Impact of glutathione on the allergenicity of the peach lipid transfer protein Pru p 3.

BACKGROUND AND OBJECTIVE: The allergenic potential of proteins can be altered under various physicochemical conditions. Glutathione (GSH) is a reducing agent that is used as an antioxidant in food products. We aimed to characterize the natural folding of peach proteins and test the allergenicity of reduced and natural Pru p 3, the major peach allergen. METHODS: Pru p 3 was purified from peach, and its conformation was analyzed by means of circular dichroism. Using a thiol fluorescent probe, reduced proteins were detected in fresh peach. GSH-reduced Pru p 3 was tested in vitro for T-cell proliferation and in vivo using skin prick testing. RESULTS: GSH-reduced Pru p 3 produced variable skin prick reactions in peach-allergic patients. The proliferative response of peripheral blood mononuclear cells from allergic patients to reduced Pru p 3 tended to be less intense, whereas secretion of the cytokines IFN-γ, IL-5, and IL-10 was comparable. In a pool of sera from peach-allergic patients, reduction hardly impaired IgE-binding. Moreover, the stability of reduced Pru p 3 to gastrointestinal digestion was similar to that of the natural form. CONCLUSIONS: GSH can at least transiently reduce Pru p 3. We found that the effect of reduction on the allergenicity of Pru p 3 varied. Therefore, as an additive, GSH does not seem to eliminate the risk of reactions for peach-allergic patients.

Value of microarray allergen assay in the management of eosinophilic oesophagitis.

BACKGROUND: Eosinophilic oesophagitis (EoE) is a disorder characterised by oesophageal dysfunction and, histologically, by eosinophilic inflammation. Although treatment, which includes dilatations, oral corticosteroids and restrictive diets, is often effective, choosing the foods to be eliminated from the diet is difficult. OBJECTIVE: Component resolved diagnostic by microarray allergen assay may be useful in detecting allergens that might be involved in the inflammatory process. METHODS: We studied 67 patients with EoE, diagnosed clinically and histologically by endoscopic biopsy. CRD analysis with microarray technology was carried out in the 67 EoE patients, 50 patients with pollen allergy without digestive symptoms, and 50 healthy controls. RESULTS: Allergies were not detected by microarray in only seven of the 67 patients with EoE. Controls with pollen allergy showed sensitisation to different groups of pollen proteins without significant differences. In EoE patients with response to some allergens, the predominant allergens were grasses group 1 and, in particular, nCyn d 1 (Cynodon dactylon) or Bermuda grass pollen in 59.5%, followed by lipid transfer proteins (LTP) of peach (19.40%), hazelnut (17.91%) and Artemisia (19.40%). CONCLUSIONS: In patients with EoE, sensitisation to plant foods and pollen is important. The proteins most frequently involved are nCyn d 1 and lipid transfer proteins, hazelnuts and walnuts. After one year of an array-guided exclusion diet and pollen-specific immunotherapy in the case of high levels of response, patients with EoE showed preliminary significant improvements.

Plum pox virus and sharka: a model potyvirus and a major disease.

TAXONOMIC RELATIONSHIPS: Plum pox virus (PPV) is a member of the genus Potyvirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. GEOGRAPHICAL DISTRIBUTION: First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. GENOMIC STRUCTURE: Typical of potyviruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5' end and a 3'-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. PATHOGENICITY FEATURES: PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the virus locally in a nonpersistent manner. SOURCES OF RESISTANCE: A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic plum, 'HoneySweet', transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.

Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

Multiyear evaluation of the durability of the resistance conferred by Ma and RMia genes to Meloidogyne incognita in Prunus under controlled conditions.

