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Background During allergen-specific sublingual immunotherapy ( SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. Objective To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. Methods Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. Results A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. Conclusions and Clinical Relevance Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level. [ABSTRACT FROM AUTHOR]

To cite this article: Poncet P, Senechal H, Clement G, Purohit A, Sutra J-P, Desvaux F-X, Wal J-M, Pauli G, Peltre G, Gougeon M-L. Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients. Allergy 2010; 65: 571–580. Background: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. Methods: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS–PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. Results: In 1D analysis, 86% of sera showed binding to Fra e 1 (18–20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to β-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. Conclusions: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization. [ABSTRACT FROM AUTHOR]

Background Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. Material and methods Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. Results The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. Discussion This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment. [ABSTRACT FROM AUTHOR]

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 65–85% allergic patients, respectively. Other allergens frequently involved in the IgE response include groups 2/3, 4, 6, 7, 10–13 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. [ABSTRACT FROM AUTHOR]

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 (O3), nitrogen dioxide (NO2) and sulphur dioxide (SO2) alone or in combination. Allergen contents were analysed by 2-dimensional immunoblot using grass pollen-sensitive patient sera. Results: For O3-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. NO2 exposure induced a decrease in Phl p 2, Phl p 5b and Phl p 6 recognition, and SO2 treatment induced a decrease in Phl p 2, Phl p 6 and Phl p 13 recognition. Moreover, samples treated with a mix of NO2/O3 or NO2/SO2 showed a higher decrease in allergen content, compared with samples treated with only one pollutant. The O3 acidification was also observed with the NO2/O3 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. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aim: Pollen cytoplasmic granules (PCG) are loaded with allergens. They are released from grass pollen grains following contact with water and can form a respirable allergenic aerosol. On the other hand, the traffic-related air pollutants NO2 and O3 are known to be involved in the current increase in the prevalence of allergic diseases via their adjuvant effects. Our objective was to determine the effects of air pollutants on the release of PCG from Phleum pratense (timothy grass) pollen. Methods:P. pratense pollen was exposed to several concentrations of NO2 and O3. The induced morphological damages were observed by environmental scanning electron microscopy, and the amount of PCG released from the pollen upon contact with water was measured. Results: The percentages of damaged grain were 6.4% in air-treated controls, 15% after treatment with the highest NO2 dose (50 ppm) and 13.5% after exposure to 0.5 ppm O3. In treated samples, a fraction of the grains spontaneously released their PCG. Upon subsequent contact with water, the remaining intact grains released more PCG than pollen exposed to air only. Conclusions: Traffic-related pollutants can trigger the release of allergen-containing granules from grass pollen, and increase the bioavailability of airborne pollen allergens. This is a new mechanism by which air pollution concurs with the current increase in the prevalence of allergic diseases. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Timothy grass ( Phleum pratense) pollen allergens are an important cause of allergic symptoms. However, pollen grains are too large to penetrate the deeper airways. Grass pollen is known to release allergen-bearing starch granules (SG) upon contact with water. These granules can create an inhalable allergenic aerosol capable of triggering an early asthmatic response and are implicated in thunderstorm-associated asthma. We studied the humoral (IgE) and bronchial lymph node cells reactivities to SG from timothy grass pollen in pollen-sensitized rats. Brown–Norway rats were sensitized (day 0) and challenged (day 21) intratracheally with intact pollen and kept immunized by pollen intranasal instillation by 4 weeks intervals during 3 months. Blood and bronchial lymph nodes were collected 7 days after the last intranasal challenge. SG were purified from fresh timothy grass pollen using 5 μm mesh filters. To determine the humoral response (IgE) to SG, we developed an original ELISA inhibition test, based on competition between pollen allergens and purified SG. The cell-mediated response to SG in the bronchial lymph node cells was determined by measuring the uptake of [3H]thymidine in a proliferation assay. An antibody response to SG was induced, and purified SG were able to inhibit the IgE ELISA absorbance by 45%. Pollen extract and intact pollen gave inhibitions of 55% and 52%, respectively. A cell-mediated response was also found, as pollen extract, intact pollen and SG triggered proliferation of bronchial lymph node cells. It was confirmed that timothy grass pollen contains allergen-loaded SG, which are released upon contact with water. These granules were shown to be recognized by pollen-sensitized rats sera and to trigger lymph node cell proliferation in these rats. These data provide new arguments supporting the implication of grass pollen SG in allergic asthma. [ABSTRACT FROM AUTHOR]

