L'armoise - AllerData - le site de l'allergologie moléculaire.

The allergenic proteins of Artemisia scoparia pollen were separated and identified with ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and RAST-inhibition techniques. The important allergenic component Artemisia VI b that constitutes 29% of total protein in the extract was purified to homogeneity. It was found to be an acidic protein with isoelectric point 3.8 and molecular weight of 14,300. It was rich in carbohydrate, but the carbohydrate portion did not appear to be important for allergenicity. In the crossed immunoelectrophoresis reference pattern of the whole pollen extract, 37 precipitin lines could be identified on the anodic side, whereas Artemisia VI b could be observed as a single precipitin line. Immunologically, the whole pollen extract of A. scoparia demonstrated shared antigenic and allergenic determinants with Ageratum conyzoides-pollen extract. The use of fast protein liquid chromatography in partial purification of allergenic components is also discussed.

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[3] - Katial RK, Lin FL, Stafford WW, Ledoux RA, Westley CR, Weber RW. Mugwort and sage (artemisia) pollen cross-reactivity: ELISA inhibition and immunoblot evaluation. Ann Allergy Asthma Immunol 1997;79:340-346

Plants of the genus Artemisia are a source of fall allergic symptoms, particularly in the western United States. Studies have characterized the allergens in one of the major species (A. vulgaris) but currently there are no cross-reactivity data on the major United States species. OBJECTIVE: The purpose of this study was to investigate the in vitro cross-reactivity among nine Artemisia species: A. frigida, A. annua, A. biennis, A. filifolia, A. tridentata, A. californica, A. gnaphalodes, A. ludoviciana, and A. vulgaris. METHODS: The cross-reactivity was demonstrated with the use of enzyme-linked immunosorbent assay (ELISA) inhibitions and immunoblotting techniques utilizing a serum pool from patients allergic to Artemisia species. RESULTS: The enzyme-linked immunosorbent assay inhibitions revealed strong cross-reactivity among all nine species with A. biennis and A. tridentata being two of the strongest inhibitors. The polyacrylamide gel electrophoresis showed a great deal of similarity in the bands among the nine species. The nitrocellulose blots showed similar IgE binding patterns among the Artemisia species with strong inhibition among all nine extracts. CONCLUSIONS: These data all demonstrate very strong in vitro cross-reactivity among the nine Artemisia species studied. Such data have significant clinical relevance, suggesting that a single Artemisia species may be sufficient for allergy skin testing and formulation of immunotherapy extracts.

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[4] - Brandys J, Grimsoen A, Nilsen BM, Smestad Paulsen B, Park HS, Hong CS. Cross-reactivity between pollen extracts from six Artemesia species. Planta Med 1993;59:221-228

Pollen extracts of six different ARTEMISIA species, A. VULGARIS, A. SCOPARIA, A. PRINCEPS, A. TRIDENTATA, A. ANNUA, and A. CAMPESTRIS were compared using SDS-PAGE, IEF, immunoblotting, and immunoelectrophoretic methods. The band patterns obtained after SDS-PAGE and IEF showed a large degree of similarity between the extracts. Immunoblotting of these gels using a pool of sera from patients allergic to A. VULGARIS gave essentially the same IgE-binding band pattern with all the extracts, demonstrating an extensive degree of cross-reactivity between A. VULGARIS and the other ARTEMISIA species. FRIE using a polyspecific antiserum against A. VULGARIS showed that all the extracts contained several antigens that were immunologically identical to antigens in A. VULGARIS extract. Antigens showing immunological identity to the important A. VULGARIS allergens Ag 12 and ART V II were present in all the extracts. The cross-reactivity between A. VULGARIS and A. PRINCEPS was further verified by screening of ten Korean and nine Norwegian individual patient sera against extracts of both species in SDS-PAGE or IEF immunoblotting. Both groups of patients had essentially the same pattern of reactivity towards both pollen extracts.

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[5] - Bousquet J, Cour P, Guerin B, Michel FB. Allergy in the Mediterranean area I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249-258

The climatic conditions of the Mediterranean area result in vegetation and pollen very different from that of the other parts of Europe. The pollen content of the atmosphere of Montpellier, southern France, was examined using a filter sampler which was shown to be more efficient than most of the current devices for air sampling. Pollen counts were subsequently compared with pollinosis of patients born and living in and around Montpellier. The mean annual pollen counts showed that grass pollens and Cupressaceae pollens (cypress and juniper) are the highest. Some Mediterranean pollens (Oleaceae, London plane, Parietaria) are also important. Plantain and oak pollens are also present in relatively large amounts. Grass pollen allergy was found to be present in 86.5% of pollen-allergic patients. It was followed by plantain, Parietaria, Oleaceae, London plane and Cupressaceae pollens which were allergenic in 13-36% of pollen-allergic patients. Oak and pine pollens were present in large quantities in the counts but few persons were sensitive to oak and none to pine. By contrast, some patients had positive skin tests to alfalfa, red clover, acacia and lime tree pollens though these pollens were almost absent from the counts. In a few cases local sources of these pollens could account for the positive skin tests but cross-sensitivities could also occur. In summary, pollinosis of the Northern Mediterranean area is intermediate between the southern part of the area and the other parts of Europe.

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[6] - Skypala I, Calderon M, Leeds A, Durham S. Pollen-food syndrome in United Kingdom subjects - which aeroallergens are commonly involved ? Allergy 2007;62(suppl. 83):358

Pollen food syndrome (PFS) is common in individuals with birch pollen sensitivity who present with hay fever symptoms in the spring. The aim of this study was to evaluate which pollen(s), apart from birch, are associated with PFS in UK-based subjects, and whether aero-allergen sensitivities differ in those subjects with springtime hay fever with/without associated PFS. Adult subjects reporting hay fever symptoms between March and May were included in the study. All subjects underwent skin prick testing with tree (birch, oak, beech, plane and three trees ˜ birch, alder and hazel), timothy grass, mugwort, latex, cat and house dust mite (D. pteronyssinus) allergens (ALK Abelló Denmark). A diagnosis of PFS was established for each subject using a combination of results from a standardised medical assessment, prick by prick testing with suspect foods and oral food challenge. 119 subjects completed the study; 92% were sensitised to one or more aeroallergens. Although all subjects reported spring and/or summer time hay fever, only 85% of the cohort were sensitised to pollen. Grass was the commonest sensitising pollen allergen (68%), followed by birch (64%), oak and three trees (53%), beech (34%), plane (26%) and mugwort (18%). Approximately half of the cohort were sensitised to cat and/or house dust mite, but less than 10% to latex. Subjects diagnosed with PFS had a significantly greater number of positive tests for birch, oak, beech (p<0.001) and grass (p<0.05) than those without PFS, but there was no significant difference between the groups for the other aeroallergens. Polysensitisation to pollen allergens occurred in 93% of the group diagnosed with PFS compared to 37% of the group without PFS. No aeroallergen sensitivities were individually or collectively significant predictors of the diagnosis of PFS although a combination of birch, grass and oak gave the best standardised beta coefficient. UK subjects with springtime hay fever are most likely to be sensitised to birch, grass and oak. The frequency of sensitivities to these allergens is greater in those who additionally have PFS. In contrast, subjects with/without PFS do not differ in their rate of sensitisation to cat and house dust mite. Sensitisation to pollen cannot predict who with springtime hay fever will have PFS, but those with PFS are more likely to be co-sensitised to several tree and grass pollens.

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[7] - Cosmes Martin PM, Moreno Ancillo A, Dominguez Noche C, Gutierrez Vivas A, Belmonte Soler J, Roure Nolla JM. [Sensitization to Castanea sativa pollen and pollinosis in northern Extremadura (Spain)]. Allergol Immunopathol (Madr) 2005;33:145-150

BACKGROUND: Castanea sativa pollen allergy has generally been considered to be uncommon and clinically insignificant. In our geographical area (Plasencia, Caceres, Spain) Castanea sativa pollen is a major pollen. OBJECTIVE: To determine the atmospheric fluctuations and prevalence of patients sensitized to Castanea pollen in our region and to compare this sensitization with sensitizations to other pollens. METHODS: Patients with respiratory symptoms attending our outpatient clinic for the first time in 2003 were studied. The patients underwent skin prick tests with commercial extracts of a battery of inhalants including Castanea sativa pollen. Serologic specific IgE to Castanea sativa pollen was determined using the CAP system (Pharmacia and Upjohn, Uppsala, Sweden). Airborne pollen counts in our city were obtained using Cour collection apparatus over a 4-year period (2000 to 2003). RESULTS: The most predominant pollens detected were (mean of the maximal weekly concentrations over 4 years in pollen grains/m3): Quercus 968, Poacea 660, Olea 325, Platanus 229, Pinus 126, Cupresaceae 117, Plantago 109, Alnus 41, Populus 40, Castanea 32. We studied 346 patients (mean age: 24.1 years). In 210 patients with a diagnosis of pollinosis, the percentages of sensitization were: Dactylis glomerata 80.4%, Olea europea 71.9%, Fraxinus excelsior 68%, Plantago lanceolata 62.8%, Chenopodium album 60.9%, Robinia pseudoacacia 49%, Artemisia vulgaris 43.8%, Platanus acerifolia 36.6%, Parietaria judaica 36.1%, Populus nigra 32.3%, Betula alba 27.6%, Quercus ilex 21.4%, Alnus glutinosa 20.9%, Cupressus arizonica 7.6% and Castanea sativa 7.1%. Fifteen patients were sensitized to Castanea sativa and 14 had seasonal rhinoconjunctivitis and asthma. Ten patients had serum specific IgE to Castanea pollen (maximum value: 17.4 Ku/l). Castanea pollen is present in our area in large amounts from the 23rd to the 28th weeks of the year, with a peak pollen count in the 25th week. CONCLUSIONS: The most important allergenic pollens in northern Extremadura were Poaceae, Olea europaea and Plantago sp. The prevalence of sensitization to Castanea sativa pollen was very low (7.1%). Most sensitized patients had asthma and polysensitization. Castanea sativa pollen is not a major cause of pollinosis in our area.

