Peach LTP Research Articles

Clinical and serological characteristics of patients allergic to LTP.

Allergy to lipid transfer proteins (LPT) is common in Mediterranean Europe, and it causes severe reactions in patients and affects multiple foods, impairing the quality of life. This study aimed to describe the clinical and sensitization profile of patients with LTP syndrome and to determine a clinical pattern of severity. Molecular diagnosis is shown in a broad population through microarrays. This study was performed at the LTP Allergy Consultation of the Reina Sofia Hospital in Murcia, Spain. We analyzed the patients' characteristics, reactions, cofactors, food implicated, quality of life, skin prick test to food and aeroallergens, and serologic parameters, such as total immunoglobulin E, peach LTP (Pru p 3 IgE) and immunoglobulin G4, and microarray Immuno Solid-phase Allergen Chip (ISAC). We related the severity of the reactions with other variables. We presented a series of 236 patients diagnosed with LTP allergy, 54.66% suffering from anaphylaxis, 36.02% from urticaria angioedema, and 9.32% from oral allergy syndrome. The most frequently implicated food was peach, producing symptoms in 70% of patients, followed by walnut in 55%, peanut in 45%, hazelnut in 44%, and apple in 38% patients. Regarding the food that provoked anaphylaxis, walnut was the most frequent instigator, along with peach, peanut, hazelnut, almond, sunflower seed, and apple. According to the severity of LPT reaction, we did not discover significant differences in gender, age, food group involved, and serologic parameters. We found differences in the presence of cofactors, with 48.84% of cofactors in patients with anaphylaxis, compared to 27.1% in patients without anaphylaxis and in family allergy background (P < 0.0001). In our series of patients, 54% presented anaphylaxis, and the foods that most frequently produced symptoms were peaches, apples, and nuts. Cofactors and family allergy backgrounds were associated with the severity of LPT reaction.

Identification of the ligand of Pru p 3, a peach LTP.

Pru p 3, a peach LTP, is located in pollinated flower styles and secreting downy hairs, transporting a derivative of camptothecin bound to phytosphingosine. Pru p 3 may inhibit a second pollination and may keep away herbivores until seed maturation. The allergen Pru p 3, a peach lipid transfer protein, has been well studied. However, its physiological function remains to be elucidated. Our results showed that Pru p 3 usually carries a lipid ligand that play an essential role in its function in plants. Using ESI-qToF, we observed that the ligand was a derivative of camptothecin binding to phytosphingosine, wich that is inserted into the hydrophobic tunnel of the protein. In addition, the described ligand displayed topoisomerase I activity inhibition and self-fluorescence, both recognized as camptothecin properties. During flower development, the highest expression of Pru p 3 was detected in the styles of pollinated flowers, in contrast to its non-expression in unpollinated pistils, where expression decreased after anthesis. During ripening, the expression of Pru p 3 were observed mainly in peel but not in pulp. In this sense, Pru p 3 protein was also localized in trichomes covering the fruit epidermis.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Response to major peach and peanut allergens in a population of children allergic to peach

Peach allergy is one of the most common allergies in subjects with reactions to plant-foods. The relationship between peach allergy and other foods and the sensitization pattern in these patients have not been studied in detail. We study the sensitization to relevant allergens, such as peach and peanut LTP, Prup3 and Arah9, and seed storage protein, Arah2. Studies were carried out in child/adolescent population from 1-20 y.o allergic to peach, in order to evaluate the relationship between peach and peanut allergy.

LTP cross-reactivity - primary sensitization to mugwort pollen LTP Art v 3, facilitates subsequent sensitisation to peach LTP Pru p 3 in mice

