Jesus F. Crespo

IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e 1

BACKGROUND Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.

Food allergy: nuts and tree nuts.

Nuts are a well-defined cause of food allergy, which affect approximately 1 % of the general population in the UK and the USA. There do appear to be differences in the frequency of nut allergy between different countries because of different dietary habits and cooking procedures. For example, in the USA and France, peanuts are one of the most frequent causes of food allergy, but in other countries, it seems to be less common. Genetic factors, in particular, appear to play a role in the development of peanut allergy. While the majority of nut allergens are seed storage proteins, other nut allergens are profilins and pathogenesis-related protein homologues, considered as panallergens because of their widespread distribution in plants. The presence of specific IgE antibodies to several nuts is a common clinical finding, but the clinical relevance of this cross-reactivity is usually limited. Allergic reactions to nuts appear to be particularly severe, sometimes even life-threatening, and fatal reactions following their ingestion have been documented. Food allergy is diagnosed by identifying an underlying immunological mechanism (i.e. allergic testing), and establishing a causal relationship between food ingestion and symptoms (i.e. oral challenges). In natural history investigations carried out in peanut-allergic children, approximately 20 % of the cases outgrew their allergy or developed oral tolerance. The treatment of nut allergies should include patient and family education about avoiding all presentations of the food and the potential for a severe reaction caused by accidental ingestion. Patients and families should be instructed how to recognise early symptoms of an allergic reaction and how to treat severe anaphylaxis promptly.

Detection and quantitation of raw fish aeroallergens from an open-air fish market

BACKGROUND IgE-mediated hypersensitivity to fish is a clinically relevant problem, particularly in several European countries. Although most allergic reactions to fish are caused by ingestion, occupational exposures to seafood allergens by inhalation have been correlated with respiratory symptoms. In Madrid, patients with fish allergy have exhibited respiratory symptoms after visits to an open-air fish market. OBJECTIVE We sought to study the possibility of passively aerosolized fish allergen in an open-air fish market through air sampling and a competitive IgE immunoassay. METHODS Air samples were collected on polytetrafluoroethylene filters by using air samplers. Samples were collected on 41 different days from both an open-air fish market and an outdoor residential area. Fish allergens were specifically quantified by competitive IgE immunoassay by using pooled sera from fish-sensitive individuals. A raw fish extract (10 mg of dry weight/mL) was used as the reference standard. RESULTS Allergen was quantified in all 39 fish market air samples (2-25 ng/m(3)). The residential air samples contained no detectable allergen. The analytic limit of detection was 2 ng, allowing detection of 0.4 ng/m(3) for the air volumes collected. A concentrated (30-fold) pool of fish market air samples was tested in serial dilutions and demonstrated an identical regression line to that of the raw fish standard. CONCLUSION By using air sampling and an immunochemical analytic technique, fish allergen is detectable in the air of an open-air fish market. Avoidance of a food allergen, such as fish, should include preventing exposure to aerosolized particles through inhalation in relevant environments.

Differences among Pollen-Allergic Patients with and without Plant Food Allergy

Background: A considerable number of pollen-allergic patients develops allergy to plant foods, which has been attributed to cross-reactivity between food and pollen allergens. The aim of this study was to analyze the differences among pollen-allergic patients with and without plant food allergy. Methods: Eight hundred and six patients were recruited from 8 different hospitals. Each clinical research group included 100 patients (50 plant food-allergic patients and 50 pollen-allergic patients). Diagnosis of pollen allergy was based on typical case history of pollen allergy and positive skin prick tests. Diagnosis of plant-food allergy was based on clear history of plant-food allergy, skin prick tests and/or plant-food challenge tests. A panel of 28 purified allergens from pollens and/or plant foods was used to quantify specific IgE (ADVIA-Centaur® platform). Results: Six hundred and sixty eight patients (83%) of the 806 evaluated had pollen allergy: 396 patients with pollen allergy alone and 272 patients with associated food and pollen allergies. A comparison of both groups showed a statistically significant increase in the food and pollen allergy subgroup in frequency of: (1) asthma (47 vs. 59%; p < 0.001); (2) positive skin test results to several pollens: Plantago,Platanus,Artemisia,Betula,Parietaria and Salsola (p < 0.001); (3) sensitization to purified allergens: Pru p 3, profilin, Pla a 1 – Pla a 2, Sal k 1, PR-10 proteins and Len c 1. Conclusion: Results showed relevant and significant differences between both groups of pollen-allergic patients depending on whether or not they suffered from plant-derived food allergy.

Heat and pressure treatments effects on peanut allergenicity.

Peanut allergy is recognized as one of the most severe food allergies. The aim of this study was to investigate the changes in IgE binding capacity of peanut proteins produced by thermal-processing methods, including autoclaving. Immunoreactivity to raw and thermally processed peanut extracts was evaluated by IgE immunoblot and skin prick test in patients with clinical allergy to peanut. Roasted peanut and autoclaved roasted peanut were selected for IgE ELISA experiments with individual sera, immunoblot experiments with antibodies against peanut allergens (Ara h 1, Ara h 2 and Ara h 3), digestion experiments, and circular dichroism spectroscopy. In vitro and in vivo experiments showed IgE immunoreactivity of roasted peanut proteins decreased significantly at extreme conditions of autoclaving. Circular dichroism experiments showed unfolding of proteins in autoclave treated samples, which makes them more susceptible to digestion. Autoclaving at 2.56atm, for 30min, produces a significant decrease of IgE-binding capacity of peanut allergens.

