Les graminées

			genre	anglais	français
BEP clade	Bambusoïdées	Bambusées		bamboos	bambous
^	Ehrhartoïdées	Oryzées	Oryza	rice	riz
Zizania	wild rice	riz sauvage
Pooïdées	Avénées	Agrostis	redtop	agrostide
Alopecurus	foxtail	vulpin
Anthoxanthum	sweet vernal	flouve
Avena	oat	avoine
Holcus	velvet	houlque
Koeleria	june	koélérie
Phalaris	canary	alpiste
Phleum	timothy	fléole
Trisetum	oatgrass	trisète
Bromées	Bromus	brome	brome
Poées	Dactylis	orchard	dactyle
Festuca	fescue	fétuque
Lolium	ryegrass	ivraie
Poa	kentucky meadow	pâturin
Triticées	Agropyron	quack	chiendent
Hordeum	barley	orge
Secale	rye	seigle
Triticum	wheat	blé
PACCAD clade	Aristidoïdées	Aristidées	Aristida	three awn
Arundinoïdées	Arunidinées	Arundo	giant reed	roseau
Cordateria	pampas	herbe pampa
Phragmites	common reed	roseau
Chloridoïdées	Cynodontées	Bouteloua	grama
Cynodon	bermuda	chiendent digité
Distichlis	salt	herbe prés salés
Eragrostis	love	éragrostide
Sporolobus	dropseed
Panicoïdées	Andropogonées	Imperata	cogon	alang alang
Saccharum	sugarcane	canne à sucre
Sorghum	johnson	sorgho
Zea	corn	maïs
Panicées	Cenchrus	english bunch
Digitaria	crab
Panicum	millet	millet
Paspalum	bahia	herbe bahia
Pennisetum	kikuyu
Stenotaphrum	buffalo

	Allergène particulier	Positivité
Groupe 1		76 à 100 %
Groupe 2		40 à 70 %
Groupe 3		37-70
Groupe 4		50-88
Groupe 5		50-90
Groupe 6	Phl p6	35-70
Groupe 7	Phl p7	3-12
	Cyn d7	35
Groupe 10		?
Groupe 11	Lol p11	65
Phl p11	32-53
Groupe 12		13-35
	Cyn d12	20-47
Groupe 13		25-75

Patient	Ratio le plus faible	avec extrait n°	Ratio le plus élevé	avec extrait n°
a	0,45	1	1,03	3
b	0,61	2	2,91	1
c	0,90	3	2,54	4
d	0,33	1	2,33	2
e	0,88	4	3,06	1
f	5,06	2	11,2	4
g	1,06	3	1,62	4
h	0,87	3	2,6	4
i	1,87	3	2,6	1
j	4,28	3	8,76	1

[1] - Sridhara S, Singh BP, Kumar L, Verma J, Gaur SN, Gangal SV. Antigenic and allergenic relationships among airborne grass pollens in India. Ann Allergy Asthma Immunol 1995;75:73-79

Pollen from grasses (Poaceae) are predominant aeroallergens throughout the world including tropical countries. Studies from USA, Europe, and Australia have shown extensive allergenic/antigenic cross reactivity among the grass pollen allergens prevalent there. No such information is available about airborne grass pollens of tropical countries. OBJECTIVE: The present study was undertaken to explore common antigenic/allergenic components, if any, of five important grass pollens of India. METHODS: Intradermal tests (ID) were performed with pollen extracts of Cenchrus, Cynodon, Imperata, Pennisetum, and Sorghum in patients with nasobronchial allergy. ELISAs were performed for estimating the allergen-specific IgE in sera of patients eliciting markedly positive ID response (2+ to 4+). To detect cross reactivity, ELISA inhibition experiments were carried out using pooled patient sera and five grasses, individually, as inhibitors with different solid phase antigens. To evaluate common antigenic components in Cenchrus, Imperata, and Pennisetum pollen extracts, rocket immunoelectrophoresis (RIE) and ELISA inhibition were conducted using rabbit antisera. RESULTS: Among 133 patients, Cynodon extract elicited markedly positive skin reactivity in most patients followed by Pennisetum, Imperata, Cenchrus, and Sorghum. A large number of patients showed markedly positive skin reactions and enhanced specific IgE levels to more than one grass pollen extract. ELISA inhibition experiments showed different degrees of cross reactivity among the grass pollens studied. Rocket immunoelectrophoresis and ELISA inhibition using rabbit antisera with homologous and heterologous pollen revealed the presence of shared antigenic components in Cenchrus, Imperata, and Pennisetum extracts. CONCLUSION: The varied dose-response curves obtained with ELISA inhibition using different inhibitors suggest the presence of both common and specific antigens/allergens in the grass pollens studied. Based on the extensive immunologic cross reactivity, among the tropical grass species, it may be possible to use mixed allergen preparations for allergy diagnosis and immunotherapy.

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[2] - Kumar L, Sridhara S, Singh BP, Gangal SV. Characterization of cogon grass (Imperata cylindrica) pollen extract and preliminary analysis of grass group 1, 4 and 5 homologues using monoclonal antibodies to Phleum pratense. Int Arch Allergy Immunol 1998;117:174-179

BACKGROUND: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. OBJECTIVES: To characterize the IgE-binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. METHODS: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme-linked immunosorbent assay (ELISA), ELISA-inhibition, thin-layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well-known group 1, 4, and 5 allergen homologues in Ic pollen extract. RESULTS: Out of 303 respiratory allergies patients skin-tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver-stained bands (pI 3.5-7.0) while SDS-PAGE resolved it into 24 Coomassie-Brilliant-Blue-stained bands (MW 100-10 kD). Immunoblotting with individual patient sera recognized 7 major IgE-binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. CONCLUSION: Ic pollen extract was characterized for the protein profile by TLIEF and SDS-PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

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[3] - Sridhara S, Kumar L, Verma J, Singh BP, Gangal SV. Studies on Cenchrus ciliaris pollen extract and cross-reactivity among tropical grasses of the family poaceae. Allergy Clin Immunol Int 2000;12:110-115

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[4] - Verma J, Singh BP, Gangal SV, Arora N, Sridhara S. Purification and partial characterization of a 67 kD cross-reactive allergen from Imperata cylindrica pollen extract. Int Arch Allergy Immunol 2000;122:251-256

Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85-16 kD. OBJECTIVES: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. METHODOLOGY: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. RESULTS: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. CONCLUSION: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants

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[5] - Sweeney M, Hosseiny S, Hunter S, Klotz SD, Gennaro RN, White RS. Immunodetection and comparison of melaleuca, bottlebrush, and bahia pollens. Int Arch Allergy Immunol 1994;105:289-296

Pollen extracts of two trees, Callistemon citrinis (bottlebrush) and Melaleuca leucadendron (melaleuca), as well as the grass Paspalum notatum (bahia) were analyzed for antigenic and allergenic cross-reactivity using SDS-PAGE and Western blotting. SDS-PAGE analysis of all three pollen extracts revealed multiple antigenic components which were reactive with rabbit antisera made to each pollen extract. Comparison of reduced and nonreduced mobility patterns suggested the possible presence of multichain proteins linked by disulfide bonds. Clinical studies demonstrated that 81% of the patients skin test positive to at least one of the pollens were also positive to the other two. Sixty-three percent of allergic individuals studied showed a high correlation between skin test results and the number of IgE-binding components analyzed by immunoblotting. These IgE-reactive components were detected in the molecular weight range of 29-66 kD. Western blot analysis detected more IgE-binding components in bahia pollen extracts as compared with the tree pollens, consistent with other reports of grass pollens being more allergenic than those from trees and weeds. Each patient's serum had a unique IgE-binding pattern, indicating heterogeneity of immune response; however, common major determinants were detected by a large percentage of the allergic patient's sera.

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[6] - Weber RW. Sorghum halepense. Ann Allergy Asthma Immunol 2004;93(1):A6

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[7] - Potter PC, Mather S, Lockey P, Ainslie G, Cadman A. IgE specific immune responses to an African grass (Kikuyu, Pennisetum clandestinum) . Clin Exp Allergy 1993;23:581-586

Kikuyu grass (Pennisetum clandestinum) is widely used as a grazing pasture in Africa and, although it is extensively cultivated as a lawn for sports fields and domestic gardens, its allergenicity has never been studied in vitro. Using an extract of Kikuyu grass pollen, polyacrylamide gel electrophoresis, Western blotting and a monoclonal anti-human IgE antibody, the specific IgE binding, in the serum of 160 allergic and non-allergic patients in the Cape Town area, to a Kikuyu grass extract was studied. IgE in the sera of 43/104 known grass-allergic individuals bound to Kikuyu grass on the Western blots. In addition, 4/28 'non-allergic' control subjects were found to have Kikuyu grass-specific IgE. Five different profiles of specific IgE reactivity to Kikuyu grass antigens were observed. In 29/43 patients, IgE bound to two dominant 48 and 70 kD allergens in the Kikuyu extract. Although a degree of cross-reactivity with Bermuda grass (Cynodon dactylis) was found in immunoabsorption studies, the 48 and 70 kD allergens appear to be unique to Kikuyu grass Exposure of heparinized blood from Kikuyu grass-positive patients to the Kikuyu extract stimulated the release of histamine from their basophils in vitro. Kikuyu grass pollen is thus identified as an important aero-allergen in South Africa.

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[8] - Esch RE. Grass pollen allergens. Clin Allergy Immunol 2004;18:185-205

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[9] - 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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[10] - Sopelete MC, Moreira PFS, Silva DAO, Cunha-Júnior JP, Vieira FAM, Sung SJ, et al. Sensitization to Lolium multiflorum Grass Pollen in Pollinosis Patients: Evaluation of Allergenic Fractions Recognized by Specific IgE Antibodies. Int Arch Allergy Immunol 2006;140:121-130

BACKGROUND: Lolium multiflorum (Lm) pollen allergens are the major causative agents for rhinoconjunctivitis in Southern Brazil. There have been no studies about the sensitization and allergenic cross-reactivity between Lm and other grass pollens. We evaluated the sensitization of Brazilian pollinosis patients to Lm pollen allergens through skin prick test (SPT) and immunoassays (ELISA and immunoblot) . METHODS: Serum samples from 60 patients with pollinosis and positive SPT to grass pollen extracts (Lm+ group), 30 patients with negative SPT to grass pollen, but positive SPT to mite extracts (Lm- group), and 30 nonatopic subjects (NA group) were tested by SPT, ELISA, and immunoblot using Lm extract. Inhibition immunoassays with Lolium perenne (Lp), mixed grass (Gmix) and Lm extracts were also performed . RESULTS: A high concordance was found between the Gmix and Lm extracts in SPT. Positivity rates in SPT were also highly concordant with IgE-ELISA results. The assay was able to detect Lm-specific IgE in >95% of Lm+ patients. A significant self- and cross-inhibition was observed in IgE-ELISA, reflecting a high cross-reactivity between the grass pollen allergens. Immunoblot revealed 13 IgE-binding Lm fractions, from which the bands 28-30 kDa and 31-34 kDa were recognized by >90% of Lm+ patients . CONCLUSION: Lm-specific IgE antibodies are highly cross-reactive with pollen proteins from other grass species. The results indicate that Lm extracts could be used in both SPT and ELISA for a more specific evaluation of IgE responses to Lm grass pollen in Brazilian pollinosis patients.

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[11] - Suphioglu C, Singh MB, Simpson RJ, Ward LD, Knox RB. Identification of canary grass (Phalaris aquatica) pollen allergens by immunoblotting: IgE and IgG antibody-binding studies. Allergy 1993;48:273-281

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and 9/24, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.

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[12] - Montero MT, Lopez C, Jimenez JA, Subiza J. Characterization of allergens from Trisetum paniceum pollen: an important aeroallergen in Mediterranean continental climatic areas. Clin Exp Allergy 1997;27:1442-1448

BACKGROUND: Trisetum paniceum is a grass plant which is characteristic of a Mediterranean continental climate and has been described as one of the major causes of type I allergy in the Madrid region. OBJECTIVES: To identify and characterize the allergens of Trisetum paniceum pollen. METHODS: Allergenic extracts were prepared by 24 h incubation of pollens in a buffered solution. Proteins were analysed by a new two-dimensional system in which agarose plates were used for isoelectric focusing. Two-dimensionally resolved proteins were electrically transferred to Immobilon membranes and the allergens immunochemically detected. Proteins from six grass pollens were bound to a membrane and incubated with a pool of serum from grass-pollen-sensitized patients. The bound IgE antibodies were then eluted and used to identify the proteins of Trisetum paniceum pollen that allergenically crossreact with allergens from other pollen grasses. RESULTS: Relative to total protein content, Trisetum paniceum pollen had a high proportion of reactive proteins. On the basis of their molecular characteristics, allergens could be classified as group 1, 2, 4 and 5 components yet included an atypical proportion of basic components. All identified allergens were crossreactive with allergens from the remaining grass pollens studied. CONCLUSIONS: Trisetum paniceum pollen contains a high proportion of allergens and these include a group of basic proteins which are not detected in other phylogenetically related pollens and could be of allergological interest.

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[13] - Cabrera M, Martinez-Cocera C, Fernandez-Caldas E, Carnes Sanchez J, Boluda L, Tejada J, et al. Trisetum paniceum (wild oats) pollen counts and aeroallergens in the ambient air of Madrid, Spain. Int Arch Allergy Immunol 2002;128:123-129

BACKGROUND: Madrid has a short but intensive grass pollen season, in which 79% of the total grass pollen load is released from the middle of May to the middle of June. The main objectives of this study were to quantify Trisetum paniceum (wild oats) aeroallergen in the atmosphere in Madrid from February to December 1996 and to correlate the aeroallergen concentrations with grass pollen counts. METHODS: Two different samplers were used to assess allergen exposure; a Burkard spore trap was used to collect pollen grains and a high-volume air sampler to collect airborne particles. A total of 182 air filters were collected and extracted in 1 ml of PBS and analysed by ELISA inhibition, using pooled sera from highly allergic individuals. RESULTS: T. paniceum aeroallergens were detected not only during the grass pollen season, but also before and after. Wild oat allergens had two main peaks of 1 and 1.9 microg/m(3), occurring in late May and July, respectively. The time series analysis established the existence of lags between the two main variables pollen counts and aeroallergen activity. Analysis of the data by the Spearman rank test and linear regression showed a weak correlation between grass allergenic activity and grass pollen counts (Spearman's rho = 0.29). Data obtained from time series analysis demonstrated that grass allergenic activity correlated strongly with current and 5-week-old grass pollen grain counts (r(2) = 0.73). CONCLUSIONS: Wild oats allergenic activity was detected during the entire year and not only during the pollen season. This fact is an important aspect to be considered in the clinical follow-up and treatment of grass pollen-sensitised patients in Madrid.

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[14] - Grote M, Vrtala S, Niederberger V, Wiermann R, Valenta R, Reichelt R. Release of allergen-bearing cytoplasm from hydrated pollen: a mechanism common to a variety of grass (Poaceae) species revealed by electron microscopy. J Allergy Clin Immunol 2001;108:109-115

BACKGROUND: The release of submicronic particles from grass pollen after rainfall was suggested to be responsible for outbreaks of grass pollen asthma. Recently, we provided evidence for the release of respirable allergen-bearing particles from hydrated ryegrass (Lolium perenne ) pollen as a possible explanation for this phenomenon. OBJECTIVE: We investigated whether water-induced release of respirable allergen-bearing particles could be a mechanism common to several members of the sweet grass family Poaceae (Gramineae). METHODS: Pollens from 6 different Poaceae species were hydrated in water and examined by means of scanning electron microscopy for release of cytoplasmic materials. Rabbit antisera raised against purified recombinant group 1 and 5 allergens were used for immunogold labeling of expelled materials by means of field emission scanning electron microscopy. In addition, group 1 and 5 allergens were immunogold-localized on ultrathin sections. RESULTS: Fresh Poaceae pollens expelled cytoplasmic materials containing group 1 and 5 allergens on hydration in water. Expulsion of submicronic particles strongly decreased after 1 month of storage. CONCLUSIONS: Our results suggest expulsion of cytoplasm after hydration as a mechanism common to pollens of important allergenic grasses. The water-induced release of respirable allergen-bearing particles from grass pollens might explain asthma attacks observed after rainfall during the grass pollen season.

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[15] - Schappi GF, Taylor PE, Pain MC, Cameron PA, Dent AW, Staff IA, et al. Concentrations of major grass group 5 allergens in pollen grains and atmospheric particles: implications for hay fever and allergic asthma sufferers sensitized to grass pollen allergens. Clin Exp Allergy 1999;29:633-641

BACKGROUND: Grass pollen allergens are the most important cause of hay fever and allergic asthma during summer in cool temperate climates. Pollen counts provide a guide to hay fever sufferers. However, grass pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet, grass pollen allergens are known to be associated with atmospheric respirable particles. OBJECTIVE: We aimed (1) to determine the concentration of group 5 major allergens in (a) pollen grains of clinically important grass species and (b) atmospheric particles (respirable and nonrespirable) and (2) to compare the atmospheric allergen load with clinical data to assess different risk factors for asthma and hay fever. METHODS: We have performed a continuous 24 h sampling of atmospheric particles greater and lower than 7.2 microm in diameter during the grass pollen season of 1996 and 1997 (17 October 1996-16 January 1997) by means of a high volume cascade impactor at a height of about 15 m above ground in Melbourne. Using Western analysis, we assessed the reactivity of major timothy grass allergen Phl p 5 specific monoclonal antibody (MoAb) against selected pollen extracts. A MoAb-based ELISA was then employed to quantify Phl p 5 and cross-reactive allergens in pollen extracts and atmospheric particles larger and smaller than 7.2 microm. RESULTS: Phl p 5-specific MoAb detected group 5 allergens in tested grass pollen extracts, indicating that the ELISA employed here determines total group 5 allergen concentrations. On average, 0.05 ng of group 5 allergens were detectable per grass pollen grain. Atmospheric group 5 allergen concentrations in particles > 7.2 microm were significantly correlated with grass pollen counts (rs = 0.842, P < 0. 001). On dry days, 37% of the total group 5 allergen load, whereas upon rainfall, 57% of the total load was detected in respirable particles. After rainfall, the number of starch granule equivalents increased up to 10-fold; starch granule equivalent is defined as a hypothetical potential number of airborne starch granules based on known pollen count data. This indicates that rainfall tended to wash out large particles and contributed to an increase in respirable particles containing group 5 allergens by bursting of pollen grains. Four day running means of group 5 allergens in respirable particles and of asthma attendances (delayed by 2 days) were shown to be significantly correlated (P < 0.001). CONCLUSION: Here we present, for the first time, an estimation of the total group 5 allergen content in respirable and nonrespirable particles in the atmosphere of Melbourne. These results highlight the different environmental risk factors for hay fever and allergic asthma in patients, as on days of rainfall following high grass pollen count, the risk for asthma sufferers is far greater than on days of high pollen count with no associated rainfall. Moreover, rainfall may also contribute to the release of allergens from fungal spores and, along with the release of free allergen molecules from pollen grains, may be able to interact with other particles such as pollutants (i.e. diesel exhaust carbon particles) to trigger allergic asthma.

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[16] - Abou Chakra OR, Sutra JP, Desvaux FX, Rogerieux F, Peltre G, Lacroix G et al. Vers une identification des allergènes hydrosolubles de granules cytoplasmiques de pollen. Rev Fr Allergol 2009;49:330-331

Le pollen des graminées est l‚un des plus abondants aéroallergènes en Europe. Ce pollen est la cause principale du rhume de foin. En contact avec l‚eau ou des polluants atmosphériques, le pollen peut libérer des microparticules dites « granules cytoplamiques de pollen » (GCP). En raison de leur petite taille (< 5 mm), les GCP sont susceptibles d‚entraîner des réactions allergiques importantes. Ils sont déjà mis en cause dans des épisodes d‚asthme associées aux orages. Peu d‚études se sont intéressées aux allergènes de ces microparticules. L‚objectif de cette étude est d‚identifier des allergènes hydrosolubles pour ces GCP et de les comparer à ceux du pollen entier. Les granules, issus du pollen de Phleum pratense, sont obtenus par choc osmotique suivi d‚une filtration et d‚une centrifugation. Les protéines des GCP et du pollen sont séparées par IEF suivie d‚un SDS-PAGE. Les allergènes des GCP et du pollen sont ensuite identifiés par immunoblotting en utilisant des sérums de patients allergiques au pollen de graminées. Pour les GCP, l‚empreinte 2D montre des spots correspondant majoritairement à des protéines de masses moléculaires (MM) relativement faibles (< 40 kDa) et de points isoélectriques hétérogènes (pI _ 4,45ˆ8,2). Pour le pollen entier, on observe un spectre de protéines plus large que celui des GCP. Les allergènes Phl p1, Phl p5 et Phl p6 apparaissent dans les empreintes des GCP et du pollen entier. Par contre, les allergènes Phl p4 et Phl p13 apparaissent uniquement dans l‚empreinte du pollen. Par ailleurs, l‚empreinte de GCP présente des spots spécifiques correspondant aux couples suivants : (23,4 kDa ; 5,85), (24,1 kDa ; 7,05) et (73 kDa ; 2,5). Les GCP présentent un nombre plus restreint d‚allergènes que ceux du pollen entier. Bien que la majorité de ces allergènes soient communs aux GCP et au pollen entier, d‚autres allergènes sont associés uniquement aux GCP.

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[17] - Niederberger V, Laffer S, Fröschl R, Kraft D, Rumpold H, Kapiotis S, et al. IgE antibodies to recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) account for a high percentage of grass pollen-specific IgE. J Allergy Clin Immunol 1998;101:258-264

BACKGROUND: Pollen from different grass species are some of the most potent elicitors of Type I allergy worldwide. The characterization of antigenic structures and IgE epitopes common to different grass species is relevant to define reagents for diagnosis and specific therapy of grass pollen allergy. OBJECTIVE: The purpose of this study was to estimate the percentage of IgE directed to common, cross-reactive, or both types of epitopes shared by recombinant pollen allergens (Phl p 1, Phl p 2, Phl p 5, and Bet v 2) and natural pollen extracts from nine different monocots (Anthoxanthum odoratum, Avena sativa, Cynodon dactylon, Lolium perenne, Phragmites australis, Poa pratensis, Secale cereale, Triticum sativum, Zea mays) by using sera from different populations. METHODS: Natural pollen extracts from nine different monocot species were characterized regarding their allergen contents by using specific antibodies and by IgE immunoblot inhibition with recombinant allergens. The percentage of grass pollen-specific IgE that was preabsorbed with a combination of recombinant timothy grass pollen allergens (Phl p 1, Phl p 2, and Phl p 5) and recombinant birch profilin (Bet v 2) was determined by ELISA in sera from 193 European, American, and Asian subjects. RESULTS: IgE to recombinant pollen allergens accounted for a mean 59% of grass pollen-specific IgE. A lower inhibition of IgE binding to certain natural extracts (C. dactylon and Z. mays) could be attributed to the absence of immunologically detectable group 5 and group 2 allergens in these species. CONCLUSION: We define four recombinant pollen allergens that account for a substantial proportion of grass pollen-specific IgE. The recombinant pollen allergens characterized may represent candidates not only for diagnosis but also for patient-tailored immunotherapy of grass pollen allergy.

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[18] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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[19] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[20] - Heinzerling L, Frew AJ, Bindslev-Jensen C, Bonini S, Bousquet J, Bresciani M, et al. Standard skin prick testing and sensitization to inhalant allergens across Europe – a survey from the GA2LEN network. Allergy 2005;60:1287-1300

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[21] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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[22] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[23] - Rossi RE, Monasterolo G. Evaluation of Recombinant and Native Timothy Pollen (rPhl p 1, 2, 5, 6, 7, 11, 12 and nPhl p 4)-Specific IgG4 Antibodies Induced by Subcutaneous Immunotherapy with Timothy Pollen Extract in Allergic Patients. Int Arch Allergy Immunol 2004;135:44-53

"BACKGROUND: Allergen immunotherapy is a widely accepted treatment for IgE-mediated allergies. The evaluation of immunotherapy-induced IgG4 antibodies based on allergen extract is questionable because the amount of allergen-extract-specific IgG4 to individual disease-eliciting allergens cannot be determined using crude allergen extracts. In this study, we examined the specific IgE and IgG4 serum binding profiles to individual Phleum pratense allergens in grass-pollen-sensitive patients who had received grass-pollen-specific immunotherapy (SIT) . METHODS: The study included 33 patients from North-West Italy. All suffered from seasonal rhinoconjunctivitis and/or asthma. A modified ""cluster"" regimen of injections of a standardized aluminium-adsorbed P.pratense extract, with once-weekly visits and 10 injections for 5 weeks followed by 3 weeks of maintenance injections was instituted. Patients' sera were analyzed for specific IgE and IgG4 reactivity to individual P. pratense allergens (recombinant Phl p 1, Phl p 2, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p12 and native Phl p 4) and natural P. pratense extract using the Pharmacia CAP system . RESULTS: IgE reactivities to new allergen components were not detected by CAP in treated patients after 15 weeks and a cumulative dose of approximately 65 microg of the major allergen Phl p 5. Patients lacking specific IgE reactivity towards individual allergens at the start of SIT did not produce significant levels of specific serum IgG4 to serum IgE-negative allergens. On the other hand, an increase in specific IgG4 only to allergens to which patients were previously sensitized was observed. Significant increases in specific IgG4 levels to rPhl p 1 (p < 0.05), 2 (p < (0.01), 5 (p < 0.0001), 6 (p < 0.0001), 7 (p < 0.05), 11 (p < 0.05) and nPhl p 4 (p < 0.01) were observed after P. pratense extract immunotherapy. No significant rPhl p 12-specific IgG4 antibody increase was documented after treatment . CONCLUSION: These findings suggest that Phl p 12 was underrepresented in the extract used, as indicated by the low specific IgG4 response induced by this grass-pollen-specific vaccine. Thus, the simple detection of specific serum IgG4 antibodies a few weeks after the start of SIT could represent a valuable tool to estimate the presence of relevant allergens in a given immunotherapeutic allergen extract."

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[24] - 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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[25] - Burton MD, Papalia L, Eusebius NP, O'Hehir RE, Rolland JM. Characterization of the human T cell response to rye grass pollen allergens Lol p 1 and Lol p 5. Allergy 2002;57:1136-1144

BACKGROUND: Knowledge of dominant T cell epitopes of major allergens recognized by allergic individuals is required to improve efficacy and safety of allergen immunotherapy. Rye grass pollen (RGP) is the most important source of seasonal aeroallergens in temperate climates and Lol p 1 and Lol p 5 are the two major IgE-reactive allergens. This study aimed to characterize the T cell response to these allergens using a large panel of RGP-sensitive individuals . METHODS: Short-term RGP-specific T cell lines (TCL) were generated from 38 RGP-sensitive subjects and stimulated with Lol p 1 and/or Lol p 5 allergens and synthetic 20-mer peptides. Proliferative responses were determined by 3H-thymidine uptake and IL-5 and IFN-gamma in culture supernatants analysed by ELISA . RESULTS: Of 17 subjects tested for reactivity to both allergens 16 (94%) responded to Lol p 1 and/or Lol p 5, establishing these as major T cell-reactive allergens. Sites of T cell reactivity were spread throughout the allergen molecules but regions of high reactivity were found. For Lol p 1 these spanned residues 19-38, 109-128, 154-173, 190-209, and for Lol p 5 37-56, 100-119, 145-164, 154-173, 190-209, 217-236 and 226-245. IL-5 and IFN-gamma were produced by T cells cultured with proliferation-inducing peptides . CONCLUSIONS: T cell responses to RGP major allergens have been extensively characterized, providing fundamental information for developing T cell-targeted immunotherapy for RGP allergy.