Non-specific lipid transfer proteins (nsLTPs) are important pollen and food allergens in the Mediterranean area. Peach LTP Pru p 3 is considered as primary sensitizer inducing LTP cross-reactive IgE antibodies. Despite the evidence on the importance of Pru p 3, it is unclear whether reactivity to multiple LTPs is due to antibody cross-reactivity or to independent sensitization events. Aim of the study was (1) to compare antigenicity of LTPs from pollen and food, and (2) to investigate whether primary sensitization to LTPs promotes secondary sensitization to LTPs from unrelated sources. IgE sensitization to peach LTP was compared in different mouse strains (C3H/HeJ, BALB/c, CBA/J). IgE high responder mice (CBA/J) were used to compare the antigenicity and IgE cross-reactivity of LTPs from pollen (mugwort/Art v 3, plane tree/Pla a 3, ragweed/Amb a 6) and foods (peach/Pru p 3, cherry/Pru av 3, hazelnut/Cor a 8). To investigate whether an established Th2 immune response to LTPs facilitates a secondary immune response and cross-reactivity to unrelated LTPs, CBA/J mice were immunized with (1) Pru p 3 followed by Art v 3, (2) Art v 3 followed by Pru p 3, and (3) a mixture of Pru p 3 and Art v 3. Induction of Pru p 3 and Art v 3 specific IgE and IgG titers, and cross-reactivity between the two LTPs were determined by ELISA. CBA and C3H mice showed strong IgE-responses to Pru p 3, whereas BALB/c mice did not. Remarkably, a species-specific immune response lacking any IgE- and IgG-cross-reactivity was observed for all tested LTPs except for Pru p 3 and Pru av 3. Although LTPs show high structural similarity, Pru p 3 and Art v 3 displayed different antigenicity. In comparison to Art v 3, Pru p 3 showed much lower IgE-sensitizing capacity. In contrast, IgE response to Pru p 3 was promoted in mice previously sensitized to Art v 3, which was even the case upon concurrent administration of both LTPs. Neither sequential immunization nor immunization with the mixture induced IgE antibodies to unrelated LTPs, e.g. Cor a 8. In summary, the immune response to LTPs in mice varied strongly between strains. The LTPs studied, displayed different immunogenic properties and did not induce cross-reactive IgE antibodies. Primary sensitization to the pollen LTP Art v 3 conditioned the animals for subsequent sensitization to the food LTP Pru p 3. The extent to which the findings are applicable to the manifestation of clinical cross-reactivity to LTPs in humans needs to be investigated.

Transport of Pru p 3 across gastrointestinal epithelium – an essential step towards the induction of food allergy?

Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated. To study the interaction of Pru p 3 with intestinal epithelium, its natural entrance, analyzing transport kinetics and cellular responses that trigger. This was achieved using Pru p 3, the peach LTP, as a model. Enterocytic monolayers were established by culturing Caco 2 cells, as a model of enterocytes, on permeable supports that separate the apical and basal compartments. Pru p 3 was added to the apical compartment, the transepithelial resistance (TEER) was measured, and the transport was quantified. The peach allergen that crossed the cell monolayer was detected in the cell fraction and in the basal medium by immunodetection with specific antibodies and the quantity was measured by ELISA assay. Pru p 3 was able to cross the monolayer without disturbing the integrity of the tight junctions. This transport was significantly higher than that of a non-allergenic peach LTP, LTP1, and occurred via lipid raft pathway. The incubation of Caco 2 cells with Pru p 3 and LTP1 produced the expression of epithelial-specific cytokines TSLP, IL33 and IL25. These results suggest that Pru p 3 was able to cross the cell monolayer by the transcellular route and then induce the production of Th2 cytokines. The results of the present study represent a step towards clarifying the importance of Pru p 3 as a sensitizer. The capacity of food allergens to cross the intestinal monolayer could explain their high allergenic capacity and its fast diffusion through the body associating to severe symptoms.

Simulated gastrointestinal digestion of Pru ar 3 apricot allergen: Assessment of allergen resistance and characterization of the peptides by ultra‐performance liquid chromatography/electrospray ionisation mass spectrometry

Non-specific lipid transfer proteins (ns-LTPs) are major food allergens of the Rosaceae family. The severity of allergic reactions often relates to resistance of the allergen to digestion. Thus, it is important to evaluate the digestibility of these proteins and characterise the peptides generated in the gastrointestinal tract. Simulated gastrointestinal digestion of purified allergen Pru ar 3 was performed using pepsin for the gastric phase in aqueous HCl at pH = 2 and chymotrypsin and trypsin for the intestinal phase in aqueous NH(4)HCO(3) at pH = 7.8. The peptide mixture obtained was analysed by ultra-performance liquid chromatography/electrospray ionisation mass spectrometry (UPLC/ESI-MS). Peptide sequences were identified by comparing their molecular mass to that obtained by in silico digestion, and were confirmed by the ions obtained by in-source fragmentation. Semi-quantification was performed for the intact protein by comparison with internal standards. The resistance to gastrointestinal digestion of Pru ar 3 allergen was evaluated to be 9%. This value is consistent with that found for grape LTP, but much lower than the resistance found for peach LTP (35%). All the peptides generated were identified by ESI-MS on the basis of their molecular mass and from the ions generated from in-source fragmentation. Apart from low molecular mass peptides, five high molecular mass peptides (4500-7000 Da) containing disulphide bridges were identified. ESI-MS of the intact protein indicated a less compact folded structure when compared to that of the homologous peach LTP. An extensive characterisation of the peptides generated from the gastrointestinal digestion of Pru ar 3 allergen was performed here for the first time via UPLC/ESI-MS analysis. The digestibility of the allergen was evaluated and compared with that of other LTPs, demonstrating that only a small amount of undigested protein remains, and that specific proteolytic action involves immunodominant epitopes. These data might explain the lower allergenicity of apricot LTP compared to peach LTP, despite their high sequence homology.

