Effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice.

Pectins are dietary fibers that are attributed with several beneficial immunomodulatory effects. Depending on the degree of esterification (DE), pectins can be classified as high methoxyl pectin (HMP) or low methoxyl pectin (LMP). The aim of this study was to investigate the effects of pectin methyl-esterification on intestinal microbiota and its immunomodulatory properties in naive mice. Supplementation of the diet with LMP or HMP induced changes in the composition of the intestinal microbiota in mice toward Bacteroides, which was mainly promoted by HMP. Metabolome analysis of stool samples from pectin-fed mice showed a different effect of the two types of pectin on the levels of short-chain fatty acids and bile acids, which was consistent with highly efficient in vivo fermentation of LMP. Analysis of serum antibody levels showed a significant increase in IgG and IgA levels by both pectins, while FACS analysis revealed a decrease of infiltrating inflammatory cells in the intestinal lamina propria by HMP. Our study revealed that the structural properties of the investigated pectins determine fermentability, effects on microbial composition, metabolite production, and modulation of immune responses. Consumption of HMP preferentially altered the gut microbiota and suppressed pro-inflammatory immune responses, suggesting a beneficial role in inflammatory diseases.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

The Flagellin:Allergen Fusion Protein rFlaA:Betv1 Induces a MyD88- and MAPK-Dependent Activation of Glucose Metabolism in Macrophages.

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differentiated from BALB/c, C57BL/6, TLR5-/-, or MyD88-/- mice were pre-treated with inhibitors, stimulated with rFlaA:Betv1 or respective controls, and analyzed for activation, cytokine secretion, metabolic state, RNA transcriptome, and modulation of allergen-specific Th2 responses. Stimulation of BMDMs with rFlaA:Betv1 resulted in MyD88-dependent production of IL-1ß, IL-6, TNF-α, IL-10, CD69 upregulation, and a pronounced shift towards glycolysis paralleled by activation of MAPK, NFκB, and mTOR signaling. Inhibition of either mTOR (rapamycin) or SAP/JNK-MAPK signaling (SP600125) resulted in dose-dependent metabolic suppression. In BMDM and T cell co-cultures, rFlaA:Betv1 stimulation suppressed rBet v 1-induced IL-5 and IL-13 secretion while inducing IFN-γ production. mRNA-Seq analyses showed HIF-1a, JAK, STAT, phagosome, NLR, NFκB, TNF, TLR, and chemokine signaling to participate in the interplay of cell activation, glycolysis, and immune response. rFlaA:Betv1 strongly activated BMDMs, resulting in MyD88-, MAPK-, and mTOR-dependent enhancement of glucose metabolism. Our results suggest macrophages are important target cells to consider during restauration of allergen tolerance during AIT.

The Dietary Fiber Pectin: Health Benefits and Potential for the Treatment of Allergies by Modulation of Gut Microbiota.

PURPOSE OF REVIEW: The incidence of allergies is increasing and has been associated with several environmental factors including westernized diets. Changes in environment and nutrition can result in dysbiosis of the skin, gut, and lung microbiota altering the production of microbial metabolites, which may in turn generate epigenetic modifications. The present review addresses studies on pectin-mediated effects on allergies, including the immune modulating mechanisms by bacterial metabolites. RECENT FINDINGS: Recently, microbiota have gained attention as target for allergy intervention, especially with prebiotics, that are able to stimulate the growth and activity of certain microorganisms. Dietary fibers, which cannot be digested in the gastrointestinal tract, can alter the gut microbiota and lead to increased local and systemic concentrations of gut microbiota-derived short chain fatty acids (SCFAs). These can promote the generation of peripheral regulatory T cells (Treg) by epigenetic modulation and suppress the inflammatory function of dendritic cells (DCs) by transcriptional modulation. The dietary fiber pectin (a plant-derived polysaccharide commonly used as gelling agent and dietary supplement) can alter the ratio of Firmicutes to Bacteroidetes in gut and lung microbiota, increasing the concentrations of SCFAs in feces and sera, and reducing the development of airway inflammation by suppressing DC function. Pectin has shown immunomodulatory effects on allergies, although the underlying mechanisms still need to be elucidated. It has been suggested that the different types of pectin may exert direct and/or indirect immunomodulatory effects through different mechanisms. However, little is known about the relation of certain pectin structures to allergies.

