Background Patients with birch pollen allergy often develop allergic reactions to plant foods. Objective To evaluate the prevalence, main symptoms, and triggers of birch pollen–related food allergy ...and the role of food-specific IgG4 antibodies in food tolerance. Methods Food-induced symptoms were evaluated in 225 individuals with birch pollen allergy by using a standardized questionnaire. IgE and IgG4 levels specific for the major birch pollen allergen Bet v 1 and birch profilin Bet v 2 and the Bet v 1 homologs in apple (Mal d 1) and hazelnut (Cor a 1) were quantified by ImmunoCAP. Mock-treated and IgG-depleted sera from patients tolerating hazelnuts in food challenges were compared for their inhibitory activity for binding of Cor a 1–IgE complexes to B cells. Results In total, 73% of the study population experienced food allergy, which was perennial in 86% of the affected individuals. The oral allergy syndrome was the main clinical manifestation. However, more than 58% of the patients also experienced food-induced rhinoconjunctivitis. Apples and hazelnuts were identified as the most frequent triggers. Food allergy correlated with IgE reactivity to Bet v 1 but not to Bet v 2. Mal d 1–specific and Cor a 1–specific IgG4 /IgE ratios were significantly higher in food-tolerant individuals than individuals with food allergy. Sera from IgG4 -positive food-tolerant patients possessed IgG-dependent IgE-inhibitory activity. Conclusion Birch pollen–related food allergy is highly prevalent and often perennial. High food allergen–specific IgG4 /IgE ratios seem associated with food tolerance, potentially because specific IgG4 blocks IgE binding to food allergens. Thus, the presence of food allergen–specific IgG4 antibodies is no diagnostic marker for birch pollen–related food allergy.
Birch pollen is an important elicitor of respiratory allergy. The major allergen, Bet v 1, binds IgE exclusively via conformational epitopes.
We identified Bet v 1–specific epitope repertoires of IgE ...and IgG from birch pollen–allergic and nonallergic subjects.
Chimeric proteins were created by grafting individual epitope-sized, contiguous surface patches of Bet v 1 onto a nonallergenic structural homolog and expressed in Escherichia coli. Binding of IgE, IgG1, and IgG4 from sera of 30 birch pollen–allergic and 11 nonallergic subjects to Bet v 1, 13 chimeric proteins, and 4 bacterial Bet v 1 homologs were measured by ELISA. The proportion of epitope-specific in-total Bet v 1–specific IgE and the cross-reactivity of Bet v 1–specific IgE with bacterial homologs were determined by competitive ELISA.
Thirteen soluble, correctly folded chimeric proteins were produced. IgE from 27 of 30 birch pollen–allergic patients bound to 1 to 12 chimeric proteins (median, 4.0), with patient-specific patterns evident. Three chimeras binding IgE from the majority of sera were identified, the grafted patches of which overlapped with previously published epitopes. Patterns of IgG1 and IgG4 binding to the chimeric proteins did not correspond to the binding patterns of IgE. Sera of 19 of 30 birch pollen–allergic patients contained low amounts of IgE to bacterial homologs. Bacterial proteins were able to partially inhibit IgE binding to Bet v 1.
Epitopes recognized by Bet v 1–specific antibodies from birch pollen–allergic patients are specific to each patient and differ between IgE, IgG1, and IgG4.
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Scope
Increasingly regulators are demanding evaluation of potential allergenicity of foods prior to marketing. Primary risks are the transfer of allergens or potentially cross‐reactive proteins into ...new foods. AllergenOnline was developed in 2005 as a peer‐reviewed bioinformatics platform to evaluate risks of new dietary proteins in genetically modified organisms (GMO) and novel foods.
Methods and results
The process used to identify suspected allergens and evaluate the evidence of allergenicity was refined between 2010 and 2015. Candidate proteins are identified from the NCBI database using keyword searches, the WHO/IUIS nomenclature database and peer reviewed publications. Criteria to classify proteins as allergens are described. Characteristics of the protein, the source and human subjects, test methods and results are evaluated by our expert panel and archived. Food, inhalant, salivary, venom, and contact allergens are included. Users access allergen sequences through links to the NCBI database and relevant references are listed online. Version 16 includes 1956 sequences from 778 taxonomic‐protein groups that are accepted with evidence of allergic serum IgE‐binding and/or biological activity.
Conclusion
AllergenOnline provides a useful peer‐reviewed tool for identifying the primary potential risks of allergy for GMOs and novel foods based on criteria described by the Codex Alimentarius Commission (2003).
The AllergenOnline database is a risk assessment tool to identify novel proteins in genetically modified organisms or processed foods that might be sufficiently identical to an allergen based on FASTA to suspect potential cross‐reactivity, requiring allergen‐specific serum IgE testing. The AllergenOnline.org is updated annually with sequences associated with published proof of allergy based on pre‐defined criteria as interpreted by a panel of experts. Criteria include definition of protein characteristics, allergic test subjects, IgE binding, and evidence of biological activity (Basophil or in vivo tests).
