Real-life evaluation of molecular multiplex IgE test methods in the diagnosis of pollen associated food allergy.

BACKGROUND: Diagnosis of food allergies is challenging, as combining information from specific IgE (sIgE)-sensitization pattern and skin prick tests (SPTs) with clinical history is necessary for a personalized management of allergic patients. The aim of this study was to compare two molecular tests, the ImmunoCAP ISAC (ISAC) and the Allergy Explorer, version 2 (ALEX2 ) in the context of pollen food syndrome (PFS) diagnosis in a real-life scenario, to assess the benefit of multiplex testing in PFS patients. METHODS: Diagnosis of food allergy was performed in 53 patients. Allergen-sIgE concentrations were measured with ISAC and ALEX2 . Results for sIgE were statistically compared with each other, with SPT results and with clinical presentation of the patients. RESULTS: Using ISAC as reference test for sIgE measurements, the average sensitivity of ALEX2 for PR-10 allergens was 83.2% and the average specificity 88.0%. If only low sIgE concentrations were included, the sensitivity was 60.8% and the specificity 91.1%. Apple and hazelnut sensitizations were confirmed in most patients by concordance of sIgE and SPT results. Significant correlations were shown between clinical symptoms and Mal d 1- and Gly m 4-sIgE levels measured by both tests and for Cor a 1-sIgE levels measured by ALEX2 . In eight patients, profilin related symptoms were supported by Hev b 8-sensitization. CONCLUSION: Multiplex testing is beneficial to understand patient-specific individual sensitization profiles and to providing personalized management recommendations. In the future, custom-designed test kits might enable reducing costs of multiplex testing for specific patient groups without compromising the diagnostic value.

Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

Pru p 9, a new allergen eliciting respiratory symptoms in subjects sensitized to peach tree pollen.

Peach tree (PT) pollen sensitization is highly prevalent in subjects living in areas where this tree is widely cultivated. None of the allergens responsible for these sensitizations have been identified so far. Our aim was to identify the most relevant PT pollen allergens and analyze their capacity for inducing respiratory symptoms. We studied sixty-two individuals sensitized to PT pollen who developed symptoms after its exposure. The IgE binding profile on peach pollen extract by means of immunoblotting using sera from these subjects was analyzed. Protein extract was fractionated by anion-exchange chromatography and HPLC, fractions run in SDS-PAGE and proteins were identified from IgE-binding bands by mass spectrometry. Several allergenic proteins in the PT pollen extract were recognized by patients' IgE: a glucan endo-1,3-beta-glucosidase-like, a polygalacturonase, an UTP-glucose-1-phosphate uridylyltransferase and a PR-1a protein. This PR-1a protein is a novel allergen frequently recognized with a molecular mass of 18 kDa, named as Pru p 9 following the WHO-IUIS nomenclature. Skin Prick Test (SPT) performed with this allergen was positive in 41% of the PT pollen-sensitized clinical cases. Most of them had rhinitis or rhinoconjunctivitis, but a significant percentage experienced asthma with seasonal symptoms during the period of PT flowering. Nasal Provocation test (NPT) with Pru p 9 was positive in all cases with positive SPT to this new allergen eliciting nasal symptoms similar to those challenged with PT pollen. We demonstrate that PT pollen can induce sensitization and allergy in an exposed population, being Pru p 9 a relevant allergen responsible of respiratory symptoms. Considering the extensive peach worldwide production with a large number of people involved, our results add a great value for the diagnosis and management of subjects allergic to this pollen.

New insights into the sensitization to nonspecific lipid transfer proteins from pollen and food: New role of allergen Ole e 7.

BACKGROUND: Ole e 7 is a nonspecific lipid transfer protein (nsLTP) from olive pollen, one of the main allergenic pollens worldwide. This allergenic nsLTP is responsible for severe symptoms in regions with high olive pollen exposure, where many Ole e 7-sensitized patients exhibit a co-sensitization to the peach nsLTP, Pru p 3. However, there is no evidence of cross-reactivity, which explains this observed co-sensitization. Therefore, the purpose of this study was to explore the relationship between Ole e 7 and Pru p 3. METHODS: A total of 48 patients sensitized to Ole e 7 and/or Pru p 3 were included in the study. Specific IgE serum levels were measured by ImmunoCAP 250 and ELISA. Inhibition assays were performed to determine the existence of cross-reactivity between both nsLTPs. Allergic response was analyzed ex vivo (basophil activation test) and in vitro (RBL-2H3 mast cell model). RESULTS: Common IgG and IgE epitopes were identified between both allergens. IgE-binding inhibition was detected in Ole e 7-monosensitized patients using rPru p 3 as inhibitor, reaching inhibition values of 25 and 100%. Ex vivo and in vitro assays revealed a response against rPru p 3 in four (31%) Ole e 7-monosensitized patients. CONCLUSIONS: Our results suggest that Ole e 7 could play a new role as primary sensitizer in regions with high olive pollen exposure, leading to the peach nsLTP sensitization. This co-sensitization process would occur because of the cross-reactivity between Ole e 7 and Pru p 3 observed in some allergic patients.

