Jorge A. Guisantes

The major allergen of Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae family

There is general consensus regarding the scarce cross‐reactivity existing between Alternaria alternata and other allergenic moulds such as Aspergillus fumigatus, Penicillium notatum or Cladosporium herbarum. However, A. alternata has been shown to have a very significant level of allergenic cross‐reactivity with other fungi belonging to the Pleosporaceae family. To date, no biological identity or homologies with other proteins have been described for Alt a 1, and it remains unclear whether the major allergen Alt a 1 contributes to the cross‐reactivity shown for these moulds. Specific quantification of Alt a 1 in culture filtrates of Stemphylium botryosum, Ulocladium botrytis, Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum and Asp. fumigatus, and immunoblotting using culture filtrate extracts from the above‐mentioned moulds and rabbit serum anti‐recombinant Alt a 1 have shown significant amounts of Alt a 1 in culture filtrates as well as antigenic components ranging from 14 to 20 kDa that strongly react with the specific serum for all taxonomically related species (Pleosporaceae family). No reactions were revealed in culture filtrates of Cladosporium, Penicillium and Aspergillus. These results restrict the cross‐reactivity phenomenon due to Alt a 1 to the scope of the taxonomical term of family.

Diagnostic value of Alt a 1, fungal enolase and manganese‐dependent superoxide dismutase in the component‐resolved diagnosis of allergy to pleosporaceae

Cite this as: I. Postigo, A. Gutiérrez‐ Rodríguez, J. Fernández, J. A. Guisantes, E. Suñén and J. Martínez, Clinical & Experimental Allergy, 2011 (41) 443–451.

Characterization of excretory–secretory products from protoscoleces of Echinococcus granulosus and evaluation of their potential for immunodiagnosis of human cystic echinococcosis

This study describes, for the first time, the characterization of excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, evaluating their usefulness in the immunodiagnosis of human cystic echinococcosis. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. This preparation contained over 20 major protein components which could be distinguished by 1-dimensional SDS-PAGE with apparent masses between 9 and 300 kDa. The culture of of protoscoleces from liver produced a greater variety of excretory-secretory protein components than those from lung. Determination of enzymatic activities of secreted proteins revealed the presence of phosphatases, lipases and glucosidases, but no proteases. These findings were compared to those obtained from somatic extracts of protoscoleces and hydatid cyst fluid products. Immunochemical characterization was performed by immunoblotting with sera from individuals infected by cystic echinococcosis (n = 15), non-hydatidic parasitoses (n = 19), various liver diseases (n = 24), lung neoplasia (n = 16), and healthy donors (n = 18). Antigens with apparent masses of 89, 74, 47/50, 32, and 20 kDa showed specificity for immunodiagnosis of human hydatidosis. The 89 and 74 kDa components corresponded to antigens not yet described in E. granulosus, whereas proteins of 41-43 kDa and 91-95 kDa were recognized by the majority of the non-hydatid sera studied.

Enzymatic activities of Alternaria alternata allergenic extracts and its major allergen (Alt a 1)

Several allergens from Alternaria alternata have been isolated allowing some of them to be identified and characterised. Despite the fact that the major allergen of A. alternata (Alt a 1) has been extensively produced by recombinant technology, its biological activity still remains unknown. In the present study, extracts from culture filtrates were used to evaluate the intra‐specific variability of the enzymes and also as a source for isolating and purifying native Alt a 1. This was purified by affinity chromatography using antibody anti‐recombinant Alt a 1 (produced in Escherichia coli). Enzyme activities were analysed by the API‐ZYM System screening method. Results demonstrated the high variability of enzyme activities among the different strains. Only activities corresponding to phosphatases, esterases and β‐glucosidase were expressed by 100% of the strains. Both native and recombinant Alt a 1 showed phosphatase and esterase activities, suggesting that the glucidic moiety of this allergen does not significantly affect its enzyme activity.

Identification of allergens homologous to Alt a 1 from Stemphylium botryosum and Ulocladium botrytis

Several studies have demonstrated that proteins homologous to the Alt a 1 major allergen of Alternaria alternata are expressed in other members of the Pleosporaceae family. However, since no direct biochemical data have been reported concerning the presence of Alt a 1 allergen homologues capable of binding IgE in the excretion-secretion products of Stemphylium and Ulocladium, our objective was to explore their presence in Stemphylium botryosum and Ulocladium botrytis. S. botryosum and U. botrytis culture filtrate extracts were analyzed by two-dimensional (2D)-electrophoresis and 2D-immunoblotting using polyclonal rabbit antibodies raised against recombinant Alt a 1, as well as five human sera from patients allergic to Alternaria. Cross-reactivity immunoassays were performed by ImmunoCAP inhibition and 2D-immunoblotting inhibition. IgE-binding proteins recognized by the rabbit antiserum raised against Alt a 1, with apparent molecular weights of 17-18 kDa and isoelectric points of 4, were identified as Alt a 1-like proteins. Alt a 1 inhibited IgE-specific binding to the Alt a 1 homologues from S. botryosum and U. botrytis. In conclusion, it was demonstrated that allergens which are homologous to Alt a 1 are expressed in the excretory-secretory materials of the phylogenetically-related species S. botryosum and U. botrytis.

