bands at 37 and 28 KDa, corresponding to an Ara h 3 fragment and Peanut Agglutinin.Discussion The study estimated a relatively high prevalence of peanut sensitization in population. Data reported here suggest that IgE sensitization in Cuban patients is focused mostly on MW bands corresponding to the major allergens Ara h 6 and Ara h 2.Conclusions Sensitization to peanut allergen is indeed relatively frequent in Cuba. The IgE profile is congruent to a sensitization pattern by ingestion of roasted peanuts and is directed to well-known major allergens. Keywords: Allergic sensitization, Peanut allergens, Food allergy, IgE specificity

BackgroundPeanut allergy is a severe and increasingly frequent disease with high medical, psychosocial, and economic burden for affected patients and wider society. A causal, safe, and effective therapy is not yet available.ObjectiveWe sought to develop an immunogenic, protective, and nonreactogenic vaccine candidate against peanut allergy based on virus-like particles (VLPs) coupled to single peanut allergens.MethodsTo generate vaccine candidates, extracts of roasted peanut (Ara R) or the single allergens Ara h 1 or Ara h 2 were coupled to immunologically optimized Cucumber Mosaic Virus–derived VLPs (CuMVtt). BALB/c mice were sensitized intraperitoneally with peanut extract absorbed to alum. Immunotherapy consisted of a single subcutaneous injection of CuMVtt coupled to Ara R, Ara h 1, or Ara h 2.ResultsThe vaccines CuMVtt-Ara R, CuMVtt-Ara h 1, and CuMVtt-Ara h 2 protected peanut-sensitized mice against anaphylaxis after intravenous challenge with the whole peanut extract. Vaccines did not cause allergic reactions in sensitized mice. CuMVtt-Ara h 1 was able to induce specific IgG antibodies, diminished local reactions after skin prick tests, and reduced the infiltration of the gastrointestinal tract by eosinophils and mast cells after oral challenge with peanut. The ability of CuMVtt-Ara h 1 to protect against challenge with the whole extract was mediated by IgG, as shown via passive IgG transfer. FcγRIIb was required for protection, indicating that immune complexes with single allergens were able to block the allergic response against the whole extract, consisting of a complex allergen mixture.ConclusionsOur data suggest that vaccination using single peanut allergens displayed on CuMVtt may represent a novel therapy against peanut allergy with a favorable safety profile.

Allergy is a widespread disease effective 10%–20% of the general population all around the world. Allergies are chronic illnesses caused by the exposure of the respiratory system to dust or pollen, the skin to chemical materials, or the digestion system to particular foods. Immunoglobulin E (IgE) antibody plays a very important role in the development of allergies in humans. The high level of total IgE in human serum reflects the presence of allergic conditions. Test kits for commercially available serum IgE measurements are based on conventional immunoassay techniques. These techniques include time-consuming procedures, expensive instruments, and hazardous labels. Currently, molecularly imprinted polymer-based sensors are utilized as an alternative artificial receptor. MIP-based sensor systems for biological applications have been widely utilized in works on biomolecular interactions, with the change in sensor signals. Herein, the development of rapid, easy-to-use and cost-effective allergen detection methods based on mass sensitive, optic, electrochemical, and nanomaterials based sensors have been reported. Finally, we present sensor systems were investigated with a molecular imprinting technique for the detection of allergens as a marker of various allergies.

Background Allergenic cross reactivity between the members of the Pooids (Lolium perenne , Phleum pratense , and Poa pratensis) and Chloridoids (Cynodon dactylon and Paspalum notatum) is well established. Studies using crude extracts in the past have demonstrated limited cross reactivity between the Pooids and the Chloridoids suggesting separate diagnosis and therapy. However, little is known regarding the molecular basis for the limited cross reactivity observed between the 2 groups of grasses. The present study was undertaken to gain insights into the molecular basis of cross allergenicity between the major allergens from rye and Bermuda grass pollens. Methods Immunoblot inhibition tests were carried out to determine the specificity of the proteins involved in cross reactivity. Crude pollen extract and bacterially expressed and purified recombinant Lol p 1and Lol p 5 from rye grass were subjected to cross inhibition experiments with crude and purified recombinant Cyn d 1 from Bermuda grass using sera from patients allergic to rye grass pollen. Results The immunoblot inhibition studies revealed a high degree of cross inhibition between the group 1 allergens. In contrast, a complete lack of inhibition was observed between Bermuda grass group 1 allergen rCyn d 1, and rye grass group 5 allergen rLol p 5. Crude rye grass extract strongly inhibited IgE reactivity to Bermuda grass, whereas crude Bermuda grass pollen extract showed a weaker inhibition. Conclusions Our data suggests that a possible explanation for the limited cross reactivity between the Pooids and Chloridoids may, in part, be due to the absence of group 5 allergen from Chloridoid grasses. This approach of using purified proteins may be applied to better characterize the cross allergenicity patterns between different grass pollen allergens.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10μg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.

