The non-proteolytic house dust mite allergen Der p 2 induce NF-κB and MAPK dependent activation of bronchial epithelial cells

Background House dust mites (HDM) are well‐known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro‐inflammatory mediators, while there is limited knowledge regarding such activity among non‐proteolytic HDM allergens. Objective To investigate whether Der p 2, a major non‐proteolytic allergen of Dermatophagoides pteronyssinus, activates respiratory epithelial cells to produce mediators involved in asthma pathogenesis and to elucidate the mechanism of such activation. Methods The human bronchial epithelial cell line BEAS‐2B, normal human bronchial epithelial (NHBE) cells and the alveolar epithelial cell line A549 were exposed to recombinant Der p 2. Following exposure, we analysed a panel of soluble mediators and cell adhesion receptors involved in asthma pathogenesis by promoting recruitment, survival and binding of inflammatory cells. The involvement of nuclear factor (NF)‐κB and mitogen‐activated protein kinases (MAPKs) was studied using specific inhibitors. Results Der p 2 activated bronchial BEAS‐2B and NHBE cells, but not alveolar A549 cells. In BEAS‐2B cells Der p 2 induced dose‐dependent up‐regulation in both mRNA level and protein secretion of granulocyte‐macrophage colony‐stimulating factor, IL‐6, IL‐8, monocyte‐chemotactic protein‐1 and macrophage inflammatory protein‐3α. Secretion as well as surface expression of intercellular adhesion molecule (ICAM)‐1 was also up‐regulated, which was associated with increased adhesion of monocytes to the epithelial cells. The release of cytokines and chemokines was regulated by NF‐κB and MAPK activation in different ways, while expression of ICAM‐1 was solely dependent on NF‐κB activation. Conclusion These results show that Der p 2 activates respiratory epithelial cells, indicating that this non‐proteolytic allergen, in addition to its immunogenic properties, can aggravate respiratory airway disease by adjuvant‐like activation of the lung epithelium.     

Heterogeneity of commercial timothy grass pollen extracts

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 μg/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.     

The high molecular mass allergen fraction of timothy grass pollen (Phleum pratense) between 50–60 kDa is comprised of two major allergens: Phl p 4 and Phl p 13

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.     

Purified Timothy grass pollen major allergen Phl p 1 may contribute to the modulation of allergic responses through a pleiotropic induction of cytokines and chemokines from airway epithelial cells

By definition, allergens are proteins with the ability to elicit powerful T helper lymphocyte type 2 (Th2) responses, culminating in immunoglobulin (Ig)E antibody production. Why specific proteins cause aberrant immune responses has remained largely unanswered. Recent data suggest that there may be several molecular paths that may affect allergenicity of proteins. The focus of this study is the response of airway epithelium to a major allergen from Phleum pratense Phl p 1. Instead of focusing on a few genes and proteins that might be affected by the major allergen, our aim was to obtain a broader view on the immune stimulatory capacity of Phl p 1. We therefore performed detailed analysis on mRNA and protein level by using a microarray approach to define Phl p 1-induced gene expression. We found that this allergen induces modulation and release of a broad range of mediators, indicating it to be a powerful trigger of the immune system. We were able to show that genes belonging to the GO cluster 'cell communication' were among the most prominent functional groups, which is also reflected in cytokines and chemokines building centres in a computational model of direct gene interaction. Further detailed comparison of grass pollen extract (GPE)- and Phl p 1-induced gene expression might be beneficial with regard to the application of single components within diagnosis and immunotherapy.     

Interaction of cigarette smoke and house dust mite allergens on inflammatory mediator release from primary cultures of human bronchial epithelial cells

