Search

Das pollenassoziierte Orale Allergie-Syndrom (OAS) ist eine Sonderform der IgE-vermittelten Nahrungsmittelallergien. Besonders bei Birkenpollenallergikern kommt es aufgrund der Ähnlichkeit der Epitope des Apfelallergens Mal d 1 und dem Birkenpollenallergen Bet v 1 häufig zu einer sog. Kreuzreaktion beim Essen handelsüblicher Supermarktäpfel. In einer prospektiven Kohortenstudie mit einem offenen Studiendesign wurde untersucht, ob eine Toleranzentwicklung bei „Apfelallergikern“ durch den regelmäßigen Konsum allergenarmer Äpfel erreicht werden kann. Hierzu wurde die Ausprägung oraler Symptome nach dem Essen einer Frucht der Sorte ‘Golden Delicious‘ vor und nach dem täglichen Konsum eines allergenarmen Apfels über 90 Tage untersucht. Die Probanden zeigten signifikant geringere Symptome beim Essen des ‘Golden Delicious‘ nach dem regelmäßigen Verzehr der allergenarmen Äpfel alter Sorten. Es erscheint möglich, durch den regelmäßigen Verzehr von Äpfeln alter Sorten mit geringerem Allergengehalt eine orale Toleranz gegenüber hochallergenen Äpfeln zu erreichen., Pollen-associated oral allergy syndrome (OAS) is a special form of IgE-mediated food allergies. Especially with birch pollen allergy sufferers, due to the similarity of the epitopes of the apple allergen Mal d 1 and the birch pollen allergen Bet v 1, there is often a so-called cross-reaction when eating commercially available supermarket apples. In a prospective cohort study with an open study design, it was examined whether tolerance development in “apple allergy sufferers” can be achieved through regular consumption of low-allergen apples. For this purpose, the expression of oral symptoms after eating a fruit of the variety ‘Golden Delicious’ before and after daily consumption of an allergen-poor apple for 90 days was examined. The subjects showed significantly less symptoms when eating the ‘Golden Delicious’ after regular consumption of the low-allergen apples of old varieties. It appears possible to achieve an oral tolerance to highly allergenic apples through regular consumption of old varieties with a lower allergen content.

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

About 50–70% of patients allergic to birch pollen suffer from sensitization after apple ingestion. Apple allergenicity was established in only few varieties. Studies were performed on apple fruits of 21 traditional and nine modern varieties organically, intensively, or integratively produced. The aim of the study was to assess whether the factors like cultivation method, maturity stage, genotype, or type of tissue place an impact on the allergenic potential of apples. To answer these questions, we used semiquantitative real-time PCR, ELISA, and immunoblotting. Apple allergen genes present divergent expression across apple cultivars. Expression of the Mal d 1.06A correlates with the Mal d 1 level and is affected by the cultivation method and maturity of the fruit. The content of the main allergen Mal d 1 varied widely across cultivars. Interestingly, in our study, the Gala variety presented a low Mal d 1 concentration regardless of the cultivation method. Based on the Mal d 1.06A expression, the Mal d 1 protein content, and the immunoreactivity assay, the Kandil Sinap, Kosztela, Rumianka from Alma-Ata, Kantówka Gdańska, Reinette Coulon, and Gala cultivars emerged as potentially hypoallergenic apple cultivars. Our study allowed distinguishing between potentially low, medium, and highly allergenic varieties.

BACKGROUND: The efficacy of an allergen-specific IgG cocktail to treat cat allergy suggests that allergen-specific IgG may be a major protective mechanism elicited by allergen immunotherapy. OBJECTIVES: Extending these findings, we tested a Bet v 1-specific antibody cocktail in birch-allergic subjects. METHODS: This was a phase 1, randomized, double-blind, study with 2 parts. Part A administered ascending doses of the Bet v 1-specific antibody cocktail REGN5713/14/15 (150-900 mg) in 32 healthy adults. Part B administered a single subcutaneous 900-mg dose or placebo in 64 birch-allergic subjects. Total nasal symptom score response to titrated birch extract nasal allergen challenge and skin prick test (SPT) with birch and alder allergen were assessed at screening and days 8, 29, 57, and 113 (SPT only); basophil activation tests (n = 26) were conducted. RESULTS: Single-dose REGN5713/14/15 significantly reduced total nasal symptom score following birch nasal allergen challenge relative to baseline. Differences in total nasal symptom score areas under the curve (0-1 hour) for subjects treated with REGN5713/14/15 versus those given placebo (day 8: -1.17, P = .001; day 29: -1.18, P = .001; day 57: -0.85, P = .024) and titration SPT with birch difference in area under the curve of mean wheal diameters for subjects treated with REGN5713/14/15 versus placebo (all P < .001) were sustained for ≥2 months; similar results were observed with alder SPT. REGN5713/14/15 was well tolerated. Basophil responsiveness to birch-related allergens was significantly decreased in subjects treated with REGN5713/14/15 versus those given placebo on days 8, 57, and 113 (all P < .01). CONCLUSIONS: Single-dose REGN5713/14/15 was well tolerated and provided a rapid (1 week) and durable (2 months) reduction in allergic symptoms after birch allergen nasal allergen challenge, potentially offering a new paradigm for the treatment of birch allergy symptoms. ispartof: JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY vol:149 issue:1 pages:189-199 ispartof: location:United States status: published

