Snail-induced anaphylaxis in patients with underlying Artemisia vulgaris pollinosis: the role of carbohydrates.

PURPOSE: The importance of carbohydrates in anaphylaxis has been described with some foods. The current work intends to obtain clinical and immunological evidence of the importance of the O-glycans for IgE binding activity in anaphylactic reactions due to Helix aspersa (HA) ingestión and Artemisia vulgaris (AV) exposition. METHODS: The studio focused on two cases of IgE-mediated anaphylaxis induced by snail ingestion in patients with underlying rhino-conjunctivitis and asthma due to AV. We performed on both patients: skin prick tests ( SPTs) with HA and AV and with a battery of aeroallergen, controlled nasal challenge and specific IgE to HA and AV, ImmunoCAP ISAC®, and a differential pattern of IgE recognition with SDS-PAGE Immunoblotting (SDSI) when these allergens have suffered an O-deglycosylation procedure. RESULTS: The patients showed positive results in SPTs, nasal challenges, and serum-specific IgE against HA and AV. In patient 1, the SDSI detected several IgE-binding proteins in AV with a molecular mass of 22, 24, and 44 kDa, whereas a band of 12 kDa was detected in HA. On the other hand, patient 2's serum revealed an IgE-binding zone between 75 and 20 kDa in the AV and a band of 24 kDa in the HA. When glycans were removed, patient 1's serum only revealed the AV's 22 and 24 kDa bands, whereas patient 2's serum did not detect any IgE-reactive protein in the HA. CONCLUSIONS: Our data suggest that O-glycosylation can be relevant in patients with anaphylaxis due to snails and allergy to Artemisia vulgaris. This new entity representing cross-reactivity between AV and HA could be named Snail-Artemisia Syndrome.

Therapeutic potential of peptides from Ole e 1 in olive-pollen allergy.

Olive-pollen allergy is one of the leading causes of respiratory allergy in Mediterranean countries and some areas of North America. Currently, allergen-specific immunotherapy is the only etiophatogenic treatment. However, this approach is not fully optimal, safe, or effective. Thus, efforts continue in the search for novel immunotherapy strategies, being one of the most promising the use of peptides derived from major allergens. This work tries to determine the therapeutic potential and safety of 5 dodecapeptides derived from the main allergen of olive-pollen allergy, Ole e 1. The immunomodulatory capacity of these peptides was studied using peripheral blood mononuclear cells (PBMCs) obtained from 19 olive-pollen-allergic patients and 10 healthy controls. We determined the capacity of these peptides to inhibit the proliferative response toward olive-pollen allergenic extract and to induce the regulatory cytokines, IL-10 and IL-35. To test the safety and absence of allergenicity of the peptides, the basophil activation was analyzed by flow-cytometry, using peripheral blood. The results showed that two of five peptides inhibited near to 30% the proliferative response against the total olive-pollen allergenic extract in olive-pollen-allergic patients. Inhibition increased to nearly 35% when the 5 peptides were used in combination. In both cases, a statistically significant induction of IL-10 and IL-35 secretion was observed in the supernatants of allergic patients PBMCs cultures. None of the 5 peptides induced basophil activation and cross-link inflammatory cell-bound IgE. In conclusion, these results open up new possibilities in the treatment of olive-pollen allergy, which could solve some of the problems facing current therapy approaches.

Tolerability and surrogate efficacy parameters of a polymerized depot mixture pollen extracts without dilutional effect.

Aim: To evaluate tolerability of subcutaneous immunotherapy, in a polymerized mixture (Olea europaea/Phleum pratense) depot presentation. Patients & methods: A total of 47 poly-allergic patients received: an abbreviated schedule with three injections at weekly intervals or a cluster schedule with two administrations in 1 day. Both treatments continued with 3 monthly maintenance administrations. Results: Two systemic reactions, (4.3%). One grade 0 and one grade I. No local reactions. Immunoglobulin levels, increased significantly at final visit versus baseline in sIgG and sIgG4; in both schedules and allergens, no significant changes in specific immunoglobulin E levels were detected. Cutaneous reactivity at final visit decreased significantly. Conclusion: Both administration schedules with polymerized mixture of O. europaea/P. pratense, presented an excellent tolerability profile and induced preliminary efficacy changes.

A Hypoallergenic Polygalacturonase Isoform from Olive Pollen Is Implicated in Pollen-Pollen Cross-Reactivity.

