Headspace control and antimicrobials: Inhibition strategies to prevent the growth of Alicyclobacillus acidoterrestris in orange juice.

Alicyclobacillus acidoterrestris can cause spoilage in orange juice that leads to consumer rejection. Six different orange juices were physiochemically characterized (pH, total soluble solids, titratable acidity, total polyphenols and vitamin C). A bottle for each sampling point per juice was filled (headspace: 40% volume) and inoculated with 102 -103 CFU per ml of A. acidoterrestris ATCC® 49025™ (heat shocked before inoculation: 75°C, 20 min). Samples were stored for 21 days at 45 ± 1°C and plate counted periodically on acidified YSG agar (pH 3·7) incubated at 45 ± 1°C for 3 days. The effect of headspace (6% versus 40% volume) on A. acidoterrestris growth was also evaluated. The effect of nisin (0·006, 0·003, 0·0015, and 0·00075%), sodium benzoate (0·1%), potassium sorbate (0·1%) and a mix of benzoate and sorbate (0·05% each) on A. acidoterrestris was additionally addressed. Alicyclobacillus acidoterrestris reached up to 107 CFU per ml in five of the six juices in less than 1 week. Headspace significantly impacted (P < 0·05) A. acidoterrestris maximum population, which reached the critical value of 5 log CFU per ml at 40% headspace. All preservatives, regardless of concentration, showed a bacteriostatic effect during 22 days of storage with no significant differences amongst treatments (P > 0·05).

Oral Immunotherapy With Egg and Milk: Changes in Peripheral Serum Cytokines Are Not Predictive Factors for Severe Adverse Reactions or for the Final Report.

BACKGROUND: Oral immunotherapy (OIT) is a new approach in patients with food allergy. Various immunological mechanisms underlie the reversal of food allergy. In this paper, we study possible changes in peripheral cytokine patterns during OIT. METHODS: Determinations of cytokines in peripheral blood were made in children who had milk or egg allergy and who received OIT. The determinations were made before and after OIT, and again following a final repeat oral challenge a month after a diet excluding the culprit food. RESULTS: No significant changes were registered in the cytokines studied (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFNγ, and TNF) at any of the 3 time points. Similarly, no differences in cytokine pattern were observed between children who had presented anaphylaxis during OIT and those who overcame or did not overcome the final oral challenge. DISCUSSION: Peripheral cytokines do not undergo significant changes during the OIT process. They are not predictors of serious adverse reactions or the final result of the OIT.

Is the Quantification of Antigen-Specific Basophil Activation a Useful Tool for Monitoring Oral Tolerance Induction in Children With Egg Allergy?

OBJECTIVES: To assess modifications in baseline specific IgE- and anti-IgE- and antigen-specific-mediated basophil activation in egg-allergic children. The values were compared before and after the children completed specific oral tolerance induction (SOTI) with egg. PATIENTS AND METHODS: We studied 28 egg-allergic children who completed SOTI with egg. The basophil activation test and specific IgE determinations with egg white, ovalbumin, and ovomucoid were performed in all 28 children. RESULTS: A decrease in antigen-specific activation with egg white, ovalbumin, and ovomucoid was observed only at the 2 lowest concentrations used (5 and 0.05 ng/mL). Baseline activation was higher in patients with multiple food allergies and in those who developed anaphylaxis during SOTI; this activation decreased in both groups after completion of SOTI. A significant decrease was also observed in specific IgE values for egg white, ovalbumin, and ovomucoid after tolerance induction. CONCLUSIONS: Food tolerance induction is a specific process for each food that can be mediated by immunologic changes such as a decrease in specific IgE values and in specific and spontaneous basophil activation.

Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

BACKGROUND: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. OBJECTIVE: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. METHODS: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cora 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. RESULTS: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP-sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P < .05). Similar rates of Cora 8 and Jug r 3 sensitization were detected by both techniques. CONCLUSIONS: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.

Is the ISAC 112 Microarray Useful in the Diagnosis of Pollinosis in Spain?

