Les ambroisies

[1] - 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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[2] - Mohapatra SS, Lockey RF, Polo F. Weed pollen allergens. Clin Allergy Immunol 2004;18:207-222

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[3] - Thibaudon M, Lachasse C. Allergenic risk to ragweed in France since 15 years. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°282

Ragweed is a plant which become very common in some parts of France. It is on the area of Lyon in the Dauphine and along the Rhone Valley that we find the must important quantities of pollen of ragweed. In these area there is more than 12% of the population who suffer of allergic disease due to ragweed pollen. In this study, we will see that we can find ragweed pollen in all the different pollen traps which are located in France by RNSA. Even, if on some of them the quantities are increasing, these quantities stay very low and under the threshold high allergenic risk. But in the areas of Lyon, wa can look at the temporal evolution of ragweed pollen, for instance the number of grain by cubic meters which was a few hundred grains in 1987 and 1.200 grains in 2002. In the same time the number of days with a real allergenic risk go from 10 in 1987 to 30 in 2002. The surveillance of the evolution of the plant is very important. For this reason, the registration of pollen counts can permit to oberve these evolutions.

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[4] - Laaidi M, Laaidi K, Besancenot JP, Thibaudon M. Ragweed in France: an invasive plant and its allergenic pollen. Ann Allergy Asthma Immunol 2003;91:195-201

BACKGROUND: Ragweed is an annual, herbaceous, wind-pollinated plant that is responsible for strong allergies. In France, it is mainly present in the region of Lyon, where it threatens the health of the population. OBJECTIVE: To analyze annual, daily, and bihourly pollen concentrations in the air to determine the characteristics of ragweed pollination and in particular its diurnal rhythm. METHODS: Ragweed pollens were sampled from 1987 to 2001 by a Hirst volumetric trap calibrated to handle a flow of 10 L/min of air, which roughly corresponds to a human breathing rhythm. Pollen counts were performed on a daily and bihourly basis, which is of particular importance in allergologic practice. RESULTS: Ragweed pollination occurs from the beginning of August to the end of September. Annual and daily levels of pollen have increased significantly since 1987. The pronounced diurnal periodicity shows a peak from 9 to 11 AM. Pollen counts increased from the early morning with temperature increase and relative humidity decrease. CONCLUSIONS: Daily pollen counts of this strongly allergic plant are above the allergic thresholds commonly defined, limiting the interest of eradication campaigns. Thus, prevention of ragweed allergy depends on informing and educating the public through reports and updates. Diurnal variations seem to be the most crucial part of such a report, allowing allergy patients to adapt their daily outdoor activities in respect to the pollen peaks and off-peaks.

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[5] - Cecchi L, Torrigiani Malaspina T, Albertini R, Zanca M, Ridolo E, Usberti I, et al. The contribution of long-distance transport to the presence of Ambrosia pollen in central northern Italy. Aerobiologia 2007;23:145-151

Abstract Ragweed is an allergenic weed of public health concern in several European countries. In Italy ragweed occurs prevalently in north-north-eastern regions, where sensitization is increasing. Because of the small diameter of pollen grains, ragweed pollen is often involved in episodes of long-range transport, as already shown in central Italy. The objective of this study was to evaluate the extent of such transport by comparing pollen and meteorological data for two northern Italian cities (Parma and Mantova) with data from Pistoia and Florence in central Italy. In 2002 and 2004 peaks in ragweed pollen levels were detected in these four cities on the same day, and concentrations of the grains were above clinical thresholds. Weather-map analysis and computation of back-trajectories showed that air masses from eastern Europe might carry ragweed pollen to a wide area of central and northern Italy. These findings suggest that episodes of long-range transport of ragweed pollen could be clinically relevant, resulting in sensitization of a large number of people. The results might provide a basis for monitoring and forecasting periods of long-distance transport with the objective of reducing their effects on allergic patients.

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[6] - Asero R, Weber B, Mistrello G, Amato S, Madonini E, Cromwell O. Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: Analysis of the allergenic differences between short and giant ragweed. J Allergy Clin Immunol 2005;116:1036-1041

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy . OBJECTIVE: To investigate allergenic differences between short and giant ragweed . METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species . RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed . CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.

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[7] - Asero R. Analysis of new respiratory allergies in patients monosensitized to airborne allergens in the area North of Milan. J Investig Allergol Clin Immunol 2004;14:208-213

