Intranasal administration of allergen increases specific IgE whereas intranasal omalizumab does not increase serum IgE levels-A pilot study.

BACKGROUND: Administration of the therapeutic anti-IgE antibody omalizumab to patients induces strong increases in IgE antibody levels. OBJECTIVE: To investigate the effect of intranasal administration of major birch pollen allergen Bet v 1, omalizumab or placebo on the levels of total and allergen-specific IgE in patients with birch pollen allergy. METHODS: Based on the fact that intranasal allergen application induces rises of systemic allergen-specific IgE, we performed a double-blind placebo-controlled pilot trial in which birch pollen allergic subjects were challenged intranasally with omalizumab, placebo or birch pollen allergen Bet v 1. Total and allergen-specific IgE, IgG and basophil sensitivity were measured before and 8 weeks after challenge. For control purposes, total, allergen-specific IgE levels and omalizumab-IgE complexes as well as specific IgG levels were studied in subjects treated subcutaneously with either omalizumab or placebo. Effects of omalizumab on IgE production by IL-4/anti-CD40-treated PBMCs from allergic patients were studied in vitro. RESULTS: Intranasal challenge with Bet v 1 induced increases in Bet v 1-specific IgE levels by a median of 59.2%, and this change differed significantly from the other treatment groups (P = .016). No relevant change in allergen-specific and total IgE levels was observed in subjects challenged with omalizumab. Addition of omalizumab did not enhance IL-4/anti-CD40-induced IgE production in vitro. Significant rises in total IgE (mean IgE before: 131.83 kU/L to mean IgE after: 505.23 kU/L) and the presence of IgE-omalizumab complexes were observed after subcutaneous administration of omalizumab. CONCLUSION: Intranasal administration of allergen induced rises of allergen-specific IgE levels, whereas intranasal administration of omalizumab did not enhance systemic total or allergen-specific IgE levels.

EAACI Molecular Allergology User's Guide.

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Poor association of allergen-specific antibody, T- and B-cell responses revealed with recombinant allergens and a CFSE dilution-based assay.

BACKGROUND: The adaptive immunity underlying allergy comprises two components, the allergen-specific antibody (i.e. IgE, IgG) and the T-cell response. These two components are responsible for different disease manifestations and can be targeted by different therapeutic approaches. Here, we investigated the association of allergen-specific antibody and T- as well as B-cell responses in pollen-allergic patients using recombinant (r) major birch pollen allergen rBet v 1 and major timothy grass pollen allergen rPhl p 5 as defined antigens. METHODS: Allergen-specific IgE and IgG antibody responses were determined by ELISA, and allergen-specific T- and B-cell responses were measured in peripheral blood mononuclear cells using a carboxyfluorescein-diacetate-succinimidylester (CFSE) dilution assay. RESULTS: CFSE staining in combination with T-cell- and B-cell-specific gating allowed discriminating between allergen-specific T-cell and B-cell responses. Interestingly, we identified patients where mainly T cells and others where mainly B cells proliferated in response to allergen stimulation. No association between the level of allergen-specific Ig responses and B- or T-cell proliferation was observed. CONCLUSION: Purified recombinant allergens in conjunction with CFSE staining allow the dissection of allergen-specific B- and T-cell responses. The dissociation of allergen-specific antibody, and B- and T-cell responses may explain the occurrence of selective IgE- and T-cell-mediated manifestations of allergic inflammation and may be important for the development of diagnostic and therapeutic strategies selectively targeting B cells and T cells.

Multiple grass mixes as opposed to single grasses for allergen immunotherapy in allergic rhinitis.

