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Background: Endotoxin (LPS) released from gram-negative bacteria causes strong immunologic and inflammatory effects and, when airborne, can contribute to respiratory conditions, such as allergic asthma., Objectives: We sought to identify the source of airborne endotoxin and the effect of this endotoxin on allergic sensitization., Methods: We determined LPS levels in outdoor air on a daily basis for 4 consecutive years in Munich (Germany) and Davos (Switzerland). Air was sampled as particulate matter (PM) greater than 10 μm (PM > 10) and PM between 2.5 and 10 μm. LPS levels were determined by using the recombinant Factor C assay., Results: More than 60% of the annual endotoxin exposure was detected in the PM > 10 fraction, showing that bacteria do not aerosolize as independent units or aggregates but adhered to large particles. In Munich 70% of annual exposure was detected between June 12th and August 28th. Multivariate modeling showed that endotoxin levels could be explained by phenological parameters (ie, plant growth). Indeed, days with high airborne endotoxin levels correlated well with the amount of Artemisia pollen in the air. Pollen collected from plants across Europe (100 locations) showed that the highest levels of endotoxin were detected on Artemisia vulgaris (mugwort) pollen, with little on other pollen. Microbiome analysis showed that LPS concentrations on mugwort pollen were related to the presence of Pseudomonas species and Pantoea species communities. In a mouse model of allergic disease, the presence of LPS on mugwort pollen was needed for allergic sensitization., Conclusions: The majority of airborne endotoxin stems from bacteria dispersed with pollen of only one plant: mugwort. This LPS was essential for inducing inflammation of the lung and allergic sensitization., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Modifiers of symptom severity in patients with allergic rhinoconjunctivitis (AR) are imprecisely characterized. The hygiene hypothesis implicates childhood microbial exposure as a protective factor. Cockroach sensitization (C+) might be a proxy for microbial exposure., Objective: We sought to determine whether C+ assayed by means of skin prick tests influenced AR symptom severity in controlled and natural settings., Methods: Total symptom scores (TSSs) were recorded by 21 participants with house dust mite allergy (M+) in the natural setting and during repeated exposures of 3 hours per day to house dust mite allergen in an allergen challenge chamber (ACC). In M+ participants the peripheral blood and nasal cells were assayed for T-cell activation and transcriptomic profiles (by using RNA sequencing), respectively. Participants allergic to mountain cedar (n = 21), oak (n = 34), and ragweed (n = 23) recorded TSSs during separate out-of-season exposures to these pollens (any pollen sensitization [P+]) in the ACC; a subset recorded TSSs in the pollination seasons., Results: The hierarchy of TSSs (highest to lowest) among M+ participants tracked the following skin prick test sensitization statuses: M+P+C- > M+P+C+ > M+P-C- > M+P-C+. In nasal cells and peripheral blood the immune/inflammatory responses were rapidly resolved in M+P+C+ compared with M+P+C- participants. Among those allergic to pollen, C+ was associated with a lower TSS during pollen challenges and the pollination season. After aggregated analysis of all 4 ACC studies, C+ status was associated with a 2.8-fold greater likelihood of a lower TSS compared with C- status (odds ratio, 2.78; 95% CI, 1.18-6.67; P = .02)., Conclusions: C+ status is associated with mitigation of AR symptom severity in adults with AR., (Published by Elsevier Inc.)

