Angel Ferrer

The use of raw or boiled crustacean extracts for the diagnosis of seafood allergic individuals.

BACKGROUND Seafood plays an important role in human nutrition and is responsible for severe hypersensitivity reactions. OBJECTIVE To evaluate how the cooking process may alter the in vivo and in vitro allergenicity of these extracts. METHODS Raw and boiled extracts of shrimps and 2 types of lobsters were manufactured. Boiled extracts were prepared after the raw material was boiled for 15 minutes in phosphate-buffered saline. Raw and boiled extracts were homogenized and extracted for 4 hours. Afterward, the extracts were centrifuged, dialyzed, filtered, and freeze-dried. Seventy-eight patients were skin prick tested with these raw and boiled extracts. Specific IgE against the 6 extracts was measured by enzyme-linked immunosorbent assay (ELISA). Immunoblots and ELISA inhibition studies were performed with a pool of sera. RESULTS In vivo results showed that boiled extracts induced statistically significant larger wheals than raw extracts. More patients with positive results were also detected with boiled extracts. In vitro experiments by direct ELISA confirmed the in vivo results. The protein content in the boiled extracts decreased, and important differences were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-reactivity experiments showed that both types of extracts retained similar allergenic characteristics, even if the immunoblots revealed some differences in IgE binding. CONCLUSIONS More patients were identified using boiled extracts of shrimp and American and spiny lobsters than with raw extracts. The wheal sizes of the skin test reactions and specific IgE levels were also significantly greater using boiled extracts. The use of boiled extracts seems to be more effective in diagnosing seafood allergy.

Allergenicity of 10 different apple varieties.

BACKGROUND More than 100 apple varieties are consumed worldwide. However, the allergenic composition of most apple varieties has not been fully characterized. OBJECTIVE To analyze the antigenic and allergenic profiles of 10 different commercially available apple varieties. METHODS Golden, Golden Perlim, Reineta, Reineta Parda, Fuji, Verdedoncella, Granny-Smith, Pink Lady, Royal Gala, and Starking apple varieties were carefully peeled. Peels were individually extracted and centrifuged, and the supernatants were collected, dialyzed, filtered, frozen, and freeze-dried. The extracts were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. Protein content was measured using the Lowry-Biuret method. Twenty-two Spanish patients with oral allergy syndrome after apple ingestion were included in the study. Specific IgE to all apple varieties and birch pollen was evaluated. Eleven patients underwent skin prick testing using extracts of Golden, Starking, and Reineta Parda varieties to determine differences in their in vivo allergenicity. RESULTS The antigenic profile of the 10 varieties showed differences using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 9-kDa band, which could correspond to Mal d 3, was visualized in all the extracts. The Starking variety had the highest protein content. Higher specific IgE values were also obtained for the Starking variety, and the lowest values were for Reineta Parda and Royal Gala. The largest wheals were obtained in all patients with the Starking variety, and the smallest with Reineta Parda. Fourteen patients (64%) recognized Mal d 3 in the Golden variety using immunoblots; 6 patients (27%), who also had specific IgE to Betula, recognized bands with higher molecular weights. CONCLUSIONS We demonstrated differences in the antigenic and allergenic profiles of the analyzed varieties and a significant variation in Mal d 3 content.

Recently introduced foods as new allergenic sources: Sensitisation to Goji berries (Lycium barbarum)

Goji berries (GB) have been introduced in Western diet. Preliminary reports have demonstrated its allergenic capacity. The objectives of the study were to investigate the frequency of sensitisation and the allergens involved. 566 individuals, with respiratory or cutaneous symptoms were skin-prick tested with GB extract. Thirty three were positive (5.8%). 94% were sensitised to other allergens. Specific IgE to GB, peach, tomato and nut-mix was measured. Thirteen individuals from 24 available sera (54.2%) had positive specific IgE. 92.3% of GB positive patients were positive to peach. Seven individuals recognised 8 bands and six recognised a 7kDa band. This band was identified as a LTP by MS/MS. Cross-reactivity was demonstrated with tomato, tobacco, nutmix, Artemisia pollen and purified Lyc e 3 and Pru p 3. GB are a new allergenic source with high prevalence of sensitisation. LTP seems to be the major allergen involved in sensitisation and cross-reactivity.