"BACKGROUND: A recent study carried out in North of Milan, an area that was recently colonized both by birch and ragweed pollen, showed that subjects developing allergies to these ""new"" allergens were much older than those becoming allergic to ""traditional"" airborne allergens, which suggests that predisposition to develop respiratory allergies is probably allergen-specific, persists throughout life, and does not occur until the patient encounters the appropriate allergen for a sufficiently long period . OBJECTIVE: The present study aimed to test this hypothesis by following-up a large cohort of patients monosensitized to airborne allergens . METHODS: The prevalence and characteristics of new sensitizations to airborne allergens after >2 years of follow-up were retrospectively assessed in 726 patients monosensitized to grass, pellitory, mite, birch, ragweed, Alternaria, mugwort, or plantain living North of Milan . RESULTS: The overall prevalence of new sensitizations was 35% (256/726), with marked differences between the subgroups ranging from 11% in Alternaria-allergic subjects to 46% in grass-allergic subjects. Patients allergic to grass, birch, or pellitory pollen showed a significantly higher propensity to become sensitized to a second airborne allergen than subjects allergic to ragweed, Alternaria, and mite. Overall, the ""new"" allergens (birch and ragweed) caused 228/256 (89%) new sensitizations detected in the whole study group. Ragweed pollen induced 90% (38/42) of new sensitizations in birch pollen allergic patients, and birch pollen induced 80% (8/10) of new sensitizations in ragweed allergic patients. No difference in age at the first visit or in duration of the follow-up between patients developing and not developing new respiratory allergies was observed . CONCLUSION: This study shows that: 1) predisposition to develop respiratory allergies is allergen-specific and persists throughout life; 2) proneness to become allergic to certain airborne allergens might be associated with primary sensitization to specific airborne allergens; 3) in monosensitized adults, sensitization to another airborne allergen that has been always present in that particular geographical area is unlikely."

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[8] - Asero R. Injection Immunotherapy with Different Airborne Allergens Did Not Prevent de novo Sensitization to Ragweed and Birch Pollen North of Milan. Int Arch Allergy Immunol 2004;133:49-54

BACKGROUND: Previous studies suggest that allergen-specific immunotherapy (SIT) with a single allergen may prevent sensitization to other airborne allergens. OBJECTIVE: To assess the effectiveness of injection SIT in preventing sensitization to birch and ragweed pollen north of Milan, a geographical area that was recently colonized by these two airborne allergens. METHODS: 691 adult patients monosensitized to grass (n = 342), pellitory (n = 76), ragweed (n = 66), birch (n = 112), and house dust mite (n = 95) were studied. 284 (41%) of them received injection SIT as part of routine outpatient care; the remaining 407 (59%) not submitted to SIT, served as controls. All patients underwent follow-up visits after no less than 2 years in order to detect possible new sensitization to birch and/or ragweed. RESULTS: 227 (33%) patients became sensitized to birch and/or ragweed pollen during the follow-up period. Surprisingly, the prevalence of new sensitizations was significantly higher among subjects receiving SIT (132/284; 46%) than among those not receiving SIT (95/407; 23%; p < 0.001). 27 subjects developed new sensitization to birch and/or ragweed pollen while undergoing SIT. These findings were consistent in all subgroups with different airborne allergies. CONCLUSION: In this study, injection SIT did not exert any preventive effect against denovosensitization to the two novel airborne allergens, birch and ragweed pollen, in adult monosensitized patients.

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[9] - Asero R. Birch and ragweed pollinosis north of Milan: a model to investigate the effects of exposure to ‘‘new’’ airborne allergens. Allergy 2002;57:1063-1066

"BACKGROUND: The effects of sudden and massive exposure of the general population to novel airborne allergens are not known. This study aimed to investigate the clinical effects of two ""new"" allergens, ragweed and birch, in an area north of Milan during the last 15 years . METHODS: We reviewed the records of 2571 monosensitized patients seen during the last 10 years in two allergy units north of Milan. Data included age at onset of allergic symptoms, and family history of allergic diseases. In this sample, 500 were allergic to grass, mite, birch, and ragweed; 293 to pellitory; 167 to mugwort; 100 to Alternaria; and 11 to plantain . RESULTS: Birch pollen-allergic patients and ragweed pollen-allergic patients showed a similar mean age at onset (35.3 years vs. 35.1 years; P = NS), but were significantly older than all other groups of patients (P < 0.001). Patients allergic to ragweed and birch pollen, 304 and 323 respectively, were >30 years at the onset of allergic symptoms. A family history of allergic disorders among first degree relatives was far less frequent among patients allergic to birch pollen (29%) or ragweed pollen (27%) than among patients sensitive to all other airborne allergens, except those allergic to mugwort pollen (P < 0.001). In both ragweed and birch groups, a positive family history was significantly more common among subjects < 30 years than in those > 30 years at onset of respiratory allergy (81/196 (41%) vs. 54/304 (18%), P < 0.001 for the ragweed group; 80/177 [45%]vs. 65/323 (20%), P < 0.001 for the birch group) . CONCLUSION: Exposure of the general population of this area to two new airborne allergens resulted in the onset of respiratory allergy in many older people who lacked any relevant predisposing factor. Although we cannot exclude the possibility that those who became allergic had been exposed to birch or ragweed pollen elsewhere, a more likely explanation is a specific susceptibility that remains viable until the subject encounters the ""right"" allergen."

