Relationship between Phenolic Compounds, Antioxidant Properties, and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica).

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.

IgE-blocking antibodies following SLIT with recombinant Mal d 1 accord with improved apple allergy.

BACKGROUND: We recently reported that 16 weeks of sublingual immunotherapy (SLIT) with recombinant (r) Mal d 1, but not rBet v 1, significantly improved birch pollen-related apple allergy. Allergen-specific IgE-blocking IgG antibodies have been associated with clinical efficacy. OBJECTIVE: We compared the quantity, quality, and IgE-blocking bioactivity of SLIT-induced Mal d 1-specific IgG antibodies in both treatment groups. METHODS: Pre- and post-SLIT sera were assessed for rMal d 1-specific IgG antibodies in ELISA and for their ability to inhibit apple allergen-induced upregulation of CD63 on basophils from nontreated individuals with birch pollen-related apple allergy. Post-SLIT sera depleted of IgG1 or IgG4 were compared for their IgE-blocking activity. IgG1 binding to rMal d 1 was competed with rMal d 1 and rBet v 1 in ELISA. RESULTS: SLIT with rMal d 1 and rBet v 1 induced comparable levels of rMal d 1-specific IgG1, IgG2, IgG3, and IgG4 antibodies. Only post-rMal d 1 SLIT sera displayed IgE-blocking activity, which was significantly reduced by depletion of IgG1 and less so by IgG4 depletion. In competition ELISA, IgG1 binding to Mal d 1 in post-rMal d 1 SLIT sera was fully inhibited with rMal d 1 but not with rBet v 1. Correspondingly, Bet v 1 was the more potent competitor for IgG1 binding to Mal d 1 in post-rBet v 1 SLIT sera. CONCLUSION: rMal d 1 SLIT for 16 weeks induced functional, primarily Mal d 1-specific IgE-blocking antibodies, whereas rBet v 1 SLIT induced Bet v 1-specific, Mal d 1-cross-reactive IgG antibodies with limited cross-blocking activity. These results provide a possible explanation for the limited effectiveness of birch pollen immunotherapy in birch pollen-related food allergy and indicate a dominant protective role of functional IgE-blocking IgG1 antibodies in the early phase of allergy treatment.

The Effect of Birch Pollen Immunotherapy on Apple and rMal d 1 Challenges in Adults with Apple Allergy.

BACKGROUND: A proportion of patients allergic to birch pollen are also allergic to pit fruit. The objective of this study was to investigate the effect of immunotherapy with birch pollen on birch-pollen-related apple allergy. METHOD: Patients with birch pollen immunotherapy underwent a skin-prick test with birch pollen, apple and rMal d 1, global assessments and nasal challenges with birch pollen, open food challenge with apple and a double-blind, placebo-controlled test with rMal d 1 at the start of and during the immunotherapy. Measurements of specific IgE in response to Bet v 1 and rMal d 1 and IgG4 in response to Bet v 1 and rMal d 1 took place. RESULTS: Six of eight patients demonstrated an improvement of nasal challenge test results and all patients improved on global assessment during the immunotherapy. The median oral dose of apple required to elicit a reaction increased but was not statistically significant. The patients showed a decrease in skin-prick test values in response to birch pollen (1.05 to 0.36), apple (0.78 to 0.25) and rMal d 1 (0.51 to 0.10) with p-values of 0.04, 0.03 and 0.06, respectively and a decrease of specific IgE in response to Bet v 1 (10.66 kU/L to 5.19 kU/L) and rMal d 1 (0.99 to 0.61 kU/L) with p-values of 0.01 and 0.05, respectively. Only the median specific IgG4 value to Bet v 1 increased from 0.05 to 1.85 mg/L (p-value of 0.02) and not to IgG4 rMal d 1 (0.07 to 0.08 kU/L). CONCLUSION: The beneficial effects of immunotherapy for birch pollen were accompanied by a limited effect on apple allergy.

Biological Evaluation of Golden Delicious Apples Exposure to UV Lights in Rats.