Peach Tree Pollen and Prunus persica 9 Sensitisation and Allergy in Children and Adolescents.

BACKGROUND: Although plant and fruit pollens are entomophilous and relevant in exposed workers, we have shown a high frequency of sensitisation and symptoms induction of peach tree pollen (PTP) and Prunus persica 9 (Pru p 9) in adults from areas of peach cultivars. METHODS: We studied the sensitisation and clinical relevance of PTP and Pru p 9 in a large group of children and adolescents aged 3-19 years. A detailed questionnaire plus skin prick testing to prevalent allergens, PTP, and Pru p 9 were carried out. The clinical relevance was established by nasal provocation test (NPT) and symptom score index. RESULTS: We evaluated 685 children (mean age 8.75 ± 3.3 years, median 9 years), 52% of them female. Sensitisation to PTP occurred in 20% of the cases following olive tree (33%) and Phleum pratense (26%). In a randomly selected subgroup of subjects sensitised to PTP, 30% were skin prick test-positive to Pru p 9. Most cases had rhinitis or rhinoconjunctivitis. NPT showed the relevance of PTP and Pru p 9 in the induction of symptoms. CONCLUSION: PTP and Pru p 9 are relevant in the induction of sensitisation and respiratory symptoms in children and adolescents. This allergen should be evaluated in children living in regions of peach tree cultivars.

Effect of pre- and post-weaning dietary supplementation with arginine and glutamine on rabbit performance and intestinal health.

BACKGROUND: The purpose of the present study was to assess if the exposure to glutamine (Gln), arginine (Arg) or their combination from pregnancy, through the maternal diet, to a post weaning supplemented diet, can stimulate litter performance, gut development and immune function. To this end does and their litters were fed the same basal diet no supplemented (control C), or supplemented with 0.4% Gln, 0.4% Arg, or 0.4 Gln + 0.4 Arg. Rabbits were weaned at 25 d of age and fed the same experimental diet as their mothers for 10 additional days (35 d of age). Bacterial translocation to mesenteric lymph nodes (MLN) at 6 d of age and intestinal histology, enzymatic activity, phenotypical and functional analysis of intraepithelial lymphocytes (IEL) from the appendix were determined at 6, 25 and 35 d of age. RESULTS: No significant differences on animal performance or mortality rates were observed among dietary treatments. However, kits from rabbit does supplemented with Gln tended (P ≤ 0.10) to reduce the translocation of total number of both aerobic and facultative anaerobic bacteria to the MLN. Also, rabbits fed the Gln supplemented diets maintained intestinal villous height at weaning compared to the non-supplemented diets (P < 0.05). The proportions of CD45+CD4+ and CD45+CD8+ IEL in the appendix were not affected by dietary means. However, in rabbits IEL at weaning dietary Gln significantly upregulated IL-2 and downregulated IL-6 expression. CONCLUSIONS: Despite a lack of effect on performance and mortality the inclusion of 0.4% Gln has a positive effect by maintaining intestinal villous height and modulating the cytokine profile at weaning. The supplementation with Arg or Arg + Gln at the selected doses in this study did not exert positive effects on rabbit intestinal health.

A relevant IgE-reactive 28kDa protein identified from Salsola kali pollen extract by proteomics is a natural degradation product of an integral 47kDa polygalaturonase.

A highly prevalent IgE-binding protein band of 28kDa is observed when Salsola kali pollen extract is incubated with individual sera from Amaranthaceae pollen sensitized patients. By an immunoproteomic analysis of S. kali pollen extract, we identified this protein band as an allergenic polygalacturonase enzyme. The allergen, named Sal k 6, exhibits a pI of 7.14 and a molecular mass of 39,554.2Da. It presents similarities to Platanaceae, Poaceae, and Cupressaceae allergenic polygalacturonases. cDNA-encoding sequence was subcloned into the pET41b vector and produced in bacteria as a His-tag fusion recombinant protein. The far-UV CD spectrum determined that rSal k 6 was folded. Immunostaining of the S. kali pollen protein extract with a rSal k 6-specific pAb and LC-MS/MS proteomic analyses confirmed the co-existence of the 28kDa band together with an allergenic band of about 47kDa in the pollen extract. Therefore, the 28kDa was assigned as a natural degradation product of the 47kDa integral polygalacturonase. The IgE-binding inhibition to S. kali pollen extract using rSal k 6 as inhibitor showed that signals directed to both protein bands of 28 and 47kDa were completely abrogated. The average prevalence of rSal k 6 among the three populations analyzed was 30%, with values correlating well with the levels of grains/m3 of Amaranthaceae pollen. Sal k 6 shares IgE epitopes with Oleaceae members (Fraxinus excelsior, Olea europaea and Syringa vulgaris), with IgE-inhibition values ranging from 20% to 60%, respectively. No IgE-inhibition was observed with plant-derived food extracts.