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[8] - García-Sellés FJ, Díaz-Perales A, Sánchez-Monge R, Alcántara M, Lombardero M, Barber D, et al. Patterns of reactivity to lipid transfer proteins of plant foods and Artemisia pollen: an in vivo study. Int Arch Allergy Immunol 2002;128:115-122

Background: Lipid transfer proteins (LTPs) are major allergens of Rosaceae fruits in the Mediterranean area. IgE-cross-reactivity has been demonstrated in vitro among LTPs from peach, apple, chestnut and Artemisia pollen. The aim of this study was to evaluate the reactivity to LTPs from peach, apple, chestnut and Artemisia pollen by means of skin prick tests (SPTs). Methods: Forty-seven patients allergic to peach (peach group), 20 patients sensitized to Artemisia pollen with no food allergies (Artemisia group), and 12 control subjects were skin tested with fresh peach, as well as with whole extracts and purified LTPs of peach, apple, chestnut and Artemisia pollen. Results: The rates of positive SPTs for peach, apple, chestnut and Artemisia LTPs were, respectively, 91, 77, 23, and 36% in the peach group, and 30, 5, 15 and 40% in the Artemisia group. No response was observed in the control subjects. SPTs with peach LTP strongly correlated with SPTs conducted with fresh peach. In the peach group, the most frequent pattern of reactivity to LTPs was the combination peach-apple (45%), followed by peach-apple-Artemisia-chestnut (21%). Significant correlations were found between peach and apple LTPs, and between Artemisia and chestnut LTPs. Positive SPTs to chestnut LTP were only observed in patients with positive SPTs to Artemisia LTP. All the patients with positive case histories to chestnut reacted to chestnut LTP. Conclusions: LTPs are plant panallergens with different patterns of cross-reactivity. They are major allergens of Rosaceae fruits and seem to be involved in allergic reactions to unrelated foodstuffs such as chestnut, probably through sensitization to the cross-reactive Artemisia LTP. Rosaceae LTPs could be useful tools for in vivo diagnosis of Rosaceae fruit allergy.

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[9] - Lombardero M, Garcia-Sellés FJ, Polo F, Jimeno L, Chamorro MJ, Garcia-Casado G, et al. Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens. Clin Exp Allergy 2004;34:1415-1421

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.

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[10] - Boehncke WH, Loeliger C, Kuehnl P, Kalbacher H, Bohm BO, Gall H. Identification of HLA-DR and -DQ alleles conferring susceptibility to pollen allergy and pollen associated food allergy. Clin Exp Allergy 1998;28:434-441

BACKGROUND: Allergenic crossreactivity of pollen and foods due to the antigeneic similarity of oligopeptides is a well established clinical phenomenon. OBJECTIVE: To determine the immunopathological relevance of antigen presentation, we analysed the HLA class-II genotype of patients with either pollen allergy or pollen associated food allergy. METHODS: One hundred and twenty patients with pollen allergy and 80 patients with pollen associated food allergy were evaluated by skin- prick tests, RAST, and HLA class-II genotyping. The control population comprised 4251 healthy blood and bone marrow donors. RESULTS: Monovalent pollen allergy was observed in 57% (n=68) of patients with pollinosis (57x grass pollen, 11x birch pollen), but only in 15% (n=12) of patients with food allergy (9x grass pollen, 3x birch pollen). Hazelnut (71%), almond (65%), walnut (44%) and apple (41%) were the most common food allergens and frequently associated with birch pollen allergy. Grass pollen allergy was associated with an increased frequency of HLA-DQB1*0301 (RR=2.3; EF=0.4; P=0.0016) when compared with the control population. HLA-DRB *08 conferred a sixfold higher risk for peanut allergy (EF=0.3; P=0.0013) and -DRB1*12 a 13-fold higher risk for carrot allergy (EF=0.3; P<0.000001). The differences on allele frequencies detected among patients with food allergies diminished or turned statistically insignificant when their genotypes were directly compared to those of patients with the corresponding pollen allergies. This was found in the case of birch pollen associated hazel nut allergy for the extended haplotype HLA-DRB1*01, -DQA1*0101, -DQB1*0501 as well as in grass pollen associated peanut allergy for HLA-DRB1*08 (from RR=6, P=0.0013 to insignificant) and in birch pollen associated carrot allergy for HLA-DRB1*12 (from RR=13, P < 0.000001 to insignificant). CONCLUSION: We were able to identify HLA class-II alleles associated with some allergies thus indicating that these alleles might confer susceptibility to the respective allergens. Similarities at the level of the HLA class-II genotype parallel the empirical finding of distinct cross-reactivity patterns thus complementing investigations of IgE specificities. Our observations provide evidence for the major importance of antigen presentation on the manifestation of distinct crossreactivity patterns.

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[11] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[12] - Bousquet J, Cour P, Guerin B, Michel FB. Allergy in the Mediterranean area I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249-258

The climatic conditions of the Mediterranean area result in vegetation and pollen very different from that of the other parts of Europe. The pollen content of the atmosphere of Montpellier, southern France, was examined using a filter sampler which was shown to be more efficient than most of the current devices for air sampling. Pollen counts were subsequently compared with pollinosis of patients born and living in and around Montpellier. The mean annual pollen counts showed that grass pollens and Cupressaceae pollens (cypress and juniper) are the highest. Some Mediterranean pollens (Oleaceae, London plane, Parietaria) are also important. Plantain and oak pollens are also present in relatively large amounts. Grass pollen allergy was found to be present in 86.5% of pollen-allergic patients. It was followed by plantain, Parietaria, Oleaceae, London plane and Cupressaceae pollens which were allergenic in 13-36% of pollen-allergic patients. Oak and pine pollens were present in large quantities in the counts but few persons were sensitive to oak and none to pine. By contrast, some patients had positive skin tests to alfalfa, red clover, acacia and lime tree pollens though these pollens were almost absent from the counts. In a few cases local sources of these pollens could account for the positive skin tests but cross-sensitivities could also occur. In summary, pollinosis of the Northern Mediterranean area is intermediate between the southern part of the area and the other parts of Europe.

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[13] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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[14] - Gadermaier G, Dedic A, Obermeyer G, Frank S, Himly M, Ferreira F. Biology of weed pollen allergens. Curr Allergy Asthma Rep 2004;4:391-400

Weeds represent a heterogeneous group of plants, usually defined by no commercial or aesthetic value. Important allergenic weeds belong to the plant families Asteraceae, Amaranthaceae, Urticaceae, Euphorbiaceae, and Plantaginaceae. Major allergens from ragweed, mugwort, feverfew, pellitory, goosefoot, Russian thistle, plantain, and Mercurialis pollen have been characterized to varying degrees. Four major families of proteins seem to be the major cause of allergic reactions to weed pollen: the ragweed Amb a 1 family of pectate lyases; the defensin-like Art v 1 family from mugwort, feverfew, and probably also from sunflower; the Ole e 1-like allergens Pla l 1 from plantain and Che a 1 from goosefoot; and the nonspecific lipid transfer proteins Par j 1 and Par j 2 from pellitory. As described for other pollens, weed pollen also contains the panallergens profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.

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[15] - Mohapatra SS, Lockey RF, Polo F. Weed pollen allergens. Clin Allergy Immunol 2004;18:207-222

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[16] - Wopfner N, Gadermaier G, Egger M, Asero R, Ebner C, Jahn-Schmid B, et al. The Spectrum of Allergens in Ragweed and Mugwort Pollen. Int Arch Allergy Immunol 2005;138:337-346

Ragweed and mugwort are important allergenic weeds belonging to the Asteraceae or Compositae plant family. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10-14% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is now rapidly increasing particularly in certain areas in France, Italy, Austria, Hungary, Croatia, and Bulgaria. Amb a 1 and Art v 1, the major allergens of ragweed and mugwort, respectively, are unrelated proteins. Amb a 1 is an acidic 38-kDa nonglycosylated protein. The natural protein undergoes proteolysis during purification and is cleaved into a 26-kDa alpha chain, which associates noncovalently with the beta chain of 12 kDa. The two-chain form seems to be immunologically indistinguishable from the full-length molecule. Art v 1 is a basic glycoprotein comprising two domains: an N-terminal cysteine-rich, defensin-like domain and a C-terminal proline/hydroxyproline-rich module. The proline/hydroxyproline-rich domain was recently shown to contain two types of glycosylation: (1) a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core substituted by a variable number (5-28) of alpha-arabinofuranose residues forming branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses, and (2) single and adjacent beta-arabinofuranoses linked to hydroxyproline. As described for other pollen, ragweed and mugwort pollen also contain the pan-allergen profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.

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[17] - de la Hoz F, Polo F, Moscoso del Prado J, Selles JG, Lombardero M, Carreira J. Purification of Art v I, a relevant allergen of Artemisia vulgaris pollen. Mol Immunol 1990;27:651-657

An allergenic protein from Artemisia vulgaris pollen has been purified to homogeneity. Its molecular weight in native conditions is 47,000. The purified allergen, hereafter denominated Art v I, is a monomeric protein It is a clinically relevant allergen since, at least, 70% of the individuals allergic to Artemisia vulgaris pollen have specific IgE in serum and in mast cells, demonstrated by ELISA and skin prick tests, respectively.

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[18] - Gadermaier G, Harrer A, Girbl T, Palazzo P, Himly M, Vogel L et al. Isoform identification and characterization of Art v 3, the lipid-transfer protein of mugwort pollen. Mol Immunol 2009;46:1919-1924

Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.

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[19] - Wopfner N, Willeroidee M, Hebenstreit D, van Ree R, Aalbers M, Briza P, et al. Molecular and immunological characterization of profilin from mugwort pollen. Biol Chem 2002;383:1779-1789

In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens. No information about the complete molecular structure of any mugwort allergen has been published so far. Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin. Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms. One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions. Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions. Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients. However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release. Profilin isolated from mugwort pollen also formed multimers. Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen. Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts

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[20] - Asero R, Mistrello G, Roncarolo D, Casarini M. Detection of allergens in plantain (Plantago lanceolata) pollen. Allergy 2000;55:1059-1062

BACKGROUND: Allergens in Plantago lanceolata have not been characterized yet. The objective was to characterize some plantain-pollen allergens and to investigate the cross-reactivity between plantain and grass pollens. METHODS: Sera from four patients monosensitive to plantain pollen and from eight grass-pollen-allergic patients showing strong skin reactivity to plantain pollen in the skin prick test (SPT) underwent immunoblot analysis with both Plantago and grass mix extract. Moreover, immunoblot inhibition experiments were done with grass mix extract as inhibitor. RESULTS: All four sera from plantain-allergic patients reacted to two distinct bands at 17 and 19 kDa, and 2/4 sera showed further reactivity to a 40-kDa protein, which in one case represented the most prominent IgE-binding allergen. Plantain-monosensitive subjects did not show any reactivity to grass-pollen extract, and preabsorption of their sera with grass-pollen extract did not cause any loss of reactivity to plantain pollen. Sera from all eight grass-pollen-allergic controls reacted to a 30-kDa protein in plantain pollen, and some sera showed cross-reactivity to higher and lower molecular-weight structures as well. In all cases, plantain reactivity was totally abolished by preabsorption of sera with grass-pollen extract. A preliminary investigation by immunoblot showed that polyclonal IgG anti-Phl p 5 (but not polyclonal Phl p 1) from rabbit reacted to a 30-kDa protein in plantain pollen. CONCLUSIONS: Three specific allergens (of 17, 19, and 40 kDa, respectively) have been detected in plantain pollen. Further studies on a larger number of patients will determine whether these proteins may be considered major allergens. Cross-reactivity between grass and plantain pollen is mainly caused by a 30-kDa protein in plantain pollen. Group 5 grass-pollen allergen is probably responsible for most grass/plantain cross-reactivity.