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[10] - Asero R. The changing pattern of ragweed allergy in the area of Milan, Italy. Allergy 2007;62:1097-1099

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[11] - Rybnicek O, Jäger S. Ambrosia (ragweed) in Europe. Allergy Clin Immunol Int 2001;13:60-66

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[12] - Cecchi L, Morabito M, Domeneghetti MP, Crisci A, Onorari M, Orlandini S. Long distance transport of ragweed pollen as a potential cause of allergy in central Italy. Ann Allergy Asthma Immunol 2006;96:86-91

BACKGROUND: Ambrosia pollen is an important allergen in North America and, as recently discovered, in some European countries. In Italy, the most affected area is the northeast, whereas ragweed has not been reported in the central and southern parts of the country. OBJECTIVE: To identify the source of ragweed pollen detected in Florence and Pistoia in central Italy. METHODS: Ragweed pollen data were collected in Florence and Pistoia for a 6-year period (1999-2004). The relationship between pollen counts and local ground prevalent wind directions was evaluated with analysis of variance and the least significant difference test. Weather conditions were also evaluated on a large-scale circulation pattern by analyzing weather maps and air mass back trajectories. RESULTS: A highly statistically significant relationship between daily prevailing wind direction and pollen count was found in the period under investigation; the ragweed pollen peaks were recorded when winds from northeast in Florence and north-northeast in Pistoia were observed. The synoptic weather situation and the path of back trajectories suggest an area around southern Hungary as a possible source of Ambrosia pollen. Furthermore, the pollen count was above the clinical threshold several times in both Florence and Pistoia. CONCLUSIONS: Several factors indicate that the detection of ragweed pollen in central Italy is due to long distance transport. Taking into consideration the high allergenicity of Ambrosia pollen, the present findings, if confirmed, suggest that the number of sensitized individuals might significantly increase in the near future.

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[13] - Mohapatra SS, Lockey RF, Polo F. Weed pollen allergens. In Lockey RF. and Ledford DK, (eds). Allergens and Allergen Immunotherapy for Allergic Diseases. 4th ed. 2008, New York: Informa Healthcare USA, Inc., p. 127-39

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[14] - Mole LE, Goodfriend L, Lapkoff CB, Kehoe JM, Capra JD. The amino acid sequence of ragweed pollen allergen Ra5. Biochemistry 1975;14:1216-1220

The complete amino acid sequence of Ra5, a ragweed pollen allergen, has been determined. Allergen Ra5 is a low molecular weight protein of 45 residues derived from Ambrosia elatior, the short ragweed. It contains no detectable carbohydrate or lipid and has four disulfide bridges. The total structure was determined on 1.4 mumol of material and indicates that structural analysis is increasingly possible on relatively small amounts of highly purified material when a combination of automated and manual sequencing techniques and highly sensitive detection systems is employed. This represents the first complete amino acid sequence of a ragweed allergen and it should provide a basis for many structure- function correlative experiments in the field of immediate hypersensitivity

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[15] - Klapper DG, Goodfriend L, Capra JD. Amino acid sequence of ragweed allergen Ra3. Biochemistry 1980;19:5729-5734

The complete amino acid sequence of ragweed pollen allergen Ra3 has been determined. The molecule consists of 101 amino acid residues and to date is the only allergen isolated from Ambrosia elatior (short ragweed) which contains carbohydrate. This particular preparation of allergen has a single, unique amino acid sequence, but there is evidence suggesting that, like Ra5, Ra3 isolated from pollen collected in diverse geographical areas shows amino acid sequence variation. The complete amino acid sequence was derived by utilizing only 10--12 mg of material (approximately 1 microM) as a result of recent technical innovations such as DEAE--glass ion exchangers and Polybrene as a useful sequencing aid. This is the second pollen allergen active in man which has been sequenced, and information resulting from these data should be useful in dissecting the molecular mechanisms involved in atopic allergy

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[16] - Rafnar T, Griffith IJ, Kuo MC, Bond JF, Rogers BL, Klapper DG. Cloning of Amb a I (antigen E), the major allergen family of short ragweed pollen. J Biol Chem 1991;266:1229-1236

To determine the structure of Amb a I (previously called antigen E), the major allergen from short ragweed, cDNA from pollen was cloned into lambda gt11 and lambda gt10. One of the three distinct clones isolated from the lambda gt11 library by screening with anti-denatured Amb a I antibodies was used to screen both libraries for other Amb a I sequences. Multiple clones were isolated and sequenced and proved to be highly homologous but nonidentical. The clones could be divided into three groups based on sequence similarity, and in accordance with the International Union of Immunological Societies-approved nomenclature (Marsh, D. G., Goodfriend, L., King, T. P., Lowenstein, H., and Platts-Mills, T. A. E. (1986) Bull. WHO 64, 767-770) they have been designated Amb a I.1, Amb a I.2, and Amb a I.3. Clones within a group have greater than 99% identity, and similarity among groups is 85-90% at the nucleotide level. The amino acid sequence of four peptides (isolated from antigen E obtained from the Research Resources Branch of the National Institutes of Health) containing 132 amino acids was identical to one of the clones (Amb a I.1). The presence of multiple naturally occurring isoelectric forms of Amb a I was demonstrated by two-dimensional gel electrophoresis and Western blotting. Southern blot analysis demonstrates the presence of multiple Amb a I-related sequences in the ragweed genome. Amb a I is therefore not a single molecule but rather a family of closely related proteins.