In almost all countries, food allergy is of growing concern affecting all age groups. Given the increased prevalence of food allergies, current research focuses on developing new treatment strategies and to predict allergenicity of novel and modified food proteins. The recent use of animal models has significantly contributed to a better understanding of the complex immunological and pathophysiological mechanisms of food allergies. Central to the development of food allergy is the allergic cascade driven by cells of the innate and adaptive immune system. These models can now be integrated into the risk assessment of possible allergenic proteins. In this review, we discuss the role of the immune system as a qualitative readout for the sensitizing potential and risk assessment of food proteins.

Environmental allergens, such as pollens and house dust mite allergens, cause sensitization and lead to allergic diseases, which make people suffer from asthma or rhinitis. These environmental allergens often become airborne in a living space, and residents are endangered to be exposed to and inhale the airborne allergens. Therefore, prevention of the exposure directly connects to prevention of allergic diseases. One of the possible ways to avoid the exposure is monitoring of airborne allergens in a living space and making a resident become aware of the environmental quality. Such a system requires a sensor, and especially with high selectivity along with a sensitivity to the allergens. For this purpose, immunosensors are the candidates due to their high selectivity, which relies on the specificity of an antibody. A challenge to use immunosensors for monitoring is difficulty of sensor surface regeneration—breaking down a formed immunocomplex and removing them from the sensor surface for a next measurement. In this chapter, we describe a reusable immunosensor for monitoring of a HDM allergen, Dermatophagoides farinae group 1 (Der f 1). The immunosensor utilizes a surface acoustic wave device and surface modification with pH-tolerant protein scaffold (ORLA85 protein) for capture antibody immobilization. Der f 1 is measured by detecting viscoelastic change induced by sandwich assay with detection antibodies. In addition, the use of gold nanoparticles for amplification of the sensor’s output signal is also discussed.

A review of cockroach allergen and its role in allergic disease is presented. Relationships among cockroach allergen exposure, allergic sensitization, and disease are described, prevalence and exposure risk are characterized, and research on reducing exposures is discussed. Studies have demonstrated that the combination of sensitization and exposure to cockroach allergens is associated with asthma morbidity, and some have concluded that levels of cockroach allergen in homes may be one of the strongest risk factors predictive of allergic sensitization and asthma morbidity in children. Other research has found that cockroach allergens are detectable in most homes in the United States and many other regions. The prevalence of cockroach allergens is particularly high in urban areas and low-income settings, but is not limited to these environments as studies have found cockroach exposure and sensitization in suburban and rural areas as well. Practical measures to reduce environmental exposures have had mixed success; many have achieved reductions in cockroach populations, but not a corresponding, sustained reduction in allergen exposure. However, a few studies have achieved a sustained reduction in allergen levels through sophisticated cockroach eradication measures. It remains of interest to demonstrate associated improvements in asthma morbidity.

Background: The diagnosis of allergic diseases with recombinant allergens allows us to detect antibodies specific for single allergens in extracts. The aim of the present study was to assess the early effect of grass pollen immunotherapy on IgE and IgG4 responses to eight purified grass pollen allergens in patients undergoing hyposensitization treatment. Methods: The sera of 22 consecutive atopic individuals undergoing cluster regimen grass pollen immunotherapy were analyzed for IgE and IgG4 antibodies specific for grass pollen allergens (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Two serum samples were taken, one before the start of therapy and one between 12 and 15 weeks after the first immunization. Immunotherapy was performed with two allergy vaccines comprising a standardized extract aluminum-adsorbed grass pollen mix and a standardized extract of grass pollen mix adsorbed onto calcium phosphate. Results: One treated patient showed a specific IgE conversion from negative (