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[21] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[22] - Ferreira F. Characterization of mugwort and ragweed pollen allergens. in "Immunobiology of allegens and allergen-specific immune responses" NFN Final Report

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[23] - Gadermaier G, Wopfner N, Wallner M, Egger M, Didierlaurent A, Regl G, et al. Array-based profiling of ragweed and mugwort pollen allergens. Allergy 2008;63:1543-1549

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons . OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA) . METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays . RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods . CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

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[24] - Ferreira F, Himly M, Hebenstreit D, Dabrowska M, Vollmann U, Kraft D, et al. Isolation and characterization of cDNA clones coding for mugwort (Artemisia vulgaris) pollen allergens. Int Arch Allergy Immunol 2001;124:77-79

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[25] - Hoidn C, Puchner E, Pertl H, Holztrattner E, Obermeyer G. Nondiffusional Release of Allergens from Pollen Grains of Artemisia vulgaris and Lilium longiflorum Depends Mainly on the Type of the Allergen. Int Arch Allergy Immunol 2005;137:27-36

BACKGROUND: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process . METHODS: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen. Using three elution media of different pH, osmolality and salt concentration mimicking various types of wet surfaces, the time-dependent elution profiles of total protein, a cell wall-bound acid phosphatase activity (acPase), allergenic (profilin, Art v 1) and nonallergenic molecules (14-3-3 protein, actin) were monitored . RESULTS: The release kinetics of total protein and cell wall-bound acPase followed an exponential decrease in both pollen species indicating a diffusion-based protein release, whereas the elution profiles of profilin, Art v 1 and 14-3-3 protein showed nondiffusion characteristics. No general dependence on pH, osmolality or salt concentration of the elution media was observable in the elution profiles. Under the applied conditions, actin was not released indicating that the pollen grains remained intact during the elution . CONCLUSION: The elution profiles of pollen allergens indicated that substantial amounts of these proteins do not diffuse from the cell wall or are released from intracellular compartments during imbibitional leakage. Instead, a mechanism seems to operate that involves translocation from the pollen cytoplasm to the extracellular environment by crossing an intact plasma membrane. Such a mechanism would probably allow the use of pharmaceuticals for inhibition of allergen release.

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[26] - Karamloo F, Wangorsch A, Kasahara H, Davin LB, Haustein D, Lewis NG, et al. Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables. Eur J Biochem 2001;268:5310-5320

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

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[27] - Dedic A, Himly M, Engel E, Richter K, Ferreira F. Structural Characterization of Art v 1, the Major Mugwort Pollen Allergen. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°378

In late summer, pollen of mugwort (Artemisia vulgaris) is one of the main causes of allergies in Europe. Among patients suffering from pollinosis, the incidence of allergic disease caused by mugwort pollen is between 10 and 14%. We have previously isolated a cDNA clone coding the major allergen of mugwort pollen Art v 1, by IgE immunoscreening of a cDNA library. Computer analysis of the deduced protein sequence predicts that Art v 1 has a typical N-terminal hydrophobic signal sequence that causes targeting to the endoplasmatic reticulum and the Golgi apparatus. The sequence of the mature form of the protein predicted by a computer algorithm was identical with the N-terminal sequence found in the natural protein. Thus, Art v 1 is a secreted glycoprotein whose N-terminus is created by removal of a typical ER signal sequence. We have also shown that highly purified samples of natural Art v 1 display a considerable degree of heterogeneity when analyzed by SDS-PAGE. Different glycosylation patterns due to isogenes coding for isoforms and/or allelic variants could be an explanation for the observed heterogeneity. Therefore, we undertook a more detailed structural characterization of Art v 1 by cDNA cloning. For this purpose, the primary cDNA clone of Art v 1 was radiolabeled and used as a hybridization probe for screening a mugwort pollen cDNA library. The isolated cDNA clones were then analyzed by DNA sequencing. Two clones were found to correspond to the original Art v 1 clone. In addition, 22 different cDNA clones coding for Art v 1 isoforms were isolated. In several cases, differences in the nucleotide sequences did not result in amino acid changes. Therefore, including the original Art v 1, eleven Art v 1 protein sequence isoforms have been identified altogether, with amino acid identities ranging from 94% (6 amino acids exchanges) to 99% (a single amino acid exchange). Interestingly, all amino acid exchanges in the isoforms were restricted to the C-terminal proline-rich domain, which carries the sugar chains of Art v 1. Production of these isoform sequences in a plant-based expression system will help to clarify if these amino acid exchanges are responsible for differences in the glycosylation pattern of the Art v 1 allergen

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[28] - Lombardero M, Garcia-Sellés FJ, Polo F, Jimeno L, Chamorro MJ, Garcia-Casado G, et al. Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens. Clin Exp Allergy 2004;34:1415-1421

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.

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[29] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[30] - Himly M, Jahn-Schmid B, Dedic A, Kelemen P, Wopfner N, Altmann F, et al. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain. FASEB J 2003;17:106-108

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

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[31] - Léonard R, Petersen BO, Himly M, Kaar W, Wopfner N, Kolarich D et al. Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding. J Biol Chem 2005;280:7932-7940

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As some allergic patients sera react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry and 800 MHz 1H and 13C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short ss1,6-galactan core which is substituted by a variable number (5-28) of alpha-arabinofuranose residues which form branched side chains with 5-, 2,5-, 3,5- and 2,3,5 substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans and we suggest to name it type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) ss-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp4 motifs in other hydroxyproline-rich glycoproteins such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation and / or digestion with alpha-arabinofuranosidase were used in ELISA and immunoblot experiments with a rabbit serum and with patients sera. While we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked ss-arabinose residues appeared to react with antibodies. Mono-alpha-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive carbohydrate determinant in plant proteins.

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[32] - Schmid-Grendelmeier P, Holzmann D, Himly M, Weichel M, Tresch S, Rückert B, et al. Native Art v 1 and recombinant Art v 1 are able to induce humoral and T cell–mediated in vitro and in vivo responses in mugwort allergy. J Allergy Clin Immunol 2003;111:1328-1336

BACKGROUND: Mugwort pollen is an important allergen source in hay fever and pollen-related food allergy. Little is known about the clinical relevance of the major mugwort allergen Art v 1 and its importance in allergy . OBJECTIVE: In this study we aimed to investigate the allergenicity of mugwort extract compared with the allergenicity of native (n)Art v 1 and recombinant (r)Art v 1, one major allergen of mugwort, in vivo and in vitro . METHODS: Thirty-two patients allergic to mugwort and 10 control subjects were investigated by means of skin prick and nasal provocation testing with different concentrations of mugwort extract, nArt v 1, and rArt v 1. nArt v 1 was purified from aqueous mugwort extract, and rArt v 1 was cloned, expressed in Escherichia coli, and then purified. The in vitro allergenicity was measured by means of ImmunoCAP, ELISA, ELISA-inhibition experiments, and T-cell proliferation assays . RESULTS: nArt v 1 and rArt v 1 were able to elicit positive in vivo and in vitro reactions. The IgE-binding capacity, as determined by means of ELISA, was slightly higher for nArt v 1 than for rArt v 1, and both allergens were able to induce T-cell proliferation in sensitized patients. However, rArt v 1 elicited a reduced response in skin and nasal provocation tests compared with nArt v 1. Compared with mugwort extract, both nArt v 1 and rArt v 1 showed lower sensitivity in patients with mugwort allergy in vivo . CONCLUSIONS: Art v 1, either in its native or recombinant form, is able to induce allergic reactions in patients with mugwort allergy. rArt v 1 induced comparable humoral and cell-mediated responses in vitro but showed reduced in vivo allergenicity compared with biochemically purified nArt v 1.

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[33] - Himly M, Jahn-Schmid B, Dedic A, Kelemen P, Wopfner N, Altmann F, et al. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain. FASEB J 2003;17:106-108

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

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[34] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[35] - Dedic A, Gadermaier G, Vogel L, Ebner C, Vieths S, Ferreira F, et al. Immune recognition of novel isoforms and domains of the mugwort pollen major allergen Art v 1. Mol Immunol 2009;46:416-421

Allergen isoforms can differ in their IgE and T cell recognition patterns, and thus might have an impact on the selection of candidates for molecule-based diagnostic and therapeutic approaches. The present study aimed at the identification and characterization of isoforms of Art v 1, the mugwort pollen major allergen. In addition, single Art v 1 domains were physicochemically and immunologically characterized. For this purpose, the Art v 1 cDNA was radiolabeled and used to screen a mugwort pollen cDNA library. Positive clones were sequenced and used for the production of recombinant proteins in Escherichia coli using the pHIS-Parallel2 vector. Protein purification was performed by affinity- and ion exchange chromatography. Antibody binding to the recombinant proteins was determined by immunoblot, ELISA, cross-inhibition experiments, and mediator release assays. We could identify 7 Art v 1 isoforms differing in 1-6 amino acid residues. Interestingly, all amino acid variations were restricted to the proline domain carrying the molecule's post-translational modifications. No significant difference in IgG or IgE reactivity could be observed between Art v 1 isoforms and the defensin domain produced in E. coli. When expressed in E. coli, the proline domain was not recognized by Art v 1-specific antibodies. Our results demonstrated that the relevant IgE epitopes of Art v 1 are located on the defensin domain and suggest the involvement of carbohydrates in the allergenicity of natural Art v 1. Plant-based expression systems could help to reveal possibly different glycosylation patterns and IgE binding properties of Art v 1 isoforms. These findings have direct implications on the development of novel tools for mugwort pollen allergy diagnosis and therapy.

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[36] - Ferreira F, Hawranek T, Gruber P, Wopfner N, Mari A. Allergic cross-reactivity: from gene to the clinic. Allergy 2004;59:243-267

A large number of allergenic proteins have now their complete cDNA sequences determined and in some cases also the 3D structures. It turned out that most allergens could be grouped into a small number of structural protein families, regardless of their biological source. Structural similarity among proteins from diverse sources is the molecular basis of allergic cross-reactivity. The clinical relevance of immunoglobulin E (IgE) cross-reactivity seems to be influenced by a number of factors including the immune response against the allergen, exposure and the allergen. As individuals are exposed to a variable number of allergenic sources bearing homologous molecules, the exact nature of the antigenic structure inducing the primary IgE immune response cannot be easily defined. In general, the 'cross-reactivity' term should be limited to defined clinical manifestations showing reactivity to a source without previous exposure. 'Co-recognition', including by definition 'cross-reactivity', could be used to describe the large majority of the IgE reactivity where co-exposure to a number of sources bearing homologous molecules do not allow unequivocal identification of the sensitizing molecule. The analysis of reactivity clusters in diagnosis allows the interpretation of the patient's reactivity profile as a result of the sensitization process, which often begins with exposure to a single allergenic molecule.