BACKGROUND: Multiple sensitization is frequent among pollen-allergic patients. The goal of this study was to determine the diagnostic accuracy of the ImmunoCAP ISAC 112 (ISAC112) microarray in allergy to pollen from several taxa and its clinical utility in a Spanish population. METHODS: Specific IgE was determined in 390 pollen-allergic patients using the ISAC112 microarray. Diagnostic accuracy (sensitivity, specificity, predictive values, and area under the ROC curve) was calculated for the diagnosis of allergy to pollen from grass (n=49), cypress (n=75), olive tree (n=33), plane tree (n=63), and pellitory of the wall (n=17) and compared with that of the singleplex ImmunoCAP immunoassay. RESULTS: The sensitivity of the ISAC112 microarray ranged from 68.2% for allergy to plane tree pollen to 93.9% for allergy to grass pollen. The specificity was >90%. The AUC for the diagnosis of allergy to plane tree pollen was 0.798, whereas the AUC for the remaining cases was ≥0.876. The accuracy of ISAC112 was higher than that of ImmunoCAP for plane tree pollen and similar for the remaining pollens. The frequency of sensitization to most species-specific allergenic components and profilins varied between the different geographical regions studied. A total of 73% of pollen-allergic patients were sensitized to species-specific components of more than 1 pollen type. CONCLUSIONS: The ISAC112 microarray is an accurate tool for the diagnosis of allergy to pollen from grass, cypress, olive tree, plane tree, and pellitory of the wall. The features of the ISAC112 microarray are similar or superior (in the case of plane tree pollen) to those of ImmunoCAP. This microarray is particularly useful for the etiologic diagnosis of pollinosis in patients sensitized to multiple pollen species whose pollination periods overlap.

Fungal Planet description sheets: 400-468.

Novel species of fungi described in the present study include the following from Australia: Vermiculariopsiella eucalypti, Mulderomyces natalis (incl. Mulderomyces gen. nov.), Fusicladium paraamoenum, Neotrimmatostroma paraexcentricum, and Pseudophloeospora eucalyptorum on leaves of Eucalyptus spp., Anungitea grevilleae (on leaves of Grevillea sp.), Pyrenochaeta acaciae (on leaves of Acacia sp.), and Brunneocarpos banksiae (incl. Brunneocarpos gen. nov.) on cones of Banksia attenuata. Novel foliicolous taxa from South Africa include Neosulcatispora strelitziae (on Strelitzia nicolai), Colletotrichum ledebouriae (on Ledebouria floridunda), Cylindrosympodioides brabejum (incl. Cylindrosympodioides gen. nov.) on Brabejum stellatifolium, Sclerostagonospora ericae (on Erica sp.), Setophoma cyperi (on Cyperus sphaerocephala), and Phaeosphaeria breonadiae (on Breonadia microcephala). Novelties described from Robben Island (South Africa) include Wojnowiciella cissampeli and Diaporthe cissampeli (both on Cissampelos capensis), Phaeotheca salicorniae (on Salicornia meyeriana), Paracylindrocarpon aloicola (incl. Paracylindrocarpon gen. nov.) on Aloe sp., and Libertasomyces myopori (incl. Libertasomyces gen. nov.) on Myoporum serratum. Several novelties are recorded from La Réunion (France), namely Phaeosphaeriopsis agapanthi (on Agapanthus sp.), Roussoella solani (on Solanum mauritianum), Vermiculariopsiella acaciae (on Acacia heterophylla), Dothiorella acacicola (on Acacia mearnsii), Chalara clidemiae (on Clidemia hirta), Cytospora tibouchinae (on Tibouchina semidecandra), Diaporthe ocoteae (on Ocotea obtusata), Castanediella eucalypticola, Phaeophleospora eucalypticola and Fusicladium eucalypticola (on Eucalyptus robusta), Lareunionomyces syzygii (incl. Lareunionomyces gen. nov.) and Parawiesneriomyces syzygii (incl. Parawiesneriomyces gen. nov.) on leaves of Syzygium jambos. Novel taxa from the USA include Meristemomyces arctostaphylos (on Arctostaphylos patula), Ochroconis dracaenae (on Dracaena reflexa), Rasamsonia columbiensis (air of a hotel conference room), Paecilomyces tabacinus (on Nicotiana tabacum), Toxicocladosporium hominis (from human broncoalveolar lavage fluid), Nothophoma macrospora (from respiratory secretion of a patient with pneumonia), and Penidiellopsis radicularis (incl. Penidiellopsis gen. nov.) from a human nail. Novel taxa described from Malaysia include Prosopidicola albizziae (on Albizzia falcataria), Proxipyricularia asari (on Asarum sp.), Diaporthe passifloricola (on Passiflora foetida), Paramycoleptodiscus albizziae (incl. Paramycoleptodiscus gen. nov.) on Albizzia falcataria, and Malaysiasca phaii (incl. Malaysiasca gen. nov.) on Phaius reflexipetalus. Two species are newly described from human patients in the Czech Republic, namely Microascus longicollis (from toenails of patient with suspected onychomycosis), and Chrysosporium echinulatum (from sole skin of patient). Furthermore, Alternaria quercicola is described on leaves of Quercus brantii (Iran), Stemphylium beticola on leaves of Beta vulgaris (The Netherlands), Scleroderma capeverdeanum on soil (Cape Verde Islands), Scleroderma dunensis on soil, and Blastobotrys meliponae from bee honey (Brazil), Ganoderma mbrekobenum on angiosperms (Ghana), Geoglossum raitviirii and Entoloma kruticianum on soil (Russia), Priceomyces vitoshaensis on Pterostichus melas (Carabidae) (Bulgaria) is the only one for which the family is listed, Ganoderma ecuadoriense on decaying wood (Ecuador), Thyrostroma cornicola on Cornus officinalis (Korea), Cercophora vinosa on decorticated branch of Salix sp. (France), Coprinus pinetorum, Coprinus littoralis and Xerocomellus poederi on soil (Spain). Two new genera from Colombia include Helminthosporiella and Uwemyces on leaves of Elaeis oleifera. Two species are described from India, namely Russula intervenosa (ectomycorrhizal with Shorea robusta), and Crinipellis odorata (on bark of Mytragyna parviflora). Novelties from Thailand include Cyphellophora gamsii (on leaf litter), Pisolithus aureosericeus and Corynascus citrinus (on soil). Two species are newly described from Citrus in Italy, namely Dendryphiella paravinosa on Citrus sinensis, and Ramularia citricola on Citrus floridana. Morphological and culture characteristics along with ITS nrDNA barcodes are provided for all taxa.