Several studies have shown that exposure to cigarette smoke and/or house dust mite (HDM) can lead to increased airway inflammation in susceptible individuals. The underlying mechanisms, however, are not defined. To investigate the interaction between cigarette smoke and HDM allergen on mediator release from primary cultures of human bronchial epithelial cells. Confluent human bronchial epithelial cell cultures were exposed to cigarette smoke in the absence or presence of HDM allergen and investigated for the release of IL-8, IL-1beta, and sICAM-1. Damage to the epithelial cells themselves was assessed by release of 51Cr. On separate occasions, we investigated the effect of PTL11028, a highly potent and selective Der p1 inhibitor, on HDM allergen-induced release of IL-8, following activation of HDM allergen by incubation with cysteine. The effect of cigarette smoke exposure on the stability of these released mediators in prepared solutions in the absence/presence of reduced glutathione was also studied. Both HDM allergens and short-term (20 min) cigarette smoke exposure led to a significantly increased release of IL-8, IL-1beta and sICAM-1 from the epithelial cell cultures. Longer exposure (1-6 h) to cigarette smoke led to a dramatic decrease in the amount of these mediators detected in the culture medium. Whilst incubation of epithelial cultures with HDM allergen did not cause any significant change in the release of 51Cr from pre-loaded cells, cigarette smoke on its own led to a marked, exposure and incubation-time dependent increase in the release of 51Cr. Incubation with HDM allergen led to a significant, dose and time-dependent increase in the release of IL-8, which was further enhanced when the allergen extract was pre-activated with cysteine. This effect was completely abrogated by PTL11028, a novel Der p1 inhibitor. Prepared solutions of various concentrations of IL-8, IL-1beta and sICAM-1 exposed to cigarette smoke demonstrated a dramatic exposure time-dependent decrease in the detectable amount of these mediators, an effect which was abrogated by GSH. HDM-induced airway inflammation may include Der p-mediated release of inflammatory mediators from epithelial cells. Additionally, short-term cigarette smoke exposure may induce airway inflammation by release of inflammatory mediators from these cells, an effect which may be potentiated by Der p allergens. Longer term cigarette smoke exposure may cause damage to epithelial cells and changes in the structure of inflammatory mediators.     

Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination

Background:  The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.
Methods:  A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice.
Results:  Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils.
Conclusion:  We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.     

Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity

BACKGROUND: The major timothy grass pollen allergen Phl p 1 is one of the most potent and frequently recognized environmental allergens. OBJECTIVE: We sought to study at a molecular and structural level the IgE recognition of Phl p 1 and its relation to allergenic activity. METHODS: Monoclonal human IgE antibody fragments specific for Phl p 1 and group 1 allergens from various grasses were isolated from a combinatorial library made of lymphocytes from patients with grass pollen allergy. Recombinant Phl p 1 fragments and the 3-dimensional structure of Phl p 1 were used to localize the major binding site for the IgE antibodies. A rPhl p 1 fragment containing this binding site was expressed in Escherichia coli, purified, and tested for IgE reactivity and allergenic activity with sera and basophils from patients with grass pollen allergy. RESULTS: Monoclonal antibodies, as well as polyclonal serum IgE, from patients with grass pollen allergy defined a C-terminal fragment of Phl p 1 that represents a sterically oriented portion on the Phl p 1 structure. This Phl p 1 portion bound most of the allergen-specific IgE antibodies and contained the majority of the allergenic activity of Phl p 1. CONCLUSION: IgE recognition of spatially clustered epitopes on allergens might be a general factor determining their allergenic activity. CLINICAL IMPLICATIONS: Geographic distribution of IgE epitopes on an allergen might influence its allergenic activity and hence explain discrepancies between diagnostic test results based on IgE serology and provocation testing. It might also form a basis for the development of low allergenic vaccines.     

Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11

BACKGROUND: Allergy 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. OBJECTIVE: To clone and characterize the 20 kDa timothy grass pollen allergen Phl p 11. METHODS: Phl 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. RESULTS: Phl p 11 cDNA defined an acidic polypeptide of 15.8 kDa 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. CONCLUSION: As 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.     

Mapping of T-cell epitopes of Phl p 5: evidence for crossreacting and non-crossreacting T-cell epitopes within Phl p 5 isoallergens

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.     

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.     

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.     