Background The immunologic mechanisms underlying sublingual immunotherapy (SLIT) are still unclear, particularly the role of regulatory T cells. Objective We sought to characterize allergen-specific ...T-cell responses during successful birch pollen SLIT. Methods Proliferation of PBMCs and PBMCs depleted of CD25+ cells obtained from 9 patients before, after 4 weeks, and after 52 weeks of SLIT was assessed in response to the major birch pollen allergen Bet v 1, the homologous apple allergen Mal d 1, or tetanus toxoid. Allergen-induced cytokine responses and FoxP3 expression of T cells were analyzed by using real-time PCR. The role of IL-10 for regulatory activity of T cells was investigated. Results After 4 weeks, higher frequencies of circulating CD4+ CD25+ T cells were detected together with increased FoxP3 and IL-10 and reduced IL-4 and IFN-γ mRNA expression levels compared with those before SLIT. Proliferation to all 3 antigens was markedly reduced but increased significantly after depletion of CD25+ cells or addition of anti–IL-10 antibodies. After 52 weeks, proliferation in response to Mal d 1 or tetanus toxoid returned to pre-SLIT levels, whereas Bet v 1–induced proliferation remained significantly suppressed and was enhanced by neither depletion of CD25+ cells nor addition of anti–IL-10 antibodies. In parallel, increased IFN-γ and reduced IL-4, IL-10, and FoxP3 mRNA expression was detected. Neither TGF-β levels nor cell-cell contact–mediated suppression of CD4+ CD25+ cells changed during the course of SLIT. Conclusion SLIT induces regulatory T-cell suppression through IL-10 during the early phase and specific nonreactivity and immune deviation of allergen-specific T cells during the later phase of therapy. Clinical implications SLIT induces immune mechanisms comparable with subcutaneous specific immunotherapy.
Acute lung injury (ALI) occurs in 23% unilateral. Models of unilateral ALI were developed and used previously without clearly demonstrating the strictly unilateral nature and severity of lung injury ...by the key parameters characterizing ALI as defined by the American Thoracic Society (ATS). Thus, the use of unilateral ALI remained rare despite the innovative approach. Therefore, we developed a unilateral model of ALI and focused on the crucial parameters characterizing ALI. This model can serve for direct comparisons between the injured and intact lungs within single animals, thus, reducing the number of animals required for valid experimental conclusions.
We established the model in nine pigs, followed by an evaluation of key parameters in six pigs (main study). Pigs were ventilated using an adapted left double-lumen tube for lung separation and two ventilators. ALI was induced in the left lung with cyclic rinsing (NaCl 0.9% + Triton® X-100), after which pigs were ventilated for different time spans to test for the timing of ALI onset. Ventilatory and metabolic parameters were evaluated, and bronchoalveolar lavage (BAL) was performed for measurements of inflammatory mediators. Finally, histopathological specimens were collected and examined in respect of characteristics defining the lung injury score (LIS) as suggested by the ATS.
After adjustments of the model (n = 9) we were able to induce strictly left unilateral ALI in all six pigs of the evaluation study. The median lung injury score was 0.72 (IQR 0.62-0.79) in the left lung vs 0.14 (IQR 0.14-0.16; p < 0.05) in the right lung, confirming unilateral ALI. A significant and sustained drop in pulmonary compliance (C
) of the left lung occurred immediately, whereas C
of the right lung remained unchanged (p < 0.05). BAL fluid concentrations of interleukin-6 and -8 were increased in both lungs.
We established a model of unilateral ALI in pigs, confirmed by histopathology, and typical changes in respiratory mechanics and an inflammatory response. This thoroughly evaluated model could serve as a basis for future studies and for comparing pathophysiological and pharmacological changes in the uninjured and injured lung within the same animal.
...whether the apo- or holoform of Pru p 3 has an impact on the IgE-binding activity is yet unknown. ...we studied a range of saturated and mono-/poly-unsaturated fatty acids and their interaction ...with Pru p 3 and investigated whether Pru p 3-ligand interaction is able to affect IgE recognition in sera from peach-allergic patients. ...no interaction of rPru p 3 with STE was detected. Because pH changes influence the presence of the OLE protonation state, we decided to investigate whether both OLE and its anion (OLE−) affect the 3-dimensional structure of Pru p 3. Because the region affected by conformational changes of Pru p 3 is the one that was identified as the major IgE epitope responsible for severe reactions,7 we decided to investigate whether conformational changes due to ligand binding lead to increased IgE-binding capacity. ...our ligand-binding assays provided interesting results regarding the binding specificity of Pru p 3, preferably binding poly- and mono-unsaturated fatty acids as compared with saturated ones.