Grass pollen allergy affects approximately 40% of allergic patients. Subcutaneous allergen immunotherapy (SCIT) is the only allergen-specific and disease-modifying treatment available. Currently available therapeutic vaccines for the treatment of grass pollen allergy are based on natural grass pollen extracts which are either made from pollen of one cross-reactive grass species or from several related grass species. Clinical studies have shown that SCIT performed with timothy grass pollen extract is effective for the treatment of grass pollen allergy. Moreover, it has been demonstrated that recombinant timothy grass pollen allergens contain the majority of relevant epitopes and can be used for SCIT in clinical trials. However, recent in vitro studies have suggested that mixes consisting of allergen extracts from several related grass species may have advantages for SCIT over single allergen extracts. Here, we review current knowledge regarding the disease-relevant allergens in grass pollen allergy, available clinical studies comparing SCIT with allergen extracts from timothy grass or from mixes of several related grass species of the Pooideae subfamily, in vitro cross-reactivity studies performed with natural allergen extracts and recombinant allergens and SCIT studies performed with recombinant timothy grass pollen allergens. In vitro and clinical studies performed with natural allergen extracts reveal no relevant advantages of using multiple grass mixes as opposed to single grass pollen extracts. Several studies analysing the molecular composition of natural allergen extracts and the molecular profile of patients' immune responses after SCIT with allergen extracts indicate that the major limitation for the production of a high quality grass pollen vaccine resides in intrinsic features of natural allergen extracts which can only be overcome with recombinant allergen-based technologies.

An assay that may predict the development of IgG enhancing allergen-specific IgE binding during birch immunotherapy.

BACKGROUND: It has been shown that birch pollen immunotherapy can induce IgG antibodies which enhance IgE binding to Bet v 1. We aimed to develop a serological assay to predict the development of antibodies which enhance IgE binding to Bet v 1 during immunotherapy. METHODS: In 18 patients treated by Bet v 1-fragment-specific immunotherapy, the effects of IgG antibodies specific for the fragments on the binding of IgE antibodies to Bet v 1 were measured by ELISA. Blocking and possible enhancing effects on IgE binding were compared with skin sensitivity to Bet v 1 after treatment. RESULTS: We found that fragment-specific IgG enhanced IgE binding to Bet v 1 in two patients who also showed an increase of skin sensitivity to Bet v 1. CONCLUSION: Our results indicate that it may be possible to develop serological tests which predict the induction of unfavourable IgG antibodies enhancing the binding of IgE to Bet v 1 during immunotherapy.

The majority of allergen-specific IgE in the blood of allergic patients does not originate from blood-derived B cells or plasma cells.

BACKGROUND: The production of allergen-specific IgE antibodies is a hallmark of IgE-mediated allergy but the contribution of blood cells to allergen-specific IgE production in allergic patients has not been studied in detail. OBJECTIVE: Aim of this study was the characterization of IgE-producing cells in the blood of allergic patients and the determination of the amount of IgE antibodies which are produced by these cells in relation to total amounts of circulating specific IgE. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from allergic patients and cell populations were purified or depleted using magnetically labelled antibodies directed against specific cell surface markers (CD19, CD20, CD22, CD27, CD38, CD126, CD138, CD203c). Allergen-specific IgE was measured in serum samples and cell culture supernatants by quantitative ImmunoCAP measurements and by ELISA using purified recombinant allergens. IgE transcripts were detected using RT-PCR with primers specific for human IgE. RESULTS: We found that allergen-specific IgE levels in PBMC supernatants correlated strongly with specific serum IgE but represented less than 1% of circulating IgE. Depletion of basophils resulted in substantial reduction of allergen-specific IgE levels in PBMC culture supernatants suggesting that an important source of allergen-specific IgE in PBMC supernatants could be IgE derived from the surface of basophils. Newly synthesized IgE was derived from CD138+ plasma cells, but not from B and B memory cells, and accounted for only approximately 0.2% of circulating IgE in blood. CONCLUSION AND CLINICAL RELEVANCE: Our finding that the majority of allergen-specific IgE in the peripheral blood is not derived from IgE-secreting cells in the blood suggests local IgE production in tissues as a major source for allergen-specific IgE and possible target for therapeutic intervention.

Basophils are not the key antigen-presenting cells in allergic patients.