Allergy is a type of abnormal immune reactions, which is triggered by environmental antigens or allergens and mediated by IgE antibodies. Now-a-days mass spectrometry is the method of choice for allergen identification based on homology searching. Here, we provide the mass spectrometry dataset associated with our previously published research article on identification of sunflower pollen allergens (Ghosh et al., 2015 [1] ). In this study allergenicity of sunflower ( Helianthus annuus ) pollen grains were primarily investigated by clinical studies followed by detailed immunobiochemical and immunoproteomic analyses. The mass spectrometry data for the identification of allergens were deposited to ProteomeXchange Consortium via PRIDE partner repository with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002397 .

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components. © 2012 Elsevier Ltd. All rights reserved., This work was supported by Projects AGL2008-01740 and AGL2011-24740. We also acknowledge CYTED P109AC-0302 and COST ACTION Infogest FA1005. G. M. is in receipt of a JAE-CSIC Ph. D. fellowship.

Proteins are ubiquitous and contribute to the technofunctional characteristics of several foods, and also serve as nutrients. However, some dietary proteins are allergens and their presence and immunological mechanisms can elicit acute response in sensitized/allergic individuals. Allergenic foods include foods from both plants and animals such as peanuts, tree nuts, wheat, soy, cow's milk, eggs, fish, and shellfish. A sizeable number of the human population is hypersensitive to immunoglobulin E (IgE)-mediated reactions and some form of food allergy, which affects their lifestyle. During the course of food processing, cross-contamination and changes in protein structure and conformation may affect their physicochemical properties and induce allergenicity. Both conformational and linear epitopes and interaction of food proteins with other ingredients may be affected by processing. These processes can lead to secondary and tertiary structure modifications and response of IgE immune system by reducing intramolecular disulfide bonds of the proteins and epitope accessibility through destruction or masking. Nonetheless, some allergens are stable to processing and it is imperative to select effective processing methods and parameters to reduce allergenicity without contributing to the formation of neoallergens (new allergenic epitopes) or increased allergenic potential. The efficacy of the deactivation technique can be established by reliable detection and identification methods. For both established and new protein sources, the need to establish their safety and threshold levels in food products are important to consumers, food manufacturers, and public health and regulatory agencies. Efforts are needed to provide food composition information with the declared allergenic ingredients or the presence of traces of allergens due to contamination or that formed during food processing, present allowable limits in the products, and determine if a manufactured food is safe for consumption. This chapter describes the current and emerging techniques for detecting and deactivating food allergens.

Immunoproteomics, IgE-inhibition assays and cDNA-cloning reveals that ash and olive allergenic protein profiles are mostly equivalent, thus explaining their high cross reactivity. Our data suggest simplifying diagnosis of patients by using indistinctly ash or olive pollen.

One of the main concerns about genetically modified foods and their potential impacts on human health is that the introduction of a new/ altered gene may putatively alter the expression of others, namely endogenous allergens. We intended to evaluate, and to compare, using quantitative real time RT-PCR technique, the expression of 5 already known maize allergens (Zea m14, Zea m25, Zea m27kD, 50kD Zein and trypsin inhibitor) in MON 810 vs. its non-transgenic counterpart, throughout seed development (10, 16 and 23 days after pollination). We have shown that none of the tested allergen genes presented differential expression, with statistic significance, along all tested seed development stages, in MON810 vs. its conventional counterpart. We have also used bidimensional gel electrophoresis followed by Western blotting with plasma from two maize allergic subjects to characterize their immunologic responses against MON 810 vs. its non-transgenic control. Immunoreactive spots were characterized by MS. We have identified fourteen new IgE-binding proteins present in both transgenic and non-transgenic maize.

Indoor and outdoor allergen sources contain several allergens. The majority are proteinaceous, although carbohydrate epitopes, particularly α-Gal, may be significant. The most important outdoor allergens are derived from the pollens and fungal spores, and phylogenetically-related species produce similar spectra of allergens. Although no unifying structural feature determines allergenicity, the biochemistry of allergens, as well as their association with non-allergen derived components may be significant. Indoor allergens are derived from dust mites, pets, cockroaches and rodents. Submicronic allergen-containing particles will penetrate into the lung and remain airborne for longer periods compared with larger particles, which will be trapped in the upper airways. Allergen avoidance can be an effective countermeasure for asthma, perennial rhinitis, and atopic dermatitis but compliance and sufficient allergen reduction may be difficult. Air pollution may occur both indoors and outdoors and exacerbate asthma symptoms, as well as contribute to the development of disease through direct, non-allergic, effects or by interactions with allergens per se .