In vivo and in vitro testing with rAni s 1 can facilitate diagnosis of Anisakis simplex allergy.

BACKGROUND: Traditional diagnostic tests such as skin prick tests (SPT) and specific IgE (slgE) against whole Anisakis simplex extract have low specificity. Consequently, allergy to A simplex is overdiagnosed. OBJECTIVE: Our aim was to compare tests used in component-resolved diagnosis. METHODS: We evaluated 34 patients with allergy to A simplex, 15 patients with acute urticaria who were sensitized to A simplex but had no clinical history of allergy to A simplex, and 10 patients allergic to seafood. SPT, slgE (ELISA and ISAC-I 12), and the basophil activation test (BAT) were performed with A simplex whole extract and the molecular components rAni s 1, rAni s 3, and nPen m 1. Sensitivity and specificity were calculated and compared with different cutoffs. RESULTS: With the A simplex whole extract, SPT, slgE, and BAT yielded specificity values of 72%, 68%, and 70%, respectively, with a cutoff (wheal size) of 11.2 mm, an slgE value of 7.9 kUAIL, and a stimulation index of 1.9. Specificity increased to 100% using the molecular component rAni s 1 with SPT, slgE by ELISA, and ISAC-112. Neither rAni s 3 sensitization nor cross-reactivity with Pen m 1 was observed in patients sensitized to A simplex. CONCLUSION: rAni s 1 is recognized by 100% of our patients and is able to distinguish between patients allergic to A simplex and patients with acute urticaria who are sensitized to A simplex but have no clinical history of allergy to this parasite.

Allergenic characterization of new mutant forms of Pru p 3 as new immunotherapy vaccines.

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

In vitro methods for diagnosing nonimmediate hypersensitivity reactions to drugs.

Nonimmediate drug hypersensitivity reactions (DHRs) are difficult to manage in daily clinical practice, mainly owing to their heterogeneous clinical manifestations and the lack of selective biological markers. In vitro methods are necessaryto establish a diagnosis, especially given the low sensitivity of skin tests and the inherent risks of drug provocation testing. In vitro evaluation of nonimmediate DHRs must include approaches that can be applied during the different phases of the reaction. During the acute phase, monitoring markers in both skin and peripheral blood helps to discriminate between immediate and nonimmediate DHRs with cutaneous responses and to distinguish between reactions that, although they present similar clinical symptoms, are produced by different immunological mechanisms and therefore have a different treatment and prognosis. During the resolution phase, in vitro testing is used to detect the response of T cells to drug stimulation; however, this approach has certain limitations, such as the lack of validated studies assessing sensitivity. Moreover, in vitro tests indicate an immune response that is not always related to a DHR. In this review, members of the Immunology and Drug Allergy Committee of the Spanish Society of Allergy and Clinical Immunology (SEAIC) provide an overview of the most widely used in vitro tests for evaluating nonimmediate DHRs.