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[37] - Gruber P, Gadermaier G, Bauer R, Weiss R, Wagner S, Léonard R et al. Role of the polypeptide backbone and post-translational modifications in cross-reactivity of Art v 1, the major mugwort pollen allergen. Biol Chem 2009;390:445-451

Artemisia vulgaris (mugwort) is one of the main causes of late summer pollinosis in Europe, with >95% of patients sensitized to the glycoallergen Art v 1. Despite the importance of this allergen, little is known about its cross-reactive behavior. Here we investigated the occurrence of conserved Art v 1 antigenic determinants in sources known to display clinically relevant cross-reactivity with mugwort pollen. For this purpose, monoclonal antibodies specific for a cysteine-stabilized epitope of the Art v 1 defensin domain and for carbohydrates attached to the proline domain were produced by hybridoma and phage display technologies. Using polyclonal Art v 1-specific rabbit sera and antibodies against both the Art v 1 carbohydrate and polypeptide moieties, we could identify cross-reactive structures in pollen from botanically related Asteraceae weeds (Artemisia absinthium, Helianthus annuus and Ambrosia sp.). Homologous allergens were also recognized by IgE from mugwort-sensitized patients and the reactivity could be decreased by serum pre-incubation with natural and recombinant Art v 1. As no cross-reactive structures could be found in foods associated with mugwort pollinosis, we conclude that Art v 1 is poorly involved in mugwort cross-reactivity to food allergens.

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[38] - Egger M, Wopfner N, Himly M, Dedic A, Bauer R, Mari A, et al. Art v 1, the Major Mugwort Allergen, Cross-Reacts With Proteins Originating From Compositae and Grass Pollen. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°1098 (113(2 suppl):S300)

Rationale Art v 1 is the major mugwort (Artemisia vulgaris) pollen allergen and therefore represents one of the main causes of late summer pollinosis in Europe. Cross-reacting specific IgE antibodies can lead to clinically significant reactions with other members of the Compositae plant family (e.g. ragweed) in other geographic areas, such as Northern America. Furthermore, previous studies revealed cross-reactivity between mugwort pollen and certain food, known as the „celery-mugwort-spice-syndrome‰. Method s : In vitro cross-reactivity between Art v 1 and proteins from other Compositae-, grass- and tree pollen and common food allergen sources was investigated by immunoblot and IgE-inhibition experiments using the following antibodies: polyclonal rabbit anti-natural Art v 1, rabbit anti-recombinant Art v 1, moAb anti-Art v 1, produced by genetic immunization, and sera from mugwort and ragweed sensitized patients. Result s : The glycoallergen Art v 1 shares epitopes with proteins originating mainly from the botanically related plants of the Compositae family (Artemisia absinthium, Ambrosia artemisifolia, Ambrosia psilostachya, Ambrosia trifida and Helianthus annuus) and with the timothy grass (Phleum pratense) major allergen Phl p 1. We could not observe cross-reactivity between Art v 1 and proteins in extracts from tree pollen (Betula verrucosa) and food (apple and celery). Conclusions : The Art v 1 cross-reactive epitopes seem to encompass both the Art v 1 polypeptide and O-glycans attached to its C-terminal domain. Cross-reactivity with the glycoprotein Phl p 1 seems to be due to structural similarities in the Art v 1 defensin like domain or to sugar chains linked to its proline rich domain.

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[39] - Nilsen BM, Sletten K, Paulsen BS, O'Neill M, van Halbeek H. Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.). J Biol Chem 1991;266:2660-2668

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).

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[40] - Himly M, Jahn-Schmid B, Dedic A, Kelemen P, Wopfner N, Altmann F, et al. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain. FASEB J 2003;17:106-108

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

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[41] - Arilla MC, Ibarrola I, Puente Y, Daza JC, Martínez A, Asturias JA. Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1. Mol Immunol 2007;44:3653-3660

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140bp that codify for a polypeptide of 15.8kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

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[42] - Nilsen BM, Sletten K, Paulsen BS, O'Neill M, van Halbeek H. Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.). J Biol Chem 1991;266:2660-2668

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).

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[43] - Arilla MC, Ibarrola I, Puente Y, Daza JC, Martínez A, Asturias JA. Cloning, expression and characterization of mugwort pollen allergen Art v 2, a pathogenesis-related protein from family group 1. Mol Immunol 2007;44:3653-3660

Mugwort (Artemisia vulgaris) belongs to the Compositae family, and is one of the main causes of allergy in late summer and autumn. The aim of the study was to characterize the allergen Art v 2 from mugwort pollen. Skin prick tests, performed in 19 patients allergic to mugwort and 10 control patients, showed an Art v 2 sensitization prevalence of 58%, whereas none false-positives were detected among control patients. Art v 2 was purified by standard chromatography and binding to Concanavalin A column and had an apparent molecular mass of 33 and 20kDa, calculated by gel permeation and SDS-PAGE under denaturing conditions, respectively, showing that the allergen is composed of two identical subunits. Art v 2-encoding cDNA was amplified by PCR using degenerate primers based on reported partial amino acid sequences. Cloned cDNA encoding Art v 2 contains 140bp that codify for a polypeptide of 15.8kDa, with a predicted pI value of 5.2, and one potential N-glycosylation site. Protein homology search demonstrated that Art v 2 share 55-42% identical residues with pathogenesis-related protein PR-1 of tomato, potato, rape, wheat and rice. Homology was also found to Ves v 5 (41% identical residues). Bacterial-expressed recombinant Art v 2 was recognized only by 21% of mugwort-allergic patients. In conclusion, Art v 2 from mugwort is the first weed pollen allergen that belongs to the pathogenesis-related protein PR-1 and its recombinant form could help molecular diagnosis of mugwort associated allergy.

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[44] - Heiss S, Fischer S, Müller WD, Weber B, Hirschwehr R, Spitzauer S, et al. Identification of a 60 kd cross-reactive allergen in pollen and plant-derived food. J Allergy Clin Immunol 1996;98:938-947

Cross-reactive IgE antibodies were found to be responsible for allergic reactions in patients allergic to pollen on ingestion of food (oral allergy syndrome). So far, the major birch pollen allergen Bet v 1 and birch profilin (Bet v 2) were identified as relevant cross-reactive allergens. OBJECTIVE: In this study we attempted to identify additional cross-reactive plant allergens, which could be responsible for food intolerance in patients allergic to pollen. METHODS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, representing a 60 kd glycoprotein, were used to detect cross-reactive allergens in other pollens and plant-derived food. The amino acid compositions of the cross-reactive structures were determined, and their resistance against trypsin treatment was investigated. In addition, IgE immunoblot inhibitions were done with the 60 kd mugwort pollen allergen. RESULTS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, cross-reacted with proteins of comparable molecular weight in fruit and vegetables. Preadsorption of patients' sera with the 60 kd mugwort allergen led to a reduction of IgE binding to components of a similar molecular weight present in different pollen (birch, timothy grass), fruit (apple, peanuts), and vegetable (celery) extracts and reduced IgE binding to apple, kiwi, and celery as determined by RAST inhibitions. CONCLUSION: A cross-reactive plant panallergen, possibly identical to the major mugwort pollen allergen, Art v 1, is described. The allergen represents a protein of approximately 60 kd present in various pollen and plant foods; which is distinct from Bet v 1 and profilin and hence may represent a novel cross-reactive allergen in the oral allergy syndrome.

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[45] - Heiss S, Fischer S, Müller WD, Weber B, Hirschwehr R, Spitzauer S, et al. Identification of a 60 kd cross-reactive allergen in pollen and plant-derived food. J Allergy Clin Immunol 1996;98:938-947

Cross-reactive IgE antibodies were found to be responsible for allergic reactions in patients allergic to pollen on ingestion of food (oral allergy syndrome). So far, the major birch pollen allergen Bet v 1 and birch profilin (Bet v 2) were identified as relevant cross-reactive allergens. OBJECTIVE: In this study we attempted to identify additional cross-reactive plant allergens, which could be responsible for food intolerance in patients allergic to pollen. METHODS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, representing a 60 kd glycoprotein, were used to detect cross-reactive allergens in other pollens and plant-derived food. The amino acid compositions of the cross-reactive structures were determined, and their resistance against trypsin treatment was investigated. In addition, IgE immunoblot inhibitions were done with the 60 kd mugwort pollen allergen. RESULTS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, cross-reacted with proteins of comparable molecular weight in fruit and vegetables. Preadsorption of patients' sera with the 60 kd mugwort allergen led to a reduction of IgE binding to components of a similar molecular weight present in different pollen (birch, timothy grass), fruit (apple, peanuts), and vegetable (celery) extracts and reduced IgE binding to apple, kiwi, and celery as determined by RAST inhibitions. CONCLUSION: A cross-reactive plant panallergen, possibly identical to the major mugwort pollen allergen, Art v 1, is described. The allergen represents a protein of approximately 60 kd present in various pollen and plant foods; which is distinct from Bet v 1 and profilin and hence may represent a novel cross-reactive allergen in the oral allergy syndrome.

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[46] - Grote M, Fischer S, Müller WD, Valenta R. In situ localization of a high molecular weight cross-reactive allergen in pollen and plant-derived food by immunogold electron microscopy. J Allergy Clin Immunol 1998;101:250-257

BACKGROUND: A high molecular weight (60 kd) allergen has been recently identified as a cross-reactive moiety in pollen and plant-derived food. While the cross-reactive allergen has been characterized by immunochemical techniques, little is known concerning its biologic properties. OBJECTIVE: In this investigation we studied the in situ localization of the 60 kd cross-reactive allergen in tree, grass, and weed pollen, as well as in plant-derived food (apple and celery). METHODS: A monoclonal antibody (3A4) that was raised against the major mugwort pollen allergen, Art v 1, was used to demonstrate the presence of related allergens in nitrocellulose-blotted pollen and plant-food extracts. The tissue localization of the cross-reactive allergen was investigated by immunogold electron microscopy. RESULTS: Monoclonal antibody 3A4 recognized IgE epitopes of the 60 kd mugwort allergen and cross-reacted with moieties of comparable molecular weights in birch and timothy grass pollen, as well as in apple and celery extracts. In pollen and plant-derived food the allergen could be localized intracellularly in ribosome-rich areas in the mitochondria and the nucleus. No labeling was observed in the pollen or cell walls or in organelles that are engaged in storage (e.g., starch granules and lipid particles). CONCLUSION: Tree, grass, and weed pollen, as well as plant-derived foods, contain a high molecular weight Art v 1-cross-reactive allergen that maps to similar cell compartments.

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[47] - Hirschwehr R, Heppner C, Spitzauer S, Sperr WR, Valent P, Berger U, et al. Identification of common allergenic structures in mugwort and ragweed pollen. J Allergy Clin Immunol 1998;101:196-206

Identification of common allergenic structures in mugwort and ragweed pollen. BACKGROUND: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens. OBJECTIVE: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens. METHODS: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests. RESULTS: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1. CONCLUSION: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions.

