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Notes: Background Artemisia vulgaris is a widespread weed in the Mediterranean area and several allergens have been detected in its pollen. One of them, Art v 3, belongs to the lipid-transfer protein (LTP) family and its prevalence in Artemisia-sensitized patients or its relationship with other LTP allergens is not clear.Objective To assess the pattern of sensitization to an array of mugwort allergens in a Mediterranean population, and to study the cross-reactivity of Art v 3 with Pru p 3 and Par j 1, relevant LTP allergens in the area.Methods Skin prick test was performed with whole extracts (A. vulgaris, Parietaria judaica and peach) and pure natural allergens Art v 1, Art v 3, Art v 60 kDa and Par j 1 in 24 mugwort-allergic patients from a Mediterranean area. In vitro assays included measurement of specific IgE and ELISA inhibition among LTP allergens.Results The three Artemisia allergens elicited a positive skin response in 70–80% of the patients. Seven patients were clearly sensitized to Par j 1 and 11 to Pru p 3. There was no correlation between Par j 1 and Pru p 3 sensitization, but a highly significant correlation was found between peach extract and Art v 3 as regards the skin response. No IgE cross-reactivity was observed between Art v 3/Par j 1 or Pru p 3/Par j 1. In contrast, Art v 3 significantly inhibited the binding to Pru p 3 of IgE from three patients' sera out of six studied, but Pru p 3 was not able to inhibit the IgE binding to Art v 3.Conclusion Art v 3 is a major mugwort allergen and in some patients with IgE to both Art v 3 and Pru p 3, Art v 3 behaves as the primary sensitizing agent.

Notes: Background Allergic reactions to pea (Pisum sativum) ingestion are frequently associated with lentil allergy in the Spanish population. Vicilin have been described as a major lentil allergen.Objective To identify the main IgE binding components from pea seeds and to study their potential cross-reactivity with lentil vicilin.Methods A serum pool or individual sera from 18 patients with pea allergy were used to detect IgE binding proteins from pea seeds by immunodetection and immunoblot inhibition assays. Protein preparations enriched in pea vicilin were obtained by gel filtration chromatography followed by reverse-phase high-performance liquid chromatography (HPLC). IgE binding components were identified by means of N-terminal amino acid sequencing. Complete cDNAs encoding pea vicilin were isolated by PCR, using primers based on the amino acid sequence of the reactive proteins.Results IgE immunodetection of crude pea extracts revealed that convicilin (63 kDa), as well as vicilin (44 kDa) and one of its proteolytic fragments (32 kDa), reacted with more than 50% of the individual sera tested. Additional proteolytic subunits of vicilin (36, 16 and 13 kDa) bound IgE from approximately 20% of the sera. The lentil vicilin allergen Len c 1 strongly inhibited the IgE binding to all components mentioned above. The characterization of cDNA clones encoding pea vicilin has allowed the deduction of its complete amino acid sequence (90% of sequence identity to Len c 1), as well as those of its reactive proteolytic processed subunits.Conclusions Vicilin and convicilin are potential major allergens from pea seeds. Furthermore, proteolytic fragments from vicilin are also relevant IgE binding pea components. All these proteins cross-react with the major lentil allergen Len c 1.