This paper reviews the in vitro digestion models developed to assess the stability digestion of food allergens, as well as the factors derived from the methodology and food structure that may affect the assay results. The adequacy of using the digestion stability of food allergens as a criterion for assessing potential allergenicity is also discussed. Data based on the traditional pepsin digestibility test in simulated gastric fluid are discussed in detail, with special attention to the influence of the pH and pepsin: allergen ratio in the pepsinolysis rate. This review points out the importance of using physiologically relevant in vitro digestion systems for evaluating digestibility of allergens. This would imply the sequential use of digestive enzymes in physiological concentrations, simulation of the stomach/small intestine environment (multi-phase models) with addition of surfactants such as phospholipids or bile salts, as well as the consideration of the gastrointestinal transit and the effect of the food matrices on the allergen digestion and subsequent absorption through the intestinal mucosa. In vitro gastrointestinal digestion protocols should be preferably combined with immunological assays in order to elucidate the role of large digestion-resistant fragments and the influence of the food matrix on the stimulation of the immune system., This work was supported by the European Union (Project MERG-CT-2004-01170) and the CSIC-Comunidad de Madrid (Project 200570M066). F.J.M. is recipient of an I3P-CSIC contract.

Publisher Summary This chapter discusses the status and future potential of plant biotechnology in reducing allergens in food. Food allergic reactions affect 6-8% of children and 1-2% of the adult population. Many allergic reactions are to plant origin, including peanuts, soy, wheat, and tree nuts. Allergic reactions are typically elicited by a defined subset of proteins that are found in abundance in food. The increased prevalence of allergic reactions coupled with severe clinical symptoms has led many scientists to explore methods of reducing allergenicity of some crops. This chapter explores the potential of reducing the allergenicity of plants used as food crops by both traditionally breeding practices and genetic engineering methods. The chapter reviews how biotechnology began and progressed, the uses of biotechnology and points out its uses in decreasing levels of known allergens or their allergenicity. The characteristics of food allergens are first explored. Traditional plant breeding methods for reducing allergenicity are discussed. Use of genetic engineering to reduce allergenic potential is also described focusing on post-transcriptional gene silencing, reduction of disulfide bonds by thioredoxin, and modification of genes encoding allergens.

Background : In Japan, hydrolyzed wheat proteins (HWP) have been reported to cause wheat-dependent exercise-induced anaphylaxis (WDEIA) by transcutaneous sensitization using HWP-containing soap. Patients develop allergic reactions not only with soap use, but also with exercise after the intake of wheat protein (WP). ω5-Gliadin and HMW-glutenin were identified as major allergens in conventional WP-WDEIA patients. However, the allergens in HWP-WDEIA have yet to be elucidated. Methods : Sera were obtained from 22 patients with HWP-sensitized WDEIA. The allergenic activities of HWP and six recombinant wheat gluten proteins, including α/β-, γ-, ω1,2- and ω5-gliadin and low- and high molecular weight (HMW)-glutenins, were characterized by immunoblot analysis and histamine releasing test. IgE-binding epitopes were identified using arrays of overlapping peptides synthesized on SPOTs membrane. Results : Immunoblot analysis showed that IgE antibodies (Abs) from HWP-WDEIA bound to α/β-, γ-, and ω1,2-gliadin. Recombinant γ-gliadin induced significant histamine release from basophils in eight of 11 patients with HWP-WDEIA. An IgE-binding epitope "QPQQPFPQ" was identified within the primary sequence of γ-gliadin, and the deamidated peptide containing the "PEEPFP" sequence bound with IgE Abs more strongly compared to the native epitope-peptide. The epitope-peptide inhibited IgE-binding to HWP, indicating that the specific IgE to HWP cross-reacts with γ-gliadin. Conclusions : HWP-WDEIA patients could be sensitized to HWP containing a PEEPFP sequence, and WDEIA symptoms after WP ingestion could partly be induced by γ-gliadin. These findings could be useful to help develop tools for diagnosis and desensitization therapy for HWP-WDEIA.