BACKGROUND: Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen-presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients. METHODS: T cells, basophils, and APCs depleted of basophils were purified from allergic patients. Co-culture systems based on purified major allergens were established to study allergen-specific T cell responses using proliferation assays. RESULTS: Only co-cultures of T cells with APCs depleted of basophils but not with basophils proliferated in response to allergen. Even addition of IL-3 to T cell-basophil co-cultures failed to induce allergen-specific T cell proliferation. CONCLUSIONS: Our data demonstrate by classical in vitro proliferation assays that basophils are not key antigen-presenting cells that promote T cell proliferation in secondary immune responses to allergen in allergic patients.

Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens.

The broad applicability of allergen-specific immunotherapy for the treatment and eventually prevention of IgE-mediated allergy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. Today, the genetic code of the most important allergens has been deciphered; recombinant allergens equalling their natural counterparts have been produced for diagnosis and immunotherapy, and a large panel of genetically modified allergens with reduced allergenic activity has been characterized to improve safety of immunotherapy and explore allergen-specific prevention strategies. Successful immunotherapy studies have been performed with recombinant allergens and hypoallergenic allergen derivatives and will lead to the registration of the first recombinant allergen-based vaccines in the near future. There is no doubt that recombinant allergen-based vaccination strategies will be generally applicable to most allergen sources, including respiratory, food and venom allergens and allow to produce safe allergy vaccines for the treatment of the most common forms of IgE-mediated allergies.

Inhibition of CD23-dependent facilitated allergen binding to B cells following vaccination with genetically modified hypoallergenic Bet v 1 molecules.

BACKGROUND: Vaccination with hypoallergenic recombinant Bet v 1 derivatives (Bet v 1 fragments and Bet v 1 trimer) is associated with the induction of IgG antibodies specific to natural Bet v 1. OBJECTIVE: To investigate whether IgG antibodies induced following vaccination with genetically modified hypoallergenic Bet v 1 derivatives are able to inhibit IgE-facilitated binding of allergen-IgE complexes to B cells. METHODS: Sera from 46 patients obtained before and after subcutaneous vaccination with Bet v 1 trimer (n=14), Bet v 1 fragments (n=11) or placebo (n=21) were incubated with recombinant (r) Bet v 1 and an indicator serum (IS) from a birch pollen-allergic patient with high CD23 binding capacity. Bet v 1 immune complexes were added to a CD23-expressing B cell line and co-operative binding of Bet v1-IgE complexes to CD23 was measured with a polyclonal anti-IgE FITC antibody using a bio-functional cellular flow cytometric assay. RESULTS: When sera from patients vaccinated with rBet v 1 derivatives were incubated with Bet v 1 and the IS, a reduction of IgE binding to CD23 was observed. This effect was not seen when sera from placebo-treated patients were used. The decrease in CD23/IgE binding was statistically significant in the trimer group [pre- vs. post-specific immunotherapy (SIT): P=0.02; trimer vs. placebo: P<0.04] but not in the Bet v 1 fragments-treated group. Trimer-treated patients had higher levels of Bet v 1-specific IgG than fragment-treated patients. The degree of inhibitory activity of IgE-facilitated allergen binding correlated with Bet v 1-specific IgG levels following SIT (R=0.492; P=0.012). CONCLUSION: Vaccination with both recombinant Bet v 1 derivatives induces Bet v 1-specific IgG antibodies, which are able to inhibit the co-operative binding of allergen-IgE complexes to CD23, and may thereby reduce allergen-specific T cell responses.

Association of allergic patients' phenotypes with IgE reactivity to recombinant pollen marker allergens.

