Barbara Bohle

OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS INDUCE IL-12, IL-18 AND IFN-GAMMA PRODUCTION IN CELLS FROM ALLERGIC INDIVIDUALS AND INHIBIT IGE SYNTHESIS IN VITRO

The effects of phosphorothioate oligonucleotides containing CpG motifs (CpG‐ODN) on cultured cells from allergic patients and non‐atopic individuals were investigated. In peripheral blood mononuclear cells (PBMC) CpG‐ODN led to a significant increase of IFN‐γ. By intracellular cytokine staining, IFN‐γ production could be attributed to NK cells and inhibition experiments indicated an IL‐12‐dependent mechanism. Moreover, CpG‐ODN increased mRNA expression of IL‐12 and IL‐18 in PBMC. In this respect, no significant difference between allergic and non‐atopic individuals was observed. Monocyte‐derived dendritic cells were identified as one IL‐12‐ and IL‐18‐producing source. In addition, stimulation of PBMC derived from atopic patients with CpG‐ODN led to a considerable increase of polyclonal IgG and IgM synthesis. In contrast, the production of total IgE was suppressed. CpG‐ODN induced a significant rise of IgG and IgM specific for allergens to which the patients were sensitized, whereas allergen‐specific IgE levels remained unchanged. Our data suggest that CpG‐ODN display a strong influence on the ongoing immune response and might represent potential adjuvants for specific immunotherapy of type I allergy.

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen‐specific serum immunoglobulin E

Background The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen‐specific IgE.

Alpha-Gal on bioprostheses: xenograft immune response in cardiac surgery

Background  The α‐Gal (Galα1,3‐Galβ1–4GlcNAc‐R) epitope is the major xenoantigen causing hyperacute rejection of pig organs transplanted into primates. Porcine bioprostheses are utilized in cardiac surgery. However, premature degeneration of bioprostheses has limited utilization in younger patients and the immune response remains elusive. We sought to investigate whether a specific α‐Gal immune response may play a role in this clinical scenario.

IL-12p70-Dependent Th1 Induction by Human B Cells Requires Combined Activation with CD40 Ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-α, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-α, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-α, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-γ production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.

EAACI Molecular Allergology User's Guide

The availability of allergen molecules (‘components’) from several protein families has advanced our understanding of immunoglobulin E (IgE)‐mediated responses and enabled ‘component‐resolved diagnosis’ (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology Users Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low‐abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross‐reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE‐mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross‐reactive panallergens from plant (lipid transfer proteins, polcalcins, PR‐10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE‐mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients.

Systemic Immunological Changes Induced by Administration of Grass Pollen Allergens via the Oral Mucosa during Sublingual Immunotherapy

Twenty–four patients suffering from grass pollen allergy underwent sublingual immunotherapy (SLIT) with standardized grass pollen extract for 1 year. In order to investigate immunological changes induced by the administration of allergens via the oral mucosa, the SLIT–spit method was applied. The cumulative dose of approximately 80 μg of major allergen (grass group 5 allergen), was relatively low. During the time of treatment, we could observe a significant increase in the levels of specific IgG and IgG4 antibodies. However, the titers of allergen–specific IgE antibodies showed a significant increase in the course of SLIT as well. Analyzing lymphoproliferative responses, a significant decrease in reactivity in response to stimulation with complete grass pollen extract (p = 0.001) and to recombinant Phl p 1 (a major allergen of timothy grass, p<0.001) could be observed, indicating the induction of immunological tolerance. Proliferative responses to a control antigen (tetanus toxoid) were not influenced by the treatment. At different time points during SLIT, allergen (Phl p 1)–specific T cell clones (TCC) were established from the peripheral blood of the patients. Cytokine production by allergen–stimulated T cells did not reveal any changes consistent with immune deviation, i.e. the ratio of Th1/Th2 TCC did not change during SLIT. In conclusion, we provide evidence that sublingual treatment leads to systemic changes in immunoreactivity to the administered allergen.