Recommendations for the use of in vitro methods to detect specific immunoglobulin E: are they comparable?

Total and specific immunoglobulin (Ig) E can be detected in vitro using several commercially available methods. The largest share of the global market for these methods is held by the ImmunoCAP technique (Thermo Fisher, previously Phadia), Immulite (Siemens), and Hytec-288 (Hycor). Most comparative studies examine Immulite and ImmunoCAP, which differ methodologically but use similar units of measurement relative to the same standard of total IgE (WHO IgE Standard 75/502). Despite their similarity, these kits differ in their quantification of specific IgE, which varies depending on the allergen studied.Thus, specific IgE results obtained with ImmunoCAP and Immulite are not interchangeable. It is important to bear this in mind, especially when determining cutoff points as predictors of a response to oral challenge with specific food allergens. The method used in practice must be the same as the one in the publication guiding clinical decision making. We analyze differences between ImmunoCAP and ISAC microarray, 2 methods from the same manufacturer used to detect IgE to specific proteins (purified or recombinant).The results show that the IgE values obtained with ImmunoCAP are not equivalent to the corresponding values obtained with the ISAC microarray system.

Analysis of mite allergic patients in a diverse territory by improved diagnostic tools.

BACKGROUND: There are few studies comparing the sensitization with mite allergens from different mite species which could potentially be the cause of allergy. OBJECTIVE: To improve the diagnosis of mite allergic patients from a diverse territory in which D. pteronyssinus/D. farinae mites together with storage mites could be present in the environment. METHODS: Four hundred and seventy-seven patients (both children and adults) from different regions, covering the main mite prevalent areas of Spain, were recruited. sIgE to eight allergens was measured together with SPT to whole mite extracts, level of mite allergen exposure, and specific IgG(4) . BAT and CAST was performed in a subgroup of patients. RESULTS: D. pteronyssinus and L. destructor were more prevalent in Atlantic areas, whereas D. farinae predominate in Mediterranean areas. About 90% of patients were sensitized to group 1 and/or group 2 allergens. Group 2 was the most prevalent, and the IgE response/intensity of sensitization in BAT was higher. sIgE to Der p 2/Der f 2 was almost fully cross-reactive, but no cross-reactivity was detected with Lep d 2. Group 1 allergens were also cross-reactive, but in some patients a species-specific response was observed. sIgE to Lep d 2 was associated with SPT results to storage mites. Sensitization to Der p 1 was more frequent in children, whereas Lep d 2 sensitization was more frequent in adults. A higher ratio IgE/IgG(4) to Der p 2 was associated with the presence of allergic asthma. CONCLUSION: An improved diagnosis algorithm has been established. Group 2 allergens seem to have a leading role in mite allergy, but as group 1 sensitization could be species-specific in some patients and its prevalence is higher in children, an adequate balance on major mite species and major allergens must be consider in the design of mite allergy vaccines.

Diagnostic utility of components in allergy to Anisakis simplex.

BACKGROUND: In our region, Anisakis allergy is responsible for 8% of acute urticarial reactions, 25% of which progress to anaphylactic shock. The poor specificity of skin tests and in vitro specific immunoglobulin (Ig) E means that Anisakis allergy is frequently overdiagnosed. OBJECTIVE: We studied the diagnostic value of 2 Anisakis allergens: rAni s 1 and rAni s 3. METHODS: Skin tests, the basophil activation test (BAT), and specific IgE determination were performed with rAni s 1 and 3 in 25 patients allergic to Anisakis, 17 atopic controls, and 10 controls with acute urticaria and positive skin test and sIgE results for Anisakis, but no allergy to Anisakis. RESULTS: For rAni s1, skin tests had a sensitivity and specificity of 100% and specific IgE had a sensitivity and specificity of 100% in the atopic control group and 90% in the urticaria control group. BAT had a sensitivity of 96.8% and a specificity of 100% in the atopic control group and 66.7% in the urticaria control group. For rAni s 3, only 1 patient had positive specific IgE results to rAni s 3. All other techniques gave negative results in patients and controls CONCLUSIONS: rAni s 1 is the major allergen of Anisakis and the target allergen when diagnosing allergy to Anisakis, rAni s 3 is not relevant when attempting to explain false-positive results.