The intestinal tract and lung represent large surface areas of the body in which there is continuous contact with an array of ingested or inhaled antigens and microorganisms. The induction of physiological tolerance to harmless antigens and commensal microorganisms is vital to avoid unwarranted excessive inflammation; yet, the ability to mount efficient inflammatory responses to pathogens remains important. This dynamic balance between tolerance to allergens and commensal microorganisms versus inflammatory responses to pathogens is coordinately regulated by innate and adaptive components of the mucosal immune system. Interactions among epithelial cells, antigen-presenting cells, and induced regulatory T cells (iTreg) mediate the development of physiological tolerance, with iTreg playing a critical role. This chapter describes the mechanisms that mediate physiological tolerance to ingested and inhaled allergens.

The in vitro gastric digestion of several food allergens (beta-lactoglobulin (BLG), alpha-lactalbumin (LA) and peanut allergens (PE)) in the presence of a catechin-enriched polyphenol extract of green tea (GTC), oxidized polyphenols and phenol oxidase processed food allergens and GTC was investigated. Pepsin-resistant proteins, such as BLG, major peanut allergens, Ara h 1 and Ara h 2, degrade faster in the presence of catechin-enriched green tea polyphenols. Phenol oxidase polymerized GTC affected adversely protein digestion of BLG and LA, but not digestion of PE proteins. Protecting effect of polyphenols correlated well with the ability of proteins to form insoluble complexes with oxidized catechins. Cross-linking of proteins and polyphenols further extended the half-lives of BLG and LA in the in vitro digestion by pepsin. Catechin-enriched green tea polyphenols of food supplements facilitate pepsin digestion of major food allergens, but hamper their digestion if oxidized and polymerized by phenol oxidase. (c) 2012 Elsevier Ltd. All rights reserved.

Helminth infections and allergic diseases elicit strong Th2 responses. The up-regulation of IL-4, IL-13, IL-5, IL-9, total IgE, eosinophils and macrophages are part of the host immune response against helminths and also compose an immunological pattern that accompanies allergic diseases. These facts together with the common features observed between allergens and parasite antigens suggest that the immune response to helminths and allergy could have developed under the same evolutionary process. However, asthma can have a positive or negative association with helminth infections, depending on several factors, such as the helminth species, the clinical status of infection, host genetics, or intensity of infection. The model of immune regulation observed in helminths can be very useful for developing alternative strategies for the prevention, treatment, and control of inflammatory diseases including allergic diseases. Helminth infections induce a complex immune regulatory network that utilizes dendritic cells, Treg cells and systematic elevated levels of IL-10 and possibly TGF-β, B reg cells and activated macrophages to achieve chronic asymptomatic conditions that can be used as a model to design the aforementioned strategies.

Several important nematode parasites have been found to express members of a gene family variously termed as venom allergen antigen homologue (vah) or Ancylostoma secreted protein (asp). In some cases these products are secreted by infective larval stages and have been suggested to be effective vaccine immunogens. We isolated the corresponding gene from the human filarial nematode, Brugia malayi, by first searching the expressed sequence tag (EST) dataset generated by the Filarial Genome Project and then using gene-specific nondegenerate primers matching the selected gene for PCR, from B. malayi cDNA libraries. We report here the full-length gene sequence, which we have designated as Bm-val-1, for vah/asp-like. The corresponding protein (Bm-VAL-1) contains 232 amino acids in a single homology unit, unlike products from some other species in which there is a tandem repeat. A putative signal sequence is present at the 5' end and there are two potential N-glycosylation sites. Murine antibodies to recombinant Bm-VAL-1 react with a 28 kDa protein in L3 extracts and recombinant Bm-VAL-1 is recognised by murine T cells primed with soluble L3 proteins. Of 82 ESTs corresponding to Bm-val-1, 72 are recorded from the infective larval (L3) stage. However, PCR on the first-strand cDNA libraries from later mammalian stages revealed some expression at most subsequent time points. Over 95% (20/21) of microfilaraemic human filariasis patients are seropositive for antibodies to Bm-VAL-1, with particularly high levels of IgG3 and IgG4 isotypes. The IgG4 subclass may indicate stimulation by adult and/or microfilarial-derived immunogens. The association of Bm-VAL-1 with the infective stage and its recognition by humans exposed to filariasis suggests that further evaluation of this antigen as a vaccine candidate should be performed. © 2001 Published by Elsevier Science B.V.