Root-knot nematodes (RKNs) (Meloidogyne spp.) are highly polyphagous pests that parasitize Prunus crops in Mediterranean climates. Breeding for RKN-resistant Prunus cultivars, as an alternative to the now-banned use of nematicides, is a real challenge, because the perennial nature of these trees increases the risk of resistance breakdown. The Ma plum resistance (R) gene, with a complete spectrum, and the RMia peach R gene, with a more restricted spectrum, both provide total control of Meloidogyne incognita, the model parthenogenetic species of the genus and the most important RKN in terms of economic losses. We investigated the durability of the resistance to this nematode conferred by these genes, comparing the results obtained with those for the tomato Mi-1 reference gene. In multiyear experiments, we applied a high and continuous nematode inoculum pressure by cultivating nematode-infested susceptible tomato plants with either Prunus accessions carrying Ma or RMia R genes, or with resistant tomato plants carrying the Mi-1 gene. Suitable conditions for Prunus development were achieved by carrying out the studies in a glasshouse, in controlled conditions allowing a short winter leaf fall and dormancy. We first assessed the plum accession 'P.2175', which is heterozygous for the Ma gene, in two successive 2-year evaluations, for resistance to two M. incognita isolates. Whatever the isolate used, no nematodes reproducing on P.2175 were detected, whereas galls and nematodes reproducing on tomato plants carrying Mi-1 were observed. In a second experiment with the most aggressive isolate, interspecific full-sib material (P.2175 × ['Garfi' almond × 'Nemared' peach]), carrying either Ma or RMia (from Nemared) or both (in the heterozygous state) or neither of these genes, was evaluated for 4 years. No virulent nematodes developed on Prunus spp. carrying R genes, whereas galling and virulent individuals were observed on Mi-1-resistant tomato plants. Thus, the resistance to M. incognita conferred by Ma in Prunus material in both a pure-plum and an interspecific genetic background, or by RMia in an interspecific background, appears to be durable, highlighting the value of these two genes for the creation of Prunus rootstock material.

Peamaclein--a new peach allergenic protein: similarities, differences and misleading features compared to Pru p 3.

BACKGROUND: Among the peach-derived allergens which are already known, the lipid transfer protein (Pru p 3) seems to be the one to exert severe allergic reactions. OBJECTIVE: To identify and characterize a new peach allergen causing a clinical picture similar to that of Pru p 3. METHODS: Patients were selected on the basis of their severe clinical reactivity and negative results to a panel of peach allergens available on the ISAC103 microarray. Several in-house and commercial preparations were compared. Several methods were used to characterize the newly identified molecule. Specific IgE and inhibition assays were performed using the Allergen micro-Beads Array (ABA) assay. RESULTS: Negative ISAC results to Pru p 3 were confirmed by additional testing in contrast with the positive results obtained by commercial Pru p 3-enriched peach peel extracts. The analyses of one of these preparations led to the identification of Peamaclein, a new allergenic protein. It is a small, basic, cysteine-rich, heat-stable, digestion-resistant protein, homologous to a potato antimicrobial peptide. Peamaclein was able to trigger positive skin test reactions and to bind IgE in the ABA assay. It displays an electrophoretic mobility and chromatographic behaviour similar to that of Pru p 3; therefore, it can be hidden in Pru p 3 preparations. In fact, Pru p 3-enriched peach peel extracts were found to contain both Pru p 3 and Peamaclein by means of comparative in vivo testing, and by biochemical and immunochemical assays. Commercially available anti-Pru p 3 polyclonal antibodies were found to have a double specificity for the two molecules. CONCLUSIONS AND CLINICAL RELEVANCE: A new allergen from peach belonging to a new family of allergenic proteins has been identified and characterized. This knowledge on Peamaclein will improve our understanding on the clinical aspects of the peach allergy and the quality of diagnostic reagents.

Evaluation of the impact of sequential microwave/ultrasound processing on the IgE binding properties of Pru p 3 in treated peach juice.

Peach lipid transfer protein (LTP) can cause severe allergic reactions to peach-allergic patients. It belongs to the nonspecific LTPs family, a class of proteins extremely resistant both to proteolytic digestion and to high temperatures. Food processing can either drop or increase the allergenicity, depending on the process and on the food. As far as peach-derived products (pulp, juice) are concerned, it has been previously shown how thermal treatment performed in an autoclave does not decrease LTP allergenicity. In this work, it was attempted to investigate whether sequential microwave and ultrasound processing could affect the allergenicity of peach juice. Incubation with specific anti-Pru p 3 serum showed how treating peach peel with microwave at 140 °C and with ultrasound does not eliminate Pru p 3 IgE binding properties. The application of MW/US protocol on peach pulp appeared to be insufficient for the reduction of IgE binding to Pru p 3.

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.

Biochemical and spectroscopic characterization of almond and cashew nut seed 11S legumins, amandin and anacardein.