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[17] - 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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[18] - Wopfner N, Bauer R, Thalhamer J, Ferreira F, Chapman M. Natural and Recombinant Amb a 1: Immunologic Analysis of IgE and Monoclonal Antibody Epitopes. J Allergy Clin Immunol 2007;119(1 suppl):S106

RATIONALE: Amb a 1, the major allergen from ragweed pollen is considered to be one of the most important allergens since more than 90% of ragweed-allergic patients react with this protein. Nevertheless neither an assay for detection of Amb a 1 nor recombinant produced Amb a 1 are available. To investigate IgE antibody response to the major allergen from ragweed monoclonal antibodies were produced against natural and recombinant Amb a 1 and a chimeric ELISA was established to compare human IgE binding of the natural and recombinant protein. METHODS: Natural and recombinant Amb a 1 were detected by immunoblotting with serum IgE and monoclonal antibodies and additionally using a two site ELISA assay. Using chimeric ELISA specific IgE antibodies to the natural and recombinant protein were quantified analyzing a panel of 89 ragweed allergic patients from four countries. RESULTS: There was a significant correlation between IgE antibodies to natural and recombinant Amb a 1 in all groups of patients but a subset of patients showed a up to ten fold greater reactivity to natural Amb a 1 than to recombinant Amb a 1. CONCLUSIONS: The chimeric ELISA allowed accurate quantification of IgE antibodies to natural and recombinant Amb a 1 and demonstrated the limitations of the E. coli expressed recombinant protein.

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[19] - Wopfner N, Bauer R, Thalhamer J, Ferreira F, Chapman M. Immunologic analysis of monoclonal and immunoglobulin E antibody epitopes on natural and recombinant Amb a 1. Clin Exp Allergy 2008;38:219-226

BACKGROUND: Amb a 1 is the major allergen from ragweed pollen and more than 90% of ragweed-allergic patients react with this protein. Although Amb a 1 was cloned and sequenced in 1991, little is known of the specificity of anti-Amb a 1 antibodies or of the immunologic properties of the recombinant allergen . OBJECTIVE: To compare binding of monoclonal antibodies (mAb) and IgE antibodies to purified natural Amb a 1 (nAmb a 1) and recombinant Amb a 1 (rAmb a 1) . METHODS: Binding of a panel of anti-Amb a 1 mAb and IgE antibodies to nAmb a 1 or rAmb a 1 was compared by immunoblotting. Chimeric ELISA was used to measure specific IgE to these allergens using 89 ragweed-allergic sera from Austria, Italy, Canada and the United States . RESULTS: The 8 mAb bound to a 38 kDa Amb a 1 band in ragweed pollen extract and a subset of 5 mAb also bound to the 26 kDa chain of nAmb a 1. A two-site ELISA was developed using a mAb pair, which was approximately 10-fold more sensitive to rAmb a 1. There was a significant correlation between IgE antibody binding to nAmb a 1 and rAmb a 1 (n=89, r=0.79, P<0.001). A subset of approximately 40% of patients showed greater reactivity to nAmb a 1 than to rAmb a 1 . CONCLUSIONS: The data suggest that there is less reactivity of human IgE to rAmb a 1 compared with nAmb a 1. The development of more sensitive, quantitative ELISA for Amb a 1 will require the production of new mAb especially directed against nAmb a 1.

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[20] - Wopfner N, Jahn-Schmid B, Schmidt G, Christ T, Hubinger G, Briza P et al. The alpha and beta subchain of Amb a 1, the major ragweed-pollen allergen show divergent reactivity at the IgE and T-cell level. Mol Immunol 2009;46:2090-2097

Ragweed is one of the most important pollen allergens in North America and parts of Europe. Although the major allergen Amb a 1 was isolated and cloned in 1991, recombinant Amb a 1 was not explored further to improve diagnosis and specific immunotherapy of ragweed-pollen allergy. In the present study the immunological properties of natural Amb a 1 and its proteolytical cleavage products was investigated in detail and compared with recombinant produced Amb a 1 variants. Characterization of natural Amb a 1 and the identification of its proteolytic fragments, designated Amb a 1alpha and Amb a 1beta, was performed by N-terminal sequencing and mass spectroscopy. Amb a 1 and fragments were further produced in Escherichia coli, purified, and immunologically characterized. Amb a 1-specific T-cell cultures were used to compare the T-cell response to the different Amb a 1 variants. Divergent immunological properties of Amb a 1alpha (aa 181-396) and Amb a 1beta (aa 26-180) were revealed. Amb a 1beta contained important IgE epitopes, whereas Amb a 1alpha showed low IgE binding. When compared to natural Amb a 1, all recombinant variants possessed >100-fold reduced IgE-mediated mediator release activity. At the T-cell level recombinant and natural Amb a 1 stimulated comparable T-cell responses and the T-cell reactivity was largely directed to the C-terminal part. The results demonstrated that recombinant Amb a 1alpha behaves as hypoallergen with reduced IgE binding but preservation of the major T-cell reactivity. In addition, recombinant Amb a 1alpha can be easily purified to homogeneity in large quantity and therefore represents an ideal candidate for specific immunotherapy.

