Marco Ispano

Comparison of results of skin prick tests (with fresh foods and commercial food extracts) and RAST in 100 patients with oral allergy syndrome

One hundred adult patients with a history of oral allergy syndrome (OAS) after ingestion of fruits and vegetables, 77 patients with hay fever and 13 with skin prick tests and RAST positive to pollens but without seasonal symptoms, and 32 normal nonallergic control subjects, had Phadebas RAST and skin prick tests with commercial extracts (CSPT) and with fresh foods (FFSPT) to assess the reliability of these three tests. Sensitivity was better with FFSPT for carrot, celery, cherry, apple, tomato, orange, and peach; better with CSPT for peanut, pea, and walnut; and better with RAST for hazelnut. Specificity, negative predictive value, and positive predictive value of the three tests were determined for apple, carrot, hazelnut, orange, pea, peanut, and tomato. Specificity in the patient groups ranged between 40% (pea) and 100% (apple) for CSPT, between 61% (peanut) and 87% (carrot) for RAST, and between 42% (carrot) and 93% (peanut) for FFSPT. However, all tests were negative in the control group. Thus, false positive results may result from cross-reactivity with pollen allergens. The diagnostic accuracy of these tests in the population with OAS proved comparable for peanut, carrot, hazelnut, and pea. FFSPT proved more sensitive than CSPT or RAST in confirming a history of OAS to certain alimentary allergens, such as apple, orange, tomato, carrot, cherry, celery, and peach.

The major allergen of peach (Prunus persica) is a lipid transfer protein

BACKGROUND Allergy to fresh fruits and vegetables is mostly observed in subjects with pollinosis, especially from birch, because of cross-reacting allergens in vegetable foods and pollens. However, allergic reactions to fruits, specifically Rosaceae fruits, have been reported in subjects without pollinosis. OBJECTIVE This study evaluated the pattern of IgE reactivity, identifying the allergen responsible in 2 groups of patients with oral allergy syndrome to peach with or without birch pollinosis. METHODS The allergenic components of peach were detected by SDS-PAGE and immunoblotting. The major peach allergen was purified by HPLC with a cation-exchange column followed by gel filtration chromatography. Its IgE-binding capacity and its homology with the protein of the crude extract were demonstrated by immunoblotting inhibition techniques. To better characterize this allergen, periodic acid-Schiff stain and isoelectrofocusing were used. The amino acid sequencing was done with a gas-phase sequencer. RESULTS SDS-PAGE and immunoblotting of the 15 patients allergic to peach, 8 without and 7 with birch pollinosis, showed that they all recognized a protein with a molecular weight of 9 kd. This was the only allergen recognized by patients not sensitized to pollen, whereas the birch pollen-sensitive patients had IgE binding to other allergenic proteins at higher molecular weights. The purified 9-kd protein retained its IgE-binding capacity, was negative to periodic acid-Schiff stain, and had an isoelectric point value of greater than 9. A search in the Swiss Prot Bank showed this was a lipid transfer protein, belonging to a group of molecules involved in the defensive system of plants. CONCLUSIONS The major allergen of peach is a 9-kd protein belonging to the group of lipid transfer proteins. This is the only allergen recognized by patients allergic to peach but not sensitized to birch pollen.

Allergenic cross-reactivity among peach, apricot, plum, and cherry in patients with oral allergy syndrome: An in vivo and in vitro study

BACKGROUND Oral allergy syndrome in response to fruits and vegetables frequently occurs as clusters of hypersensitivity to members of the same botanical family, for which the immunologic basis lies in a number of common allergens, most of them still unidentified. OBJECTIVE This study was designed to assess the in vivo and in vitro cross-reactivity between fruits of the Prunoideae subfamily (i.e., peach, cherry, apricot, and plum) and to identify their major allergens and the cross-reactivity of the peach extract with grass and birch pollen. METHODS The in vivo study was conducted by skin prick tests and open food challenges with fresh fruits in 23 patients with oral allergy syndrome for peach and positive skin prick test and RAST results for the other Prunoideae. In vitro sodium dodecylsulfate-polyacrylamide gel electrophoresis was followed by immunoblotting and immunoblotting-inhibition. RESULTS A 13 kd component was identified as the only major allergen common to all the Prunoideae, the other major allergens were found at 14 kd in peach and at 30 kd in cherry. Immunoblotting inhibition showed wide cross-reactivity within the Prunoideae, whereas grass and birch pollen partially inhibited the peach blotting. CONCLUSIONS Clinical cross-reactivity to Prunoideae is essentially due to a common 13 kd IgE-binding component, which seems to be the most important major allergen of this subfamily, not shared with grass and birch pollen.