Native, undenatured amandin and anacardein secondary structures were estimated to be, respectively, 56.4 and 49% ß-sheet, 14 and 23.7% α-helix, and 29.6 and 27.4% random coil. Circular dichroic (CD) and fluorescence spectroscopy were used to assess structural changes in amandin and anacardein subjected to denaturing treatments that included heat (100 °C, 5 min), guanidium HCl (GuHCl), urea, sodium dodecyl sulfate (SDS), and reducing agent, 2% v/v ß-mercaptoethanol (ßME) + heat. Mouse monoclonal antibodies (mAbs) 4C10 and 4F10 directed against amandin and 1F5 and 4C3 directed against anacardein were used to assess the influence of denaturing treatments on the immunoreactivity of amandin and anacardein. Among the denaturing treatments investigated, SDS and ß-ME caused a significant reduction in the immunoreactivity of amandin and anacardein when probed with mAb 4C10 and 4C3, respectively.

Immune-enhancing effect of fermented Maesil (Prunus mume Siebold & Zucc.) with probiotics against Bordetella bronchiseptica in mice.

Maesil (Prunus mume) has long been used as a traditional drug and healthy food in East Asian countries. It possesses a number of beneficial biological activities including potential antimicrobial effects against pathogens. Probiotics also have antibacterial effects. Moreover, some probiotics have an important role in regulating the immune system. The present study evaluated the immune enhancing effects of fermented Maesil with probiotics (Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus acidophilus) in mice, especially against Bordetella bronchiseptica, as an initial step towards the development of feed supplements for the promotion of immune activity and prevention of disease, especially in pigs. Continuous ingestion of fermented Maesil with probiotics markedly increased the macrophage ratio in peripheral blood and the T lymphocyte ratio in the spleen. In addition, antibody production against formalin-killed B. bronchiseptica significantly increased in the mice fed fermented Maesil compared with the control group. The number of leukocytes was significantly higher in the bronchio-alveolar lavage obtained from the fermented Maesil-fed animals compared to it in the control group at day 3 (maximal peak time) after experimental B. bronchiseptica infection. Moreover, at 7 day post-infection, relative messenger RNA expression levels of tumor necrosis factor- α and interferon-γ were significantly increased in splenocytes of mice fed fermented Maesil compared with those in the control group. Taken together, these findings suggest that feed containing fermented Maesil with probiotics enhances immune activity in mice, especially against B. bronchiseptica, via the potent stimulation of non-specific immune responses.

Immunomodulatory and antiviral activity of almond skins.

The elimination of a viral infection requires a proinflammatory host response (type 1 immunity), characterized by activation of mononuclear cells and production of proinflammatory cytokines, such as interferons (IFNs), tumor necrosis factor (TNF)-alpha and interleukin (IL)-12. On the other hand, IL-4 and IL-10 play a role in decreasing the inflammatory response supported by helper T (Th)1 cells. In this study we evaluated the effects of almond skins on the release of cytokines by peripheral blood mononuclear cells (PBMC), either infected or not with herpes simplex virus type 2 (HSV-2). Natural (NS) and blanched almond skins (BS) were subjected to simulated gastric and duodenal digestion and used at not cytotoxic concentrations. NS induced a significant decrease in HSV-2 replication, whereas extracts obtained from BS did not significantly influence the viral replication. High levels of cytokines production, such as IFN-alpha (38+/-5.3 pg/ml), IL-12 (215+/-17.1 pg/ml), IFN-gamma (5+/-0.7 IU/ml), TNF-alpha (3940+/-201.0 pg/ml), were detected. Moreover, IL-10 (210+/-12.2 pg/ml) and IL-4 (170+/-21.4 pg/ml), representative of Th2 responses, were found. Our data suggest that almond skins improve the immune surveillance of PBMC towards viral infection, both by triggering the Th1 and Th2 subsets.

Crystal structure of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

Seed storage proteins are accumulated during seed development and act as a reserve of nutrition for seed germination and young sprout growth. Plant seeds play an important role in human nutrition by providing a relatively inexpensive source of protein. However, many plant foods contain allergenic proteins, and the number of people suffering from food allergies has increased rapidly in recent years. The 11S globulins are the most widespread seed storage proteins, present in monocotyledonous and dicotyledonous seeds as well as in gymnosperms (conifers) and other spermatophytes. This family of proteins accounts for a number of known major food allergens. They are of interest to both the public and industry due to food safety concerns. Because of the interests in the structural basis of the allergenicity of food allergens, we sought to determine the crystal structure of Pru1, the major component of the 11 S storage protein from almonds. The structure was refined to 2.4 A, and the R/Rfree for the final refined structure is 17.2/22.9. Pru1 is a hexamer made of two trimers. Most of the back-to-back trimer-trimer association was contributed by monomer-monomer interactions. An alpha helix (helix 6) at the C-terminal end of the acidic domain of one of the interacting monomers lies at the cleft of the two protomers. The residues in this helix correspond to a flexible region in the peanut allergen Ara h 3 that encompasses a previously defined linear IgE epitope.

