Raquel Moya

Cross-reactivity among non-specific lipid-transfer proteins from food and pollen allergenic sources.

Non-specific lipid-transfer proteins (nsLTPs) are a family of pan-allergens present in foods and pollen. However, sequence homology among them is limited. The objective of this study was to evaluate the IgE-mediated cross-reactivity between nsLTPs from different sources and evaluate the allergenic properties of LTPs from peach (Pru p 3) and pellitory (Par j 1/Par j 2), major fruit and pollen allergens. Both proteins were purified and characterised. Cross-reactivity studies among nsLTPs from different foods and pollens were performed by immunoblot inhibition using sera specific to peach or pellitory pollen. Cross-reactivity with Pru p 3 was observed in hazelnut, onion, corn, peanut and apple while in pollens, none of the extracts was inhibited with Par j 1/2. In conclusion, Pru p 3 did not inhibit LTPs from most fruits. Therefore, although Pru p 3 covers the largest number of epitopes, diagnosis with only this allergen may not detect all LTP sensitivities.

In vivo diagnosis with purified tropomyosin in mite and shellfish allergic patients

BACKGROUND Tropomyosin is the most studied shellfish allergen and has been involved in cross-reactivity among different invertebrates (crustacean, mollusks, mites, insects, and nematodes). OBJECTIVE To determine the relevance of tropomyosin in mite- and shellfish-sensitized patients using tropomyosin skin testing. METHODS Patients were divided into 3 groups: group M included mite allergic patients (ie, individuals with respiratory symptoms and a positive result on skin prick testing [SPT] to house dust mites), group S included shellfish allergic patients (ie, individuals who reported symptoms with shellfish), and group MS included mite- and shellfish allergic patients (ie, individuals who simultaneously fulfilled the inclusion criteria for groups M and S). Tropomyosin was purified from shrimp, characterized, and used in SPT for diagnosis in the patient population. RESULTS Eight hundred fifty patients were included in the study: 790 (92.9%) in group M, 21 (2.5%) in group S, and 39 (4.6%) in group MS. Tropomyosin was purified from shrimp with a purity higher than 95%. Forty-two individuals tested positive to tropomyosin: the prevalence was 2.7% in group M, 28.6% in group S, and 38.5% in patients of group MS. Twenty-one (50%) of the tropomyosin-positive individuals had symptoms with shellfish, and 3 (14.3%) reported anaphylaxis. CONCLUSION The prevalence of tropomyosin was low in mite-sensitized patients (2.7 %) and high in shellfish allergic patients (28.6%). The higher prevalence of tropomyosin was found in patients sensitized to both mite and shellfish (38.5%). The selection of tropomyosin-sensitized patients by SPT might help in the choice of appropriate treatments or management for these patients.

Immunoproteomic characterization of a Dermatophagoides farinae extract used in the treatment of canine atopic dermatitis

BACKGROUND Canine atopic dermatitis is a pruritic allergic skin disease. House dust mites have been identified as the main non-seasonal responsible agent. Unlike in human allergic patients, groups 1 and 2 antigens have been described as minor allergens in dogs, while groups 15 and 18 are considered the major allergens. Despite these differences, allergic dogs have traditionally been treated using extracts intended for human immunotherapy. OBJECTIVES To investigate the immunological characteristics and the allergen reactivity of dogs with atopic dermatitis using a Dermatophagoides farinae commercial extract. METHODS Eighteen dogs diagnosed with atopic dermatitis and 3 healthy control dogs from the Iberian Peninsula were included in the study. All the animals were older than 12 months, from both sexes and different breeds and showed positive specific IgE against D. farinae (>2500 ELISA Absorbance Units). The D. farinae allergenic extract used in this study was manufactured and characterized. The allergenic profile of the dogs was investigated by immunoblot and specific IgE, IgG, IgG1 and IgG2 measured by direct ELISA. Allergen identity was confirmed by immunoblot inhibition and mass spectrometry analyses. RESULTS The results confirmed the relevance of groups 15 and 18 antigens, but also groups 1, 2 and other medium molecular weight allergens in the sensitization of dogs with atopic dermatitis. Immunoblot inhibition and mass spectrometry assays confirmed these results. Relevant allergens were quantified by scanning densitometry (Der f 1: 17μg/mg, Der f 2: 20.3μg/mg, Der f 15: 18.1μg/mg and Der f 18: 9.4μg/mg). Concerning immunoglobulins profile, differences in IgE and IgG1 levels were observed between non-atopic and atopic dogs. CONCLUSIONS The commercial D. farinae extract characterized in this study contains the major allergens involved in the sensitization of dogs with atopic dermatitis, representing a suitable candidate for its use in the diagnosis and immunotherapy of mite allergic dogs.