Sensitization Prevalence, Antibody Cross-Reactivity and Immunogenic Peptide Profile of Api g 2, the Non-Specific Lipid Transfer Protein 1 of Celery

BackgroundCelery (Apium graveolens) represents a relevant allergen source that can elicit severe reactions in the adult population. To investigate the sensitization prevalence and cross-reactivity of Api g 2 from celery stalks in a Mediterranean population and in a mouse model.Methodology786 non-randomized subjects from Italy were screened for IgE reactivity to rApi g 2, rArt v 3 (mugwort pollen LTP) and nPru p 3 (peach LTP) using an allergen microarray. Clinical data of 32 selected patients with reactivity to LTP under investigation were evaluated. Specific IgE titers and cross-inhibitions were performed in ELISA and allergen microarray. Balb/c mice were immunized with purified LTPs; IgG titers were determined in ELISA and mediator release was examined using RBL-2H3 cells. Simulated endolysosomal digestion was performed using microsomes obtained from human DCs.ResultsIgE testing showed a sensitization prevalence of 25.6% to Api g 2, 18.6% to Art v 3, and 28.6% to Pru p 3 and frequent co-sensitization and correlating IgE-reactivity was observed. 10/32 patients suffering from LTP-related allergy reported symptoms upon consumption of celery stalks which mainly presented as OAS. Considerable IgE cross-reactivity was observed between Api g 2, Art v 3, and Pru p 3 with varying inhibition degrees of individual patients' sera. Simulating LTP mono-sensitization in a mouse model showed development of more congruent antibody specificities between Api g 2 and Art v 3. Notably, biologically relevant murine IgE cross-reactivity was restricted to the latter and diverse from Pru p 3 epitopes. Endolysosomal processing of LTP showed generation of similar clusters, which presumably represent T-cell peptides.ConclusionsApi g 2 represents a relevant celery stalk allergen in the LTP-sensitized population. The molecule displays common B cell epitopes and endolysosomal peptides that encompass T cell epitopes with pollen and plant-food derived LTP.

Hypersensitivity to fennel is frequent in peach allergic patients and LTP is a major fennel allergen

Methods within a population of patients with peach allergic symptoms of variable severity we investigated which patients also had documented allergy to fresh fennel (Clinical Trials.gov, protocol ID NCT00715156). We examined the type of allergic reaction to fennel by means of a clinical questionnaire, skin prick test, prickprick with fresh fennel, open challenge and IgE-specific levels to fennel and to the following recombinant allergens: anti-rPru p 1, 3, 4, anti-rBet v 1, 2 and 4. We compared the different clinical reactions to fennel between patients with mild symptoms to peach (group A, 21pts) and patients with severe systemic symptoms to peach (group B, 16 pts). SDS-Page and IgE immunoblotting were performed with fennel extract and the Nterminal sequences of the allergenic molecules were determined analyzing the proteins eluted by SDS-gel on a protein sequencer. Immunoblotting inhibition was performed to evaluate the cross-reactivity between fennel and peach extract.

IgE Reactivity To Food And Latex Components In Sera From Chestnut Allergic Individuals From The Mediterranean Area

RATIONALE: Knowledge concerning allergens associated with chestnut allergy is limited. Many studies have focused on allergens related to the latex-fruit syndrome as many chestnut allergic individuals also suffer from simultaneous allergy to latex. Studies regarding chestnut allergens not linked to latex allergy are less common. In the present study, IgE responses to allergens related to either latex- or food allergy in sera from chestnut allergic individuals from the Mediterranean area were studied. METHODS: Specific IgE to 12 allergenic components were analysed in sera from 13 individuals diagnosed with chestnut allergy. Inhibition analysis was performed with peach LTP (Pru p 3) and chestnut extract. RESULTS: Specific IgE responses to Pru p 3, Hev b 5, Hev b 6 and Hev b 11 were detected. Six individuals were found to have IgE to Pru p 3, but only one had concomitant allergy to latex. High level inhibition (83-98%) of the chestnut IgE response was obtained with Pru p 3. Nine individuals were found to have IgE to latex components and, out of these, seven had a concomitant allergy to latex. Eight individuals had IgE responses to Hev b 6, related to the chitinase of chestnut. Inhibition of the latex IgE responses was obtained with chestnut extract. CONCLUSIONS: Chestnut allergic individuals from the Mediterranean area appear to have two main patterns of IgE responses. Individuals diagnosed with only chestnut allergy, mainly had responses to LTP while individuals with concomitant latex allergy had reponses to Hev b 6, probably indicating a response directed to chestnut specific chitinase.