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[21] - 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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[22] - 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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[23] - Roebber M, Klapper DG, Goodfriend L, Bias WB, Hsu SH, Marsh DG. Immunochemical and genetic studies of Amb.t. V (Ra5G), an Ra5 homologue from giant ragweed pollen. J Immunol 1985;134:3062-3069

Giant ragweed pollen allergen Amb.t. V (Ra5G), a homologue of short ragweed pollen Amb.a. V (Ra5S), was isolated in ultrapure form from a 16-min extract of ragweed pollen by a combination of molecular sieving through an Amicon hollow fiber cartridge (H1P5), cation-exchange chromatography, and gel filtration. The size was found to be 4400 daltons (D) by amino acid analysis and 6000 D by SDS-PAGE, and the pI was 8.3 as determined by isoelectric focusing. There was no cross-reactivity detected between the two Amb. V antigens by immunodiffusion and IEP with the use of hyperimmune antisera raised against crude or highly purified antigens. Cross-reactivity between the two Amb. V antigens was further investigated by inhibition double antibody radioimmunoassay by using the sera of nine selected ragweed-allergic patients who had recently been immunized with either mixed short-giant ragweed pollen extract or with short ragweed extract alone and who had IgG antibodies (Ab) to Amb.t. V and generally to Amb.a. V. Unlabeled Amb.t. V did not inhibit the binding of 125I-Amb.a. V to the IgG Ab in any of the sera tested. Conversely, unlabeled Amb.a. V produced some inhibition of the binding of 125I-Amb.t. V to the patients' IgG Ab, primarily in those patients who had received immunotherapy with short ragweed alone. This weak cross-reactivity was probably a result of the primary structural homology between the two protein allergens. The sera from two groups of ragweed-allergic individuals were investigated for the presence of IgG and IgE Ab to Amb.t. V. The presence of IgG Ab was found to be associated both with previous (or current) immunotherapy with giant ragweed extract and with HLA-Dw2. The HLA association is of interest in view of the previously established association between Dw2 and response toward the homologue Amb.a. V. The result suggests the existence of a similar genetic control at the primary level of antigenic recognition of the two Amb. V antigens.

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[24] - Ghosh B, Rafnar T, Perry MP, Bassolino-Klimas D, Metzler WJ, Klapper DG, et al. Immunologic and molecular characterization of Amb p V allergens from Ambrosia psilostachya (western Ragweed) pollen. J Immunol 1994;152:2882-2889

We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.

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[25] - 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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[26] - 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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[27] - 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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[28] - 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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[29] - 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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[30] - Wopfner N, van Ree R, Ebner C, Mari A, Ferreira F. Weed pollen profilins. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°292

Ambrosia (ragweed) and Artemisia (mugwort) are one of the major sources of atopic allergens worldwide. Mugwort is widely spread in central Europe and parts of Asia and is one of the main causes of hay fever in late summer and autumn. Approximately 10% of patients suffering from pollinosis are sensitized by mugwort pollen. Ragweed pollen represents the major source of allergenic proteins in the United States, with a prevalence of about 50% in atopic individuals. Profilins are small actin-binding proteins with molecular masses of approximately 14 kDa involved in regulation of the actin-polymerization in cells. Profilins are present in almost all eukaryotic cells and because of their ubiquitous occurrence they were termed cross-reactive plant pan-allergens. Here we report the isolation and characterization of cDNA clones coding for two profilin isoforms from mugwort and ragweed pollen. Recombinant ragweed pollen profilin was expressed in E. coli and isolated from the soluble fraction of the bacterial lysate as a highly pure 6xhis tagged fusion protein via affinity chromatography. Mugwort pollen profilin was also produced in E.coli but as non-fusion protein and purified with affinity chromatography using a poly-(L-proline)-CNBr-activated Sepharose (PLP- Sepharose) column. Approximately 15-26% of the ragweed pollen-allergic and about 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. Mugwort and ragweed profilins were purified to homogeneity and structural analysis indicated that the proteins exist in solution as dimers and tetramers stabilized by sulphydryl 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.