Cloning and characterization of tropomyosin from the mite Chortoglyphus arcuatus.

Tropomyosin is a pan-allergen that shares a high homology among species. It is involved in cross-reactivity among mites, crustaceans, mollusks and insects. The objectives were to express and purify recombinant tropomyosin from the storage mite Chortoglyphus arcuatus, and to investigate the homology and cross-reactivity with tropomyosin from other invertebrates. Recombinant C. arcuatus tropomyosin (r-Cho a 10) was selected from a library by screening with a pool of patient sera. r-Cho a 10 (UniProt: H2DFL1) was sequenced, expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Polyclonal anti-tropomyosin antibodies were produced in mice. IgE recognition of r-Cho a 10 was checked by immunoblot. Immunoblot inhibition assays were used to identify the native tropomyosin in the complete extract from C. arcuatus and study cross-reactivity between r-Cho a 10 and Der p 10. Identification of tropomyosin in other allergenic sources was performed by immunoblot. r-Cho a 10 showed a high homology (54-96%) with other tropomyosins from different allergenic sources. IgE recognition was observed using a pool of sera from sensitized individuals. Tropomyosins from different extracts were identified not only in the whole C. arcuatus extract but also in Dermatophagoides pteronyssinus, shrimp, mussel, cockroach and Anisakis extracts with polyclonal α-Cho a 10. r-Cho a 10 completely inhibited the recognition of Der p 10. Recombinant C. arcuatus tropomyosin maintained its capacity to recognize IgE. r-Cho a 10 was used to prove cross-reactivity among tropomyosins from other invertebrate species, including mites. This is the first C. arcuatus allergen included in the WHO/IUIS (World Health Organization/International Union of Immunological Societies) Allergen Nomenclature database.

In vitro evidence of efficacy and safety of a polymerized cat dander extract for allergen immunotherapy

BackgroundAllergy to cat epithelia is highly prevalent, being the major recommendation for allergy sufferers its avoidance. However, this is not always feasible. Allergen specific immunotherapy is therefore recommended for these patients. The use of polymerized allergen extracts, allergoids, would allow to achieve the high allergen doses suggested to be effective while maintaining safety.ResultsCat native extract and its depigmented allergoid were manufactured and biochemically and immunochemically characterized. Protein and chromatographic profiles showed significant modification of the depigmented allergoid with respect to its corresponding native extract. However, the presence of different allergens (Fel d 1, Fel d 2, Fel d 3, Fel d 4 and Fel d 7) was confirmed in the allergoid. Differences in IgE-binding capacity were observed as loss of biological potency and lower stability of the IgE-allergen complex on surface plasmon resonance. The allergoid induced production of IgG antibodies able to block IgE-binding to native extract. Finally, studies carried out with peripheral-blood mononuclear cells from cat allergic patients showed that the allergoid induced IFN-γ and IL-10 production similar to that induced by native extract.ConclusionsCat depigmented allergoid induced production of cytokines involved in a Th1 and Treg response, was able to induce production of IgG-antibodies that blocks IgE-binding to cat native extract, and showed reduced interaction with IgE, suggesting greater safety than native extract while maintaining in vitro efficacy.