Identification of actinidin as the major allergen of kiwi fruit

BACKGROUND Allergic reactions to fruits and vegetables are among the most frequent food allergies in adults. Kiwi fruit (Actinidia chinensis) is commonly involved, causing local mucosal, systemic, or both types of symptoms by an IgE-mediated mechanism. In a previous study on 30 patients allergic to kiwi, we identified a major allergen of 30 kd against which all sera tested clearly reacted. Other allergens were detected at 12, 24, and 28 kd. OBJECTIVE The aim of this study was to fully characterize the major kiwi fruit allergen of 30 kd. METHODS Allergens were separated and purified by high-performance liquid chromatography with anion-exchange columns. The purity of the single proteins was checked by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and their allergenicity was checked by immunoblotting with a pool of sera from patients allergic to kiwi. The allergens were characterized by isoelectrofocusing and amino acid sequencing, and periodic acid-Schiff stain was used to detect glycoproteins. RESULTS Proteins of 30, 28, 24, and 17 kd were purified by high-performance liquid chromatography. IgE binding indicated the 30 kd protein, which showed an isoelectric point of 3.5, as the major allergen of kiwi. Determination of its partial amino acid sequence and comparison with the Swiss Protein Bank showed that this was actinidin, the main protein component of kiwi. The 24 and 28 kd proteins had the same N-terminal sequence, which did not correspond to any known protein. The 17 kd protein had a blocked N-terminal sequence. CONCLUSIONS These results demonstrate that the major allergen of kiwi fruit, Act c 1, is actinidin, a proteolytic enzyme belonging to the class of thiol-proteases. Two other allergens of 24 and 28 kd appear identical on amino acid sequencing.

Clinical role of a lipid transfer protein that acts as a new apple-specific allergen

BACKGROUND Allergy to apple is commonly associated with birch pollinosis because the two share homologous allergens. However, some patients have apple allergy but no birch pollinosis, suggesting that there are allergens that do not cross-react with birch. OBJECTIVE The aim of the study was to evaluate the IgE reactivity pattern to an apple extract in subjects with allergic reactions to apple, with and without birch hay fever. METHODS Forty-three patients with oral allergy syndrome for apple and positive open food challenge, skin prick test, and serum specific IgE antibodies to apple were admitted to the study. Thirty-two had birch pollinosis (documented by specific IgE for birch) and 11 were not allergic to birch. The IgE reactivity pattern to apple extract was identified by SDS-PAGE and immunoblotting. The consistent allergen, a 9-kd protein, was then purified by HPLC and characterized by periodic acid-Schiff staining, isoelectric point, and N-terminal amino acid sequencing. RESULTS The sera from 28% of patients allergic to apple with birch pollinosis, but from all patients allergic only to apple, recognized the 9-kd protein. This protein has an isoelectric point of 7.5 and is not glycosylated. Determination of its partial amino acid sequence showed that it belongs to the family of lipid transfer proteins, which act as major allergens in Prunoideae fruits. CONCLUSIONS These results indicate that a lipid transfer protein is an important allergen in patients allergic to apple but not to birch pollen. The prevalent IgE reactivity to this allergen in subjects with no birch pollinosis and the physicochemical characteristics of this protein suggest that sensitization may occur through the oral route.

Sensitization to the major allergen of Brazil nut is correlated with the clinical expression of allergy

BACKGROUND Only a few studies have investigated the clinical role of food allergens, especially the relationship between sensitization to a given allergen and occurrence of adverse reactions when eating the relevant food item. OBJECTIVE This study evaluated the clinical role of the allergens of Brazil nut by comparing the patterns of IgE binding in sera from 11 patients with anaphylaxis after eating Brazil nuts with those from 10 subjects with no symptoms to this food item. Both groups had specific IgE to Brazil nut. METHODS Allergens in the in-house extract of Brazil nut were identified by SDS-PAGE/immunoblotting, the major allergen was purified by HPLC, and its N-terminal sequence was determined by a protein sequencer. RESULTS SDS-PAGE/immunoblotting detected a number of allergenic components with molecular weights ranging from 4 to 58 kd. All sera from symptomatic patients recognized a 9-kd allergen corresponding (as established by amino acid sequencing) to a 2S albumin already described as a major allergen of Brazil nut, whereas the other allergens each bound IgE from less than 50% of sera. No sera from asymptomatic subjects showed IgE binding to the 9-kd allergen, but they did recognize components from 25 to 58 kd, which are minor allergens. CONCLUSIONS These findings indicate that the allergen underlying clinical reactions to Brazil nut is a 2S albumin of 9 kd and that in vitro reactivity to this allergen identifies subjects who react in vivo to ingestion of this food.