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[26] - Corti V, Cattaneo A, Bachi A, Rossi RE, Monasterolo G, Paolucci C, et al. Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening. Proteomics 2005;5:729-736

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.

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[27] - Pires APT, Viseu R, Inacio FFC. Recombinant allergens and immunoprinting as a tool for the follow-up of IgE sensitization along the years. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°881

Introduction: Although allergic diseases risk are largely determined by genetic factors the environment essential to define the profile of the allergic patients` sensitisation. No studies have been published relating the IgE sensitisation to the molecular allergens and their evolution along the years. Aim: Our aim was to study the evolution of the IgE sensitisation to grass pollen molecular allergens in a group of allergic patients with clinical allergy to grass pollens. Material and Methods: We studied 23 grass pollen allergic patients living in the same geographic area for at least 5 years. All patients were allergic to grass pollen. Specific IgE to Dactylis glomerata (Dac g), Phleum pratensis (Phl p), rPhl p 1, rPhl p 2, rPhl p 4, rPhl p 5, rPhl p 7, rPhl p 12 and simple IgE immunopring pattern (group I) or intermediate IgE immunoprinting pattern to Dac g and Phl p. Blood samples were collected with a medium interval of 4 years. We considered that a patient has a simple IgE immunoprinting pattern when he recognised 2 allergen groups of Dac g or Phl p and intermediate IgE immunoprinting pattern when he recognised 3 allergen groups. Results: In group I (19 patients) 7 recognized the allergens of group 1 they presented a specific IgE average to Dac g of 2,8 KU/l, 3 recognized the allergens of group 4 they presented a specific IgE average to Dac g of 25,9 KU/l, 1 recognized the allergens of group 5 he presented a specific IgE to Dac g of 1,74 KU/ l, 8 recognized the allergens of group 1 and group 4 they presented a specific IgE average to Dac g of 6,4 KU/l. In group II (4 patients) all patients recognized the allergens of the groups 1, 2, 4 they presented a specific IgE average to Dac g of 15,5 KU/l. In group 1, most of the patients (14) maintained simple IgE immunoprinting pattern along the years; only 5 patients increased the IgE immunoprinting pattern recognition and increased the specific IgE to Dac g. Concerning group II we also observed no change in the recognition of molecular allergens. Conclusion: In this study we could observe that the most of the allergic patients to grass pollen allergens do not change the pattern of molecular allergen recognition along the years. This means that there are genetic constraints that do not allow the recognition of other allergens. The environment is an important factor to allergen sensitivity but the genetic background determines wich are the molecular allergens to which patients will be sensitised

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[28] - Ball T, Fuchs T, Kraft D, Valenta R. Lessons from the antibody recognition of the major Timothy grass pollen allergen Phl p 1. Int Arch Allergy Immunol 1999;118:208-209

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[29] - van Ree R, Van Leeuwen WA, Dieges PH, Van Wijk RG, De Jong N, Brewczyski PZ, et al. Measurement of IgE antibodies against purified grass pollen allergens (Lol p 1, 2, 3 and 5) during immunotherapy. Clin Exp Allergy 1997;27:68-74

BACKGROUND: IgE titres tend to rise early after the start of immunotherapy, followed by a decline to pre-immunotherapy levels or lower. OBJECTIVES: We were interested to know whether the early increase in IgE antibodies includes new specificities of IgE, and whether these responses persist. METHODS: Sera of 64 patients undergoing grass pollen immunotherapy were tested for IgE against four purified grass pollen allergens: Lol p 1, 2, 3, and 5. At least two serum samples were taken, one before the start of therapy and one between 5 and 18 months after the first immunization (mean: 10 months). RESULTS: The mean IgE responses to Lol p 1, 2 and 3 showed a moderate but not significant increase. In contrast, the mean IgE response to Lol p 5 showed a significant decrease of > 30%. IgE against total Lohum perenne pollen extract moderately increased (> 20%), showing that a RAST for total pollen is not always indicative for the development of IgE against its major allergens. For > 40% of the patients it was found that IgE against one or more of the four allergens increased, while IgE against the remaining allergen(s) decreased. For 10 sera the ratio of IgE titres against at least two allergens changed by at least a factor of 5. The changes in specific IgE also included conversions from negative (< 0.1 RU) to positive (0.6 to 5.0 RU) for five patients. For two patients, the induction of these 'new' IgE antibodies against major allergens was shown to result in a response that was persistent over several years. CONCLUSION: Although active induction of new IgE specificities by immunotherapy was not really proven, the observations in this study indicate that monitoring of IgE against purified (major) allergens is necessary to evaluate changes in specific IgE in a reliable way.

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[30] - Jutel M, Jaeger L, Suck R, Meyer H, Fiebig H, Cromwell O. Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 2005;116:608-613

BACKGROUND: Allergen-specific immunotherapy uses aqueous extracts of natural source materials as a basis for preparations to down regulate the allergic response. Recombinant DNA technology has enabled the cloning of many allergens, thus facilitating investigations aimed at improving efficacy and safety of immunotherapy . OBJECTIVE: To determine the effectiveness of a mixture of 5 recombinant grass pollen allergens in reducing symptoms and need for symptomatic medication in patients allergic to grass pollen . METHODS: A randomized, double-blind, placebo-controlled study of subcutaneous injection immunotherapy was performed in subjects with allergic rhinoconjunctivitis, with or without asthma. Primary endpoint was a symptom medication score compiled from separate symptom and medication scores. Secondary endpoints included a rhinitis quality of life questionnaire, conjunctival provocation, and specific antibody responses . RESULTS: The symptom medication score showed significant improvements in subjects receiving recombinant allergens as opposed to placebo, with reductions in both symptoms and medication usage. The rhinitis quality of life questionnaire revealed clinically relevant significant improvements in overall assessment and in 5 of 7 separate domains, and conjunctival provocation showed a clear trend in favor of active treatment. All treated subjects developed strong allergen-specific IgG(1) and IgG(4) antibody responses. Some patients were not sensitized to Ph l p 5 but nevertheless developed strong IgG antibody responses to that allergen . CONCLUSION: A recombinant allergen vaccine can be a effective and safe treatment to ameliorate symptoms of allergic rhinitis. The clinical benefit is associated with modification of the specific immune response with promotion of IgG(4) and reduction of IgE antibodies consistent with the induction of IL-10-producing regulatory T cells.

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[31] - Ball T, Fuchs T, Kraft D, Valenta R. Lessons from the antibody recognition of the major Timothy grass pollen allergen Phl p 1. Int Arch Allergy Immunol 1999;118:208-209

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[32] - Lelong M, Thibaudon M, Thelliez PH. [Is it necessary to test children having summer respiratory problems with cereal pollens?]. Allerg Immunol (Paris) 1989;21:394-395

We tested 100 children with clinical signs that occurred in spring or summer. Prick tests were made with extracts of wild grass pollens (Dactylis, Phleum, Lolium) and with separate cereal pollens (Wheat, oats, Sweet-corn Barley and Rye). In 20 children whose test was negative for wild grass pollen, we saw no sensitization to cereals. In 80 children with grass pollenosis, Barley and Rye are the cereals that gave the most positive prick tests, whilst sweet corn is the cereal with most negative tests.

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[33] - Gonzalo-Garijo MA, Pérez-Calderon R, Muñoz-Rodriguez A, Tormo-Molina R, Silva-Palacios I. Hypersensitivity to maize pollen. Allergy 2004;59:365

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[34] - Parisot L. Sensibilisation aux pollens de maïs et de colza en France. Alim'Inter 2005;10:198-199

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[35] - Petersen A, Dresselhaus T, Grobe K, Becker WM. Proteome analysis of maize pollen for allergy-relevant components. Proteomics 2006;6:6317-6325

Over the last few decades, the cultivation of maize (Zea mays) has strongly increased in Central Europe. We therefore decided to study the allergen composition and the allergenic potency of its pollen in comparison with pollen from timothy grass (Phleum pratense), a typical representative of the native grasses. We found that 65% of the sera reactive to timothy pollen also bound to maize pollen proteins. By using 2-DE immunoblotting, followed by incubation with mAbs directed against known allergens or protein sequencing, those IgE-reactive components were further classified. Although novel, maize-specific pollen allergens could not be found, the presence of crossreacting allergens belonging to groups 1 and 13 (Zea m 1 and 13), both having high IgE prevalence, as well as the presence of the less important group 3 and 12 allergens was found. The structural variability of Zea m 1 and Zea m 13 was determined by sequencing clones isolated from a maize pollen cDNA library. This revealed sequence identities of 72 and 70%, respectively, to the corresponding Phl p 1 and Phl p 13 allergens of timothy grass pollen. IgE-crossreactivity was further studied using immunoblot inhibition tests. Here, timothy pollen extract completely blocked IgE binding to maize, whereas maize pollen extract blocked IgE reactivity to only some timothy pollen allergens.

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[36] - Hrabina M, Peltre G, Van Ree R, Moingeon P. Grass pollen allergens. Clin Exp Allergy Rev 2008;8:7-11

To date, eleven groups of grass pollen allergens eliciting a specific IgE response in atopic individuals have been identified. Groups 1 and 5 allergens are the most critical (major) pollen allergens leading to the sensitization of 90% and 65ˆ85% allergic patients, respectively. Other allergens frequently involved in the IgE response include groups 2/3, 4, 6, 7, 10ˆ13 allergens. Allergens found in various Pooideae exhibit high homology in terms of their amino acid sequence composition, which translates into significant cross-reactivity in terms of antibody (IgE and IgG) as well as T cell responses. Nevertheless, for a given allergen group, there is evidence of both interspecies (i.e. differences in amino acid sequences) and intraspecies (multigenes, post-translational modification, mRNA splicing or editing) molecular variability.

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[37] - Andersson K, Lidholm J. Characteristics and Immunobiology of Grass Pollen Allergens. Int Arch Allergy Immunol 2003;130:87-107

Grass pollens are one of the most important airborne allergen sources worldwide. About 20 species from five subfamilies are considered to be the most frequent causes of grass pollen allergy, and the allergenic relationships among them closely follow their phylogenetic relationships. The allergic immune response to pollen of several grass species has been studied extensively over more than three decades. Eleven groups of allergens have been identified and described, in most cases from more than one species. The allergens range from 6 to 60 kD in apparent molecular weight and display a variety of physicochemical properties and structures. The most complete set of allergens has so far been isolated and cloned from Phleum pratense (timothy grass) pollen. Based on the prevalence of IgE antibody recognition among grass pollen-sensitized individuals, several allergens qualify as major, but members of two groups, groups 1 and 5, have been shown to dominate the immune response to grass pollen extract. Isoform variation has been detected in members of several of the allergen groups, which in some cases can be linked to observed genetic differences. N-linked glycosylation occurs in members of at least three groups. Carbohydrate- reactive IgE antibodies have been attributed to grass pollen sensitization and found to cross-react with glycan structures from other allergen sources, particularly vegetable foods. Another cause of extensive cross-reactivity are the group 12 allergens (profilins), which belong to a family of proteins highly conserved throughout the plant kingdom and present in all tissues. Members of eight allergen groups have been cloned and expressed as recombinant proteins capable of specific IgE binding. This development now allows diagnostic dissection of the immune response to grass pollen with potential benefits for specific immunotherapy.

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[38] - Esch RE. Grass 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. 107-26

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[39] - Mohapatra SS, Lockey RF, Shirley S. Immunobiology of grass pollen allergens. Curr Allergy Asthma Rep 2005;5:381-387

Among pollen allergens, grass pollen allergens are some of the most frequent contributors to allergic symptoms. Substantial progress has been made since the 1960s in the identification and characterization of the grass allergens. Members of this group belong to the Poaceae family, and have been classified into 13 distinct groups based on their structure, and their biological and immunologic properties. The major contributors to allergy and, hence, most studied among the grass allergens, are those belonging to groups 1 and 5. This review is focused on the structure and immunobiology of the grass allergens and highlights how recent advances in the field have contributed to superior diagnosis and immunotherapy for allergy to grass pollens.

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[40] - Suphioglu C. What are the important allergens in grass pollen that are linked to human allergic disease ? Clin Exp Allergy 2000;30:1335-1341

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[41] - Metz-Favre C, Pauli G, Castro L, Valenta R, de Blay F. Demonstration of Bet v 2 responsibility in birch-induced symptoms. Allergy 2009;64(Suppl. 90):508

Background: Clinical relevance of profilin in respiratory symptoms has not yet been demonstrated. Bet v 1 is recognized as the major allergen in birch pollinosis in France. Case report: A 54-year-old man, born in Maurice island, had suffered from grass pollinosis for 13 years and had been desensitised with grass pollen for 4 years. He also complained of oral symptoms when eating melon, longan and avocado. Recently he complained of rhinitis in April (birch season). Skin prick tests were positive with grass, birch, ash, artemisia and plantago pollens as well as with melon, longan and avocado. Specific IgE serum measurements were performed only with recombinant allergens : they were significantly high to grass pollen molecular allergens (anti-rPhl p 1= 9,6 kU/l; anti-rPhl p 5 = 17.3 kU/l, anti-rPhl p 2= 4.3 kU/L, anti-rPhlp 12=1.6 kU/l). For birch molecular allergens, specific IgE serum levels were negative to Bet v 1, 4, and 6 but positive to Bet v 2 (1.4 kU/l). For ash pollen molecular allergens specific IgE against nOle e 1 were found at a low level of 0.21 kU/l. The nasal provocation test with birch extract (Allerbio-ALK, France) was positive in a dose dependent manner. In order to verify that grass profilin is the inducer of profilin sensitization we performed ELISA inhibition studies with the patient‚s serum. The inhibition experiment confirmed that the primary sensitizer was Phl p 12 because it was able to inhibit the IgE binding to Bet v 2 better than Bet v 2 did with Phl p 12. Conclusion: Sensitization to major allergens (Phl p 1,2, and 5) from grass pollen confirms grass pollinosis. Food allergy may be related to profilins. Sensitization to grass pollen and food profilins probably induced sensitization to birch profilin, as well as the subsequent symptoms during birch pollen season and the positive nasal challenge with birch pollen extract.

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[42] - Lafosse-Marin S, Leduc V, Aparicio C, Guérin L. Paspalum notatum (Bahia grass) pollen sensitization in Martinique (France). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1384

Background: Paspalum notatum (Bahia grass) is a grass which naturally grows in tropical and subtropical climates. It is considered as a very vigourous and aggressive grass, not easily eradicated. One aim of this work was to know if there is one relationship between Bahia grass sensitization in Martinique (French Antilles) and long stay in temperate countries (mainly in metropolitan France) during the grass pollen period. Moreover, even though various studies have demonstrated the importance of Bahia grass as an aeroallergen, it is to be considered that no allergens from Bahia grass pollen have been yet characterized. Methods: Skin prick tests were performed with Paspalum pollen SPT solution on 407 patients. Sera from six Bahia grass sensitized patients were used for allergens identification by IEF, SDS-PAGE and 2D-PAGE followed by immunoblotting. Monospecific antibodies directed against grass group 1, grass group 5 and profilin were used to study potential cross-reactivity between Paspalum and temperate grass pollens. Results: Of the 407 tested patients, 44 (10.8%) patients showed positive SPT to Paspalum extract. Skin prick test to Paspalum pollen extract showed wheals ranging from 3 to 11 mm. RAST measured by CAP RAST ranged class 2 to 6. Paspalum notatum pollen was extracted (1/20 p/v) in NaHCO3-NaCl buffer at 4°C overnight. IEF immunoblotting showed a very basic allergen (pI>10) and numerous bands located between 3.5 and 7.0. SDS-PAGE immunoblotting showed allergens located at 54 kDa and 29 kDa, the last being the major one. Reducing conditions increased the 29 kDa to a molecule with apparent molecular mass of 31 kDa, suggesting intra-chain disulfide bond(s). Direct ELISA and SDS-PAGE immunoblotting with monospecific rabbit antibodies showed that Paspalum notatum pollen contains a profilin and cross- reactive grass group 1 and group 5 allergens. Profilin was not identified as an important allergen from Paspalum pollen. Conclusions: Our study showed that important allergens from grass pollen (profilin, group 1 and 5) were detected in Paspalum notatum pollen suggesting that cross- reactivity with temperate grass pollen could occur.

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[43] - Pires APT, Viseu R, Inacio FFC. Recombinant allergens and immunoprinting as a tool for the follow-up of IgE sensitization along the years. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°881

Introduction: Although allergic diseases risk are largely determined by genetic factors the environment essential to define the profile of the allergic patients` sensitisation. No studies have been published relating the IgE sensitisation to the molecular allergens and their evolution along the years. Aim: Our aim was to study the evolution of the IgE sensitisation to grass pollen molecular allergens in a group of allergic patients with clinical allergy to grass pollens. Material and Methods: We studied 23 grass pollen allergic patients living in the same geographic area for at least 5 years. All patients were allergic to grass pollen. Specific IgE to Dactylis glomerata (Dac g), Phleum pratensis (Phl p), rPhl p 1, rPhl p 2, rPhl p 4, rPhl p 5, rPhl p 7, rPhl p 12 and simple IgE immunopring pattern (group I) or intermediate IgE immunoprinting pattern to Dac g and Phl p. Blood samples were collected with a medium interval of 4 years. We considered that a patient has a simple IgE immunoprinting pattern when he recognised 2 allergen groups of Dac g or Phl p and intermediate IgE immunoprinting pattern when he recognised 3 allergen groups. Results: In group I (19 patients) 7 recognized the allergens of group 1 they presented a specific IgE average to Dac g of 2,8 KU/l, 3 recognized the allergens of group 4 they presented a specific IgE average to Dac g of 25,9 KU/l, 1 recognized the allergens of group 5 he presented a specific IgE to Dac g of 1,74 KU/ l, 8 recognized the allergens of group 1 and group 4 they presented a specific IgE average to Dac g of 6,4 KU/l. In group II (4 patients) all patients recognized the allergens of the groups 1, 2, 4 they presented a specific IgE average to Dac g of 15,5 KU/l. In group 1, most of the patients (14) maintained simple IgE immunoprinting pattern along the years; only 5 patients increased the IgE immunoprinting pattern recognition and increased the specific IgE to Dac g. Concerning group II we also observed no change in the recognition of molecular allergens. Conclusion: In this study we could observe that the most of the allergic patients to grass pollen allergens do not change the pattern of molecular allergen recognition along the years. This means that there are genetic constraints that do not allow the recognition of other allergens. The environment is an important factor to allergen sensitivity but the genetic background determines wich are the molecular allergens to which patients will be sensitised

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[44] - Pauli G. Allergènes recombinants et immunothérapie. Rev Fr Allergol Immunol Clin 2007;47:133-138

La mise au point de nouvelles techniques de désensibilisation utilisant des produits issus de la biologie moléculaire nécessite le contrôle de leur efficacité par des essais cliniques appropriés. Un aperçu des principales molécules proposées par les recherches fondamentales au cours des dernières années est délivré : allergènes recombinants correspondant à des molécules d'allergènes pertinents du fait de leur forte prévalence de sensibilisation, dérivés hypoallergéniques d'allergènes recombinants (polymères, fragments, molécules avec des mutations ou des délétions d'acides aminés), molécules hybrides, épitopes T, épitopes B, molécules conjuguées à des CpG ou d'autres co-stimulateurs. L'activité biologique d'un grand nombre de ces molécules a été démontrée in vivo. La sélection de molécules candidates pour l'immunothérapie a d'abord comporté des études préliminaires effectuées en ouvert sur un nombre restreint de patients. Les auteurs rapportent ici les résultats de cinq études de désensibilisations effectuées par injection, réalisées en double insu, le groupe témoin étant un placebo et/ou un groupe traité par l'extrait naturel (dans deux études). Les résultats cliniques les plus significatifs sur le plan statistique ont été observés avec des allergènes recombinants non modifiés, sans survenue d'accidents systémiques sévères. Dans les cinq études, on relève une augmentation importante des IgG spécifiques vis-à-vis de l'allergène naturel et vis-à-vis des allergènes recombinants. La réduction des tests cutanés dans les groupes traités par rapport au groupe placebo est significative dans deux études sur trois. Des études comportant un nombre de patients plus important sont maintenant souhaitables pour répondre aux exigences réglementaires du développement clinique des allergènes recombinants.

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[45] - Godfrin D, Sénéchal H, Desvaux F, Clément G, Wal J, Hennion M, et al. Immuno-detection of non-water soluble grass pollen (Dactylis glomerata) allergens. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°926

Background: In order to describe the whole grass pollen allergen repertoire we studied non water-soluble allergens and quantified the specific antibodies (Ab) directed to them. The classical solubilizing protocol of non water-soluble proteins (TUC: Thiourea 2M, Urea 7M and CHAPS 5%) includes agents that inhibit adsorption on microtitration plates and blotting membranes, preventing detection and quantification of Ab with classical ELISA and blot methods. Methods: After water-soluble proteins removal from Dactylis glomerata pollen, the remaining pollen pellet was submitted to extraction with TUC. By an adaptation of ELISA method, quantification of Ab to TUC-soluble proteins was performed. These allergens were analyzed by isoelectrofocusing (IEF) separations followed by immunoblots, revealed by IgEs from grass pollen sensitive patient sera. Results: To prevent the TUC inhibitor effect on ELISA, we tried several organic components (ethanol, isopropanol, acetonitrile and trichloroacetic acid) to make the TUC-soluble proteins sticking to the ELISA plates. Ethanol was shown to be the most appropriate one. This method allowed us to detect down to 50 nanograms/mL of TUC-soluble proteins. 33 sensitive patient sera were tested by this new ELISA and by blots. Fourteen sera showed strong IgE responses to both water and TUC-soluble allergens, 14 others had a strong recognition of water-soluble allergens but a weak one for TUC-soluble allergens. Three sera were more reactive with TUC-soluble allergens than water-soluble ones. Most positive sera for these new non water-soluble allergens had IgE titers to grass pollen higher than 15kU/L. However some negative sera had IgE titers higher than 100. Conclusion: Our new ELISA and blots showed that besides water-soluble allergens grass pollen sensitive patients can be reactive to new TUC-soluble allergens. These allergens should be further characterized and identified. The clinical relevance of these new allergens is under investigation.

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[46] - Metz-Favre C, Linhart B, Purohit A, de Blay F, Valenta R, Pauli G. Profils de sensibilisation au pollen de graminées en Alsace. Rev Fr Allergol Immunol Clin 2006;46:356

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[47] - Wöhrl S, Vigl K, Zehetmayer S, Hiller R, Jarisch R, Prinz M, et al. The Performance of a New Component-Based Allergen- Microarray in Clinical Practice. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°754

RATIONALE: Currently the diagnosis of IgE-mediated allergy is based on the allergen-specific history and diagnostic procedures using natural allergen extracts for in vivo and in vitro tests. The aim of the study was to comparatively analyse a new allergen-microarray and the 'quasi-standard' ImmunoCAP(r) for their clinical relevance in patients with allergic rhinoconjunctivitis to 5 aeroallergens [house dust mite (HDM), cat, birch, grass and mugwort pollen] in a prospective, double-centre study METHODS: We enrolled 120 subjects at the two study centers. Allergic patients were defined as having an allergen-specific history plus a concomitant SPT to natural allergen extracts and specific serum IgE was measured by both methods. Each allergen was analysed separately RESULTS: The microarray performed equally well in ROC analyses when compared to the CAP in cat (23 allergic vs. 97 non-allergic, ROC area under the curve microarray: 0.950 vs. CAP: 0.894, p = 0.211), birch (31/89, 0.908 vs. 0.878, p = 0.483) and grass pollen (47/73, 0.923 vs 0.915, p = 0.770). It was slightly less sensitive in HDM allergic subjects (26 allergic vs. 94 non-allergic, ROC area microarray 0.808 vs. CAP 0.911, p = 0.053) and performed poorer in the mugwort pollen-allergic patients (17/103, 0.723 vs. 0.879, p = 0.032) CONCLUSIONS: Component-based testing and the whole-allergen CAP are equally relevant in the diagnosis of grass-, birch- and cat-allergic patients. Although slightly less sensitive, the microarray is sufficient for the diagnosis of HDM-allergic patients, but needs better and/or additional components for the detection of mugwort allergy.

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[48] - Rossi RE, Monasterolo G, Monasterolo S. Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen. Allergy 2001;56:1180-1185

Background: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis. Methods: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens. Results: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed. Conclusions: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.

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[49] - Kazemi-Shirazi L, Niederberger V, Linhart B, Lidholm J, Kraft D, Valenta R. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-268

During the past decade an increasing number of recombinant allergens have become available, representing a significant proportion of the epitope complexity of natural allergen extracts. Component-resolved diagnosis with recombinant allergens reveals the antibody reactivity profile of allergic patients and identifies the disease-eliciting allergen molecules. This article exemplifies how recombinant allergen molecules with high cross-reactive potential can be used as marker allergens to identify allergic patients who are cross-sensitized to a variety of allergen sources. It further demonstrates how the use of allergens with a restricted distribution in a certain group of allergen sources may allow the identification of patients who have been genuinely sensitized by a particular allergen molecule. Drawing from those examples, it is suggested how diagnostic tests based on such recombinant marker allergens may be used to improve the choice and monitoring of currently available forms of specific immunotherapy.

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[50] - Stumvoll S, Lidholm J, Thunberg R, DeWitt AM, Eibensteiner P, Swoboda I, et al. Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential. Biol Chem 2002;383:1383-1396

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

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[51] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[52] - Ong EK, Griffith IJ, Knox RB, Singh MB. Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from ryegrass pollen that demonstrates specific antigenic immunoreactivity. Gene 1993;134:235-240

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572] Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.

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[53] - Marknell DeWitt Å, Niederberger V, Lehtonen P, Spitzauer S, Sperr WR, Valent P, et al. Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11. Clin Exp Allergy 2002;32:1329-1340

BackgroundAllergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. ObjectiveTo clone and characterize the 20kDa timothy grass pollen allergen Phl p 11. MethodsPhl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. ResultsPhl p 11 cDNA defined an acidic polypeptide of 15.8kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n=184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. ConclusionAs the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.