The Role of Lipid Transfer Proteins as Food and Pollen Allergens Outside the Mediterranean Area.

PURPOSE OF REVIEW: To provide an overview of the prevalence and clinical manifestation of non-specific lipid transfer proteins (LTP)-mediated allergies outside the Mediterranean area and to address potential reasons for the different geographical significance of LTP-driven allergies. RECENT FINDINGS: LTPs are major allergens in the Mediterranean area, which frequently can elicit severe reactions. Pru p 3 the LTP from peach is reported as genuine allergen and is considered a prototypic marker for LTP-mediated allergies. However, both food and pollen LTP allergies exist outside the Mediterranean area, but with lower clinical significance, different immunogenicity, and less clarified role. Evidence has been reported that in areas with high exposure to pollen, in particular to mugwort, pollen-derived LTPs can act as a primary sensitizer to trigger secondary food allergies. Co-sensitization to unrelated allergens might be causative for less severe reactions in response to LTPs. However, the reason for the geographical different sensitization patterns to LTPs remains unclear.

Identification and Characterization of IgE-Reactive Proteins and a New Allergen (Cic a 1.01) from Chickpea (Cicer arietinum).

SCOPE: Chickpea (Cicer arietinum) allergy has frequently been reported particularly in Spain and India. Nevertheless, chickpea allergens are poorly characterized. The authors aim to identify and characterize potential allergens from chickpea. METHODS AND RESULTS: Candidate proteins are selected by an in silico approach or immunoglobuline E (IgE)-testing. Potential allergens are prepared as recombinant or natural proteins and characterized for structural integrity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD)-spectroscopy, and mass spectrometry (MS) analysis. IgE-sensitization pattern of Spanish chickpea allergic and German peanut and birch pollen sensitized patients are investigated using chickpea extracts and purified proteins. Chickpea allergic patients show individual and heterogeneous IgE-sensitization profiles with extracts from raw and boiled chickpeas. Chickpea proteins pathogenesis related protein family 10 (PR-10), a late embryogenesis abundant protein (LEA/DC-8), and a vicilin-containing fraction, but not 2S albumin, shows IgE reactivity with sera from chickpea, birch pollen, and peanut sensitized patients. Remarkably, allergenic vicilin, DC-8, and PR-10 are detected in the extract of boiled chickpeas. CONCLUSION: Several IgE-reactive chickpea allergens are identified. For the first time a yet not classified DC-8 protein is characterized as minor allergen (Cic a 1). Finally, the data suggest a potential risk for peanut allergic patients by IgE cross-reactivity with homologous chickpea proteins.

Critical role of mammalian target of rapamycin for IL-10 dendritic cell induction by a flagellin A conjugate in preventing allergic sensitization.

BACKGROUND: Fusion proteins incorporating the Toll-like receptor 5 ligand flagellin are currently undergoing clinical trials as vaccine candidates for many diseases. OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSION: These results provide evidence that mammalian target of rapamycin is a key player involved in prevention of TH2 responses by flagellin A conjugate vaccines.

Conjugation of wildtype and hypoallergenic mugwort allergen Art v 1 to flagellin induces IL-10-DC and suppresses allergen-specific TH2-responses in vivo.

Allergies to weed pollen including members of the Compositae family, such as mugwort, ragweed, and feverfew are spreading worldwide. To efficiently treat these newly arising allergies, allergen specific immunotherapy needs to be improved. Therefore, we generated novel vaccine candidates consisting of the TLR5-ligand Flagellin A from Listeria and the major mugwort allergen Art v 1 including either the wild type Art v 1 sequence (rFlaA:Artv1) or a hypoallergenic variant (rFlaA:Artv1hyp) with reduced IgE-binding capacity. Immune modulating capacity of these constructs and respective controls was evaluated in vitro and in vivo. Incorporation of hypoallergenic Art v 1 derivative did not interfere with the resulting fusion proteins' immune stimulatory capacity. Both rFlaA:Artv1 and rFlaA:Artv1hyp induced a prominent, mTOR-dependent, IL-10 secretion from murine dendritic cells, and suppressed allergen-specific TH2-cytokine secretion in vitro and in vivo. Both conjugates retained the capacity to induce rFlaA-specific antibody responses while efficiently inducing production of Art v 1-specific IgG1 and IgG2a antibodies in mice. Interestingly, only the suppression of TH2-cytokine secretion by rFlaA:Artv1 (but not rFlaA:Artv1hyp) was paralleled by a strong secretion of IFN-γ. In summary, we provided evidence that incorporating hypoallergens into flagellin:allergen fusion proteins is a suitable strategy to further improve these promising vaccine candidates.

