Valerio Pravettoni

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.

Wheat IgE-Mediated Food Allergy in European Patients: α-Amylase Inhibitors, Lipid Transfer Proteins and Low-Molecular-Weight Glutenins

BACKGROUND Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osbornes three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. METHODS Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osbornes protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. RESULTS The most important wheat allergens were the alpha-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. CONCLUSIONS The alpha-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.Background: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne’s three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. Methods: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne’s protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. Results: The most important wheat allergens were the α-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. Conclusions: The α-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

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.

The major allergen of sesame seeds (Sesamum indicum) is a 2S albumin.

BACKGROUND Allergic reactions induced by ingestion of foods containing sesame seeds are a well recognized cause of severe food-induced anaphylaxis. OBJECTIVE This study aimed to identify and characterize the clinically most important major allergen of sesame seeds. METHODS Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and IgE immunoblotting were performed on sera of 10 patients selected for severe and documented allergic reaction after eating food containing sesame. The major allergen was purified by gel filtration and characterized by isoelectric point (pI), glycosylation and amino acid sequencing. RESULTS All the patients had positive IgE antibodies and skin prick tests (SPTs) to sesame. The major, clinically most important allergen was a protein with molecular mass of about 9000. It was not glycosylated, the amino acid sequence showed it was a 2S albumin with a pI of 7.3; the small and the large subunits, forming the whole protein, showed pI values of 6.5 and 6.0.

Evidence for a lipid transfer protein as the major allergen of apricot

BACKGROUND Apricots are widely grown in Europe, and allergic reactions are becoming more common, especially oral allergy syndrome. Apricot belongs to the botanical subfamily of Prunoideae, which includes peach, the major allergen of which was identified as a 9-kd protein, a lipid transfer protein (LTP). OBJECTIVE The aim of the study was to evaluate the IgE reactivity pattern to an apricot extract in subjects with allergic reactions to apricot, as demonstrated by a positive oral challenge response. METHODS Thirty patients were investigated. All the patients displayed oral allergy syndrome (2 with systemic reactions) to apricot, with positive open food challenge responses, skin prick test responses, and serum-specific IgE antibodies to apricot. The IgE reactivity pattern to apricot extract was identified by using SDS-PAGE and immunoblotting. The major allergen, a 9-kd protein, was then purified by HPLC and characterized by periodic acid-Schiff stain, isoelectric point determination, and N-terminal amino acid sequencing. RESULTS The sera from all patients allergic to apricot recognized the 9-kd protein, whereas none of the other allergens, with molecular weights from 15 to 80 kd, acted as a major allergen. The 9-kd allergen has an isoelectric point of 8.7 and is not glycosylated. Determination of the N-terminal 34 amino acid sequence showed that it belongs to the LTP family, with a 94% homology with the LTP from peach. IgE blotting of the apricot extract was completely inhibited by the 9-kd purified LTP from peach. CONCLUSIONS The major allergen of apricot is an LTP, which is highly cross-reactive with the LTP from peach.

Incidence of anaphylaxis in the emergency department of a general hospital in Milan.

OBJECTIVE To evaluate incidence and causes of anaphylactic reactions in the emergency room (E.R.) of a general hospital in Milan during a 2-year period. METHODS We retrospectively studied the computerized records of patients discharged from an E.R. with a diagnosis of anaphylactic reaction. Anaphylaxis was established on the presence of at least two cutaneous, respiratory, gastrointestinal or cardiovascular system symptoms. RESULTS During 1997 and 1998, out of 38 685 patients referred to the E.R., 13 had severe anaphylaxis with loss of consciousness (LOC) and 127 had anaphylactic symptoms, without LOC. Of the 13 patients with LOC, a possible cause was identified in 12 (five foods, six drugs, one hair dye). In the other 127 patients anaphylaxis was related to foods in 49 cases (38.5%), drugs in 44 (34.6%), unknown causes in 29 (22.8%), hymenoptera stings in two (1.5%), and other causes in three (2.3%). CONCLUSION The incidence of anaphylactic reactions was 0.4% and mainly affected females and atopic subjects. Foods, particularly fruits and vegetables, appeared to be the most important cause; other important causes were non steroidal antiinflammatory drugs and beta-lactam antibiotics.

Characterization of the major allergen of plum as a lipid transfer protein

BACKGROUND Allergy to Prunoideae fruit (plum, peach, cherry and apricot) is one of the most frequent food allergies in southern Europe. All these fruits cross-react in vivo and in vitro, as they share their major allergen, a 9 kD lipid transfer protein (LTP). OBJECTIVE The aim of the study was the identification and molecular characterization of the major allergen of plum. METHODS The IgE pattern of reactivity to plums was investigated by SDS-PAGE and immunoblotting with the sera of 23 patients. The identified major allergen was purified by HPLC, using a cationic-exchange column followed by gel-filtration. Further characterization was achieved by periodic-Schiff stain, isoelectrofocusing and N-terminal amino acid sequencing. RESULTS AND CONCLUSIONS The major allergen of plum is a 9 kD lipid transfer protein, not glycosylated and with a basic character (pI>9), highly homologous to the major allergen of peach.