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[54] - Swoboda I, Grote M, Verdino P, Keller W, Singh MB, De Weerd N, et al. Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles. J Immunol 2004;172:6490-6500

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

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[55] - Laffer S, Vrtala S, Duchene M, van Ree R, Kraft D, Scheiner O, et al. IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens. J Allergy Clin Immunol 1994;94:88-94

A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens. In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed. Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen-specific IgE was estimated. Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I. The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to est imate the percentage of specific IgE that individuals produce against certain allergens. The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen-specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy

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[56] - Bascones Nestar O, Carretero Anibarro P, Reinares Ten C, Iparraguirre Castro A, Amo Vazquez de la Torre A, Juste Picon S, et al. Grass pollen recombinant allergens profile from Burgos population. Allergy 2008;63(suppl. 88):224

Background: Sensitivity to grass pollen allergens is a common feature among Spanish patients with seasonal pollen allergy. In this in vitro study, we examined the IgE profiles to grass pollen recombinant allergens in grass-sensitive patients from the population of Burgos in the north of Spain. Methods: The study included 75 patients. All suffered from seasonal rhinoconjunctivitis and/or asthma due to grass pollen allergy. Their sera were analyzed for specific IgE reactivity to individual grass pollen allergens (recombinant Phl p 1, Phl p 5, Phl p 7 and Phl p 12). Results: All patients recognized almost one of Phl p 1 and/or Phl p 5 allergens. Phl p 1 specific IgE antibodies were found in 90,6% of the patients and 65,3% of the sera were positives to Phl p 5, whereas reactivity to Phl p 12 and Phl p 7 were rare in all the patients (17,3-10,6% respectively). Conclusions: In grass pollen allergy diagnosis it is so important to do in vivo as in vitro tests. Specific IgE determination using recombinant allergens show specificity and sensitivity values close to 100%. Most grass polen allergic patients from the north of Spain are sensitised to r Phl p 1. An accurate diagnosis is basic to choose a correct and specific treatment to each patient, and ensure the success of the immunotherapy

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[57] - Metz-Fabre C, Linhart B, Focke-Tejkl M, Purohit A, de Blay F, Valenta R, et al. Skin test diagnosis of grass pollen allergy with a recombinant hybrid molecule. J Allergy Clin Immunol 2007;120:315-321

BACKGROUND: A recombinant hybrid molecule (HM) consisting of 4 major allergens from timothy grass (Phl p 1, 2, 5, and 6) was expressed in Escherichia coli, purified, and characterized regarding its immunologic properties . OBJECTIVE: We sought to determine whether the recombinant HM can be used for the diagnosis of grass pollen allergy by means of skin testing . METHODS: Skin prick testing was performed in 32 patients with grass pollen allergy and in 9 control individuals by using increasing concentrations (4, 12, 36, and 108 mug/mL) of the HM and using commercial grass pollen extract. Specific IgE reactivities against the HM, grass pollen extract, and a panel of purified grass pollen allergens (recombinant Phl p 1, 2, 5, 6, 7, 12, and 13 and natural Phl p 4) were measured by means of ELISA, and timothy grass pollen-specific IgE levels were determined by using ImmunoCAP . RESULTS: Grass pollen allergy was diagnosed in all patients by means of skin testing with the HM. No false-positive skin test responses were obtained in the control individuals. There was an excellent correlation between IgE levels obtained with the HM and natural grass pollen extract measured by means of ELISA (r = 0.98, P < .0001) and by means of ImmunoCAP (r = 0.98, P < .0001) . CONCLUSIONS: The recombinant HM permitted accurate and specific in vivo diagnosis of grass pollen allergy in all tested patients. It can be considered a well-defined tool for the diagnosis and perhaps for immunotherapy of grass pollen allergy. CLINICAL IMPLICATIONS: A recombinant HM can replace traditional allergen extracts for skin test-based diagnosis of grass pollen allergy.

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[58] - Westritschnig K, Horak F, Swoboda I, Balic N, Spitzauer S, Kundi M, et al. Different allergenic activity of grass pollen allergens revealed by skin testing. Eur J Clin Invest 2008;38:260-267

BACKGROUND: Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. MATERIAL AND METHODS: Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. RESULTS: Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. CONCLUSION: Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

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[59] - Petersen A, Dresselhaus T, Grobe K, Becker WM. Proteome analysis of maize pollen for allergy-relevant components. Proteomics 2006;6:6317-6325

Over the last few decades, the cultivation of maize (Zea mays) has strongly increased in Central Europe. We therefore decided to study the allergen composition and the allergenic potency of its pollen in comparison with pollen from timothy grass (Phleum pratense), a typical representative of the native grasses. We found that 65% of the sera reactive to timothy pollen also bound to maize pollen proteins. By using 2-DE immunoblotting, followed by incubation with mAbs directed against known allergens or protein sequencing, those IgE-reactive components were further classified. Although novel, maize-specific pollen allergens could not be found, the presence of crossreacting allergens belonging to groups 1 and 13 (Zea m 1 and 13), both having high IgE prevalence, as well as the presence of the less important group 3 and 12 allergens was found. The structural variability of Zea m 1 and Zea m 13 was determined by sequencing clones isolated from a maize pollen cDNA library. This revealed sequence identities of 72 and 70%, respectively, to the corresponding Phl p 1 and Phl p 13 allergens of timothy grass pollen. IgE-crossreactivity was further studied using immunoblot inhibition tests. Here, timothy pollen extract completely blocked IgE binding to maize, whereas maize pollen extract blocked IgE reactivity to only some timothy pollen allergens.

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[60] - Martínez A, Asturias JA, Monteseirín J, Moreno V, García-Cubillana A, Hernández M, et al. The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen. Allergy 2002;57(suppl. 71):17-23

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the aminoacid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

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[61] - Asturias JA, Arilla MC, Gomez-Bayon N, Martinez J, Martinez A, Palacios R. Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen. Clin Exp Allergy 1997;27:1307-1313

BACKGROUND: Profilin, an actin-binding protein, was previously described as a panallergen which is involved in about 20% of the crossreactivity found among pollen and food allergic patients. This allergen is usually under-represented in natural extracts used for allergy diagnosis. OBJECTIVES: To obtain an immunologically active and soluble recombinant profilin from Cynodon dactylon pollen which could be used for diagnostic and therapy. METHODS: Isolation of cDNA clones was performed by polymerase chain reaction amplification using degenerate primers. Expression in Escherichia coli BL21 (DE3) was carried out using vector pKN172, and the expressed product was isolated by affinity chromatography on poly L-proline-Sepharose. RESULTS: Four cDNA inserts coding for Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) were cloned and sequenced. Full-length C. dactylon profilin gene was expressed in Escherichia coli as non fusion protein. Induced cells could produce high amounts of recombinant Cyn d 12, and after a single purification step on poly (L-proline)-Sepharose, up to 45 mg of pure allergen per litre culture could be obtained. The reactivity of recombinant Cyn d 12 with IgE antibodies present in sera from Bermuda grass-allergic patients is comparable to that of the natural Bermuda grass allergen. Recombinant Bermuda grass pollen profilin was shown to share B-epitopes with sunflower profilin. CONCLUSIONS: Our results showed that this heterologous expression system and purification procedure are suitable for the production of large amounts of pure allergen which can be used for the characterization of allergenic epitopes recognized by T and B cells and finally for diagnostic and therapeutic purposes.

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[62] - Davies J, Dang T, Pettit B, Voskamp A, Rolland J, O'Hehir R. Characterisation of the group 13 pollen allergen of the subtropical bahia grass, Paspalum notatum. Allergy 2009;64(Suppl. 90):108-109

Background: The subtropical Bahia grass, Paspalum notatum, is an important allergen source with a prolonged period of pollination triggering allergic rhinitis and asthma in sensitized patients. We recently cloned and characterised the group 1 allergen of Bahia grass pollen (BaGP); recombinant Pas n 1 reacted with IgE from 47 (85%) of 55 grass pollen-allergic sera and potently activated basophils of grass pollen-allergic donors. A 55 kDa component of BaGP also has strong reactivity with serum IgE from 11 of 21 (52%) of BaGPallergic patients. Here we aimed to identify and characterise the 55 kDa allergen of BaGP. Methods: BaGP was separated by two-dimensional gel electrophoresis and immunoblotted with grass pollen-allergic patient serum for IgE reactivity with the 55 kDa spot. The N-terminal peptide of this protein was sequenced and oligonucleotide primers were designed to obtain the cDNA of this allergen by rapid amplification of cDNA ends. The allergen was purified from BaGP by ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography and tested for IgE reactivity with a panel of sera by ELISA, inhibition of IgE reactivity with BaGP by western blotting and activation of basophils. Results: Two protein spots at 55 kDa with neutral pH were reactive with IgE of a grass pollen-allergic serum donor. The N-terminal peptide sequence of the predominant 55 kDa spot had similarity with the group 13 allergens of maize and timothy grass pollen. The cDNA sequence for Pas n 13 encodes a protein of 423 amino acids including a 28 residue signal peptide and two predicted N linked glycosylation sites. Pas n 13 purified from BaGP was reactive with serum IgE of 35 of 71 (49.3%) grass pollen-allergic patients. Purified Pas n 13 inhibited serum IgE reactivity with the 55 kDa allergen of BaGP and the allergenic activity of Pas n 13 was demonstrated by activation of basophils of grass pollen-allergic donors. Conclusion: The cDNA encoding the group 13 allergen of BaGP has been amplified, cloned and sequenced. Pas n 13 is a clinically significant pollen allergen of the subtropical Bahia grass and is a valuable reagent for improved diagnosis and specific immunotherapy of grass pollen allergy. Immunological characterisation of the major allergens from BaGP is important given the phylogenetic distance of Bahia grass from the well known temperate grass species including Timothy and ryegrass.

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[63] - Davies JM, Mittag D, Dang TD, Symons K, Voskamp A, Rolland JM, et al. Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen. Mol Immunol 2008;46:286-293

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the beta-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

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[64] - van Ree R, Fernández-Rivas M, Cuevas M, van Wijngaarden M, Aalberse RC. Pollen-related allergy to peach and apple: an important role for profilin. J Allergy Clin Immunol 1995;95:726-734

Birch pollinosis is often accompanied by allergy to fruits such as peach and apple. Bet v I is of major importance as cross-reactive allergen for this combined allergy. We studied a group of patients with combined grass pollinosis and fruit allergy from an area virtually without birch trees. OBJECTIVE: The aim of this study was to investigate the possible involvement of profilin and carbohydrate groups as cross-reactive structures in pollen and fruits. METHODS: RAST inhibition was performed to measure cross-reactive IgE to pollen and fruits. The presence of IgE against profilin was determined in a RAST with purified grass profilin, and IgE against carbohydrate structures was determined in a RAST with proteinase K-digested grass pollen extract. The biologic activity of IgE in response to profilin was tested by in vitro histamine release and skin prick tests. RESULTS: IgE against fruits was shown to be largely cross-reactive with grass pollen. The majority of the patients had IgE against profilin (12 of 16) and carbohydrate structures (9 of 10). Profilin was shown to have biologic activity, in both histamine release and skin prick tests. CONCLUSION: Profilin is an important allergen for patients with combined grass pollen/fruit allergy in areas without birch trees.

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[65] - Fernández-Rivas M, Van Ree R, Cuevas M. Allergy to Rosaceae fruits without related pollinosis. J Allergy Clin Immunol 1997;100:728-733

BACKGROUND: Rosaceae fruit allergy is frequently associated with birch pollinosis in Central and Northern Europe and with grass pollen allergy in Central Spain. The main cross-reactive structures involved for birch pollinosis are Bet v 1 and profilin, and for grass pollinosis they are profilin and carbohydrate determinants. Rosaceae fruit allergy can occasionally be observed in patients without pollinosis . OBJECTIVE: We investigated the clinical presentation and the allergens involved in allergy to Rosaceae fruit without pollinosis . METHODS: Eleven patients from Central Spain allergic to apples, peaches, and/or pears but not to pollens were compared with 22 control subjects with combined grass pollen and fruit allergy. Skin prick tests and RASTs to apple, peach, and pear were performed. Cross-allergenicity was studied by RAST inhibition. Bet v 1 was tested with an indirect RAST, and profilin was tested in skin prick tests, histamine release, and RAST . RESULTS: Rosaceae fruit allergy without pollinosis is severe with 82% of patients reporting systemic symptoms, mainly anaphylaxis (73%), whereas oral symptoms are less frequent (64%). Anaphylactic shock was observed in 36% of patients. The fruit allergens involved showed cross-reactivity among Rosaceae species but were not related to profilin or Bet v 1. Ninety-one percent of patients with combined grass pollinosis and fruit allergy reported oral allergy, 45% reported systemic symptoms, 18% reported anaphylaxis, and 9% reported anaphylactic shock . CONCLUSION: Allergy to Rosaceae fruits in patients without a related pollen allergy is a severe clinical entity. Profilin- and Bet v 1-related structures are not involved in Rosaceae fruit allergy without pollinosis.

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[66] - Diez-Gomez ML, Quirce S, Cuevas M, Sanchez-Fernandez C, Baz G, Moradiellos FJ, et al. Fruit-pollen-latex cross-reactivity: implication of profilin (Bet v 2). Allergy 1999;54:951-961

An association between allergy to fruits and latex, and between pollen and plant-derived food has been described. The cross-reactive structures responsible for these associations have not yet been completely elucidated. METHODS: IgE reactivity to the recombinant allergens Bet v 1 and Bet v 2, different pollens, natural latex, papain, and bromelain was investigated in 29 patients with allergy to fruits or vegetables who lived in an area without birch trees. RESULTS: Exactly 79.3% of patients were allergic to grass pollen, and two of them had clinical allergy to latex. Serum IgE reactivity (CAP) to birch pollen was found in 65% of patients, to Bet v 2 in 51.7%, to Bet v 1 in 3.4%, to latex in 58.6%, to bromelain in 51.7%, and to papain in 17.2% of patients. All subjects with positive IgE to Bet v 2 had also reactivity to latex, grass, olive tree, birch, and mugwort pollens. The six patients not allergic to pollen did not show IgE reactivity to latex, Bet v 1, or Bet v 2. A significant correlation was found between CAP to latex with Bet v 2 (r=0.86, P<0.001), with birch (r=0.86, P<0.001), and with ryegrass (r=0.81, P<0.001). Immunoblotting using nine sera with positive CAP to birch pollen showed IgE-binding to a 15-kDa band that was recognized by antiprofilin monoclonal antibody. Bet v 2 CAP could be inhibited up to 52% by ryegrass and up to 23% by mugwort. CAP to latex was almost completely inhibited by ryegrass pollen with sera from five subjects without symptoms due to latex, whereas no inhibition was observed with serum from one patient with allergy to latex. CONCLUSIONS: Patients with allergy to plant-derived food and associated pollinosis showed a high frequency of IgE reactivity to Bet v 2, which may cause positive serum IgE determinations to latex and birch pollen due to the presence of cross-reactive epitopes. IgE reactivity to Bet v 2 may serve as an indicator of broad sensitization.

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[67] - Petersen A, Vieths S, Aulepp H, Schlaak M, Becker WM. Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens. J Allergy Clin Immunol 1996;98:805-815

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.

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[68] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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[69] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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[70] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[71] - Mari A. Revisiting the allergy diagnosis of IgE-mediated diseases using the allergenic molecule perspective: new concepts defining the allergic sensitisation pattern. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°721

The use of allergenic extracts for the allergy diagnosis of IgE-mediated diseases led the classification and the definition of the atopic reactivity pattern. Reactivity to a single allergenic extract, containing one or several allergenic components, defined the monosensitivity, whereas the reactivity to two or more allergenic sources defined the multiple sensitivity. Furthermore, listing the number of sources any subject reacted to, defined the potential triggering sources. The identification of allergenic molecules is leading to a reassessment of several aspects of the diagnostic and therapeutic approach to the allergic diseases. The aim of the study was to re-examine the concept of monosensitization and to re-evaluate the multiple reactivity by comparing the extract SPT and IgE reactivity pattern with the patterns obtained by means of allergenic molecules. 721 grass pollen allergic subjects (skin test positive to Phleum pratense) were selected and stratified after skin testing with 23 pollen extracts including common, less common and rare sources (reported in IAAI 2001;125:57-65). Sera were tested for IgE to Phleum pratense and to eight of its allergenic components (rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12). IgE detection was performed using the CAP system (Pharmacia, Sweden). Sera were positive to at least one of the allergenic molecules in 93% and 97% of the subjects considering those positive to the in vivo or to the in vitro test respectively. Isolated allergenic molecule reactivity within the whole grass allergic group were as follows (n/%): rPhl p 1=43/6, rPhl p 2=4/0.6, nPhl p 4=8/1.1, rPhl p 5=1/0.1, rPhl p 6=0, rPhl p 7=3/0.4, rPhl p 11=13/2, rPhl p 12=2/0.3. When grass allergic subject with associated reactivity to at least another pollen source were excluded figures were as follows: (n/%): rPhl p 1=16/2, rPhl p 2=1/0.1, nPhl p 4=3/0.4, rPhl p 5=1/0.1, rPhl p 7=0, rPhl p 11=3/0.4, rPhl p 12=0. Allergenic molecule monosensitization is a quite rare phenomenon accounting only for the 3.2% in the grass allergy model (24 subjects). Though rare, there could be another form of monosensitization: subjects reacting to a panallergen, showing a broad reactivity to all the sources containing the shared molecule, but without a reactivity to family-restricted molecules. The allergenic molecule pattern of reactivity should thus re-address the diagnostic pathway, and re-define criteria for immunotherapy.

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[72] - Metz-Favre C, Linhart B, Purohit A, de Blay F, Valenta R, Pauli G. Profils de sensibilisation au pollen de graminées en Alsace. Rev Fr Allergol Immunol Clin 2006;46:356

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[73] - Mari A. Revisiting the allergy diagnosis of IgE-mediated diseases using the allergenic molecule perspective: new concepts defining the allergic sensitisation pattern. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°721

The use of allergenic extracts for the allergy diagnosis of IgE-mediated diseases led the classification and the definition of the atopic reactivity pattern. Reactivity to a single allergenic extract, containing one or several allergenic components, defined the monosensitivity, whereas the reactivity to two or more allergenic sources defined the multiple sensitivity. Furthermore, listing the number of sources any subject reacted to, defined the potential triggering sources. The identification of allergenic molecules is leading to a reassessment of several aspects of the diagnostic and therapeutic approach to the allergic diseases. The aim of the study was to re-examine the concept of monosensitization and to re-evaluate the multiple reactivity by comparing the extract SPT and IgE reactivity pattern with the patterns obtained by means of allergenic molecules. 721 grass pollen allergic subjects (skin test positive to Phleum pratense) were selected and stratified after skin testing with 23 pollen extracts including common, less common and rare sources (reported in IAAI 2001;125:57-65). Sera were tested for IgE to Phleum pratense and to eight of its allergenic components (rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12). IgE detection was performed using the CAP system (Pharmacia, Sweden). Sera were positive to at least one of the allergenic molecules in 93% and 97% of the subjects considering those positive to the in vivo or to the in vitro test respectively. Isolated allergenic molecule reactivity within the whole grass allergic group were as follows (n/%): rPhl p 1=43/6, rPhl p 2=4/0.6, nPhl p 4=8/1.1, rPhl p 5=1/0.1, rPhl p 6=0, rPhl p 7=3/0.4, rPhl p 11=13/2, rPhl p 12=2/0.3. When grass allergic subject with associated reactivity to at least another pollen source were excluded figures were as follows: (n/%): rPhl p 1=16/2, rPhl p 2=1/0.1, nPhl p 4=3/0.4, rPhl p 5=1/0.1, rPhl p 7=0, rPhl p 11=3/0.4, rPhl p 12=0. Allergenic molecule monosensitization is a quite rare phenomenon accounting only for the 3.2% in the grass allergy model (24 subjects). Though rare, there could be another form of monosensitization: subjects reacting to a panallergen, showing a broad reactivity to all the sources containing the shared molecule, but without a reactivity to family-restricted molecules. The allergenic molecule pattern of reactivity should thus re-address the diagnostic pathway, and re-define criteria for immunotherapy.

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[74] - Marth K, Focke M, Flicker S, Valenta R. Human monoclonal antibody-based quantification of group 2 grass pollen allergens. J Allergy Clin Immunol 2004;113:470-474

BACKGROUND: Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients . OBJECTIVE: The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens . METHODS: We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen-allergic patient. This Fab was converted into a complete human IgG1 antibody and used together with rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPh1 p 2, which can be detected with peroxidase-labeled streptavidin . RESULTS: The competition-ELISA measured rPh1 p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data . CONCLUSIONS: The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.

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[75] - Ramirez J, Obispo TM, Duffort O, Carpizo JA, Chamorro MJ, Barber D, et al. Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA - Correlation with biologic activity. Allergy 1997;52:806-813

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

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[76] - Matthiesen F, Lowenstein H. Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses. Clin Exp Allergy 1991;21:309-320

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.

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[77] - Lièvre K, Aparicio C, Lallemand K, Kosmalski D, Leduc V. Le pollen de fléole (Phleum pratense L.) est-il représentatif de l’ensemble des pollens de graminées ?. Rev Fr Allergol 2009;49:333

La polysensibilisation au sein de la famille des graminées est fréquente et expliquée par la présence d‚allergènes majeurs présentant de fortes similitudes de séquence. Cette étude a pour but d‚évaluer la représentativité d‚un pollen de graminée (fléole) par rapport à l‚ensemble des allergènes de pollens de 17 graminées fourragères et céréalières. Résultats.ˆ Les profils allergéniques des pollens de 17 graminées ont été comparés par SDS-PAGE et immuno-empreinte. À l‚aide d‚anticorps polyclonaux spécifiques de Phl p 1 et Phl p 5, les allergènes majeurs des groupes 1 et 5 ont été identifiés dans l‚ensemble des pollens étudiés, excepté pour le maïs et le cynodon pour lesquels le groupe 5 n‚est pas reconnu. L‚utilisation d‚un pool de sérums de 4 patients allergiques a permis de mettre en évidence d‚autres allergènes (groupe 2, 4, 12 et 13). La fixation des IgE de ce pool de sérums sur les extraits allergéniques des 17 pollens de graminées est complètement inhibée par l‚extrait de fléole, montrant ainsi une communauté épitopique entre tous les allergènes de ces pollens. Cela confirme que le pollen de fléole est bien un représentant valide des 15 pollens de graminées, dans le cadre du diagnostic et de la désensibilisation. Conclusion.ˆ Le pollen de fléole est représentatif de la plupart des pollens de graminées pour les allergènes majeurs. Les anticorps polyclonaux utilisés sont spécifiques de plusieurs épitopes, au même titre que les IgE des patients allergiques, ce qui permet de mimer au mieux leur cross-reactivité, contrairement aux anticorpsmonoclonaux souvent trop spécifiques.L‚implication d‚allergènes moins représentés dans des sensibilisations plus spécifiques ne doit cependant pas être écartée, en particulier dans le cadre de réactions allergiques où un allergènemineur serait impliqué, justifiant la disponibilité des autres pollens de graminées.

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[78] - Ramirez J, Obispo TM, Duffort O, Carpizo JA, Chamorro MJ, Barber D, et al. Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA - Correlation with biologic activity. Allergy 1997;52:806-813

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

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[79] - Kazemi-Shirazi L, Niederberger V, Linhart B, Lidholm J, Kraft D, Valenta R. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-268

During the past decade an increasing number of recombinant allergens have become available, representing a significant proportion of the epitope complexity of natural allergen extracts. Component-resolved diagnosis with recombinant allergens reveals the antibody reactivity profile of allergic patients and identifies the disease-eliciting allergen molecules. This article exemplifies how recombinant allergen molecules with high cross-reactive potential can be used as marker allergens to identify allergic patients who are cross-sensitized to a variety of allergen sources. It further demonstrates how the use of allergens with a restricted distribution in a certain group of allergen sources may allow the identification of patients who have been genuinely sensitized by a particular allergen molecule. Drawing from those examples, it is suggested how diagnostic tests based on such recombinant marker allergens may be used to improve the choice and monitoring of currently available forms of specific immunotherapy.

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[80] - Matthiesen F, Lowenstein H. Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses. Clin Exp Allergy 1991;21:309-320

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.

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[81] - Lafosse-Marin S, Leduc V, Aparicio C, Guérin L. Paspalum notatum (Bahia grass) pollen sensitization in Martinique (France). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1384

Background: Paspalum notatum (Bahia grass) is a grass which naturally grows in tropical and subtropical climates. It is considered as a very vigourous and aggressive grass, not easily eradicated. One aim of this work was to know if there is one relationship between Bahia grass sensitization in Martinique (French Antilles) and long stay in temperate countries (mainly in metropolitan France) during the grass pollen period. Moreover, even though various studies have demonstrated the importance of Bahia grass as an aeroallergen, it is to be considered that no allergens from Bahia grass pollen have been yet characterized. Methods: Skin prick tests were performed with Paspalum pollen SPT solution on 407 patients. Sera from six Bahia grass sensitized patients were used for allergens identification by IEF, SDS-PAGE and 2D-PAGE followed by immunoblotting. Monospecific antibodies directed against grass group 1, grass group 5 and profilin were used to study potential cross-reactivity between Paspalum and temperate grass pollens. Results: Of the 407 tested patients, 44 (10.8%) patients showed positive SPT to Paspalum extract. Skin prick test to Paspalum pollen extract showed wheals ranging from 3 to 11 mm. RAST measured by CAP RAST ranged class 2 to 6. Paspalum notatum pollen was extracted (1/20 p/v) in NaHCO3-NaCl buffer at 4°C overnight. IEF immunoblotting showed a very basic allergen (pI>10) and numerous bands located between 3.5 and 7.0. SDS-PAGE immunoblotting showed allergens located at 54 kDa and 29 kDa, the last being the major one. Reducing conditions increased the 29 kDa to a molecule with apparent molecular mass of 31 kDa, suggesting intra-chain disulfide bond(s). Direct ELISA and SDS-PAGE immunoblotting with monospecific rabbit antibodies showed that Paspalum notatum pollen contains a profilin and cross- reactive grass group 1 and group 5 allergens. Profilin was not identified as an important allergen from Paspalum pollen. Conclusions: Our study showed that important allergens from grass pollen (profilin, group 1 and 5) were detected in Paspalum notatum pollen suggesting that cross- reactivity with temperate grass pollen could occur.

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[82] - Davies JM, Bright ML, Rolland JM, O'Hehir RE. Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass. Allergy 2005;60:251-255

BACKGROUND: Perennial Ryegrass is a major cause of rhinitis in spring and early summer. Bahia grass, Paspalum notatum, flowers late into summer and could account for allergic rhinitis at this time. We determined the frequency of serum immunoglobulin (Ig)E reactivity with Bahia grass in Ryegrass pollen allergic patients and investigated IgE cross-reactivity between Bahia and Ryegrass . METHODS: Serum from 33 Ryegrass pollen allergic patients and 12 nonatopic donors were tested for IgE reactivity with Bahia and Ryegrass pollen extracts (PE) by enzyme-linked immunosorbent assay (ELISA), western blotting and inhibition ELISA. Allergen-specific antibodies from a pool of sera from allergic donors were affinity purified and tested for IgE cross-reactivity . RESULTS: Seventy-eight per cent of the sera had IgE reactivity with Bahia grass, but more weakly than with Ryegrass. Antibodies eluted from the major Ryegrass pollen allergens, Lol p 1 and Lol p 5, showed IgE reactivity with allergens of Ryegrass and Canary but not Bahia or Bermuda grasses. Timothy, Canary and Ryegrass inhibited IgE reactivity with Ryegrass and Bahia grass, whereas Bahia, Johnson and Bermuda grass did not inhibit IgE reactivity with Ryegrass . CONCLUSIONS: The majority of Ryegrass allergic patients also showed serum IgE reactivity with Bahia grass PE. However, Bahia grass and Ryegrass had only limited IgE cross-reactivity indicating that Bahia grass should be considered in diagnosis and treatment of patients with hay fever late in the grass pollen season.