Background: Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry. Methods: Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing andMALDI-TOF mass spectrometry analysis. Results: Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis. Conclusion: The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to detect antigens and allergens in egg‐white byproduct ingredients and after their incorporation in processed pork meat pastes. Commercially prepared foods may have potentially allergenic ingredients (egg, milk, soybean, wheat, and peanut) added in processing. Since allergic patients may react to unidentified ingredients, it is important to assess the allergenic potency of these food proteins added during processing. Egg white was chosen as an experimental model, since egg is one of the most prevalent allergens in food hypersensitivity. Methods: Experimental pork meat pastes containing egg white underwent pasteurization and sterilization. Ingredients derived from egg‐white or paste extracts were isoelectrofocused and then blotted onto cyanogen bromide‐activated nitrocellulose membranes. Egg‐white antigens were identified in ingredients and in meat products with rabbit anti‐egg‐white antiserum by isoelectric focusing immunoblotting. Allergens were identified with sera from sensitized patients. A sensitive ELISA test was developed to detect egg‐white proteins in raw, pasteurized, and sterilized meat products. Results: Antigens and allergens in four egg‐white byproducts were detected. Egg‐white antigens were detectable in all ingredients and meat pastes by ELISA. Allergens were detected in ingredients and in raw and pasteurized products by immunoprint techniques and ELISA. Conclusions: Masked egg‐white allergens are recognized by human serum IgE after pasteurization. Egg‐white antigens are detectable in sterilized meat by ELISA techniques. Ingestion of processed foods could entail a risk of allergic reactions for sensitized consumers. Abbreviations. ELISA: enzyme‐linked immunosorbent assay; MaRG‐AP: mouse anti‐rabbit IgG alkaline phosphatase conjugate; RaEggW: anti‐hen's egg‐white antiserum; GaHE‐AP: goat... [ABSTRACT FROM AUTHOR]

IgE-anti-IgE complexes were formed by preincubation of a human myeloma IgE with a monoclonal anti-human IgE antibody at different concentrations. Binding of IgE onto the anti-IgE inhibited the histamine release capacity of anti-IgE from basophils. The IgE cell-binding capacity was altered by the IgE/anti-IgE ratio in the preincubation buffer. Heating of the complexes gave a partial recovery of the anti-IgE degranulating capacity, suggesting that the monoclonal anti-IgE is specific for an epitope present on the Dɛ2 domain. [ABSTRACT FROM AUTHOR]

The thennoinactivation kinetics of IgE were studied in experimental models revealing the antigenic properties and the basophil-sensitizing capacity of these immunoglobulins. A pool of human sera containing anti-Dactylis gloinerata (Dg) IgE was heated from 5 mm up to 4 hr at 56°. The IgE antigenicity was tested by two polyclonal 125I-labelled anti-IgE antibodies; one anti-IgE was specific of the whole Fc∊ region, while the other had a specificity restricted to the D∊2 domain. Radioimmunoassays showed that the D∊2 epitopes were more rapidly altered than the D∊1 epitopes. The capacity of IgE to bind to basophil Fc∊ receptors was assayed by passive sensitization experiments. Basophil sensitivity towards the Dg pollen extract was tested by histamine release experiments in the presence of this allergen. A progressive decrease in cell sensitivity was observed when IgE samples used for cell sensitization were heated for longer than 5 mm. Thermoinactivation kinetics of IgE revealed an unexpected increase in the apparent quantity and biological activity of IgE heated for 5 mm at 56°. This fact could be due to auto-anti-IgE antibodies linked to the unheated IgE and which interfere with the biological activities of lgE and their quantification. [ABSTRACT FROM AUTHOR]

