Gerlinde Hofstetter

Characterization of Bet v 1‐related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

The Major Cow Milk Allergen Bos d 5 Manipulates T-Helper Cells Depending on Its Load with Siderophore-Bound Iron

The mechanisms of allergic sensitization to milk are still elusive. The major allergen Bos d 5 belongs to the lipocalin-family and thus is able to transport numerous ligands. In this study we investigated its ability to bind to iron-siderophore complexes and tested the immune-modulatory properties of Bos d 5 in either forms. Structural and in silico docking analysis of Bos d 5 revealed that Bos d 5 is able to bind to iron via catechol-based flavonoids (quercetin, myricetin, luteolin) that act as siderophores as confirmed by spectral-analysis and iron staining. Calculated dissociation constants of docking analyses were below 1 µM by virtual addition of iron. When incubated with human peripheral blood mononuclear cells (PBMCs), only the apo-form of Bos d 5 led to an increase of CD4+positive cells and significantly elevated IL13 and IFNγ-levels. In contrast, holo-Bos d 5 decreased numbers of CD4 expressing cells and induced apoptosis. Taken together, our data give evidence that Bos d 5 is capable of binding iron via siderophores. Moreover, our data support for the first time the notion that the form of application (apo- or holo-form) is decisive for the subsequent immune response. The apo-form promotes Th2 cells and inflammation, whereas the holo-form appears to be immunosuppressive.

Structural similarities of human and mammalian lipocalins, and their function in innate immunity and allergy.

Owners and their domestic animals via skin shedding and secretions, mutually exchange microbiomes, potential pathogens and innate immune molecules. Among the latter especially lipocalins are multifaceted: they may have an immunomodulatory function and, furthermore, they represent one of the most important animal allergen families. The amino acid identities, as well as their structures by superposition modeling were compared among human lipocalins, hLCN1 and hLCN2, and most important animal lipocalin allergens, such as Can f 1, Can f 2 and Can f 4 from dog, Fel d 4 from cats, Bos d 5 from cows milk, Equ c 1 from horses, and Mus m 1 from mice, all of them representing major allergens. The β‐barrel fold with a central molecular pocket is similar among human and animal lipocalins. Thereby, lipocalins are able to transport a variety of biological ligands in their highly conserved calyx‐like cavity, among them siderophores with the strongest known capability to complex iron (Fe3+). Levels of human lipocalins are elevated in nonallergic inflammation and cancer, associated with innate immunoregulatory functions that critically depend on ligand load. Accordingly, deficient loading of lipocalin allergens establishes their capacity to induce Th2 hypersensitivity. Our similarity analysis of human and mammalian lipocalins highlights their function in innate immunity and allergy.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome

Birch pollen‐associated plant food allergy is caused by Bet v 1‐specific IgE, but presence of cross‐reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen‐plant food syndrome has not been investigated in detail.

Vig r 6, the cytokinin‐specific binding protein from mung bean (Vigna radiata) sprouts, cross‐reacts with Bet v 1‐related allergens and binds IgE from birch pollen allergic patients’ sera

Scope Birch pollen associated allergy to mung bean sprouts is caused by cross-reactivity between the birch pollen allergen Bet v 1 and the mung bean allergen Vig r 1. We aimed to determine the allergenicity of the cytokinin-specific binding protein from mung bean (Vig r 6), another allergen related to Bet v 1 with only 31% sequence identity. Methods and results Bet v 1, Gly m 4, Vig r 1, and Vig r 6 were produced in Escherichia coli. In an ELISA, 73 and 32% of Bet v 1-sensitized birch-allergic patients’ sera (n = 60) showed IgE binding to Vig r 1 and Vig r 6, respectively. Of 19 patients who reported allergic reactions or had positive prick-to-prick tests to mung bean sprouts, 79% showed IgE binding to Vig r 1 and 63% showed IgE binding to Vig r 6. Bet v 1 completely inhibited IgE binding to both mung bean allergens. Vig r 6 showed partial cross-reactivity with Vig r 1 and activated basophils sensitized with mung bean allergic patients’ sera. Conclusion We demonstrated IgE cross-reactivity despite low sequence identity between Vig r 6 and other Bet v 1-related allergens. Thus, IgE binding to Vig r 6 may contribute to birch pollinosis-associated mung bean sprout allergy.

Crosstalk of carcinoembryonic antigen and transforming growth factor-β via their receptors: comparing human and canine cancer

Abstract There is accumulating evidence that the transforming growth factor beta (TGF-β) and nuclear factor kappa-B (NFκB) pathways are tightly connected and play a key role in malignant transformation in cancer. Immune infiltration by regulatory T- and B-lymphocytes (Tregs, Bregs) has recently gained increased attention for being an important source of TGF-β. There is a plethora of studies examining the pro-tumorigenic functions of carcinoembryonic antigen (CEA), but its receptor CEAR is far less studied. So far, there is a single connecting report that TGF-β also may signal through CEAR. The crosstalk between cancer tissues is further complicated by the expression of CEAR and TGF-β receptors in stromal cells, and implications of TGF-β in epithelial–mesenchymal transition. Furthermore, tumor-infiltrating Tregs and Bregs may directly instruct cancer cells by secreting TGF-β binding to their CEAR. Therefore, both TGF-β and CEA may act synergistically in breast cancer and cause disease progression, and NFκB could be a common crossing point between their signaling. CEAR, TGF-β1–3, TGF-β-R types I–III and NFκB class I and II molecules have an outstanding human–canine sequence identity, and only a canine CEA homolog has not yet been identified. For these reasons, the dog may be a valid translational model patient for investigating the crosstalk of the interconnected CEA and TGF-β networks.

Specificity of Mimotope-Induced Anti-High Molecular Weight-Melanoma Associated Antigen (HMW-MAA) Antibodies Does Not Ensure Biological Activity

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope

The major cow’s milk allergen Bos d 5 belongs to the lipocalin protein family, with an intramolecular pocket for hydrophobic ligands. We investigated whether Bos d 5 when loaded with the active vitamin A metabolite retinoic acid (RA), would elicit differential immune responses compared to the unloaded state. By in silico docking an affinity energy of −7.8 kcal/mol was calculated for RA into Bos d 5. Loading of RA to Bos d 5 could be achieved in vitro, as demonstrated by ANS displacement assay, but had no effect on serum IgE binding in tolerant or challenge-positive milk allergic children. Bioinformatic analysis revealed that RA binds to the immunodominant T-cell epitope region of Bos d 5. In accordance, Bos d 5 significantly suppressed the CD3+ CD4+ cell numbers, proliferative response and IL-10, IL-13 and IFN-γ secretion from stimulated human PBMCs only when complexed with RA. This phenomenon was neither associated with apoptosis of T-cells nor with the activation of Foxp3+ T-cells, but correlated likely with enhanced stability to lysosomal digestion due to a predicted overlap of Cathepsin S cleavage sites with the RA binding site. Taken together, proper loading of Bos d 5 with RA may suppress its immunogenicity and prevent its allergenicity.

Random mutagenesis and phage display technology as a tool for identifying ige epitopes of the birch pollen allergen Bet v 1

Background IgE-binding epitopes of the major birch pollen allergen Bet v 1 are dependent on the native structure of the allergen. To date, only a single IgE epitope has been identified. We aimed to identify additional epitopes by directed in vitro evolution of non-IgE-binding structural homologues to proteins carrying single IgE-binding patches. For that purpose, we chose cytokinin-specific binding protein (CSBP) from Vigna radiata (mung bean), a structural homologue of Bet v 1 with only 31% sequence identity, as a template subjected to a combination of random mutagenesis and phage display.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer

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.