BACKGROUND AND OBJECTIVE: Anthocyanin is responsible for the red color of apple. Ultraviolet light plays a key role in activating the genes responsible for anthocyanin biosynthesis. However, the most important concern is using UV light irradiation on fruit to increase anthocyanins level and its nutritional quality. In this study, the accumulation of anthocyanin in green apple using UV-B and UV-C was investigated and its biological influence was evaluated in rats. MATERIAL AND METHOD: Green Golden delicious apples were irradiated with doses of UV-C and UV-B light for a period of 3 h/day each for 3 days. Two Groups of rats were fed on balanced diet or balanced diet supplemented with 10% apple exposure to UV (AP-UV) for a month. RESULTS: The HPTLC and spectrophotometric determination of anthocyanin revealed that color development was significantly increased by 90% in treated apple compared to the control apples. Histological difference was observed between the 2 groups. Plasma levels of uric acid, the activity of transaminases (ALT and AST) as well as malondialdehyde (MDA) were significantly elevated in AP-UV rats. Plasma total cholesterol, triglycerides and creatinine level did not differ among the 2 groups. Liver MDA and catalase levels were eminent in AP-UV rats compared to control. Gene expression of selected inflammatory cytokines (TNF-α, IL-6 and IL-1ß) was significantly up-regulated in liver of AP-UV rats in comparison to control rats. CONCLUSION: The result revealed that there is a health-hazard linked to feeding rats on diet containing irradiated-apple with UV-B and UV-C, which represented by body weight reduction, inflammation development, liver function and oxidative stress elevation.

Lipid transfer protein sensitization in an apple-allergic patient: a case report from northern Europe.

Summary: We describe a case of a woman who developed three separate episodes of urticaria and ana-phylaxis during exercise after consuming an apple, with immunological evidence that nonspecific lipid transfer proteins (LTP) may have been responsible for these reactions. LTP sensitivity can cause life-threatening allergies and anaphylaxis. Although LTP sensitization is common in Mediterranean countries, the frequency of knowledge and diagnoses is increasing in Europe. Despite the geographic differences, LTP allergy should be kept on sight when facing severe anaphylaxis after consuming LTP-included food.

Dietary Interventions in Pollen-Related Food Allergy.

In practice, it remains unclear what the best dietary approach is in subjects with pollen-related food allergy (PRFA). Our objective was to evaluate the effect of (1) dietary avoidance advice, (2) oral immunotherapy (OIT), (3) (heat) processing, and (4) consumption of hypoallergenic cultivars on frequency, severity, and eliciting dose of pollen-related food allergic reactions. A systematic search was conducted in PubMed, Embase, and Cochrane. All studies performing an in vivo investigation of one of the four interventions in adults with PRFA were included. Each study was assessed for quality and validity. Available data on frequency, severity, and eliciting dose of allergic reactions were extracted. Ten studies matched the eligibility criteria. No studies were retrieved on dietary avoidance advice. Two studies (N = 92) on apple OIT reported that tolerance was induced in 63% and 81% of subjects. Four studies (total N = 116) focused on heat processing. Heating was found to completely eradicate symptoms in 15⁻71% of hazelnut allergic and 46% of celery allergic individuals. Four studies (N = 60) comparing low to high allergenic apple cultivars revealed that Santana (and possibly Elise) apples seemed to cause milder reactions than Golden Delicious. In the awareness that overall level of evidence was low, we conclude that OIT, heat processing, and hypoallergenic cultivars may diminish or completely prevent allergic reactions in some but not all subjects with PRFA.

Comparative analysis of allergen genes and pro-inflammatory factors in pollen and fruit of apple varieties.

Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A2 (PLA2) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA2 and ROS producing enzyme are lower in the hypo-allergenic variety 'Durello di Forlì' in comparison with the high-allergenic genotypes 'Gala' and 'Florina'. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity.

Birch pollen immunotherapy inhibits anaphylaxis to the cross-reactive apple allergen Mal d 1 in mice.

BACKGROUND: Cross-reactive apple allergy is a common co-morbidity of birch pollen allergy, caused by the presence of a Bet v 1 homologue allergen in apple, Mal d 1. Treatment of tree pollen hay fever by immunotherapy is well established, but its effect on the accompanying apple allergy is debated. OBJECTIVE: To establish a mouse model of birch pollen induced cross-reactivity to Mal d 1 and investigate the effect of birch pollen immunotherapy on the cross-reactivity to Mal d 1. METHODS: Respiratory allergy was induced in Balb/c mice by intraperitoneal exposure to alum-adsorbed birch pollen extract (BPE) in combination with short or prolonged intranasal exposure to BPE. To evaluate the response to Mal d 1, mice were exposed intraperitoneally to Mal d 1. Immunoglobulin responses and cytokine production by splenocytes were measured by ELISA. Allergic symptoms were evaluated by measuring airway hyper-reactivity and hypothermia as a surrogate marker for anaphylaxis. Immunotherapy was performed subcutaneously with alum-adsorbed BPE. RESULTS: Mice exposed to BPE develop cross-reactive IgE to Mal d 1. Early after exposure to BPE, this response is still weak and does not yet translate into anaphylaxis. Interestingly, later re-challenge with BPE increased cross-reactivity to a level where Mal d 1 exposure induced anaphylaxis. Cross-sensitization can also be induced by systemic Mal d 1 exposure. Birch pollen immunotherapy significantly reduced the anaphylactic response of mice to Mal d 1. CONCLUSION & CLINICAL RELEVANCE: A mouse model mimicking birch pollen induced cross-reactivity to Mal d 1 was successfully established. In this model, birch pollen immunotherapy significantly ameliorated the anaphylaxis induced by Mal d 1. Our experimental data suggest that boosting of Mal d 1 recognizing immunoglobulins by BP SCIT is important for the amelioration of apple allergy in human.