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[48] - Himly M, Steiner R, van Ree R, Ebner C, Ferreira F. Comparison of the molecular and immunological properties of natural and recombinant Art v1, the major allergen of Artemisia vulgaris. AAAAI 57th Annual Meeting, New Orleans, 16-21 March, 2001, Poster n°1037

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[49] - Lombardero M, Garcia-Sellés FJ, Polo F, Jimeno L, Chamorro MJ, Garcia-Casado G, et al. Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens. Clin Exp Allergy 2004;34:1415-1421

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.

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[50] - Mahler V, Fischer S, Heiss S, Duchêne M, Kraft D, Valenta R. cDna cloning and characterization of a cross-reactive birch pollen allergen: identification as a pectin esterase. Int Arch Allergy Immunol 2001;124:64-66

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[51] - Grote M, Stumvoll S, Reichelt R, Lidholm J, Valenta R. Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by immunogold electron microscopy. Biol Chem 2002;383:1441-1445

Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by immunogold electron microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.

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[52] - Ganglberger E, Radauer C, Grimm R, Hoffmann-Sommergruber K, Breiteneder H, Scheiner O, et al. N-terminal sequences of high molecular weight allergens from celery tuber. Clin Exp Allergy 2000;30:566-570

Celery tuber is an important source of food allergens. Low molecular weight celery allergens were identified as homologues of Bet v 1 and profilin. Little is known about the relevant allergens with molecular weights between 45 and 60 kDa, which cross-react with other plant food and pollen allergens. OBJECTIVE: The aim of this study was to isolate cross-reactive, high molecular weight allergens from celery and to characterize them by N-terminal sequencing. METHODS: High molecular weight allergens of celery were identified by immunoglobulin (Ig) E immunoblotting with patients' sera, and the IgE-binding patterns were compared with those of the monoclonal antibirch pollen antibody BIP3, as well as of a polyclonal rabbit anti-Art v 1 antiserum. Two independent methods, elution from preparative SDS-PAGE or anion exchange chromatography, were used to purify the IgE-binding celery proteins of interest. The isolated proteins were examined by N-terminal sequencing and IgE-immunoblots. RESULTS: Celery allergens with molecular masses of 55, 58 and 63 kDa, which were also recognized by the monoclonal BIP3 antibody and a polyclonal anti-Art v 1 antiserum, were isolated. The 63-kDa allergen was N-terminally blocked. The 55- and 58-kDa compounds yielded the same N-terminus, which showed no homology to known proteins in the databases. CONCLUSION: The combination of two independent protein separation techniques, immunoblotting and N-terminal sequencing, identified an N-terminus of two allergens in the 60-kDa molecular weight region. Our data will be helpful for the definite molecular characterization of these important cross-reactive molecules.

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[53] - Fischer S, Grote M, Fahlbusch B, Müller WD, Kraft D, Valenta R. Characterization of Phl p 4, a major timothy grass (Phleum pratense) pollen allergen. J Allergy Clin Immunol 1996;98:189-198

Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens. OBJECTIVE: In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens. METHODS: Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunoelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE- inhibition studies. RESULTS: Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunoelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients' IgE. CONCLUSION: Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy.

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[54] - Bauer L, Ebner C, Hirschwehr R, Wüthrich B, Pichler C, Fritsch R, et al. IgE cross-reactivity between birch pollen, mugwort pollen and celery is due to at least three distinct cross-reacting allergens: immunoblot investigation of the birch-mugwort-celery syndrome. Clin Exp Allergy 1996;26:1161-1170

Allergy to celery is often associated with sensitization to birch and/or mugwort pollen. OBJECTIVE and METHODS: In a multi-centre study, sera from 23 patients suffering from type 1 allergy to celery and 15 patients with positive celery RAST but no clinical sensitization were compared. To examine whether cross-reactivity between celery and mugwort pollen includes cross-sensitization to birch pollen allergens, we determined cross-reacting structures in birch pollen, mugwort pollen and celery by means of immunoblotting. Inhibition studies were performed by preincubation of sera with extracts of birch pollen, mugwort pollen, and celery. RESULTS: We identified three groups of proteins--homologues of Bet v 1 and birch profilin (Bet v 2) as well as a group of proteins with a molecular range of 46 to 60 kD--displaying IgE-cross-reactivity, which were shared by birch pollen and celery. Two of these groups of allergens (profilin and the 46 to 60 kD proteins) were also present in mugwort pollen. In this paper we demonstrate that most cross-reacting allergens present in mugwort pollen and celery can also be detected in birch pollen extract. CONCLUSION: Therefore we propose, from a serological point of view, to extend the mugwort-celery syndrome to the birch-mugwort-celery syndrome.

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[55] - Poncet P, Senechal H, Clement G, Godfrin D, Wal JM, Peltre G, et al. Allergy to ash (Fraxinus excelsior) pollen: allergomic study. Allergy Clin Immunol Int 2005;17(Suppl. 1):217

Background : In Europe, sensitization to ash pollen (Oleaceae family) is underestimated because of a lack of precise pollinisation calendar, lack of standardized extract for diagnosis and wide cross reactivities with other pollens. Objective and methods : To identify the panel of ash pollen allergens and to decipher the IgE response, sixty two sera from patients diagnosed for ash, olive or grass allergies were screened by IgE immunoblot of an aquaeous extract of ash pollen separated by SDS-PAGE. Ten sera were selected for 2-D IgE immunoblot analysis followed by mass spectrometry for protein identification. Results : IgE reactive bands were detected in 44 sera. Thirty seven showed binding to proteins of Mr 18-20kDa, Fra e 1 (major ash pollen allergen). Twelve sera recognized a 14kDa band, Fra e 2 (profilin), 2 sera, a 10kDa protein, Fra e 3 and 12, proteins >30kDa. Specific and private patterns of several spots were observed for each of individually tested sera in 2-D IgE immunoblots. When overlapped, 5 zones of IgE reactivity were determined. (1) 15 acidic spots at 43kDa, (2) 10 neutro-basic spots at 45kDa, (3) 6 neutro-acidic spots at 32kDa, (4) a series of doublets, pI 4.8 to 8.5 at 20kDa and (5) one spot pI< 4.75 at 14kDa. Forty three spots (allergens and non-allergens) were collected for protein identification by mass spectrometry. MALDI-TOF analysis of trypsin in-gel-digested proteins revealed that proteins from zone 4 corresponded to isoforms of Fra e 1. The protein of zone 5 belongs to the profilin family and the spots of zone 2 are isoforms of a sugar transport protein. Besides these allergens, 10 proteins that did not exhibit IgE reactivity were identified with significant scores and sequence coverages. Conclusion : Ninety two percent of the ash-sensitized patients have IgE against Fra e 1 and 46% against Fra e 2. Cross-reactivities of IgE from grasssensitized patients does not involve Fra e 1. Using the highly resolving power of 2D electrophoresis coupled to the sensitivity of mass spectrometry we show that Fra e 1 exhibited a wider heterogeneity of pI than expected, and that IgE reactivities against high Mr allergens may be resolved in 3 regions. Our data contribute to a better delineation of ash pollen allergens and pattern of sensitization. The diagnostic and therapeutic relevance of these identified allergens may help to predict the evolution of symptoms and to refine the treatment of ash allergy.

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[56] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[57] - Lombardero M, Garcia-Sellés FJ, Polo F, Jimeno L, Chamorro MJ, Garcia-Casado G, et al. Prevalence of sensitization to Artemisia allergens Art v 1, Art v 3 and Art v 60 kDa. Cross-reactivity among Art v 3 and other relevant lipid-transfer protein allergens. Clin Exp Allergy 2004;34:1415-1421

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.

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[58] - de la Hoz F, Polo F, Moscoso del Prado J, Selles JG, Lombardero M, Carreira J. Purification of Art v I, a relevant allergen of Artemisia vulgaris pollen. Mol Immunol 1990;27:651-657

An allergenic protein from Artemisia vulgaris pollen has been purified to homogeneity. Its molecular weight in native conditions is 47,000. The purified allergen, hereafter denominated Art v I, is a monomeric protein It is a clinically relevant allergen since, at least, 70% of the individuals allergic to Artemisia vulgaris pollen have specific IgE in serum and in mast cells, demonstrated by ELISA and skin prick tests, respectively.

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[59] - Hirschwehr R, Heppner C, Spitzauer S, Sperr WR, Valent P, Berger U, et al. Identification of common allergenic structures in mugwort and ragweed pollen. J Allergy Clin Immunol 1998;101:196-206

Identification of common allergenic structures in mugwort and ragweed pollen. BACKGROUND: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens. OBJECTIVE: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens. METHODS: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests. RESULTS: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1. CONCLUSION: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions.

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[60] - Egger M, Himly M, Dedic A, Bauer R, Mari A, van Ree R, et al. Is Art v 1, the major allergen of mugwort pollen, a cross-reactive allergen ? EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°280

Background: Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen and therefore represents, like other allergens originating from Compositae (e.g. mugwort, ragweed) one of the main causes of pollinosis in late summer in Europe. Previous studies revealed cross-reactivity between members of Compositae plant family. In addition, cross-reactivity between mugwort pollen and certain foods known as the "celery-mugwort-spice-syndrome" and latex glycosylated allergens have been reported. Objectives: The aim of this study was to investigate in vitro cross-reactivity between mugwort Art v 1 and proteins originating from other Compositae pollen (Artemisia absinthium, Ambrosia artemisifolia, Ambrosia psilostachya, Ambrosia trifida, Helianthus annus ), grass pollen (Phleum pratense ), tree pollen (Betula verrucosa ) and common food allergen sources (apple, celery). Methods: Cross-reactivity was investigated by immunoblot using the following antibodies: polyclonal rabbit anti-natural Art v 1, rabbit anti-recombinant Art v 1, moAb anti- Art v 1 produced by genetic immunization, and sera from mugwort and ragweed sensitized patients. Results: Using different antibodies and sera from allergic patients, we found that Art v 1 share epitopes with proteins originating mainly from other Compositae pollen (Artemisia absinthium, Ambrosia artemisifolia, Ambrosia psilostachya, Ambrosia trifida, Helianthus annus ) and from grass pollen (Phleum pratense ). We did not observe cross-reactivity between Art v 1 and proteins in extracts from tree pollen (Betula verrucosa ) and foods (celery and apple). Conclusions: Art v 1 is a glycoallergen sharing epitopes with proteins found in other Compositae pollen. These cross-reactive epitopes seem to encompass both the Art v 1 polypeptide and O-glycans attached to its C-terminal domain.