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[31] - Tao AL, He SH. Bridging PCR and partially overlapping primers for novel allergen gene cloning and expression insert decoration. World J Gastroenterol 2004;10:2103-2108

AIM: To obtain the entire gene open reading frame (ORF) and to construct the expression vectors for recombinant allergen production . METHODS: Gene fragments corresponding to the gene specific region and the cDNA ends of pollen allergens of short ragweed (Rg, Ambrosia artemisiifolia L.) were obtained by pan-degenerate primer-based PCR and rapid amplification of the cDNA ends (RACE), and the products were mixed to serve as the bridging PCR (BPCR) template. The full-length gene was then obtained. Partially overlapping primer-based PCR (POP-PCR) method was developed to overcome the other problem, i.e., the non-specific amplification of the ORF with routine long primers for expression insert decoration. Northern blot was conducted to confirm pollen sources of the gene. The full-length coding region was evaluated for its gene function by homologue search in GenBank database and Western blotting of the recombinant protein Amb a 8(D106) expressed in Escherichia coli pET-44 system . RESULTS: The full-length cDNA sequence of Amb a 8(D106) was obtained by using the above procedure and deduced to encode a 131 amino acid polypeptide. Multiple sequence alignment exhibited the gene D106 sharing a homology as high as 54-89% and 79-89% to profilin from pollen and food sources, respectively. The expression vector of the allergen gene D106 was successfully constructed by employing the combined method of BPCR and POP-PCR. Recombinant allergen rAmb a 8(D106) was then successfully generated. The allergenicity was hallmarked by immunoblotting with the allergic serum samples and its RNA source was confirmed by Northern blot . CONCLUSION: The combined procedure of POP-PCR and BPCR is a powerful method for full-length allergen gene retrieval and expression insert decoration, which would be useful for recombinant allergen production and subsequent diagnosis and immunotherapy of pollen and food allergy.

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[32] - Wopfner N, Gruber P, van Ree R, Mari A, Ferreira F. A Novel Two EF-Hand Ca2+-Binding Allergen From Ragweed Pollen. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°1100

Ragweed pollen represents the major source of allergenic proteins in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is not a major cause of pollinosis, but it is rapidly increasing. Here we reported the isolation of cDNAs coding for a pollen-specific two EF-hand calcium binding allergen from ragweed pollen showing homology to the calcium binding allergens from Bet v 4, Phl p 7, and other polcalcins. Calcium binding proteins (CBP) contain a variable number of EF-hand sequence motifs, which consist of an a-helix, a loop around the calcium ion and a second a-helix. Furthermore, CBPs were also described as relevant cross-reactive pollen allergens. Twenty-two cDNA clones (coding for nine isoforms) have been identified by IgE immunoscreening as CBPs containing two EF-hand domains. Two of nine isoforms were expressed in E.coli as soluble his tagged fusion proteins and purified to homogeneity. Immunoblots using rabbit anti-Phl p 7 antiserum demonstrated no cross-reactivity of the Phl p 7 antiserum with the Bet v 4 homologues in ragweed, although the sequence homology between these proteins was well above 50%. Testing various calcium-binding proteins with a large panel of pollen allergic patients primary sensitized to grass and tree pollen we found that most of the patients recognized Phl p 7 and Bet v 4. Only a small percentage displayed IgE antibodies reacting with ragweed CBPs. The observed limited cross-reactivity is probably explained by the fact that sequence homology among CBPs is extremely variable and mostly confined to the EF-hand domains.

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[33] - 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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[34] - 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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[35] - 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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[36] - Gunawan H, Takai T, Ikeda S, Okumura K, Ogawa H. Protease Activity of Allergenic Pollen of Cedar, Cypress, Juniper, Birch, and Ragweed. Allergol Int 2008;57:83-91

Background: Pollen is an important trigger of allergic rhinitis, conjunctivitis, and/or asthma, and an exacerbating factor in atopic dermatitis. Although it is proposed that protease activity from allergen sources, such as mites, enhances allergenicity, little information is available on that from relevant allergenic pollens such as Japanese cedar and Japanese cypress pollens, which are the major cause of pollinosis in Japan. Methods: We analyzed the protease activities derived from allergenic pollen of Japanese cedar, Japanese cypress, and Rocky mountain juniper, which belong to the Cupressaceae/Taxodiaceae family, and white birch and short ragweed, using synthetic substrates and class-specific inhibitors. Results: We found that the pollen of the three members of the Cupressaceae/Taxodiaceae family contained serine protease activity, that the pollen of white birch and short ragweed contained not only serine protease activity but also cysteine protease activity, that all five types of pollen tested contained at least one other type of serine protease, whose sensitivity to a serine protease-specific inhibitor was relatively low, and that the content and releasability of the pollen-derived proteases differed according to the plant families. Conclusions: Clinically relevant allergenic pollens tested in the present study can release serine and/or cysteine endopeptidases. Information on the spectrum of the endopeptidase activities from these allergenic pollen grains will be useful for investigating their contribution to the pathogenesis of allergies.