Structural stability of Amandin, a major allergen from almond (Prunus dulcis), and its acidic and basic polypeptides.

Information relating to the resistance of food allergens to thermal and/or chemical denaturation is critical if a reduction in protein allergenicity is to be achieved through food-processing means. This study examined the changes in the secondary structure of an almond allergen, amandin, and its acidic and basic polypeptides as a result of thermal and chemical denaturation. Amandin ( approximately 370 kDa) was purified by cryoprecipitation followed by gel filtration chromatography and subjected to thermal (13-96 degrees C) and chemical (urea and dithiothreitol) treatments. Changes in the secondary structure of the protein were followed using circular dichroism spectroscopy. The secondary structure of the hexameric amandin did not undergo remarkable changes at temperatures up to 90 degrees C, although protein aggregation was observed. In the presence of a reducing agent, irreversible denaturation occurred with the following experimental values: T(m) = 72.53 degrees C (transition temperature), DeltaH = 87.40 kcal/mol (unfolding enthalpy), and C(p) = 2.48 kcal/(mol degrees C) (heat capacity). The concentration of urea needed to achieve 50% denaturation was 2.59 M, and the Gibbs free energy of chemical denaturation was calculated to be DeltaG = 3.82 kcal/mol. The basic and acidic polypeptides of amandin had lower thermal stabilities than the multimeric protein.

Peach ( Prunus persica L. Batsch) allergen-encoding genes are developmentally regulated and affected by fruit load and light radiation.

The fruits of Rosaceae species may frequently induce allergic reactions in both adults and children, especially in the Mediterranean area. In peach, true allergens and cross-reactive proteins may cause hypersensitive reactions involving a wide diversity of symptoms. Three known classes of allergenic proteins, namely, Pru p 1, Pru p 3, and Pru p 4, have been reported to be mostly involved, but an exhaustive survey of the proteins determining the overall allergenic potential, their biological functions, and the factors affecting the expression of the related genes is still missing. In the present study, the expression profiles of some selected genes encoding peach allergen isoforms were studied during fruit growth and development and upon different fruit load and light radiation regimens. The results indicate that the majority of allergen-encoding genes are expressed at their maximum during the ripening stage, therefore representing a potential risk for peach consumers. Nevertheless, enhancing the light radiation and decreasing the fruit load achieved a reduction of the transcription rate of most genes and a possible decrease of the overall allergenic potential at harvest. According to these data, new growing practices could be set up to obtain hypoallergenic peach fruits and eventually combined with the cultivation of hypoallergenic genotypes to obtain a significant reduction of the allergenic potential.

Immunostimulatory effects of oriental plum (Prunus salicina Lindl.).

The fruit of the plum tree (Prunus salicina Lindl.) has been used as a traditional medicinal food in humans to enhance immunity against infectious agents and to treat cancers. However, limited information exists on the mechanisms responsible for its immune enhancing properties. In this study, the immunostimulatory effects of a methanol extract of plum fruit following methanol evaporation and dissolving in PBS were assessed by in vitro lymphocyte proliferation, tumor cell cytotoxicity, and nitric oxide (NO) production. The crude methanol extract stimulated spleen lymphocyte proliferation and NO production by cultured macrophages, and inhibited the viability of tumor cells, significantly greater than media controls. Sequential gel filtration chromatographic separation of the extract on Sephadex G-25 and Sephacryl S-200 gel filtration columns resulted in a more purified preparation that retained the ability to induce lymphoproliferation, tumor killing, and NO production. These results suggest that Prunus salicina contains immunostimulatory components that potentially may be useful in human and veterinary medicine.

T-cell epitopes of the major peach allergen, Pru p 3: Identification and differential T-cell response of peach-allergic and non-allergic subjects.

