IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.

BACKGROUND: House dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. OBJECTIVE: Identification, characterization, and evaluation of diagnostic utility of a new important HDM allergen was performed. METHODS: A cDNA coding for a new Dermatophagoides pteronyssinus (Dp) allergen, Der p 37, was isolated from a Dp expression library with allergic patients' IgE antibodies. Recombinant Der p 37 (rDer p 37) expressed in Escherichia coli was purified, then characterized by mass spectrometry, circular dichroism, and IgE reactivity by ImmunoCAP ISAC technology with sera from 111 clinically defined HDM-allergic patients. The allergenic activity of rDer p 37 was studied by basophil activation and CD4+ T-cell responses by carboxyfluorescein diacetate succinimidyl ester dilution assays. Specific antibodies raised against rDer p 37 were used for the ultrastructural localization of Der p 37 in mites by immunogold transmission electron microscopy. RESULTS: Der p 37, a 26 kDa allergen with homology to chitin-binding proteins, is immunologically distinct from Der p 15, 18, and 23. It is located in the peritrophic membrane of fecal pellets. Der p 37 reacted with IgE antibodies from a third of HDM-allergic patients and induced specific basophil- and CD4+ T-cell activation. Der p 37 IgE-positive patients had significantly higher IgE levels to major HDM allergens, reacted with more HDM allergens, and had a higher risk (odds ratio = 3.1) of asthma compared to Der p 37-negative patients. CONCLUSIONS: Der p 37, a new Dp allergen recognized by a third of HDM-allergic patients, may serve as a surrogate marker for severe HDM sensitization and asthma.

Identification of Der p 23, a peritrophin-like protein, as a new major Dermatophagoides pteronyssinus allergen associated with the peritrophic matrix of mite fecal pellets.

The house dust mite (HDM) Dermatophagoides pteronyssinus is one of most important allergen sources and a major elicitor of allergic asthma. We screened a D. pteronyssinus expression cDNA library with IgE Abs from HDM allergic patients. A cDNA coding for a new major allergen was isolated, which showed sequence homology to peritrophins, which contain chitin-binding domains and are part of the peritrophic matrix lining the gut of arthropods. The mature Der p 23 allergen was expressed in Escherichia coli as an 8-kDa protein without its hydrophobic leader sequence and purified to homogeneity. It reacted with IgE Abs from 74% of D. pteronyssinus allergic patients (n = 347) at levels comparable to the two major HDM allergens, Der p 1 and Der p 2. Thus, Der p 23 represents a new major D. pteronyssinus allergen. Furthermore, rDer p 23 exhibited high allergenic activity as demonstrated by upregulation of CD203c expression on basophils from D. pteronyssinus allergic patients. Immunogold electron microscopy localized the allergen in the peritrophic matrix lining the midgut of D. pteronyssinus as well as on the surface of the fecal pellets. Thus, we identified a new major D. pteronyssinus allergen as peritrophin-like protein. The high allergenic activity of Der p 23 and its frequent recognition as respiratory allergen may be explained by the fact that it becomes airborne and respirable through its association with mite feces. Der p 23 may be an essential component for diagnosis and specific immunotherapy of HDM allergy.

Disruption of allergenic activity of the major grass pollen allergen Phl p 2 by reassembly as a mosaic protein.

The recognition of conformational epitopes on respiratory allergens by IgE Abs is a key event in allergic inflammation. We report a molecular strategy for the conversion of allergens into vaccines with reduced allergenic activity, which is based on the reassembly of non-IgE-reactive fragments in the form of mosaic proteins. This evolution process is exemplified for timothy grass pollen-derived Phl p 2, a major allergen for more than 200 million allergic patients. In a first step, the allergen was disrupted into peptide fragments lacking IgE reactivity. cDNAs coding for these peptides were reassembled in altered order and expressed as a recombinant mosaic molecule. The mosaic molecule had lost the three-dimensional structure, the IgE reactivity, and allergenic activity of the wild-type allergen, but it induced high levels of allergen-specific IgG Abs upon immunization. These IgG Abs crossreacted with group 2 allergens from other grass species and inhibited allergic patients' IgE binding to the wild-type allergen. The mosaic strategy is a general strategy for the reduction of allergenic activity of protein allergens and can be used to convert harmful allergens into safe vaccines.

Electron tomography of structures in the wall of hazel pollen grains.

The three-dimensional structure of channels and bacula cavities in the wall of hazel pollen grains was investigated by automated electron tomography in order to explore their role in the release of allergen proteins from the pollen grains. 3D reconstructions of 100-150 nm thick resin-embedded sections, stabilized by thin platinum-carbon coating, revealed that the channels aimed directly towards the surface of the grain and that the bacula cavities were randomly sized and merged into larger ensembles. The number and the dimensions of the ensembles were quantitatively determined by neighboring voxel analysis on thresholded reconstructed volumes. To simulate the allergen release, allergen proteins were approximated by a hard sphere model of a diameter corresponding to the largest dimension of the known 3D structure of the major birch allergen, Bet v 1, whose amino acid sequence is highly similar to the amino acid sequence of the major hazel allergen, Cor a 1. The analysis of positions where the hard sphere fits into the resolved channels and bacula cavity structures revealed that unbound allergens could freely traverse through the channels and that the bacula cavities support the path of the allergens towards the surface of the grain.