Shock anafiláctico con manzana. Síndrome LTP / Anaphylactic shock caused by apple. LTP syndrome

Presentamos el caso de una paciente atendida en urgencias del centro de salud por un cuadro clínico de shock anafiláctico desencadenado tras la ingesta de manzana y realización posterior de ejercicio físico. En alergia a alimentos, hay que tener presente que en algunas personas es necesaria la presencia de determinados cofactores como el ejercicio físico o la ingesta de determinados fármacos como los AINEs para que suceda una reacción alérgica. Se sospecha que el mecanismo responsable consiste en que aceleren la absorción del alimento en el intestino y así lleguen a modular la severidad de los síntomas. Por este motivo cuando las LTP (proteína de transferencia de lípidos) están implicadas, si los cofactores no se detectan y previenen, pueden representar un serio riesgo para el desarrollo de episodios de anafilaxia severos o fatales (AU)

We report the case of a patient treated in an emergency department of a health center for a clinical picture of anaphylactic shock triggered after ingestion of apple and subsequent practice of physical exercise. In food allergy, it must be remembered that in some people the allergic reaction requires the presence of certain cofactors, such as physical exercise or use of certain prescription drugs such as NSAIDs. It is suspected that the mechanism responsible for this consists in accelerating the absorption of food in the intestine and thereby modulating the severity of symptoms. That is why when lipid transfer proteins (LTP) are involved, if the cofactors are not detected and prevented, they can pose a serious risk for developing severe or fatal episodes of anaphylaxis (AU)

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen-allergic patients with apple allergy.

It is still unclear whether allergen-specific immunotherapy (AIT) with birch pollen improves birch pollen-related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen-related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1-homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen-allergic patients with apple allergy. The dose of 1.6 µg induced oral allergy syndromes in 26.4%, 3.2 µg in 15.3%, 6.3 µg in 27.8%, 12.5 µg in 8.3%, 25 µg in 11.1%, and 50 µg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 µg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose-defined sublingual challenge tests in a high number of birch pollen-allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen-related food allergy.

Immunotherapy (oral and sublingual) for food allergy to fruits.

BACKGROUND: Food allergy is an abnormal immunological response following exposure (usually ingestion) to a food. Elimination of the allergen is the principle treatment for food allergy, including allergy to fruit. Accidental ingestion of allergenic foods can result in severe anaphylactic reactions. Allergen-specific immunotherapy (SIT) is a specific treatment, when the avoidance of allergenic foods is problematic. Recently, studies have been conducted on different types of immunotherapy for the treatment of food allergy, including oral (OIT) and sublingual immunotherapy (SLIT). OBJECTIVES: To determine the efficacy and safety of oral and sublingual immunotherapy in children and adults with food allergy to fruits, when compared with placebo or an elimination strategy. SEARCH METHODS: The Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, CINAHL, and AMED were searched for published results along with trial registries and the Journal of Negative Results in BioMedicine for grey literature. The date of the most recent search was July 2015. SELECTION CRITERIA: Randomised controlled trials (RCTs) comparing OIT or SLIT with placebo or an elimination diet were included. Participants were children or adults diagnosed with food allergy who presented immediate fruit reactions. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by the Cochrane Collaboration. We assessed treatment effect through risk ratios (RRs) for dichotomous outcomes. MAIN RESULTS: We identified two RCTs (N=89) eligible for inclusion. These RCTs addressed oral or sublingual immunotherapy, both in adults, with an allergy to apple or peach respectively. Both studies enrolled a small number of participants and used different methods to provide these differing types of immunotherapy. Both studies were judged to be at high risk of bias in at least one domain. Overall, the quality of evidence was judged to be very low due to the small number of studies and participants and possible bias. The studies were clinically heterogeneous and hence we did not pool the results. A study comparing SLIT with placebo for allergy to peach did not detect a significant difference between the number of patients desensitised at six months following a double-blind placebo-controlled food challenge (RR 1.16, 95% confidence interval (CI) 0.49 to 2.74). The second study, comparing OIT versus no treatment for apple allergy, found an effect on desensitisation in favour of the intervention using an oral provocation test at eight months, but results were imprecise (RR 17.50, 95% CI 1.13 to 270.19). Neither study reported data on evidence of immunologic tolerance. In both studies, the incidence of mild and moderate adverse events was higher in the intervention groups than in the controls. In the study comparing SLIT with placebo, patients in the intervention group experienced significantly more local adverse reactions than participants in the control group (RR 3.21, 95% CI 1.51 to 6.82), though there was not a significant difference in the number of participants experiencing systemic adverse reactions (RR 0.81, 95% CI 0.22 to 3.02). In the study of OIT, two of the 25 participants in the intervention group reported relevant side effects, whereas no participants in the control group reported relevant side effects. AUTHORS' CONCLUSIONS: There is insufficient evidence for using OIT or SLIT to treat allergy to fruit, specifically related to peach and apple. Mild or moderate adverse reactions were reported more frequently in people receiving OIT or SLIT. However, these reactions could be treated successfully with medications.

