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.

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.

Occupational allergic multiorgan disease induced by wheat flour

Bakers are repeatedly exposed to wheat flour (WF) and may develop sensitization and occupational rhinoconjunctivitis and/or asthma to WF allergens.1 Several wheat proteins have been identified as causative allergens of occupational respiratory allergy in bakery workers.1 Testing of IgE reactivity in patients with different clinical profiles of wheat allergy (food allergy, wheat-dependent exercise-induced anaphylaxis, and baker's asthma) to salt-soluble and salt-insoluble protein fractions from WF revealed a high degree of heterogeneity in the recognized allergens. However, mainly salt-soluble proteins (albumins, globulins) seem to be associated with baker's asthma, and prolamins (gliadins, glutenins) with wheat-dependent exercise-induced anaphylaxis, whereas both protein fractions reacted to IgE from food-allergic patients.1 Notwithstanding, gliadins have also been incriminated as causative allergens in baker's asthma.2 We report on a 31-year-old woman who had been exposed to WF practically since birth because her family owned a bakery housed in the same home where they lived. She moved from this house when she was 25 years, but she continued working every day in the family bakery. In the last 8 years she had suffered from work-related nasal and ocular symptoms such as itching, watery eyes, sneezing, nasal stuffiness, and rhinorrhea. These symptoms markedly improved when away from work and worsened at work. In the last 5 years, she had also experienced dysphagia with frequent choking, especially when ingesting meats or cephalopods, which had partially improved with omeprazole therapy. Two years before referral to our clinic, she began to have dry cough and breathlessness, which she also attributed to her work environment. Upper and lower respiratory tract symptoms increased when sifting the WF and making the dough. The patient did not experience gastrointestinal symptoms with ingestion of cereal products. Skin prick test results were positive to grass (mean wheal, 6 mm), cypress (5 mm) and Russian thistle pollen (4 mm), WF (4 mm), and peach lipid transfer protein (6 mm) and were negative to rice flour, corn flour, profilin, mites, molds, and animal dander. Skin prick test with a homemade WF extract (10% wt/vol) was strongly positive (15 mm). Serologic tests yielded the following results: eosinophil cationic protein, 47 ?g/L; total serum IgE, 74 kU/L; specific IgE (ImmunoCAP; ThermoFisher, Uppsala, Sweden) to WF, 7.4 kU/L; barley flour, 1.24 kU/L; and corn, gluten, alpha-amylase, peach, and apple, less than 0.35 kU/L. Specific IgE binding to microarrayed purified WF allergens (WDAI-0.19, WDAI-0.53, WTAI-CM1, WTAI-CM2, WTAI-CM3, WTAI-CM16, WTAI-CM17, Tri a 14, profilin, ?-5-gliadin, Tri a Bd 36 and Tri a TLP, and gliadin and glutamine fractions) was assessed as described elsewhere.3 The patient's serum specifically recognized ?-5-gliadin and the gliadin fraction, and no IgE reactivity was observed to other wheat allergens. Spirometry revealed a forced vital capacity of 3.88 L (88%), an FEV1 of 3.04 L (87%), and FEV1/forced vital capacity of 83%. A methacholine inhalation test was performed following an abbreviated protocol,4 and the results were expressed as PD20 in cumulative dose (mg) of methacholine. Methacholine inhalation challenge test result was positive (0.24 mg cumulative dose) when she was working, and after a 3-month period away from work and with no visits to the bakery house, it gave a negative result. A chest x-ray was normal. Specific inhalation challenge test was carried out in the hospital laboratory by tipping WF from one tray to another for 15 minutes. Spirometry was performed at baseline and at 2, 5, 10, 15, 20, 30, 45, and 60 minutes after the challenge with WF. Peak expiratory flow was measured at baseline and then hourly over 24 hours (respecting sleeping time). A 12% fall in FEV1 was observed at 20 minutes and a 26% drop in peak expiratory flow at 9 hours after exposure to WF,

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.

Super-Resolution in Respiratory Synchronized Positron Emission Tomography

Respiratory motion is a major source of reduced quality in positron emission tomography (PET). In order to minimize its effects, the use of respiratory synchronized acquisitions, leading to gated frames, has been suggested. Such frames, however, are of low signal-to-noise ratio (SNR) as they contain reduced statistics. Super-resolution (SR) techniques make use of the motion in a sequence of images in order to improve their quality. They aim at enhancing a low-resolution image belonging to a sequence of images representing different views of the same scene. In this work, a maximum a posteriori (MAP) super-resolution algorithm has been implemented and applied to respiratory gated PET images for motion compensation. An edge preserving Huber regularization term was used to ensure convergence. Motion fields were recovered using a B-spline based elastic registration algorithm. The performance of the SR algorithm was evaluated through the use of both simulated and clinical datasets by assessing image SNR, as well as the contrast, position and extent of the different lesions. Results were compared to summing the registered synchronized frames on both simulated and clinical datasets. The super-resolution image had higher SNR (by a factor of over 4 on average) and lesion contrast (by a factor of 2) than the single respiratory synchronized frame using the same reconstruction matrix size. In comparison to the motion corrected or the motion free images a similar SNR was obtained, while improvements of up to 20% in the recovered lesion size and contrast were measured. Finally, the recovered lesion locations on the SR images were systematically closer to the true simulated lesion positions. These observations concerning the SNR, lesion contrast and size were confirmed on two clinical datasets included in the study. In conclusion, the use of SR techniques applied to respiratory motion synchronized images lead to motion compensation combined with improved image SNR and contrast, without any increase in the overall acquisition times.

Occupational asthma caused by IgE-mediated sensitization to multiple woods

Wood is a natural material that is able to trigger rhinitis and asthma in exposed subjects in occupational settings. This has been described with both soft and hard woods.1,2 Involvement of both low- and high-molecular-weight allergens has been reported, and the relevance of these is related with the wood type.1 There are cases where protein may be the responsible allergen. Crossreactivity between obeche and ramin woods3 and between obeche and latex4 has been shown. However, to the best of our knowledge, this is the first report of a multiple IgE-mediated sensitization to different woods that caused occupational respiratory symptoms in the same worker.

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.

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.

Transport of Pru p 3 across gastrointestinal epithelium - an essential step towards the induction of food allergy?

Background Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. Objective To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. Methods This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. Results The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. Conclusion These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. Clinical relevance The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Component-resolved diagnosis of wheat flour allergy in baker's asthma.

Baker's asthma is one of the most common types of occupational asthma and its prevalence is increasing in the last years. Diagnosis of occupational asthma is complex. The poor specificity of current diagnostic approaches may be associated with insufficient purity of wheat extracts or lack of inclusion of major allergens in them. In this work, we use microarray technology to characterize the allergenic profiles of baker's asthma patients from three regions in Spain and to analyze the influence of other environmental allergens on the sensitization pattern.

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.

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.

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.