Sensitization and Allergy to Cannabis sativa Leaves in a Population of Tomato (Lycopersicon esculentum)-Sensitized Patients

Background: Cases of allergy to Cannabis sativa have occasionally been reported, but both the allergenic profile and eventual cross-reactivity pattern remain unknown. Objective: To analyze the allergenic profile of a population of patients from Spain sensitized to C. sativa and to characterize the C. sativa leaf extract. Methods: A total of 32 subjects were enrolled in the study: group A, 10 individuals sensitized to tomato, reporting reactions by contact or inhalation to Cannabis; group B, 14 individuals sensitized to tomato, without reactions to Cannabis; group C, 8 individuals not sensitized to tomato and without reactions to Cannabis. Sensitivity to Cannabis, tomato and peach peel, Platanus hybrida and Artemisia vulgaris pollen extracts was measured by skin tests and specific IgE. Individual immunoblots and inhibition experiments with a pool of sera were conducted. Results: All tomato-sensitized subjects (and 1 negative) had positive skin tests to C. sativa leaves and hashish. Specific IgE to C. sativa and peach peel was more common than to tomato. Immunoblot experiments showed 2 prominent bands of 10 and 14 kDa and 2 weakly recognized bands of 30 and 45 kDa. Tomato, peach and A. vulgaris extracts inhibited most of the bands present in C. sativa. P. hybrida inhibited only the high-molecular-weight bands. Conclusion: Sensitization to C. sativa with or without symptoms is frequent among patients in Spain sensitized to tomato. C. sativa leaves are a potential allergenic source and their allergens may cross-react with other allergenic sources from plants (fruit peels and pollen).

Identification and quantification of tomato allergens: in vitro characterization of six different varieties

BACKGROUND The prevalence of sensitization to tomato has been estimated in 6.5% in a Mediterranean population. OBJECTIVES To characterize the allergenic profile of 6 commonly ingested varieties of tomato and to identify and quantify the most representative allergens. METHODS Rama, Rambo, Canario, Kumato, Pera and Raf tomato varieties were carefully peeled and extracts from peel and pulp prepared. The extracts were characterized by SDS-PAGE and immunoblot. Lyc e 3 and osmotin-like protein (OLP) were identified by LC MS/MS and PG2A by immunoblot with a specific polyclonal antibody. Protein content and allergen concentration (Lyc e 3 and PG2A) were measured by scanning densitometry after identification. One hundred and ninety-one patients were enrolled in the study. Specific IgE to all tomato extracts was evaluated by direct ELISA. RESULTS Eighty (41.7%) individuals reported symptoms after contact/ingestion with tomato, most of them oral symptoms. Protein profile and allergen quantification of the 6 varieties showed differences in the allergen composition. Lyc e 3 was visualized in all extracts, being the most abundant in all cases. PG2A and OLP were identified in all peel extracts and in some pulp extracts. Higher specific IgE values were obtained for the Kumato variety. Immunoblot showed the existence of several bands with IgE-binding capacity, especially at 12 and 45-50 kDa. CONCLUSIONS The study demonstrates differences in the antigenic and allergenic profiles of the analyzed varieties and variations in their allergenic composition, affecting the diagnostic capacity. LTP and PG2A are important tomato allergens in our population but other IgE-binding capacity proteins are also involved.

Occupational asthma to carmine in a butcher.

Hypersensitivity to carmine (E120) has been identified as a cause of food intolerance and occupational asthma. We present a case of occupational asthma following exposure to carmine in a manufacturer of sausages and review the literature. Case Report: A 42-year-old non-atopic male presented with a 5-year history of rhinoconjunctivitis and asthma on occupational exposure to food additive dusts. Symptoms increased after work. The patient had been exposed for more than 20 years. Methods: Skin prick tests were performed with a battery of common inhalant allergens and spices. Cochineal, carmine lake and additive mixes used by the patient were extracted and subsequently used for skin prick test, bronchial provocation and in vitro measurements (specific IgE, Western blot and chromatographic fractionation). Results: Prick tests were positive to carmine and carmine-containing additives; carmine-specific IgE and bronchial challenge tests were also positive (PC20 = 0.0004 mg/ml and 1.6 kU/l). Western blot showed IgE binding to bands of about 30 kDa on cochineal extract and a diffuse pattern at 40–97 kDa on carmine. This result was confirmed by gel filtration chromatography and dot blot. Carmine completely inhibited IgE binding to cochineal extract. Discussion: Carmine is a potential sensitizer in an occupational setting: 18 cases of occupational asthma have been described to date. Carmine allergens are poorly defined; in general, proteins from cochineal not removed by the extraction process are considered as the main allergens in carmine. Our results are consistent with this, but show that these proteins may be subject to chemical modification.