Background Blocking antibodies are defined as antibodies that compete with IgE for binding to allergens due to their specificity for those allergens. Thus, they may inhibit allergen-induced basophil and mast cell IgE-dependent mediator release both in vivo and in vitro. Objective The present study was designed to evaluate the ability of antibodies isolated from human plasma samples on a Dactylis glomerata (Cocksfoot) pollen affinity-column to inhibit the Dactylis pollen-induced histamine release from human basophils (BHR) in vitro. Methods Antibodies from Ig pools containing either high or low IgG4 anti-Dactylis pollen were purified on a Dactylis pollen affinity-column and then separated on an antihuman IgE column. Obtained Ig fractions were incubated for 30 min with Dactylis pollen allergens prior to incubation with basophils from Dactylis pollen-allergic donors. Cell supernatants were assessed for histamine content and the inhibition of BHR was calculated. Results Unlike control non-isolated Igs, the antibodies isolated on the Dactylis pollen column were able to inhibit efficiently and in a dose-dependent manner Dactylis pollen-induced BHR. The inhibitory activity was increased in isolated antibody samples that had high IgG4 levels. Antibodies isolated on the Dactylis pollen column, however, consisted not only of true allergen-specific (potentially blocking) antibodies but also of autoanti-IgE-binding to allergen-specific IgE and mistaken for allergen-specific antibodies, thus opening to question the involvement of the true allergen-specific antibodies in the BHR-inhibitory activity. Unlike the true allergen-specific antibodies, the autoanti-IgE were retained on and eluted from the anti-lgE column. Results showed that both the autoanti-IgE-depleted and the autoanti-IgE-containing fractions accounted for the inhibition observed with the related non-depleted sample that had been isolated on the Dactylis pollen column. Conclusion For the first time, the true blocking activity of allergen-specific antibodies is demonstrated, that is. in the absence of the autoanti-IgE which can also inhibit BHR. [ABSTRACT FROM AUTHOR]

Since it has been shown that autoanti-IgE may be mistaken for antiallergen antibodies, thus appearing as pseudo-allergen-specific antibodies, it is crucial to separate true-from pseudo-allergen-specific antibodies and to determine to what extent autoanti-IgE appeared as pseudo-allergen-specific antibodies. For this purpose, human Ig pools were affinity-purified successively on a grass-pollen column and then on an antihuman-IgE column. IgG1-4, IgA, and IgM antibodies that were eluted from the grass-pollen column separated into pseudo- (∼ 30-40%) and true-allergen-specific antibodies that were coretained and not coretained, respectively, with the IgE on the anti-IgE column. Levels of autoanti-IgE were determined in individual plasma samples by surface plasmon resonance and statistically compared to the concentrations of allergen-specific antibodies obtained previously in the same plasma samples. A positive correlation between IgM autoanti-IgE levels and grass-pollen- "specific" IgM concentrations (P< 0.0002), and negative correlations between IgA autoanti-IgE and both IgE anti-grass pollen and IgG2 autoanti-IgE levels (P< 0.03, in both cases) were observed for the first time. This supports the contentions that: (1) autoanti-IgE antibodies appeared as pseudo-grass-pollen-specific antibodies, (2) they hid IgE antibodies when the latter were measured, and (3) they compete with one another in binding IgE. Lastly, a model of large Ig complexes is discussed. [ABSTRACT FROM AUTHOR]

Discusses the biochemical features of grain legume allergens in humans and animals. Soybean, peanut and lupin allergens; Differences between legume food allergens and legume inhalent allergens; Cross-allergenicity between legume allergens and other allergens; Decreasing the allergenicity of legumes; Legume allergens in farm and laboratory animals.