BACKGROUND: During the last decade allergen molecules from several allergen sources have been produced by recombinant DNA technology. The aim of this study was to investigate whether IgE reactivity to recombinant pollen allergens with broad and narrow cross-reactivity is associated with clinical phenotypes of allergic sensitization. METHODS: Serum IgE reactivity to a panel of six recombinant birch and grass pollen allergens was measured by ELISA in pollen sensitized patients from Central Europe to define groups of patients with exclusive IgE reactivity to rBet v 1, with exclusive reactivity to major grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5) and with IgE reactivity to cross-reactive pollen allergens (rBet v 2, rPhl p 7). Patients' clinical phenotypes were recorded. IgE responses to tree, grass and weed pollen as well as plant food extracts were evaluated in vitro by CAP-FEIA and clinical sensitivities were confirmed in vivo by skin prick testing. RESULTS: IgE reactivity to the recombinant major birch pollen allergen, rBet v 1, was associated with sensitization to pollen from birch, taxonomically related trees and to certain plant-derived food. Reactivity to the recombinant timothy grass pollen allergens, rPhl p 1, rPhl p 2, rPhl p 5, indicated sensitization to pollen from grasses. Patients reacting with the highly cross-reactive allergen rPhl p 7 were polysensitized to pollen from unrelated trees, grasses and weeds and rBet v 2-positive patients were polysensitized to pollen and plant-derived food from unrelated plants. CONCLUSIONS: IgE reactivity to recombinant marker allergens is associated with clinical phenotypes of allergic sensitization and may be useful for the selection of treatment strategies.

Cigarette smoke facilitates allergen penetration across respiratory epithelium.

BACKGROUND: The association between cigarette smoke exposure and allergic airway disease is a matter for debate. We sought to investigate in an in vitro system whether active smoking reduces the integrity and barrier function of the respiratory epithelium and thus facilitates allergen penetration. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate for the intact respiratory epithelium. The cell monolayer was exposed to standardized cigarette smoke extract (CSE). The extent and effects of trans-epithelial allergen penetration were measured using 125I-labelled purified major respiratory allergens (rBet v 1, rPhl p 5 and rDer p 2) and histamine release experiments. RESULTS: Exposure of cells to concentrations of CSE similar to those found in smokers induced the development of para-cellular gaps and a decrease in trans-epithelial resistance. CSE exposure induced a more than threefold increase in allergen penetration. Increased subepithelial allergen concentrations provoked a substantial augmentation of histamine release from sensitized basophils. CONCLUSIONS: Our results indicate that cigarette smoke is a potent factor capable of reducing the barrier function of the respiratory epithelium for allergens and may contribute to increased allergic inflammation, exacerbation of allergic disease and boosting of IgE memory.

Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity.

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives.

BACKGROUND: Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. OBJECTIVE: To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen-induced allergic symptoms. METHODS: A three-arm double-blind placebo-controlled immunotherapy study was conducted with one pre-seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol-treated population (n=84). In addition, skin and nasal provocation responses and allergen-specific antibodies were assessed. RESULTS: There were trends towards improvement in the subjects' well-being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end-point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen-specific antibody responses. Local injection-site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side-effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. CONCLUSION: Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well-being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.

Skin test results but not serology reflect immediate type respiratory sensitivity: a study performed with recombinant allergen molecules.

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.

[Incidence of ovarian hyperstimulation syndrome in in-vitro-fertilization treatment over a period of 11 years]. / Beobachtungen zum Auftreten des ovariellen Uberstimulationssyndroms bei der Behandlung mit In-vitro-Fertilisation in einem Zeitraum von 11 Jahren.

A total of 2915 cycles of controlled ovarian hyperstimulation (COH) were performed from 1981 to 1992 in an in-vitro fertilization--embryo transfer (IVFET) programme at our institute. In 325 (11.2%) cycles an ovarian hyperstimulation syndrome (OHSS) developed. It was defined if more than 10 follicles with a size of > 15 mm were found on the day of follicle puncture. OHSS was found to be more frequent in the 3rd quarter of the years. Soltriol or melatonin may play a role in this context. Additionally, we were able to confirm some risk factors that were previously reported to be associated with the OHSS: use of gonadotropin-releasing hormone agonist (GnRHa) protocols, and young age (< 35 years). The pregnancy rate was found to be higher among OHSS cases.