Lipid transfer proteins (LTPs), particularly peach Pru p 3, are the most relevant plant food allergens in the South of Europe, and, therefore, their allergic properties have been extensively studied. However, neither T-cell epitopes nor their effect on the patients' T-cell response has been investigated in any member of the LTP panallergen family. The objective of the present study was to map the major T-cell epitopes of Pru p 3, as well as to evaluate their induced T-cell response in peach-allergic versus control subjects. Thus, peripheral blood mononuclear cells (PBMCs) from 18 peach-allergic patients and Pru p 3-specific T-cell lines (TCLs) from 9 of them were cultured with Pru p 3 and with a panel of 17 derived peptides (10-mer overlapping in 5 amino acids representing the full sequence of Pru p 3). Proliferation in 5-day assays was carried out via tritiated-thymidine incorporation, while IL4 and IFNgamma production was assessed via sandwich enzyme-linked immunosorbent tests (ELISA) of TCL culture supernatants. The results were compared to those obtained from 10 non-peach allergic control volunteers. Two consecutive peptides showed the highest activation capacity. About 74% of PBMCs and TCLs recognized them, forming a single T-epitope: Pru p 3(65-80). Additionally, other specific T-cell epitopes were observed. Pru p 3(25-35) was detected by more than 60% of TCLs from peach-allergic patients, and Pru p 3(45-55) only activated PBMCs from control subjects. Interestingly, TCLs from patients were associated with a Th2-type, whereas control TCLs presented a Th1-type cytokine response. The major immunogenic T-cell epitope identified in Pru p 3, Pru p 3(65-80), is a good candidate to develop new vaccines for hypersensitivity reactions associated with LTP allergens from Rosaceae fruits.

Purification, crystallization and preliminary X-ray characterization of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin.

The 11S globulins from plant seeds account for a number of major food allergens. Because of the interest in the structural basis underlying the allergenicity of food allergens, we sought to crystallize the main 11S seed storage protein from almond ( Prunus dulcis). Prunin-1 (Pru1) was purified from defatted almond flour by water extraction, cryoprecipitation, followed by sequential anion exchange, hydrophobic interaction, and size exclusion chromatography. Single crystals of Pru1 were obtained in a screening with a crystal screen kit, using the hanging-drop vapor diffusion method. Diffraction quality crystals were grown after optimization. The Pru1 crystals diffracted to at least 3.0 A and belong to the tetragonal space group P4(1)22, with unit cell parameters of a = b = 150.912 A, c = 165.248 A. Self-rotation functions and molecular replacement calculations showed that there are three molecules in the asymmetry unit with water content of 51.41%. The three Pru1 protomers are related by a noncrystallographic 3-fold axis and they form a doughnut-shaped trimer. Two prunin trimers form a homohexamer. Elucidation of prunin structure will allow further characterization of the allergenic features of the 11S protein allergens at the molecular level.

Two different profiles of peach allergy in the north of Spain.

BACKGROUND: Peach allergy has two different patterns: central Europe with oral allergy syndrome (OAS) related to a primary sensitization to birch pollen Bet v 1 and profilins and southern Europe with mostly systemic symptoms, in many cases due to sensitization to lipid-transfer proteins. METHODS: Thirty peach-allergic patients with positive skin and food challenge tests and 29 control subjects were included. Skin prick tests (SPT) with inhalant allergens, commercial peach and apple extracts and native Pru p 3 were performed. In vitro specific immunoglobulin (Ig) E to grass pollen, birch pollen, peach, apple, rBet v 1, rBet v 2 and rPhl p 12 was determined by CAP, and rBet v 1, rMal d 1, rMal d 4, rMal d 3 and rPru p 3 using the ADVIA-Centaur platform. Basophil activation test (BAT) with commercial peach extract, commercial apple extract, nPru p 3, rMal d 3, rMal d 1 and rMal d 4 was also performed. RESULTS: Pru p 3 was the major allergen in the patient group from northern Spain. Sensitization to this allergen was found in 100% of the patients with systemic symptoms or contact urticaria. Only 60% of OAS patients were sensitized to Pru p 3, being all of them sensitized to profilins and 60% of them to allergens of the Bet v 1 family. Specific IgE determination and BAT using recombinant allergens (rPru p 3) show specificity and sensitivity values close to 100%. CONCLUSIONS: Most peach-allergic patients coming from the north of Spain present systemic symptoms after ingestion of peach, Pru p 3 being the main allergen. Patients with OAS present profilin-Bet v 1-related sensitization. Thus, in the north of Spain our patients show a mixed central-south Europe pattern with LTP-profilin-Bet v 1 sensitization depending on the symptoms presented. The use of natural and recombinant plant allergens, allows establishing the sensitization patterns to the different allergens studied.

IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens.

Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals.

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.