Uriticaria and angioderma after ingestion of grapes

Case report We report the case of a 28-year-old female who developed acute generalized urticaria and facial angioedema one hour after ingesting grapes of several varieties. The reaction was treated at Emergency Room level with parenteral administration of corticoids and anti-histamines. She previously ingested grapes and other fresh fruits with no reaction and denied rhinitis complaints. Skin prick tests with a large battery of aeroallergens, including latex, were positive to peach LTP, peach, apple, and plum but were negative with grape commercial extract. Prick by prick procedure performed with the pulp and peel of a variety of red and white grapes yielded positive results, as well as with fresh cherry. Specific IgE (kUA/l; ImmunoCAP, Phadia) were present for peach (1.16), peach LTP (1.79), apple (1.17), plum (1.36) and cherry (0.8) and were negative for grape.

Specific sublingual immunotherapy with peach LTP (Pru p 3). One year treatment: a case report

AbstractIntroductionFood allergy is an increasing problem with limited therapeutic approaches apart from to the eviction diet.Case presentationA 40-year-old female patient with food allergy symptoms was polysensitized to almost all vegetable food since the age of 36; the onset of symptoms was during pregnancy. The allergological study demonstrated positive skin prick tests (SPT) to nuts, legumes, cereals, spices, several fresh fruits including peach, and other groups of vegetable foods however, it was negative to common aeroallergens. Serum specific IgE levels were negative (&lt;0.35 kU/L) to profilin and carbohydrate determinants, but positive to Pru p 3 (3.5 kU/L). Positive double-blind placebo-controlled food challenge to peach confirmed the allergic disease. She received specific sublingual immunotherapy with native Pru p 3 at a concentration of 40 μg/ml with 5 administrations per week and a cumulative dose of 200 μg of nPru p 3 per month. After an ultra-rush build-up phase concluded in one day she continued therapy during a year with 5 administrations per week. The clinical evolution and laboratory studies demonstrated an early reduction on SPT reactions with no relevant changes on serum specific IgE, IgG, IgG1and IgG4to Pru p 3 during the immunotherapy period. The challenge test was negative 4 months after the beginning of the SLIT. Regarding clinical response she markedly improved after the first month of treatment, and by the 3th month she had no major vegetable dietary restrictions, except for nuts and pepper.ConclusionThese results demonstrate the excellent efficacy and safety of sublingual specific protein immunotherapy developed according to the patient specific sensitivity profile to Pru p3.

Specific sublingual immunotherapy with peach LTP (Pru p 3). One year treatment: a case report.

IntroductionFood allergy is an increasing problem with limited therapeutic approaches apart from to the eviction diet.Case presentationA 40-year-old female patient with food allergy symptoms was polysensitized to almost all vegetable food since the age of 36; the onset of symptoms was during pregnancy. The allergological study demonstrated positive skin prick tests (SPT) to nuts, legumes, cereals, spices, several fresh fruits including peach, and other groups of vegetable foods however, it was negative to common aeroallergens. Serum specific IgE levels were negative (<0.35 kU/L) to profilin and carbohydrate determinants, but positive to Pru p 3 (3.5 kU/L). Positive double-blind placebo-controlled food challenge to peach confirmed the allergic disease. She received specific sublingual immunotherapy with native Pru p 3 at a concentration of 40 μg/ml with 5 administrations per week and a cumulative dose of 200 μg of nPru p 3 per month. After an ultra-rush build-up phase concluded in one day she continued therapy during a year with 5 administrations per week. The clinical evolution and laboratory studies demonstrated an early reduction on SPT reactions with no relevant changes on serum specific IgE, IgG, IgG1 and IgG4 to Pru p 3 during the immunotherapy period. The challenge test was negative 4 months after the beginning of the SLIT. Regarding clinical response she markedly improved after the first month of treatment, and by the 3th month she had no major vegetable dietary restrictions, except for nuts and pepper.ConclusionThese results demonstrate the excellent efficacy and safety of sublingual specific protein immunotherapy developed according to the patient specific sensitivity profile to Pru p3.