Identification of the ligand of Pru p 3, a peach LTP.

KEY MESSAGE: Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

Allergen-Associated Immunomodulators: Modifying Allergy Outcome.

The prevalence of allergies is increasing since mid twentieth century; however the underlying causes of this increase are not fully clear. Understanding the mechanism by which a harmless protein becomes an allergen provides us with the basis to prevent and treat these diseases. Although most studies on allergen immunogenicity have traditionally focused on structural properties of the proteins, it is increasingly clear that allergenicity cannot be determined only based on structural features of the allergenic proteins. In fact, allergens do not encounter human facings as isolated molecules but contained in complex mixtures of proteins, carbohydrates and lipids, such as pollen grains or foods. As a result, attention has lately been directed to examine whether allergen-associated molecules exhibit immune-regulatory properties. The present review aims to illustrate some examples of how non-protein molecules accompanying the allergen can modulate allergic responses.

A recombinant Sal k 1 isoform as an alternative to the polymorphic allergen from Salsola kali pollen for allergy diagnosis.

BACKGROUND: The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Challenges for allergy diagnosis in regions with complex pollen exposures.

Over the past few decades, significant scientific progress has influenced clinical allergy practice. The biological standardization of extracts was followed by the massive identification and characterization of new allergens and their progressive use as diagnostic tools including allergen micro arrays that facilitate the simultaneous testing of more than 100 allergen components. Specific diagnosis is the basis of allergy practice and is always aiming to select the best therapeutic or avoidance intervention. As a consequence, redundant or irrelevant information might be adding unnecessary cost and complexity to daily clinical practice. A rational use of the different diagnostic alternatives would allow a significant improvement in the diagnosis and treatment of allergic patients, especially for those residing in complex pollen exposure areas.

Food allergy: management, diagnosis and treatment strategies.

Food allergy is an increasing problem in western countries, with strict avoidance being the only available reliable treatment. However, accidental ingestion can occur and anaphylactic reactions still happen. In recent years, many efforts have been made to better understand the humoral and cellular mechanisms involved in food allergy, and to improve the strategies for diagnosis and treatment. This review focuses on IgE-mediated food hypersensitivity and provides an overview of the diagnostic strategies and treatment advances. Specific immunotherapy, including different routes of administration and allergen sources, such as natural, recombinant and T-cell epitopes, are analyzed in detail. Other treatments such as anti-IgE monoclonal antibody therapy, adjuvant therapy and Chinese herbs will also be described.

Graph based study of allergen cross-reactivity of plant lipid transfer proteins (LTPs) using microarray in a multicenter study.

The study of cross-reactivity in allergy is key to both understanding. the allergic response of many patients and providing them with a rational treatment In the present study, protein microarrays and a co-sensitization graph approach were used in conjunction with an allergen microarray immunoassay. This enabled us to include a wide number of proteins and a large number of patients, and to study sensitization profiles among members of the LTP family. Fourteen LTPs from the most frequent plant food-induced allergies in the geographical area studied were printed into a microarray specifically designed for this research. 212 patients with fruit allergy and 117 food-tolerant pollen allergic subjects were recruited from seven regions of Spain with different pollen profiles, and their sera were tested with allergen microarray. This approach has proven itself to be a good tool to study cross-reactivity between members of LTP family, and could become a useful strategy to analyze other families of allergens.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray.

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Plant lipid transfer protein allergens: no cross-reactivity between those from foods and olive and Parietaria pollen.