Introducing chemists to food allergy

Adverse reactions to food may be toxic or non toxic, depending on the susceptibility to a certain food; non toxic reactions that involve immune mechanisms are termed allergy if they are IgE‐mediated. If no immunological mechanism is responsible, it is termed intolerance. The following disorders are considered a consequence of food allergy: gastrointestinal reactions (oral allergy syndrome, vomiting, diarrhea, protein‐induced enterocolitic syndrome, eosinophilic gastroenteritis); respiratory reactions (rhinitis, asthma, laryngeal edema); cutaneous reactions (urticaria‐angioedema, atopic dermatitis); anaphylaxis. There is much recent evidence to consider celiac disease an immunological disorder. Food allergy diagnosis is based on history, SPT, specific IgE, food challenges. DBPCFC is fundamental for diagnosing true food allergy; patients who have had anaphylaxis to food must not undergo DBPCFC. Rapidly progressive respiratory reactions and anaphylactic shock are life‐threatening reactions that can be caused by food allergy. The doses of food inducing anaphylaxis can be very low, therefore commercial cross‐contamination with an unsuspected food during food processing can be risky for the food allergic patient. The prevention of severe anaphylactic food reactions may lie in interdisciplinary collaboration among allergologists, chemists, food technologists, and experts in food industry research.

Complete amino acid sequence determination of the major allergen of peach (Prunus persica) Pru p 1

Abstract The major protein allergen of peach (Prunus persica), Pru p1, has recently been identified as a lipid transfer protein (LTP). The complete primary structure of Pru p1, obtained by direct amino acid sequence and liquid chromatography-mass spectrometry (LC-MS) analyses with the purified protein, is described here. The protein consists of 91 amino acids with a calculated molecular mass of 9178 Da. The amino acid sequence contains eight strictly conserved cysteines, as do all known LTPs, but secondary structure predictions failed to classify the peach 9 kDa protein as an ‘all-alpha type’, due to the high frequency of amino acids (nine prolines) disrupting alpha helices. Although the sequence similarity with maize LTP is only 63%, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP 16 are identical and 7 similar in the peach homolog, supporting the hypothesis of a similar function.

New allergens in fruits and vegetables

regards the charac- terization of allergens, advances in allergology have served so far to introduce allergenic extracts capable of improving diagnostic and therapeutic performances rather than leading to a deep know- ledge of the etiopathogenesis of allergy. Currently, the concept that there are no peculiar properties making an antigen an efficient allergen is disputed, since recent data suggest that some allergens can directly induce an IgE response. This seems to be true of phospholipase

Diagnostic problems due to cross‐reactions in food allergy

The immunochemical cross-reactivity of allergens is a common problem of diagnostic procedures, especially in food allergy (1). Cross-reactivity can elicit positive in vitro or in vivo allergy tests in subjects who do not display any clinical symptom, thus giving rise to the so-called asymptomatic sensitization. Cross-reactivity is an in vitro phenomenon caused by IgE antibodies directed against epitopes expressed in molecular structures from different allergenic sources, and does not always produce clinical symptoms. Cross-reactions in food allergy mainly affect specificity. Nonetheless, the positive tests in subjects who do not have symptoms cannot be simply classified as “false-positive results”, since they do not occur in nonatopic control subjects (2); therefore, their significance has yet to be fully understood. Cross-reactive allergens can be divided into three groups on the basis of the appearance/absence of resulting clinical symptoms. Group I comprises allergens which provoke clear clinical symptoms (Table l), most of which are taxonomically related, and clinical syndromes in which respiratory and food allergies are combined, such as the “birch-fruit’’ or “bird-egg’’ syndromes (3). Group I1 comprises cross-reactive allergens that do not always produce symptoms. Examples of these are profilins in the pollen-vegetable foods allergy association; cysteine proteases contained in kiwi fruit, papaya, pineapple, and mites; and the cross-reacting allergens in the “latex-fruit” syndrome. Group I11 comprises those cross-reactive allergens that do not produce symptoms, and thus seem to represent a mere in vitro phenomenon. Examples are legumes, cereals, and several differOr C. Ortolani Bizzozzero Division Niguarda Ca Granda Hospital Milan Italy