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[37] - Perrick D, Stafford CT, Armstrong E, DuRant RH. Modification of the fluorescent allergosorbent test as an inhibition assay for determination of cross-reactivity among aeroallergens. J Allergy Clin Immunol 1991;87:98-103

The fluorescent allergosorbent test was adapted as an inhibition assay to determine cross-reactivity between aeroallergens. With this method, similar antigenic determinants were found between short ragweed and giant ragweed, cocklebur, lamb's-quarter, rough pigweed, marsh elder, and goldenrod. Cocklebur and giant ragweed were highly potent in their ability to competitively bind to short ragweed IgE. The other pollens demonstrated lower potency of cross-reacting antigens. The fluorescent allergosorbent test-inhibition assay appears to be a useful method to determine cross-reactivity among aeroallergens.

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[38] - Leiferman KM, Gleich GJ, Jones RT. The cross-reactivity of IgE antibodies with pollen allergens. II. Analyses of various species of ragweed and other fall weed pollens. J Allergy Clin Immunol 1976;58:140-148

Although the antigenic composition of short ragweed pollen has been extensively investigated, very few studies have analyzed the allergenic cross-reactivity among various types of ragweed pollens. Using a serum pool from patients sensitive to short ragweed, we studied the cross-reactivity of IgE antibodies to six ragweeds by the radioallergosorbent test. Extracts were analyzed for their inhibitory activities with solid-phase allergens prepared from all of the ragweed pollens. Also, samples of serum were absorbed with the various solid-phase allergens and the reactivity of the remaining IgE antibodies was determined. Two patterns of reactivity were observed. Short, giant, western, and false ragweeds displayed comparable reactivity in both inhibition and absorption experiments. Slender and southern ragweed were considerably less active, indicating that they lacked allergenic groupings possessed by the other species. These same patterns of cross-reactivity were found using ragweed pollens from four commercial sources. Knowledge of these patterns of cross-allergenicity is of importance for diagnosis and treatment of sensitive patients as well as for in vitro standardization of extracts.

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[39] - Asero R, Weber B, Mistrello G, Amato S, Madonini E, Cromwell O. Giant ragweed specific immunotherapy is not effective in a proportion of patients sensitized to short ragweed: Analysis of the allergenic differences between short and giant ragweed. J Allergy Clin Immunol 2005;116:1036-1041

BACKGROUND: Short ragweed and giant ragweed pollen allergens are considered largely cross-reactive, and it is generally believed that 1 species is sufficient for skin testing and immunotherapy. However, in the area north of Milan (a zone widely invaded only by short ragweed), about 50% of patients submitted to injection specific immunotherapy with giant ragweed showed little or no clinical response, but showed an excellent outcome if they were shifted to short ragweed specific immunotherapy . OBJECTIVE: To investigate allergenic differences between short and giant ragweed . METHODS: IgE reactivity to short ragweed of sera from 16 patients allergic to ragweed was assessed by immunoblot before and after absorption with short and giant ragweed. Moreover, 41 ragweed-monosensitive patients underwent skin prick test with both ragweed species . RESULTS: In several cases, preabsorption of sera with giant ragweed extract was unable to inhibit IgE reactivity fully against both a 43-kd allergen and other allergens at different molecular weights in short ragweed. On skin prick test, short ragweed induced larger wheals than giant ragweed in the majority of patients, and 6 of 41 (15%) patients were strongly short ragweed-positive but giant ragweed-negative. The immunoblot with the serum from 1 of these subjects showed a strong IgE reactivity to short ragweed at about 43 kd in the absence of any reactivity to giant ragweed . CONCLUSION: Short and giant ragweed are not allergenically equivalent. Allergenic differences involve both the major allergens Amb a 1-2/Amb t 1-2 and some minor allergens. In patients allergic to ragweed, both diagnosis in vivo and immunotherapy should always be performed by using the ragweed species present in that specific geographic area.

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[40] - Leiferman KM, Gleich GJ, Jones RT. The cross-reactivity of IgE antibodies with pollen allergens. II. Analyses of various species of ragweed and other fall weed pollens. J Allergy Clin Immunol 1976;58:140-148

Although the antigenic composition of short ragweed pollen has been extensively investigated, very few studies have analyzed the allergenic cross-reactivity among various types of ragweed pollens. Using a serum pool from patients sensitive to short ragweed, we studied the cross-reactivity of IgE antibodies to six ragweeds by the radioallergosorbent test. Extracts were analyzed for their inhibitory activities with solid-phase allergens prepared from all of the ragweed pollens. Also, samples of serum were absorbed with the various solid-phase allergens and the reactivity of the remaining IgE antibodies was determined. Two patterns of reactivity were observed. Short, giant, western, and false ragweeds displayed comparable reactivity in both inhibition and absorption experiments. Slender and southern ragweed were considerably less active, indicating that they lacked allergenic groupings possessed by the other species. These same patterns of cross-reactivity were found using ragweed pollens from four commercial sources. Knowledge of these patterns of cross-allergenicity is of importance for diagnosis and treatment of sensitive patients as well as for in vitro standardization of extracts.

