Donatella Fortunato

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.

Efficacy of donkey's milk in treating highly problematic cow's milk allergic children: An in vivo and in vitro study

Successful therapy in cows milk protein allergy rests on completely eliminating cows milk proteins from the childs diet: it is thus necessary to provide a replacement food. This prospective study investigated tolerance of donkeys milk in a population of 46 selected children with cows milk protein allergy, for whom it was not possible to use any cows milk substitute. Thirty‐eight children (82.6%) liked and tolerated donkeys milk at the challenge and for the entire duration of follow‐up. Catch‐up growth was observed in all subjects with growth deficit during cows milk proteins challenge. The degree of cross‐reactivity of immunoglobulin E (IgE) with donkeys milk proteins was very weak and aspecific. Donkeys milk was found to be a valid alternative to both IgE‐mediated and non‐IgE‐mediated cows milk proteins allergy, including in terms of palatability and weight‐height gain.

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.

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.

Lipid-transfer protein is the major maize allergen maintaining IgE-binding activity after cooking at 100°C, as demonstrated in anaphylactic patients and patients with positive double-blind, placebo-controlled food challenge results

BACKGROUND In a previous study a 9-kd lipid-transfer protein (LTP) was identified as the major allergen of raw maize in a population of 22 anaphylactic patients. However, the stability of this protein in cooked maize is unknown. OBJECTIVE We investigated the allergenicity of 5 maize hybrids and its modification after different thermal treatments by using sera from anaphylactic patients and patients with positive double-blind, placebo-controlled food challenges. METHODS Five maize hybrids were extracted by using different methods, obtaining the water-soluble, zein, total zein, glutelin, and total protein fractions. The IgE-binding capacity of the different extracts, both raw and after thermal treatment, was investigated by means of SDS-PAGE immunoblotting. A 9-kd heat-stable allergen was purified by means of HPLC and sequenced. Changes in its secondary structure during and after heating from 25 degrees C to 100 degrees C were monitored by means of circular dichroism. RESULTS All raw maize hybrids showed similar protein and IgE-binding profiles. The SDS-PAGE of all the heat-treated hybrids demonstrated a decreased number of stained bands in respect to the raw samples. The IgE immunoblotting demonstrated that the major allergen of the water-soluble, total zein, total protein, and glutelin fractions was a 9-kd protein identified by means of amino acid sequence as an LTP and a sub-tilisin-chymotrypsin inhibitor (in total zein fraction). The IgE-binding capacity of this 9-kd protein remained unchanged after thermal treatments, even though circular dichroism demonstrated an altered secondary structure. CONCLUSIONS Maize LTP maintains its IgE-binding capacity after heat treatment, thus being the most eligible candidate for a causative role in severe anaphylactic reactions to both raw and cooked maize.

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.

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.

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.

Maize food allergy: lipid-transfer proteins, endochitinases, and alpha-zein precursor are relevant maize allergens in double-blind placebo-controlled maize-challenge-positive patients.

Italian patients with maize anaphylaxis have been shown to have IgE toward two major maize allergens: an alpha-amylase inhibitor and a 9-kDa LTP. A complete study on maize food allergens in patients with positive maize double-blind, placebo-controlled food challenge (DBPCFC) is lacking. The objective was to utilize the three maize protein fractions to identify and characterize the most relevant IgE-binding proteins recognized by the sera of Italian and Swiss patients with either a positive maize-DBPCFC or a history of maize-induced anaphylaxis. Osborne’s protein fractions of maize were extracted to obtain water-soluble, total zein, and total protein fractions. Protein IgE-binding capacity was investigated by SDS-PAGE immunoblotting using the sera from DBPCFC-positive patients and from patients with maize-induced anaphylaxis. Purified maize LTP was used to inhibit the IgE immunoblotting of the three protein fractions. IgE immunoblotting demonstrated that the 9-kDa LTP was recognized by all the Italian patients and by none of the Swiss patients. Other allergens were: 14-kDa α-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-β precursor, and 26 kDa zein-α precursor; a newly described allergen, the globulin-2 precursor, identified in the total protein fraction. It is noteworthy that maize LTP and endochitinase were cross-reactive with grape LTP and one grape endochitinase. LTP was found to be the only major allergen in Italian patients with either positive maize challenge or a history of maize-induced anaphylaxis. We have identified other maize allergens in subjects with maize food allergy, as grape cross-reactive endochitinase, however, the clinical significance of these proteins needs to be investigated in larger groups of patients with allergy to these food items.

Determination of the primary structure of two lipid transfer proteins from apricot (Prunus armeniaca).

It has been recently demonstrated that the major allergen of apricot is a protein of molecular mass (Mr) 9000 belonging to the family of Lipid Transfer Protein. The aim of this study was the determination of the primary structure of apricot LTP by micro-sequencing and mass spectrometric analyses. Apricot LTP is a 91 amino acids protein like peach and almond LTPs with a sequence identity of 91% and 94%, respectively. Like for the peach LTP, 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 apricot LTP, supporting the hypothesis of a similar function.