Notes: Lipid-transfer proteins (LTPs), but not Bet v 1 homologues, have been identified as major allergens of apple and peach in the Rosaceae fruit-allergic population in the Mediterranean area. Many of these patients show cosensitization to mugwort pollen. LTPs have an ubiquitous distribution in tissues of many plant species, and have been proposed as a novel type of plant panallergens.〈section xml:id="abs1-2"〉〈title type="main"〉ObjectiveWe sought to isolate LTPs from Artemisia pollen and from a plant food not belonging to the Rosaceae family, such as chestnut nut, and to compare their amino acid sequences and IgE-binding capacities with those of apple and peach LTPs.〈section xml:id="abs1-3"〉〈title type="main"〉MethodsAllergens (LTPs) were isolated by different chromatographic methods (gel-filtration, ion exchange and/or reverse-phase HPLC), and characterized by N-terminal amino acid sequencing and MALDI analysis. Specific IgE-quantification and immunodetection, as well as immunoblot and ELISA inhibition assays, were carried out using sera from patients allergic to both apple and peach.〈section xml:id="abs1-4"〉〈title type="main"〉ResultsPurified LTPs from Artemisia pollen and from chestnut seed showed molecular masses about 9 700d, and 43–50% sequence identity with the equivalent allergens of apple and peach in the first 30 N-terminal residues, which comprise about one third of the total amino acid sequence. A similar degree of sequence identity (50%) was found between the Artemisia and chestnut proteins. Both isolated LTPs bound specific IgE of sera from Rosaceae fruits allergic patients. However, substantially lower values of specific IgE-binding and maximum ELISA inhibition percentages were obtained for Artemisia and chestnut LTPs when compared to those from apple and peach.〈section xml:id="abs1-5"〉〈title type="main"〉ConclusionLTPs from Artemisia pollen and chestnut crossreact with allergens (LTPs) of Rosaceae fruits, but significant differences in specific IgE-binding capacities were observed among members of the plant LTP family. Thus, further studies are needed to evaluate the clinical significance of the observed cross-reactivities of plant LTPs.

Notes: Background Putative wheat allergens with molecular sizes around 35kDa have been repeatedly detected in immunoblots using sera from patients with baker's asthma. However, none of these allergens had been previously isolated.Objective To purify and characterize a major component of the 35 kDa aliergenic band which seems to be present in flour from diploid. tetraploid (pasta) and hexaploid (bread) wheats.Methods Gel-filtration chromatography followed by RP-HPLC have allowed the purification of a new wheat allergen. Immunodetection with sera from allergic patients and with a specific serum for asparagine-linked complex glycans, as well as amino acid sequencing, were used to identify the isolated protein.Results A 36 kDa allergen has been purified from wheat flour. Its N-terminal and internal sequences, N-linked glycans, and enzymatic activity indicated that this allergen is a seed-specific peroxidase. Sera from six out of 10 patients hypersensitive to wheat flour displayed positive reactions when assayed against the purified dot-blotted allergen.Conclusion Seed-specific peroxidases from cereals (flours) seem to be important allergens in hypersensitive diseases associated with the manipulation of these plant materials. A new enzyme has thus been involved in IgE-mediated diseases.

Notes: Several members of the plant non-specific lipid transfer protein (LTP) family have been identified as relevant allergens in foods and pollens. These allergens are highly resistant to both heat treatment and proteolytic digestion. These characteristics have been related with the induction of severe systemic reactions in many patients, and with the possibility of being primary sensitizers by the oral route. A specific geographical distribution pattern of sensitization to LTP allergens has been uncovered. This allergen family is particularly important in the Mediterranean area, but shows a very limited incidence in Central and Northern Europe. The potential role in the plant, as well as the biochemical and allergenic properties of the LTP family, are reviewed here.

Notes: Background Cereals are among the major foods that account for food hypersensitivity reactions. Salt-soluble proteins appear to be the most important allergens contributing to the asthmatic response. In contrast, very limited information is available regarding cereal allergens responsible for allergic reactions after ingestion of cereal proteins.Objective The aim of this study was to evaluate the allergenic reactivity of ingested and inhaled cereal allergens in different ages, in order to investigate if the response to different allergens would depend on the sensitization route.Methods We included 66 patients in three groups. Group 1: 40 children aged 3 to 6 months who suffered from diarrhoea, vomiting, eczema or weight loss after the introduction of cereal formula in their diet and in which a possibility of coeliac disease was discarded. Group 2: 18 adults with food allergy due to cereals tested by prick tests, specific IgE and food challenge. Group 3: eight patients previously diagnosed as having baker's asthma. Sera pool samples were collected from each group of patients and IgE immunoblotting was performed.Results We found an important sensitization to cereal in the 40 children. The most important allergens were wheat followed by barley and rye. Among the adults with cereal allergy, sensitization to other allergens was common, especially to Lolium perenne (rye grass) pollen. Immunoblotting showed similar allergenic detection in the three groups.Conclusion Clinically significant reactivity to cereal may be observed in early life. Inhalation and ingestion routes causing cereal allergy seem to involve similar allergens. The diet control was more effective in children. The possibility of cereal allergy after the introduction of cereal formula during the lactation period should not be underestimated.