Immunogold electron microscopic localization of the 2 EF-hand calcium-binding pollen allergen Phl p 7 and its homologues in pollens of grasses, weeds and trees.

BACKGROUND: The 2 EF-hand calcium-binding allergen from timothy grass pollen, Phl p 7, contains the majority of relevant IgE epitopes among calcium-binding allergens occurring in pollen species of different plants. OBJECTIVE: To describe the ultrastructural localization of Phl p 7 allergen in timothy grass pollen and its homologues in a broad spectrum of allergologically relevant pollens from grasses (timothy grass, rye grass), trees (birch, alder, olive) and weeds (mugwort, ribwort, ragweed) commonly growing in Europe. MATERIALS AND METHODS: Mature pollens from 8 different plant species were collected and anhydrously prepared for transmission electron microscopy. In ultrathin sections, allergens were localized using an antibody prepared against a Phl p 7-derived peptide comprising the C-terminal half of the Phl p 7 wild-type molecule in combination with a secondary antibody coupled to 10-nm colloidal gold particles. RESULTS: Phl p 7 and Phl p 7 homologues were detected in pollen from each of the 8 pollen species investigated. The allergens were found in the cytoplasm of the pollen grains (cytoplasmic matrix, mitochondria, nuclei) and in the pollen wall (preferably the exine). Reserve materials were unlabeled. CONCLUSIONS: The 2 EF-hand calcium-binding allergen Phl p 7 from timothy grass and its homologues can be localized in all pollen species under investigation. This finding confirms that Phl p 7 is a marker allergen for sensitization of patients to a novel family of 2 EF-hand calcium-binding pollen allergens occurring in a number of important allergenic plants in Europe.

Characterization of folded recombinant Der p 5, a potential diagnostic marker allergen for house dust mite allergy.

BACKGROUND: Der p 5 was reported as an important allergen in Dermatophagoides pteronyssinus, which is particularly recognized by patients suffering from asthma. The aim of this study was to produce, by recombinant DNA technology, a folded Der p 5 allergen for diagnostic, therapeutic and preventive purposes. METHODS: Der p 5-encoding cDNA was isolated from a lambda gt11 D. pteronyssinus expression cDNA library and expressed in Escherichia coli. rDer p 5 was purified to homogeneity and characterized by mass spectroscopy and circular dichroism. IgE reactivity was tested with sera from 117 mite-allergic patients and in a basophil histamine release experiment. Der p 5-specific rabbit IgG antibodies were produced for the ultrastructural localization of the allergen in mites by immunogold electron microscopy as well as for cross-reactivity studies. RESULTS: rDer p 5 is a heat-stable protein with predominantly alpha-helical secondary structure which reacted with IgE from 31% of mite-allergic patients' sera and showed no relevant cross-reactivity to group 5 allergens from storage mites and tropical mites. rDer p 5-specific rabbit IgG antibodies inhibited mite-allergic patients' IgE binding to Der p 5 and allowed to localize the allergen in secretory granules of midgut epithelial cells of house dust mites. CONCLUSIONS: The described rDer p 5 molecule may be useful for diagnosis and immunotherapy of house dust mite-allergic patients.

Molecular and immunological characterization of a wheat serine proteinase inhibitor as a novel allergen in baker's asthma.

IgE-mediated sensitization to wheat flour belongs to the most frequent causes of occupational asthma. A cDNA library from wheat seeds was constructed and screened with serum IgE from baker's asthma patients. One IgE-reactive phage clone contained a full-length cDNA coding for an allergen with a molecular mass of 9.9 kDa and an isoelectric point of 6. According to sequence analysis it represents a member of the potato inhibitor I family, a group of serine proteinase inhibitors, and thus is the first allergen belonging to the group 6 pathogenesis-related proteins. The recombinant wheat seed proteinase inhibitor was expressed in Escherichia coli and purified to homogeneity. According to circular dichroism analysis, it represented a soluble and folded protein with high thermal stability containing mainly beta-sheets, random coils, and an alpha-helical element. The recombinant allergen showed allergenic activity in basophil histamine release assays and reacted specifically with IgE from 3 of 22 baker's asthma patients, but not with IgE from grass pollen allergic patients or patients suffering from food allergy to wheat. Allergen-specific Abs were raised to localize the allergen by immunogold electron microscopy in the starchy endosperm and the aleuron layer. The allergen is mainly expressed in mature wheat seeds and, despite an approximately 50% sequence identity, showed no relevant cross-reactivity with allergens from other plant-derived food sources such as maize, rice, beans, or potatoes. Recombinant wheat serine proteinase inhibitor, when used in combination with other specific allergens, may be useful for the diagnosis and therapy of IgE-mediated baker's asthma.

Bundles of hexagonally arranged tubules in timothy grass pollen: detection of a novel pollen component using anhydrous fixation and image analysis techniques in transmission electron microscopy.