First successful reduction of clinical allergenicity of food by genetic modification: Mal d 1-silenced apples cause fewer allergy symptoms than the wild-type cultivar.

BACKGROUND: Genetic modification of allergenic foods such as apple has the potential to reduce their clinical allergenicity, but this has never been studied by oral challenges in allergic individuals. METHODS: We performed oral food challenges in 21 apple-allergic individuals with Elstar apples which had undergone gene silencing of the major allergen of apple, Mal d 1, by RNA interference. Downregulation of Mal d 1 gene expression in the apples was verified by qRT-PCR. Clinical responses to the genetically modified apples were compared to those seen with the wild-type Elstar using a visual analogue scale (VAS). RESULTS: Gene silencing produced two genetically modified apple lines expressing Mal d 1.02 and other Mal d 1 gene mRNA levels which were extensively downregulated, that is only 0.1-16.4% (e-DR1) and 0.2-9.9% (e-DR2) of those of the wild-type Elstar, respectively. Challenges with these downregulated apple lines produced significantly less intense maximal symptoms to the first dose (Vmax1) than with Elstar (Vmax1 Elstar 3.0 mm vs 0.0 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2 mm vs 0.2 mm for e-DR1, P = 0.017 and 0.0 mm for e-DR2, P = 0.043). Only one subject (5%) remained symptom-free when challenged with the Elstar apple, whereas 43% did so with e-DR1 and 63% with e-DR2. CONCLUSION: These data show that mRNA silencing of Mal d 1 results in a marked reduction of Mal d 1 gene expression in the fruit and reduction of symptoms when these apples are ingested by allergic subjects. Approximately half of the subjects developed no symptoms whatsoever, and virtually all subjects wished to consume the apple again in the future.

Public attitudes to GM foods. The balancing of risks and gains.

In the paper we study the variables influencing attitudes to the use of two biotechnologies related to gene transfer within apples. Using Eurobarometer 73.1 survey data on biotechnology, science and technology, with 15,650 respondents, we study the extent these attitudes are determined by socio-economic and other variables. We found that attitudes to the risks and gains are determined by socio-economic variables and also by the individual's knowledge, scientific background, their parent's education in science and their religion. Perceptions of naturalness and of environmental impact combined with perceived risks and gains in determining overall approval, proxied by views on whether the technologies should be encouraged, for GMTs. However there are substantial differences in attitudes to transgenesis and cisgenesis.

A Randomised trial to investigate the erosive effect of hot drinks.

Research on dental erosion has largely been undertaken at room temperature despite fruit juice drinks often being consumed at elevated temperatures in the United Kingdom, notably during periods of convalesce. The aim of the study was to evaluate the erosive potential of two fruit juices containing acidic non-alcoholic drinks at elevated temperatures in situ on human enamel after 5, 10 and 15 days. A commercially available conventional apple and blackcurrant fruit juice drink was compared to a blackcurrant juice drink modified to have low erosive potential, and mineral water consumed at approximately 59 °C. Twenty-one healthy volunteers aged 18 or over participated in a single-centre, single-blind (blinded to the investigator), three-treatment crossover study. Subjects were randomised to a treatment sequence using a Latin square design. Subjects wore upper removable appliances containing one human enamel specimen from 9 am to 5 pm for 15 days for each beverage. Measurements of enamel loss were recorded after 5, 10 and 15 days by contact surface profilometry. The low erosive blackcurrant drink caused significantly less enamel loss (P < 0·05) than the commercially available conventional apple and blackcurrant fruit juice drink and was not statistically significantly different to mineral water at any of the time points in the study. Consuming the modified low erosive blackcurrant drink at an elevated temperature resulted in negligible enamel loss in situ, consistent with room temperature findings.