Notes: Background Class I chitinases are the major panallergens in fruits associated with the latex–fruit syndrome. These enzymes contain an N-terminal hevein-like domain homologous to latex hevein, and a larger catalytic domain. The role of these domains in their allergenic capacity is still controversial.Objective We sought to evaluate the role of both domains of class I chitinases in their IgE-binding properties, using Cas s 5, the major allergen from chestnut, as a model.Methods Recombinant Cas s 5 and its deleted form, lacking the hevein-like domain, designated rCat, were expressed in Pichia pastoris using the pPIC 9 vector. Both recombinant products were purified from the supernatants of transformed yeast cultures by gel-filtration and cation-exchange chromatography. The isolated proteins were characterized by N-terminal sequencing, enzymatic activity and N-glycosylation tests, anti-chitinase and specific IgE immunodetection. Immunoblot, RAST and CAP inhibition assays were also performed.Results Both purified rCas s 5 and rCat showed the expected N-terminal amino acid sequences and an enzymatic activity similar to that of their natural counterparts isolated from chestnut seeds, and were strongly recognized by anti-chitinase antibodies. In contrast, only rCas s 5, but not rCat, bound specific IgE from sera of patients suffering from the latex–fruit syndrome, and fully inhibited IgE-binding to natural Cas s 5 in immunoblot inhibition assays. Latex hevein also exerted a strong immunoblot inhibition of IgE-binding to chestnut Cas s 5. RAST and CAP inhibition using whole chestnut extract on the solid phase, rendered inhibition levels around 70–90% for rCas s 5 and 60% for rCat, in contrast to the immunoblotting results.Conclusions Recombinant Cas s 5 behaves like natural Cas s 5 in IgE-binding assays in vitro. The hevein-like domain of allergenic class I chitinases seems to include all their main IgE-binding epitopes when tested by immunodetection and immunoblot inhibition experiments. RAST and CAP inhibition assays, on the contrary, suggest that relevant epitopes are also harboured in the catalytic domain of these allergens.

Notes: Background A number of wheat and barley flour proteins that belong to the cereal α-amylase/trypsin inhibitor family have been identified as major allergens associated with baker's asthma. However, the allergenic role of this protein family had not been investigated in rye.Objective To study the allergenicity of flve purified proteins from rye flour which belong to the same inhibitor family, as well as to compare their properties with those of their wheat and barley homologues.Methods In vivo skin-prick tests were carried out in 21 patients with radioallergo-sorbent test (RAST) 2–3 to rye and allergic sensitization mainly to this cereal flour. In addition, sera from all these patients were used to assay the IgE binding capacity of dot blotted purified proteins.Results Three of the rye proteins tested, namely Sec c 1, RDAI-1 and RDAI-3, provoked positive skin-prick tests in more than 50% of patients, although their in vitro reactivity was lower. Different reactivities were found for the rye components compared with their wheat and barley homologues. Statistical analyses showed a significant correlation between the results of in vivo and in vitro tests for seven out of the nine purified proteins considered in this study.Conclusion Members of the rye α-amylase inhibitor family are main allergens involved in allergic reactions to cereal flours. However, different allergenic behaviours were found between homologous allergens from rye, barley, and wheat.

Notes: Eleven purified members of the α-amylase/trypsin inhibitor family from wheat and barlcy that showed very different IgE-binding capacities when previously assayed in vitro. were used in double blind in vivo diagnostic tests to further evaluate their allergenic activity. These tests were carried out in 31 patients who showed allergic scnsilization to wheat flour as veritied by skin test, RAST and challenge test. The three members of the protein family with highest igl; binding in vitro (the glycosylated subunits of tetrameric α-amylase inhibitors CM16* from wheat and CMb* from barley, and the barley monomeric inhibitor BMAI-1) were found to be the strongest allergens as indicated by-skin sensitivity in prick tests.