Immunomodulatory properties of Lactobacillus plantarum and its use as a recombinant vaccine against mite allergy

Background:  Selected lactic acid bacteria were reported to prevent atopic dermatitis and experimental asthma but the mechanisms of their immunomodulatory effects are not fully elucidated. In this study, the signaling pathways triggered by Lactobacillus plantarum NCIMB8826 were investigated and the potential use of this strain producing a variant of the mite allergen Der p 1 as live vaccine vehicle was evaluated.
Methods:  Mouse bone marrow-derived dendritic cells were stimulated with wild-type or a L. plantarum teichoic acid mutant to evaluate the secretion of cytokines. A recombinant L. plantarum expressing Der p 1 was engineered, its in vitro immunomodulatory properties were characterized and its prophylactic potential was evaluated in a Der p 1-sensitization murine model.
Results:  Mouse dendritic cells stimulated by L. plantarum triggered the release of interleukin-10 (IL-10), IL-12 p40, IL-12 p70 and tumor necrosis factor-alpha (TNF-α). IL-12 p40 secretion was dependent on nuclear factor-κB (NF-κB), mitogen-activated protein (MAP) kinases, Toll-like receptor 2 (TLR2), TLR9 and on the bacterial teichoic acid composition. Recombinant L. plantarum producing Der p 1 exhibited similar immunostimulatory properties as wild-type. Prophylactic intranasal pretreatment of mice with this recombinant strain prevented the development of the typical Th2-biased allergic response by a drastic reduction of specific IgE and the induction of protective allergen-specific IgG2a antibodies. Moreover, both wild-type or recombinant L. plantarum reduced airway eosinophilia following aerosolized allergen exposure and IL-5 secretion upon allergen restimulation.
Conclusion:  By combining both Th1-type immunostimulatory properties and an efficient allergen delivery capacity, recombinant L. plantarum producing Der p 1 represents a promising vaccine against house dust mite allergy.     

Timothy grass (Phleum pratense L.) pollen as allergen carriers and initiators of an allergic response

Contrary to indoor allergen exposure (e.g. house dust mite), there is no reliable quantitative association between pollen exposure and symptoms of allergic diseases. Therefore we studied localization and release of major allergens from timothy grass (Phleum pratense L.) pollen using different methods and pollen grain sources. Localization of major allergens Phl p 5 and Phl p 1 was visualized by field emission scanning electron microscopy after anhydrous fixation and immunogold silver staining in a three-dimensional reconstruction; Phl p 5 was found in the cytoplasm and on the exine, Phl p 1 in the intine. No allergens were found inside the starch granules. Allergen liberation from pollen grains was studied in vitro under physiological conditions (30 min, 37 degrees C) at pH 6. 0, 7.4 and 9.0. Besides total protein measurements in the supernatant, major allergens were determined by immunoblot, Phl p 5 was quantitated by ELISA. There were striking differences in total protein and major allergen release between freshly collected and commercially available grass pollen grains as well as among freshly collected pollen between rural meadows and areas near high-traffic roads. There was a significantly different release of total protein being lowest in supernatants from commercially available pollen grains (rural/traffic vs. commercial, p<0.001), and of Phl p 5 major allergen (rural>traffic>commercial, p<0.005). Therefore, allergen bioavailability seems to be an important parameter in order to establish reliable dose-response relationships for the outdoor allergen response. Pollen grains incubated in aqueous protein-free buffer solution were also found to secrete significant amounts of eicosanoids namely prostaglandin E2 and leukotriene B4. Pollen grains thus do not act only as allergen carriers but also might have important implications on early events as initiators of allergy.     

Conversion of grass pollen allergen-specific human IgE into a protective IgG(1) antibody

More than 100 million individuals exhibit IgE-mediated allergic reactions against Phl p 2, a major allergen from timothy grass pollen. We isolated cDNA coding for three Phl p 2-specific human IgE antibodies from a combinatorial library, which was constructed from lymphocytes of a grass pollen-allergic patient. Recombinant Phl p 2-specific IgE antibody fragments (Fab) recognized a fragment comprising the 64 N-terminal amino acids of Phl p 2 and cross-reacted with group 2 allergens from seven grass species. cDNA coding for the variable regions of one of the IgE Fab were cloned into aplasmid vector expressing the constant region of human IgG(1) to obtain a complete, recombinant Phl p 2-specific human IgG(1). This antibody blocked the binding of grass pollen-allergic patients IgE (n=26; mean inhibition: 58%) to Phl p 2 and caused a 100-fold reduction of Phl p 2-induced basophil histamine release. The recombinant human Phl p 2-specific IgG(1) may be used for environmental allergen detection, for standardization of diagnostic as well as therapeutic grass pollen allergen preparations and for passive therapy of grass pollen allergy.     