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[83] - Smith PM, Ong EK, Knox RB, Singh MB. Immunological relationships among group I and group V allergens from grass pollen. Mol Immunol 1994;31:491-498

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

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[84] - Tordesillas L, Sanchez-Monge R, Cuesta-Herranz J, Compes E, Garcia-Carrasco B, Lombardero M, et al. A search for peach Pru P 3-homologous lipid transfer proteins in pollens. Allergy 2008;63(suppl. 88):117-118

Background: Two different patterns of sensitisation to Rosaceae fruits have been described in the Centre/North (Bet v 1-like as main allergens) and South (Lipid Transfer Proteins, LTPs, as major allergens) of Europe. Sensitisation to distinctive pollens have been claimed as a putative source of these differential profiles. Objetive: To explore the presence of putative Pru p 3-like allergens in Ambrosia artemissifolia (ragweed), Artemisia vulgaris (mugwort), Betula verrucosa (birch), Dactylis glomerata (barnyard grass), Helianthus annus (sunflower), Parietaria judaica (pellitory), Phleum pratensis (timothy), Plantago lanceolata (plantain) and Platanus occidentalis (plane tree) pollens. Methods: PBS extracts from the different pollens were separated by SDS-PAGE and immunodetected with anti-Pru p 3 polyclonal antibodies or IgE of sera from peach allergic patients (with associated pollinosis). ELISA-inhibition assays using purified Pru p3 and mugwort Art v 3 allergens as inhibitors were also performed. Pollen RNA samples and a cDNA encoding Pru p 3 probe were used in Northern analysis. Results: Pru p 3-like proteins were detected only in mugwort, but not in the other pollens analyzed, using either anti-Pru p 3 antibodies or sera from allergic patients. The cDNA-Pru p 3 probe hybridized with specific RNA bands from mugwort and plane tree pollens, but no band was found in the rest of pollens. Conclusion: Pru p 3-like LTPs were present in mugwort and plane tree pollens, as previously reported, but were not detected in ragweed, mugwort, birch, barnyard grass, sunflower, pellitory, timothy, plantain and plane tree pollens. A limited structural similarity with Pru p 3 and or even null levels of putative homologous allergens could explain the lack of detection of Pru p 3-like LTPs in most pollen analyzed.

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[85] - Cosgrove DJ. Loosening of plant cell walls by expansins. Nature 2000;407:321-326

Plant cell walls are the starting materials for many commercial products, from lumber, paper and textiles to thickeners, films and explosives. The cell wall is secreted by each cell in the plant body, forming a thin fibreglass-like network with remarkable strength and flexibility. During growth, plant cells secrete a protein called expansin, which unlocks the network of wall polysaccharides, permitting turgor-driven cell enlargement. Germinating grass pollen also secretes an unusual expansin that loosens maternal cell walls to aid penetration of the stigma by the pollen tube. Expansin's action has puzzling implications for plant cell-wall structure. The recent explosion of gene sequences and expression data has given new hints of additional biological functions for expansins.

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[86] - Grobe K, Poppelmann M, Becker WM, Petersen A. Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases. Eur J Biochem 2002;269:2083-2092

Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group I pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties.

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[87] - Grobe K, Becker WM, Schlaak M, Petersen A. Grass group I allergens (beta-expansins) are novel, papain-related proteinases. Eur J Biochem 1999;263:33-40

Expansins are a family of proteins that catalyse long-term extension of isolated plant cell walls due to an as yet unknown biochemical mechanism. They are divided into two groups, the alpha-expansins and beta-expansins, the latter group consisting of grass group I allergens and their vegetative homologs. These grass group I allergens, to which more than 95% of patients allergic to grass pollen possess IgE antibodies, are highly immunologically crossreactive glycoproteins exclusively expressed in pollen of all grasses. Alignments of the amino-acid sequences of grass group I allergens derived from diverse grass species reveal up to 95% homology. It is therefore likely that these molecules share a similar biological function. The major grass group I allergen from timothy grass (Phleum pratense), Phl p 1, was chosen as a model glycoprotein and expressed in the methylotrophic yeast Pichia pastoris to obtain a post-translationally modified and functionally active allergen. The recombinant allergen exhibited proteolytic activity when assayed with various test systems and substrates, which was also subsequently demonstrated with the natural protein, nPhl p 1. These observations are confirmed by amino-acid alignments of Phl p 1 with three functionally important sequence motifs surrounding the active-site amino acids of the C1 (papain-like) family of cysteine proteinases. Moreover, the significantly homologous alpha-expansins mostly share the functionally important C1 sequence motifs. This leads us to propose a C1 cysteine proteinase function for grass group I allergens, which may mediate plant cell wall growth and possibly contributes to the allergenicity of the molecule.

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[88] - Petersen A, Grobe K, Schramm G, Vieths S, Altmann F, Schlaak M, et al. Implications of the grass group I allergens on the sensitization and provocation process. Int Arch Allergy Immunol 1999;118:411-413

Grass pollen allergens of group I are particularly important because of their high IgE prevalence and occurrence in all grass species. Four independent IgE-binding regions and one continous epitope were identified. The posttranslational modifications on the molecule increased allergenicity. Phl p 1 is a cysteine protease, as determined by specific substrates, inhibitors and consensus sequence motifs. In analogy to other allergens and/or proteases, we deduce that Phl p 1 might enhance the permeability of the epithelium, influence T helper cells to bias Th2, and increase the IgE production of plasma cells. Thus, the group I allergens seem to be the crucial components in a pollen extract which can mediate sensitization and enhance the triggering of symptoms leading to the persistence of a grass pollen allergy.

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[89] - Petersen A, Grobe K, Schramm G, Gehlhar K, Vieths S, Altmann F, et al. Structure and function can determine important features in allergenicity: investigations on the group 1 allergens of the grasses. Internet Symp Food Allergens 1999;1(3):95-101

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[90] - Cosgrove DJ. Loosening of plant cell walls by expansins. Nature 2000;407:321-326

Plant cell walls are the starting materials for many commercial products, from lumber, paper and textiles to thickeners, films and explosives. The cell wall is secreted by each cell in the plant body, forming a thin fibreglass-like network with remarkable strength and flexibility. During growth, plant cells secrete a protein called expansin, which unlocks the network of wall polysaccharides, permitting turgor-driven cell enlargement. Germinating grass pollen also secretes an unusual expansin that loosens maternal cell walls to aid penetration of the stigma by the pollen tube. Expansin's action has puzzling implications for plant cell-wall structure. The recent explosion of gene sequences and expression data has given new hints of additional biological functions for expansins.

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[91] - Cosgrove DJ. New genes and new biological roles for expansins. Curr Opin Plant Biol 2000;3:73-78

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.

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[92] - Barre A, Rougé P. Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: structural basis for the cellulose recognition and associated allergenic properties. Biochem Biophys Res Commun 2002;296:1346-1351

A three-dimensional model of the cellulose-binding domain of the rye-grass pollen allergen Lol pI built by homology modeling is proposed as a structural scaffold for expansins and other expansin-related proteins. A groove and an extended strip of aromatic and polar residues presumably account for the cellulose-binding properties of the protein domain. Two of the four predicted T-cell epitopes readily exposed on the surface of the cellulose-binding domain match with previously reported IgE-binding regions. A close structural relationship occurs between the cellulose-binding and allergenic properties

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[93] - Röschmann K, Farhat K, König P, Suck R, Ulmer AJ, Petersen A. Timothy grass pollen major allergen Phl p 1 activates respiratory epithelial cells by a non-protease mechanism. Clin Exp Allergy 2009;39:1358-1369

BACKGROUND: Group 1 allergens from grass pollen (e.g. Phl p 1, the major allergen of timothy grass Phleum pratense) cause IgE reactivity in about 95% of allergic subjects and exist in all grass species. The respiratory epithelium represents a first line of contact of the immune system with airborne allergens, functions as physical barrier and is an important immunological regulation system . OBJECTIVE: The aim of this study was to investigate the interaction of Phl p 1 with human respiratory epithelium to elucidate the contribution of epithelial cells to the development of allergic reactions . METHODS: Purified Phl p 1 was used to stimulate A549 cells and transient transfected HEK293 cells. mRNA level of different mediators were investigated by real-time PCR, release of the mediators was determined by ELISA. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and an ex vivo model of the murine trachea were used to investigate a potential proteolytic activity of Phl p 1 . RESULTS: Phl p 1 activates respiratory epithelial cells as measured by induction of IL-6, IL-8 and TGF-beta mRNA and release. Phl p 1, in contrast to Der p 1 from the house dust mite, does not exert proteolytic activity, as investigated by microscopic observation and MTT test. In an ex vivo model of the murine trachea we were able to show that Der p 1, in contrast to Phl p 1, enhances the transportation velocity of particles by the trachea, presumably by ATP released from the injured epithelium . CONCLUSION: We conclude that under physiological conditions Phl p 1 affects tracheal epithelial cells through a non-proteolytic activity. Enhancement of TGF-beta expression induced by Phl p 1 together with the increased release of IL-6 and IL-8 might provide an indirect mechanism through which the allergen may cross the epithelial barrier and attracts immunocompetent cells.

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[94] - Chang ZN, Liu CC, Tam MF, Peng HJ, Tsai JJ, Han SH. Characterization of the isoforms of the group I allergen of Cynodon dactylon. J Allergy Clin Immunol 1995;95:1206-1214

The group I allergen of Cynodon dactylon, Cyn d I, was found to consist of four to 10 isoforms. METHODS: We studied the isoforms with the use of two-dimensional gel electrophoresis. The antigenic difference of the isoforms was evaluated by radioimmunoprecipitation with monoclonal antibodies (MAbs). The acidic isoforms and the basic and neutral isoforms were further isolated by MAb-affinity chromatography for RAST and competitive RAST. In addition, the N-terminal sequence was evaluated by microsequencing. RESULTS: A total of 11 isoforms were found in Cyn d I in extracts prepared from different sources of Bermuda grass pollen (BGP) They were either acidic (Cyn d I-A, I-B, I-C, I-D, I-E, I-F, I-G, I-H, and I-I), neutral (Cyn d I-X), or basic (Cyn d I-J). Cyn d I-G, with an isoelectric point of approximately 6.4, was constantly present in all the pollen preparations, whereas the content of the basic Cyn d I-J varied from less than 5% to greater than 20%. The molecular weight of the basic and neutral isoforms were slightly lower than those of the acidic isoforms. All isoforms shared a common antigenic determinant(s) recognizable by MAb 4-37, and the basic and neutral isoforms possessed a unique antigenic determinant(s) recognizable by MAb 1-61. RAST showed that both the acidic Cyn d I and the basic and neutral Cyn d I were recognized by human IgE in the pooled sera of persons allergic to BGP. Competitive RAST showed a high crossreactivity between the acidic and the basic and neutral isoforms. A 95% sequence identity also existed between the N-terminal 20 amino acid residues of basic Cyn d I-J and the dominant acidic isoform Cyn d I-G. CONCLUSIONS: The present study disclosed that basic Cyn d I-J is an important allergen and that the content of this isoform varies in different lots of BGP.

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[95] - van Oort E, de Heer PG, Dieker M, van Leeuwen AW, Aalberse RC, van Ree R. Characterization of natural Dac g 1 variants: An alternative to recombinant group 1 allergens. J Allergy Clin Immunol 2004;114:1124-1130

Background Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. Objective : Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. Method s : Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. Result s : Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. Conclusions : Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.

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[96] - Petersen A, Schramm G, Schlaak M, Becker WM. Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen. Clin Exp Allergy 1998;28:315-321

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.

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[97] - Au LC, Lin ST, Peng HJ, Liang CC, Lee SS, Liao CD, et al. Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens. Allergy 2002;57:215-220

Background:Cynd1, the major allergen of Bermuda grass pollen, contains some acidic/basic isoforms. The N-terminal amino acid sequences of some acidic Cynd1 isoforms were found to be different from those of Cynd1 cDNA clones identified previously. Methods:A predicted 17-meric oligonucleotide probe was designed to fish the unidentified isoallergen cDNAs out of BGP cDNA library. The reactive clones were isolated and verified by sequencing. Two of them were expressed in the yeast Pichia pastoris to obtain recombinant Cynd1 proteins. Results:All four cDNA clones encode the full-length Cynd1 with mature proteins of 244 amino acid residues. A 97-99% identity was found among the deduced amino acids of these four clones while an 86% identity was elicited between the four clones and the ones previously identified. The predicted isoelectric focusing (pI) values of the newly identified Cynd1s are acidic while pIs of the previously identified Cynd1s are basic. The two recombinant acidic Cynd1 proteins possess the epitopes recognized by mouse and rabbit polyclonal anti-Cynd1 antibodies, and have human IgE-binding capacity as revealed by immunodot assay. Conclusions:The present study identified full-length cDNAs encoding new isoallergens of Cynd1, and separated Cynd1 gene into an acidic group and a basic group.

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[98] - Davies JM, Mittag D, Dang TD, Symons K, Voskamp A, Rolland JM, et al. Molecular cloning, expression and immunological characterisation of Pas n 1, the major allergen of Bahia grass Paspalum notatum pollen. Mol Immunol 2008;46:286-293

Bahia grass, Paspalum notatum, is a clinically important subtropical grass with a prolonged pollination season from spring to autumn. We aimed to clone and characterise the major Bahia grass pollen allergen, Pas n 1. Grass pollen-allergic patients presenting to a tertiary hospital allergy clinic were tested for IgE reactivity with Bahia grass pollen extract by skin prick testing, ImmunoCAP, ELISA and immunoblotting. Using primers deduced from the N-terminal peptide sequence of a group 1 allergen of Bahia grass pollen extract separated by two-dimensional gel electrophoresis, the complete Pas n 1 cDNA was obtained by rapid amplification of cDNA ends and cloned. Biological relevance of recombinant Pas n 1 expressed in Escherichia coli was assessed by serum IgE reactivity and basophil activation. Twenty-nine of 34 (85%) consecutive patients presenting with grass pollen allergy were skin prick test positive to Bahia grass pollen. The Pas n 1 cDNA has sequence homology with the beta-expansin 1 glycoprotein family and is more closely related to the maize pollen group 1 allergen (85% identity) than to ryegrass Lol p 1 or Timothy grass Phl p 1 (64 and 66% identity, respectively). rPas n 1 reacted with serum IgE in 47 of 55 (85%) Bahia grass pollen-allergic patients, activated basophils and inhibited serum IgE reactivity with the 29kDa band of Bahia grass pollen extract. In conclusion the cDNA for the major group 1 allergen of the subtropical Bahia grass pollen, Pas n 1, was identified and cloned. rPas n 1 is immunologically active and is a valuable reagent for diagnosis and specific immunotherapy of grass pollen allergy.

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[99] - Smith PM, Ong EK, Knox RB, Singh MB. Immunological relationships among group I and group V allergens from grass pollen. Mol Immunol 1994;31:491-498

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

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[100] - Han SH, Chang ZN, Chi CW, Perng HJ, Liu CC, Tsai JJ, et al. Use of monoclonal antibodies to isolate and characterize Cyn d I, the major allergen of Bermuda grass pollen. J Allergy Clin Immunol 1993;92:549-558

Cyn d I has been found to be the major allergen of Bermuda grass (Cynodon dactylon) pollen, but its exact nature remains to be clarified. METHODS: Cyn d I, the major allergen of Bermuda grass (Cynodon dactylon) pollen, was purified by monoclonal antibody (MoAb) affinity chromatography, and its biochemical and immunologic properties were characterized. Anti-Cyn d I MoAb 4-37, which recognizes all of the isoallergens of Cyn d I, was chosen as the immunosorbent. RESULTS: The purified protein has an amino acid composition similar to that of the group I allergens of other grass pollens. It appears as a single 34 kd band or as a mixture of 34 and 29 kd polypeptides in sodium dodecylsulfate-polyacrylamide gel electrophoresis analysis. The hydrophobicity of these two polypeptides is similar because they have the same retention time on a C18 reverse-phase column when a trifluoroacetic acid/H2O/CH3CN buffer system is used. The N-terminal amino acid sequence of the 34 kd component has a 60% homology with residues of 1-25 of Lol p I, whereas that of the 29 kd component has a 68% homology with residues 31-68 of Lol p I. In addition, this 29 kd polypeptide can be recognized by another anti-Cyn d I MoAb 1-61. CONCLUSIONS: These results suggest that the 29 kd component is derived from Cyn d I. In spite of the similarity in the amino acid composition between Cyn d I and group I allergens of other grass pollens, none of our four anti-Cyn d I MoAbs cross-reacted with 10 other grass pollens tested, including ryegrass pollen. Despite biochemical similarity with other group I allergens, the B-cell epitopes on Cyn d I are different from those on other grass pollens.

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[101] - Giroux F, Cano Y, Malandain H. Intérêt diagnostique de l’allergène naturel nCyn d 1 (pollen de chiendent digité, Cynodon dactylon). Rev Fr Allergol 2009;49:330

Objectif.ˆ Le chiendent digité, Cynodon dactylon, est une graminée prospérant surtout en climat chaud. Cette graminée appartient à une sous-famille distante de celle des Pooïdées (dactyle, fléole. . .), de sorte qu‚elle est parfois ajoutée aux Pooïdées dans le protocole de désensibilisation. Il est depuis peu possible de tester in vitro l‚IgE-réactivité vis-à-vis de nCyn d 1, un allergène du chiendent, homologue de Phl p 1 (fléole), etc. Ce test permet-il de préciser le diagnostic et d‚éclairer le choix d‚inclure ou non Cynodon dans le protocole de désensibilisation ? Méthodes.ˆ L‚étude a porté sur 18 patients polliniques aux graminées. La technique CAP Phadia1 a été utilisée pour mesurer l‚IgE-réactivité à nCyn d 1, à rPhl p 1 et à un témoin d‚IgE-réactivité vis-à-vis des épitopes glucidiques (CCD), la broméline. L‚interférence due aux IgE anti-CCD a été évaluée à l‚aide d‚une méthode d‚immuno-absorption et en étudiant également 3 sujets allergiques aux venins d‚hyménoptères et positifs en broméline. Résultats.ˆ Tous les patients polliniques sont positifs pour nCyn d 1,même après immuno-absorption des IgE anti-CCD. L‚allergène Cyn d 1 croise donc avec les allergènes homologues du groupe 1 des graminées, comme Phl p 1. Et si le patient présente une IgE-réactivité vis-à-vis des CCD, la réponse en nCyn d 1 est interférée, surtout quand le rapport rPhl p 1ˆbroméline est faible. Conclusion.ˆ Il semble difficile de tirer parti du test in vitro pour nCyn d 1 en France où les pollens de chiendent digité sont très minoritaires parmi les pollens de graminées : en effet, une IgE-réactivité à nCyn d 1 sera avant tout le reflet de la sensibilisation aux allergènes dominants dans l‚environnement (Phl p 1 fléole, Dac g 1 dactyle, Lol p 1 ivraie. . .) et ne permettra pas de juger s‚il est utile ou non d‚associer Cynodon dactylon dans le protocole de désensibilisation du patient. De plus, le test pour nCyn d 1 est interféré par la présence d‚IgE anti- CCD.

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[102] - van Ree R, Foetisch K, Focke-Tejkl M, van Leeuwen A, Aalbers M, Valenta R, et al. Comparison of IgE-binding potency and biological activity of natural and recombinant major allergens from birch, grass and olive pollen and house dust mite, using sera from eight European countries. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°26

Background: Major allergens of the most important inhalant allergen sources have been purified and produced as recombinant allergens. These molecules are candidates to be used as reference materials for allergen standardisation. Objective: To be able to make a choice between natural (nat) and recombinant (rec) molecules as reference materials, both versions of Bet v 1, Phl p 1, Phl p 5, Ole e 1, Der p 1 and 2 and Der f 1 and 2 were produced to allow characterisation of physico-chemical properties and immune reactivity. In this study, the aim was to compare IgE-binding potency and biological activity. Methods: Sera (n=975) from patients with respiratory allergy to birch, grass, olive pollen and/or house dust mite were collected at 11 clinical centres in Europe. Each serum with a SPT > 5mm for any of the 4 allergen sources was tested by RAST for specific IgE against nat and rec major allergen from the respective allergen sources. A selection of 25 sera with IgE against these major allergens was used in a histamine release test (stripped basophil protocol) and a dot-blot to further compare nat and rec allergens. Results: Frequencies of recognition of all eight major allergens were > 80% with Spearman-rank correlations between nat and rec >0.9. IgE-binding to nat and rec Bet v 1 was close to identical (mean ratio: 1.0; p>0.2). For Phl p 1, binding to the rec was 0.6 times weaker (p<0.001). This is caused by incorrect folding of the recombinant molecule. For Phl p 5, two rec isoforms were tested, Phl p 5a (ratio to nat: 0.7) and Phl p 5b (ratio:0.5). A significant number of sera with IgE antibodies to the nat allergen were negative for Phl p 5b. This was not observed for Phl p 5a. For Ole e 1 the ratio rec/nat was 0.6. For both house dust mite group 1 allergens this was 0.5 and for Der p 2 and Der f 2, 0.7 and 0.8 respectively (p<0.001). Dotblot analyses gave very similar results with especially for rec Phl p 1 lower recognition intensities. Assessment of biological activity confirmed differences between nat and rec allergens. Interestingly, a two-fold lower IgE-binding potency in RAST translated into a 100-fold lower biological activity. Conclusion: Both nat and rec major allergens have good IgE-binding potencies, although differences were observed (£ two-fold). These minor differences in IgE-binding potency, however translates into a much stronger decrease in biological activity.

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[103] - van Ree R, Van Leeuwen A, Aalberse RC. How far can we simplify in vitro diagnostics for grass pollen allergy ? A study with 17 whole pollen extracts and purified natural and recombinant major allergens. J Allergy Clin Immunol 1998;102:184-190

BACKGROUND: Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species. Their complex composition hampers accurate standardization . OBJECTIVE: The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens . METHODS: Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species. Cross-reactivity of IgE responses was studied by means of RAST inhibition. Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5 . RESULTS: Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%). The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata. IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays). With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species. Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5. IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE. For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy. With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results . CONCLUSION: One grass species is sufficient for in vitro diagnosis of grass pollen allergy. With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected. Around 80% of the IgE response to grass pollen is directed to these major allergens. Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts.

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[104] - van Oort E, de Heer PG, Dieker M, van Leeuwen AW, Aalberse RC, van Ree R. Characterization of natural Dac g 1 variants: An alternative to recombinant group 1 allergens. J Allergy Clin Immunol 2004;114:1124-1130

Background Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. Objective : Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. Method s : Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. Result s : Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. Conclusions : Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.

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[105] - 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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[106] - Dolecek C, Vrtala S, Laffer S, Steinberger P, Kraft D, Scheiner O, et al. Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen. FEBS Lett 1993;335:299-304

Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C-terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE-binding capacity as the natural allergen.

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[107] - Barre A, Rougé P. Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: structural basis for the cellulose recognition and associated allergenic properties. Biochem Biophys Res Commun 2002;296:1346-1351

A three-dimensional model of the cellulose-binding domain of the rye-grass pollen allergen Lol pI built by homology modeling is proposed as a structural scaffold for expansins and other expansin-related proteins. A groove and an extended strip of aromatic and polar residues presumably account for the cellulose-binding properties of the protein domain. Two of the four predicted T-cell epitopes readily exposed on the surface of the cellulose-binding domain match with previously reported IgE-binding regions. A close structural relationship occurs between the cellulose-binding and allergenic properties

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[108] - Schweimer K, Petersen A, Suck R, Becker WM, Rösch P, Matecko I. Solution structure of Phl p 3, a major allergen from Timothy grass pollen. Biol Chem 2008;389:919-923

Abstract The major 97 amino acid Timothy grass (Phleum pratense) allergen Phl p 3 was recently isolated from Timothy grass pollen extract, and sequence comparison classifies this protein as a group 3 allergen. The solution structure as determined by nuclear magnetic resonance spectroscopy of Phl p 3 shows this protein to consist of a core of hydrophobic amino acid side chains from two beta-sheets of five and four antiparallel beta-strands, respectively. This conformation is highly similar to the published crystal structure of Phl p 2 and strongly resembles the known conformation of the carboxy-terminal domain of Phl p 1, the major difference being the loop orientations. Phl p 2 and Phl p 3 show virtually identical immunoreactivity, and comparison of the charged surface amino acids of the two proteins gives initial clues as to the IgE recognition epitopes of these proteins.

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[109] - Petersen A, Suck R, Lindner B, Georgieva D, Ernst M, Notbohm H, et al. Phl p 3: Structural and immunological characterization of a major allergen of timothy grass pollen. Clin Exp Allergy 2006;36:840-849

BACKGROUND: The relevant importance of individual allergens for allergic sensitization is only partially understood. More detailed information on allergen structure and how it influences immunological responses can lead to better diagnosis of disease and improved preparations for allergen-specific immunotherapy. Grass pollen contains several different allergens, and although the group 3 allergens have been classified long ago, their structure and allergenicity have been poorly investigated . OBJECTIVE: To characterize Phl p 3 from timothy grass pollen and compare it with Phl p 2 with respect to biochemical structure and allergenicity . METHODS: Natural Phl p 2 and Phl p 3 were separated from a pollen extract by chromatography and characterized by 2D electrophoresis and protein sequencing. The complete sequences were determined by DNA cloning and detected in natural pollen extracts by mass spectrometry. Further comparisons of the allergens were made for IgE-binding and cross-reactivity, allergenicity was determined by basophil CD203c activation and skin prick test and 3D structures were compared by molecular modelling . RESULTS: Phl p 3 reveals molecular masses of 10.958 and 10.973 kDa and pIs of 8.9 and 9.3, respectively, Phl p 2 a molecular mass of 10.816 kDa and a pI of 4.6. The sequence identity is 58%. In spite of these differences in the primary structures, both allergens reveal similar conformational structures, resulting in similar immunological and allergological moieties . CONCLUSIONS: The group 3 and group 2 allergens are major allergens with similar 3D structures. Although they differ considerably in their protein sequences and their pIs, they show only a slightly higher immunological reactivity for Phl p 3 on the B-cell level (conformational epitopes). But distinct differences between the sequences may influence reactivity at the T cell level.

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[110] - van Ree R, van Leeuwen WA, van den Berg M, Weller HH, Aalberse RC. IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Identification of the C-termini of Lol p I, II, and III as cross-reactive structures. Allergy 1994;49:254-261

In this study, the homologous C-termini of Lol p I, Lol p II, and Lol p III were shown to contain cross-reactive B-cell epitopes. This was demonstrated by inhibition studies with purified Lol p I, II, and III and synthetic peptides of their C-termini. It was ruled out that the observed cross-reactivity was caused by cross-contamination of the purified allergens. Both human IgE and IgG bound to the C-terminus of Lol p I. These antibodies were cross-reactive with Lol p II and, more specifically, with its C-terminus. Within a small panel of allergic patients, no cross-reactivity with Lol p III was found. A hyperimmune polyclonal rabbit antiserum against Lol p I also recognized the Lol p I C-terminus. As for human antibodies, cross-reactivity with Lol p II and its C-terminus was demonstrated. Cross-reactivity with Lol p III was demonstrated with C-terminal peptides, but not with native Lol p III. A polyclonal rabbit antiserum against Lol p II bound to the C-terminal peptides of both Lol p II and III. This binding was inhibited with Lol p I, confirming that cross-reactive structures exist not only on the C-termini of Lol p II and Lol p I, but also of Lol p III and Lol p I. The existence of cross-reactivity between Lol p I and Lol p II and III possibly contributes to the frequently observed cosensitization for these allergens in grass-pollen-allergic patients.