Background: Allergies to grass pollen are the number one cause of outdoor hay fever. The human immune system reacts with symptoms to allergen from pollen., Objective: We investigated the natural variability in release of the major group 5 allergen from grass pollen across Europe., Methods: Airborne pollen and allergens were simultaneously collected daily with a volumetric spore trap and a high-volume cascade impactor at 10 sites across Europe for 3 consecutive years. Group 5 allergen levels were determined with a Phl p 5-specific ELISA in 2 fractions of ambient air: particulate matter of greater than 10 μm in diameter and particulate matter greater than 2.5 μm and less than 10 μm in diameter. Mediator release by ambient air was determined in FcεRI-humanized basophils. The origin of pollen was modeled and condensed to pollen potency maps., Results: On average, grass pollen released 2.3 pg of Phl p 5 per pollen. Allergen release per pollen (potency) varied substantially, ranging from less than 1 to 9 pg of Phl p 5 per pollen (5% to 95% percentile). The main variation was locally day to day. Average potency maps across Europe varied between years. Mediator release from basophilic granulocytes correlated better with allergen levels per cubic meter (r(2) = 0.80, P < .001) than with pollen grains per cubic meter (r(2) = 0.61, P < .001). In addition, pollen released different amounts of allergen in the non-pollen-bearing fraction of ambient air, depending on humidity., Conclusion: Across Europe, the same amount of pollen released substantially different amounts of group 5 grass pollen allergen. This variation in allergen release is in addition to variations in pollen counts. Molecular aerobiology (ie, determining allergen in ambient air) might be a valuable addition to pollen counting., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Sensitization to wheat flour plays an important role in the development and diagnosis of baker's asthma., Objectives: We evaluated wheat allergen components as sensitizers for bakers with work-related complaints, with consideration of cross-reactivity to grass pollen., Methods: Nineteen recombinant wheat flour proteins and 2 cross-reactive carbohydrate determinants were tested by using CAP-FEIA in sera of 101 bakers with wheat flour allergy (40 German, 37 Dutch, and 24 Spanish) and 29 pollen-sensitized control subjects with wheat-specific IgE but without occupational exposure. IgE binding to the single components was inhibited with wheat flour, rye flour, and grass pollen. The diagnostic efficiencies of IgE tests with single allergens and combinations were evaluated by assessing their ability to discriminate between patients with baker's allergy and control subjects based on receiver operating characteristic analyses., Results: Eighty percent of bakers had specific IgE levels of 0.35 kUA/L or greater and 91% had specific IgE levels of 0.1 kUA/L or greater to at least one of the 21 allergens. The highest frequencies of IgE binding were found for thiol reductase (Tri a 27) and the wheat dimeric α-amylase inhibitor 0.19 (Tri a 28). Cross-reactivity to grass pollen was proved for 9 components, and cross-reactivity to rye flour was proved for 18 components. A combination of IgE tests to 5 components, Tri a 27, Tri a 28, tetrameric α-amylase inhibitor CM2 (Tri a 29.02), serine protease inhibitor-like allergen (Tri a 39), and 1-cys-peroxiredoxin (Tri a 32), produced the maximal area under the curve (AUC = 0.84) in receiver operating characteristic analyses, but this was still lower than the AUC for wheat- or rye flour-specific IgE (AUC = 0.89 or 0.88, respectively)., Conclusions: Component-resolved diagnostics help to distinguish between sensitization caused by occupational flour exposure and wheat seropositivity based on cross-reactivity to grass pollen. For routine diagnosis of baker's allergy, however, allergen-specific IgE tests with whole wheat and rye flour extracts remain mandatory because of superior diagnostic sensitivity., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Sensitization to profilins and other cross-reacting molecules might hinder proper specific immunotherapy (SIT) prescription in polysensitized patients with pollen-related allergic rhinitis (AR). In these patients, component-resolved diagnosis (CRD) might modify SIT prescription by improving the identification of the disease-eliciting pollen sources., Objectives: We sought to measure the effect of CRD on SIT prescription in children with pollen-related AR., Methods: Children (n = 651) with moderate-to-severe pollen-related AR were recruited between May 2009 and June 2011 in 16 Italian outpatient clinics. Skin prick test (SPT) reactivity to grass, cypress, olive, mugwort, pellitory, and/or Betulaceae pollen was considered clinically relevant if symptoms occurred during the corresponding peak pollen season. IgE sensitization to Phl p 1, Phl p 5, Bet v 1, Cup a 1, Art v 1, Ole e 1, Par j 2, and Phl p 12 (profilin) was measured by using ImmunoCAP. SIT prescription was modeled on SPT responses first and then remodeled considering also CRD according to GA(2)LEN-European Academy of Allergology and Clinical Immunology guidelines and the opinions of 14 pediatric allergists., Results: No IgE to the respective major allergens was detected in significant proportions of patients with supposed clinically relevant sensitization to mugwort (45/65 [69%]), Betulaceae (146/252 [60%]), pellitory (78/257 [30%]), olive (111/390 [28%]), cypress (28/184 [15%]), and grass (56/568 [10%]). IgE to profilins, polcalcins, or both could justify 173 (37%) of 464 of these SPT reactions. After CRD, the SPT-based decision on SIT prescription or composition was changed in 277 (42%) of 651 or 315 (48%) of 651 children according to the European or American approach, respectively, and in 305 (47%) of 651 children according to the opinion of the 14 local pediatric allergists., Conclusions: In children with pollen-related AR, applying CRD leads to changes in a large proportion of SIT prescriptions as opposed to relying on clinical history and SPT alone. The hypothesis that CRD-guided prescription improves SIT efficacy deserves to be tested., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally unrelated allergens., Objective: We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4., Methods: Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays., Results: The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein., Conclusion: The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris-derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Clinical experiments demonstrate that the asthmatic response to an aeroallergen can be enhanced by prior exposure to an air pollutant., Objective: We sought to compare the effects of ambient aeroallergens on hospitalization for asthma between high and low air pollution days in 11 large Canadian cities., Methods: Daily time-series analysis was used, and results were adjusted for day of the week, temperature, barometric pressure, and relative humidity., Results: The relative risk of admission for an interquartile increase in tree pollen levels was 1.124 (95% CI, 1.101-1.147) on days of lower values of fine particulate matter with a median aerodynamic diameter less than or equal to 2.5 μm (PM(2.5)) compared with 1.179 (95% CI, 1.149-1.21) on days of higher PM(2.5) values. Significant (P ≤ .05) differences in the relative risks of admission between lower versus higher values of particulate matter with a median aerodynamic diameter less than or equal to 10 μm in diameter were 1.149 (95% CI, 1.118-1.181) versus 1.210 (95% CI, 1.161-1.261) for ascomycetes, 1.112 (95% CI, 1.085-1.14) versus 1.302 (95% CI, 1.242-1.364) for basidiomycetes, 1.159 (95% CI, 1.125-1.195) versus 1.149 (95% CI, 1.129-1.169) for deuteromycetes, and 1.061 (95% CI, 1.016-1.107) versus 1.117 (95% CI, 1.092-1.143) for weeds., Conclusion: We identified an association between aeroallergens and hospitalizations for asthma, which was enhanced on days of higher air pollution. Minimizing exposure to air pollution might reduce allergic exacerbations of asthma., (Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Cross-reactive carbohydrate determinants (CCDs) are probably the most widely occurring IgE epitopes. Approximately one fifth of patients with allergy develop IgE antibodies against such glycans. However, they appear to be of low clinical significance., Objective: We wanted to elucidate the reasons for this lack of clinical symptoms on contact with CCD allergens by determination of the binding affinities of patients' IgE and IgG antibodies., Methods: IgE and IgG against CCDs were affinity-purified from sera of selected patients. The binding affinity to defined glyco-epitopes was measured by surface plasmon resonance., Results: From a pool of CCD-positive sera, we isolated 0.1 and 25 microg CCD-specific IgE and IgG, respectively. The binding affinity of purified IgE antibodies to core alpha1,3-fucosylated glycans was in the 10(-10) mol/L range. The affinity was highest when both fucose and xylose were present, whereas xylosylation alone did not cause IgE binding. CCD-specific IgG exhibited a dissociation constant of approximately 10(-8) mol/L. IgG(4) amounted to only 20% of the CCD-specific IgG (as well as total IgG)., Conclusion: Low binding affinity of anti-CCD IgE cannot be the reason for the observed clinical insignificance of IgE against plant/insect glycan epitopes. Notably, the affinity of IgG to CCDs is higher than that to protein allergens, and it may therefore function as blocking antibody.

Background: We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids, such as phytoprostanes, that modulate human dendritic cell (DC) function in a way that results in an enhanced T(H)2 polarization in vitro., Objective: Here we analyzed the immunomodulatory capacities of Betula alba (white birch) aqueous pollen extracts (Bet-APEs) and pollen-associated phytoprostanes in the murine system in vitro and in vivo., Methods: DC function was analyzed in vitro by using BALB/c bone marrow-derived DCs. T-cell responses were analyzed with DO11.10 peptide 323-339 from chicken ovalbumin (OVA)-specific CD4 T cells as responder cells. For in vivo studies, OVA-specific CD4 T cells were adoptively transferred into BALB/c mice. Twenty-four hours later, mice were challenged by means of intranasal application of OVA in the absence or presence of Bet-APEs or phytoprostanes. Polarization of T-cell responses in vivo was analyzed in draining lymph node cells., Results: In vitro Bet-APEs and E(1)-phytoprostanes dose-dependently inhibited LPS-induced IL-12p70 of DCs. In addition, Bet-APEs induced a T(H)2 polarization in vitro. Similarly, intranasal instillation of Bet-APEs in vivo, together with the antigen, lead to increased IL-4, IL-5, and IL-13 secretion and decreased IFN-gamma secretion from antigen-specific T cells in the draining lymph nodes. In contrast, intranasal E1- and F1-phytoprostanes downregulated both T(H)1 and T(H)2 cytokine production in vivo., Conclusion: Pollen release water-soluble factors that display T(H)2-polarizing capacities in vivo independently of E(1)- and F(1)-phytoprostanes., Clinical Implications: Identification of the underlying mechanisms might open new approaches for pharmacologic intervention.