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.     

Immune reactivity of candidate reference materials

Immune reactivity is a key issue in the evaluation of the quality of recombinant allergens as potential reference materials. Within the frame of the CREATE project, the immune reactivity of the natural and recombinant versions of the major allergens of birch pollen (Bet v 1), grass pollen (Phl p 1 and 5), olive pollen (Ole e 1), and house dust mite (Der p 1 and 2, and Der f 1 and 2) was analysed. The IgE binding capacity of the allergens was studied by direct RAST and RAST inhibition, and their biological activity by basophil histamine release, using sera of allergic patients selected across Europe. For birch pollen, rBet v 1 is an excellent mimic of the natural allergen. For grass pollen, rPhl p 1 showed a significant lower IgE reactivity and was not considered a suitable candidate, whereas rPhl p 5a exhibited an immune reactivity closer to that of its natural counterpart. For olive, rOle e 1 had a lower IgE binding capacity in RAST but a higher biological activity in histamine release. For house dust mite, recombinant group 1 allergens were significantly less potent than their natural counterparts, but recombinant group 2 allergens were close mimics of their natural homologues.     

A recombinant allergen chimer as novel mucosal vaccine candidate for prevention of multi-sensitivities

BACKGROUND: As conventional immunotherapy is less efficacious in patients with allergic multi-sensitivities compared with mono-sensitized subjects, new intervention strategies are needed. Therefore, an allergen chimer was genetically engineered for treatment of multi-sensitization with birch and grass pollen on the basis of mucosal tolerance induction. METHODS: The major birch pollen allergen Bet v 1 served as a scaffold for N- and C-terminal linkage of the immunodominant peptides of the grass pollen allergens Phl p 1 and Phl p 5 and this new construct was cloned and expressed in Escherichia coli. After purification, physicochemical and immunological characterization the chimer was used for intranasal tolerance induction prior to poly-sensitization with Bet v 1, Phl p 1 and Phl p 5. RESULTS: The immunological characterization revealed that the conformation of Bet v 1 within the chimer was comparable to that of natural as well as recombinant Bet v 1. The chimer was immunogenic in mice for T and B cell responses to the three allergens. Intranasal application of the chimer prior to poly-sensitization significantly suppressed humoral and cellular allergen-specific Th2 responses and prevented development of airway inflammation upon allergen challenge. Moreover, local allergen-specific IgA antibodies were induced by the chimer. The mechanisms of poly-tolerance induction seemed to be mediated by regulatory cytokines, since TGF-beta and IL-10 mRNA in splenocytes were upregulated and tolerance was transferable with these cells. CONCLUSION: The data indicate that such allergen chimers harboring several unrelated allergens or allergen peptides could serve as mucosal polyvalent vaccines for prevention of multi-sensitivities.     

The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

Modifications of Phleum pratense grass pollen allergens following artificial exposure to gaseous air pollutants (O(3), NO(2), SO(2))

BACKGROUND: Air pollution is frequently proposed as a potential cause of the increased incidence of allergy in industrialised countries. Our objective was to investigate the impact of the major gaseous air pollutants on grass pollen allergens. METHODS: Timothy grass pollen was exposed to ozone (O(3)), nitrogen dioxide (NO(2)) and sulphur dioxide (SO(2)) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. RESULTS: For O(3)-treated pollen, immunoblotting showed an acidification of allergens Phl p 1b, Phl p 4, Phl p 5 and Phl p 6 and an IgE recognition decrease in Phl p 1, Phl p 2, Phl p 6 and Phl p 13. NO(2) exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO(2) treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO(2)/O(3) or NO(2)/SO(2) showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O(3) acidification was also observed with the NO(2)/O(3) mix. CONCLUSION: Exposure of pollen to gaseous pollutants induced a decrease in allergen detection in pollen extracts. This decrease could be due to a mechanical loss of allergens from the altered pollen grains and/or post-translational modifications affecting allergen recognition by IgE.