Identification and implication of an allergenic PR-10 protein from walnut in birch pollen associated walnut allergy.

SCOPE: English walnut (Juglans regia) belongs to the most important allergenic tree nuts. Co-sensitization with birch (Betula verrucosa) pollen has been reported. We aimed to identify a walnut allergen homologous to the major birch pollen allergen Bet v 1. METHODS AND RESULTS: A cDNA encoding a Bet v 1-homologous allergen (Jug r 5) in walnut kernels was cloned by RT-PCR. Jug r 5 was expressed in Escherichia coli, purified by column chromatography and characterized by circular dichroism spectroscopy. Specific IgE levels to walnut, Bet v 1, and Jug r 5 in birch pollen allergics (n = 16) with concomitant walnut allergy were measured by ImmunoCAP: 44% of the patients were tested positive to walnut while 94% were reactive to Jug r 5, and 100% to Bet v 1. Jug r 5 and Bet v 1 allergens showed bidirectional IgE cross-reactivity by competitive ELISA and were capable of inducing histamine release from effector cells. Immunoblot competition experiments demonstrated the presence of IgE-reactive Jug r 5 in walnut extract, but at low levels. CONCLUSION: A Bet v 1-like allergen was identified in walnut. Diagnostic use of Jug r 5 will compensate for the low sensitivity of walnut extract for patients with birch pollen associated walnut allergy.

Cor a 1-reactive T cells and IgE are predominantly cross-reactive to Bet v 1 in patients with birch pollen-associated food allergy to hazelnut.

BACKGROUND: IgE- and T-cell cross-reactivity contribute to the birch pollen-food syndrome. OBJECTIVES: We performed a comprehensive analysis of T-cell cross-reactivity in primary cell cultures, facilitating the identification of allergen-specific T-cell subpopulations from individual patients. METHODS: Patients with birch pollen allergy and associated food allergy to hazelnuts, carrots, or both were analyzed for IgE cross-reactivity, T-cell responses, and T-cell cross-reactivity to recombinant Bet v 1.0101 (Bet v 1; birch), Cor a 1.0401 (Cor a 1; hazelnut), and Dau c 1.0104 (Dau c 1; carrot). A novel flow cytometry-based method using a 2-step staining process with fluorescent dyes was established to identify subpopulations of cross-reactive T cells. RESULTS: IgE-binding inhibition tests of individual sera revealed that the vast majority of Cor a 1-reactive IgE was cross-reactive to Bet v 1, whereas Bet v 1-reactive IgE was only partially inhibited by preincubation with Cor a 1. Primary stimulation of T cells with Bet v 1 or Cor a 1 resulted in a significant increase in specific responses to Cor a 1 or Bet v 1 after secondary stimulation, respectively, indicating T-cell cross-reactivity between birch and hazelnut allergens in all patients of the study cohort. Preactivation with Dau c 1 induced less pronounced effects. A novel flow cytometry-based proliferation assay identified a predominant Cor a 1/Bet v 1-cross-reactive T-cell subpopulation within highly Bet v 1/Cor a 1-responsive T cells. CONCLUSION: Analysis of primary allergen-specific T cells combined with flow cytometry-based proliferation assays facilitates investigation of allergen-specific T-cell subpopulations in subjects and might be helpful to evaluate the effect of birch-specific immunotherapy on pollen-associated food allergies.

Rice-induced anaphylaxis: IgE-mediated allergy against a 56-kDa glycoprotein.