BACKGROUND: Cross-reactivity reactions between allergenic polygalacturonases (PGs) from different biological sources, especially foods and pollens from the Oleaceae family, have been described using Salsola kali PG (Sal k 6). No PG from olive pollen has been characterized to date, hampering further knowledge about cross-reactions through PGs. OBJECTIVES: The aim of this work was to determine the potential allergenicity of the PG from olive pollen and clarify its role in cross-reactivity. METHODS: A cDNA-encoding olive pollen PG sequence was subcloned into the pET41b vector and used to transform BL21(DE3) Escherichia coli cells to produce a His-tag fusion recombinant protein. The allergenic properties of olive pollen PG were determined by immunoblotting and ELISA in comparison to Sal k 6. The cross-reactivity potential of the protein with other pollen sources was analyzed by inhibition immunoassays. RESULTS: The existence of other isoforms of Ole e 14 with different allergenicity was confirmed by proteomics and a meta-analysis of the recently reported olive genome. Sal k 6 showed a higher IgE recognition than Ole e 14 regardless of patient sensitization, suggesting the existence of more allergenic Ole e 14 isoforms in olive pollen. IgG and IgE inhibition assays supported the existence of cross-reactions between them and with other PGs from Oleaceae and Poaceae plant families. CONCLUSIONS: A new allergen from olive pollen, Ole e 14, has been identified, produced as a recombinant isoform, and structurally and immunologically characterized. Its role in cross-reactivity has been confirmed and, due to its smaller IgE binding capacity, it could have an important role for therapeutic purposes.

A recombinant isoform of the Ole e 7 olive pollen allergen assembled by de novo mass spectrometry retains the allergenic ability of the natural allergen.

The allergenic non-specific lipid transfer protein Ole e 7 from olive pollen is a major allergen associated with severe symptoms in areas with high olive pollen levels. Despite its clinical importance, its cloning and recombinant production has been unable by classical approaches. This study aimed at determining by mass-spectrometry based proteomics its complete amino acid sequence for its subsequent expression and characterization. To this end, the natural protein was in-2D-gel tryptic digested, and CID and HCD fragmentation spectra obtained by nLC-MS/MS analyzed using PEAKS software. Thirteen out of the 457 de novo sequenced peptides obtained allowed assembling its full-length amino acid sequence. Then, Ole e 7-encoding cDNA was synthesized and cloned in pPICZαA vector for its expression in Pichia pastoris yeast. The analyses by Circular Dichroism, and WB, ELISA and cell-based tests using sera and blood from olive pollen-sensitized patients showed that rOle e 7 mostly retained the structural, allergenic and antigenic properties of the natural allergen. In summary, rOle e 7 allergen assembled by de novo peptide sequencing by MS behaved immunologically similar to the natural allergen scarcely isolated from pollen. SIGNIFICANCE: Olive pollen is an important cause of allergy. The non-specific lipid binding protein Ole e 7 is a major allergen with a high incidence and a phenotype associated to severe clinical symptoms. Despite its relevance, its cloning and recombinant expression has been unable by classical techniques. Here, we have inferred the primary amino acid sequence of Ole e 7 by mass-spectrometry. We separated Ole e 7 isolated from pollen by 2DE. After in-gel digestion with trypsin and a direct analysis by nLC-MS/MS in an LTQ-Orbitrap Velos, we got the complete de novo sequenced peptides repertoire that allowed the assembling of the primary sequence of Ole e 7. After its protein expression, purification to homogeneity, and structural and immunological characterization using sera from olive pollen allergic patients and cell-based assays, we observed that the recombinant allergen retained the antigenic and allergenic properties of the natural allergen. Collectively, we show that the recombinant protein assembled by proteomics would be suitable for a better in vitro diagnosis of olive pollen allergic patients.

High-throughput screening of T7 phage display and protein microarrays as a methodological approach for the identification of IgE-reactive components.