Preliminary study of the presence of antibodies against excretory-secretory antigens from protoscoleces of Echinococcus granulosus in dogs with intestinal echinococcosis

The aim of the present study was to analyze the antibody response against excretory-secretory antigens (ES-Ag) from Echinococcus granulosus protoscoleces, using sera from dogs infected with E. granulosus and other helminths. ES-Ag were obtained from the first 50 h maintenance of protoscoleces in vitro. Immunochemical characterization was performed by immunoblotting with sera from dogs naturally infected with E. granulosus (n = 12), sera from dogs infected with helminths other than E. granulosus (n = 30), and helminth-free dog sera (n = 20). These findings were compared to those obtained from a somatic extract of protoscoleces (S-Ag). ES-Ag only showed four cross-reacting proteins of 65, 61, 54, and 45-46 kDa. Antigens with apparent masses of 89 and 50 kDa in ES-Ag and of 130 and 67 kDa in S-Ag were identified by sera of dogs infected with E. granulosus only, whereas a protein of 41-43 kDa was recognised by the majority of the sera from dogs with non-echinococcal infection. Employing ELISA to study the same sera, S-Ag revealed higher immunoreactivity than ES-Ag, but also showed higher cross-reactivity levels when sera from dogs with non-echinococcal infection were assayed in immunoblotting.

Cross-Reactions Between Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) Related to the Different Growth Phases of Cultures

Abstract The allergenic cross-reactivity of both inter- and intraspecies of house dust mites, Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961, taking into account the allergenic differences that exist throughout the growth curves, was evaluated by means of RAST-inhibition, using sera from patients allergic to these mites. The results demonstrate that extracts obtained from mite cultures during the maximum exponential growth phase are the best source of reagents to better discriminate cross-reactivity studies. The analyses obtained from this work, together with those obtained in previous reports, help to define the ideal conditions related to the allergenic diversity, avidity, and cross-reactivity of specific antibodies for the elaboration of allergenic extracts as a tool for use in diagnosis and specific treatment of IgE-mediated hypersensitivity caused by house dust mites.

Profilin cross-reactive panallergen causes latex sensitization in the pediatric population allergic to pollen

BACKGROUND Component-resolved diagnostics (CRD) has been demonstrated to be an excellent new tool for improving the current diagnosis of allergies, and it allows differentiation between polysensitization and cross-reactivity. OBJECTIVE To demonstrate the role of cross-reactive pollen allergens in pediatric patients living in areas with large amounts of airborne grass pollen grains who are sensitive to grass pollen and latex. METHODS Serum samples were obtained from 106 children between 3 and 14 years of age diagnosed with allergies to pollen based on clinical history, skin prick tests, and specific immunoglobulin E (IgE). None of them had allergy symptoms to latex or fruits. From these 106 children, 56 patients revealed positive results to Phleum-specific major allergens but not to cross-reactive allergens. The other 50 patients who showed positive specific IgE to Phleum-specific major allergens and to cross-reactive pollen allergens also showed positive results to latex allergens. CRD was carried out by specific IgE quantification using a fluoro-enzyme immunoassay (ImmunoCAPT System). RESULTS Results demonstrated a positive significant relationship between the specific IgE to Hev b 8 and Phl p 12 and also between the specific IgE to Hev b 8 and latex extract in the group of patients sensitized to species-specific and cross-reactive Phleum allergens. Positive significant relationships were also found between profilin and avocado or peach sensitizations. No other latex allergens gave positive results. CONCLUSION The apparent sensitization to latex in pediatric patients allergic to grass pollen is caused by the cross-reactive profilin panallergen; however, it is appears not to be clinically relevant.

