Elide A. Pastorello

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.

Efficacy of recombinant birch pollen vaccine for the treatment of birch-allergic rhinoconjunctivitis

BACKGROUND Recombinant DNA technology has the potential to produce allergen-specific immunotherapy vaccines with defined composition. OBJECTIVE To evaluate the effectiveness of a new recombinant birch pollen allergen vaccine in patients with birch pollen allergy. METHODS A multicenter, randomized, double-blind, placebo-controlled trial was undertaken to compare the following 3 vaccines in 134 adults with birch pollen allergy: recombinant birch pollen allergen vaccine (rBet v 1a), licensed birch pollen extract, natural purified birch pollen allergen (nBet v 1), and placebo. Patients received 12 weekly injections followed by monthly injections of the maintenance dose containing 15 microg Bet v 1 for 2 years. RESULTS Significant reductions (about 50%) in rhinoconjunctivitis symptoms (rBet v 1, P = .0002; nBet v 1, P = .0006; birch extract, P = .0024), rescue medication (rBet v 1, P = .0011; nBet v 1, P = .0025; birch extract, P = .0063), and skin sensitivities (P < .0001) were observed in the 3 actively treated groups compared with placebo during 2 consecutive pollen seasons. Clinical improvement was accompanied by marked increases in Bet v 1-specific IgG levels, which were higher in the rBet v 1-treated group than in the birch and nBet v 1-treated groups. New IgE specificities were induced in 3 of 29 patients treated with birch pollen extract, but in none of the 32 rBet v 1-treated or 29 nBet v 1-treated patients. No severe systemic adverse events were observed in the rBet v 1-treated group. CONCLUSION The rBet v 1-based vaccine was safe and effective in treating birch pollen allergy, and induced a highly specific immune response.

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.

Studies on the relationship between the level of specific IgE antibodies and the clinical expression of allergy: I. Definition of levels distinguishing patients with symptomatic from patients with asymptomatic allergy to common aeroallergens

BACKGROUND The detection of specific IgE antibodies to environmental allergens does not always coincide with a diagnosis of clinically evident allergic disease, because some patients with positive skin and/or in vitro test results have no symptoms related to the allergen or allergens that induced the antibodies. OBJECTIVE In a multicenter study the optimal cutoff values for specific IgE antibody levels and skin test results that could discriminate between patients with symptomatic and those with asymptomatic allergy were determined. METHODS IgE antibodies specific for a panel of common aeroallergens were assayed with the Pharmacia CAP System (Pharmacia, Uppsala, Sweden) in two groups of patients, a group of 267 patients with symptomatic allergy and a group of 232 with asymptomatic allergy--both with positive skin prick test results--and in a group of 243 healthy, nonallergic control subjects. The cutoff values were established by receiver operating characteristic analysis. RESULTS A significantly higher mean specific IgE antibody value was found in patients with symptomatic allergy compared with patients with asymptomatic allergy (p < 0.001) and in patients with symptomatic allergy compared with healthy control subjects (p < 0.001). The optimal CAP System cutoff value between patients with symptomatic and those with asymptomatic allergy was 11.7 kU/L, and when seasonal allergens were compared with perennial allergens, the cutoffs were 10.7 kU/L and 8.4 kU/L, respectively. The optimal cutoff value for the skin prick test was a wheel area of 32 mm2 for seasonal allergens and 31 mm2 for perennial allergens. The skin test had a lower diagnostic value (sum of sensitivity and specificity) than the CAP System. CONCLUSIONS Cutoff values for specific serum IgE antibody levels are likely to be useful in clinical practice to distinguish symptomatic from asymptomatic allergy in patients with positive skin test results.

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.