Blocking antibodies induced by allergen-specific immunotherapy ameliorate allergic airway disease in a human/mouse chimeric model.

BACKGROUND: Allergen-specific immunotherapy (AIT) induces specific blocking antibodies (Ab), which are claimed to prevent IgE-mediated reactions to allergens. Additionally, AIT modulates cellular responses to allergens, for example, by desensitizing effector cells, inducing regulatory T and B lymphocytes and immune deviation. It is still enigmatic which of these mechanisms mediate(s) clinical tolerance. We sought to address the role of AIT-induced blocking Ab separately from cellular responses in a chimeric human/mouse model of respiratory allergy. METHODS: Nonobese diabetic severe combined immunodeficient γc-/- (NSG) mice received intraperitoneally allergen-reactive PBMC from birch pollen-allergic patients together with birch pollen extract and human IL-4. Engraftment was assessed by flow cytometry. Airway hyperresponsiveness (AHR) and bronchial inflammation were analyzed after intranasal challenges with allergen or PBS. Sera collected from patients before and during AIT with birch pollen were added to the allergen prior to intranasal challenge. The IgE-blocking activity of post-AIT sera was assessed in vitro. RESULTS: Human cells were detected in cell suspensions of murine lungs and spleens indicating successful humanization. Humanized mice displayed a more pronounced AHR and bronchial inflammation when challenged with allergen compared to negative controls. Post-AIT sera exerted IgE-blocking activity. In contrast to pre-AIT sera, the presence of heterologous and autologous post-AIT sera significantly reduced the allergic airway inflammation and matched their IgE-blocking activity determined in vitro. CONCLUSION: Our data demonstrate that post-AIT sera with IgE-blocking activity ameliorate allergic airway inflammation in a human/mouse chimeric model of respiratory allergy independently of AIT-induced cellular changes.

The quantity and quality of α-gal-specific antibodies differ in individuals with and without delayed red meat allergy.

BACKGROUND: IgG to galactose-α-1,3-galactose (α-gal) are highly abundant natural antibodies (Ab) in humans. α-Gal-specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3-7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their α-gal-specific IgE and IgG responses in more detail. METHODS: α-Gal-specific IgE was determined by ImmunoCAP. IgE reactivity to meat extract and bovine gamma globulin (BGG) was assessed by immunoblotting and ELISA, respectively. In some experiments, sera were pre-incubated with α-gal or protein G to deplete IgG Ab. α-Gal-specific IgG1-4 Ab in individuals with and without meat allergy were assessed by ELISA. RESULTS: In immunoblots, BGG was the most frequently recognized meat protein. Binding of IgE and IgG to BGG was confirmed by ELISA and completely abolished after pre-incubation with α-gal. Neither the depletion of autologous α-gal-specific IgG Ab nor the addition of α-gal-specific IgG Ab from nonallergic individuals changed the IgE recognition of BGG of meat-allergic patients. Meat-allergic patients showed significantly higher α-gal-specific IgG1 and IgG3 Ab than nonallergic individuals, whereas the latter showed significantly higher levels of α-gal-specific IgG4 Ab. CONCLUSION: Patients with delayed meat allergy display IgE and IgG Ab that selectively recognize the α-gal epitope on BGG. Their enhanced α-gal-specific IgE levels are accompanied by high levels of α-gal-specific IgG1 devoid of IgE-blocking activity. This subclass distribution is atypical for food allergies and distinct from natural α-gal IgG responses in nonallergic individuals.

Characterization of the T-cell response to Dau c 1, the Bet v 1-homolog in carrot.

BACKGROUND: In contrast to other Bet v 1-related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process, we sought to characterize the T-cell response to Dau c 1 and its cross-reactivity with Bet v 1. METHODS: Dau c 1-specific T-cell lines (TCL) and clones (TCC) established from PBMC of birch pollen-allergic patients with carrot allergy were analyzed for reactivity to Bet v 1, epitope specificity, allergen-induced cytokine secretion, and expression of integrins α4ß7 and α4ß1, critical for gut and lung homing, respectively. mRNA expression of GATA3 and Tbet was analyzed in sorted CD3+ CD4+ CFSElow cells proliferating upon stimulation of PBMC with Dau c 1 or Bet v 1. Dau c 1 was incubated with endolysosomal proteases, and the resulting fragments were identified by mass spectrometry. RESULTS: Among 14 distinct T-cell-activating regions, Dau c 1139-153 was recognized by 55% of the patients. Only 6 of 15 (40%) Dau c 1-specific TCL and 9 of 21 (43%) TCC reacted with Bet v 1. Bet v 1-nonreactive TCC were mainly Th1-like and showed a higher expression of the integrin ß7 and a significantly lower expression of the integrin ß1 than Bet v 1-positive TCC. A Th1-like response was also detected in Dau c 1-reactive CD3+ CD4+ CFSElow cells. Full-length Dau c 1 was still detectable after 48 h of endolysosomal degradation. Proteolytic fragments of Dau c 1 matched its T-cell-activating regions. CONCLUSION: Dau c 1 displays several characteristics of sensitizing allergens, namely a major T-cell-activating region, low susceptibility to endolysosomal degradation, and induction of a Bet v 1-independent T-cell response. These cellular insights confirm that the major carrot allergen has a special status among Bet v 1-related food allergens.