Expulsion of allergen-containing materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy.

BACKGROUND: Several studies demonstrated episodes of grass pollen-induced allergic asthma after heavy rainfalls. It has been hypothesized that these asthma attacks might be due to the release of respirable allergen-bearing particles from pollen cytoplasm. OBJECTIVE: In this study we investigated the release mechanism of the most potent and frequently recognized grass pollen allergens, group 1 and group 5, from freshly harvested and subsequently hydrated rye grass pollen at the ultrastructural level. METHODS: Rabbit antisera against purified recombinant group 1 and group 5 allergens were used to investigate, by using field emission scanning and transmission immunogold electron microscopy, the allergen release from rye grass pollen grains into isotonic aqueous solutions or water. RESULTS: Pollen grains exposed to isotonic aqueous solutions remained intact and released allergens by means of diffusion. However, pollen grains hydrated in distilled water or rainwater expelled starch grains and cytoplasmic debris of respirable size. Group 1 and group 5 allergens were observed on and within these materials. CONCLUSIONS: Exposure of rye grass pollen to water leads to an expulsion of subcellular allergen-containing pollen components of respirable size. Our ultrastructural data thus support the idea that this release of allergen-containing respirable pollen materials may be a cause of asthma attacks after heavy rainfalls.

The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species.

BACKGROUND: Ash, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. OBJECTIVE: The aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen METHODS: The IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. RESULTS: The allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. CONCLUSIONS: Allergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.

Release of allergen-bearing cytoplasm from hydrated pollen: a mechanism common to a variety of grass (Poaceae) species revealed by electron microscopy.

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.

Comparison of genetically engineered hypoallergenic rBet v 1 derivatives with rBet v 1 wild-type by skin prick and intradermal testing: results obtained in a French population.

BACKGROUND: Bet v 1, the major allergen in birch pollen, is recognized by more than 90% of patients allergic to birch in northern and central Europe. Immunotherapy is commonly performed with birch pollen extracts. Recently, hypoallergenic derivatives of Bet v 1 (rBet v 1 fragments, rBet v 1 dimer and trimer) were constructed and purified. OBJECTIVE: Our aim was to compare the allergenic activity of wild-type rBet v 1 with recombinant Bet v 1 derivatives (rBet v 1 fragments, dimer and trimer) with potentially reduced anaphylactic activity by skin testing in a French population. METHODS: Among the 36 birch pollen allergic patients included in the study, 29 were tested by skin prick testing and 30 by intradermal injections with purified monosubstances: rBet v 1 fragments (F1: aa1-74 and F2: aa75-160), Bet v 1 dimer and trimer. Intradermal tests were performed by the end-point intradermal titration method. Eight of the intradermally-tested patients were previously hyposensitized. Tests were performed over a period of 6 months (before, during and after birch pollen season); Bet v 1-specific IgE and IgG4 subclass responses were measured by immunoblotting and ELISA. RESULTS: All patients showed lower reactivity with the modified rBet v 1 allergens, both in skin prick and intradermal tests. In 25 and 23 out of 29 patients the lowest concentration of fragment 1 and 2, respectively, resulting in a positive prick test was 100-fold higher than the lowest concentration of monomer resulting in a positive prick test. For dimer it was 100-fold or more in 25 out of 29 patients, and for trimer it was 100-fold or superior in 26 out of 29 patients. By intradermal testing, the end-point concentration was 160-fold higher for trimer than for monomer in 24 patients and 40-fold higher in five patients. For the two fragments the end-point concentration was 160-fold higher in 20 out of 22 patients. CONCLUSION: Genetically modified hypoallergenic derivatives of the major birch pollen allergen, Bet v 1 showed reduced capacity to induce immediate type skin reactions. They may represent candidate molecules for immunotherapy of birch pollen allergy with reduced risk of anaphylactic side-effects.