Grass pollen allergic patients are naturally exposed and sensitized to multiple pollens from various Pooideae species. The question arises as to whether such patients should be desensitized with extracts based on a single pollen or a pollen mixture. Neither conventional diagnosis based on IgE reactivity nor pollen counts enable the identification of which grass species are involved in patient sensitization. Significant cross-immunogenicity is observed between allergens from Pooideae pollens due to their conserved amino acid sequences (e.g. >90% for group 1, 55–80% for group 5, 30–60% for other allergens, including minor allergens). Nevertheless, pollen allergens also contain species-specific T or B cell epitopes, and there is evidence that at least 50% of allergic patients are sensitized to such distinct epitopes. In addition, substantial quantitative differences exist in the allergen (e.g. group 1, 2/3, 4, 5, 6, 10, 13) content of pollens obtained from distinct grass species. In this context, we recommend a vaccine consisting of a combination of pollens from five common and well characterized Pooideae including Anthoxanthum odoratum, Dactylis glomerata, Lolium perenne, Phleum pratense and Poa pratensis for desensitization purposes. This 5-grass mixture is recommended because, (i) it has been validated, in terms of safety and efficacy, by established clinical practice; (ii) it better reflects natural exposure and sensitization conditions at the molecular level than a single pollen; and (iii) it provides a consistent and well-balanced composition of critical allergens, while extending the repertoire of T and B cell epitopes. [ABSTRACT FROM AUTHOR]

Summary Background Health effects due to air pollution arizing from motor vehicles are a major public and political concern world-wide. Epidemiological studies have shown that the manifestations of asthma are increased by air pollution in already affected individuals. Objective To investigate the potential role of air-polluted tunnel dust (traffic particulate matter, TPM) or pure carbon core particles in the initiation and persistence of experimental allergic inflammation. Methods BP2 mice were immunized with birch pollen alone (group B) or pollen together with TPM (group A), or with birch pollen and Al(OH)3 (group C), or with birch pollen and carbon core particles (group D). Before methacholine challenge they were challenged intranasally and thereafter bronchial hyper-reactivity (BHR) was evaluated in a whole-body plethysmograph. Levels of Th2 cytokines, fibronectin and lactate dehydrogenase (LDH) were determined, and differential counts were performed in the bronchoalveolar lavage (BAL) fluid. Sera were collected for determination of antibody titres and cytokine levels. Results Specific IgE titres, BHR, the number of recruited eosinophils and levels of fibronectin and LDH in BAL were increased in mice immunized and challenged with a mixture of birch pollen and TPM. However, mice immunized with birch pollen alone and challenged intranasally with pollen or a mixture of pollen and TPM demonstrated the highest levels of IL-4 and IL-5. Conclusion This study highlights the importance of the exposure to a combination of particulate matters and pollen allergens, in the induction of allergic disease in the airways, and we have demonstrated that polluted tunnel dust has an effect on both the inflammatory and immunological components of experimental allergy. Immunization and challenge with carbon core particles together with birch pollen increased neither the BHR nor the specific IgE production significantly. Our results therefore strongly suggest that it is... [ABSTRACT FROM AUTHOR]

Latex allergy is now known to be related to type I hypersensitivity. In latex products, several reports have identified at least 10 allergens. However, 56 polypeptides have been shown to react to IgE by two-dimensional gel electrophoresis and immunoprints. All studies using immunoprints have analyzed a relatively small number of sera. The aim of the present study was to analyze the specificity and the isotypic expression of a wide sample of latex-sensitive patients. Latex proteins were separated by SDS–PAGE in an 8–18% gel gradient, transferred onto a nitrocellulose sheet by Western blot, and revealed by serum from latex-sensitive patients. The presence of IgE, IgA, IgG1, IgG2, IgG3, and IgG4 was analyzed for each of the 54 sera. The results showed Hev b 3 to be the most frequently recognized protein, followed by Hev b 1, Hev b 7, and molecules of 94 kDa and 6–8 kDa. The most common isotypic association was IgE-IgG1, frequently with IgA. IgG4 was also present in a little less than half of the IgE-positive sera. IgG3 was also expressed but frequently recognized nonallergenic molecules. IgG2 was present in only two cases. Thus, as several authors have proposed a protective role for IgG in the allergic reaction, it may be of interest to compare these results with the course of the disease. [ABSTRACT FROM AUTHOR]