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[111] - Marth K, Focke M, Flicker S, Valenta R. Human monoclonal antibody-based quantification of group 2 grass pollen allergens. J Allergy Clin Immunol 2004;113:470-474

BACKGROUND: Grasses belong to the most potent allergen sources worldwide. Group 2 grass pollen allergens are recognized by more than 100 million allergic patients . OBJECTIVE: The aim was to develop an assay for the specific detection and quantification of group 2 grass pollen allergens . METHODS: We have isolated a monoclonal human IgE Fab specific for group 2 grass pollen allergens by combinatorial cloning from lymphocytes of a grass pollen-allergic patient. This Fab was converted into a complete human IgG1 antibody and used together with rPh1 p 2 to develop a competitive ELISA for the specific measurement of group 2 allergens. ELISA plate-bound purified recombinant human Ph1 p 2-specific IgG1 is incubated with a constant amount of biotinylated rPh1 p 2 competing with increasing concentrations of group 2 allergens to be determined. Defined concentrations of purified rPhl p 2 are used to establish a standard curve. The concentration of unlabeled group 2 allergens can thus be deduced from the displacement of biotinylated rPh1 p 2, which can be detected with peroxidase-labeled streptavidin . RESULTS: The competition-ELISA measured rPh1 p 2 concentrations ranging from 10 ng/mL to 500 ng/mL and allowed to quantify group 2 allergens from 9 different grass families. The results were in good agreement with immunoblot data . CONCLUSIONS: The described assay can be used for standardization of diagnostic and therapeutic vaccines as well as for the quantification of group 2 allergens in environmental samples.

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[112] - Leduc-Brodard V, Inacio F, Jaquinod M, Forest E, David B, Peltre G. Characterization of Dac g 4, a major basic allergen from Dactylis glomerata pollen. J Allergy Clin Immunol 1996;98:1065-1072

Monoclonal antibodies were produced against Dac g 4, a purified major basic allergen from Dactylis glomerata pollen. Their ability to be used for immunopurification of Dac g 4 was studied on a BIAcore apparatus (Pharmacia). The allergen was purified by affinity chromatography with one monoclonal antibody. Its precise molecular mass, 59,185 +/- 30 d, was determined by mass spectrometry. Its isoelectric point is 10.4. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting showed that Dac g 4-related proteins of similar molecular mass were detected in the majority of allergenic grass pollen species. By double-site ELISAs, we have estimated that Dac g 4 represents about 6% of the total proteins from a water-soluble extract. One monoclonal antibody (mAb H) recognized a 60 kd cross-reactive protein in other grass pollens, though none in any of the tree or weed pollens tested. Inhibition studies of IgE antibody binding to Dac g 4 with pollen extracts confirmed the presence of cross-reactive allergens in Secale cereale, Lolium perenne, Festuca elatior, Holcus lanatus, Bromus arvensis, Poa pratense, Hordeum sativum, and Phleum pratense.

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[113] - Marknell DeWitt A, Andersson K, Peltre G, Lidholm J. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy 2006;36:77-86

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein . OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4 . METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies . RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner . CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

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[114] - Nandy A, Petersen A, Wald M, Suck R, Kahlert H, Weber B, et al. Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense). Biochem Biophys Res Commun 2005;337:563-570

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

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[115] - 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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[116] - 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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[117] - 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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[118] - Marknell DeWitt A, Andersson K, Peltre G, Lidholm J. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy 2006;36:77-86

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein . OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4 . METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies . RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner . CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

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[119] - 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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[120] - Gavrovic-Jankulovic M, Cirkovic T, Bukilica M, Fahlbusch B, Petrovic S, Jankov RM. Isolation and partial characterization of Fes p 4 allergen. J Investig Allergol Clin Immunol 2000;10:361-367

More than 75% of grass pollen-allergic patients produce specific IgE antibodies against group-4 allergens. Purification and characterization of different grass group-4 allergens should help to further understand their allergenicity. In this study, an attempt was made to isolate and characterize Fes p 4 allergen by several biochemical and immunochemical methods. Fes p 4 was purified by a combination of chromatographic techniques (gel permeation and ion exchange chromatography). Isolated protein revealed four main spots at a molecular weight of 60 kDa and a pI ranging from 8.7 to 9.1. Eight sera were selected from patients with positive result of skin prick test to the mixture of grass pollen extracts. ELISA inhibition technique was used to study Fes p 4-specific IgE in the patients' sera. ELISA to Festuca pratensis was inhibited up to 80% by F. pratensis pollen extract and up to 48% by Fes p 4. 2D-PAGE-immunoblot was used to identify allergenic and antigenic components of Fes p 4 with patients' IgE and monoclonal antibodies (MABs). Three components of purified protein expressed IgE binding ability. Two MABs which recognized unrelated regions on Phl p 4, bound three components of Fes p 4. The role of the carbohydrate moiety in allergenicity was examined with individual patient sera by using periodate-treated Fes p 4. Six out of eight patients reduced IgE binding to periodate-treated allergen. Isolated Fes R 4 glycoprotein consisted of four components, three of which were allergenic, and share common epitopes specific for grass group-4 homologs. The results of periodate oxidation of Fes p 4 suggest that the carbohydrate moiety is involved in IgE binding.

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[121] - Liaw S, Lee DY, Chow L, Lau GX, Su SN. Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein. Biochem Biophys Res Commun 2001;280:738-743

(erratum appears in Biochem Biophys Res Commun 2001 Mar 16;281(5):1359) Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.

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[122] - Nandy A, Petersen A, Wald M, Suck R, Kahlert H, Weber B, et al. Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense). Biochem Biophys Res Commun 2005;337:563-570

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

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[123] - Nandy A, Wald M, Graefe L, Cromwell O, Fiebig H. DNA sequences of group 4 allergens from rye, wheat, barley and Lolium perenne: Comparison with isoforms of Phleum pratense Phl p 4. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°484

Background: Grass pollen allergy is one of the most important allergic diseases world-wide. Several grass species grown in meadows, like P. pratense and L. perenne, contribute to allergic sensitisations, but also allergens from extensively cultured cereals, especially rye, make a profound contribution to the development of allergy. The group 4 major allergen of P. pratense, Phl p 4, is recognised by more than 70 % of grass allergic patients. IgE-binding cross-reactivity has been described for some group 4 allergens of different grass species, but on the DNA-level only the Phl p 4 gene could be deciphered until now. Method: Based on the DNA sequence of Phl p 4 several PCR-primer sequences with cross-reactivity to DNA sequences of related species could be designed. The group 4 DNA sequences of L. perenne (Lol p 4), Secale cereale (Sec c 4), Hordeum vulgare (Hor v 4), and Triticum aestivum (Tri a 4) have been amplified, cloned and sequenced. Results: The Pooideae group 4 allergens represent a family of basic proteins with molecular weights of about 55 kDa and calculated pI values far above 8. In rye, barley and P. pratense distinct isoforms with amino acid identities of 88 to 94 % could be detected. Additionally these isoforms exist in different minor variants. The interspecies homology is settled in the range from 83 % (Phl p 4 to Triticeae species) to 95 % (Sec c 4 to Tri a 4). Conclusion: The group 4 allergens represent a family of proteins that are conserved among different grass species. The occurrence of cross-reacting isoforms in distinct species with amino acid homologies that are comparable to those of different group 4 molecules across the species border is remarkable. Since recombinant group 4 allergens may be important for a future recombinant allergen based specific immunotherapy, strong efforts should be made to evaluate the cross-reactive therapeutic potential of the different group 4 allergens and their isoforms.

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[124] - Nandy A, Petersen A, Wald M, Suck R, Kahlert H, Weber B, et al. Primary structure, recombinant expression, and molecular characterization of Phl p 4, a major allergen of timothy grass (Phleum pratense). Biochem Biophys Res Commun 2005;337:563-570

Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.

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[125] - Marknell DeWitt A, Andersson K, Peltre G, Lidholm J. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy 2006;36:77-86

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein . OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4 . METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies . RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner . CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

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[126] - Su SN, Lau GX, Yang SY, Shen HD, Tsai JJ, Han SH. Isolation and partial characterization of Bermuda grass-pollen allergen: BG-60b. Proc Natl Sci Counc Repub China B 1990;14:85-90

Our earlier studies have shown that the pollen of Bermuda grass (Cynodon dactylon)-pollen contain at least 12 IgE-binding proteins which can be analyzed by the immunoblot technique. One of the highly active components was found to be a basic protein with a molecular weight of 60,000 daltons, designated as BG-60. This component was showed to consist of a group of proteins. One of them, BG-60a (pI 9.7), has been isolated and characterized. In this study, we have further isolated and characterized the second component of the antigen, designated as BG-60b. Its purity was demonstrated by gel electrophoresis experiment and antigen-antibody precipitation studies. The antigen is of glycoprotein nature with a pI of 10.0. It exhibits IgE-binding activity and shows cross-reactivity to antigen BG-60a in double diffusion. Its chemical and physical properties are similar to antigen BG-60a

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[127] - Nandy A, Wald M, Cornelissen G, Oltersdorf N, Weber B, Kahlert H, et al. Timothy grass pollen allergen Phl p 4: Purification and characterisation of N-glycosylation site variants expressed in Pichia pastoris. Allergy 2007;62(suppl. 83):183

Background Phleum pratense Phl p 4 is a major allergen of timothy grass pollen with reported IgE frequencies of up to 88 %. The calculated molecular mass of Phl p 4 (56 kDa) differs from the apparent molecular mass of natural Phl p 4 (~ 65 kDa) observed in SDS-PAGE as a result of post-translational glycosylation. Two N-glycosylation consensus sequences are present in the Phl p 4.0201 sequence. Expression in E. coli yields a recombinant protein of the expected calculated size, but so far a correct folding could not be achieved. Expression in the methylotrophic yeast P. pastoris yields folded, soluble, but glycosylated recombinant Phl p 4. P. pastoris is known to add mannose rich carbohydrate moieties that differ from those of plant derived proteins at N-glycosylation sites. The development of non-glycosylated recombinant Phl p 4 is needed to determine true IgE-frequencies based on protein IgE epitopes and for the formulation of a pharmaceutical grade recombinant compound for allergen specific immunotherapy. Method Site directed mutagenesis was performed using a PCR method to generate three site-directed N-linked glycosylation consensus site variants of Phl p 4: N61Q, N330Q, and N61Q/N330Q. The variants were expressed in P. pastoris fused to the Saccharomyces cerevisae a-factor signal sequence to achieve secretion of the recombinant proteins into the culture media. The recombinant variants were compared by SDS-PAGE, IgE-binding assays and by two-site quantification ELISA using monoclonal antibodies 3C4 und 5H1. Results The P. pastoris derived Phl p 4 glycosylation-site variants were purified to homogeneity. The glycosylation at N330 proved to be responsible for the majority of post-translational added carbohydrates as observed by the absence of the high molecular weight smear of Phl p 4 N330Q in SDS-PAGE. The Phl p 4 N61Q/N330Q double mutant appeared at the expected calculated size of ~56 kDa in SDS-PAGE. IgE binding assays using allergic patients' sera showed a good correlation of the reactivity of natural and recombinant Phl p 4. In contrast to natural Phl p 4 the recombinant hyper-glycosylated Phl p 4 displayed a drastically reduced reactivity in a two-site binding ELISA using mAb 3C4 and 5H1. Conclusion Recombinant Phl p 4 glycosylation-site variants expressed in P. pastoris are promising candidates for diagnostic applications and for specific immunotherapy.

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[128] - Stumvoll S, Lidholm J, Thunberg R, DeWitt AM, Eibensteiner P, Swoboda I, et al. Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential. Biol Chem 2002;383:1383-1396

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

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[129] - Gavrovic-Jankulovic M, Cirkovic T, Bukilica M, Fahlbusch B, Petrovic S, Jankov RM. Isolation and partial characterization of Fes p 4 allergen. J Investig Allergol Clin Immunol 2000;10:361-367

More than 75% of grass pollen-allergic patients produce specific IgE antibodies against group-4 allergens. Purification and characterization of different grass group-4 allergens should help to further understand their allergenicity. In this study, an attempt was made to isolate and characterize Fes p 4 allergen by several biochemical and immunochemical methods. Fes p 4 was purified by a combination of chromatographic techniques (gel permeation and ion exchange chromatography). Isolated protein revealed four main spots at a molecular weight of 60 kDa and a pI ranging from 8.7 to 9.1. Eight sera were selected from patients with positive result of skin prick test to the mixture of grass pollen extracts. ELISA inhibition technique was used to study Fes p 4-specific IgE in the patients' sera. ELISA to Festuca pratensis was inhibited up to 80% by F. pratensis pollen extract and up to 48% by Fes p 4. 2D-PAGE-immunoblot was used to identify allergenic and antigenic components of Fes p 4 with patients' IgE and monoclonal antibodies (MABs). Three components of purified protein expressed IgE binding ability. Two MABs which recognized unrelated regions on Phl p 4, bound three components of Fes p 4. The role of the carbohydrate moiety in allergenicity was examined with individual patient sera by using periodate-treated Fes p 4. Six out of eight patients reduced IgE binding to periodate-treated allergen. Isolated Fes R 4 glycoprotein consisted of four components, three of which were allergenic, and share common epitopes specific for grass group-4 homologs. The results of periodate oxidation of Fes p 4 suggest that the carbohydrate moiety is involved in IgE binding.

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[130] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[131] - Smith PM, Ong EK, Knox RB, Singh MB. Immunological relationships among group I and group V allergens from grass pollen. Mol Immunol 1994;31:491-498

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

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[132] - Bufe A, Uhlig U, Scholzen T, Matousek J, Schlaak M, Weber W. A nonspecific, single-stranded nuclease activity with characteristics of a topoisomerase found in a major grass pollen allergen: possible biological significance. Biol Chem 1999;380:1009-1016

The major allergen from timothy grass pollen, Phlp5b (Phleum pratense), was shown to exhibit ribonuclease activity. It turned out that the C-terminal portion of this molecule was the biologically active domain. Here evidence is presented that the allergen is a single-stranded, sugar-nonspecific nuclease with topoisomerase activity. An isomerase-specific active site was identified, and a non-active mutant was constructed by site directed mutagenesis, and showed no nucleolytic activity. In contrast to the wild type (WT), the mutant did not dimerize. Although the binding capacity of IgE antibodies toward the mutant was reduced as compared to the WT, the allergenic activity was retained. We conclude that the allergen Phlp5b is a single-stranded nuclease with an unusual topoisomerase-like activity. This biological activity is not by itself connected to the allergenicity of the molecule. Whether the enzymatic activity is responsible for the induction of the allergic sensitization and inflammation remains an open question.

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[133] - Ong EK, Griffith IJ, Knox RB, Singh MB. Cloning of a cDNA encoding a group-V (group-IX) allergen isoform from ryegrass pollen that demonstrates specific antigenic immunoreactivity. Gene 1993;134:235-240

We have isolated and characterized the cDNA clone, 19R, that encodes an isoform of a major rye-grass pollen allergen, Lol p V [previously referred to as Lol p 1b; Singh et al., Proc. Natl. Acad. Sci. USA 88 (1991) 1384-1388; and Lol p IX; Suphioglu et al., Lancet 339 (1992) 569-572] Clone 19R was isolated from a rye-grass pollen cDNA expression library using grass pollen-specific immunoglobulin E (IgE) antibodies (Ab) from an allergic serum pool. The nucleotide (nt) sequence of clone 19R potentially encodes a 33.8-kDa protein of 339 amino acids (aa). It possesses a leader peptide essentially identical to the previously characterized isoform of Lol p V (Lol p VA). This indicates a mature processed 31.3-kDa protein of 314 aa, correlating well with the size of the polypeptides revealed by Western analysis of pollen proteins using IgE Ab affinity purified from recombinant fusion protein (reFP) encoded by clone 19R as solid matrix There is no N-glycosylation motif. The protein encoded by clone 19R, designated Lol p VB, has 66.4% identity and 80.4% similarity with Lol p VA. However, a Lol p VA-specific monoclonal Ab, FMC A7, does not recognize reFP encoded by clone 19R, indicating that Lol p VB does not share this epitope. Cross-reactivity studies using affinity purified IgE Ab showed that both isoforms share similar allergenic epitopes. Immunoblot analysis using sera from a population of 30 patients showed that 80% possess IgE Ab that recognize both Lol p V isoforms. Variation occurred in the signal intensities of IgE binding.

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[134] - Becker WM, Bufe A, Petersen A, Schlaak M. Molecular characterization of timothy grass pollen group V allergens. Int Arch Allergy Immunol 1995;107:242-244

Phl p V is the dominant allergen of timothy grass (Phleum pratense) with two isoforms having the apparent molecular weights of 38 (Phl p Va) and 32 kD (Phl p Vb) under Western blot conditions. Two-dimensional electrophoresis/immunoblotting reveals that each isoform is split into at least four isoallergens. Structural differences in the isoforms are shown by N-terminal sequencing (only 60% identity), by reaction patterns of monoclonal antibodies and, more convincingly, by enzymic degradation of purified isoforms followed by immunologic fingerprinting. These findings are confirmed by the deduced primary protein structure of cloned Phl p Va and Phl p Vb. Experiments with IgE--affinity-purified by immobilized recombinant allergens or their fragments--reveal identical epitopes and at least one different epitope between the isoforms. Furthermore, on Phl p Va we can localize different IgE-reactive epitopes at the C terminus as well as the N terminus. By probing serum from 11 patients on recombinant C- or N-terminal fragments, an individual reaction pattern was found. Testing the histamine liberation potency of the fragments, we found the N-terminal fragment of Phl p Va to be superior to that of the C-terminal fragment or the whole molecule. These results give insights into the variability of allergens, the individuality of human reaction patterns to epitopes and the alteration of allergenicity to higher or lower levels by fragmentation. [References: 9]

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[135] - Suphioglu C, Singh MB. Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen. Clin Exp Allergy 1995;25:853-865

BACKGROUND: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. OBJECTIVE: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. METHODS: A canary grass pollen lambda gt11 cDNA expression library was constructed and screened with sera of grass- pollen-sensitive patients. IgE-reactive clones were isolated, sub- cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. RESULTS: One of the clones encoded the group 1 a llergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol l 1, from velvet grass pollen and 86.6% with Phl p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6-95.5% nucleotide and 40.1-81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. CONCLUSIONS: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens

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[136] - Ramirez J, Obispo TM, Duffort O, Carpizo JA, Chamorro MJ, Barber D, et al. Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA - Correlation with biologic activity. Allergy 1997;52:806-813

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

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[137] - Roberts AM, van Ree R, Emly J, Cardy SM, Rottier MMA, Walker MR. N-terminal amino acid sequence homologies of group V grass pollen allergens. Int Arch Allergy Immunol 1992;98:178-180

In an earlier study, we presented data regarding the immunoaffinity purification and N-terminal sequencing of a major pollen allergen from orchard/cocks-foot grass (Dactylis glomerata), now identified as the group V allergen Dac g V. In this paper, we have extended our investigations to include group V allergens from other grass species. Our data confirm the presence of group V-restricted characteristic N-terminal amino acid sequences containing a high alanine and hydroxyproline (P') rather than proline (P) content, and based upon two conserved elements (ADAGY and TPA/TP'A).

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[138] - Suphioglu C, Mawdsley D, Schappi G, Gruehn S, de Leon M, Rolland JM, et al. Molecular cloning, expression and immunological characterisation of Lol p 5C, a novel allergen isoform of rye grass pollen demonstrating high IgE reactivity. FEBS Lett 1999;462:435-441

A novel isoform of a major rye grass pollen allergen Lol p 5 was isolated from a cDNA expression library. The new isoform, Lol p 5C, shares 95% amino acid sequence identity with Lol p 5A. Both isoforms demonstrated shared antigenic activity but different allergenic activities. Recombinant Lol p 5C demonstrated 100% IgE reactivity in 22 rye grass pollen sensitive patients. In comparison, recombinant Lol p 5A showed IgE reactivity in less than 64% of the patients. Therefore, Lol p 5C represents a novel and highly IgE-reactive isoform allergen of rye grass pollen.

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[139] - Schramm G, Bufe A, Petersen A, Schlaak M, Becker WM. Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus). Eur J Biochem 1998;252:200-206

Group V grass pollen allergens belong to the major grass pollen allergens causing reactions of type I allergy. cDNAs coding for two isoforms of the group V allergen of velvet grass pollen (Holcus lanatus), a widespread grass species, were isolated from a cDNA library by hybridization with a 5'-terminal reverse-transcribed PCR-derived cDNA probe. Amino acid sequences of the two isoforms, designated rHol l 5.01 and rHol l 5.02, revealed high similarity between them (78% identity) and to group V allergens and their isoforms found in other grass species. Recombinant Hol l 5 isoforms were expressed in Escherichia coli and purified as fusion proteins. To compare their immunological reactivities with group-V-specific monoclonal antibodies and patients' IgE, immunoblotting, ELISA and histamine release assay were performed. Interestingly, monoclonal antibody Bo9, specific for group Vb isoforms of timothy grass, bound only to isoform rHol l 5.01, not to rHol l 5.02. On the other side, IgE reactivities of patients' sera revealed no differences between the two isoforms when investigated by immunoblotting and only slight differences when investigated by ELISA. In histamine release assay both isoforms released comparable amounts of histamine from basophils of four individual patients. Thus, the two group V isoforms of velvet grass pollen exhibit differential binding when tested with monoclonal antibodies, i.e. different structure of single epitopes, but negligible differences concerning overall IgE-binding capacity and histamine-releasing capacity.

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[140] - Rajashankar K, Bufe A, Weber W, Eschenburg S, Lindner B, Betzel C. Structure of the functional domain of the major grass-pollen allergen Phlp 5b. Acta Crystallogr D Biol Crystallogr 2002;58:1175-1181

The major allergen Phlp 5b from timothy grass pollen induces allergic rhinitis and bronchial asthma in millions of allergic patients worldwide. As an important step towards understanding the interactions between the pollen protein and components of the human immune system, the structure of the C-terminal key domain of Phlp 5b has been determined at 2.0 A resolution and refined to an R value of 19.7%. This is the first known allergen composed entirely of alpha-helices. The protein forms a dimer stabilized by one intermolecular disulfide bridge. Sequence homology suggests that at least all group V and group VI grass-pollen allergens belong to this new class of 'four-helix-bundle allergens'.

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[141] - Meno K, Ipsen H, Gajhede M, Spangfort M. Molecular characterisation of the major timothy grass pollen allergen Phl p 5. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°911

The group 5 allergen from timothy grass (Phleum pratense, Phl p 5) is one of the major allergens in grass pollen. Approximately 80% of patients with grass pollen allergy produce IgE antibodies against Phl p 5. The amino acid composition of Phl p 5 is unique containing almost 30 mole% alanine and no conserved cysteine residues. The amino acid sequence has no similarity to any other known allergen or protein in general except for the highly homologous Phl p 6 allergen and other grass group 5 allergens. Two recombinant isoforms of Phl p 5 (rPhl p 5.01, formerly rPhl p 5a) were purified and characterised. The multimerisation state in solution was investigated by size exclusion chromatography and by dynamic light scattering, and both techniques indicated that rPhl p 5.01 exists as a dimer in solution. Circular dichroism (CD) analysis performed at acidic, neutral, and basic pH under conditions where rPhl p 5.01 was stable showed that the secondary structure of rPhl p 5.01 is dominated by a-helices, which is consistent with the result obtained with natural Phl p 5 (nPhl p 5) purified from grass pollen. When monitoring the thermal denaturation of rPhl p 5.01 using CD, the unfolding appears monophasic with a melting temperature of at least 62-65°C at the three pH values investigated. The nPhl p 5/rPhl p 5.01 proteins are unstable and degrade in solution generating a range of smaller fragments. The identity of a substantial number of the degradation products was identified by means of mass spectroscopy for the two recombinant isoforms. Furthermore, the temperature and pH dependence of the degradation was determined for the two rPhl p 5.01 isoforms and for nPhl p 5, and the results indicated that these proteins have a considerably lower stability at acidic pH and a decreasing stability with temperature. The dimerisation and degradation of Phl p 5 has important implications for antibody binding and use of grass pollen allergen extracts in allergy vaccination.

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[142] - Maglio O, Saldanha JW, Vrtala S, Spitzauer S, Valenta R, Pastore A. A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle. Protein Eng 2002;15:635-642

Phl p 5, a 29 kDa major allergen from timothy grass pollen, is one of the most reactive members of group 5 allergens. Its sequence comprises two repeats of a novel alanine-rich motif (AR) whose structure and allergenic response are still mostly unkown. We report here a structural characterization of an immunodominant fragment of Phl p 5, Phl p 5(56-165) which comprises the first AR repeat. Recombinant (r)Phl p 5(56-165) was expressed in Escherichia coli, purified to homogeneity and shown to be sufficient to react with serum IgE from 90% of grass pollen allergic patients. Using NMR spectroscopy, we show conclusively that the fragment forms a compact globular domain which is, however, prone to degradation with time. The rPhl p 5(56-165) fold consists of a four-helix bundle held together by hydrophobic interactions between the aromatic rings and aliphatic side chains. This evidence gives clear indications about the structure of the full-length Phl p 5 and provides a rational basis for finding ways to stabilize the fold and designing therapeutic vaccines against grass pollen allergy.

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[143] - Subroto T, Van Koningsveld GA, Schreuder HA, Soedjanaatmadja UMS, Beintema JJ. Chitinase and beta-1,3-glucanase in the lutoid-body fraction of Hevea latex. Phytochemistry 1996;43:29-37

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a beta-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charg neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.

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[144] - Maglio O, Saldanha JW, Vrtala S, Spitzauer S, Valenta R, Pastore A. A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle. Protein Eng 2002;15:635-642

Phl p 5, a 29 kDa major allergen from timothy grass pollen, is one of the most reactive members of group 5 allergens. Its sequence comprises two repeats of a novel alanine-rich motif (AR) whose structure and allergenic response are still mostly unkown. We report here a structural characterization of an immunodominant fragment of Phl p 5, Phl p 5(56-165) which comprises the first AR repeat. Recombinant (r)Phl p 5(56-165) was expressed in Escherichia coli, purified to homogeneity and shown to be sufficient to react with serum IgE from 90% of grass pollen allergic patients. Using NMR spectroscopy, we show conclusively that the fragment forms a compact globular domain which is, however, prone to degradation with time. The rPhl p 5(56-165) fold consists of a four-helix bundle held together by hydrophobic interactions between the aromatic rings and aliphatic side chains. This evidence gives clear indications about the structure of the full-length Phl p 5 and provides a rational basis for finding ways to stabilize the fold and designing therapeutic vaccines against grass pollen allergy.

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[145] - Vrtala S, Fischer S, Grote M, Vangelista L, Pastore A, Sperr WR, et al. Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen. J Immunol 1999;163:5489-5496

Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.