Prevalence of allergic sensitization to conifer pollen in a high cypress exposure area

Background Sensitization to Finales (Cupressaceae and Pinaceae) has increased dramatically in recent years. The prevalence of sensitization in different geographic areas is related to exposure to specific pollens. Objectives To investigate the prevalence of allergy to different conifer pollens, describe the characteristics of patients with such allergy, and identify the involved allergens. Methods Patients were recruited at five hospitals near Madrid. Extracts from conifer pollen were prepared and used in skin-prick testing. Wheal sizes were recorded, and serum samples obtained from patients with positive reactions to Cupressus arizonica and/or Pinus pinea. The specific immunoglobulin E value to C. arizonica and Cup a 1 was determined. Individual immunoblots for each patient and with a pool of sera were performed. Allergenic proteins were sequenced by using liquid chromatography-tandem mass spectrometry. Results Of 499 individuals included in the study, 17 (14%) had positive skin-prick test results to some conifer pollen extracts. Sixty-four patients had positive results to C. arizonica (prevalence 12.8%) and 11 had positive results to P. pinea (2.2%). All the patients had respiratory symptoms (61.4% during the C. arizonica pollination period), and 62.9% had asthma. Approximately 86% of the patients had positive specific immunoglobulin E results to C. arizonica and 923% had positive results to Cup a 1. Fourteen different bands were recognized by immunoblot; the most frequent bands were those detected at 43, 18, 16, and 14 kDa. All sequenced proteins corresponded to Cup a 1. Conclusion Allergy to conifer pollen could be considered a relevant cause of respiratory allergy in central Spain. Asthma was more frequent than in other studies. We only identified Cup a 1 as involved in sensitization.

Preclinical safety and immunological efficacy of Alternaria alternata polymerized extracts: Efficacy and safety of Alternaria alternata allergoid extract

Alternaria alternata is a widespread fungi whose allergy is a risk factor for asthma development. The use of a polymerized allergen extract (allergoid) may be safer than native extract based treatments while maintaining efficacy. The objective of this study was to characterize biochemically and immunochemically a new Alternaria alternata allergoid.

Purification and immunochemical characterization of Pla l 2, the profilin from Plantago lanceolata

&NA; Profilins are small actin‐binding proteins found in eukaryotes and involved in cell development, cytokinesis, membrane trafficking, and cell motility. From an allergenic point of view, profilins are panallergens usually involved in allergic polysensitization, although they are generally recognized as minor allergens. The objectives of this study were to identify and characterize the profilin from Plantago lanceolata pollen and to investigate the cross‐reactivity between profilins from different pollen allergenic sources. Profilins from P. lancelolata (Pla l 2) and palm tree pollen (Pho d 2) were purified by affinity chromatography, deeply characterized and identified by mass spectrometry. Pla l 2 allergenicity was confirmed by immunoblot with serum samples from a patient population sensitized to profilin. Immunoblot inhibition was performed to study IgG reactivity between different pollen profilins. IgE cross‐reactivity was demonstrated by ImmunoCAP inhibition. Pla l 2 is the second P. lanceolata allergen included in the IUIS Allergen Nomenclature database. Four peptides from purified Pla l 2 were identified with percentages of homology with other pollen profilins between 73 and 86%. Eighty‐six percent (21/24) of the patient population recognized Pla l 2. The allergenic relatedness between Pla l 2, Pho d 2 and six pollen profilins was confirmed, and IgE cross‐reactivity of Pla l 2 with rBet v 2 and rPhl p 12 was demonstrated. Pla l 2 is the profilin from P. lanceolata. The demonstrated allergenicity of this protein and its cross‐reactivity with other pollen profilins support its use in profilin diagnostic assays. HighlightsPla l 2 is a new allergen from Plantago lanceolata included in the IUIS.The profilin from P. lanceolata pollen was purified and characterized.Pla l 2 showed a high homology (73–86%) with other pollen profilins.Cross‐reactivity between pollen profilins has been investigated.Pla l 2 could be used in profilin diagnostic assays.