IgE antibodies circulate in the blood of individuals sensitized to food allergens. This chapter overviews in vivo and in vitro assays that employ human IgE antibodies as reagents for the detection and quantification of select allergens in extracts of foods. More specifically, judicious application of dilutions of physiological food extracts into the skin of humans known to be allergic to a specific food allows detection and semi-quantitative measurement of the allergen in food extracts. Alternatively, human serum containing IgE antibodies to known food allergens can be used as reagents in a competitive inhibition allergosorbent assay to quantify allergen in unknown food extracts. Performance characteristics, assay limitations and assay quality control are discussed for in vivo and in vitro assays.

Background IgG antibody to allergens in the serum of pollinosis patients is not routinely measured, because there are no simple methods for assaying small amounts of specific IgG in the serum ; so far only IgE and IgG4 antibodies have been assayed. In this study, we used a reverse-sandwich ELISA for measuring specific non-IgE,-IgG4 (probably, mainly IgG1) to Japanese cedar pollen allergens, and compared the antibody-positive rates and geometric mean antibody titers between pollinosis patients and non-patients (healthy individuals). Methods Antibodies to two major allergens of Japanese cedar, Cry j 1 and Cry j 2, were assayed by the following methods : specific non-IgE,-IgG4 was measured by a reverse-sandwich ELISA ; specific IgE and IgG4 were measured by indirect ELISAs. Results We detected specific non-IgE,-IgG4 in both the patients and non-patients. In comparison to the IgE antibody, which was detected in a small proportion of the non-patients with low titers, the non-IgE,-IgG4 antibody was present in a higher proportion with higher titers among both the patients and non-patients. Conclusions Healthy individuals had specific non-IgE,-IgG4 to Japanese cedar allergens in a higher proportion than the specific IgE. The non-IgE,-IgG4 antibody assay may be useful in studies on the prevalence of allergen-specific antibody responders and may help in clarifying the natural history of pollinosis.

The polymerase chain reaction (PCR) allows identifying and quantifying the culprit allergenic food for verification of compliance with food labelling requirements or the identification of so-called hidden allergens. For more than one decade, highly specific and sensitive nucleic acid-based methods for allergen detection, especially applications of PCR, have been under development. Published and commercial methods are available. Meanwhile, PCR has been introduced as official method for allergen detection in Japan and Germany. This chapter highlights the prospects of PCR for allergen detection, achievable levels of sensitivity and specificity, and its potential for quantitative analysis and multi-allergen detection.

In the last decades allergies are increasing in the western countries and different protein families were identified as determinants of allergenicity. In apple fruit, one of the most important fruit crop in the world, four allergen families (Mal d 1–4) have been identified so far, including pathogenesis-related proteins 10, thaumatin-like proteins, lipid transfer proteins and profilins. Moreover, it has been demonstrated that patient sensitivity differs in relation to apple variety, fruit tissue, cultivation and conservation conditions, increasing the difficulties to associate genetic, molecular and biochemical data with the results of clinical tests. Mapping studies and the availability of the complete apple genome sequence confirmed that allergens are organized in large families with many different isoforms, the contribution of which to allergenicity is still largely unknown. The unravelling of allergy determinants is crucial for different reasons: • to better understand environmental influences on allergenicity. • to produce genetically modified plants with a reduced allergen content through a targeted-gene silencing approach. • to drive future breeding programs towards hypoallergenic apples through the exploitation of natural variability. Finally, the above-mentioned knowledge will be essential to allow the consumption of this fruit by individuals who are allergic to apples.

Publisher Summary This chapter examines the influence of allergen exposure during development. Exposure to allergen can occur during fetal, neonatal, and juvenile years, which are critical times for immunological and structural development of the lung. Exposure of the fetus to allergens inhaled by the mother during pregnancy can result in its sensitization to those allergens. The interaction of environment and genetic background during in utero life of a human infant may determine the future allergic status of the child and adult. The exposures that occur in the first months of life are very likely crucial to phenotypic development of the child and subsequently the adult. Thus, exposures to allergens, such as pollens, may facilitate future development of allergic disease, especially in the atopy prone genotype. However, exposure to certain other antigens during this critical time of development can influence or modulate the immune response. Several infectious diseases are thought to have a role in either facilitating development of allergic sensitization to inhaled allergen during the neonatal period or subverting the allergic response.