BACKGROUND: Although rice (Oryza sativa) is one of the most common cereals produced and consumed around the world, there have been only a few reports on immediate hypersensitivity reactions after ingestion of rice. Few clinical studies on rice allergy in Asia have been reported concerning rhinitis, asthma and atopic dermatitis. In this case study, we identify allergens presumably responsible for anaphylaxis after ingestion of rice in a German patient. METHODS: Prick-to-prick tests, determination of specific IgE and the basophil activation test (BAT) were performed to confirm IgE-mediated allergy. IgE reactivity was further analyzed by immunoblotting of protein extracts from cooked commercial rice products. Rice allergens were purified, subjected to N-terminal sequencing and characterized by IgE binding and IgE inhibition assays using additional sera from 8 subjects with sensitization to rice and/or a history of hypersensitivity symptoms after rice ingestion. RESULTS: Prick-to-prick tests were positive to raw and cooked rice (basmati rice and long-grain rice) and preparations of different rice extracts. Specific IgE against rice (f9) was 1.87 kU(A)/l. The BAT showed specific IgE-mediated activation of basophils after stimulation with rice extracts. Four IgE-reactive rice proteins with an apparent molecular weight of 49, 52, 56 and 98 kDa were identified. Interestingly, only binding to the 56-kDa glycoprotein was at least partially independent from cross-reactive carbohydrate determinants (CCD), whereas IgE binding to the other rice proteins was completely inhibited by pre-incubation with the CCD MUXF derived from bromelain. CONCLUSIONS: Yet unidentified high-molecular-weight allergens from rice seeds, predominantly a 56-kDa glycoprotein, seem to be responsible for anaphylaxis after consumption of rice in a German patient.

Design of tomato fruits with reduced allergenicity by dsRNAi-mediated inhibition of ns-LTP (Lyc e 3) expression.

Plant genetic engineering has the potential to introduce new allergenic proteins into foods but, at the same time, it can be used to remove established allergens. Here, we report the molecular characterization of Lyc e 3, a new tomato (Lycopersicon esculentum) allergen, and the efficient down-regulation of its expression in transgenic tomato plants. Following the identification of an immunoglobulin E (IgE)-binding 9-kDa polypeptide in tomato peel, designated Lyc e 3, its partial amino acid sequence was determined by N-terminal protein sequencing. Sequence comparison revealed that Lyc e 3 encodes a nonspecific lipid transfer protein (ns-LTP). In plants, ns-LTPs are encoded by large gene families which differ in primary amino acid sequence, expression and proposed cellular function. To identify Lyc e 3 encoding complementary DNAs (cDNAs), public tomato expressed sequence tag (EST) databases were screened for ns-LTP sequences. Following this strategy, two cDNAs, LTPG1 and LTPG2, with high homology to the N-terminal sequence of Lyc e 3, were identified. Ectopic expression of LTPG1 and LTPG2 in Escherichia coli, followed by immunoblotting, verified their IgE reactivity. Subsequently, transgenic tomato plants constitutively expressing LTPG1- or LTPG2-specific double-stranded RNA interference (dsRNAi) constructs were created and tested for the suppression of Lyc e 3 accumulation. Efficient silencing of Lyc e 3 was documented by Northern and Western blotting. In both cases, Lyc e 3 accumulation was decreased to levels below the detection limit (less than 0.5% of the wild-type protein). The allergenic potential of Lyc e 3-deficient tomato fruits was tested by measuring histamine release from sensitized human basophils stimulated with transgenic and parental lines. These assays revealed a strong (10- to 100-fold) decrease in histamine release of human basophils challenged with transgenic fruit extracts when compared with control extracts. These results demonstrate the feasibility of creating low allergenic tomato fruits by means of dsRNAi inhibition.

Bet v 1142-156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens.