Background: Levels of the Clara cell 16-kd protein (CC16) are lower in plasma and bronchoalveolar lavage fluid from adults with asthma relative to those seen in healthy control subjects, and CC16 inhibits the T(H)2 cytokine production from murine T cells., Objective: We sought to determine the plasma levels of CC16 in infants and to investigate whether CC16 might inhibit the T(H)2 cytokine production from neonatal T cells., Methods: Cord blood and blood samples at 4, 18, and 36 months of age were taken from 64 children prospectively, and CC16 levels were analyzed in plasma. Cord monocyte-derived dendritic cells (DCs) were pulsed with birch allergen extract alone or together with CC16 or prostaglandin D(2) receptor inhibitors, after which autologous naive CD4(+) T cells were added to the DCs. The production of IL-5, IL-13, and IFN-gamma was measured by means of ELISA and flow cytometry., Results: The plasma levels of CC16 in children peaked at 4 months. CC16 did not directly affect the cytokine production from human T(H)2 cells. However, CC16 inhibited birch pollen extract-stimulated T(H)2 differentiation of naive T cells through the DC. Inhibition of the prostaglandin D(2) receptors did not consistently result in suppressed T(H)2 differentiation., Conclusion: The production of CC16 seems to peak early in life, and CC16 has an inhibitory effect on T(H)2 cell differentiation from human infants by affecting DCs., Clinical Implications: CC16 is an immunoregulatory protein, and its inhibitory effect on T(H)2 cell differentiation might be of importance in the pathogenesis of allergy in infants.

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

Background: Cross-reactivity between the major birch pollen allergen, Bet v 1, and the apple protein, Mal d 1, frequently causes food allergy., Objective: To investigate the effects of successful sublingual immunotherapy (SLIT) with birch pollen extract on apple allergy and the immune response to Bet v 1 and Mal d 1., Methods: Before and after 1 year of SLIT, Bet v 1-sensitized patients with oral allergy syndrome to apple underwent nasal challenges with birch pollen and double-blind placebo-controlled food challenges with apple. Bet v 1-specific and Mal d 1-specific serum antibody levels and proliferation in PBMCs and allergen-specific T-cell lines (TCLs) were determined. Bet v 1-specific TCLs were mapped for T-cell epitopes., Results: In 9 patients with improved nasal provocation scores to birch pollen, apple-induced oral allergy syndrome was not significantly reduced. Bet v 1-specific IgE and IgG(4) levels significantly increased. Bet v 1-specific T-cell responses to all epitopes and those cross-reactive with Mal d 1 significantly decreased. However, neither Mal d 1-specific IgE and IgG(4) levels nor Mal d 1-induced T-cell proliferation changed significantly. In contrast, Mal d 1-specific TCLs showed increased responses to Mal d 1 after 1 year of SLIT., Conclusion: This longitudinal study indicates that pollen SLIT does not efficiently alter the immune response to pollen-related food allergens, which may explain why pollen-associated food allergy is frequently not ameliorated by pollen immunotherapy even if respiratory symptoms significantly improve., Clinical Implications: SLIT with birch pollen may have no clinical effect on associated apple allergy.

Background: Parietaria judaica pollen is a common cause of pollinosis in the Mediterranean area., Objective: This study sought to purify and characterize the peptidase responsible for the majority of proteolytic activity present in the pollen extract of P judaica, and to investigate its contribution to the allergic response., Methods: A serial of chromatographic steps was applied to isolate the peptidase from P judaica's pollen, and its biochemical properties were determined. Bioactive peptides present in the airways were incubated with the peptidase, and their degradation was visualized by direct protein sequencing. In addition, we measured the cellular detachment, by methylene blue binding assay, of an airway-derived epithelial cell line (A549) in the presence of the peptidase, and visualized, by Western blot, the degradation of proteins from intercellular junctions., Results: We purified a 98-kDa peptidase from the pollen of P judaica that was classified as an aminopeptidase on the basis of its biochemical properties and internal amino acid sequence. The aminopeptidase was able to degrade bioactive peptides. Moreover, the aminopeptidase caused cellular detachment of A549 cell line and degradation of occludin and E-cadherin., Conclusion: Our results suggest that the P judaica aminopeptidase can alter the integrity of the epithelium barrier by degrading occludin as well as E-cadherin. In addition, P judaica aminopeptidase can degrade bioactive peptides, which can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs., Clinical Implications: The novel aminopeptidase described here could constitute a relevant therapeutic target in the treatment of allergic disorders induced by the pollen of P judaica.

Background: Individuals with birch pollen allergy frequently experience hypersensitivity reactions to certain foods, primarily because of IgE antibodies specific for the major birch pollen allergen Bet v 1 that cross-react with homologous food allergens., Objective: We sought to characterize the major T-cell epitopes of Bet v 1 and to investigate their involvement in the cellular cross-reactivity with homologous food allergens., Methods: T-cell epitope mapping of Bet v 1 was performed by testing Bet v 1-specific T-cell lines derived from 57 individuals with birch pollen allergy, with overlapping peptides representing the entire allergen. T-cell lines and T-cell clones were stimulated with Bet v 1-related major allergens from apple (Mal d 1), cherry (Pru av 1), hazelnut (Cor a 1), celery (Api g 1), carrot (Dau c 1), and soybean (Gly m 4) and with peptides deduced from the C-terminal amino acid sequences of these molecules. Results Bet v 1 142-156 , positioned in the highly conserved C-terminal region of Bet v 1, was identified as the major T-cell epitope recognized by 61% of individuals. Most T lymphocytes specific for Bet v 1 142-156 were activated by one or more homologous food proteins or the respective peptides, as indicated by proliferation and cytokine production., Conclusion: The major T-cell epitope of Bet v 1, Bet v 1 142-156 , plays an important role in the cellular cross-reactivity between this respiratory allergen and related food allergens. Thus T lymphocytes specific for Bet v 1 142-156 might be activated by various Bet v 1-related food allergens in vivo, even out of the pollen season.