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[41] - 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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[42] - Stokes J, Kessler R, Philip G, Casale TB. Ragweed Skin Test Responsiveness Correlates with Specific Immunoglobulin E Levels. Allergy Asthma Proc 2005;26:103-107

Evaluation for allergic rhinitis requires an objective measure of atopy. Serum eosinophils, total and specific immunoglobulin E (IgE), and skin testing have been used as this measure. The objective of our study was to examine the relationship between in vitro allergy tests and in vivo responsiveness. We compared eosinophil counts, total IgE, and the specific IgE radioallergosorbent test (RAST) measurements to end point skin test titrations with ragweed. Forty subjects >18 years of age with at least a 2-year history of moderate to severe ragweed-allergic rhinitis and a positive skin-prick test to ragweed participated in this study with 33 subjects having data for all measurements. End point skin tests were performed by intradermal injection of 0.03 mL of threefold dilutions of standardized short ragweed extract into the forearm. Ragweed-specific IgE was significantly correlated to end point wheal and erythema concentrations. The results were similar whether the end point wheal (r = -0.714; p < 0.001) or erythema (r = -0.862; p < 0.001) concentration was used, and the correlation between these two values was significant (r = 0.97; p < 0.001). However, 8 of 33 subjects had a negative specific IgE RAST value for ragweed. There was a significant relationship between ragweed-specific IgE and total IgE (r = 0.72; p < 0.01). No significant correlations were found between blood eosinophils and either total IgE, ragweed-specific IgE, and end point wheal or erythema concentrations. Skin test responsiveness to ragweed correlated with in vitro ragweed-specific IgE levels, but these tests are not equivalent indicators of the degree of IgE-mediated sensitivity.

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[43] - Sosnovikova L, Lobanov K. Relation of clinical severity of allergy and specific serum IgE. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1292

Purpose: The purpose of this study was to evaluate ragweed-specific IgE in adults with rhinoconjunctivitis and/or asthma due to the sensitization of Ambrosia artemisiafolia pollen and the association between IgE values and the severity of allergy and/or asthma. Methods: Specific serum IgE antibodies to ragweed pollen were measured in 83 adults with positive skin prick test to this allergen before and during peak pollen season using Pharmacia CAP System. Relative clinical symptoms of rhiniconjunctivitis and/or asthma were assessed using 0-3 scale and lung function tests were monitored every 2 weeks. Results: It has been shown that there were no significant differences in ragweed-specific IgE concentrations between patients with isolated rhinoconjunctivitis and its concomitance with asthma as well as in patients with pollen asthma of different severity. All patients demonstrated significant increase in specific serum IgE during peak pollen season but also this increase did not correlate with the severity of allergy and asthma symptoms. We failed to reveal that the initial specific IgE values before pollen season may be a useful predictor of asthma onset. Conclusion: We conclude that the severity of symptoms in seasonal allergic airway diseases was not associated with the level of serum specific IgE and may depend on other factors.

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[44] - 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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[45] - Wopfner N, Bauer R, Thalhamer J, Ferreira F, Chapman M. Immunologic analysis of monoclonal and immunoglobulin E antibody epitopes on natural and recombinant Amb a 1. Clin Exp Allergy 2008;38:219-226

BACKGROUND: Amb a 1 is the major allergen from ragweed pollen and more than 90% of ragweed-allergic patients react with this protein. Although Amb a 1 was cloned and sequenced in 1991, little is known of the specificity of anti-Amb a 1 antibodies or of the immunologic properties of the recombinant allergen . OBJECTIVE: To compare binding of monoclonal antibodies (mAb) and IgE antibodies to purified natural Amb a 1 (nAmb a 1) and recombinant Amb a 1 (rAmb a 1) . METHODS: Binding of a panel of anti-Amb a 1 mAb and IgE antibodies to nAmb a 1 or rAmb a 1 was compared by immunoblotting. Chimeric ELISA was used to measure specific IgE to these allergens using 89 ragweed-allergic sera from Austria, Italy, Canada and the United States . RESULTS: The 8 mAb bound to a 38 kDa Amb a 1 band in ragweed pollen extract and a subset of 5 mAb also bound to the 26 kDa chain of nAmb a 1. A two-site ELISA was developed using a mAb pair, which was approximately 10-fold more sensitive to rAmb a 1. There was a significant correlation between IgE antibody binding to nAmb a 1 and rAmb a 1 (n=89, r=0.79, P<0.001). A subset of approximately 40% of patients showed greater reactivity to nAmb a 1 than to rAmb a 1 . CONCLUSIONS: The data suggest that there is less reactivity of human IgE to rAmb a 1 compared with nAmb a 1. The development of more sensitive, quantitative ELISA for Amb a 1 will require the production of new mAb especially directed against nAmb a 1.

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[46] - 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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[47] - 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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Les allergènes des pollens d’ambroisie

Réactivité croisée des pollens d’ambroisie

Ambroisie : diagnostic

Ambroisie et CCD

Rubriques

Dans la même rubrique

Les Amaranthacées

L’armoise

Les Astéracées (Composées)

Les plantains

Les Euphorbiacées

Les Pariétaires