BACKGROUND: Individuals with birch pollen allergy frequently experience hypersensitivity reactions to certain foods, primarily because of IgE antibodies specific for the major birch pollen allergen Bet v 1 that cross-react with homologous food allergens. OBJECTIVE: We sought to characterize the major T-cell epitopes of Bet v 1 and to investigate their involvement in the cellular cross-reactivity with homologous food allergens. METHODS: T-cell epitope mapping of Bet v 1 was performed by testing Bet v 1-specific T-cell lines derived from 57 individuals with birch pollen allergy, with overlapping peptides representing the entire allergen. T-cell lines and T-cell clones were stimulated with Bet v 1-related major allergens from apple (Mal d 1), cherry (Pru av 1), hazelnut (Cor a 1), celery (Api g 1), carrot (Dau c 1), and soybean (Gly m 4) and with peptides deduced from the C-terminal amino acid sequences of these molecules. Results Bet v 1 142-156 , positioned in the highly conserved C-terminal region of Bet v 1, was identified as the major T-cell epitope recognized by 61% of individuals. Most T lymphocytes specific for Bet v 1 142-156 were activated by one or more homologous food proteins or the respective peptides, as indicated by proliferation and cytokine production. CONCLUSION: The major T-cell epitope of Bet v 1, Bet v 1 142-156 , plays an important role in the cellular cross-reactivity between this respiratory allergen and related food allergens. Thus T lymphocytes specific for Bet v 1 142-156 might be activated by various Bet v 1-related food allergens in vivo, even out of the pollen season.

Molecular basis of pollen-related food allergy: identification of a second cross-reactive IgE epitope on Pru av 1, the major cherry (Prunus avium) allergen.

Birch (Betula verrucosa) pollen-associated food allergy is a well-characterized syndrome, which is due to the cross-reactivity of IgE antibodies to homologous allergens in various foods. One crossreacting area on the major birch pollen allergen Bet v 1 and its homologue in cherry (Prunus avium) Pru av 1 has already been identified. This is the so-called 'P-loop' region, which encompasses amino acid residues around position 45 and is found on the two virtually identical tertiary protein structures. We tried to determine an additional IgE cross-reacting patch on Pru av 1 and Bet v 1. The putative IgE-binding region on Pru av 1 was localized with a mAb (monoclonal antibody) that was generated against Bet v 1, and cross-reacts with several Bet v 1 homologues in food and inhibits the binding of patients' IgE to Pru av 1. mAb reactivity pattern was analysed and amino acid positions 28 and 108 of Pru av 1 were selected and mutated by site-directed mutagenesis. The Pru av 1 mutants were produced as recombinant proteins and characterized for their folding, mAb- and IgE-binding capacity and allergenic potency with a cellular assay using the humanized rat basophilic leukaemia cell line RBL-25/30. Amino acid position 28 is involved in a second major IgE-binding region on Pru av 1 and probably on Bet v 1. The identification of this second major IgE-binding region is an essential prerequisite to understand the phenomenon of cross-reactivity and its clinical consequences, and to produce hypoallergenic proteins for an improved immunotherapy of type I allergy.

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics.

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.

Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. OBJECTIVE: To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. METHODS: Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. RESULTS: Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. CONCLUSION: In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.

Monoclonal IgE antibodies against birch pollen allergens: novel tools for biological characterization and standardization of allergens.

BACKGROUND: IgE antibodies are key players in immediate hypersensitivity reactions. Allergen characterization and standardization is usually based on the sera of allergic patients, whereas monoclonal IgE antibodies specific for clinically relevant allergens are very rare. OBJECTIVE: The aim of this study was to establish IgE mAbs specific for birch pollen allergens, because these are important inhalant allergens. METHODS: IgE-producing hybridomas were identified by using the highly sensitive rat basophilic leukemia cell mediator release assay with enhanced allergen stimulation by additional cross-linking with birch pollen-specific IgG antibodies. The obtained IgE mAbs were characterized by immunologic methods and by cDNA sequencing. RESULTS: Seven IgE mAbs specific for the birch pollen allergens Bet v 1 or Bet v 6 were obtained and were all biologically active in mast cell-based assays. Mediator release experiments with mAb combinations indicated that 2 different epitope regions were recognized on Bet v 1, whereas the 2 Bet v 6-specific mAbs bound to the same epitope region. After sensitization of rat basophilic leukemia cells with IgE mAbs, different amounts of Bet v 1 or Bet v 6 were detected in commercial diagnostic allergen reagents, whereas sensitization with polyclonal IgE resulted in similar allergenic potency of all products. CONCLUSIONS: IgE mAbs represent promising novel tools for allergen characterization and component-resolved standardization of allergen extracts.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.