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[146] - Vrtala S, Fischer S, Grote M, Vangelista L, Pastore A, Sperr WR, et al. Molecular, immunological, and structural characterization of Phl p 6, a major allergen and P-particle-associated protein from Timothy grass (Phleum pratense) pollen. J Immunol 1999;163:5489-5496

Due to the wide distribution and heavy pollen production of grasses, approximately 50% of allergic patients are sensitized against grass pollen allergens. cDNAs coding for two isoforms and four fragments of a major timothy grass (Phleum pratense) pollen allergen, Phl p 6, were isolated by IgE immunoscreening from a pollen expression cDNA library. Recombinant Phl p 6 (rPhl p 6), an acidic protein of 11.8 kDa, was purified to homogeneity as assessed by mass spectrometry and exhibited almost exclusive alpha-helical secondary structure as determined by circular dichroism spectroscopy. Phl p 6 reacted with serum IgE from 75% of grass pollen-allergic patients (n = 171). IgE binding experiments with rPhl p 6 fragments indicated that the N terminus of the allergen is required for IgE recognition. Purified rPhl p 6 elicited dose-dependent basophil histamine release and immediate type skin reactions in patients allergic to grass pollen. A rabbit antiserum raised against purified rPhl p 6 identified it as a pollen-specific protein that, by immunogold electron microscopy, was localized on the polysaccharide-containing wall-precursor bodies (P-particles). The association of Phl p 6 with P-particles may facilitate its intrusion into the deeper airways and thus be responsible for the high prevalence of IgE recognition of Phl p 6. Recombinant native-like Phl p 6 can be used for in vitro as well as in vivo diagnoses of grass pollen allergy, whereas N-terminal deletion mutants with reduced IgE binding capacity may represent candidates for immunotherapy of grass pollen allergy with a low risk of anaphylactic side effects.

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[147] - Støvhase R, Lund G, Lenhard T, Henmar H, Friberg L, Giselsson A, et al. Allergenic cross-reactivity of Phl p 5, Phl p 6 and their derived recombinant protein fragments. Allergy 2007;62(suppl. 83):157

Rationale: Phlp5 and Phlp6 from P.pratense share a high degree of sequence similarity. We have investigated to what extent the allergen activity of these two proteins relies on immunological cross reactivity or independent sensitizations. Methods: IgE abs were measured in inhibition assays using biotinylated Phlp5 or -6, respectively. Basophil activation assays and generation of specific T-cell lines were done with fresh blood from five grass-allergic individuals. Recombinant proteins were expressed in P.pastoris and characterized with respect to purity(HPLC, MS), folding(CD) and immunogenicity(CRIE, mouse immunizations). Results: All sera contained anti-Phlp5 IgE abs. Three reactivity patterns were found:A)(n=2) IgE antibodies reactive towards Phlp5 and -6 were detected and full basophil activation was obtained with both allergens. Competition of biotinylated Phlp5 with Phlp6 (and vice versa) resulted in partial loss of binding, indicating the presence of cross reacting IgE. B)(n=1) contained no detectable anti-Phlp6 IgEs, but gave rise to full basophil activation when challenged with Phlp5 and Phlp6, respectively. C)(n=2) contained no detectable anti-Phlp6 IgE and gave rise to no or strongly reduced basophil activation when challenged with Phlp6. Sera with reactivity pattern B and C exhibited no inhibition of IgE binding to biotinylated-Phl p5 with Phlp6. In IgE-inhibition assays with pooled sera, recombinant Phlp5 and -6 fragments (N-terminal-, C-terminal-,Phlp5/6 chimeras) gave rise to ca.50% inhibition, relative to intact Phlp5, of IgE-binding to biotinylated Phlp5, indicating that Phlp5 IgE-epitopes are equally distributed on the surface of the protein. T-cell epitopes were found throughout the aa-sequences of both allergens. In basophil activation assays with blood from all five patients the fragments gave rise to similar or reduced activation compared to intact Phlp5. Conclusions: Analyses of serum IgE from grass pollen allergic patients revealed the presence of IgE specific for Phlp5, cross-reactive between Phlp5 and -6, and specific for Phlp6, respectively. This indicates that cross-reactivity of patients‚ sera is determined by the recognition of a subset of epitopes/surface areas on the two allergens, which are subject to patient-to-patient variation. The partial inhibition of IgE binding to biotinylated Phlp6 by Phlp5 is indicative of Phlp6-specific IgEs and suggests that pattern A stems from concurrent sensitization to Phlp5 and -6.

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[148] - Petersen A, Bufe A, Schlaak M, Becker WM. Characterization of the allergen group VI in timothy grass pollen (Phl p 6). I. Immunological and biochemical studies. Int Arch Allergy Immunol 1995;108:49-54

For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pI 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phl p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients' serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes.

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[149] - Petersen A, Bufe A, Schramm G, Schlaak M, Becker WM. Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. : cDNA cloning of Phl p 6 and structural comparison to grass group V. Int Arch Allergy Immunol 1995;108:55-59

The complete primary structure of the major allergen Phl p 6 was determined by cDNA cloning. A cDNA library of timothy grass pollen was screened by a Phl p 6 directed mouse antiserum. Six clones were obtained. From the cDNA data we deduced a protein sequence of 110 amino acids. While the size of the leader sequences varied considerably between the different clones, only one amino acid exchange affected the mature proteins. Sequence comparison of group VI and V allergens showed a high degree of homology in the N- and C-terminal regions. The structural data strongly indicate that group VI and V are independent groups of allergens, which have derived from a common precursor gene.

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[150] - Blume C, Lindner B, Becker WM, Petersen A. Microheterogeneity of the major grass group 6 allergen Phl p 6: Analysis by mass spectrometry. Proteomics 2004;4:1366-1371

The precise structural characterization of allergens is a basic requirement to improve diagnostics and to find therapeutic strategies against allergic disorders. Natural grass pollen allergens exhibit a wide variety of isoforms and it is still unknown whether this microheterogeneity is essential for the allergic reaction or has a functional effect on sensitization. Well-defined recombinant allergens are considered to replace natural allergens for clinical trials. For the major timothy grass pollen allergen Phl p 6 (approximately 12 kDa) and a recombinant rPhl p 6 we determined the structural microheterogeneity by two-dimensional electrophoresis (2-DE), high-resolution electrospray ionization-Fourier transform-mass spectrometry (ESI-FT-MS) of the intact molecules, and by tryptic peptide mass fingerprinting using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Natural Phl p 6 is a mixture of mainly two isoforms that differ by two amino acids leading to a mass difference of 5 Da. For each of this two isoforms six variants were identified with modifications at the C- and/or N-terminus. The recombinant Phl p 6 comprises the same structure as one of the main isoforms indicating that it represents a major part of the natural Phl p 6.

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[151] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[152] - Niederberger V, Hayek B, Vrtala S, Laffer S, Twardosz A, Vangelista L, et al. Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7. FASEB J 1999;13:843-856

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.

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[153] - Smith PM, Xu H, Swoboda I, Singh MB. Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d7: IgE cross-reactivity with oilseed rape pollen allergen Bra r1. Int Arch Allergy Immunol 1997;114:265-271

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

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[154] - Rossi RE, Monasterolo G, Monasterolo S. Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen. Allergy 2001;56:1180-1185

Background: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis. Methods: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens. Results: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed. Conclusions: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.

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[155] - Verdino P, Westritschnig K, Valenta R, Keller W. The cross-reactive calcium-binding pollen allergen, Phl p 7, reveals a novel dimer assembly. EMBO J 2002;21:5007-5016

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.

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[156] - Verdino P, Westritschnig K, Valenta R, Keller W. The cross-reactive calcium-binding pollen allergen, Phl p 7, reveals a novel dimer assembly. EMBO J 2002;21:5007-5016

The timothy grass pollen allergen Phl p 7 assembles most of the IgE epitopes of a novel family of 2 EF-hand calcium-binding proteins and therefore represents a diagnostic marker allergen and vaccine candidate for immunotherapy. Here we report the first three-dimensional structure of a representative of the 2 EF-hand allergen family, Phl p 7, in the calcium-bound form. The protein occurs as a novel dimer assembly with unique features: in contrast to well known EF-hand proteins such as calmodulin, parvalbumin or the S100 proteins, Phl p 7 adopts an extended conformation. Two protein monomers assemble in a head-to-tail arrangement with domain-swapped EF-hand pairing. The intertwined dimer adopts a barrel-like structure with an extended hydrophobic cavity providing a ligand-binding site. Calcium binding acts as a conformational switch between an open and a closed dimeric form of Phl p 7. These findings are interesting in the context of lipid- and calcium-dependent pollen tube growth. Furthermore, the structure of Phl p 7 allows for the rational development of vaccine strategies for treatment of sensitized allergic patients.

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[157] - Smith PM, Xu H, Swoboda I, Singh MB. Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d7: IgE cross-reactivity with oilseed rape pollen allergen Bra r1. Int Arch Allergy Immunol 1997;114:265-271

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

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[158] - Suphioglu C, Ferreira F, Knox RB. Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen. FEBS Lett 1997;402:167-172

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+-binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+-binding proteins.

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[159] - Niederberger V, Hayek B, Vrtala S, Laffer S, Twardosz A, Vangelista L, et al. Calcium-dependent immunoglobulin E recognition of the apo- and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7. FASEB J 1999;13:843-856

Type I allergy, an immunodisorder that affects almost 20% of the population worldwide, is based on the immunoglobulin E (IgE) recognition of per se innocuous antigens (allergens). Pollen from wind-pollinated plants belong to the most potent allergen sources. We report the isolation of a cDNA coding for a 8.6 kDa two EF-hand calcium binding allergen, Phl p 7, from a timothy grass (Phleum pratense) pollen expression cDNA library, using serum IgE from a grass pollen allergic patient. Sequence analysis identified Phl p 7 as a member of a recently discovered subfamily of pollen-specific calcium binding proteins. Recombinant Phl p 7 was expressed in Escherichia coli and purified to homogeneity as determined by mass spectroscopy. Approximately 10% of pollen allergic patients displayed IgE reactivity to rPhl p 7 and Phl p 7-homologous allergens present in pollens of monocotyledonic and dicotyledonic plants. Circular dichroism analysis of the calcium-bound and apo-rPhl p 7 indicated that differences in IgE recognition may be due to calcium-induced changes in the protein conformation. The fact that patients mount IgE antibodies against different protein conformations is interpreted as a footprint of a preferential sensitization against either form. The biological activity of rPhl p 7 was demonstrated by its ability to induce basophil histamine release and immediate type skin reactions in sensitized individuals. In conclusion, IgE binding to Phl p 7 represents an example for the conformation-dependent IgE recognition of an allergen. Recombinant Phl p 7 may be used for diagnosis and perhaps treatment of a group of patients who suffer from allergy to pollens of many unrelated plant species.

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[160] - Ekramoddoullah AK, Kisil FT, Sehon AH. Allergenic cross-reactivity of cytochromes c of Kentucky bluegrass and perennial ryegrass pollens. Mol Immunol 1982;19:1527-1534

The allergenic cross-reactivity of cytochromes c isolated from Kentucky bluegrass (KBG) and ryegrass (RG) pollens was demonstrated by the finding that both cytochromes elicited PCA reactions of comparable titers in rats sensitized with murine reaginic antiserum to either KBG or RG cytochrome c. However, allergenic differences were revealed at limiting doses of the cytochromes c; under these conditions RG cytochrome c elicited PCA reactions only with the homologous reaginic serum, whereas KBG cytochrome c elicited PCA reactions with both murine reaginic sera. Moreover, in experiments involving inhibition of PCA, RG cytochrome c failed to neutralize some of the IgE antibodies of the antiserum to KBG cytochrome c, whereas KBG cytochrome c neutralized all IgE antibodies of both murine reaginic antisera. From these results it may be concluded that: (1) the two cytochromes c share common allergenic determinants, and (2) the KBG cytochrome c possesses additional allergenic determinant(s) not present on RG cytochrome c. The radioallergosorbent test, utilizing KBG cytochrome c discs and a pool of sera of individuals allergic to KBG, was inhibited to the extent of 87 or 41% by the addition of 10 micrograms of KBG or RG cytochrome c, respectively. By contrast both cytochromes c at 10 micrograms were equally effective in the inhibition (92%) of the binding of the IgE antibodies in this serum pool to RG cytochrome c allergosorbent discs. These experiments confirmed that the two cytochromes share common allergenic determinants.

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[161] - Sharma V, Singh BP, Gaur SN, Arora N. Molecular and immunological characterization of cytochrome c: a potential cross-reactive allergen in fungi and grasses. Allergy 2008;63:189-197

BACKGROUND: Recombinant allergens are required for component-resolved diagnosis/therapy of allergic disorders. The study was aimed to express and characterize an allergenic protein from Curvularia lunata and study its cross-reactivity . METHODS: A clone encoding a 12-kDa protein screened from the cDNA library of C. lunata was sequenced and expressed in pET22b+ vector. The purified protein was characterized by biophysical and immunological methods . RESULTS: The sequence of gene encoding a 12-kDa protein showed homology to cytochrome c. It was expressed in Escherichia coli yielding 0.5 mg protein/l culture and designated as Cur l 3. The absorption and circular dichroism spectrum of Cur l 3 were similar to horse cytochrome c and the protein reacted with cytochrome c antibody. ELISA with different fungal-positive patients' sera showed > or = 3 times specific IgE to Cur l 3 compared with healthy controls. Mice anti-Cur l 3 reacted with tropical and temperate grass extracts. Protein also reacted with grass-positive patients' sera. In vitro stimulation of peripheral blood mononuclear cells from C. lunata, fungi or grass-positive patients with it released significant levels of Th2 cytokines. In vivo testing of this protein in allergic patients showed marked positive skin reactivity in 60% fungal and 43% grass-positive cases. Cross inhibition assays (EC(50)) demonstrated allergenic cross-reactivity of Cur l 3 with fungi and grasses . CONCLUSIONS: Cytochrome c, a major allergen from C. lunata was cloned, sequenced and expressed. It was identified as a cross-reactive allergen among fungi and grasses and has potential for clinical application.

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[162] - van Ree R, Hoffman DR, van Dijk W, Brodard V, Mahieu K, Koeleman CA, et al. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins. J Allergy Clin Immunol 1995;95:970-978

Monoclonal antibodies were obtained against an unknown allergen from Lolium perenne grass pollen. The allergen had an apparent molecular mass of 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Earlier immunoblotting studies had shown that carbohydrate-specific IgG antibodies recognize an antigen of similar size. OBJECTIVE: We sought to characterize the allergen biochemically and immunologically. METHODS: The amino acid sequence of the allergen was determined by automated Edman degradation, and its monosaccharide composition was determined by gas chromatographic analysis. A panel of 270 grass pollen-positive sera was assessed in a RAST with the purified allergen. Protease digestion (proteinase K) and chemical deglycosylation (trifluoromethane sulfonic acid) were used to distinguish between carbohydrate and peptide epitopes for IgE antibodies. RESULTS: The allergen was shown to be a glycoprotein with a molecular mass of 16 kd, of which 8% is carbohydrate. Its amino acid sequence shares 32% homology with soybean trypsin inhibitor (Kunitz) but lacks its active site. No homology was found with known grass pollen allergens, hence it was designated Lol p XI. A high degree of homology (44%) was found with a tree pollen allergen, Ole e I, the major allergen of olive pollen. More than 65% of grass pollen-positive sera had IgE against Lol p XI. IgE reactivity was demonstrated both with the carbohydrate moiety and the peptide backbone. CONCLUSIONS: Lol p XI is a new major grass pollen allergen carrying an IgE-binding carbohydrate determinant. Lol p XI is structurally related to the major allergen from olive pollen.

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[163] - Lombardero M, Obispo T, Calabozo B, Lezaún A, Polo F, Barber D. Cross-reactivity between olive and other species. Role of Ole e 1-related proteins. Allergy 2002;57(suppl. 71):29-34

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[164] - Marknell DeWitt Å, Niederberger V, Lehtonen P, Spitzauer S, Sperr WR, Valent P, et al. Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11. Clin Exp Allergy 2002;32:1329-1340

BackgroundAllergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. ObjectiveTo clone and characterize the 20kDa timothy grass pollen allergen Phl p 11. MethodsPhl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. ResultsPhl p 11 cDNA defined an acidic polypeptide of 15.8kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n=184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. ConclusionAs the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.

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[165] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[166] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[167] - Rossi RE, Monasterolo G, Monasterolo S. Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen. Allergy 2001;56:1180-1185

Background: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis. Methods: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens. Results: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed. Conclusions: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.

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[168] - Wensing M, Akkerdaas JH, van Leeuwen A, Stapel SO, Bruijnzeel-Koomen CAFM, Aalberse RC, et al. IgE to Bet v 1 and profilin: Crossreactivity patterns and clinical relevance. J Allergy Clin Immunol 2002;110:435-442

BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants . OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance . METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition . RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon . CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.

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[169] - Schierz J, Burow G. Determination of antibody pattern with recombinant allergen components in pollen allergic patients. Allergy Clin Immunol Int 2005;17(Suppl. 1):205

Quantitative determination of specific IgE antibodies to recombinant allergen components allows to establish the individual sensitization profiles of allergic patients. The presence of IgE antibodies to crossreactive components can be used to predict sensitization to related allergen sources. The aim of the study is to determine the IgE antibody pattern in sera of grass and birch pollen allergic patients. Methods: Serum samples from 151 patients were analyzed for IgE antibodies to recombinant major allergens of timothy and birch, Phl p1, Phl p5, Bet v1, the profilins Phl p12, Bet v2, and the calcium-binding proteins Phl p7 and Bet v4 with UniCAP 100 System and the recombinant ImmunoCAP allergens (Pharmacia Diagnostics, Freiburg, Germany). Serum samples with levels >5kU/l to timothy and/or birch pollen were selected. Results: Serum samples of 44 patients were positive for the major allergens of both timothy and birch pollen, Phl p1, Phl p5, Bet v1, and negative for Phl p7, Phl p12, Bet v2 and Bet v4. 51 samples were only positive for Phl p1 and Phl p5, negative for birch, 30 samples were only positive for Bet v1, negative for timothy. 6 samples positive for timothy, levels between 5.7-9.4 kU/l were only positive for the additionally tested component Phl p4. One sample positive for timothy and rye pollen was only positive for Phl p7 and Phl p12, but negative for the major components. In 19 samples IgE antibodies to both timothy and birch pollen major allergens but also to profilins and calcium-binding proteins could be detected, partly with lower levels for the major allergen components. Conclusion: In 125 of 151 samples IgE antibodies were found to only the major allergen components of timothy and/or birch pollen. In 26 samples antibodies to profilins and/or calcium-binding proteins were detected, partly with negative or lower levels for the major allergens. The measurement of IgE antibodies with recombinant allergen components is useful for the detection of individual sensitization pattern to predict crossreactivity and might be of importance for the selection of the patients for specific immunotherapy.

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[170] - Asero R. Plant Food Allergies: A Suggested Approach to Allergen-Resolved Diagnosis in the Clinical Practice by Identifying Easily Available Sensitization Markers. Int Arch Allergy Immunol 2005;138:1-11

"BACKGROUND: Molecular biology techniques have led to the identification of a number of allergens in vegetable foods, but due to the lack of purified food proteins for routine diagnostic use, the detection of sensitizing allergens remains a nearly impossible task in most clinical settings. The allergen-resolved diagnosis of food allergy is essential because each plant-derived food may contain a number of different allergens showing different physical/chemical characteristics that strongly influence the clinical expression of allergy; moreover, many allergens may cross-react with homologue proteins present in botanically unrelated vegetable foods . OBJECTIVE: Through a review of the available literature, this study aimed to detect ""markers"" of sensitization to specific plant food allergens that are easily accessible in the clinical practice . RESULTS: There are several ""markers"" of sensitization to different allergenic proteins in vegetable foods that can be helpful in the clinical practice. Specific algorithms for patients allergic to Rosaceae and to tree nuts were built . CONCLUSION: Clinical allergologists lacking the assistance of an advanced molecular biology lab may take advantage of some specific clinical data as well as of some ""markers"" in the difficult task of correctly diagnosing patients with plant food allergy and to provide them the best preventive advice"

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[171] - Suck R, Hagen S, Cromwell O, Fiebig H. The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50-60 kDa is comprised of two major allergens: Phl p4 and Phl p13. Clin Exp Allergy 2000;30:1395-1402

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.

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[172] - Petersen A, Suck R, Hagen S, Cromwell O, Fiebig H, Becker WM. Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability. J Allergy Clin Immunol 2001;107:856-862

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

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[173] - Petersen A, Suck R, Hagen S, Cromwell O, Fiebig H, Becker WM. Group 13 grass allergens: structural variability between different grass species and analysis of proteolytic stability. J Allergy Clin Immunol 2001;107:856-862

BACKGROUND: Determination of the allergen composition of an extract is essential for the improvement of hyposensitization therapy. Surprisingly, although grass pollen extracts have been studied intensively for 20 years, a further major allergen, Phl p 13, was detected recently in timothy grass pollen. OBJECTIVES: We sought to determine the occurrence and importance of group 13 allergens in various grass species and to investigate their proteolytic stability. METHODS: The group 13 allergens were determined by means of 2-dimensional PAGE blotting with patient sera and group 13-specific mAbs. The allergens were isolated chromatographically from several pollen extracts and analyzed by means of microsequencing. Cross-reactivity among various grass species was studied by using Western blots and immunoblot inhibition tests. The stability of the allergens was tested under defined extraction conditions. RESULTS: Group 13 allergens are detectable in all common grasses and show IgE cross-reactivity among them. The allergenic components were identified in the neutral pH range with molecular masses of 50 to 60 kd, and in the case of Phl p 13, maximal binding of the isoforms was observed at 55 kd and at an isoelectric point of 6 to 7.5. Protein sequencing clearly confirms structural identities between different grass species, although individual variations are found. If low-molecular-mass components were depleted by means of gel filtration, a rapid degradation of group 13 allergens was observed. This is in contrast to other pollen allergens described thus far. CONCLUSION: Group 13 allergens are widespread and are major allergens in the grasses. Predicted from their primary structures, these allergens are polygalacturonases. This class of enzymes is already known from microorganisms, and these enzymes are recognized as potential inducers of asthma. Our studies indicate that the group 13 allergens show a considerable microheterogeneity and degradation, especially after depletion of low-molecular-mass components. One has to be aware of this pivotal fact when soluble grass pollen extracts are prepared for diagnostics and hyposensitization therapy.

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[174] - Swoboda I, Grote M, Verdino P, Keller W, Singh MB, De Weerd N, et al. Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles. J Immunol 2004;172:6490-6500

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

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[175] - Suck R, Petersen A, Hagen S, Cromwell O, Becker WM, Fiebig H. cDNA cloning and expression of a newly recognized high molecular mass allergen Phl p13 from Timothy grass pollen (Phleum pratense). Clin Exp Allergy 2000;30:324-332

Grass pollen extracts contain a range of different allergenic components that can be classified as having low, middle or high molecular mass. Almost 75% of patients allergic to grass pollen display immunoglobulin (Ig) E-reactivity to allergens in the high molecular mass range of 55-60 kDa. These proteins have not yet been fully characterized on the protein and DNA level. OBJECTIVE: The aim of this study was to identify and characterize an allergen of the high molecular mass fraction of Phleum pratense pollen by N-terminal protein sequencing and molecular cloning. METHODS: A previously uncharacterized allergen which migrates as a double band with a molecular mass of 55-60 kDa was biochemically purified and investigated by N-terminal sequencing. Subsequently, a DNA primer was designed to amplify the corresponding cDNA using PCR. The cloned cDNA and deduced amino acid sequence were compared with sequence data bases. Immunoblots carrying the recombinant expression product were developed with monoclonal antibodies and sera derived from allergic subjects. The IgE-binding capacity of natural and recombinant allergen was determined using EAST. RESULTS: The nucleic acid sequence as well as the deduced amino acid sequence consisting of 394 amino acids indicated homology with pollen specific polygalacturonases. Four potential sites for glycosylation and 16 cysteine residues were found. The recombinant expression product exhibited the same molecular size as the natural allergen and was clearly IgE-reactive. CONCLUSION: The newly characterized allergen Phl p 13, which shows homology with polygalacturonases, is clearly different from the allergen designated as Phl p 4 and therefore the high molecular mass fraction is composed of at least two different allergens. A possible reason why this important allergen has not been detected until now is that Phl p 13 and Phl p 4 are hardly separable by one dimensional SDS-PAGE.

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[176] - Suck R, Hagen S, Cromwell O, Fiebig H. The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50-60 kDa is comprised of two major allergens: Phl p4 and Phl p13. Clin Exp Allergy 2000;30:1395-1402

BACKGROUND: More than 70% of the patients allergic to grass pollen exhibit IgE-reactivity against the high molecular mass fraction between 50 and 60 kDa of timothy grass pollen extracts. One allergen from this fraction is Phl p 4 that has been described as a basic glycoprotein. A new 55/60 kDa allergen, Phl p 13, has recently been purified and characterized at the cDNA level. OBJECTIVE: The relative importance of the two high molecular mass allergens has been characterized with respect to their IgE-binding frequency and capacity. METHODS: Both high molecular mass allergens were biochemically purified and subjected to nitrocellulose strips. About 306 sera obtained from subjects allergic to grass pollens were used to determine specific IgE-binding frequency to Phl p 4 and Phl p 13. IgE-binding of allergens was quantified by ELISA measurements. Pre-adsorption of sera with purified allergens and subsequent incubation of nitrocellulose-blotted timothy grass pollen extract was performed to determine whether or not Phl p 4 and Phl p 13 represent the whole high molecular mass allergen fraction. Proteolytic stability of both allergens was investigated by addition of protease Glu-C. RESULTS: More than 50% of 300 patients displayed IgE-binding with both allergens. Clear differences concerning the immunological properties of Phl p 4 and Phl p 13 were confirmed by individual IgE reactivities. Quantification of specific IgE for both allergens revealed comparable values. For complete inhibiton of IgE-binding in the high molecular mass range preincubation of sera with both allergens was necessary. Interestingly, inhibition of strong reacting sera with Phl p 13 eliminated not only reactivity of the 55/60 kDa double band, but in addition a 'background smear'. Whilst undenatured Phl p 4 was resistent to proteolytic digestion with Glu-C, native Phl p 13 was degraded rapidly. CONCLUSION: Phl p 4 and Phl p 13 are immunologically different and must both be considered as major allergens. They are judged to be important candidates for potential recombinant therapeutics that may provide a basis for improved immunotherapy.

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[177] - Chow LP, Chiu LL, Khoo KH, Peng HJ, Yang SY, Huang SW, et al. Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen. FEBS J 2005;272:6218-6227

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18 411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man(3)GlcNAc(2)) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.

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[178] - Chow LP, Chiu LL, Khoo KH, Peng HJ, Yang SY, Huang SW, et al. Purification and structural analysis of the novel glycoprotein allergen Cyn d 24, a pathogenesis-related protein PR-1, from Bermuda grass pollen. FEBS J 2005;272:6218-6227

Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18 411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man(3)GlcNAc(2)) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1.

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[179] - Wu WC, Tam MF, Peng HJ, Tsai LC, Chi CW, Chang ZN. Isolation and partial characterization of a 46-kd allergen of Bermuda grass pollen. J Biomed Sci 2001;8:342-348

Cyn d Bd46K, a 46-kD component of Bermuda grass (Cynodon dactylon) pollen, had been identified as an allergenic constituent. In the present study two-dimensional (2D) gel electrophoresis illustrated the presence of five acidic isoforms in Cyn d Bd46K, and this molecule was purified by monoclonal antibody (MAb) affinity chromatography for further characterization. Using a digoxigenin-labeled lectin-binding assay, the elucidating protein was disclosed to be a glycoprotein with terminal mannose. The involvement of a carbohydrate moiety in the allergenicity and antigenicity of the elucidated molecule was demonstrated with sodium-periodate-treated Cyn d Bd46K, which reduced binding to its specific MAb and human IgE. We were unable to identify the N-terminal amino acid sequences of Cyn d Bd46K, but some internal amino acid sequences were disclosed by microsequencing some fragments cleaved by Achromobacter protease I and fractionated by reversed-phase column chromatography. The amino acid sequences of 4 identified Cyn d Bd46K internal peptide fragments were found to be 25-71% identical with that of cytochrome c oxidase III from corn grass pollen. The present study provided important information for future experiments on the molecular cloning of the elucidated allergen.