The transfer of pollen, whether it is transported by insects or carried by the wind, from floral anther to recipient stigma is the critical reproductive event among higher plants. In this scenario, the pollen grain functions as a fully constituted life-cycle stage, capable of growth (albeit limited) and delivery of gametes. Pollen is prepared for this role by an intricate developmental process with dual sources of structural elements and chemical constituents, including allergens. The resulting complexity relates, at least in part, to the requirements of an unforgiving recognition process at stigmatic surfaces and of active growth before the achievement of gametic union. Recently, the basic participants in pollen-stigma interactions have been defined, and they provide a striking counterpoint to human histocompatibility concerns. Pollen development offers a useful tableau in terms of which to reexamine forces affecting pollen prevalence and their interactions. Development also provides clues to the sources and significance of more minute bioaerosols now known to carry pollen allergens.

Background: Birch pollen is a common allergen in northern, central, and eastern Europe. Earlier studies of specific immunotherapy using birch pollen extract were not placebo-controlled or were only preseasonal. Long-term, placebo-controlled studies with subcutaneously administered standardized birch pollen extract are lacking., Objective: The aim of this study was to evaluate the effect of immunotherapy with birch pollen extract on airway symptoms and use of medication in adult birch pollen-allergic patients in a double-blind, placebo-controlled trial., Methods: Forty-nine patients with histories of birch pollen allergy from the upper and lower airways, positive skin prick test and conjunctival provocation test results, and in vitro specific IgE to birch pollen (Betula verrucosa ) extract were included. Immunotherapy with birch pollen extract was given during 2 consecutive years in a double-blind, randomized, placebo-controlled study. Clinical symptom scores from the upper and lower airways and use of rescue medication were registered throughout the pollen season., Results: Forty-six patients reached the maintenance dose and were maintained on that dose during the 2-year study. The median symptom scores during the 1997 and 1998 seasons were 1.3 and 2.6, respectively, in the specific immunotherapy group and 2.1 and 4.3, respectively, in the placebo group. The differences between the groups were significant (P =.05 in 1997 and P =.005 in 1998). The placebo group used significantly more rescue medication during both seasons than the specific immunotherapy group (P =.004 for 1997 and P =.004 for 1998)., Conclusion: Specific immunotherapy with birch pollen extract is an effective and safe treatment for reducing clinical allergy symptoms and medication use in birch pollen-allergic patients during the pollen season.

Background: Under natural exposure conditions, pollen grains function as allergen carriers that release allergens from internal binding sites on contact with the aqueous phase of mucosa membranes. In addition, we recently demonstrated that pollen are a rich source of eicosanoid-like mediators, which are rapidly released on contact with the aqueous phase., Objective: The current study was designed to characterize the biochemical nature of pollen-derived lipid mediators in more detail and to delineate their biologic activity on polymorphonuclear granulocytes (PMNs)., Methods: Aqueous and lipid extracts from Phleum pratense L and Betula alba L pollen were analyzed by means of HPLC. PMNs were exposed to aqueous extracts or lipid fractions from pollen or to HPLC-purified lipid mediators identified in pollen extracts. Effects on PMNs were tested with transwell migration, calcium mobilization, and surface expression of CD11b., Results: Aqueous pollen extracts (APEs) contained predominantly monohydroxylated products derived of linoleic acid and linolenic acid. In chemotaxis assays PMNs displayed significant migration to APEs. Lipid extracts from pollen and the HPLC fraction containing 13-hydroxy-octadecadienoic acid/hydroxy-linoleic acid and 13-hydroxy-octadecatrienoic acid/hydroxy-linolenic acid induced migratory responses, although to a lesser degree than the APEs. In addition, APE, as well as lipid, extracts induced PMN activation, as documented by means of calcium mobilization and upregulation of CD11b., Conclusion: Pollen grains release mediators that recruit and activate PMNs in vitro. Similar mechanisms may be effective in vivo, suggesting that pollen-derived lipid mediators may act as adjuvants in the elicitation phase of allergic reactions.

Background: Calcium-binding plant allergens can be grouped in different families according to the number of calcium-binding domains (EF hands)., Objective: We sought to identify pollens containing crossreactive calcium-binding allergens and to investigate structural and immunologic similarities of members belonging to different families of calcium-binding allergens., Methods: By means of multiple sequence alignment and molecular modeling, we searched for structural similarities among pollen allergens with 2 (Phl p 7, timothy grass; Aln g 4, alder), 3 (Bet v 3, birch) and 4 EF hands (Jun o 4, prickly juniper). Purified recombinant Aln g 4 and Jun o 4 were used to determine the prevalence of IgE recognition in 210 patients sensitized to different pollens and to search, by means of ELISA competition, for the presence of cross-reactive epitopes in pollens from 16 unrelated plant species. IgE cross-reactivity among the allergen families was studied with purified rPhl p 7, rAln g 4, rBet v 3, and rJun o 4 and 2 synthetic peptides comprising the N-terminal and C-terminal EF hands of Phl p 7 by means of ELISA competition., Results: Structural similarities were found by using molecular modeling among the allergens with 2, 3, and 4 EF hands. Pollens from 16 unrelated plants contained Aln g 4- and Jun o 4-related epitopes. Twenty-two percent of the patients with multiple pollen sensitization reacted to at least one of the calcium-binding allergens. A hierarchy of IgE cross-reactivity (rPhl p7 > rAln g 4 > rJun o 4 > rBet v 3) could be established that identified rPhl p 7 as the EF-hand allergen containing most IgE epitopes in the population studied., Conclusion: The demonstration that members of different families of calcium-binding plant allergens share similarities suggests that it may be possible to use representative molecules for the diagnosis and therapy of allergies to EF-hand allergens.

Background: Carbohydrates expressed on allergens are known to be important for allergenicity. However, little is known about whether the carbohydrates drive the T(H)2 response., Objective: We sought to determine a role for carbohydrates expressed on Cry j 1, which is the major allergen of Cryptomeria japonica pollen and causes the most prevalent pollinosis in Japan, in in vitro cellular responses., Methods: Carbohydrates on Cry j 1 were destroyed by periodate-oxidation under mild conditions. Proliferative responses and cytokine productions against native, periodate-treated, and mock-treated Cry j 1 were compared in peripheral blood mononuclear cells, Cry j 1-specific T-cell lines, and clones from patients with Japanese cedar pollinosis., Results: We found that peripheral blood mononuclear cells from patients with Japanese cedar pollinosis displayed a significant decrease in proliferation and IL-5 production in response to periodate-treated Cry j 1 in comparison with native and mock-treated Cry j 1. Decreased proliferative responses against periodate-treated Cry j 1 were also seen in polyclonal T-cell lines, and the responses showed a heterogeneity. In addition, Cry j 1-specific CD4+ T-cell clones also displayed a significant decrease in proliferation and IL-4 and IL-5 production-but not IFN-gamma production-in comparison with the control antigens. However, most of the clones showed decreased but positive proliferative responses against periodate-treated Cry j 1. Blockade of the mannose receptor had no effect on cellular responses., Conclusion: The results suggest that carbohydrates on Cry j 1 play a major role in promoting Cry j 1-specific T(H)2 response in vitro, though they are not major targets as T-cell epitopes.

