Molecular, proteomic and immunological parameters of allergens provide inclusion criteria for new candidates within established grass and tree homologous groups.
ABSTRACT: BACKGROUND:Our knowledge of allergen structure and function continues to rise and new scientific data on the homology and cross-reactivity of allergen sources should be considered to extend the work of Lorenz et al., 2009 (Int Arch Allergy Immunol. 148(1):1-1, 2009) and the concept of homologous groups. In addition to this, sophisticated techniques such as mass spectrometry (MS) are increasingly utilised to better characterise the complex mix and nature of allergen extracts. METHODS:Homology models were used of Fag s 1 (Beech) and Cyn d 1 (Bermuda grass) and compared with template crystal structures of Bet v 1 and Phl p 1 from the 'exemplar' species of Birch and Timothy grass, respectively. ELISA experiments were performed to assess cross-reactivity of Beech (tree) and Bermuda (grass) extracts to rabbit sera raised to either "3-Tree" (Birch, Alder and Hazel) extract or "Grass" (12-grass mix extract), respectively. The comparability of biochemical stability of different allergen sources was assessed through statistical methods for a range of tree and grass species. RESULTS:Allergen cross-reactivity and/or structural homology have been described providing justification for inclusion of Beech within the Birch homologous tree group. Data from Bermuda grass (Cyn d 1) provides further justification for the inclusion of this species into the homologous group of the sweet grasses. However, further characterisation of relevant allergens from Bermuda grass and, in particular, comparison of cross-reactive patterns between subjects specifically in areas with high abundance of both Pooideae and Chloridoideae is sought. CONCLUSION:MS allows the possibility to identify individual proteins or allergens from complex mixes by mass and/or sequence, and this has been extensively applied to the allergen field. New data on the homology, cross-reactivity and biological parameters of allergen sources have been considered to extend the work of Lorenz et al., 2009 in the context of tree and grass species. The concept of homologous groups is certainly dynamic allowing the flexibility and potential in streamlining quality parameters, such as stability profiles, due to extrapolation of exemplar data to a wider range of allergens.
Project description:BACKGROUND:Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass. METHODS:A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay. RESULTS:Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120-170 and 224-244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction. CONCLUSION:Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.
Project description:<h4>Background</h4>Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles.<h4>Methods</h4>Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic.<h4>Results</h4>Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet.<h4>Conclusion</h4>In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.
Project description:<h4>Background</h4>Diagnosis of pollen allergies is mainly based on test allergens for skin prick testing. In the minimum battery of test inhalant allergens recommended by the Global Allergy and Asthma European Network 10 pollen allergens are included. Complementary other pollen allergens may need to be considered; however, respective awareness may not always be granted. Furthermore, at least in Germany, the situation may be even more complicated by the fact that test allergens need regulatory approval. A decline in commercially available test allergens may result in a diagnostic gap regarding patients with non-frequent allergies. How many patients with non-frequent pollen allergies would be affected by this gap? The data presented here partly answer this question.<h4>Methods</h4>The study consisted of a descriptive and an analytical part. In the descriptive part, sensitization to frequent pollen allergens (alder, hazel, birch, sweet grasses; according to the German Therapy Allergen Ordinance) and to respective non-frequent pollen allergens (cypress, Japanese cedar, ash, plane tree, olive, Bermuda grass, wall pellitory, plantain, goosefoot, mugwort, ragweed, and saltwort) was measured in adult patients with physician-diagnosed allergic rhinitis from two German federal states, namely North-Rhine Westphalia (<i>n</i> = 360) and Bavaria (<i>n</i> = 339), using skin prick testing and/or ISAC technology. Furthermore, respective regional pollen data were assessed. In the analytical part, sensitization data were correlated with each other and with anamnestic data on symptom periods.<h4>Results</h4>Sensitization to frequent pollen allergens ranged from 45% (sIgE to Aln g 1/Alder, NRW) to 72% (prick test reactivity to birch, NRW). Sensitization to non-frequent pollen allergens ranged from 0% (sIgE to Amb a 1/ragweed, NRW) to 41% (prick test reactivity to olive, Bavaria). Sensitization data partly correlated with each other and in connection with symptom periods showed a partly similar seasonal pattern as pollen data.<h4>Conclusions</h4>Sensitization to non-frequent pollen allergens have to be considered when examining patients with respective seasonal symptoms, and test (and respective therapy) allergens for non-frequent pollen allergies need to be available. Further prerequisites for adequate patient management would be a nationwide pollen monitoring system giving continuous pollen data and a systematic sensitization monitoring at patient level.
