Allergenic risk assessment of cowpea and its cross-reactivity with pea and peanut.

BACKGROUND: Novel protein sources can represent a risk for allergic consumers. The aim of this study was to evaluate the allergenicity of cowpea (Vigna unguiculata), an increasingly consumed legume and potential new industrial food ingredient which may put legume-allergic patients at risk. METHODS: Children with allergy to legumes associated to peanut (LP group: n = 13) or without peanut allergy (L group: n = 14) were recruited and sensitization to several legumes including cowpea was assessed by prick tests and detection of specific IgE (sIgE). Cowpea protein extract was analyzed by SDS-PAGE and immunoblotting, IgE-reactive spots were subjected to mass spectrometry. IgE-cross-reactivity between cowpea, pea, and peanut was determined using ELISA inhibition assays. Basophil activation tests were performed to evaluate sensitivity and reactivity of patient basophils toward legumes. RESULTS: Prick tests and sIgE levels to cowpea were positive in 8/14 and 4/13 patients of the L group and in 9/13 and 10/13 patients of the LP group, respectively. Four major IgE-binding proteins were identified as vicilins and seed albumin. Cowpea extract and its vicilin fraction strongly inhibited IgE-binding to pea and peanut extract. Peanut, lentil, and pea were the strongest activators of basophils, followed by cowpea, soybean, mung bean, and lupin. CONCLUSION: A majority of patients with legume allergy were sensitized to cowpea proteins. Four novel allergens were identified in cowpea, among which storage proteins were playing an important role in IgE-cross-reactivity, exposing legume-allergic patients to the risk of clinical cross-reactivity to cowpea and thus adding cowpea to the group of nonpriority legumes that are not subjected to allergen labeling such as chickpea, pea, and lentil.

Mammalian derived lipocalin and secretoglobin respiratory allergens strongly bind ligands with potentially immune modulating properties.

Allergens from furry animals frequently cause sensitization and respiratory allergic diseases. Most relevant mammalian respiratory allergens belong either to the protein family of lipocalins or secretoglobins. Their mechanism of sensitization remains largely unresolved. Mammalian lipocalin and secretoglobin allergens are associated with a function in chemical communication that involves abundant secretion into the environment, high stability and the ability to transport small volatile compounds. These properties are likely to contribute concomitantly to their allergenic potential. In this study, we aim to further elucidate the physiological function of lipocalin and secretoglobin allergens and link it to their sensitizing capacity, by analyzing their ligand-binding characteristics. We produced eight major mammalian respiratory allergens from four pet species in E.coli and compared their ligand-binding affinities to forty-nine ligands of different chemical classes by using a fluorescence-quenching assay. Furthermore, we solved the crystal-structure of the major guinea pig allergen Cav p 1, a typical lipocalin. Recombinant lipocalin and secretoglobin allergens are of high thermal stability with melting temperatures ranging from 65 to 90°C and strongly bind ligands with dissociation constants in the low micromolar range, particularly fatty acids, fatty alcohols and the terpene alcohol farnesol, that are associated with potential semiochemical and/or immune-modulating functions. Through the systematic screening of respiratory mammalian lipocalin and secretoglobin allergens with a large panel of potential ligands, we observed that total amino acid composition, as well as cavity shape and volume direct affinities to ligands of different chemical classes. Therefore, we were able to categorize lipocalin allergens over their ligand-binding profile into three sub-groups of a lipocalin clade that is associated with functions in chemical communication, thus strengthening the function of major mammalian respiratory allergens as semiochemical carriers. The promiscuous binding capability of hydrophobic ligands from environmental sources warrants further investigation regarding their impact on a molecule's allergenicity.

Allergen Content of Therapeutic Preparations for Allergen-Specific Immunotherapy of European Paper Wasp Venom Allergy.

Allergy to Polistes dominula (European paper wasp) venom is of particular relevance in Southern Europe, potentially becoming a threat in other regions in the near future, and can be effectively cured by venom immunotherapy (VIT). As allergen content in extracts may vary and have an impact on diagnostic and therapeutic approaches, the aim was to compare five therapeutic preparations for VIT of P. dominula venom allergy available in Spain. Products from five different suppliers were analyzed by SDS-PAGE and LC-MS/MS and compared with a reference venom sample. Three products with P. dominula venom and one product with a venom mixture of American Polistes species showed a comparable band pattern in SDS-PAGE as the reference sample and the bands of the major allergens phospholipase A1 and antigen 5 were assignable. The other product, which consists of a mixture of American Polistes species, exhibited the typical band pattern in one, but not in another sample from a second batch. All annotated P. dominula allergens were detected at comparable levels in LC-MS/MS analysis of products containing P. dominula venom. Due to a lack of genomic information on the American Polistes species, the remaining products were not analyzed by this method. The major Polistes allergens were present in comparable amounts in the majority, but not in all investigated samples of venom preparations for VIT of P. dominula venom allergy.

Animal Allergens, Endotoxin, and ß-(1,3)-Glucan in Small Animal Practices: Exposure Levels at Work and in Homes of Veterinary Staff.