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

Background: Allergen immunotherapy is inconvenient and associated with the risk of anaphylaxis. Efforts to improve the safety of immunotherapy by means of chemical modification of allergens have not been successful because it greatly reduced their antigenicity. Recently, immunostimulatory DNA sequences (ISS or CpG motifs) have been shown to act as strong T(H)1 response-inducing adjuvants., Objective: We sought to determine whether conjugation of ISS to the major short ragweed allergen Amb a 1 results in enhanced immunotherapeutic potential in mice and decreased allergenicity in human subjects., Methods: A 22-mer ISS oligodeoxynucleotide (ISS-ODN) was coupled to Amb a 1 and used for immunization of mice, rabbits, and monkeys., Results: In mice the Amb a 1-ISS conjugate induced a T(H)1 response (IFN-gamma secretion), whereas Amb a 1 induced a T(H)2 response (IL-5 secretion). The T(H)1 response was not observed with an Amb a 1-non-ISS conjugate. Coinjection of Amb a 1 with ISS-ODN was much less effective in inducing a T(H)1 response. In mice primed for a T(H)2 response, injection with Amb a 1-ISS conjugate induced a de novo T(H)1 response and suppressed IgE antibody formation after challenge with Amb a 1. Amb a 1-ISS conjugate induced high-titer anti-Amb a 1 IgG antibodies in rabbits and cynomolgus monkeys, whereas Amb a 1 alone or Amb a 1 coinjected with ISS-ODN did not induce a detectable response. Amb a 1-ISS conjugate was less allergenic than Amb a 1 alone, as shown by a 30-fold lower histamine release from human basophils of patients with ragweed allergy, whereas mixing ISS-ODN with Amb a 1 did not reduce histamine release., Conclusion: Amb a 1-ISS conjugate has an enhanced T(H)1-biased immunogenicity and reduced allergenicity. It may offer a more effective and safer approach for allergen immunotherapy than currently available methods.

Background: Five birch pollen allergens have been identified so far. In a previous study we detected new birch pollen allergens with an isoelectric point in the range 9.0 to 9.3, present only in extracts prepared at controlled basic pH., Objective: The purpose of the current study was to purify and characterize those allergens., Methods: The target allergens were purified by ion exchange and hydrophobic interaction chromatography. Analyses were carried out by SDS-PAGE, isoelectric focusing, immunoblotting, and amino acid sequencing. The in vivo reactivity of the allergens was evaluated by skin testing., Results: An 18-kd protein, which we named Bet v 7, was purified. This 18-kd protein corresponded to 3 bands on isoelectric-focusing immunoblots that probably represent isoforms. On immunoblots up to 20.8% of birch pollen-allergic patients recognized those allergens. The clinical relevance of Bet v 7 was demonstrated by positive immediate-type skin testing on a patient allergic to birch pollen. Sequencing of an internal peptide yielded an amino acid sequence showing high homology with various plant cyclophilins. The rotamase activity of the protein, inhibited by cyclosporin A, further confirmed that Bet v 7 belongs to the group of cyclophilins., Conclusion: We have purified a novel allergen of birch pollen, Bet v 7, belonging to the cyclophilin family. Because cyclophilins are highly conserved proteins over the phylogeny, we may postulate that Bet v 7 is a member of a new family of panallergens.

Background: Pollen from trees of the order Fagales are important allergen sources in most parts of the world. Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens. The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli., Objective: The purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen-specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen., Methods: The presence of rBet v 1- and rBet v 2-homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen-specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments. The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts., Results: Natural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2. A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen-specific IgE on average. Most of the tree pollen-specific IgE was directed against rBet v 1., Conclusion: rBet v 1 and rBet v 2 contain most of the Fagales pollen-specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy.

Unlabelled: Identification of common allergenic structures in mugwort and ragweed pollen., Background: Despite the rare occurrence of ragweed in Middle Europe, a surprisingly high number of patients allergic to mugwort, a frequently encountered weed, display IgE reactivity against ragweed pollen allergens., Objective: The aim of this study was to investigate whether the high prevalence of IgE reactivity against ragweed in patients allergic to mugwort is caused by the presence of common allergenic determinants. We also sought to characterize any cross-reactive allergens., Methods: Common allergenic structures in mugwort and ragweed pollen were characterized by qualitative IgE immunoblot inhibition experiments performed with natural allergen extracts and recombinant allergens. The degree of cross-reactivity was estimated by quantitative CAP-FEIA competitions. The clinical significance of cross-reactive IgE antibodies was studied with histamine release experiments and nasal provocation tests., Results: Mugwort and ragweed RAST values were significantly correlated in a population of 82 Austrian patients allergic to mugwort. IgE antibodies cross-reacted with allergens of comparable molecular weight that were present in both extracts. By using recombinant birch profilin and specific antisera for IgE inhibition experiments, profilin was identified as one of the cross-reactive components in mugwort and ragweed pollen. Preincubation of sera from patients allergic to mugwort with mugwort extract inhibited IgE binding to ragweed pollen extract greater than 80%. Mugwort and ragweed pollen extract induced comparable histamine release and reduction of nasal air flow in a patient with IgE reactivity against the major mugwort allergen Art v 1., Conclusion: In addition to profilin, mugwort and ragweed pollen contain a number of cross-reactive allergens, among them the major mugwort allergen Art v 1. Cross-reactive IgE antibodies can lead to clinically significant allergic reactions.