Project description:BACKGROUND:Placebo control in allergen immunotherapy (AIT) trials presents ethical and blinding concerns. We tested a trial design with an "active allergen placebo," as proposed by ARIA-GA2 LEN, to investigate in a double-blind trial the efficacy and safety of AIT in dual-allergic patients (grass and birch pollen) using active untargeted treatments as controls. METHODS:We randomized 95 patients to receive either grass (N = 47) or birch AIT (N = 48). Patients were exposed to both allergens in an allergen challenge chamber (ACC) before and after 9 months of AIT. Targeted (ACC-allergen = AIT-allergen) and untargeted (ACC-allergen ≠ AIT-allergen) treatment effects were assessed. RESULTS:Immunotherapy reduced significantly the mean (95% confidence interval) area under the curve of total nasal symptom score (targeted effects) by -13.55 (-17.56, -9.54; P < 0.001) after grass and -9.81 (-14.13, -5.50; P < 0.001) after birch AIT. Differences in targeted vs untargeted effects between AIT groups (utility of control group) were statistically significant for both grass (P = 0.02) and birch (P = 0.02) allergens. Targeted vs untargeted differences within-treatment groups (specificity of ACC measurement) were significant for grass AIT (P < 0.001) but not significant for birch AIT group (P = 0.24). Specific immunoglobulin G4 to both allergens increased significantly (P < 0.001) after targeted treatment, while remained unchanged for untargeted treatments. Both treatments were well tolerated. CONCLUSIONS:Immunotherapies for both grass and birch allergens were efficacious and safe. The study confirms the specificity of AIT. Untargeted treatment groups could serve as controls in future AIT trials.
Project description:<h4>Background</h4>Allergen exposure leads to allergen sensitization in susceptible individuals and this might influence allergic rhinitis (AR) phenotype expression. We investigated whether sensitization patterns vary in a country with subtropical and tropical regions and if sensitization patterns relate to AR phenotypes or age.<h4>Methods</h4>In a national, cross-sectional study AR patients (2-70 y) seen by allergists underwent blinded skin prick testing with a panel of 18 allergens and completed a validated questionnaire on AR phenotypes.<h4>Results</h4>628 patients were recruited. The major sensitizing allergen was house dust mite (HDM) (56%), followed by Bermuda grass (26%), ash (24%), oak (23%) and mesquite (21%) pollen, cat (22%) and cockroach (21%). Patients living in the tropical region were almost exclusively sensitized to HDM (87%). In the central agricultural zones sensitization is primarily to grass and tree pollen. Nationwide, most study subjects had perennial (82.2%), intermittent (56.5%) and moderate-severe (84.7%) AR. Sensitization was not related to the intermittent-persistent AR classification or to AR severity; seasonal AR was associated with tree (p?<?0.05) and grass pollen sensitization (p?<?0.01). HDM sensitization was more frequent in children (0-11 y) and adolescents (12-17 y) (subtropical region: p?<?0.0005; tropical region p?<?0.05), but pollen sensitization becomes more important in the adult patients visiting allergists (Adults vs children?+?adolescents for tree pollen: p?<?0.0001, weeds: p?<?0.0005).<h4>Conclusions</h4>In a country with (sub)tropical climate zones SPT sensitization patterns varied according to climatological zones; they were different from those found in Europe, HDM sensitization far outweighing pollen allergies and Bermuda grass and Ash pollen being the main grass and tree allergens, respectively. Pollen sensitization was related to SAR, but no relation between sensitization and intermittent-persistent AR or AR severity could be detected. Sensitization patterns vary with age (child HDM, adult pollen). Clinical implications of our findings are dual: only a few allergens -some region specific- cover the majority of sensitizations in (sub)tropical climate zones. This is of major importance for allergen manufacturers and immunotherapy planning. Secondly, patient selection in clinical trials should be based on the intermittent-persistent and severity classifications, rather than on the seasonal-perennial AR subtypes, especially when conducted in (sub)tropical countries.
Project description:Protein allergens can be related by cross-reactivity. Allergens that share relevant sequence can cross-react, those lacking sufficient similarity in their IgE antibody-binding epitopes do not cross-react. Cross-reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross-reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE-binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E-value) was used to identify a threshold specific to identifying cross-reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross-reactive or other homologous allergens. A FASTA expectation value range (1 × 10<sup>-5</sup> -1 × 10<sup>-6</sup> ) was identified that could act as a threshold to help identify cross-reactivity potential.
