Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

Background: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. Objective: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. Methods: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cor a 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. Results: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP?sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P<.05). Similar rates of Cor a 8 and Jug r 3 sensitization were detected by both techniques. Conclusions: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.

Clinical presentation, allergens, and management of wheat allergy

IgE-mediated allergy to wheat proteins can be caused by exposure through ingestion, inhalation, or skin/mucosal contact, and can affect various populations and age groups. Respiratory allergy to wheat proteins is commonly observed in adult patients occupationally exposed to flour, whereas wheat food allergy is more common in children. Wheat allergy is of growing importance for patients with recurrent anaphylaxis, especially when exercise related. The diagnosis of wheat allergy relies on a consistent clinical history, skin prick testing with well-characterized extracts and specific IgE tests. The accuracy of wheat allergy diagnosis may be improved by measuring IgE responses to several wheat components. However, a high degree of heterogeneity has been found in the recognition pattern of allergens among patient groups with different clinical profiles, as well as within each group. Thus, oral provocation with wheat or the implicated cereal is the reference test for the definitive diagnosis of ingested wheat/cereal allergy.

Effect of Pru p 3 on dendritic cell maturation and T-lymphocyte proliferation in peach allergic patients.

BACKGROUND: Pru p 3 is the major peach allergen and the most frequent cause of food allergy in adults in the Mediterranean area. Although its allergenicity is well characterized, its ability to generate a T-cell response is not completely known. OBJECTIVE: To investigate the influence of Pru p 3 allergen on dendritic cell (DC) maturation and specific T-cell response (T(H)1/T(H)2) in peach allergic patients. METHODS: Peach allergic patients (n = 11) and tolerant controls (n = 14) were included in the study. Monocyte-derived DC maturation after incubation with Pru p 3 was evaluated by the increase of maturational markers (CD80, CD86, and CD83) by flow cytometry. Lymphocyte proliferation was evaluated by coculturing monocyte-derived DCs and 5,6-carboxyfluorescein diacetate N-succinimidyl ester-stained lymphocytes with different concentrations of Pru p 3 (25, 10, and 1 ?g/mL) by flow cytometry and cytokine production. RESULTS: Pru p 3 induced a significant increase in the CD80, CD86, and CD83 expression on stimulated DCs from patients compared with controls. The lymphocyte proliferative response after Pru p 3 stimulation was also significantly higher along with an increase in interleukin 8 in patients compared with tolerant controls. CONCLUSION: Pru p 3 allergen induces changes in DC maturational status mainly in peach allergic patients. An increase in lymphocyte proliferative response accompanied with a different cytokine pattern was also observed compared with healthy controls.

Pru p 3 acts as a strong sensitizer for peanut allergy in Spain

Pru p 3 has been suggested to be the primary sensitizing allergen in patients with peanut allergy in the Mediterranean area. We aimed to confirm this hypothesis, studying 79 subjects.

Tri a 14, wheat Lipid Transfer Protein, a marker of wheat respiratory allergy

Over 30 wheat allergens have been associated to baker’s asthma and much of them have been also implied in food allergy. Few of them have rendered as major allergens. Tri a 14, wheat LTP, has been associated to baker’s asthma as major allergen in patients that can consume peach and wheat derived foodstuffs. In Spanish baker’s asthma patients, 60% showed positive response to Tri a 14 and 45% to Pru p 3. However, the cross-reactivity between peach and wheat has been unusual in allergic population (1,8). Moreover, wheat allergy is not so often as should be attending to the high consume.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias está aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la población, pero las causas de este aumento aún no están claras. Encontrar el origen del mecanismo por el cual una proteína inofensiva se convierte en capaz de inducir una respuesta alérgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterización de alérgenos relevantes ha ayudado a mejorar el manejo clínico y a aclarar los mecanismos básicos de las reacciones alérgicas, todavía queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (proteínas de transferencia de lípidos –LTPs- y taumatinas –TLPs-) y estudiando los mecanismos que median la sensibilización y la reactividad cruzada para mejorar tanto el diagnóstico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panalérgenos; y estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas. Para estudiar la reactividad cruzada entre miembros de la misma familia de proteínas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilización a alérgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional más relevante de origen alérgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con alérgenos y analizando los resultados por la teoría de grafos. En relación al estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas, se llevaron a cabo estudios sobre la interacción de los alérgenos alimentarios con células del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de moléculas co-transportadas con los alérgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y alérgeno principal del melocotón) se selección como modelo para llevarlo a cabo. Por otra parte, se analizó el papel de moléculas activadoras del sistema inmune producidas por patógenos en la inducción de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, alérgeno mayor de dicho hongo, en la sensibilización a Act d 2, alérgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigación innovadora aportando resultados de gran utilidad tanto para la mejora del diagnóstico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins –LTPs-, and Thaumatin-like Proteins –TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Diagnosis and Management of Grain-Induced Asthma