Background: The inhalation of olive (Olea europaea) pollen is an important cause of allergic respiratory diseases in southern Europe and California., Objective: The aims of this study were to characterize the allergenic composition of O. europaea pollen collected in California and to purify two important allergens., Methods: One hundred grams of O. europaea pollen was extracted dialyzed in 10 kd cut-off membranes and lyophilized. Allergen characterization was done by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Two allergens were isolated by gel filtration, ion exchange, and hydrophobic interaction chromatography and sequenced., Results: Ole e 4 has an apparent molecular weight, under reducing conditions, of 32 kd and pIs between 4.65 and 5.1. The N-terminal was blocked and the analysis of the amino acid sequence of two internal regions revealed no homology with other known proteins. Ole e 5 has a molecular weight of 16 kd and pIs between 5.1 and 6.5. The amino acid sequence of the N-terminal showed a high degree of homology with superoxide dismutase of several plant species. Ole e 4 and Ole e 5 had an IgE binding frequency by immunoblot of 80% and 35%, respectively., Conclusions: Olive pollen extracts have a heterogeneous composition, with several important allergens. One of these allergens showed a high degree of homology with a superoxide dismutase.

Background: A high molecular weight (60 kd) allergen has been recently identified as a cross-reactive moiety in pollen and plant-derived food. While the cross-reactive allergen has been characterized by immunochemical techniques, little is known concerning its biologic properties., Objective: In this investigation we studied the in situ localization of the 60 kd cross-reactive allergen in tree, grass, and weed pollen, as well as in plant-derived food (apple and celery)., Methods: A monoclonal antibody (3A4) that was raised against the major mugwort pollen allergen, Art v 1, was used to demonstrate the presence of related allergens in nitrocellulose-blotted pollen and plant-food extracts. The tissue localization of the cross-reactive allergen was investigated by immunogold electron microscopy., Results: Monoclonal antibody 3A4 recognized IgE epitopes of the 60 kd mugwort allergen and cross-reacted with moieties of comparable molecular weights in birch and timothy grass pollen, as well as in apple and celery extracts. In pollen and plant-derived food the allergen could be localized intracellularly in ribosome-rich areas in the mitochondria and the nucleus. No labeling was observed in the pollen or cell walls or in organelles that are engaged in storage (e.g., starch granules and lipid particles)., Conclusion: Tree, grass, and weed pollen, as well as plant-derived foods, contain a high molecular weight Art v 1-cross-reactive allergen that maps to similar cell compartments.

Background: Birch tree pollen allergens are an important cause of early spring hay fever and allergic asthma. Pollen counts provide a guide for individuals with birch pollen allergy. However, birch pollen, because of its size, has a low probability of entering the lower airways to trigger asthma. Yet birch pollen allergens are known to be associated with respirable particles present in the atmosphere., Objective: We sought to determine the concentration of major allergen Bet v 1 in birch pollen and respirable particles in the atmosphere during the birch pollen season., Methods: We used a two-site monoclonal antibody-based assay (ELISA) to quantitate Bet v 1 in pollen extracts and high-volume air sampler filters collecting particles larger and smaller than 7.2 microm., Results: Bet v 1 (0.006 ng) is detectable per birch pollen grain, of which 0.004 ng is present in aqueous extracts (13.9% of soluble proteins). Atmospheric Bet v 1 concentrations are correlated with birch pollen counts. Heavy rainfall tended to wash out pollen and particles, indicated by a mean daily Bet v 1 concentration of 0.12 ng/m3 (20 pollen equivalents), but light rainfall produced a dramatic increase in allergen-loaded respirable particles with Bet v 1 concentrations of 1.2 ng/m3 (200 pollen equivalents)., Conclusion: These results highlight the different environmental risk factors for hay fever and allergic asthma in patients sensitized to Bet v 1. Light rainfall causes an increase in respirable particles; hence, this is an important risk factor for asthma.

Background: Hop Japanese (Hop J) pollens are abundant in the air of Korea during the autumn season. Their significance as a source of allergic sensitization is still unclear., Objectives: We sought to detect the sensitization rate to Hop J pollen as an inhalant allergen and to identify its allergenic components., Method: We carried out skin prick tests with Hop J pollen extract in 1287 patients with respiratory allergy who visited our hospital over the course of 1 year. The serum-specific IgE antibody to Hop J pollen antigen was detected by ELISA, and its binding specificity was confirmed by the ELISA inhibition test. To confirm the respiratory sensitization, bronchoprovocation tests were performed in 16 asthmatic patients sensitive to this pollen. To characterize allergenic components, Hop J pollen extract was analyzed by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis followed by IgE immunoblotting., Results: A positive result on the skin prick test (> or = 2+ of the antigen to histamine ratio) was noted in 79 (6.1%) patients. The serum-specific IgE antibody was detected in 18 (41.9%) patients among 43 positive reactors tested. The ELISA inhibition test with the addition of Hop J pollen extract showed a dose-dependent response. Minimal inhibitions were noted with addition of ragweed and mugwort pollen extracts. Nine asthmatic patients showed significant bronchoconstriction after inhalation of the Hop J pollen extract (five early and four dual asthmatic responders), and all of them had high specific IgE binding. Immunoblot analysis revealed 12 IgE-binding components ranging from 13 to 89 kd. Three bands (13 kd, 74 kd, and 80 kd) were bound to the IgE among the sera tested from more than 50% of the patients., Conclusion: We believe that the Hop J pollen should be considered as a relevant allergen during the autumn season and thus included in skin test batteries in Korea. Some patients diagnosed as having "intrinsic" asthma or rhinitis might be sensitized to this pollen and other previously unknown allergens.

Background: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule., Objective: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1., Methods: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients., Results: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids)., Conclusions: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

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

Background: The airborne pollen of the Compositae weed, Parthenium hysterophorus, is a major cause of allergic rhinitis in the Indian subcontinent and in certain parts of the southern United States and western Australia. Earlier studies have identified a 31 kd protein as the major allergen in Parthenium pollen., Objective: This study was undertaken to carry out the purification, immunochemical characterization, sequencing, and epitope analysis of this major allergen, designated as Par h I., Methods: The IgE-binding activity of the allergen was evaluated by immunoblot and inhibition ELISAs. Pronase digestion, periodate oxidation, and chemical deglycosylation were performed to determine the role of peptide and carbohydrate components of the allergen in IgE binding., Results: The data provide evidence for the involvement of carbohydrate moieties on Par h 1 in its IgE-binding ability. The N-terminal 91 amino acid sequence of Par h 1 shows 81% identity with a protein from sunflower anther, and the hydroxyproline-rich region of Par h 1 is 30% to 40% identical to similar stretches in extensins, a class of hydroxyproline-rich cell wall glycoproteins from different plant species. IgE antibodies in the sera of individuals allergic to Parthenium cross-reacted with a 50 kd hydroxyproline-arabinose-rich extensin precursor from potato tuber, and this binding was periodate-sensitive., Conclusions: It appears that a group of soluble plant glycoproteins, which are related to the ubiquitous extensins, have certain carbohydrate-containing IgE-binding epitopes that may contribute to allergenic cross-reactivity among specific pollens and foods.