Project description:BACKGROUND:In the temperate climate zone of the Northern hemisphere, Fagales pollen allergy represents the main cause of winter/spring pollinosis. Among Fagales trees, pollen allergies are strongly associated within the Betulaceae and the Fagaceae families. It is widely accepted that Fagales pollen allergies are initiated by sensitization against Bet v 1, the birch pollen major allergen, although evidence is accumulating that the allergenic activity of some Bet v 1-like molecules has been underestimated. OBJECTIVE:To investigate the allergenic potential of the clinically most important Fagales pollen allergens from birch, alder, hazel, hornbeam, hop-hornbeam, oak, beech and chestnut. METHODS:To obtain the full spectrum of allergens, the three previously unavailable members of the Bet v 1-family, hop-hornbeam Ost c 1, chestnut Cas s 1 and beech Fag s 1, were identified in the respective pollen extracts, cloned and produced as recombinant proteins in E. coli. Together with recombinant Bet v 1, Aln g 1, Car b 1, Cor a 1 and Que a 1, the molecules were characterized physicochemically, mediator release assays were performed and IgE cross-reactivity was evaluated by ELISA and Immuno Solid-phase Allergen Chip (ISAC) IgE inhibition assays. RESULTS:All allergens showed the typical Bet v 1-like secondary structure elements, and they were all able to bind serum IgE from Fagales allergic donors. Strong IgE binding was observed for Betuloideae and Coryloideae allergens, however, cross-reactivity between the two subfamilies was limited as explored by inhibition experiments. In contrast, IgE binding to members of the Fagaceae could be strongly inhibited by serum pre-incubation with allergens of the Betuloideae subfamily. CONCLUSIONS AND CLINICAL RELEVANCE:The data suggest that Bet v 1-like allergens of the Betuloideae and Coryloideae subfamily might have the potential to induce IgE antibodies with different specificities, while allergic reactions towards Fagaceae allergens are the result of IgE cross-reactivity.
Project description:Fucosylated glycans on pathogens are known to shape the immune response through their interaction with pattern recognition receptors, such as C-type lectin receptors (CLRs), on dendritic cells (DCs). Similar fucosylated structures are also commonly found in a variety of allergens, but their functional significance remains unclear. To test a hypothesis that allergen-associated glycans serve as the molecular patterns in functional interaction with CLRs, an enzyme-linked immunosorbent assay-based binding assay was performed to determine the binding activity of purified allergens and allergen extracts. THP-1 cells and monocyte-derived DCs (MDDCs) were investigated as a model for testing the functional effects of allergen-CLR interaction using enzyme-linked immunosorbent assay, Western blotting, and flow cytometry. Significant and saturable bindings of allergens and allergen extracts with variable binding activities to DC-specific ICAM3-grabbing non-integrin (DC-SIGN) and its related receptor, L-SIGN, were found. These include bovine serum albumin coupled with a common glycoform (fucosylated glycan lacking the alpha1,3-linked mannose) of allergens and a panel of purified allergens, including BG60 (Cyn dBG-60; Bermuda grass pollen) and Der p2 (house dust mite). The binding activity was calcium-dependent and inhibitable by fucose and Lewis-x trisaccharides (Le(x)). In THP-1 cells and human MDDCs, BG60-DC-SIGN interaction led to the activation of Raf-1 and ERK kinases and the induction of tumor necrosis factor-alpha expression. This effect could be blocked, in part, by Raf-1 inhibitor or anti-DC-SIGN antibodies and was significantly reduced in cells with DC-SIGN knockdown. These results suggest that allergens are able to interact with DC-SIGN and induce tumor necrosis factor-alpha expression in MDDCs via, in part, Raf-1 signaling pathways.
Project description:<h4>Background</h4>Atopic dermatitis (AD) is a complex chronic inflammatory disease where allergens can act as specific triggering factors.<h4>Aim</h4>To characterize the specificities of IgE-reactivity in patients with AD to a broad panel of exogenous allergens including microbial and human antigens.<h4>Methodology</h4>Adult patients with AD were grouped according to the SCORAD index, into severe (n = 53) and moderate AD (n = 126). As controls 43 patients were included with seborrhoeic eczema and 97 individuals without history of allergy or skin diseases. Specific IgE reactivity was assessed in plasma using Phadiatop®, ImmunoCap™, micro-arrayed allergens, dot-blotted recombinant Malassezia sympodialis allergens, and immune-blotted microbial and human proteins.<h4>Results</h4>IgE reactivity was detected in 92% of patients with severe and 83% of patients with moderate AD. Sensitization to cat allergens occurred most frequently, followed by sensitization to birch pollen, grass pollen, and to the skin commensal yeast M. sympodialis. Patients with severe AD showed a significantly higher frequency of IgE reactivity to allergens like cat (rFel d 1) and house dust mite (rDer p 4 and 10), to Staphylococcus aureus, M. sympodialis, and to human antigens. In contrast, there were no significant differences in the frequencies of IgE reactivity to the grass pollen allergens rPhl p 1, 2, 5b, and 6 between the two AD groups. Furthermore the IgE reactivity profile of patients with severe AD was more spread towards several different allergen molecules as compared to patients with moderate AD.<h4>Conclusion</h4>We have revealed a hitherto unknown difference regarding the molecular sensitization profile in patients with severe and moderate AD. Molecular profiling towards allergen components may provide a basis for future investigations aiming to explore the environmental, genetic and epigenetic factors which could be responsible for the different appearance and severity of disease phenotypes in AD.
Project description:An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor Fc?RI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.