Mite allergens Derpl and Derfl were tested in 200 house dust samples using a nitrocellulose membrane- based dot-blot assay. The assay enables the mite allergens in a dust extract to be detected with results that can be visually read semiquantitatively within 30min. Positive results obtained with this method were easily observed as brown dots. The Derp1 and Derf1 allergens were also precisely quantitated by a two-site monoclonal antibody based enzyme-linked immunosorbent assay. Results of Derp1 and Derf1 mite allergens measured from the two methods were compared. The semiquantitative analysis using Spearman's correlation yielded a correlation coefficient of 0.892 (P

In cases of occupational asthma due to reactive chemicals, an appreciable number of patients have persistent asthmatic symptoms and airway inflammation even after several years' avoidance of the cause agents. A case of late respiratory systemic syndrome (LRSS) caused by phthalic anhydride is reported. The patient showed a progression of bronchial asthma due to house dust mite and developed a new bronchial sensitization to horse hair during the 5 year follow-up period. The patient was diagnosed in September 1990 as having LRSS due to phthalic anhydride. He was atopic and had worked in a factory preparing materials for paints for 8 years. After leaving his workplace and commencing treatment with anti-asthmatic medications, his asthmatic symptoms and airway hyper-responsiveness were much improved for 1year (PC20 methacholine level was increased from 0.44 to 4.4 mg/mL). For several months before the second admission (October 1992) his asthmatic symptoms were again aggravated and methacholine PC20 decreased to 1.1mg/mL without improvement in the following 3 years. The level of serum specific IgE antibody to phthalic anhydride has been gradually decreasing, while specific IgE to two types of house dust mite and horse hair has been increasing year by year during the last 3 years' follow-up period. These findings suggest that chronic exposure to inhalant allergens can induce the progression of allergen-induced airway inflammation, and result in new bronchial sensitization to inhalant allergens. Careful follow-up study is needed to detect new developments of allergen-induced bronchoconstriction in occupational asthma patients with persistent asthmatic symptoms.

Allergic diseases are characterized by excessive and inappropriate responses to allergens leading to a Th2 skewed immune environment. While the exact mechanisms involved are not completely understood, Th2 polarization is likely to involve both dendritic cell (DC) and T cell dependent factors. Furthermore, allergens themselves are now no longer viewed as inert proteins, but have their own biological properties that can skew immune responses. Such "intrinsic properties" of allergens may influence DC function, and this subsequently has profound effects on allergic inflammation. This chapter will focus on common aeroallergens such as house dust mite (HDM) and pollens, and the mechanisms by which these allergens alter DC cytokine, prostaglandin, and chemokine synthesis and the expression of costimulatory molecules. Some of the effects of allergens on DC differ between atopic and healthy subjects, while other DC responses are similar in both groups. This chapter will also discuss how HDM and pollen allergens activate DC, and the mounting evidence to suggest that pathogen recognition receptors, in particular the C-type lectins, play an important role in the dysregulated allergic immune response. While most of the population is exposed to common aeroallergens, only a proportion will develop allergic disease and our understanding of allergen-DC-T cell interactions and mechanisms involved in this process is increasing. In the future this should allow for more targeted therapies to reduce the impact of allergic disease.

Publisher Summary The effects of processing on the allergenicity of foods can be viewed either from the diversity of technological processes or the common physicochemical effects that accompany them. This chapter presents the latter approach and provides an overview of the impact that food processing has on major types of allergenic proteins, both regarding the types of modification and their impact on allergenicity (particularly elicitation potential). The chapter highlights the different ways in which food processing can modify food protein structure, including both events involved in unfolding and aggregation and those relating to covalent modification, in particular non-enzymatic glycation reactions. There are no clear rules regarding how different allergens respond to food processing, with some, such as the Bet v 1 family of allergens found in fruits, clearly having their allergenicity destroyed by cooking, while for many others it is unaltered. On the other hand the allergenicity of certain foods, such as peanut, may even increase following food processing. Such complexity makes managing allergens in foods difficult but shows the importance of understanding the molecular basis of the effects of processing if food manufacturers are to move toward knowledge-based ways of managing allergen risks during processing. This is also important in supporting the allergenic risk assessment process which forms part of the regulatory framework pertaining to novel foods and processes.