Background: Cross-reactive IgE antibodies were found to be responsible for allergic reactions in patients allergic to pollen on ingestion of food (oral allergy syndrome). So far, the major birch pollen allergen Bet v 1 and birch profilin (Bet v 2) were identified as relevant cross-reactive allergens., Objective: In this study we attempted to identify additional cross-reactive plant allergens, which could be responsible for food intolerance in patients allergic to pollen., Methods: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, representing a 60 kd glycoprotein, were used to detect cross-reactive allergens in other pollens and plant-derived food. The amino acid compositions of the cross-reactive structures were determined, and their resistance against trypsin treatment was investigated. In addition, IgE immunoblot inhibitions were done with the 60 kd mugwort pollen allergen., Results: Monoclonal antibodies specific for the major mugwort pollen allergen, Art v 1, cross-reacted with proteins of comparable molecular weight in fruit and vegetables. Preadsorption of patients' sera with the 60 kd mugwort allergen led to a reduction of IgE binding to components of a similar molecular weight present in different pollen (birch, timothy grass), fruit (apple, peanuts), and vegetable (celery) extracts and reduced IgE binding to apple, kiwi, and celery as determined by RAST inhibitions., Conclusion: A cross-reactive plant panallergen, possibly identical to the major mugwort pollen allergen, Art v 1, is described. The allergen represents a protein of approximately 60 kd present in various pollen and plant foods; which is distinct from Bet v 1 and profilin and hence may represent a novel cross-reactive allergen in the oral allergy syndrome.

Background: In a previous study we showed that antigen BG60 of Bermuda grass pollen contains isoallergens. Because the yield of purified isoallergens was low when a chromatofocusing technique was used, it was difficult to carry out further studies, such as determination of carbohydrate composition and structure., Objective: The aim of this study was to establish a procedure to purify antigen BG60 proteins as a group and to characterize this group's physicochemical and immunologic properties., Methods: A combination of chromatographic techniques (ion-exchange, gel filtration, blue gel affinity, and reverse-phase high-performance liquid chromatography) was used for the purification of BG60. Immunoblot and ELISA techniques were used to study BG60-specific IgE and IgG antibodies in patients' sera. The role of the carbohydrate moiety in antigenicity and allergenicity was examined with monoclonal antibodies and allergic sera by using periodate-treated BG60. Its carbohydrate composition was analyzed by high-performance anion-exchange chromatography with a pulsed amperometric detector., Results: Homogeneity of BG60 was demonstrated by a single sharp peak in reverse-phase high-performance liquid chromatography, a single band in sodium dodecylsulfate-polyacrylamide gel electrophoresis, and only one band stained by anti-BG60 monoclonal antibody. BG60-specific IgE and IgG antibodies were shown to be present in allergic sera. Six plant lectins were found to react with BG60. On periodate treatment, BG60 reduced binding toward its monoclonal antibody and human IgE and IgG. Carbohydrate composition analysis showed that BG60 contains three kinds of sugars: mannose, N-acetylglucosamine, and fucose (in a ratio of approximately 3:2:1) and a minute amount of xylose. The carbohydrate content is approximately 7.5%, and peptide content is about 92.5%., Conclusion: A procedure was established for the purification of a large quantity of the BG60 antigen. The results suggest that the carbohydrate moiety of antigen BG60 may play an important role in the immune response.

Background: Only a few food allergens have as yet been identified, mainly because of the difficulty of obtaining a sufficient number of patients who are clinically sensitized to a given food. This is more feasible in the case of the oral allergy syndrome (OAS), a common form of food allergy, which is especially prevalent in patients with pollinosis., Objective: We designed a study to identify the allergens of kiwi fruit (Actinidia chinensis) by analyzing the sera of patients with OAS for kiwi and to examine the cross-reactivity of these allergens with timothy and birch pollen allergens., Methods: Twenty-seven patients with OAS for kiwi, a positive skin prick test response and serum IgE antibody to kiwi, and a positive open kiwi challenge test result and three patients who had OAS with severe systemic symptoms, which excluded a challenge test, were included in this study. The different polypeptide components of an extract of fresh kiwi were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and analyzed by IgE immunoblotting with sera from these patients. Cross-reactivity with the two pollen extracts was assessed by inhibition of the immunoblots with pooled and individual patients' sera., Results: Twelve IgE-binding components with molecular weights ranging from 12 to 64 kd were identified in the kiwi extract, but only a 30 kd component acted as major allergen, being recognized by sera of 100% of these patients. Inhibition of kiwi immunoblots with timothy and birch pollen extracts demonstrated strong cross-reactivity with some of the kiwi allergens, suggesting complete identity between certain food and pollen allergens; whereas others, particularly the 30 kd allergen, were only partially inhibited, suggesting much weaker cross-reactivity., Conclusions: Kiwi fruit contains a large number of allergens widely cross-reacting with allergens in grass and birch pollen extracts. Nevertheless, the major allergen at 30 kd appears to be specific for kiwi.

Background: Group 4 grass pollen allergens represent glycoproteins with a molecular weight of 50 to 60 kd, which are present in many grass species. Almost 75% of patients allergic to grass pollen display IgE reactivity to group 4 allergens, which hence can be regarded as major grass pollen allergens., Objective: In this study attempts were made to obtain information regarding the immunologic properties, localization, and occurrence of Phl p 4 and related allergens., Methods: Phl p 4 was detected in timothy grass pollen extracts by immunoblotting with serum IgE and monoclonal antibodies and was localized in pollen by immunoelectron microscopy. A peptide sequence from Phl p 4 was obtained by amino acid sequencing. The resistance of Phl p 4 against trypsin was analyzed after trypsin treatment of timothy grass pollen extracts with serum IgE and monoclonal antibodies. Cross-reactivities between Phl p 4 and Amb a 1, the major allergen of ragweed, were studied by using monoclonal antibodies and by IgE- inhibition studies., Results: Phl p 4 was characterized as a trypsin-resistant major timothy grass pollen allergen. By immunoelectron microscopy Phl p 4 was localized in the exine, cytoplasm, and amyloplast of timothy grass pollen. significant sequence similarities of a Phl p 4 10 amino acid peptide with Amb a 1, the major ragweed allergen, could be found. The immunologic similarity of Phl p 4 and Amb a 1 was confirmed by cross-reactivity of monoclonal antibodies and patients' IgE., Conclusion: Phl p 4 represents a trypsin-resistant major timothy grass pollen allergen with immunologic similarities to the major ragweed allergen Amb a 1 and therefore must be considered an important cross-reactive component in grass pollen and weed pollen allergy.