Characterization and improvement of apple extracts for the diagnosis of apple IgE-mediated allergy

BACKGROUND The production process of reliable fruit extracts is not well established. OBJECTIVES To improve the overall quality of apple extracts by reducing protein loss during the manufacturing process and to evaluate the improved extracts using in vivo and in vitro experiments. METHODS Two types of extracts were prepared from peels of Golden Delicious apples (Malus domesticus). Extract A was extracted, 1:2 wt/vol, for 30 minutes at 40 degrees C in 0.01 M phosphate-buffered saline, and extract B was extracted, 1:2 wt/vol, in phosphate-buffered saline with 20% polyvinylpolypyrrolidone and 2-mmol/L EDTA. Both extracts were filtered, dialyzed in 3.5-kDa dialysis membranes, and lyophilized. The antigenic and allergenic profiles were analyzed using immunoblot and enzyme-linked immunosorbent assay. Nine patients clinically sensitive to apples and 12 controls underwent skin testing with both extracts. RESULTS Extracts A and B had dry weight yields of 0.71% and 1.86% and protein contents of 104.6 and 257 microg/mg of freeze-dried material, respectively. A steady and progressive loss of protein, greater in extract A than in extract B, was detected at different intervals during the manufacturing process of both extracts. Extract B produced larger wheal sizes than extract A (P = .008). Enzyme-linked immunosorbent assay inhibition results confirmed that extract B had a greater inhibition capacity than extract A. CONCLUSIONS A progressive loss of protein content occurs during the manufacturing of apple extracts. Wheal sizes induced by extract B were significantly larger than those induced by extract A and prick-by-prick solutions. Extract B was also more potent in vitro than extract A.

Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method

BackgroundCommercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed.The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method.MethodsTwo groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (≥ 7 mm2) were compared between groups.ResultsIn group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 ± 40.19 mm2 compared with 44.25 ± 36.68 mm2 induced by the peel extract (Not significant), and 17.79 ± 9.39 mm2 induced by the pulp extract (p < 0.01).In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 ± 13.12 mm2 compared with 33.17 ± 17.55 mm2 induced by peel extract (Not significant), and 13.33 ± 4.80 mm2 induced by pulp extract (p < 0.05 with peel extract and prick-prick).ConclusionCanary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.

Allergic bronchopulmonary cladosporiosis in a child

Since the first descript ion of allergic bronchopu lmona ry aspergillosis (ABPA) in 1952, m a n y repor ts in the l i terature have increased the knowledge about immunolog ic mechanisms, clinical presentat ion, diagnostic criteria, and t r ea tmen t of the disease. 1-3 Aspergillus fumigatus is the mos t comm o n fungus involved, but o ther Aspergillus species and o the r fungi have been r epor t ed as causat ive agents. 4-a2 Candida albicans, 4, 11 Mucor spp., 12 Helminthosporium spp., 5 Stemphylium spp., 6 Torulopsis spp., 7 Curvularia lunata, 8, 22 Rhizopus spp.,l°, 11 Penicillium spp., n Drechslera spp., 8 and Pseudallescher/a spp? 1 are fungi involved in allergic b ronchopul mona ry mycosis ( A B P M ) (Table I). Diagnostic criteria for A B P A are well established in adult patients, 1 but there are few reports of this disease in children. 2,3,9, 22,13 We present a case of childhood A B P M caused by C. herbarum. To our knowledge, this is the only published case of childhood A B P M caused by this fungus. Remiss ion was achieved by adequa te cor t icosteroid t rea tment .

Allergenic Differences among Pollens of Three Salsola Species

Background: Sensitivity to Chenopodiaceae is a frequent cause of allergic respiratory diseases in geographic areas where sensitization to Salsola kali and Chenopodium album has been reported. The objective of this study was to evaluate the pattern of sensitization to 3 Salsola species in patients residing on the Mediterranean coast of south-eastern Spain. Methods:S. kali, S. vermiculata and S. oppositifolia pollen extracts were prepared. Patients reporting respiratory and/or cutaneous symptoms were skin prick tested with the 3 Salsola extracts. Individuals with positive skin prick tests to at least 1 of the 3 Salsola species were included. Specific IgE was determined by direct ELISA. SDS-PAGE and 2-D analysis were conducted to elucidate the protein profile. The allergenic profile was investigated by immunoblot. Inhibition experiments were conducted to establish cross-reactivity between different species. Results: 246 patients were included. 237 patients (96.3%) tested positive to S. oppositifolia, 189 (76.8%) to S. kali and 185 (75.2%) to S. vermiculata. Protein profile and immunoblot demonstrated similar patterns in all extracts, except in low-molecular-weight allergens of S. oppositifolia. Immunoblot inhibition experiments demonstrated that most high-molecular-weight allergens of S. oppositifolia were inhibited by S. kali whereas low-molecular-weight allergens were totally inhibited only by C. album. Conclusions: This study confirms the allergenic importance of other Salsola species, especially S. oppositifolia. We have demonstrated that the 3 species show a high degree of cross-reactivity, but S. oppositifolia shares more allergenic similarities with C. album than S. kali.