Grain-induced asthma is a frequent occupational allergic disease mainly caused by inhalation of cereal flour or powder. The main professions affected are bakers, confectioners, pastry factory workers, millers, farmers, and cereal handlers. This disorder is usually due to an IgE-mediated allergic response to inhalation of cereal flour proteins. The major causative allergens of grain-related asthma are proteins derived from wheat, rye and barley flour, although baking additives, such as fungal α-amylase are also important. This review deals with the current diagnosis and treatment of grain-induced asthma, emphasizing the role of cereal allergens as molecular tools to enhance diagnosis and management of this disorder. Asthma-like symptoms caused by endotoxin exposure among grain workers are beyond the scope of this review. Progress is being made in the characterization of grain and bakery allergens, particularly cereal-derived allergens, as well as in the standardization of allergy tests. Salt-soluble proteins (albumins plus globulins), particularly members of the α-amylase/trypsin inhibitor family, thioredoxins, peroxidase, lipid transfer protein and other soluble enzymes show the strongest IgE reactivities in wheat flour. In addition, prolamins (not extractable by salt solutions) have also been claimed as potential allergens. However, the large variability of IgE-binding patterns of cereal proteins among patients with grain-induced asthma, together with the great differences in the concentrations of potential allergens observed in commercial cereal extracts used for diagnosis, highlight the necessity to standardize and improve the diagnostic tools. Removal from exposure to the offending agents is the cornerstone of the management of grain-induced asthma. The availability of purified allergens should be very helpful for a more refined diagnosis, and new immunomodulatory treatments, including allergen immunotherapy and biological drugs, should aid in the management of patients with this disorder.

Allergy to Uncommon Pets: New Allergies but the Same Allergens.

The prevalence of exotic pet allergies has been increasing over the last decade. Years ago, the main allergy-causing domestic animals were dogs and cats, although nowadays there is an increasing number of allergic diseases related to insects, rodents, amphibians, fish, and birds, among others. The current socio-economic situation, in which more and more people have to live in small apartments, might be related to this tendency. The main allergic symptoms related to exotic pets are the same as those described for dog and cat allergy: respiratory symptoms. Animal allergens are therefore, important sensitizing agents and an important risk factor for asthma. There are three main protein families implicated in these allergies, which are the lipocalin superfamily, serum albumin family, and secretoglobin superfamily. Detailed knowledge of the characteristics of allergens is crucial to improvement treatment of uncommon-pet allergies.

Wheat allergy in celiac children

Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cereals (rye, barley, and oats) which are responsible for different immunologic responses with different clinical expressions of disease. The spectrum of gluten-related disorders has been classified according to pathogenic, clinical, and epidemiological differences in three main forms: (i) wheat allergy (WA), an IgE-mediated disease; (ii) autoimmune disease, including celiac disease (CD), dermatitis herpetiformis, and gluten ataxia; and (iii) possibly immune-mediated, gluten sensitivity [1]. WA is an immunologic Th2 response with typical manifestations which can vary from dermatological, respiratory, and/or intestinal to anaphylactic reactions. In contrast, CD is an autoimmune disorder, a gliadin-specific T-cell response which is enhanced by the action of intestinal tissue transglutaminase (tTG), with a wide clinical spectrum including symptomatic cases with either intestinal (e.g., chronic diarrhea, weight loss) or extraintestinal features (e.g., anemia, osteoporosis, neurologic disturbances) and silent forms that are occasionally discovered as a result of serological screening [1]. We studied wheat allergy in two children with early diagnosis of CD, who developed immediate allergic symptoms after eating small amounts of wheat flour.

Is the performance of ImmunoCAP ISAC 112 sufficient to diagnose peach and apple allergies?

Food allergies constitute a public health issue, with a reported overall estimated prevalence of 6% in Europe1 and Rosacea as the main allergenic fruits among adults.2 The commercial microarray ImmunoCAP ISAC 112 (Thermofisher, Uppsala, Sweden) is a semiquantitative and reproducible in vitro diagnostic tool used for the determination of specific IgE (sIgE).3 However, its panel of allergens does not have the best accuracy when it comes to determining fruit allergies in the Mediterranean area: the inclusion of the thaumatinlike protein (TLP) Pru p 2 or the apple lipid transfer protein (LTP) Mal d 3 has been proposed to improve the diagnosis of peach4 and apple5 allergies, respectively, in the Mediterranean basin. We sought to determine the usefulness of a component-resolved microarray for the diagnosis of peach and apple allergies in the Mediterranean area.

A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from Salsola kali Pollen for Allergy Diagnosis

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.

The major allergens of birch pollen and cow milk, Bet v 1 and Bos d 5, are structurally related to human licocalin 2, enabling them to manipulate T-helper cells depending on their load with siderophore-bound iron

We conclude that Bet v 1 and Bos d 5 not only structurally mimic human LCN2, but also functionally by their ability to bind iron via siderophores. The apo-forms promote Th2 cells, whereas the holo-forms appear to be immunosuppressive. These results provide for the first time a functional understanding on the principle of allergenicity of major allergens from entirely independent sources, like birch and milk.