In comparison with other helminths that afflict humans on a global scale, Ascaris lumbricoides and ascariasis are relatively under-researched in terms of the proteins and other products to which the parasites expose their hosts. The reputation of the parasite as a source of potent allergens, and the recognition that immune hypersensitivity in the lung caused by migrating larvae is a significant problem, has served to focus attention on immune responses to Ascaris. Research has increasingly focused on the parasite’s products and responses to them in experimental rodents, and in pigs as permissive hosts for the full life-cycle of Ascaris suum. In parallel, immunoepidemiology of ascariasis in humans has been developing, often aimed at understanding how the infection polarizes immune responses. This review deals with the antigens of the parasite, how the immune repertoire to them varies dramatically between infected humans, the genetic loci that are probably involved, and its unpredictability. New work on the molecular structure of the unusual proteins produced by Ascaris and other nematodes is also covered, including the well-known ABA-1 allergen. Emphasis is also placed on a poorly understood aspect of Ascaris immunobiology, prompted by a few tantalizing observations, namely the potential diversity of surface and secreted antigens of the human parasite, and how such polymorphisms could be crucial to understanding the chronicity of ascariasis in the face of continuous loss and recruitment of the parasites themselves.

Olive pollinosis represents a major public health problem, prevalence of which is growing worldwide. Update 14 olive pollen allergens have been characterized and recombinant produced, which is required for developing standardization, diagnosis, and immunotherapy strategies. Olive pollen allergens are carried by airborne nanovesicles released by pollen during germination and subsequent tube growth. An in vitro primary normal human bronchial epithelial cell-based model has been established to study the role of the functional state of bronchial epithelium in the aeroallergen-specific response. Novel designs for pollen allergy treatment are required; thus prophylactic nasal vaccines based on exosomes and hypoallergens have been assayed in an olive pollinosis mouse model. Moreover, the immunomodulatory capacity of Ole e 1-derived peptides has been analyzed in peripheral blood mononuclear cells from allergic patients. Finally, an olive fruit thaumatin-like protein has been involved in occupational asthma, and the olive seed allergenicity has been assayed to promote as superfood candidate.

Publisher Summary This chapter explores the role of mass spectrometry in the identification of particular allergens and of the reaction the human body stages to the flowers and other parts of the elderberry tree. Nine patients with a long history in summer hay fever were tested for symptoms after inhalative and dietary contact with elderberry products. All of them reported rhinoconjunctivitis; four of them even exhibited asthmatic symptoms. As patients may be exposed to the allergens hailing from Sambucus nigra via the oral route—flowers and fruits have been used in plant remedies and food for centuries—it has been of special interest that one patient developed upper-airway obstruction when drinking elderberry juices. Four patients showed strong reactions after skin prick tests (SPT), medium response was observed in two cases, and negative results were received for three persons including the patient exhibiting airway obstruction. IgE serum levels measured by radioallergosorbent tests (RAST) also varied significantly. In some cases no serum IgE was detectable; in another case up to 4080 kU/L was measured. The fuzzy characteristic of the immuno-detected spots after 2D gel electrophoresis pointed out that the allergy elicitor may be just one protein, posttranslationally modified, such as by carbohydrate moieties, or that the immunological response results from highly homologous proteins with just minor variation in their polypeptide sequence.

Publisher Summary This chapter focuses on allergy and its mechanisms with emphasis on the relevance of allergen in the pathogenesis of asthma. Allergens are recognized as potential causes of allergy and thus asthma. Atopy is the tendency to develop immunoglobulin E (IgE) antibodies to commonly encountered environmental allergens by natural exposure in which the route of entry of allergen is across intact mucosal surfaces. Inhaled complete allergens that provoke asthma by IgE- mediated mechanisms are soluble organic high-molecular-weight protein or protein-containing molecules, which are derived from the plant or animal kingdoms. The important inhalant allergens commonly include pollen, fungal spores, animal danders, and household mites or insects. Other allergens often encountered in occupational settings include various plant parts, insect dusts, and bacterial enzymes. Chronic or recurrent allergen exposure leads to persistent asthma. Early anti-inflammatory therapeutic strategies can result in improved long-term outcomes in allergic asthma and in the primary prevention of allergic asthma.