BACKGROUND: Cross-reactivity among plant food allergens belonging to the nonspecific lipid transfer protein (LTP) family is well known. In contrast, the relationship among these allergens and their putative homologs from olive (Ole e 7) and Parietaria (Par j 1) pollen has not been clarified. METHODS: Sera with specific IgE to LTP allergens were obtained from peach-, mustard- and olive pollen-allergic patients. Purified LTP allergens from foods (peach, apple, mustard and wheat) and pollens (olive, mugwort and Parietaria) were tested by ELISA and ELISA-inhibition assays. RESULTS: Plant food LTP-allergic patients showed a significantly higher number of sera (89-100 vs. 33-64%) with specific IgE and mean specific IgE levels (0.30-1.56 vs. 0.21-0.34 OD units) to the 4 food LTP allergens tested than to olive Ole e 7 and Parietaria Par j 1 pollen. ELISA-inhibition assays indicated cross-inhibition between food LTP allergens but no cross-reactivity between these allergens and Ole e 7 and Par j 1, or, even more, between the LTP allergens from olive and Parietaria pollen. CONCLUSIONS: LTP allergens from olive and Parietaria pollen cross-react neither with allergenic LTPs from plant foods nor between themselves. Therefore, both pollens do not seem to be related with the LTP syndrome.

Pollen and plant food profilin allergens show equivalent IgE reactivity.

BACKGROUND: Profilins are commonly involved in polysensitization of allergic patients; therefore, appropriate markers should be used in component-resolved diagnosis. OBJECTIVE: To evaluate the immunological equivalence between profilins from pollens and plant-derived foods, to be used in component-resolved diagnosis. METHODS: Specific immunoglobulin (Ig) G antibodies against pollen and fruit profilins, as well as sera from patients allergic to mustard, melon, or olive pollen, were used. Purified profilins from mustard seeds, fruit melon, and chenopod and birch pollen were assayed in immunoblotting, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition assays. RESULTS: Significant correlation was found in the response of purified profilins by ELISA and immunoblotting for both specific IgG and IgE. The highest levels of IgE binding were obtained for olive pollen-allergic patients, which could be related to the route of sensitization. The responses of individual patients to profilins were also similar and independent of the sensitizing source. The inhibition between pairs of allergens was generally higher than 70%, indicating that profilins share most of the IgE epitopes. Modeling of mimotopes in the conformational structure of the implicated profilins supports their strong cross-reactivity obtained experimentally. CONCLUSIONS: No correlation exists between the level of IgE response of individual patients to specific profilins and the corresponding theoretical sensitizing source, suggesting that the sensitization could be attributable to any profilin present in the environment of the patients. This would bear out the use of most profilins as a common marker for polysensitization in component-resolved diagnosis and for therapeutic approaches.

A new lipid transfer protein homolog identified as an IgE-binding antigen from Japanese cedar pollen.

Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal rhinitis and conjunctivitis in Japan, and an understanding of its full allergen repertoire is prerequisite for the development of future molecular diagnostics and immunotherapeutic strategies. Here we report the identification of a new C. japonica pollen IgE-binding antigen (CJP-8) homologous to lipid transfer proteins (LTPs), a class of plant cross-reactive allergens found in foods, latex, and pollen grains. The cjp-8 cDNA encodes a 165-amino acid polypeptide possessing the conserved eight cysteines characteristic of plant LTP family members. Escherichia coli-expressed recombinant CJP-8 (r-CJP-8) reacted with IgE antibody from Japanese cedar pollinosis patients at a 37.5% frequency (6/16).

Anaphylaxis to wheat flour-derived foodstuffs and the lipid transfer protein syndrome: a potential role of wheat lipid transfer protein Tri a 14.

BACKGROUND: Food allergy to wheat-derived foodstuffs is on the rise. Tri a 14, a wheat flour lipid transfer protein (LTP) allergen, has been described as a major allergen associated with baker's asthma and wheat food allergy. Cross-reactivity among LTP allergens leads to the so-called 'LTP syndrome'. METHODS: Eight adult patients showing anaphylaxis after ingestion of wheat-derived foodstuffs were selected. A homemade wheat extract, purified natural (n) and recombinant (r) Tri a 14, and peach fruit and Artemisia pollen LTP allergens Pru p 3 and Art v 3 were subjected to skin prick test, specific IgE determination (ELISA) and IgE immunodetection assays. RESULTS: All tests were positive in the 8 selected patients with the homemade extract. Positive skin prick test responses to nTri a 14, Pru p 3 and Art v 3 were found in 5/8, 6/8 and 4/4 patients, respectively. Specific IgE determined by ELISA assays was detected in 6 to nTri a 14 and rTri a 14, in 4 to Pru p 3 and in 3 to Art v 3 out of 8 individual sera tested, whereas all these sera showed IgE binding to nTri a 14 and Pru p 3 in immunodetection after SDS-PAGE separation. CONCLUSIONS: Tri a 14 seems to be a relevant allergen in patients with anaphylaxis after ingestion of wheat flour foodstuffs, according to in vitro and in vivo results. Clinical history of the analyzed patients, together with sensitization to peach Pru p 3 and Artemisia pollen Art v 3, suggests that 6 of them suffer from LTP syndrome.