Allergenic cross-reactivity between members of the Fagales family (birch, alder, hazel, and beech) and between members of the Oleaceae family (ash, olive, lilac, and privet) is well known, but little is known about possible cross-reactivity between these two groups of trees, in particular between birch and ash, both of which flower in the spring. Various immunochemical methods including RAST inhibition, Western blot, and Western blot inhibition have been used in this study to show that there is partial cross-reactivity between birch and ash pollens. Enzyme allergosorbent test measurements were performed on sera from 35 patients with hay fever in spring by using birch and ash pollen allergen disks. The major allergen of birch, Bet v 1, was readily detectable in the birch pollen extract, but a homologous allergen in the ash pollen extract was barely detectable. Common allergens could be determined in the high molecular weight region. Ash pollen should be included in diagnostic procedures for spring pollinosis and should be considered for use in specific immunotherapy.

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

Background: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5., Objective: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli, the cDNAs were inserted into expression plasmids., Methods: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays., Results: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils., Conclusion: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.

We generated T cell clones specific to a Japanese cedar pollen allergen (Cry j 1) and investigated effects of altered T cell receptor (TCR) ligand on changes of T cell responses. One of these Cry j 1-specific T cell clones established from patients with Japanese cedar pollinosis, ST1.9, recognized an antigenic peptide Cry j 1 p335-346 in the context of HLA-DRA+DRB3*0301 molecules and secreted interleukin-4 dominantly, with a smaller amount of interferon-gamma. ST1.9 represented one of the major T cell clones specific to Cry j 1 in the donor, because a short-term cultured polyclonal T cell line specific to Cry j 1 exhibited the same character as the ST1.9. We synthesized various analog peptides derived from Cry j 1 p335-346 with single amino acid substitutions and determined key residues for interactions between TCR of ST1.9 and HLA-DR molecules. We also analyzed changes in the responses of ST1.9 to Cry j 1 p335-346-derived analog peptides. Of interest was that the substitution of 339threonine to valine resulted in a significant increase in interferon-gamma production, with no remarkable changes either in proliferative response or interleukin-4 production. Analog peptides carrying the substitutions of 339threonine to glycine or glutamine revealed TCR antagonism, without changes in their binding affinities to the DR molecule. Therefore single amino acid substitutions on an allergen peptide carrying the T cell epitope may suppress helper-T-dependent class switch pressure to IgE in B cells either by inducing increased interferon-gamma production or by inhibiting proliferative responses in helper-T cells.

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

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

Species of the Cupressaceae family are an important cause of respiratory allergies in countries with a Mediterranean climate. An allergenic extract from Cupressus arizonica pollen was prepared with two extraction steps followed by ammonium sulfate precipitation, giving a protein yield of about 3%. Cupressus arizonica pollen extract was also characterized by means of sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by IgE and IgG immunoblotting and lectin blotting. IgE reactivity was restricted to six components, whereas IgG binding showed a more complex pattern. A 43 kd component, predominant both in its intensity and frequency of recognition by human IgE antibodies, was identified as the major allergen of C. arizonica. Four of the six IgE-binding components, including the major allergen, seem to be glycoproteins, as confirmed by the lectin blotting analysis. The extract produced inhouse was used to set up an immunoenzymatic test to evaluate the specific IgE binding in a panel of sera from 33 immunotherapy-free subjects who were monosensitized to cypress pollen. The percent of positivity obtained was much higher than that reported in the literature for commercial immunoassays.

Previous data showed that the major pollen allergens from trees of the order Fagales, in particular alder, birch, hazel, and hornbeam, are highly interrelated. As only the complete amino acid sequence of Bet v I, the major allergen from birch, has been known, it was of interest to obtain the primary structure of other major allergens of this group, to attribute IgE-binding properties to certain features of the amino acid sequences of those allergens. cDNA was synthesized from alder pollen mRNA, sequence-specifically amplified by polymerase chain reaction and cloned into plasmid bluescript. Comparison of the deduced amino acid sequences of Aln g I and Bet v I revealed a 86.8% homology. The Aln g I encoding cDNA was subcloned into pKK223-3 and expressed in Escherichia coli as a full-length nonfusion protein. The recombinant Aln g I bound IgE from tree pollen-allergic patients and was shown to share IgE-epitopes with Bet v I by inhibition studies with recombinant Bet v I. Computer-aided calculations predicted epitopes in both Aln g I and Bet v I at the same position; the Bet v I molecule was predicted to possess two additional epitopes near the N-terminus of the molecule.

It is known that most patients with type I allergy to tree pollens also suffer from intolerance to nuts. To identify allergenic structures common to hazel pollen and hazelnuts, cross-reactivity of patients' IgE was investigated. With use of immunoblotting,.serum IgE from 25 patients displaying type I allergic reactions to tree pollens and intolerance to hazelnuts (group I) bound to the 17 kd major hazel pollen allergen Cor a I (100%) and to the 14 kd hazel pollen profilin (16%). IgE binding to proteins of comparable molecular weights in hazelnut extracts was found (18 kd and 14 kd), suggesting that proteins similar to Cor a I and hazel profilin might be also expressed in hazelnuts. In contrast, only four sera (22%) from 18 patients (group II) with tree pollen allergy but without any case history of nut hypersensitivity showed IgE binding to the 18 kd protein of hazelnut extract, and none of these sera exhibited IgE reactivity to the hazelnut profilin. To characterize the hazel pollen and hazelnut allergens, purified recombinant Bet v I (major birch pollen allergen) and purified recombinant Bet v II (birch profilin), respectively, were used for IgE-inhibition experiments. Binding of IgE from patients (with nut allergy) to the blotted hazelnut allergens could be blocked by preincubation of patients' sera with the recombinant proteins. Furthermore, the 18 kd protein of hazelnut extract was purified and induced specific release of histamine from basophils of a patient suffering nut hypersensitivity but not from a healthy control donor. A rabbit antibody raised against celery profilin identified the 14 kd proteins in hazel pollen and hazelnuts as profilin. Our experiments suggest a protein with IgE binding properties similar to the major allergens from pollens of hazel, Cor a I, and of birch, Bet v I, as predominant allergens in hazelnuts, and show that the plant pan-allergen profilin can be detected in both hazel pollen and hazelnut extracts.