Identification of a plane pollen lipid transfer protein (Pla a 3) and its immunological relation to the peach lipid‐transfer protein, Pru p 3

An association between plane tree pollen allergy and plant food allergy has been described, but the cross-reacting allergens have not yet been identified. The aim of this study was the identification of homologous non-specific lipid-transfer proteins (nsLTPs) in plane pollen, and to investigate its immunological relationship with the peach LTP, Pru p 3. Three different patient groups were recruited in Spain: 22 plane pollen-allergic patients without food allergy (A), 36 plane pollen-allergic patients with peach allergy (B) and 10 peach-allergic patients without plane pollen allergy (C). Proteins from plane pollen extract were fractionated by ion-exchange and reversed-phase chromatography. Further methods applied were N-terminal amino acid sequence analysis, immunoblotting, enzyme allergosorbent test, CAP and basophil histamine release assays. A 10 kDa IgE-reactive protein was purified from plane pollen and identified as nsLTP. Pla a 3 was characterized as a minor allergen (27.3%) in plane pollen-allergic patients without food allergy (A) and as a major allergen in plane pollen-allergic patients with peach allergy (B) showing a prevalence of IgE-reactivity of 63.8%. Group B contained patients sensitized to Pru p 3 without IgE-reactivity to plane-LTP (16.6%). By contrast, Pla a 3 IgE-reactive patients without sensitization to Pru p 3 could be found (16.6%). The sera of patients sensitized to both LTPs (50%), Pla a 3 and Pru p 3, showed different biological activity in histamine release assay: depending on individual patient's sera tested, Pla a 3 showed a similar, a stronger or a weaker allergenic potency in comparison with Pru p 3. Plane LTP is a major allergen in plane pollen-allergic patients with peach allergy recruited in the Mediterranean area. The results of histamine release tests and different IgE-binding profiles pointed towards the existence of species-specific IgE epitopes. Likewise, no general conclusion on the sensitizer could be made.

The role of profilin and lipid transfer protein in strawberry allergy in the Mediterranean area

In contrast to other Rosaceae fruit, only few cases of patients with adverse reactions to strawberry are listed in literature. OBJECTIVE To identify allergenic proteins in strawberry and to express and characterize recombinant strawberry lipid transfer protein (LTP; rFra a 3). Established apple-allergic patients were recruited on the basis of a reported allergic reaction to strawberry (n=28, confirmed by double-blind placebo-controlled food challenge in four patients) or on the basis of IgE reactivity to LTP (n=34). Sensitization to purified natural and recombinant allergens was assessed by RAST, immunoblot (inhibition) and basophil histamine release (BHR). A strawberry cDNA library was screened for genes homologous to known fruit allergens. Fra a 3 was cloned and expressed in the yeast Pichia pastoris and compared with peach and apple LTP by RAST, immunoblot-inhibition and BHR tests. Genes homologous to Bet v 1, Bet v 6, profilin and LTP were identified in a strawberry cDNA library. In BHR the rFra a 3 induced histamine release at a 100-fold higher concentration than peach LTP. RAST inhibition showed high cross-reactivity to peach and apple LTP, although IgE reactivity was lower by a factor 5. On strawberry immunoblot, patients' IgE showed reactivity to a Bet v 1 homologue, profilin, LTP and high-molecular weight bands. In addition to a Bet v 1 homologue, strawberry also contains IgE-binding profilin and LTP. The rFra a 3 has less allergenic potency than peach and apple LTP, and therefore is an interesting tool for future immunotherapy. Fra a 3 does not seem to be clinically relevant.

Determination of the primary structure of two lipid transfer proteins from apricot ( Prunus armeniaca)

It has been recently demonstrated that the major allergen of apricot is a protein of molecular mass ( M r) 9000 belonging to the family of Lipid Transfer Protein. The aim of this study was the determination of the primary structure of apricot LTP by micro-sequencing and mass spectrometric analyses. Apricot LTP is a 91 amino acids protein like peach and almond LTPs with a sequence identity of 91% and 94%, respectively. Like for the peach LTP, out of the 25 amino acids forming the inner surface of the tunnel-like hydrophobic cavity in maize ns-LTP, 16 are identical and 7 similar in the apricot LTP, supporting the hypothesis of a similar function.