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen-specific immunotherapy (AIT) with birch pollen improves birch pollen-related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen-related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1-homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen-allergic patients with apple allergy. The dose of 1.6 µg induced oral allergy syndromes in 26.4%, 3.2 µg in 15.3%, 6.3 µg in 27.8%, 12.5 µg in 8.3%, 25 µg in 11.1%, and 50 µg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 µg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose-defined sublingual challenge tests in a high number of birch pollen-allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen-related food allergy.

Human blood basophils do not act as antigen-presenting cells for the major birch pollen allergen Bet v 1.

BACKGROUND: Several studies in mice have recently shown that basophils can act as antigen-presenting cells (APC) inducing Th2-mediated immune responses against parasites or protease allergens. The aim of this study was to investigate whether human basophils function as APC for the major birch pollen allergen Bet v 1. METHODS: Fluorescently labeled Bet v 1 was used to assess surface binding and internalization of allergen by basophils and different types of APC from birch pollen-allergic and nonallergic individuals. Sorted basophils were analyzed in terms of up-regulation of MHC class II and co-stimulatory molecules in the absence and presence of IL-3 and IFN-γ by flow cytometry. Expression of proteins crucial for antigen presentation, namely cathepsin S and invariant chain, was determined. Basophils were used as APC in co-culture experiments with Bet v 1-specific T-cell clones (TCCs). RESULTS: Basophils from birch pollen-allergic donors very efficiently bound Bet v 1 through IgE/FcεRI complexes on their surface. In contrast to professional APC, basophils did not internalize allergen and expressed marginal levels of cathepsin S and invariant chain. HLA-DP, HLA-DQ, CD80/CD86, and CD40 were absent from purified basophils even when stimulated with IL-3 plus IFN-γ. IL-3/IFN-γ marginally up-regulated HLA-DR. Bet v 1-pulsed basophils failed to induce proliferative and cytokine responses in Bet v 1-specific, HLA-DR-restricted TCCs. CONCLUSION: Human basophils neither internalize, process nor present Bet v 1. Because Bet v 1 is a highly relevant allergen, we conclude that basophils play no role as APC in IgE-mediated allergy in humans.

Pru p 3, the nonspecific lipid transfer protein from peach, dominates the immune response to its homolog in hazelnut.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) are important food allergens. Often, patients allergic to the nsLTP in peach suffer from allergy to hazelnuts. We aimed to analyse the T-cell response to Cor a 8, the nsLTP in hazelnut and its immunological cross-reactivity with the nsLTP in peach, Pru p 3. METHODS: Cor a 8-reactive T-cell lines (TCL) established from patients allergic to hazelnut and peach were stimulated with 12-mer peptides representing the complete amino acid sequence of Cor a 8 to identify its T-cell-activating regions and with Pru p 3 to investigate cellular cross-reactivity. T-cell clones specific for different major T-cell-activating regions of Pru p 3 were stimulated with Cor a 8. Both nsLTPs were subjected to endolysosomal degradation assays. Immunoglobulin E (IgE) cross-reactivity between Cor a 8 and Pru p 3 was assessed in inhibition enzyme-linked immunosorbent assay. RESULTS: No major T-cell-activating region was found among 26 T-cell-reactive peptides identified in Cor a 8. Although generated with Cor a 8, 62% of the TCL responded more strongly to Pru p 3. This cross-reactivity was mediated by T cells specific for the immunodominant region Pru p 3(61-75) . Peptide clusters encompassing this region were generated during lysosomal degradation of both nsLTPs. Cor a 8 was more rapidly degraded by lysosomal proteases than Pru p 3. Pre-incubation of sera with Pru p 3 completely abolished IgE binding to Cor a 8, which was not the case vice versa. CONCLUSIONS: T-cell reactivity to Cor a 8 is predominantly based on cross-reactivity with Pru p 3, indicating that the latter initiates sensitisation to its homolog in hazelnut. The limited allergenic potential of Cor a 8 seems to be associated with rapid lysosomal degradation during allergen processing and the lack of major T-cell-activating regions.

[Endocarditis prophylaxis in children and adolescents : implementation of the current guidelines in the Department of Paediatric Surgery at the Medical University Graz]. / Endokarditisprophylaxe bei Kindern und Jugendlichen : Umsetzung der aktuellen Leitlinien an der Universitätsklinik für Kinderchirurgie in Graz.

The timely administration of endocarditis prophylaxis means an additional stress situation for many children and their parents in an already stressful preoperative period. In addition it causes an increased organizational effort most of all in the day care department. Over many decades the use of prophylactic antibiotics to prevent infective endocarditis was recommended in patients with underlying cardiac conditions undergoing medical procedures which could lead to bacteraemia. However, transient bacteraemia occurs commonly during routine daily activities such as cleaning teeth or chewing. Most cases of endocarditis are not related to a medical procedure. There are currently no randomized and carefully controlled human trials to definitely prove the effectiveness and efficiency of endocarditis prophylaxis. Therefore, the new guidelines recommend the use of antibiotic prophylaxis only for cardiac conditions associated with the highest risk of adverse outcome from endocarditis. In paediatric surgery and paediatric anaesthesiology this applies mainly to patients with congenital heart disease. The implementation of the new guidelines in the Department of Paediatric Surgery at the Medical University Graz is illustrated in the following article.