High prevalence of lipid transfer protein sensitization in apple allergic patients with systemic symptoms.

BACKGROUND: Apple allergy manifests as two main groups of clinical entities reflecting different patterns of allergen sensitization: oral allergy syndrome (OAS) and generalized symptoms (GS). OBJECTIVE: We analysed the sensitization profile to a wide panel of different components of food allergens (rMal d 1, Mal d 2, rMal d 3, rMal d 4, rPru p 3, rBet v 1 and Pho d 2) for a population of Mediterranean patients with OAS and GS to apple. METHODS: Patients (N = 81) with a history of apple allergy that could be confirmed by positive prick-prick test and/or double-blind-placebo-controlled food challenge (DBPCFC), were included. Skin prick test (SPT) and ELISA were performed using a panel of inhalant, fruit and nut allergens. ELISA and ELISA inhibition studies were performed in order to analyse the sensitization patterns. RESULTS: Thirty-five cases (43.2%) had OAS and 46 (56.8%) GS. SPT showed a significantly higher number of positive results with peach, cherry and hazelnut in those with GS. ELISA showed a significantly high percentage of positive cases to rMal d 3, rMal d 4, rPru p 3 and Pho d 2 in patients with OAS and GS compared to controls, and to rBet v 1 in patients with OAS vs controls and between OAS and GS patients. Three different patterns of recognition were detected: positive to LTP (rMal d 3 or rPru p 3), positive to profilin (rMal d 4 and Pho d 2), or positive to both. There were also patients with rMal d 1 recognition who showed cross-reactivity to rBet v 1. CONCLUSION: In an apple allergy population with a high incidence of pollinosis different patterns of sensitization may occur. LTP is most often involved in those with GS. Profilin, though more prevalent in patients with OAS, has been shown to sensitise patients with both types of symptoms.

Effects of long-term storage on Mal d 1 content of four apple cultivars with initial low Mal d 1 content.

BACKGROUND: Up to 6.6% of adults and 11.5% of children aged between 0 and 6 years are reporting a perceived fruit allergy. In the last group 8.5% perceived reactions against apple. To keep allergic reactions as low as possible and to make apple consumption more tolerable for patients allergic to apple, the Mal d 1 content of four apple cultivars ('Boskoop', 'Elise', 'Pinova', 'Santana' and 'Topaz') were analyzed, to identify apple cultivars with only a low Mal d 1 content. Further, the development of Mal d 1 content of low-allergenic cultivars should be investigated during long-term storage. RESULTS: The lowest Mal d 1 content was measured for fruits of the cultivar 'Santana' at harvest, followed by 'Pinova' and 'Elise' after storage without 1-MCP. Nearly all fruits treated with 1-MCP had a significantly lower Mal d 1 content than fruits without 1-MCP, except for 'Boskoop' fruits after 44 weeks of storage. Apple cultivars with a low Mal d 1 content are not a cross of 'Golden Delicious' with another cultivar, with the exception of 'Pinova' which is a cultivar from the so-called 'Pi-series', which have a moderate resistance to scab and mildew. First experiments on tomatoes and peanuts have shown a reduction of allergic proteins in older and child generations after transfer of RNA. This reduction is probably based on an alteration of gene expression regulating the synthesis of allergic proteins, leading to changes in protein composition in the fruit. CONCLUSION: Individuals allergic to apple should choose the described cultivars with relative low Mal d 1 content. If breeders want to breed cultivars with low allergy, they should avoid crossings with 'Golden Delicious'.

Where to prick the apple for skin testing?

Mal d 1 is not equally distributed over the apple. We aimed to examine the influence of the location of pricking in the apple on prick-to-prick skin prick test (PTP) results. PTPs were performed in autumn 2007 and spring 2008, before the birch pollen season, in 32 Dutch adults with symptoms of oral allergy to fresh apple, using apples harvested in autumn 2007. PTPs with fresh intact and unpeeled Pink Lady, Golden Delicious, Elise, Santana and Modi apples were performed using material obtained from approximately 2 cm near the stalk (top), and the middle region. All PTP responses were greater when performed with apple material near the stalk than from the middle region. In 2007, these differences were statistically significant for Pink Lady, Golden Delicious and Elise, and in 2008, for Pink Lady and Modi. When performing PTPs, the apple should be pricked near the stalk rather than in the middle.