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[61] - Asero R, Wopfner N, Gruber P, Gadermaier G, Ferreira F. Artemisia and Ambrosia hypersensitivity: co-sensitization or co-recognition ? Clin Exp Allergy 2006;36:658-665

BACKGROUND: Ragweed and mugwort have nearly identical flowering periods. Clinical and serological studies showed that ragweed and mugwort sensitization are often associated and this poses relevant clinical problems in patients for whom specific immunotherapy is warranted . OBJECTIVE: To establish whether the concurrent ragweed and mugwort pollen hypersensitivity is the result of co-sensitization or of co-recognition by using purified recombinant allergens . METHODS: Sensitization to ragweed and mugwort pollen was assessed by skin prick test (SPT) in all patients reporting allergic symptoms in August and September. IgE reactivity of sera from 42 patients (26 Amb+/Art+, 14 Amb+/Art-, and two Amb-/Art+) to ragweed and mugwort pollen extract as well as to several recombinant ragweed (rAmb a 1, rAmb a 5, rAmb a 6, rAmb a 8, rAmb a 9, and Amb a 10) and mugwort (rArt v 1, rArt v 4, rArt v 5, rArt v 6, and three EF-hand calcium-binding protein) allergens was detected by dot-blot and ELISA analyses . RESULTS: IgE reactivity of 372 weed pollen-allergic patients was studied. Mugwort reactivity was strongly associated with ragweed hypersensitivity: only 10/147 (7%) mugwort-hypersensitive patients were not sensitized to ragweed, whereas 225/362 (62%) ragweed-hypersensitive patients were not sensitized to mugwort. In vitro, 90% of ragweed-allergic patients reacted with rAmb a 1. Reactivity to other ragweed allergens ranged between 20% and 35%. Forty-six percent of the mugwort-sensitized patients recognized rArt v 1%, 25% reacted to Art v 4, Art v 5, and Art v 6, and 7% recognized the three-EF hand calcium-binding protein. Immunoblot inhibition experiments showed that pre-incubation with ragweed pollen extract only weakly decreased IgE reactivity to mugwort allergens . CONCLUSION: Patients showing both ragweed- and mugwort-positive SPT and/or RAST are co-sensitized. Future studies will establish whether IgE reactivity translates into clinical symptoms and, hence, if co-sensitized patients should undergo specific immunotherapy with extracts of both mugwort and ragweed pollen.

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[62] - Asero R, Wopfner N, Gruber P, Gadermaier G, Ferreira F. Artemisia and Ambrosia hypersensitivity: co-sensitization or co-recognition ? Clin Exp Allergy 2006;36:658-665

BACKGROUND: Ragweed and mugwort have nearly identical flowering periods. Clinical and serological studies showed that ragweed and mugwort sensitization are often associated and this poses relevant clinical problems in patients for whom specific immunotherapy is warranted . OBJECTIVE: To establish whether the concurrent ragweed and mugwort pollen hypersensitivity is the result of co-sensitization or of co-recognition by using purified recombinant allergens . METHODS: Sensitization to ragweed and mugwort pollen was assessed by skin prick test (SPT) in all patients reporting allergic symptoms in August and September. IgE reactivity of sera from 42 patients (26 Amb+/Art+, 14 Amb+/Art-, and two Amb-/Art+) to ragweed and mugwort pollen extract as well as to several recombinant ragweed (rAmb a 1, rAmb a 5, rAmb a 6, rAmb a 8, rAmb a 9, and Amb a 10) and mugwort (rArt v 1, rArt v 4, rArt v 5, rArt v 6, and three EF-hand calcium-binding protein) allergens was detected by dot-blot and ELISA analyses . RESULTS: IgE reactivity of 372 weed pollen-allergic patients was studied. Mugwort reactivity was strongly associated with ragweed hypersensitivity: only 10/147 (7%) mugwort-hypersensitive patients were not sensitized to ragweed, whereas 225/362 (62%) ragweed-hypersensitive patients were not sensitized to mugwort. In vitro, 90% of ragweed-allergic patients reacted with rAmb a 1. Reactivity to other ragweed allergens ranged between 20% and 35%. Forty-six percent of the mugwort-sensitized patients recognized rArt v 1%, 25% reacted to Art v 4, Art v 5, and Art v 6, and 7% recognized the three-EF hand calcium-binding protein. Immunoblot inhibition experiments showed that pre-incubation with ragweed pollen extract only weakly decreased IgE reactivity to mugwort allergens . CONCLUSION: Patients showing both ragweed- and mugwort-positive SPT and/or RAST are co-sensitized. Future studies will establish whether IgE reactivity translates into clinical symptoms and, hence, if co-sensitized patients should undergo specific immunotherapy with extracts of both mugwort and ragweed pollen.

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[63] - Wopfner N, Gruber P, Wallner M, Briza P, Ebner C, Mari A, et al. Molecular and immunological characterization of novel weed pollen pan-allergens. Allergy 2008;63:872-881

BACKGROUND: Pan-allergens like profilins, calcium-binding proteins (CBPs), and nonspecific lipid transfer proteins have been suggested as possible specific markers for multiple pollen sensitizations, and could be used to predict cross-sensitization/poly-sensitization to several pollen allergens. Therefore, the purification and characterization of cross-reacting allergens in pollen is an extremely important task towards correct allergy diagnosis . METHODS: New pan-allergens were identified by screening a ragweed pollen cDNA library with sera of patients allergic to mugwort pollen. Resulting proteins were cloned, expressed, purified and characterized . RESULTS: We report complete cDNA sequences of two profilin isoforms (Amb a 8.01 and Amb a 8.02), two isoforms of a 2EF-hand CBP (Amb a 9.01 and Amb a 9.02), a new 3EF-hand CBP (Amb a 10) from ragweed pollen and a 2EF-hand CBP from mugwort (Art v 5). All these proteins were expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological means . CONCLUSIONS: The identified proteins are novel pan-allergens and can be used as diagnostic markers for polysensitization and used in component-resolved diagnosis.

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[64] - Gadermaier G, Wopfner N, Wallner M, Egger M, Didierlaurent A, Regl G, et al. Array-based profiling of ragweed and mugwort pollen allergens. Allergy 2008;63:1543-1549

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons . OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA) . METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays . RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods . CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

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[65] - Léonard R, Wopfner N, Stadlmann J, Pabst M, Himly M, Petersen B et al. A new allergen from ragweed has homology to Art v 1 from mugwort pollen. Allergy 2009;64(Suppl. 90):252

Background: Originating from Northern America, ragweed (e.g. Ambrosia artemisiifolia) is increasingly becoming a major weed pollen allergen in central Europe. Ragweed and mugwort pollen allergic patients exhibit a considerable degree of cross-sensitization and several allergens are similar. However, a homolog to the major mugwort allergen Art v 1 has not yet been described for ragweed. Here, we report on the characterization and molecular cloning of this new allergen. Methods: Natural Art v 1-homologue was purified from short ragweed pollen and characterized by N-terminal sequencing, mass spectrometry and NMR. Recombinant protein was expressed in E. coli. IgE binding was measured by ELISA using sera from mugwort and/ or ragweed sensitized patients. Result: The Art v 1-homologue migrated with an apparent mass of about 30 kDa on SDS-PAGE. By MALDI-TOF MS, the - true - mass of the allergen was found to be only 18 kDa with a very broad distribution. One third of this mass is accounted for by galactose and arabinose in a ratio of 1 to 17. These sugars are primarily organized in small polysaccharides or oligosaccharides. Mono-arabinosyl-residues are rare and hence the O-glycan determinant found on Art v 1 is absent from its ragweed homolog. N-terminal and internal sequencing revealed homology of the new ragweed allergen with Art v 1 and enabled to clone its cDNA. Natural as well as recombinant Art v 1-homologue was recognized by IgE from 54 of 94 ragweed-sensitized patients, whereby 25 gave a reading five-times higher than the control sera. Conclusion: Ragweed pollen contains an arabinogalactan-protein allergen recognized by a significant number of weed pollen allergic individuals. Unlike Art v 1, the homologous glycoprotein from ragweed does not contain mono-arabinosyl patches recognized by patients' IgE.

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[66] - Mutschlechner S, Mari A, van Ree R, Didierlaurent A, Ebner C, Ferreira F, et al. Patterns of Art v 1 recognition in mugwort- and ragweed- sensitized patients. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1357

The family Compositae or Asteraceae is known to include several allergenic plants, and among them Ambrosia (ragweed) and Artemisia (mugwort) are of major importance. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is rapidly increasing and is now abundant in the Rhone valley (France), northern Italy, eastern parts of Austria, Hungary and Bulgaria. Several studies showed that mugwort and ragweed share common allergenic structures but the antigen(s) responsible for these allergic cross-reactions are not yet identified. In this study, we investigated the patterns of Art v1- recognition in mugwort and ragweed sensitized patients. Art v 1, the major allergen of mugwort pollen, was recently characterized in our lab. IgE immunoblots were performed using sera from 270 mugwort- sensitized patients (Austrians) and 137 ragweed-sensitized patients (83 Canadians, 22 Italians, 32 Americans) and pollen extracts from mugwort and ragweed, as well as purified natural and recombinant Art v1. Among mugwort-sensitized patients, 77% recognized purified Art v 1, 67% reacted with a 27 - 28 kDa protein in ragweed, and 40% recognized Amb a1, the major allergen from ragweed pollen. In the ragweed-sensitized group, 30% reacted with purified mugwort Art v 1, 53% reacted with a 27 - 28 kDa protein from ragweed, and 90% reacted with Amb a 1. Our results showed that mugwort and ragweed share common allergens. However, their recognition pattern by mugwort- and ragweed- sensitized patients pointed to a limited cross-reactivity.

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[67] - Ackermann-Liebrich U, Schindler C, Frei P, Probst-Hensch NM, Imboden M, Gemperli A et al. Sensitisation to Ambrosia in Switzerland: a public health threat on the wait. Swiss Med Wkly 2009;139:70-75

BACKGROUND: Ambrosia artemisiifolia (short name = Ambrosia common ragweed) pollen is a potent allergen and has recently been found in Switzerland, spreading from the southwest of the country. The aim of this study is to describe Ambrosia sensitisation rates in the population-based SAPALDIA cohort (Swiss Study on Air Pollution And Lung Diseases In Adults) and to test whether an increase in these rates could be observed. METHODS: Among the 6345 participants from 8 areas who provided blood samples in 1991 and 2002, 5823 had valid results for specific IgE against common inhalant allergens tested with Phadiatop(R). In 2002 Ambrosia sensitisation was measured and positive tests were analysed for Artemisia vulgaris (mugwort). Blood samples taken in 1991 in Ticino and Geneva were also tested for Ambrosia. RESULTS: Sensitisation rate (Phadiatop(R)) did not increase significantly between the two surveys and sensitisation was found in 30% of the participants. A proportion of 7.9% showed specific IgE to Ambrosia pollen. The sensitisation rate in Lugano and Geneva had not changed substantially since 1991. Among those sensitised to Ambrosia 82% also showed specific IgE against Artemisia, suggesting a high rate of cross-reactivity. Only 1.3% were sensitized to Ambrosia alone. The incidence of asthma or hay fever in participants with specific IgE to Ambrosia pollen was not higher than in the general study population. CONCLUSION: Currently Ambrosia pollen does not appear to be an important cause of inhalant allergies in Switzerland. Sensitisation rates are low and have not increased since 1991. Due to cross-reactivity Ambrosia sensitisation may be a consequence of primary sensitisation to Artemisia. Elimination of Ambrosia plants is nevertheless mandatory to avoid a future increase.