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[180] - Su SN, Yang SY, Peng HJ, Chow LP. A New High Molecular Weight, Basic IgE-Binding Protein from Bermuda Grass Pollen. J Allergy Clin Immunol 2008;121:S175

RATIONALE: Bermuda grass pollen is an important outdoor allergen source, which contains a very complexity of allergens.We have previously characterized a high molecular weight, basic allergen, BG60. The aim of this study was to characterize a new high molecular weight, basic IgEbinding protein from Bermuda grass pollen. METHODS: Ion exchange chromatography was used to purify the high molecular weight, basic IgE binding protein. Immunoblot and ELISAwere used to study the IgE-binding activity. Lectin binding assay was used to examine the lectin binding activity of the new IgE binding protein. Periodate oxidation was carried out to study the role of carbohydrate moiety in IgE and IgG binding. N-terminal sequence was analyzed using solid phase sequencer. RT-PCR was used to clone the cDNA of a high molecular, basic IgE binding protein. RESULTS: The protein has an apparent molecular weight about 66 kDa, which binds Con-A only indicating the presence of the terminal mannosyl residues. Monoclonal antibody showed cross-reactivity to pollen proteins from other grasses. Sodium periodate treated protein reduced IgE and IgG binding activities. The cDNA encoded a mature protein of 529 amino acids with a theoretical pI value of 8.82 and a MWof 59,339. The amino acid sequence deduced from the cDNA clone showed high sequence homology to copper binding protein/ascobate oxidase. CONCLUSION: A new high molecular weight, basic IgE binding protein was identified and isolated. The results obtained in this study indicate that the carbohydrate moiety is involved in the IgE and IgG binding.

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[181] - Turcich MP, Hamilton DA, Mascarenhas JP. Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases. Plant Mol Biol 1993;23:1061-1065

A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis. When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2). The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp. The two genes map to different chromosomes.

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[182] - Hrabina M, Peltre G, Van Ree R, Moingeon P. Grass pollen allergens. Clin Exp Allergy Rev 2008;8:7-11

To date, eleven groups of grass pollen allergens eliciting a specific IgE response in atopic individuals have been identified. Groups 1 and 5 allergens are the most critical (major) pollen allergens leading to the sensitization of 90% and 65ˆ85% allergic patients, respectively. Other allergens frequently involved in the IgE response include groups 2/3, 4, 6, 7, 10ˆ13 allergens. Allergens found in various Pooideae exhibit high homology in terms of their amino acid sequence composition, which translates into significant cross-reactivity in terms of antibody (IgE and IgG) as well as T cell responses. Nevertheless, for a given allergen group, there is evidence of both interspecies (i.e. differences in amino acid sequences) and intraspecies (multigenes, post-translational modification, mRNA splicing or editing) molecular variability.

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[183] - Westritschnig K, Horak F, Swoboda I, Balic N, Spitzauer S, Kundi M, et al. Different allergenic activity of grass pollen allergens revealed by skin testing. Eur J Clin Invest 2008;38:260-267

BACKGROUND: Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. MATERIAL AND METHODS: Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. RESULTS: Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. CONCLUSION: Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

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[184] - Leiferman KM, Gleich GJ. The cross-reactivity of IgE antibodies with pollen allergens. I. Analyses of various species of grass pollens. J Allergy Clin Immunol 1976;58:129-139

Atopic patients with histories of grass pollen allergy often are sensitive to a variety of species of grasses. Using a serum pool from patients sensitive to June grass, we analyzed the reactivity of IgE antibodies to seven grasses by the radioallergosorbent test. Extracts were analyzed for their inhibitory activities with solid-phase allergens prepared from all of the grass pollen. Also samples of serum were exhaustively absorved with solid-phase allergens and the supernatants tested to determine the reactivity of the remaining IgE antibodies. Three patterns of reactivity were observed: (1) June, orchard, meadow fescue, and perennial rye grasses displayed similar reactivity in both inhibition and absorption studies; (2) sweet vernal and Bermuda grasses were considerably less reactive with the serum pool, indicating that they lacked antigenic determinants possessed by the other grasses; and (3) timothy grass possessed unique antigenic determinants. 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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[185] - Kalveram KJ, Forck G. Cross-reactivity between grass and corn pollen antigens. Int Arch Allergy Appl Immunol 1978;57:549-553

Analogous reactions of grass and corn pollen extracts in skin tests on patients suffering from pollinosis might suggest an antigenic relationship between grass and corn pollens. This problem was studied using the RAST inhibition test. Tests were performed with cellulose discs labelled with commercial skin test extracts containing grass, rye, wheat, barley, oat and maize pollens. Different mutual inhibitions were measured showing various grades of antigenic relationship. Only grass pollen antigens could strongly inhibit all other antigen-antibody reactions. Thus, we suppose that the investigated grass pollen extract also contains all antigens typical of corn pollen. Therefore, exclusive use of this extract seems to be possible in diagnosis and perhaps therapy of combined grass and corn pollen allergy.

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[186] - Johansen N, Weber RW, Ipsen H, Barber D, Broge L, Hejl C. Extensive IgE Cross-Reactivity towards the Pooideae Grasses Substantiated for a Large Number of Grass-Pollen-Sensitized Subjects. Int Arch Allergy Immunol 2009;150:325-334

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[187] - Mari A. Multiple pollen sensitization: a molecular approach to the diagnosis. Int Arch Allergy Immunol 2001;125:57-65

BACKGROUND: Sensitization to multiple pollen species is a frequent diagnostic event. Several allergenic molecules with a high level of homology have been identified in divergent pollen families and named panallergens. METHODS: We sought to define the criteria to evaluate the prevalence of the multiple pollen sensitization, to identify specific markers of this condition, and to correlate them with the underlying allergic disease. Patients presenting an allergic respiratory disease underwent skin testing with 23 pollens. Patients fulfilling predefined selection criteria were grouped and classified as having multiple pollen sensitization. Patients in each subgroup were tested for IgE to rBet v 2, rJun o 2, rBet v 1, rPhl p 5 and bromelain. Demographical, allergological and clinical data were recorded in the subgroup of patients with multiple pollen sensitization. RESULTS: Seventeen percent of the pollen-sensitized patients formed the multiple pollen-sensitized subgroup. These subjects were positive for most of the pollen species tested regardless of known exposure to them. None of the subjects sensitized to less than six pollen species were positive to panallergens, whereas 55% of the sera of the multiple pollen-sensitized group were positive to rBet v 2, and 15% to rJun o 2. IgE to rBet v 1 and rPhl p 5 were found positive in all the subgroups. Age, gender, bronchial asthma, oral allergy syndrome, skin test reactivity and previous specific immunotherapy differed significantly when these two subsets were considered. CONCLUSIONS: Allergy diagnosis based on allergenic molecules is crucial in the patient with multiple pollen sensitization. This condition appears to be determined by the sensitization to defined allergenic components (panallergens) rather than by pollen of multiple species as such. Detection of IgE to nonpanallergenic molecules allows to identify more relevant allergenic sources. Clinical aspects of the underlying allergic disease (e.g. asthma and oral allergy syndrome) seem to be differently related to IgE reactivity to panallergens

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

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[189] - Valenta R, Vrtala S, Ebner C, Kraft D, Scheiner O. Diagnosis of grass pollen allergy with recombinant timothy grass (Phleum pratense) pollen allergens. Int Arch Allergy Immunol 1992;97:287-294

In order to establish a test system for grass pollen allergy based on the use of recombinant allergens we chose timothy grass (Phleum pratense), a widely spread grass, as a model. From a lambda gt11 cDNA expression library that we had constructed from pollen RNA of timothy grass (P. pratense), we had obtained with serum IgE from a grass pollen- allergic individual 60 IgE-binding clones. By differential testing with sera from different grass pollen-allergic patients, we selected three distinct clones encoding Phl p I (group I), Phl p V (group V) and profilin from timothy grass, which when used together allowed the diagnosis of grass pollen allergy in 97 out of 98 tested grass pollen- allergic patients employing a simple plaque lift technique. This recombinant test based on plaque lifts containing allergen-beta- galactosidase fusion proteins was compared with IgE immunoblots using crude pollen protein extracts from timothy grass. Both methods were in good agreement with RAST scores and clinical data, and proofe d to be useful for the diagnosis of grass pollen allergy. Our results further indicate that a limited panel of only two recombinant grass pollen allergens, Phl p I and Phl p V, together with the plant panallergen profilin could be sufficient for the diagnosis and possibly immunotherapy of grass pollen allergy

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[190] - Mari A. Skin test with a timothy grass (Phleum pratense) pollen extract vs. IgE to a timothy extract vs. IgE to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12: epidemiological and diagnostic data. Clin Exp Allergy 2003;33:43-51

The diagnostic approach to grass pollen allergy is now possible by detecting specific IgE to its allergenic components. ObjectiveTo compare the IgE reactivity to a timothy grass pollen extract with the IgE reactivity to eight allergenic components from the same source (Phl p 1, 2, 4, 5, 6, 7, 11, 12). Both were compared with the skin test reactivity to a timothy grass extract. MethodsA population survey was carried out by means of the skin test to identify grass-allergic subjects, and to characterize them in terms of demographic and allergological parameters. Seven hundred and forty-nine sera were available for IgE detection to a timothy extract, to the recombinant Phl p 1, 2, 5, 6, 7, 11, 12, and to native Phl p 4 and bromelain. Results were stratified by means of demographic and allergy parameters. ResultsNinety-five per cent of the sera had detectable IgE to the timothy extract. Prevalence of IgE reactivity increased from 86.8% to 93.3% as the number of combined reactive molecules rose from 2 to 8. Adjusted prevalences for each allergen were: rPhl p 1 = 83%, rPhl p 2 = 55%, nPhl p 4 = 70%, rPhl p 5 = 50%, rPhl p 6 = 44%, rPhl p 7 = 7%, rPhl p11 = 43%, rPhl p 12 = 15%. Isolated reactivity to rPhl p 1 was 6%, whereas it was negligible for the remaining molecules. IgE reactivity prevalence and mean values differed when patients were stratified on the basis of their associated pollen reactivity and their skin test reactivity grade. No differences were found when age, symptom type and duration were considered. Up to eight-fold higher IgE concentrations were found when the sum of IgE to molecules was compared with IgE to the extract. Testing for the IgE reactivity to the glycan of the native Phl p 4 allergen showed a possible interference with prevalence and value estimation. Higher prevalence values were found in previously immunotherapy-treated patients. ConclusionsThe use of a complete panel of grass allergenic molecules can mimic the current use of allergenic extracts, but new relevant information, such as individual pattern of reactivity, adjusted prevalence, correct specific IgE concentration, can be achieved only by means of discrete allergenic molecules.

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[191] - Metz-Fabre C, Linhart B, Focke-Tejkl M, Purohit A, de Blay F, Valenta R, et al. Skin test diagnosis of grass pollen allergy with a recombinant hybrid molecule. J Allergy Clin Immunol 2007;120:315-321

BACKGROUND: A recombinant hybrid molecule (HM) consisting of 4 major allergens from timothy grass (Phl p 1, 2, 5, and 6) was expressed in Escherichia coli, purified, and characterized regarding its immunologic properties . OBJECTIVE: We sought to determine whether the recombinant HM can be used for the diagnosis of grass pollen allergy by means of skin testing . METHODS: Skin prick testing was performed in 32 patients with grass pollen allergy and in 9 control individuals by using increasing concentrations (4, 12, 36, and 108 mug/mL) of the HM and using commercial grass pollen extract. Specific IgE reactivities against the HM, grass pollen extract, and a panel of purified grass pollen allergens (recombinant Phl p 1, 2, 5, 6, 7, 12, and 13 and natural Phl p 4) were measured by means of ELISA, and timothy grass pollen-specific IgE levels were determined by using ImmunoCAP . RESULTS: Grass pollen allergy was diagnosed in all patients by means of skin testing with the HM. No false-positive skin test responses were obtained in the control individuals. There was an excellent correlation between IgE levels obtained with the HM and natural grass pollen extract measured by means of ELISA (r = 0.98, P < .0001) and by means of ImmunoCAP (r = 0.98, P < .0001) . CONCLUSIONS: The recombinant HM permitted accurate and specific in vivo diagnosis of grass pollen allergy in all tested patients. It can be considered a well-defined tool for the diagnosis and perhaps for immunotherapy of grass pollen allergy. CLINICAL IMPLICATIONS: A recombinant HM can replace traditional allergen extracts for skin test-based diagnosis of grass pollen allergy.

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[192] - Niederberger V, Stubner P, Spitzauer S, Kraft D, Valenta R, Ehrenberger K, et al. Skin test results but not serology reflect immediate type respiratory sensitivity: a study performed with recombinant allergen molecules. J Investig Dermatol 2001;117:848-851

The diagnosis of type I allergy, an IgE-antibody-mediated hypersensitivity disease affecting more than 25% of the population, is based on the measurement of allergen-specific serum IgE levels and provocation testing. Whether the determination of allergen- specific serum IgE levels can replace in vivo provocation testing for allergy diagnosis is a controversial issue. We used purified recombinant timothy grass and birch pollen allergens to compare by skin prick and nasal provocation testing as well as by serology in vivo sensitivity with antibody-binding capacity in 24 pollen allergic patients and eight control individuals. Results from biologic tests were correlated with each other and with allergen-specific IgE and IgG1-4 levels. IgE-reactive allergens induced immediate skin and nasal reactions, but the intensity of the allergic tissue reactions was not correlated with either the levels of allergen-specific IgE or the levels of allergen-specific IgG antibodies. Less frequently detected allergens with low IgE-binding capacity were able to induce strong allergic reactions comparable to those caused by major allergens with high IgE-binding capacity. In contrast, skin test and nasal provocation results were significantly correlated (r = 0.63, p < 0.01). Our study thus demonstrates on a molecular level that skin testing provides a better reflection of immediate type respiratory sensitivity than serologic measurements. These results have implications for allergy diagnosis and, in particular, for the selection of relevant allergen components for specific immunotherapy.

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[193] - Focke M, Marth K, Flicker S, Valenta R. Heterogeneity of commercial timothy grass pollen extracts. Clin Exp Allergy 2008;38:1400-1408

BACKGROUND: The diagnosis and specific immunotherapy of allergy is currently performed with allergen extracts prepared from natural allergen sources . OBJECTIVE: To analyse commercial timothy grass pollen allergen extracts used for in vivo diagnosis regarding their qualitative and quantitative allergen composition and in vivo biological activity . METHODS: Antibodies specific for eight timothy grass pollen allergens (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 12, Phl p 13) were used to detect these allergens in timothy grass pollen extracts from four manufacturers by immunoblotting. ELISA assays were developed and used to quantify the three major allergens (Phl p 1, Phl p 2, Phl p 5) in the extracts. The magnitude of skin responses to the four extracts was studied by skin prick testing in 10 grass pollen-allergic patients . RESULTS: The allergen extracts showed broad variations in protein compositions and amounts (24.1-197.7 microg/mL extract). Several allergens could not be detected in certain extracts or appeared degraded. A considerable variability regarding the contents of major allergens was found (Phl p 1: 32-384 ng/mL; Phl p 2: 1128-6530 ng/mL, Phl p 5: 40-793 ng/mL). Heterogeneous skin test results were obtained with the extracts in grass pollen-allergic patients . CONCLUSIONS: Timothy grass pollen extracts from different manufacturers exhibit a considerable heterogeneity regarding the presence of individual allergens and hence yield varying in vivo test results. Problems related to the use of natural grass pollen allergen extracts may be circumvented by using defined recombinant grass pollen allergens.

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[194] - Malandain H. IgE-reactive carbohydrate epitopes--classification, cross-reactivity, and clinical impact. Eur Ann Allergy Clin Immunol 2005;37:122-128

A glycan-related IgE-reactivity has been demonstrated in most allergen sources, especially in plant kingdom. Recent progress in glycobiology has allowed a clearer classification of these glyco-epitopes. Unlike classical peptide chain-based epitopes, glyco-epitopes can share significant structural homologies beyond the limits of protein families. These glycoepitopes are thus prone to extensive cross-reactivity. They have been called Cross-reactive Carbohydrate Determinants or CCD. Many of these glyco-epitopes behave as "panepitopes", leading to cross-reactivity between products as distant as pollens and hymenoptera venoms. But CCD are not universally cross-reactive and they rather cluster into subgroups such as plant CCD or fungal CCD. Because a monovalent IgE-binding is sufficient in serum-based assays, glyco-epitopes and CCD are classically considered as a potential source of positive in vitro results without clinical significance. But some authors recently demonstrated that glyco-epitopes could induce a response at the cell level and suggested that they might play a role in vivo. As long as in vitro assays include glycan- and CCD-related IgE responses, laboratory results should be carefully interpreted in the light of existing knowledge about the glycomes of natural products. IgE-reactivity of the patient's serum can also be tested towards a glycoprotein model such as bromelain.

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[195] - Aalberse RC, van Ree R. Crossreactive carbohydrate determinants. Clin Rev Allergy Immunol 1997;15:375-387

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[196] - Bridts CH, Ebo DG, Hagendorens MM, de Clerck LS, Stevens WJ. Cross-Reactive Carbohydrate Determinants (CCD): Mimickers of Allergy. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°379

RATIONALE: To investigate the prevalence of anti-CCD-IgE in healthy controls and patients allergic to pollen, house dust mite (HDM), pets, natural rubber latex (NRL) and hymenoptera venom. To study the contribution of anti-CCD-IgE as a cause of clinically irrelevant IgE for NRL and apple. METHODS: IgE antibodies were quantified by Immuno-CAP. Skin prick tests (SPT) were performed with HAL extracts, except for bromelain (500 µg/mL, Sigma-Aldrich). Patients were identified as anti-CCD-IgE positive if they had a negative SPT and positive IgE for bromelain. Sera containing IgE against apple or NRL were classified as true-positive or false-positive according to the presence or absence of an oral allergy syndrome (OAS) or NRL-induced anaphylaxis. RESULTS: No anti-CCD-IgE was found in controls (n=12), patients monosensitized to NRL (n=19), pets (n=7), HDM (n=28) or birch pollen (n=32). In contrast, anti-CCD-IgE was present in 21% (4/19) of patients with grass pollinosis, 25% (4/20) of patients with combined grass and birch pollinosis, in 4/8 of patients with combined grass, tree and weed pollinosis (p=0.03), and in 14% (4/28) of patients with venom anaphylaxis. False-positive NRL individuals had a higher prevalence of anti-CCD-IgE than NRL allergic patients (78% (18/23) versus 0% (0/19), p<0.001). In individuals with a false-positive IgE to apple, the frequency of anti-CCD-IgE was higher than in OAS patients (38% (15/40) versus 11% (3/27), p=0.03). CONCLUSIONS: Sensitization to CCD, caused by grass pollen or hymenoptera venom allergens, can mimic NRL and apple allergy. Patients monosensitized to NRL or birch pollen showed no anti-CCD-IgE.

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[197] - Petersen A, Schramm G, Schlaak M, Becker WM. Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen. Clin Exp Allergy 1998;28:315-321

BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.

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[198] - Johansen N, Weber RW, Ipsen H, Barber D, Broge L, Hejl C. Extensive IgE Cross-Reactivity towards the Pooideae Grasses Substantiated for a Large Number of Grass-Pollen-Sensitized Subjects. Int Arch Allergy Immunol 2009;150:325-334

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[199] - Marknell DeWitt A, Andersson K, Peltre G, Lidholm J. Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy 2006;36:77-86

BACKGROUND: Timothy grass pollen is a common cause of respiratory allergy in the temperate regions. The major group 4 allergen, Phl p 4, has previously been purified and studied biochemically and immunologically, but has so far not been produced and characterized as a recombinant protein . OBJECTIVE: To clone and characterize timothy grass pollen allergen Phl p 4 . METHODS: Full-length Phl p 4 cDNA was cloned using a PCR-based strategy including 3'-and 5'-RACE. Recombinant Phl p 4 was expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Its immunological activity was investigated using experimental ImmunoCAP tests, sera from Phl p 4 sensitized individuals and Phl p 4 reactive polyclonal and monoclonal animal antibodies . RESULTS: Five full-length Phl p 4 cDNA clones were analysed. Sequence deviations between the clones were present at nine amino acid positions, and the consensus sequence comprised an open reading frame of 525 amino acids, including a predicted 25-residue signal peptide. The calculated molecular weight of the deduced mature protein was 55.6 kDa and the isoelectric point 9.9, both consistent with previously observed properties of purified nPhl p 4. Close sequence similarity was found to genomic clones from several other Pooideae grass species and to Bermuda grass pollen allergen BG60. Further, similarity was found to members of the berberine bridge enzyme (BBE) family, including celery allergen Api g 5. Recombinant Phl p 4 bound specific immunoglobulin (Ig)E from 31 of 32 nPhl p 4-reactive sera, and the IgE binding to rPhl p 4 could be inhibited by nPhl p 4 in a dose-dependent manner . CONCLUSIONS: Full-length Phl p 4 cDNA was cloned and showed sequence similarity to members of the BBE family. Recombinant Phl p 4 was produced and shared epitopes with natural Phl p 4.

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[200] - van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, et al. beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451-11458

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

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[201] - Ferrara R, Mari A. IgE to Crossreactive Carbohydrate Determinants: A Demographical and Allergological Study. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°629

IgE to crossreactive carbohydrate determinants (CCD) have been described in patients with allergic diseases. A prevalence up to 40% of positive CCD-IgE have been reported in pollen allergic subjects, but extended epidemiological studies have never been carried out. The aim of the present study was to evaluate the distribution of CCD-IgE in a cohort of 1557 subjects with allergic or allergy-like respiratory diseases. Subjects with respiratory symptoms underwent SPT with a panel of allergenic sources and with purified bromelain. IgE to bromelain were detected in each subject (CAP, Pharmacia), and a discrepant result between positive IgE to bromelain and a negative SPT to the same glycoprotein was assumed to detect the presence of CCD-IgE. Specific IgE to SPT-negative allergenic sources were randomly detected. Positive and negative CCD-IgE subgroups were comparatively evaluated for total IgE. Four subjects had positive SPT to bromelain. The overall prevalence of CCD-IgE was 22%. Distribution of the IgE values ranged between 0.4 and 94 kU/L, most being classes I-III values. A higher prevalence of male subjects was recorded in the positive CCD-IgE subset (61 vs 47 %). Differences were recorded when age distribution was considered. 30% of the CCD-IgE positive subjects were found among the pediatric subgroup, even in subjects as young as three, whereas 22% were among CCD-IgE negative subset. Positive CCD-IgE were mainly found among pollen allergic patients (313, 32%), but 20 (8%) allergic to non-pollen sources and 17 (5%) non-allergic patients were found positive. Comparative evaluation of specific IgE to SPT-negative allergenic sources in CCD-IgE positive and negative subjects gave the following results (% values): Birch 62 vs 11; Juniper 52 vs 9; Olive 72 vs 17; Grass, Tymothy 52 vs 5; Pellitory 79 vs 21; Mugwort 78 vs 19; Ragweed 78 vs 32; Dermat. p. 28 vs 3; Cat 35 vs 3; Horse 53 vs 15; Blatella g. 53 vs 8; Alternaria 32 vs 5; Candida 40 vs 6; Latex 67 vs 19; Bee venom 46 vs 10; Peanuts 71 vs 16. IgE values to allergenic extracts correlated well with IgE to bromelain. Higher mean total IgE values were recorded in the positive CCD-IgE subgroup and differed significantly when compared with the negative one. Nevertheless, a large number of subjects with high total IgE values and negative CCD-IgE were found. IgE to CCD is a relevant occurrence in the pollen allergic population. Data on CCD-IgE to SPT-negative allergenic sources further confirm the absence of biological activity of such determinants. IgE to CCD should be always detected in order to correctly evaluate the results of serum IgE determination. Research laboratory use of allergic sera should always take into account the presence of CCD-IgE.

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[202] - Rossi RE, Monasterolo G, Monasterolo S. Measurement of IgE antibodies against purified grass-pollen allergens (Phl p 1, 2, 3, 4, 5, 6, 7, 11, and 12) in sera of patients allergic to grass pollen. Allergy 2001;56:1180-1185

Background: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis. Methods: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens. Results: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed. Conclusions: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.

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[203] - Wilson IBH, Harthill JE, Mullin NP, Ashford DA, Altmann F. Core alpha-1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 1998;8:651-661

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.

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[204] - van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, et al. beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451-11458

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

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[205] - Altmann F. The role of protein-glycosylation in allergy. Int Arch Allergy Immunol 2007;142:99-115

The asparagine-linked carbohydrate moieties of plant and insect glycoproteins are the most abundant environmental immune determinants. They are the structural basis of what is known as cross-reactive carbohydrate determinants (CCDs). Despite some structural variation, the two main motifs are the xylose and the core-3-linked fucose, which form the essential part of two independent epitopes. Plants contain both epitopes, insect glycoproteins only fucose. These epitopes and other fucosylated determinants are also found in helminth parasites where they exert remarkable immunomodulatory effects. About 20% or more of allergic patients generate specific anti-glycan IgE, which is often accompanied by IgG. Even though antibody-binding glycoproteins are widespread in pollens, foods and insect venoms, CCDs do not appear to cause clinical symptoms in most, if not all patients. When IgE binding is solely due to CCDs, a glycoprotein allergen thus can be rated as clinical irrelevant allergen. Low binding affinity between IgE and plant N-glycans now drops out as a plausible explanation for the benign nature of CCDs. This rather may result from blocking antibodies induced by an incidental 'immune therapy' ('glyco-specific immune therapy') exerted by everyday contact with plant materials, e.g. fruits or vegetables. The need to detect and suppress anti-CCD IgE without interference from peptide epitopes can be best met by artificial glycoprotein allergens. Hydroxyproline-linked arabinose (single beta-arabinofuranosyl residues) has been identified as a new IgE-binding carbohydrate epitope in the major mugwort allergen. However, currently the occurrence of this O-glycan determinant appears to be rather restricted.

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[206] - 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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[207] - Brecker L, Wicklein D, Moll H, Fuchs EC, Becker WM, Petersen A. Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.). Carbohydr Res 2005;340:657-663

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.

