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Abstract: Background: Worldwide, pollen of the weed mugwort (Artemisia vulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. Objective: We sought to characterize IgE epitopes of Art v 1–sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. Methods: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. Results: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients’ IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. Conclusions: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.
Keywords: mugwort pollen allergy, IgE epitope, allergen-specific immunotherapy
Published in DiRROS: 31.08.2022; Views: 155; Downloads: 56
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Abstract: Many questions concerning responders (R) and nonresponders (NR) in severe eosinophilic asthma (SEA) after blocking the IL-5 (interleukin 5) pathway are still not clear, especially regarding the early parameters of response to biologics in personalized treatment strategies. We evaluated 17 SEA patients treated with anti-IL-5 biologics (16 patients mepolizumab, one patient benralizumab) before the introduction of biologics, and at a week 16 follow-up. Clinical, cellular and immunological parameters in peripheral blood were measured in R and NR. Sputum induction with the measurement of cellular and immunological parameters was performed at 16 weeks only. There were 12 R and 5 NR to biologics. After 16 weeks, there was a significant improvement in percentages of FEV1 (p = 0.001), and asthma control test (ACT) (p = 0.001) in the R group, but not in NR. After 16 weeks, the eosinophils in induced sputum were 27.0% in NR and 4.5% in R (p = 0.05), with no difference in IL-5 concentrations (p = 0.743). Peripheral eosinophilia decreased significantly in NR (p = 0.032) and R (p = 0.002). In patients with SEA on anti-IL-5 therapy, there was a marked difference in airway eosinophilic inflammation between R and NR already at 16 weeks, after anti-IL-5 introduction.
Published in DiRROS: 13.01.2022; Views: 509; Downloads: 354
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Abstract: Background: The role of chemokines in anaphylaxis is unclear. Methods: We prospectively recruited 49 patients presenting to the emergency department with an acute episode of anaphylaxis and 28 healthy subjects. We measured serum levels of the chemokines CCL2, CCL5, CCL7, CCL8, CCL11, CCL13, CCL17, CCL21, CCL22, CCL24, and CCL26, tryptase, the absolute number of circulating basophils, monocytes, lymphocytes, and PMNs, and whole blood FCER1A, CPA3 and HDC gene expression at two time points: during the anaphylactic episode and in convalescent samples collected approximately 3 months later. We then investigated the in vitro chemotactic activity of chemokines induced during anaphylaxis for the in vitro migration of the corresponding cells. Results: Only CCL2 chemokine levels were signifcantly increased in anaphylaxis samples (median 514 pg/ml) compared to convalescent samples (284 pg/ml, P<0.0001) and healthy subjects (279 pg/ml, P<0.0001); there was no signifcant diference in any of the other chemokines. There was a signifcant positive correlation between the rates of increase of serum CCL2 (median [range]: 106.0% [-44.7% to 557.4%]) and tryptase (133.8% [-6.6% to 893.4%]; r=0.68, P<0.0001) and between the acute concentration of serum CCL2 and the acute concentration of serum tryptase (r=0.77, P<0.0001). The number of circulating basophils, but not other blood cells, signifcantly decreased during anaphylaxis (median 5.0 vs. 19.1 cells/[micro]l in convalescent samples; P<0.0001); a decrease in whole-blood gene expression of basophil markers (PKeywords: anaphylaxis, chemokines, tryptases, basophils, chemotaxis, CCL2, cell migration
Published in DiRROS: 18.01.2021; Views: 952; Downloads: 490
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Abstract: Background: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity. Objectives: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE. Methods: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-FiveTM insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes. Results: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2. Conclusion: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.
Keywords: allergy and immunology, allergens, glycoproteins, molecular diagnostic technique, recombinant glycoproteins, molecular allergology, component-resolved diagnosis
Published in DiRROS: 16.10.2020; Views: 1057; Downloads: 807
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