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[68] - Weber RW. Patterns of pollen cross-allergenicity. J Allergy Clin Immunol 2003;112:229-239

Knowledge of patterns of pollen cross-reactivity is crucial for diagnostics and especially for formulation of immunotherapy vaccines in times of diminishing availability of pollen extract constituents. As phylogenetic relationships have become better clarified, it becomes apparent that cross-reactivity does reflect taxonomy in the very great majority of cases. Contradictory observations of unexpected cross-reactivity between unrelated plants, sometimes remarkably distant ones, require explanation. There are many proteins, presumably performing vital functions, that are tightly preserved throughout the evolutionary tree from plants to animals, such as profilins, lipid transfer proteins, and pathogenesis-related proteins. These might function as panallergens. The small differences that exist between these ubiquitous proteins explain why these are frequently minor allergens not reacting in the majority of allergic sera. This review summarizes cross-reactivity studies with both crude pollen extracts and purified or recombinant allergenic proteins. The patterns of cross-allergenicity that emerge should be helpful in guiding both diagnostic and therapeutic decisions.

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[69] - Mohapatra SS, Lockey RF, Polo F. Weed pollen allergens. Clin Allergy Immunol 2004;18:207-222

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[70] - Heiss S, Fischer S, Müller WD, Weber B, Hirschwehr R, Spitzauer S, et al. Identification of a 60 kd cross-reactive allergen in pollen and plant-derived food. J Allergy Clin Immunol 1996;98:938-947

Cross-reactive IgE antibodies were found to be responsible for allergic reactions in patients allergic to pollen on ingestion of food (oral allergy syndrome). So far, the major birch pollen allergen Bet v 1 and birch profilin (Bet v 2) were identified as relevant cross-reactive allergens. OBJECTIVE: In this study we attempted to identify additional cross-reactive plant allergens, which could be responsible for food intolerance in patients allergic to pollen. METHODS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, representing a 60 kd glycoprotein, were used to detect cross-reactive allergens in other pollens and plant-derived food. The amino acid compositions of the cross-reactive structures were determined, and their resistance against trypsin treatment was investigated. In addition, IgE immunoblot inhibitions were done with the 60 kd mugwort pollen allergen. RESULTS: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, cross-reacted with proteins of comparable molecular weight in fruit and vegetables. Preadsorption of patients' sera with the 60 kd mugwort allergen led to a reduction of IgE binding to components of a similar molecular weight present in different pollen (birch, timothy grass), fruit (apple, peanuts), and vegetable (celery) extracts and reduced IgE binding to apple, kiwi, and celery as determined by RAST inhibitions. CONCLUSION: A cross-reactive plant panallergen, possibly identical to the major mugwort pollen allergen, Art v 1, is described. The allergen represents a protein of approximately 60 kd present in various pollen and plant foods; which is distinct from Bet v 1 and profilin and hence may represent a novel cross-reactive allergen in the oral allergy syndrome.

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[71] - Vallier P, Dechamp C, Vial O, Deviller P. A study of allergens in celery with cross-sensitivity to mugwort and birch pollens. Clin Allergy 1988;18:491-500

Sixty-one sera with positive RAST to mugwort pollen (Artemisiae vulgaris) were submitted to RASTs for birch pollen (Betula verrucosa) and celery (Apium graveolens). In 36 cases RAST results were positive for celery. In addition, 23 sera presented specific IgE to birch pollen. The binding of specific IgE to individual allergens in celery, mugwort pollen and birch pollen was studied by the immunoblotting technique. This involved electrophoretic separation of allergenic extracts, electrotransfer of proteins onto nitrocellulose sheets and sensitive immunoenzymatic detection. Eighteen sera had specific IgE binding to two celery components of molecular weight around 15 kD. All these sera also detected a 15 kD allergen in mugwort and two allergens in birch of 14 kD and 16 kD molecular weight. The sera that did not detect the 15 kD bands in celery failed to react with both the 15 kD mugwort component and the 14 and 16 kD birch components. Specific cross-inhibitions of the detection of these allergens on immunoblots were obtained by pre-incubation of the sera with crude extract of the three species. These results strongly suggest that such allergens display some structural identity and that they could be at the origin of some cases of crossed hypersensitivity to celery, mugwort pollen and birch pollen

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[72] - Gonzalez EM, Villalba M, Rodriguez R. Allergenic cross-reactivity of olive pollen. Allergy 2000;55:658-663

BACKGROUND: Sera of patients allergic to olive (Olea europaea) pollen were used to analyze the IgE cross-reactivity between olive-pollen extract and other pollens obtained from phylogenetically unrelated species. METHODS: We used IgE immunostaining of pollen extracts blotted to nitrocellulose membranes after SDS-PAGE and inhibition analysis of this binding. RESULTS: A high inhibition of the IgE binding on olive-pollen extract was exhibited by birch, mugwort, pine, and cypress pollens, suggesting that these extracts contain proteins which share common epitopes and thus can be recognized by olive-allergic sera. IgE binding to Gramineae pollen extracts was not inhibited by olive-pollen extract, indicating a primary sensitization of the patients to these species. From the inhibition assays, the presence of an allergen of 45 kDa in the olive pollen, which has no homologous counterparts in other allergenic species, has been inferred. CONCLUSIONS: Olive pollen contains allergens which cross-react with pollens from unrelated species, a fact that could simplify the diagnosis and treatment of pollinosis.

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[73] - van Ree R, Voitenko V, van Leeuwen WA, Aalberse RC. Profilin is a cross-reactive allergen in pollen and vegetable foods. Int Arch Allergy Immunol 1992;98:97-104

Sera with IgE antibodies against grass pollen often contain IgE against vegetable foods. We investigated the role of the ubiquitous protein profilin in this cross-reactivity. Profilin was purified from Lolium perenne grass pollen by means of affinity purification with Sepharose-coupled poly(L-proline). This solid phase was also used as capturing agent for profilin from pollen and food extracts for application in a radioallergosorbent test. It was shown that profilin is an allergen in grass pollen and in a wide range of vegetable foods, like potato and celery. Within a grass-pollen-sensitive population, patients with IgE to vegetable foods have a high incidence of antibodies against profilin. IgE antibodies against grass pollen profilin were shown to be cross-reactive with respect to vegetable foods

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[74] - Stumvoll S, Westritschnig K, Lidholm J, Spitzauer S, Colombo P, Duro G, et al. Identification of cross-reactive and genuine Parietaria judaica pollen allergens. J Allergy Clin Immunol 2003;111:974-979

BACKGROUND: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. OBJECTIVE: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). METHODS: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n = 42; Scandinavia, n = 8; United States, n = 19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. RESULTS: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. CONCLUSION: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract.

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[75] - Miralles JC, Caravaca F, Guillén F, Lombardero M, Negro JM. Cross-reactivity between Platanus pollen and vegetables. Allergy 2002;57:146-149

Background: Several associations have been described between tree and plant pollens and certain foods. The objective of this study is to verify whether there is cross-reactivity between Platanus pollen and vegetable origin foods. Methods: We selected 56 patients allergic to vegetable foods and subjected them to cutaneous tests with aeroallergens and vegetable foods. A statistical analysis was performed to evaluate the association of Platanus pollen with foods and with other aeroallergens. Later, a specific IgE determination was performed as well as a RAST (radioallergosorbent) inhibition experiment, to verify the existence of cross-reactivity in vitro. Results: In the cutaneous tests we found a positive correlation between Platanus pollen and hazelnut, peanut, banana and celery. The results of the RAST inhibition experiment indicate an important cross-reactivity between the pollen of Platanus acerifolia and hazelnut and banana fruit, and an intermediate cross-reactivity with celery and peanut. Conclusion: We have described an association between the pollen of the Platanus tree and some vegetable foods such as hazelnut, banana, peanut and celery. This association could be explained by the in vitro IgE cross-reactivity detected.

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[76] - Bublin M, Radauer C, Wilson IB, Kraft D, Scheiner O, Breiteneder H, et al. Cross-reactive N-glycans of Api g 5, a high molecular weight glycoprotein allergen from celery, are required for immunoglobulin E binding and activation of effector cells from allergic patients. FASEB J 2003;17:1697-1699

Allergy diagnosis relying on the determination of specific IgE is frequently complicated by the presence of cross-reacting IgE of unclear clinical relevance. Particularly, the anaphylactogenic activity of IgE directed to cross-reactive carbohydrate moieties of glycoproteins from plants and invertebrates has been a matter of debate. In this study, we present the biochemical and immunological characterization of Api g 5, a glycoprotein allergen from celery with homology to FAD containing oxidases. Carbohydrate analysis of the allergen revealed the presence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. Chemical deglycosylation of the protein completely abolished binding of serum IgE from all 14 patients tested. Likewise, basophils from a patient allergic to mugwort pollen and celery were stimulated only by native Api g 5, whereas the deglycosylated allergen did not trigger release of histamine. IgE inhibition immunoblots showed that native Api g 5 other than the deglycosylated protein completely inhibited IgE binding to high molecular weight allergens in protein extracts from birch pollen, mugwort pollen, and celery. A similar inhibition was accomplished using the IgE binding oligosaccharide, MUXF, coupled to bovine serum albumin. All these observations taken together confer convincing evidence that IgE directed to cross-reactive carbohydrates is capable of eliciting allergic reactions in vivo.

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[77] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[78] - Mortz CG, Andersen KE, Bindslev-Jensen C. The prevalence of peanut sensitization and the association to pollen sensitization in a cohort of unselected adolescents – The Odense Adolescence Cohort Study on Atopic Diseases and Dermatitis (TOACS). Pediatr Allergy Immunol 2005;16:501-506

In the last decade an increased occurrence of peanut hypersensitivity and severe anaphylactic reactions to peanut have been reported. However, few prevalence studies have been performed in unselected populations. This study evaluated the point prevalence of peanut hypersensitivity in Danish adolescents. The point prevalence of peanut allergy confirmed by oral challenge was estimated to 0.5%. The number of adolescents sensitized to peanut by specific immunoglobulin E (IgE) (CAP FEIA) and skin prick test (SPT) were higher (5.8% resp. 3.4%). In adolescents without clinically relevant peanut sensitization most cases were sensitized to grass pollen and the IgE class for grass was higher than for peanut. A correlation between peanut and pollen (grass) sensitization is therefore plausible. Before a positive SPT or specific IgE measurement to peanut is considered clinically relevant in a patient, the case history should be evaluated together with examination for pollen sensitization.