Background Recombinant fusion proteins of flagellin and antigens have been demonstrated to induce strong innate and adaptive immune responses. Such fusion proteins can enhance the efficacy of ...allergen-specific immunotherapy. Objective We sought to characterize different fusion proteins of flagellin and the major birch pollen allergen Bet v 1 for suitability as allergy vaccines. Methods A truncated version of flagellin (NtCFlg) was genetically fused to the N- or C-terminus of Bet v 1. Toll-like receptor (TLR) 5 binding was assessed with HEK293 cells expressing TLR5. Upregulation of CD40, CD80, CD83, and CD86 on monocyte-derived dendritic cells from allergic patients was analyzed by using flow cytometry. The T cell–stimulatory capacity of the fusion proteins was assessed with naive and Bet v 1–specific T cells. IgE binding was tested in inhibition ELISAs and basophil activation tests. Mice were immunized with the fusion proteins in the absence and presence of aluminum hydroxide. Cellular and antibody responses were monitored. Murine antibodies were tested for blocking capacity in basophil activation tests. Results Both fusion proteins matured monocyte-derived dendritic cells through TLR5. Compared with Bet v 1, the fusion proteins showed stronger T cell–stimulatory and reduced IgE-binding capacity and induced murine Bet v 1–specific antibodies in the absence of aluminum hydroxide. However, only antibodies induced by means of immunization with NtCFlg fused to the C-terminus of Bet v 1 inhibited binding of patients' IgE antibodies to Bet v 1. Conclusion Bet v 1–flagellin fusion proteins show enhanced immunogenicity, reduced allergenicity, and intrinsic adjuvanticity and thus represent promising vaccines for birch pollen allergen-specific immunotherapy. However, the sequential order of allergen and adjuvant within a fusion protein determines its immunologic characteristics.
Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases.
We studied the mechanisms of immune ...modulation by a flagellin:allergen fusion protein containing the Toll-like receptor 5 ligand flagellin A from Listeria monocytogenes and the birch pollen allergen Bet v 1 (recombinant flagellin A rFlaA:Betv1).
BALB/c mice were vaccinated with rFlaA:Betv1 in an experimental Bet v 1 sensitization model. Myeloid dendritic cells (mDCs) were differentiated from mouse bone marrow, and PBMCs were isolated from subjects with birch pollen allergy. Cells were stimulated with equimolar amounts of rFlaA, rBet v 1, rFlaA plus rBet v 1, or the rFlaA:Betv1 conjugate and analyzed for cell activation, cytokine secretion, and metabolic state.
rFlaA:Betv1 displayed strong immune-modulating properties both in vivo and in vitro, as characterized by secretion of both proinflammatory and anti-inflammatory cytokines from murine mDCs and PBMCs from patients with birch allergy. rFlaA:Betv1 suppressed TH2 responses from Bet v 1–specific CD4+ T cells and prevented allergic sensitization in a mouse allergy model. Aggregation of rFlaA:Betv1 resulted in stronger protein uptake accompanied by an increased resistance to microsomal digestion. Remarkably, rFlaA:Betv1 induced activation of mammalian target of rapamycin, which increased the metabolic activity of the stimulated mDCs. rFlaA:Betv1-mediated IL-10 secretion, but not proinflammatory cytokine secretion, was inhibited by rapamycin in mDCs.
These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.
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Medical digital twins are computational disease models for drug discovery and treatment. Unresolved problems include how to organize and prioritize between disease-associated changes in digital ...twins, on cellulome- and genome-wide scales. We present a dynamic framework that can be used to model such changes and thereby prioritize upstream regulators (URs) for biomarker- and drug discovery.
We started with seasonal allergic rhinitis (SAR) as a disease model, by analyses of in vitro allergen-stimulated peripheral blood mononuclear cells (PBMC) from SAR patients. Time-series a single-cell RNA-sequencing (scRNA-seq) data of these cells were used to construct multicellular network models (MNMs) at each time point of molecular interactions between cell types. We hypothesized that predicted molecular interactions between cell types in the MNMs could be traced to find an UR gene, at an early time point. We performed bioinformatic and functional studies of the MNMs to develop a scalable framework to prioritize UR genes. This framework was tested on a single-cell and bulk-profiling data from SAR and other inflammatory diseases.
Our scRNA-seq-based time-series MNMs of SAR showed thousands of differentially expressed genes (DEGs) across multiple cell types, which varied between time points. Instead of a single-UR gene in each MNM, we found multiple URs dispersed across the cell types. Thus, at each time point, the MNMs formed multi-directional networks. The absence of linear hierarchies and time-dependent variations in MNMs complicated the prioritization of URs. For example, the expression and functions of Th2 cytokines, which are approved drug targets in allergies, varied across cell types, and time points. Our analyses of bulk- and single-cell data from other inflammatory diseases also revealed multi-directional networks that showed stage-dependent variations. We therefore developed a quantitative approach to prioritize URs: we ranked the URs based on their predicted effects on downstream target cells. Experimental and bioinformatic analyses supported that this kind of ranking is a tractable approach for prioritizing URs.
We present a scalable framework for modeling dynamic changes in digital twins, on cellulome- and genome-wide scales, to prioritize UR genes for biomarker and drug discovery.
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