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[208] - Kochuyt AM, Van Hoeyveld EM, Stevens EA. Prevalence and clinical relevance of specific immunoglobulin E to pollen caused by sting-induced specific immunoglobulin E to cross-reacting carbohydrate determinants in Hymenoptera venoms. Clin Exp Allergy 2005;35:441-447

Summary Background Hymenoptera stings can induce specific IgE (sIgE) to carbohydrate determinants (CD) on venom glycoproteins that cross-react with CD in pollen. sIgE to such cross-reacting CD (CCD) are believed to have little or no biological activity and thus may cause misdiagnosis of pollen sensitization after a sting. Objective To determine the prevalence of multiple false positive CAP results to pollen because of sting induced anti-CCD sIgE in Hymenoptera venom (HV) allergic patients and to investigate the association of such anti-CCD sIgE with features of 'atopy'. Methods Skin prick tests (SPT) and CAP tests with grass, tree and weed pollen and with house dust mite (HDM) were carried out prospectively in 259 HV allergic patients and CAP tests with honeybee (HBV) and yellow jacket (YJV) venom were performed. Patients with negative pollen SPT associated with positive CAP tests to all three pollen groups were operationally defined as 'CCD positive'. We investigated in selected 'CCD positive' patients the presence of anti-CCD sIgE by CAP tests with bromelain and studied the identity of CD in HVs and pollen by mutual sIgE inhibition tests with CD from proteinase treated HBV (HBV-CD) and Lolium perenne (Lol-CD) extracts. Results sIgE to all three pollen groups without positive SPT or history was found in 16% of 259 patients. The presence of anti-CCD sIgE was substantiated by positive CAP tests with bromelain in 14/14 and by inhibition of all pollen CAP tests with HBV-CD in 8/9 and with Lol-CD in 2/2 patients. Double venom (DV) positive CAP tests were present in 93% of 'CCD positive' patients and were in some associated with DV skin test positivity and allergy. The prevalence of 'CCD positivity' was significantly higher among HBV (23%) than among YJV (11%) allergic patients, but was also unexpectedly high among those with DV allergy (47%). 'CCD positive' patients were younger, had a higher total IgE and more sIgE to HDM than 'CCD negative' patients. Conclusion We have shown that the risk in HV allergic patients for misdiagnosis of multivalent pollen sensitization is 16%, and we have confirmed that sting induced anti-pollen sIgE are directed to similar CD in venoms and pollen. We found evidence that the recognition of CCD might be related to the 'atopic' trait. Importantly, a positive bromelain CAP test does not exclude clinical reactivity to both venoms in 'CCD positive' HV allergic patients.

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[209] - 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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[210] - Kazemi-Shirazi L, Niederberger V, Linhart B, Lidholm J, Kraft D, Valenta R. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-268

During the past decade an increasing number of recombinant allergens have become available, representing a significant proportion of the epitope complexity of natural allergen extracts. Component-resolved diagnosis with recombinant allergens reveals the antibody reactivity profile of allergic patients and identifies the disease-eliciting allergen molecules. This article exemplifies how recombinant allergen molecules with high cross-reactive potential can be used as marker allergens to identify allergic patients who are cross-sensitized to a variety of allergen sources. It further demonstrates how the use of allergens with a restricted distribution in a certain group of allergen sources may allow the identification of patients who have been genuinely sensitized by a particular allergen molecule. Drawing from those examples, it is suggested how diagnostic tests based on such recombinant marker allergens may be used to improve the choice and monitoring of currently available forms of specific immunotherapy.

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[211] - Valenta R, Twaroch T, Swoboda I. Component-resolved diagnosis to optimize allergen-specific immunotherapy in the Mediterranean area. J Investig Allergol Clin Immunol 2007;17(suppl. 1):88-92

Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment for allergy. It can prevent progression of the disease and has a long-lasting therapeutic effect. Since SIT is allergen-specific, the identification of the disease-eliciting allergen is an essential prerequisite for the accurate prescription of treatment. Diagnostic tests based on allergen extracts consist of mixtures of various allergens of which some are specific for the allergen source and others occur as cross-reactive allergens in various unrelated allergen sources. It may therefore be difficult and sometimes impossible to identify the disease-causing allergen with such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollens from olive, grasses, and Parietaria in the Mediterranean area is frequently treated with SIT. Here, we describe allergen molecules from these sources that can be used for component-resolved diagnosis of allergy to facilitate the selection of patients for SIT and monitor the immunological effects of treatment.

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[212] - Pecora S, Burastero S, Milani M, Asero R, Pagani M. Clinical relevance of profilin sensitisation detected by skin prick test: a multicenter prospective study in patients with grass pollinosis. Allergy 2009;64(Suppl. 90):125

Rationale: Sensitisation to Profilin, a pan-allergen, is observed in 40% of grass allergic patients. So far the are no data regarding the clinical relevance of sensitization to profilin, in term of severity of symptoms in these patients. A purified natural date palm profilin (Pho-d-2) Skin Prick Test (SPT) has been recently developped. We investigated the prevalence of profilin sensitisation and compared symptom and symptomatic drug scores during pollen season in patients with grass rhinoconjuctivitis in relation with profilin sensitisation. Patients and Methods: 50 consecutive patients with rhinoconjunctivitis and/or asthma due to grass pollen (28 men, mean age 34 years) were enrolled. SPT for grass and profilin were evaluated before pollen season 2008. Symptom score during peak pollen season (May 2008) was rated evaluating 6-item for nose and eye symptoms using a 4-point ordinal scale (0= no symptoms; 3=severe symptoms). Medication score was evaluated assessing the use of oral/topic antihistamine or corticosteroid drugs. Results: All patients were positive for grass SPT. A total of 21 subjects (44%) were positive to profilin SPT (wheal diameter >3 mm: P+). In P + patients, seasonal symptom score was significantly (P=0.01) higher in comparison with P- subjects (155 vs. 71). Medication score, mean (SD) was 39(28) in P+ patients and 13(16) in P- subjects (P=0.0025). Conclusion: Our study confirms that in grass allergic patients profilin sensitization detected by a specific SPT is detected in 40% of the patients. In these patients profilin sensitisation is also associated with an higher symptom and medication score during pollen season.

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

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[214] - Giroux F, Cano Y, Malandain H. Intérêt diagnostique de l’allergène naturel nCyn d 1 (pollen de chiendent digité, Cynodon dactylon). Rev Fr Allergol 2009;49:330

Objectif.ˆ Le chiendent digité, Cynodon dactylon, est une graminée prospérant surtout en climat chaud. Cette graminée appartient à une sous-famille distante de celle des Pooïdées (dactyle, fléole. . .), de sorte qu‚elle est parfois ajoutée aux Pooïdées dans le protocole de désensibilisation. Il est depuis peu possible de tester in vitro l‚IgE-réactivité vis-à-vis de nCyn d 1, un allergène du chiendent, homologue de Phl p 1 (fléole), etc. Ce test permet-il de préciser le diagnostic et d‚éclairer le choix d‚inclure ou non Cynodon dans le protocole de désensibilisation ? Méthodes.ˆ L‚étude a porté sur 18 patients polliniques aux graminées. La technique CAP Phadia1 a été utilisée pour mesurer l‚IgE-réactivité à nCyn d 1, à rPhl p 1 et à un témoin d‚IgE-réactivité vis-à-vis des épitopes glucidiques (CCD), la broméline. L‚interférence due aux IgE anti-CCD a été évaluée à l‚aide d‚une méthode d‚immuno-absorption et en étudiant également 3 sujets allergiques aux venins d‚hyménoptères et positifs en broméline. Résultats.ˆ Tous les patients polliniques sont positifs pour nCyn d 1,même après immuno-absorption des IgE anti-CCD. L‚allergène Cyn d 1 croise donc avec les allergènes homologues du groupe 1 des graminées, comme Phl p 1. Et si le patient présente une IgE-réactivité vis-à-vis des CCD, la réponse en nCyn d 1 est interférée, surtout quand le rapport rPhl p 1ˆbroméline est faible. Conclusion.ˆ Il semble difficile de tirer parti du test in vitro pour nCyn d 1 en France où les pollens de chiendent digité sont très minoritaires parmi les pollens de graminées : en effet, une IgE-réactivité à nCyn d 1 sera avant tout le reflet de la sensibilisation aux allergènes dominants dans l‚environnement (Phl p 1 fléole, Dac g 1 dactyle, Lol p 1 ivraie. . .) et ne permettra pas de juger s‚il est utile ou non d‚associer Cynodon dactylon dans le protocole de désensibilisation du patient. De plus, le test pour nCyn d 1 est interféré par la présence d‚IgE anti- CCD.

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[215] - Rossi RE, Monasterolo G, Prina P, Coco G, Operti D, Rossi L. IgE Profiles of Bermuda Grass Pollen Sensitised Patients Evaluated by Phleum Pratense Allergens Phl p 1, 2, 4, 5, 6 , 7, 11, 12.. Allergol Int 2008;57:1-8

Background: Despite the difference in geographical dominance of certain grasses, a high degree of allergenic similarity or cross-reactivity between Bermuda grass pollen (BGP) and timothy grass pollen (TGP) has been previously demonstrated. The aim of the present study was to ascertain the sensitisation to TGP in 411 patients known for their reactivity to BGP extracts by analysing their reactivity to crude timothy pollen extract and timothy pollen purified allergens, establishing their specific IgE-profiles. Methods: Using the immunoenzymatic CAP method we evaluated IgE-specific antibodies for BGP- and TGP- extracts and the timothy recombinant (r) and natural (n) allergens rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and rPhl p 12. Results: BGP-IgE positive patients (median = 8.0kUA/l, 2.8-22.2kUA/l 25th-75th percentile) simultaneously had IgE positive results for TGP (100% of subjects)(median = 48.9kUA/l, 19.8- > 100kUA/l 25th-75th percentile) and high prevalence of sensitization to 6/8 Phleum pratense allergens (Phl p 1, 2, 4, 5, 6, 11, markers of genuine sensitisation to TGP) other than profilin and calcium binding protein. More than 72% of BGP allergic patients were co-sensitised to rPhl p 1, rPhl p 2, nPhl p 4, rPhl p 5, rPhl p 6. A decrease of total and specific IgE with patients' age was observed. Conclusions: Our data show that all BGP-allergic patients simultaneously exhibit higher IgE antibody levels to recombinant and natural P. pratense allergens as well as to crude TGP extract. This suggests that when choosing an immunotherapeutic regimen for BGP-sensitised patients (after establishing their IgE profile via purified TGP-allergens), subcutaneous or sublingual TGP-extract vaccines in appropriate doses, in order to influence T epitope specificity, might be beneficial. Though extremely uncommon, in cases where a patient is exclusively BGP allergen-sensitised, BGP-extract therapy is the appropriate therapeutic response.

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

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[217] - Lièvre K, Aparicio C, Lallemand K, Kosmalski D, Leduc V. Le pollen de fléole (Phleum pratense L.) est-il représentatif de l’ensemble des pollens de graminées ?. Rev Fr Allergol 2009;49:333

La polysensibilisation au sein de la famille des graminées est fréquente et expliquée par la présence d‚allergènes majeurs présentant de fortes similitudes de séquence. Cette étude a pour but d‚évaluer la représentativité d‚un pollen de graminée (fléole) par rapport à l‚ensemble des allergènes de pollens de 17 graminées fourragères et céréalières. Résultats.ˆ Les profils allergéniques des pollens de 17 graminées ont été comparés par SDS-PAGE et immuno-empreinte. À l‚aide d‚anticorps polyclonaux spécifiques de Phl p 1 et Phl p 5, les allergènes majeurs des groupes 1 et 5 ont été identifiés dans l‚ensemble des pollens étudiés, excepté pour le maïs et le cynodon pour lesquels le groupe 5 n‚est pas reconnu. L‚utilisation d‚un pool de sérums de 4 patients allergiques a permis de mettre en évidence d‚autres allergènes (groupe 2, 4, 12 et 13). La fixation des IgE de ce pool de sérums sur les extraits allergéniques des 17 pollens de graminées est complètement inhibée par l‚extrait de fléole, montrant ainsi une communauté épitopique entre tous les allergènes de ces pollens. Cela confirme que le pollen de fléole est bien un représentant valide des 15 pollens de graminées, dans le cadre du diagnostic et de la désensibilisation. Conclusion.ˆ Le pollen de fléole est représentatif de la plupart des pollens de graminées pour les allergènes majeurs. Les anticorps polyclonaux utilisés sont spécifiques de plusieurs épitopes, au même titre que les IgE des patients allergiques, ce qui permet de mimer au mieux leur cross-reactivité, contrairement aux anticorpsmonoclonaux souvent trop spécifiques.L‚implication d‚allergènes moins représentés dans des sensibilisations plus spécifiques ne doit cependant pas être écartée, en particulier dans le cadre de réactions allergiques où un allergènemineur serait impliqué, justifiant la disponibilité des autres pollens de graminées.

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[218] - Johansen N, Weber RW, Ipsen H, Barber D, Broge L, Hejl C. Extensive IgE Cross-Reactivity towards the Pooideae Grasses Substantiated for a Large Number of Grass-Pollen-Sensitized Subjects. Int Arch Allergy Immunol 2009;150:325-334

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[219] - Moingeon P, Hrabina M, Bergmann KC, Jaeger S, Frati F, Bordas V, et al. Specific Immunotherapy for Common Grass Pollen Allergies: Pertinence of a Five Grass Pollen Vaccine. Int Arch Allergy Immunol 2008;146:338-342

Patients throughout Europe are concomitantly exposed to multiple pollens from distinct Pooideae species. Given the overlap in pollination calendars and similar grain morphology, it is not possible to identify which grass species are present in the environment from pollen counts. Furthermore, neither serum IgE reactivity nor skin prick testing allow the identification of which grass species are involved in patient sensitisation. Due to their high level of amino acid sequence homology (e.g., >90% for group 1, 55-80% for group 5), significant cross-immunogenicity is observed between allergens from Pooideae pollens. Nevertheless, pollen allergens also contain species-specific T or B cell epitopes, and substantial quantitative differences exist in allergen (e.g., groups 1 and 5) composition between pollens from distinct grass species. In this context, a mixture of pollens from common and well-characterised Pooideae such as Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis is suitable for immunotherapy purposes because (1) it has been validated, both in terms of safety and efficacy, by established clinical practice; (2) it reflects natural exposure and sensitisation conditions; (3) it ensures a consistent and well-balanced composition of critical allergens, thus extending the repertoire of T and B cell epitopes present in the vaccine.

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[220] - Chabre H, Gouyon B, Batard T, Adam A, Nony E, Berrouet C, et al. Considerable variability of grass pollen major and minor allergens between Pooideae species: implications for vaccine design. Allergy 2008;63(suppl. 88):220-221

Background: Patients throughout Europe are concomitantly exposed and sensitised to multiple pollen allergens from distinct Pooideae species. Despite a high level of amino acid sequence homology (e.g. 490% for group 1, 5580% for group 5), potential quantitative and qualitative differences between allergens from common grass species remain to investigated. Methods: Group 1 and group 5 allergens were purified by chromatography from Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis pollens and analysed by mass spectrometry. MHC class II restricted T cell epitopes within group 1 allergens were analyzed using bioinformatics and direct HLA binding assays. Species-specific epitopes within minor allergens were defined using rabbit anti sera, or PF2D proteomic analyses. Results: Group 5 allergens obtained from various grasses exhibit different molecular weights and ratios between isoforms a and b. Mass spectrometry analysis reveals an unsuspected variability, in the form of variants and glycoforms, for both group 1 and group 5 major allergens. Within group 1, up to fourteen potential MHC class II restricted T cell epitopes have been identified, out of which only one is conserved among grass species. Substantial quantitative differences (at least ten fold) also exist in group 1 and 5 content between pollens from distinct grass species. IgE binding experiments conducted with purified group 1 and group 5 allergens or with natural extracts resolved by IEF reveal that 10-20% patients exhibit IgE responses to allergen epitopes restricted to a given grass species. Serologic and proteomic analyses further confirm the existence of grass species restricted epitopes for group 2, 3, 4, 6 and 11 allergens. Conclusion: Molecular analyses reveal that grass pollen major and minor allergens bear both shared and species-restricted immune epitopes. As a consequence, specific immunotherapy for grass pollen allergy should be performed with a mixture of pollens from common Pooideae, in order to better reflect natural exposure and sensitisation conditions at a molecular and submolecular (epitope) level.

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[221] - Würtzen P, Wissenbach M, Ipsen H, Bufe A, Arnved J, Van Neerven RJ. Highly heterogeneous Phl p 5-specific T cells from patients with allergic rhinitis differentially recognize recombinant Phl p 5 isoallergens. J Allergy Clin Immunol 1999;104:115-122

BACKGROUND: The prevalence of atopic allergy to Poaceae grasses poses a serious health problem. OBJECTIVE: To evaluate the T cell-activating capacity of recombinant grass pollen allergens suggested for immunotherapy, we characterized the T-cell response of allergic patients to the Phleum pratense (Phl p) major allergen, Phl p 5. METHODS: Thirty-eight Phl p 5-specific CD4(+) T-cell clones (TCCs) were isolated from the peripheral blood of 3 patients with allergic rhinitis and were characterized with respect to cross-reactivity, HLA restriction, cytokine profiles, and isoallergen specificity when tested with natural Phl p 5 and 5 rPhl p 5 isoallergens (4 Phl p 5a and 1 Phl p 5b). RESULTS: The TCCs were highly cross-reactive with group 5 allergens of related grasses, and the different cross-reactivity patterns found indicate that several T-cell epitopes are present in Phl p 5. Most TCCs displayed a TH0-like cytokine profile, whereas a few TCCs belonged to the TH2 or TH1 subset. The TCCs were restricted by HLA- DR (24/38 TCCs), HLA-DQ (11/38 TCCs), or HLA-DP (3/38 TCCs). Interestingly, 4 of the 34 TCCs tested reacted exclusively with the 4 rPhl p 5a isoforms; 8 of 34 TCCs were rPhl p 5b specific, and 3 of 34 TCCs reacted with all isoforms; 19 of 34 TCCs did not react with any of the rPhl p 5 isoallergens. Moreover, the overall isoform recognition pattern differed considerably among patients. CONCLUSION: These results demonstrate that Phl p 5-specific T cells are highly heterogeneous and that individual TCCs, and individual patients, differentially recognize isoallergens. The differential isoallergen recognition for Phl p 5- specific T cells suggests, if confirmed in larger patient groups, that a combination of 2 or more rPhl p 5 isoallergens may be needed to replace the natural grass allergen for immunotherapy

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[222] - Müller WD, Karamfilov T, Fahlbusch B, Vogelsang H, Jäger L. Analysis of human T cell clones reactive with group V grass pollen allergens. Int Arch Allergy Immunol 1994;105:391-396

Twenty-seven T cell clones (TCC) reactive with group V allergens of Phleum pratense (Phlp V) were established from the peripheral blood of 3 patients allergic to grass pollen. Twenty-four TCC showed the helper cell phenotype CD3+, CD4+, CD8-; the remaining clones were CD3+CD4-CD8+. T cell recognition of Phlp V was exclusively HLA-DR restricted. Many of the Phlp V reactive TCC (19 of 27; 70%) were stimulated additionally by other group V allergens isolated from Lolium perenne, Poa pratensis, and Dactylis glomerata. These data indicate the existence of cross-reacting T cell epitopes among group V allergens of different grasses. The Phlp V triggered cytokine production demonstrated in 13 out of 24 CD4+ TCC a Th2-like pattern (high interleukin 4/gamma-interferon ratios) suggesting group V allergens as important targets of grass pollen specific IgE.

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[223] - Schenk S, Breiteneder H, Susani M, Najafian N, Laffer S, Duchêne M, et al. T-cell epitopes of Phl p 1, major pollen allergen of timothy grass (Phleum pratense): evidence for crossreacting and non-crossreacting T-cell epitopes within grass group I allergens. J Allergy Clin Immunol 1995;96:986-996

The use of peptides representing T-cell epitopes of allergens is a modern concept for improvement of specific immunotherapy. A prerequisite for this approach is the identification of T-cell epitopes of atopic allergens. METHODS: T-cell lines and 40 T-cell clones (TCC) specific for Phl p 1, the group I allergen of timothy grass (Phleum pratense), were established from the peripheral blood of nine patients allergic to grass pollen and mapped for epitope specificity by using overlapping dodecapeptides. Phenotype and cytokine production profile of TCC were investigated. Representative TCC were analyzed for HLA-restriction, T-cell receptor V beta gene usage, and crossreactivity with grass pollen extracts from Dactylis glomerata, Poa pratensis, Lolium perenne, Secale cereale, and selected amino acid sequence-derived peptides. RESULTS: Patients displayed IgE binding to all grass species investigated. Forty TCC were established. Fifteen T-cell epitopes could be identified on Phl p 1. Of 40 TCC, 39 displayed the helper cell (Th) phenotype; one clone was CD8+. Specific stimulation induced a Th2-like type of cytokine production in 20 of 39 TCC. Crossreactivity studies revealed crossreacting and non-crossreacting T-cell epitopes. CONCLUSION: Phl p 1, a major grass pollen allergen, harbors multiple T-cell epitopes. Species-specific and crossreacting T-cell epitopes exist among group I allergens of grasses. Epitope recognition patterns could not be correlated with particular HLA haplotypes. A restricted T-cell receptor V beta gene usage was not observed.

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[224] - Chabre H, Gouyon B, Batard T, Adam A, Nony E, Berrouet C, et al. Considerable variability of grass pollen major and minor allergens between Pooideae species: implications for vaccine design. Allergy 2008;63(suppl. 88):220-221

Background: Patients throughout Europe are concomitantly exposed and sensitised to multiple pollen allergens from distinct Pooideae species. Despite a high level of amino acid sequence homology (e.g. 490% for group 1, 5580% for group 5), potential quantitative and qualitative differences between allergens from common grass species remain to investigated. Methods: Group 1 and group 5 allergens were purified by chromatography from Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis pollens and analysed by mass spectrometry. MHC class II restricted T cell epitopes within group 1 allergens were analyzed using bioinformatics and direct HLA binding assays. Species-specific epitopes within minor allergens were defined using rabbit anti sera, or PF2D proteomic analyses. Results: Group 5 allergens obtained from various grasses exhibit different molecular weights and ratios between isoforms a and b. Mass spectrometry analysis reveals an unsuspected variability, in the form of variants and glycoforms, for both group 1 and group 5 major allergens. Within group 1, up to fourteen potential MHC class II restricted T cell epitopes have been identified, out of which only one is conserved among grass species. Substantial quantitative differences (at least ten fold) also exist in group 1 and 5 content between pollens from distinct grass species. IgE binding experiments conducted with purified group 1 and group 5 allergens or with natural extracts resolved by IEF reveal that 10-20% patients exhibit IgE responses to allergen epitopes restricted to a given grass species. Serologic and proteomic analyses further confirm the existence of grass species restricted epitopes for group 2, 3, 4, 6 and 11 allergens. Conclusion: Molecular analyses reveal that grass pollen major and minor allergens bear both shared and species-restricted immune epitopes. As a consequence, specific immunotherapy for grass pollen allergy should be performed with a mixture of pollens from common Pooideae, in order to better reflect natural exposure and sensitisation conditions at a molecular and submolecular (epitope) level.

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[225] - Müller WD, Karamfilov T, Kahlert H, Stuwe HT, Fahlbusch B, Cromwell O, et al. Mapping of T-cell epitopes of Phl p 5: evidence for crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens. Clin Exp Allergy 1998;28:1538-1548

BACKGROUND: Group 5 allergens represent major grass pollen allergens because of their high sensitization indices. The identification of T-cell epitopes of these allergens is a prerequisite for the design of immunotherapeutic strategies based on peptide vaccination or modified allergens with conserved T-cell epitopes. OBJECTIVE: This study was undertaken to determine T-cell epitopes on Phl p 5 major pollen allergen of timothy grass (Phleumn pratense). METHODS: T-cell lines (TCLs) and T-cell clones (TCCs), specific to Phl p 5, were established from the peripheral blood of 18 patients allergic to grass pollen. All TCCs were mapped for epitope specificities using 178 overlapping dodecapeptides representing the primary structures of two isoforms of Phl p 5 (Phl p 5a and Phl p 5b). Phenotype and cytokine production profiles of TCCs were tested. Selected TCCs were analysed for HLA class II restriction. RESULTS: A total of 82 TCCs were isolated. All TCCs displayed the helper cell (TH) phenotype. Their reactivity with two recombinant expressed isoforms of Phl p 5a and Phl p 5b was heterogeneous. The epitope specificity of the TCCs was then revealed. Nineteen T-cell epitopes could be identified on Phl p 5. Eighty-one percent of mapped TCCs recognized three T-cell reactive regions on the Phl p 5 allergen. Some TCCs were reactive with isoepitopes presenting on Phl p 5a as well as Phl p 5b. Allergen-specific stimulation induced a TH0-like type of cytokine production in 25 of 50 TCCs. Almost all TCCs secreted high concentrations of interleukin-13. CONCLUSION: Phl p 5, a major grass pollen allergen, contains several T-cell epitopes. Some epitope regions were recognized by several patients. Epitope recognition pattern could not be correlated with special HLA class II haplotypes. T-cell stimulating isoepitopes were found at corresponding regions of Phl p 5a and Phl p 5b isoforms.

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[226] - Hejl C, Wurtzen PA, Kleine-Tebbe J, Johansen N, Broge L, Ipsen H. Phleum pratense alone is sufficient for allergen-specific immunotherapy against allergy to Pooideae grass pollens. Clin Exp Allergy 2009;39:752-759

BACKGROUND: Specific immunotherapy is the only causal treatment of allergy available today. Traditionally, therapeutic products based on either a single grass species or a mix of such extracts are used for grass pollen immunotherapy. Investigations comparing the immunological response to these allergen preparations are needed to ensure optimal treatment. The objective of this study was to investigate patterns of T and B cell cross-reactivity to Pooideae single-species extracts and to extract mixes . METHODS: IgG4 induced by immunotherapy with Phleum pratense extract was investigated for cross-reactivity using nine single-species extracts and four mixes. For the mixes, studies of IgE cross-reactivity were also performed. T cell cross-reactivity was investigated in lines specific to nPhl p 1 or nPhl p 5 allergens, and the amounts of group 1 and 5 allergens in the extracts were quantified by a single radial immunodiffusion . RESULTS: The levels of treatment-induced IgG4 detected by all the extracts displayed a clear correlation to that detected by the P. pratense pollen extract. The IgE studies confirmed the cross-reactivity of P. pratense-specific B cells towards the allergens contained in the mixes, and the T cell studies demonstrated cross-reactivity towards group 1 and 5 major allergens in extracts of six temperate grass species . CONCLUSION: Extensive T and B cell cross-reactivity was observed towards the allergens of the Pooideae grasses, and the degree of B cell cross-reactivity was independent of the number of species included in the extract mixes. This implies that treatment with pollen extract of just one Pooideae species will affect the allergic responses caused by any of the temperate grasses in this subfamily.

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[227] - Würtzen PA, van Neerven RJ, Arnved J, Ipsen H, Sparholt SH. Dissection of the grass allergen-specific immune response in patients with allergies and control subjects: T-cell proliferation in patients does not correlate with specific serum IgE and skin reactivity. J Allergy Clin Immunol 1998;101:241-249

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[228] - Würtzen PA, van Neerven RJ, Arnved J, Ipsen H, Sparholt SH. Dissection of the grass allergen-specific immune response in patients with allergies and control subjects: T-cell proliferation in patients does not correlate with specific serum IgE and skin reactivity. J Allergy Clin Immunol 1998;101:241-249

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Pollinose aux Graminées

Les allergènes de pollens de graminées

Réactivité croisée entre pollens de graminées

Réactivités croisée entre pollens de graminées et d’autres pollens

Réactivités croisées entre pollens de graminées et aliments

Le diagnostic d’une sensibilisation aux graminées

Pollens de graminées et CCD

Graminées et immuno-thérapie

Rubriques