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[79] - Movérare R, Holtz A, Brostedt P, af Geijerstam E, Larsson H, Lundberg M, et al. Study of IgE antibody responses to the major mugwort pollen allergen Art v 1 using ImmunoCAP. Allergy 2007;62(suppl. 83):150-151

Allergy to mugwort pollen is a major problem. About 95% of mugwort allergic patients have IgE antibodies to the major allergen Art v 1. Component resolved diagnostics (CRD) using individual allergens are helpful to determine if a positive IgE antibody response to a whole pollen extract might be due to sensitization to major allergens or to cross-reactive panallergens such as profilins and polcalcins. Therefore, our aim was to develop a test for Art v 1-specific IgE antibodies. We used cation exchange chromatography (Sepharose SP FF) and gelfiltration in crude purification of the major mugwort pollen allergen Art v 1. Balb/c mice were hyper-immunized with the Art v 1 preparation and the spleen cells were used for the development of hybridoma cells. Hybridoma cells producing antibodies against the Art v 1 preparation in ELISA were cloned and their antibody reactivity to mugwort pollen extract was determined using immunoblotting. One hybridoma produced monoclonal antibodies against a ~28-30 kD band, which was assumed to correspond to Art v 1, and was therefore considered to be Art v 1-specific. Art v 1-specific monoclonal antibodies were produced in roller bottle cultures and purified using rProtein A affinity chromatography. The antibodies were then coupled to NHS-activated Sepharose HP and used for affinity purification of nArt v 1 from pollen extract. The affinity-purified nArt v 1 was more than 95% pure, and no contaminating proteins were observed by SDS-PAGE. The identity of Art v 1 was determined using MALDI-TOF mass spectrometer. Experimental ImmunoCAP with affinity-purified nArt v 1 was developed and used to study IgE antibody reactivity. There was a high correlation between nArt v 1 ImmunoCAP and mugwort pollen ImmunoCAP using selected Art v 1-monospecific human samples, which indicates a preserved immunoreactivity of the coupled nArt v 1. The prevalence of Art v 1 sensitization was studied in 10 mugwort skin prick positive subjects and was found to be 50%. The subjects without Art v 1 sensitization had high levels of IgE antibodies to ragweed pollen, indicating that IgE antibodies induced to cross-reactive allergens in ragweed pollen could be responsible for their mugwort sensitization. CRD using nArt v 1 ImmunoCAP might be useful when studying the IgE response to mugwort pollen in different populations and when selecting patients for appropriate specific immunotherapy using pollen allergen vaccines.

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[80] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[81] - Gadermaier G, Wopfner N, Wallner M, Egger M, Didierlaurent A, Regl G, et al. Array-based profiling of ragweed and mugwort pollen allergens. Allergy 2008;63:1543-1549

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons . OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA) . METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays . RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods . CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

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[82] - Asero R, Wopfner N, Gruber P, Gadermaier G, Ferreira F. Artemisia and Ambrosia hypersensitivity: co-sensitization or co-recognition ? Clin Exp Allergy 2006;36:658-665

BACKGROUND: Ragweed and mugwort have nearly identical flowering periods. Clinical and serological studies showed that ragweed and mugwort sensitization are often associated and this poses relevant clinical problems in patients for whom specific immunotherapy is warranted . OBJECTIVE: To establish whether the concurrent ragweed and mugwort pollen hypersensitivity is the result of co-sensitization or of co-recognition by using purified recombinant allergens . METHODS: Sensitization to ragweed and mugwort pollen was assessed by skin prick test (SPT) in all patients reporting allergic symptoms in August and September. IgE reactivity of sera from 42 patients (26 Amb+/Art+, 14 Amb+/Art-, and two Amb-/Art+) to ragweed and mugwort pollen extract as well as to several recombinant ragweed (rAmb a 1, rAmb a 5, rAmb a 6, rAmb a 8, rAmb a 9, and Amb a 10) and mugwort (rArt v 1, rArt v 4, rArt v 5, rArt v 6, and three EF-hand calcium-binding protein) allergens was detected by dot-blot and ELISA analyses . RESULTS: IgE reactivity of 372 weed pollen-allergic patients was studied. Mugwort reactivity was strongly associated with ragweed hypersensitivity: only 10/147 (7%) mugwort-hypersensitive patients were not sensitized to ragweed, whereas 225/362 (62%) ragweed-hypersensitive patients were not sensitized to mugwort. In vitro, 90% of ragweed-allergic patients reacted with rAmb a 1. Reactivity to other ragweed allergens ranged between 20% and 35%. Forty-six percent of the mugwort-sensitized patients recognized rArt v 1%, 25% reacted to Art v 4, Art v 5, and Art v 6, and 7% recognized the three-EF hand calcium-binding protein. Immunoblot inhibition experiments showed that pre-incubation with ragweed pollen extract only weakly decreased IgE reactivity to mugwort allergens . CONCLUSION: Patients showing both ragweed- and mugwort-positive SPT and/or RAST are co-sensitized. Future studies will establish whether IgE reactivity translates into clinical symptoms and, hence, if co-sensitized patients should undergo specific immunotherapy with extracts of both mugwort and ragweed pollen.

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[83] - Himly M, Jahn-Schmid B, Dedic A, Kelemen P, Wopfner N, Altmann F, et al. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain. FASEB J 2003;17:106-108

In late summer, pollen grains originating from Compositae weeds (e.g., mugwort, ragweed) are a major source of allergens worldwide. Here, we report the isolation of a cDNA clone coding for Art v 1, the major allergen of mugwort pollen. Sequence analysis showed that Art v 1 is a secreted allergen with an N-terminal cysteine-rich domain homologous to plant defensins and a C-terminal proline-rich region containing several (Ser/Ala)(Pro)2-4 repeats. Structural analysis showed that some of the proline residues in the C-terminal domain of Art v 1 are posttranslationally modified by hydroxylation and O-glycosylation. The O-glycans are composed of 3 galactoses and 9-16 arabinoses linked to a hydroxyproline and represent a new type of plant O-glycan. A 3-D structural model of Art v 1 was generated showing a characteristic "head and tail" structure. Evaluation of the antibody binding properties of natural and recombinant Art v 1 produced in Escherichia coli revealed the involvement of the defensin fold and posttranslational modifications in the formation of epitopes recognized by IgE antibodies from allergic patients. However, posttranslational modifications did not influence T-cell recognition. Thus, recombinant nonglycosylated Art v 1 is a good starting template for engineering hypoallergenic vaccines for weed-pollen therapy.

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[84] - Oberhuber C, Ma Y, Wopfner N, Gadermaier G, Dedic A, Niggemann B, et al. Prevalence of IgE-Binding to Art v 1, Art v 4 and Amb a 1 in Mugwort-Allergic Patients. Int Arch Allergy Immunol 2008;145:94-101

BACKGROUND: Mugwort (Artemisia vulgaris) represents an important source of weed pollen allergens. The objectives of the present study were (i) to analyze the IgE binding profiles in a group of mugwort-allergic patients, (ii) to identify individual marker allergens crucial for the diagnosis of mugwort allergy and (iii) to identify potential crossreactive allergens present in ragweed (Ambrosia artemisiifolia) pollen extract . METHODS: Sera from 100 pediatric mugwort-allergic patients were analyzed for their IgE binding pattern to natural mugwort and ragweed pollen proteins, purified natural and recombinant Art v 1, recombinant Art v 4 and recombinant Amb a 1 using immunoblots and ELISA . RESULTS: 91% of the patients' sera tested displayed IgE binding to one or more mugwort pollen allergens in ELISA and 88% were positive in immunoblot. Purified natural Art v 1 was recognized by 79%, the recombinant protein by 39% of the patients tested and purified recombinant Art v 4 by 34% of the patients' sera. 67% of the sera displayed crossreactive IgE to one or more ragweed pollen allergens. Recombinant Amb a 1 was noted in only 14% of the mugwort-allergic sera . CONCLUSIONS: Allergen-specific in vitro diagnosis was performed in 100 pediatric mugwort-allergic serum samples. Using two allergens (Art v 1 and Art v 4), 91% of the patients could be identified as mugwort pollen-sensitized patients by IgE in vitro tests. Crossreactivity to ragweed pollen allergens was demonstrated by in vitro experiments, suggesting a new important and potent allergen source expanding across Europe.

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[85] - Giroux F, Cano Y, Malandain H. Validation of a simple method to overcome the interference of cross-reactive carbohydrate determinants (CCD) in specific IgE assays. Allergy 2009;64(Suppl. 90):8-9

Background: Some patients develop IgE reactivity to glyco-epitopes, ie. carbohydrate chains present on glycoproteins. This is especially the case for patients allergic to pollens or to Hymenoptera venoms. These glyco-epitopes are often cross-reactive, leading to so-called IgE anti-CCD (Cross-reactive Carbohydrate Determinants). Several studies failed to find clinical relevance for these IgE. But in vitro diagnostic tests do not differenciate between peptide- and glycan-specific IgE (sIgE) and their results can be interfered by the presence of IgE anti-CCD in the patient's serum. We sought to design a serum pretreatment to overcome this drawback. We especially aimed at simplifying the process and minimalizing cost and serum dilution so that the method could be suited to most daily routine situations. METHODS: Serum pretreatment: a reagent made of heat-inactivated bromelain and horseradish peroxidase (HRP) was added to the serum. After a waiting time, sIgE reactivity was directly measured on UniCAP (Phadia). To set up the method we used 2 sera positive to IgE anti- CCD (CAP bromelain >0,35 kU/l) (Eur Ann Allergy Clin Immunol 2007;39:216) and a pool of negative sera (total IgE <2 UI/ml). For validating the method, we selected 40 sera from our serum bank, 13 being positive to IgE anti-CCD. We studied 7 allergen sources (peanuts, white of egg, milk, cat ,dog, mite, cod) and 6 non glycosylated recombinant allergens (rBet v 1, rBet v 2, rPhl p 1, rPhl p 7, rPhl p 12, rFel d 1). RESULTS: The best compromise between the highest capture of IgE anti-CCD and the lowest serum dilution was to mix 4 parts of serum with 1 part of reagent (bromelain and HRP 25 g/l, a.a.). Serum pretreatment did not generate false positive IgE-reactivity in negative sera, nor significantly modified positive results for milk, cat, dog, mite, cod, white of egg, and non glycosylated allergens (n = 54 comparisons). It did not modify peanut results for 7 sera with bromelain <0,5 kU/l, and corrected peanut diagnostic for 7 patients with falsely high peanut sIgE (bromelain 1.5-28 kU/l). CONCLUSION: We think our method could be applied to daily lab routine. Components of the pretreatment reagent are ready-to-use, stable (>1 month, 4°C), and cheap (<0.1 •/test). Serum dilution is minimal so that CCD capture can be performed with IgE reactivity as low as 0,5 kU/ l. The method could prove especially useful to CCD-prone sIgE tests such as peanuts, latex and Hymenoptera venoms.

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