Olive pollen and yellow mustard seeds are major allergenic sources with high clinical relevance. To aid with the identification of IgE-reactive components, the development of sensitive methodological approaches is required. Here, we have combined T7 phage display and protein microarrays for the identification of allergenic peptides and mimotopes from olive pollen and mustard seeds. The identification of these allergenic sequences involved the construction and biopanning of T7 phage display libraries of mustard seeds and olive pollen using sera from allergic patients to both biological sources together with the construction of phage microarrays printed with 1536 monoclonal phages from the third/four rounds of biopanning. The screening of the phage microarrays with individual sera from allergic patients enabled the identification of 10 and 9 IgE-reactive unique amino acid sequences from olive pollen and mustard seeds, respectively. Five immunoreactive amino acid sequences displayed on phages were selected for their expression as His6-GST tag fusion proteins and validation. After immunological characterization, we assessed the IgE-reactivity of the constructs. Our results show that protein microarrays printed with T7 phages displaying peptides from allergenic sources might be used to identify allergenic components -peptides, proteins or mimotopes- through their screening with specific IgE antibodies from allergic patients.

An Enzymatically Active ß-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.

Endo-ß-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic ß-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that ß-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen ß-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long ß-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of ß-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

Therapeutic targets for olive pollen allergy defined by gene markers modulated by Ole e 1-derived peptides.

Two regions of Ole e 1, the major olive-pollen allergen, have been characterized as T-cell epitopes, one as immunodominant region (aa91-130) and the other, as mainly recognized by non-allergic subjects (aa10-31). This report tries to characterize the specific relevance of these epitopes in the allergic response to olive pollen by analyzing the secreted cytokines and the gene expression profiles induced after specific stimulation of peripheral blood mononuclear cells (PBMCs). PBMCs from olive pollen-allergic and non-allergic control subjects were stimulated with olive-pollen extract and Ole e 1 dodecapeptides containing relevant T-cell epitopes. Levels of cytokines were measured in cellular supernatants and gene expression was determined by microarrays, on the RNAs extracted from PBMCs. One hundred eighty-nine differential genes (fold change >2 or <-2, P<0.05) were validated by qRT-PCR in a large population. It was not possible to define a pattern of response according the overall cytokine results but interesting differences were observed, mainly in the regulatory cytokines. Principal component (PCA) gene-expression analysis defined clusters that correlated with the experimental conditions in the group of allergic subjects. Gene expression and functional analyses revealed differential genes and pathways among the experimental conditions. A set of 51 genes (many essential to T-cell tolerance and homeostasis) correlated with the response to aa10-31 of Ole e 1. In conclusion, two peptides derived from Ole e 1 could regulate the immune response in allergic patients, by gene-expression modification of several regulation-related genes. These results open new research ways to the regulation of allergy by Oleaceae family members.

Pectin methylesterases of pollen tissue, a major allergen in olive tree.

Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

Allergenic contribution of the IgE-reactive domains of the 1,3-beta-glucanase Ole e 9: diagnostic value in olive pollen allergy.

BACKGROUND: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. OBJECTIVE: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. METHODS: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. RESULTS: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. CONCLUSIONS: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

Analysis of IgE and IgG B-cell immunodominant regions of Ole e 1, the main allergen from olive pollen.

Ole e 1 is a major allergen from olive pollen with an IgE-binding frequency around 80% among allergic population. Its diagnostic value has been demonstrated, and cross-reactive allergens have been found in ash, lilac and privet. We sought to determine IgE- and IgG-binding regions of Ole e 1. Ole e 1-specific polyclonal antiserum and sera from patients allergic to olive pollen were used to analyze IgG and IgE epitopes, respectively. Short overlapping synthetic peptides covering the complete sequence of Ole e 1 and point mutants of these peptides bound to membranes, as well as long recombinant peptides fused to GST were used in dot blot immunostaining and ELISA. Skin prick tests were performed on 14 allergic patients to assay the response in vivo to the recombinant fusion peptides. Residues at positions 8-11, 29, 32, 33, 55-59, 70, 107-110, 112, 120, 123, 141 of Ole e 1 sequence were found to be antigenically relevant in the IgG-binding. Although amino acids K137, L138, G139, Y141 and P142 were involved in the IgE-recognition of a pool of sera from allergic individuals, the response to the IgEs seemed to be preferentially conformational. IgE-binding capability of recombinant GST-fused peptide T114-M145 was demonstrated by in vivo (prick test) and in vitro (ELISA) experiments. Major IgG and IgE-binding regions of Ole e 1 have been identified being the C-terminal an immunodominant region. These data could help to design hypoallergenic forms of the allergen.

A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies [correction of interspecie] cross-reactivity.

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.