OBJECTIVES: In veterinary settings, high exposures to animal allergens and microbial agents can be expected. However, occupational exposure levels are largely unknown. The objective of this study was to estimate the allergen, endotoxin, and ß-(1,3)-glucan concentrations in small animal practices and in the homes of practice employees. METHODS: Dust samples were collected using electrostatic dust fall collectors in diverse rooms of 36 small animal practices, as well as in employees' homes. Major animal allergens (Fel d 1, Can f 1, Ory c 3, Cav p 1, Equ c 1, Bos d 2), domestic mite (DM) allergens, and ß-(1,3)-glucan levels were measured using enzyme immunoassays. Endotoxin was determined using the Limulus amoebocyte lysate assay. Influences on exposure levels were analyzed using multilevel models. RESULTS: The levels of Can f 1, Fel d 1, Ory c 3, and Cav p 1 were up to 30 times higher in practices compared with homes without animals, but significantly lower compared with the homes with the respective pet. Although horses were not treated in the practices, Equ c 1 was found in 87.5% of samples, with the highest concentrations measured in changing rooms. DM levels were significantly lower in practices than in all private homes, and endotoxin levels were similar to those in homes with pets. In the practice itself, exposure levels were significantly influenced by animal presence, type of the room, and area per employee; whereas, room volume and diverse cleaning measures had mostly no effect. CONCLUSIONS: Exposure to animal allergens is high in veterinary practices, but it does not reach levels of households with pets. Domestic mite allergen and endotoxin exposure seem to be low for workers in veterinary practices. The high Equ c 1 detection rate strongly indicates dispersal of allergens, most likely through clothing and hair.

Component-resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals.

BACKGROUND: Furry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea-pigs still relies on animal dander extracts which do not allow to define the primary sensitization source. OBJECTIVE: To identify major guinea-pig allergens and to evaluate their potential as marker allergens for in vitro IgE-diagnosis in comparison with dander extracts. METHODS: A group of patients allergic to guinea-pig (n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea-pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA. RESULTS: The combination of 4 guinea-pig lipocalin allergens, including 2 new lipocalins, Cav p 1.0201 and Cav p 6.0101, and the previously characterized lipocalins Cav p 2 and Cav p 3, enabled the identification of 90% of all patients allergic to guinea-pig. The vast majority had specific IgE to Cav p 1 (83%). Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE-cross-reactive with these allergens. In the group of cat- and dog-allergic patients, 73% had also specific IgE to guinea-pig dander. However, only 27% of the cat /dog-allergic patients had specific IgE to any of the non-cross-reactive guinea-pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea-pig dander could be explained by IgE-cross-reactivity among serum albumins and certain lipocalins. CONCLUSIONS AND CLINICAL RELEVANCE: The availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea-pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component-resolved IgE-diagnosis of allergy to small furry pets.

Shedding Light on the Venom Proteomes of the Allergy-Relevant Hymenoptera Polistes dominula (European Paper Wasp) and Vespula spp. (Yellow Jacket).

Allergic reactions to stings of Hymenoptera species can have serious or even fatal consequences. If the identification of the culprit insect is possible, venom-specific immunotherapy effectively cures Hymenoptera venom allergies. Although component-resolved diagnostics has strongly evolved in recent years, the differentiation between allergies to closely related species such as Polistes dominula and Vespula spp. is still challenging. In order to generate the basis for new diagnostic and therapeutic strategies, this study aims at resolving the venom proteomes (venomes) of these species. The venoms of P. dominula and Vespula spp. (V. germanica, V. vulgaris) were analyzed by liquid chromatography-mass spectrometry. Resulting proteins were characterized regarding their function, localization and biochemical properties. The analyses yielded 157 proteins in Vespula spp. and 100 in P. dominula venom; 48 proteins, including annotated allergens, were found in both samples. In addition to a variety of venom trace molecules, new allergen candidates such as icarapin-like protein and phospholipase A2 were identified. This study elucidates the venomes of closely related allergy-eliciting Hymenoptera species. The data indicates that relying on marker allergens to differentiate between P. dominula and Vespula spp. venom allergy is probably insufficient and that strategies using cross-reactive major allergens could be more promising.

Identification and isolation of a Fel d 1-like molecule as a major rabbit allergen.

BACKGROUND: Rabbits are increasingly kept as domestic pets. Several rabbit allergens have been characterized. However, their sequences are still elusive, and none of these molecules are available for diagnosis. OBJECTIVE: We sought to isolate major allergens from the rabbit Oryctolagus cuniculus and to investigate their importance in sensitized patients. METHODS: Proteins were extracted from rabbit hair, and IgE-reactive proteins were purified by using sequential chromatography. Allergens were characterized by means of N-terminal sequencing and mass spectrometry. IgE reactivity to a new allergen was analyzed in sera of 35 patients sensitized to rabbits in a domestic setting. A model of the crystal structure of the isolated proteins was constructed. RESULTS: A new IgE-reactive allergen, Ory c 3, was identified as rabbit lipophilin. The molecule that belongs to the secretoglobin family is a heterodimer of 18 to 19 kDa composed of 2 polypeptide chains, CL2 and AL. CL2 has a predicted N-linked glycosylation site confirmed by using mass spectrometry. Of the 35 patients with rabbit allergy studied, 27 (77%) had IgE to both the glycosylated and deglycosylated Ory c 3 heterodimer. Allergenicity of Ory c 3 was confirmed by using skin prick tests and the basophil activation assay. Modeling of the structure revealed a marked homology to Fel d 1, the major cat allergen. However, no IgE cross-reactivity was detected between Fel d 1 and Ory c 3. CONCLUSION: The rabbit lipophilin heterodimer AL-CL2 has been identified as a major rabbit allergen. After Fel d 1, Ory c 3 is the second mammalian secretoglobin shown to be a major allergen.