Influence of thermal processing on IgE reactivity to lentil and chickpea proteins.

In the last years, legume proteins are gaining importance as food ingredients because of their nutraceutical properties. However, legumes are also considered relevant in the development of food allergies through ingestion. Peanuts and soybeans are important food allergens in Western countries, while lentil and chickpea allergy are more relevant in the Mediterranean area. Information about the effects of thermal-processing procedures at various temperatures and conditions is scarce; therefore, the effect of these procedures on legume allergenic properties is not defined so far. The SDS-PAGE and IgE-immunoblotting patterns of chickpeas and lentils were analyzed before and after boiling (up to 60 min) and autoclaving (1.2 and 2.6 atm, up to 30 min). The results indicated that some of these treatments reduce IgE binding to lentil and chickpea, the most important being harsh autoclaving. However, several extremely resistant immunoreactive proteins still remained in these legumes even after this extreme treatment.

Influence of Enzymatic Hydrolysis on the Allergenicity of Roasted Peanut Protein Extract

Background: Peanut allergy is recognized as one of the most severe food allergies. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as the soybean, chickpea and lentil. Nevertheless, there are only a few studies carried out with sera from patients with a well-documented allergy. Methods: Roasted peanut protein extract was hydrolyzed by the sequential and individual action of 2 food-grade enzymes, an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to roasted peanut extract and hydrolyzed samples was evaluated by means of IgE immunoblot, ELISA and 2-dimensional electrophoresis using sera from 5 patients with a clinical allergy to peanuts and anti-Ara h 1, anti-Ara h 2 and anti-Ara h 3 immunoblots. Results: Immunoblot and ELISA assays showed an important decrease of IgE reactivity and Ara h 1, Ara h 2 and Ara h 3 levels in the first 30 min of hydrolyzation with Alcalase. In contrast, individual treatment with Flavourzyme caused an increase in IgE reactivity detected by ELISA at 30 min and led to a 65% inhibition of IgE reactivity at the end of the assay (300 min). Ara h 1 and the basic subunit of Ara h 3 were still recognized after treatment with Flavourzyme for 300 min. Conclusion: Hydrolysis with the endoprotease Alcalase decreases IgE reactivity in the soluble protein fraction of roasted peanut better than hydrolysis with the exoprotease Flavourzyme.

Allergenic properties and differential response of walnut subjected to processing treatments

The aim of this study was to investigate changes in walnut allergenicity after processing treatments by in vitro techniques and physiologically relevant assays. The allergenicity of walnuts subjected to high hydrostatic pressure and thermal/pressure treatments was evaluated by IgE-immunoblot and antibodies against walnut major allergen Jug r 4. The ability of processed walnut to cross-link IgE on effector cells was evaluated using a rat basophil leukaemia cell line and by skin prick testing. Susceptibility to gastric and duodenal digestion was also evaluated. The results showed that walnuts subjected to pressure treatment at 256 kPa, 138 °C, were able to diminish the IgE cross-linking capacity on effector cells more efficiently than high pressure treated walnuts. IgE immunoblot confirmed these results. Moreover, higher susceptibility to digestion of pressure treated walnut proteins was observed. The use of processed walnuts with decreased IgE binding capacity could be a potential strategy for walnut tolerance induction.

Characterization of lupin major allergens (Lupinus albus L.)

White lupin is considered to be a rich source of protein with a notable content of lysine and is being increasingly used in bakery, confectionery, snacks and pastry products due to its multifunctional properties, in addition to its potential hypocholesterolemic and hypoglycemic properties. However, lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut since the risk of immunological cross-reactivity between lupin and peanut is higher than with other legumes. Previously, we had identified two proteins as major lupin allergens (34.5 and 20 kDa) as determined by IgE immunoblotting using sera of 23 patients with lupin-specific IgE. The aim of this study was to purify and characterize the two major lupin allergens. The results using in vitro IgE-binding studies and MS analysis have shown that the 34.5 kDa allergen (Lup-1) is a conglutin β (vicilin-like protein) while the 20 kDa allergen (Lup-2) corresponds to the conglutin α fraction (legumin-like protein). The high level of amino acid sequence homology of Lup-1 and Lup-2 with the major allergens of some legumes explains the IgE cross-reactivity and clinical cross-reactivity of lupin and other legumes.

Allergic Reactions Following Skin Contact With Fish

We evaluated the clinical characteristics found in 29 children who showed allergic symptoms after direct and/or indirect contact with fish, from 197 children diagnosed with IgE-mediated fish hypersensitivity. Allergic reactions to fish through ingestion began in most patients (79%) within the first 24 months of life. All the patients showed cutaneous symptoms, either alone or associated with other clinical manifestations. Whiff and hake were the species more frequently implicated in eliciting clinical manifestations upon ingestion. After diagnosis, all patients were placed on a strict fish avoidance diet. During this period of avoidance, 29 patients reported allergic reactions (mean age 5 years, 6 months; SD 4 years, 2 months) after incidental skin contact with fish. Clinical manifestations after touching fish were only cutaneous in 28 of the 29 studied patients. One showed local urticaria with wheezing. The majority reported two or more episodes. Incidental skin contact with fish could play an important role in accidental encounters with fish particles in children on a fish avoidance diet for fish IgE-mediated hypersensitivity. Such exposures could result in immunologic immediate contact reactions and in some cases could lead to systemic reactions (contact urticaria syndrome).