Asthma induced by latex from ‘Christmas flower’ (Euphorbia pulcherrima)

Christmas flower or poinsettia [Euphorbia pulcherrima (Ep)] is widely used as an ornamental plant during Christmas season in many countries. It belongs to the family Euphorbiaceae that includes other allergenic plants such as Hevea brasiliensis (Hb) or rubber tree. There are few reports of contact dermatitis induced by this plant (1, 2). We studied a 6-year-old male who presented with rhinitis and asthma upon exposure to several poinsettia plants for two consecutive Christmas seasons. He clearly improved when he was out of his apartment. At the time of the first study he did not suffer from any other respiratory symptoms. Three years after the first Christmas-related episode, he developed mild rhinoconjunctivitis during spring time. He was able to eat banana, kiwifruit and chestnut without any ill effects. He had never eaten avocado. Red and green leaves and latex from Ep, and natural rubber latex were extracted in phosphate-buffered saline (PBS), dialysed in a 3.5-kDa cut-off dialysis membrane and freeze-dried, that was later reconstituted at a concentration of 10 to 1 mg/ml (25 mg of protein per 100 mg of raw material). Healthy, atopic and rubber latex (H. brasiliensis) allergic patients were studied as controls. The patient had positive skin-prick test (SPT) to grass pollen and to Ep red leaves (5 mm)and latex extracts (7 mm),whereas it was negative to green leaf extract and other common aeroallergens in our area. Natural rubber latex extract fromHb elicited a 2-mm wheal. Prick-prick tests with kiwifruit, banana, avocado and chestnut were negative. In five healthy control patients, SPT with Ep extracts were negative, but were positive in five of eight patients diagnosed with rhinitis and asthma induced by latex fromHb. Baseline PC20 methacholine was 14.3 mg/ml. Pre-challenge induced sputum samples showed no eosinophils. Both performed as previously described (3). After parent’s consent, bronchial challenge by tidal volume method with Ep extract at a concentration of 0.31 mg/ml elicited an isolated immediate asthmatic response, with a 20% fall in forced expiratory volume (FEV)1. Twenty-four hours after the specific bronchial challenge, PC20 methacholine decreased to 2.5 mg/ml and 5% of eosinophils was seen in induced sputum sample. Rubbing and latex glove use test were negative. In one patient diagnosed with rhinitis and asthma induced by Hb latex, who also had a positive SPT to Ep, the bronchial challenge test with Ep extract was negative. Total IgE was 703 kU/l. Specific IgE (Pharmacia CAP System, Uppsala, Sweden) was positive to Ep latex (6.65 kU/l), Hb latex (8.24 kU/l), banana (6.28 kU/l) and chestnut (9.3 kU/l). ImmunoCAP inhibition experiments demonstrated a high degree of allergenic cross-reactivity between latex extracts of Hb and Ep. IgE immunoblots performed under reducing conditions revealed major IgEbinding bands in the Ep latex extract (Fig. 1). The most intense reaction was observed at 36, 25 and 19 kDa bands. Patient’s serum also revealed several IgEbinding bands in the Hb extract (Fig. 1). Case reports of IgE-mediated rhinitis and asthma induced by latex from ornamental plants, and dust samples from the floor, such as Ficus benjamina, have been previously described (4). To the best of our knowledge, this is the first case report of IgE-mediated rhinitis and asthma induced by latex of Christmas flower or poinsettia. Although extensive in vitro cross-reactivity with natural rubber latex and related fruits was demonstrated, the patient had no symptoms with these products.

Bovine serum albumin contained in culture medium used in artificial insemination is an important anaphylaxis risk factor

OBJECTIVE To analyze the cause of the anaphylactic reaction after a standard artificial insemination process in a patient diagnosed with asthma. DESIGN Case report. SETTING Residencia Sanitaria Virgen de la Arrixaca (Murcia, Spain) and University of the Basque Country (Vitoria, Spain). PATIENT(S) A 30-year-old woman with a previous medical history compatible with respiratory allergy who suffered an anaphylactic reaction after an artificial insemination with spermatozoids in capable medium (Upgraded B2 INRA medium; Laboratories CCD, Paris, France). INTERVENTION(S) Cutaneous tests and specific IgE levels to inhalant allergens, grass and Olea pollens, and insemination medium were performed. MAIN OUTCOME MEASURE(S) Specific IgE levels to mammal epithelia and bovine serum albumin (BSA). RESULT(S) Skin prick tests were positive for inhalant allergens such as mites, cat, dog, horse, and rabbit epithelia, grasses and Olea pollens, and the insemination medium. The beta-lactamic tests were negative. The determination of specific IgE demonstrated positive values to mammal epithelia and mammal serum albumins including BSA. CONCLUSION(S) We report a case of an anaphylactic reaction to the BSA included in the insemination culture medium induced by a subclinical sensitivity to serum albumins of mammal epithelia. A previous testing with the medium is recommended and specific testing might be needed in women who have a history of animal epithelium allergies.