The impact of pollen-related food allergens on pollen allergy

Patients with birch pollen allergy frequently develop hypersensitivity reactions to certain foods, e.g. apples, celery, carrots and hazelnuts. These reactions are mainly caused by IgE‐antibodies specific for the major birch pollen allergen, Bet v 1, which cross‐react with homologous proteins in these foods. Analyzing the T‐cell response to Bet v 1‐related food allergens revealed that these dietary proteins contain several distinct T‐cell epitopes and activate Bet v 1‐specific T cells to proliferate and produce cytokines. Several of these cross‐reactive T‐cell epitopes were not destroyed by simulated gastrointestinal digestion of food allergens and stimulated Bet v 1‐specific T cells despite nonreactivity with IgE antibodies. Similarly, cooked food allergens did not elicit IgE‐mediated symptoms (oral allergy syndromes) but caused T‐cell‐mediated late‐phase reactions (deterioration of atopic eczema) in birch pollen‐allergic patients with atopic dermatitis because thermal processing affected their conformational structure and not the primary amino acid sequence. Thus, T‐cell cross‐reactivity between Bet v 1 and related food allergens occurs independently of IgE‐cross‐reactivity in vitro and in vivo. We speculate that symptom‐free consumption of pollen‐related food allergens may have implications for the pollen‐specific immune response of allergic individuals.

Bet v 1, the major birch pollen allergen, and Mal d 1, the major apple allergen, cross-react at the level of allergen-specific T helper cells☆☆☆★★★

BACKGROUND Food allergy to apple is frequent in individuals allergic to tree pollen. The major allergens of birch, Bet v 1, and apple, Mal d 1, have been cloned and sequenced and display a high degree of sequence identity, leading to IgE cross-reactivity. OBJECTIVE We sought to investigate cross-reactivity between Bet v 1 and Mal d 1 at the level of allergen-specific T lymphocytes. METHODS PBMCs of 13 patients allergic to birch pollen with oral allergy syndrome to apple were stimulated with rBet v 1 and rMal d 1, respectively, thereby establishing allergen-specific T-cell lines and T-cell clones. rMal d 1-specific T-cell cultures were tested for reactivity with rBet v 1, and rBet v 1-specific T cells were analyzed for reactivity with apple allergen. Cytokine production patterns in response to specific stimulation were evaluated. A selection of cross-reacting T-cell clones was mapped for epitope specificity by the use of overlapping Bet v 1- derived peptides. RESULTS Nineteen Mal d 1-specific T-cell clones were produced, 79% of which cross-reacted with Bet v 1. Eight of 18 Bet v 1-specific T-cell clones cross-reacted with Mal d 1. Six peptides representing cross-reactive T-cell epitopes could be identified. The respective fragments from birch and apple displayed approximately 50% amino acid identity. Seventy percent of the cross-reactive T-cell clones revealed a T(H2)-like cytokine production pattern. CONCLUSION The results indicate that cross-reactivity between apple and birch pollen leading to the clinical oral allergy syndrome occurs not only at the serologic, but also at the cellular level.

Birch pollen–related food allergy: Clinical aspects and the role of allergen-specific IgE and IgG4 antibodies

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 IgG(4) antibodies in food tolerance. METHODS Food-induced symptoms were evaluated in 225 individuals with birch pollen allergy by using a standardized questionnaire. IgE and IgG(4) 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 IgG(4)/IgE ratios were significantly higher in food-tolerant individuals than individuals with food allergy. Sera from IgG(4)-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 IgG(4)/IgE ratios seem associated with food tolerance, potentially because specific IgG(4) blocks IgE binding to food allergens. Thus, the presence of food allergen-specific IgG(4) antibodies is no diagnostic marker for birch pollen-related food allergy.

Assessment of Bet v 1-Specific CD4 + T Cell Responses in Allergic and Nonallergic Individuals Using MHC Class II Peptide Tetramers

In this study, we used HLA-DRB1*0101, DRB1*0401, and DRB1*1501 peptide tetramers combined with cytokine surface capture assays to characterize CD4+ T cell responses against the immunodominant T cell epitope (peptide 141–155) from the major birch pollen allergen Bet v 1, in both healthy and allergic individuals. We could detect Bet v 1-specific T cells in the PBMC of 20 birch pollen allergic patients, but also in 9 of 9 healthy individuals tested. Analysis at a single-cell level revealed that allergen-specific CD4+ T cells from healthy individuals secrete IFN-γ and IL-10 in response to the allergen, whereas cells from allergic patients are bona fide Th2 cells (producing mostly IL-5, some IL-10, but no IFN-γ), as corroborated by patterns of cytokines produced by T cell clones. A fraction of Bet v 1-specific cells isolated from healthy, but not allergic, individuals also expresses CTLA-4, glucocorticoid-induced TNF receptor, and Foxp 3, indicating that they represent regulatory T cells. In this model of seasonal exposure to allergen, we also demonstrate the tremendous dynamics of T cell responses in both allergic and nonallergic individuals during the peak pollen season, with an expansion of Bet v 1-specific precursors from 10−6 to 10−3 among circulating CD4+ T lymphocytes. Allergy vaccines should be designed to recapitulate such naturally protective Th1/regulatory T cell responses observed in healthy individuals.