Pollen from timothy grass (Phleum pratense L.) was subjected to various aqueous and non-aqueous fixation and preparation protocols for transmission electron microscopy. Only in the cytoplasm of anhydrously prepared pollen grains were conspicuous inclusions observed that range in size from less than 1 mum up to 8 or 10 mum. These bodies have so far not been described in the literature. Higher magnifications show that these inclusions consist of bundles of hexagonally arranged small tubules. In order to obtain details of the ultrastructure of this novel pollen component, TEM micrographs of ultrathin sections of hexagonally arranged tubules were analyzed using Fourier transform techniques of image analysis. It was found that the tubules form groups with quasi-periodic hexagonal arrangement, with an average centre-to-centre spacing between the neighbouring tubules of approximately 42 nm. Individual tubules are formed by 12 or 13 particles. The outer diameter of the tubules ranges between 22 and 24 nm. From our experiments, we conclude that the quasi-periodic hexagonally arranged tubules forming conspicuous cytoplasmic inclusions in dry timothy grass pollen grains are structurally similar to microtubules.

Group 13 allergens as environmental and immunological markers for grass pollen allergy: studies by immunogold field emission scanning and transmission electron microscopy.

BACKGROUND: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen. METHODS: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13. The release kinetics of Phl p 13 from timothy grass pollen hydrated for 0.5 min to 3 h were analyzed by immunoblotting. Phl p 13 was localized in dry and hydrated grass pollen grains by immunogold field emission scanning and transmission electron microscopy. RESULTS: Group 13 allergens were detected in all 12 wild and cultivated grass genera representing the major subfamilies of the Poaceae. Ultrastructurally, the allergen was located in the wall and in the cytoplasm of timothy grass pollen grains. In the cytoplasm, Phl p 13 was associated with polysaccharide particles and as yet undescribed stacks of microtubule-like structures. After hydration in rain water, pollen grains expel cytoplasmic particles of respirable size containing Phl p 13, which becomes detectable in aqueous supernatants already after 0.5 min. CONCLUSIONS: Group 13 allergens represent one set of marker allergens which specifically occur in pollen of the major grass subfamilies and are rapidly released in association with respirable particles after pollen hydration. They may be considered as environmental markers for grass pollen exposure and group 13-specific IgE antibodies as immunological markers for genuine grass pollen sensitization.

Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy.

BACKGROUND: Pollen from early-flowering trees (eg, birch, alder, hazel) represent major seasonal allergen sources. The effects of rain on the release of allergens from tree pollen has thus far not been studied at the ultrastructural level. OBJECTIVE: This study was designed to investigate the effects of rain on the morphology of pollens from early-flowering trees and of potential rain-induced mechanisms of allergen release. METHODS: Freshly collected pollen grains (birch, alder, and hazel) were exposed under controlled conditions to rainwater. Changes of pollen morphology and the release of allergens were analyzed by scanning electron microscopy. The release of allergen-bearing submicronic particles was studied by field emission scanning electron microscopy and transmission electron microscopy in conjunction with immunogold staining by using antibodies with specificity for the major allergens. RESULTS: Scanning electron microscopy showed that freshly isolated pollen grains from birch, alder, and hazel have abortive germination in rainwater. Abortive pollen germination is characterized by the formation of short pollen tubes, which rupture at their tips and release micronic and submicronic particles containing major allergens. Immunogold transmission electron microscopy provided evidence that the allergens are transported through the pollen tubes during germination. CONCLUSIONS: Rainwater-induced release of allergen-bearing submicronic particles from abortively germinated tree pollens may represent a mechanism of allergen release, with important implications on the induction of asthma as well as on current methods for measuring environmental allergen exposure.

Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by immunogold electron microscopy.

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.

Molecular characterization of recombinant T1, a non-allergenic periwinkle (Catharanthus roseus) protein, with sequence similarity to the Bet v 1 plant allergen family.

More than 25% of the population suffer from Type I allergy, an IgE-mediated hypersensitivity disease. Allergens with homology to the major birch ( Betula verrucosa ) pollen allergen, Bet v 1, belong to the most potent elicitors of IgE-mediated allergies. T1, a cytokinin-inducible cytoplasmic periwinkle ( Catharanthus roseus ) protein, with significant sequence similarity to members of the Bet v 1 plant allergen family, was expressed in Escherichia coli. Recombinant T1 (rT1) did not react with IgE antibodies from allergic patients, and failed to induce basophil histamine release and immediate-type skin reactions in Bet v 1-allergic patients. Antibodies raised against purified rT1 could be used for in situ localization of natural T1 by immunogold electron microscopy, but did not cross-react with most of the Bet v 1-related allergens. CD analysis showed significant differences regarding secondary structure and thermal denaturation behaviour between rT1 and recombinant Bet v 1, suggesting that these structural differences are responsible for the different allergenicity of the proteins. T1 represents a non-allergenic member of the Bet v 1 family that may be used to study structural requirements of allergenicity and to engineer hypo-allergenic plants by replacing Bet v 1-related allergens for primary prevention of allergy.

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

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.