Can component-based microarray replace fluorescent enzimoimmunoassay in the diagnosis of grass and cypress pollen allergy?

BACKGROUND: Few data on the diagnostic accuracy in pollinosis of the microarray ISAC of allergens are available. OBJECTIVE: We aim to comparatively analyse ISAC CRD103 with the whole-extract ImmunoCAP in grass and cypress pollen allergy, evaluating the suitability of the manufacturer's recommended cut-off points for both techniques. METHODS: We studied 120 atopic patients grouped into grass and cypress pollen-allergic patients and controls based on clinical history and skin prick tests. Specific IgE against Phleum pratense and Cupressus arizonica by ImmunoCAP and ISAC CRD103 were performed on all subjects. RESULTS: In the grass pollen group (43 allergic/26 controls), both microarray and CAP showed high sensitivity (Se) and specificity (Sp) values (ISAC: Se 97.7, Sp 92.3; CAP: Se 95.3, Sp 96.1) for recommended cut-off points. Comparing the optimal (ISAC: 0.4 ISU; CAP: 0.33 kU/L) with the recommended cut-off points within the same technique, diagnostic agreement was observed in both techniques. Thus, CAP and ISAC showed similar diagnostic performance in grass pollen allergy when using recommended cut-off points. In cypress pollen group (12 allergic/92 controls), the microarray (Se: 91.7, Sp 91.3) showed similar Se but significantly higher Sp (P=0.034) than CAP (Se: 91.7, Sp: 80.4) using recommended cut-off points. However, although diagnostic performance of the microarray did not change when comparing the optimal (0.82 ISU) with the recommended cut-off point, CAP improved diagnosis of cypress pollen allergy, when applying the optimal (0.66 kU/L)(CAP Se: 91.7, Sp: 89.1) instead of the manufacturer's recommended cut-off point. Thus, when the most suitable cut-off point for both techniques (ISAC: 0.3 ISU; CAP: 0.66 kU/L) is selected, microarray and CAP provide equivalent diagnoses. CONCLUSIONS AND CLINICAL RELEVANCE: Component-based microarray ISAC CRD103 and whole-allergen CAP showed high Se and Sp diagnosing equally grass and cypress pollen allergy. The cut-off point for each allergen should be properly applied for both techniques.

Lupin allergy: a hidden killer in the home.

This review addresses the problem of lupin sensitization in the home environment. We summarize the data currently available on allergy to lupin, which has become, in recent years, a hidden killer in our homes. Since 2006, when lupin was included in European regulations as a food whose presence must be declared, the situation may have changed. Nevertheless, we must take into account the possibility of undeclared allergenic ingredients or the presence of 'hidden' allergens, given that contamination during food production processes may be a great risk for sensitized individuals. Furthermore, the United States, Japan, Australia and New Zealand still do not include lupin among the ingredients that must be listed on foodstuff labelling. Our responsibility is to educate the public so that they are aware of the danger and look for lupin in the labels of products that run the risk of containing it. Lupin allergy can manifest itself in isolation or in parallel to peanut allergy. Identification of the proteins causing possible cross-reactivity is complicated, and new structural studies are needed. To date, it has not been possible to clearly identify the allergens responsible for isolated lupin sensitization in relation to parallel and/or cross-sensitization between lupin and peanut. Most of the allergenic proteins of lupin are α- and ß-conglutins, with a lesser presence of γ- and δ-conglutins. A ß-conglutin corresponding to Lup an 1, with a sequence similar to Ara h 1, has been identified as a major allergen of lupin in patients with allergy following lupin ingestion.

Characterization of peach thaumatin-like proteins and their identification as major peach allergens.

BACKGROUND: Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. OBJECTIVE: To identify members of the TLP family in peach fruit and to characterize putative allergens. METHODS: Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N-terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (beta-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). RESULTS: Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N-asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed beta-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. CONCLUSION: Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area.