Birkenpollen-assoziierte Nahrungsmittelallergien und atopische Krankheiten haben in den letzten Jahrzehnten weltweit zugenommen, das belegen zahlreiche wissenschaftliche Studien. In der vorliegenden Arbeit liegt der Fokus auf der Kreuzallergie zwischen Äpfeln und Birkenpollen. Im Zuge eines wissenschaftlichen Forschungsprojekts am Institut für Pathophysiologie und Allergieforschung der Medizinischen Universität Wien im Jahr 2019 stand die Identifizierung von Apfelsorten, die potentiell als allergenarm eingestuft werden können, im Vordergrund. Eine gezielte Identifizierung allergenarmer Apfelsorten kann allergischen Personen den symptomfreien Genuss von Äpfeln ermöglichen und damit insgesamt zu einer ausgewogenen und gesunden Ernährung beitragen. Um allergikerfreundliche Apfelsorten zu identifizieren, wurde eine Studie mit insgesamt 35 Proband*innen durchgeführt, die sowohl an einer Birkenpollenallergie als auch an einer Apfelallergie leiden. Anhand verschiedener Tests, wie einer Untersuchung der allergischen Reaktion nach dem Apfelkonsum, einem Skin Prick Test und einem IgE-Immuno-Blot, wurden die allergenen bzw. hypoallergenen Eigenschaften der getesteten Apfelsorten (Bay 4069/Gräfin Goldach, Bay 4152, Bay 4210/Sonnenglanz, Santana, Golden Delicious (Bay), Golden Delicious (Spar)) untersucht. Abgesehen vom Nachweis von Antigenen mittels Immuno-Blot kamen dafür im Labor zum einen biochemische Methoden, wie beispielsweise der BCA-Test, der Western Blot und die SDS-PAGE, und zum anderen Färbemethoden, wie die Silberfärbung und die Coomassie Blau-Färbung, zum Einsatz. Die Ergebnisse der durchgeführten Tests wurden in der vorliegenden Arbeit besonders auf die in der wissenschaftlichen Literatur häufig zu findende Annahme, dass neuere Apfelsorten wie Golden Delicious stärkere allergische Reaktionen auslösen als ältere, untersucht. Dabei zeigte sich, dass die Sorte Golden Delicious im Vergleich mit den anderen getesteten Apfelsorten tatsächlich starke allergene Eigenschaften aufweist. Sowohl beim SPT als auch bei der Testung der oralen Symptome zeigten die Proband*innen im Schnitt deutlich erkennbare allergische Reaktionen auf Golden Delicious Äpfel, wohingegen die Reaktionen auf die Sorte Bay 4090/Gräfin Goldach nur sehr schwach ausfielen. Abgesehen von den Ergebnissen der Studie verweist die abschließende Diskussion in dieser Arbeit auch auf die Rolle von Mal d 1 in Äpfeln, auf gesundheitliche Aspekte bzw. mögliche Schlussfolgerungen für Apfelallergiker*innen und auf offene Fragen und zukünftige Forschungsfelder in Hinblick auf die Forschung zum Birken-Apfel-Syndrom. Birch pollen-associated food allergies and atopic diseases have increased worldwide in recent decades, as evidenced by numerous scientific studies. The following paper focuses on the cross allergy between apples and birch pollen. In the course of a scientific research project at the Institute of Pathophysiology and Allergy Research of the Medical University of Vienna in 2019, the focus was on the identification of apple varieties that can potentially be classified as low in allergens. Targeted identification of low-allergen apple varieties may allow allergic individuals to enjoy apples without symptoms and thus contribute to a balanced and healthy diet overall. In order to identify allergy-friendly apple varieties, a study was conducted with a total of 35 subjects suffering from both birch pollen allergy and apple allergy. The allergenic or hypoallergenic properties of the tested apple varieties (Bay 4069/Gräfin Goldach, Bay 4152, Bay 4210/Sonnenglanz, Santana, Golden Delicious (Bay), Golden Delicious (Spar)) were investigated using various tests, such as an examination of the allergic reaction after apple consumption, a skin prick test and an IgE immuno-blot. Apart from the detection of antigens by immuno-blot, biochemical methods such as the BCA test, Western blot and SDS-PAGE, and staining methods such as silver staining and Coomassie blue staining were used in the laboratory. In the present work, the results of the tests performed were particularly examined for the assumption, frequently found in the scientific literature, that newer apple varieties such as Golden Delicious cause stronger allergic reactions than older ones. It was found that the Golden Delicious variety does indeed exhibit strong allergenic properties compared to the other apple varieties tested. In the SPT as well as in the testing of oral symptoms, the test persons showed on average clearly recognizable allergic reactions to Golden Delicious apples, whereas the reactions to the variety Bay 4090/Gräfin Goldach were only very weak. Apart from the results of the study, the final discussion in this paper also refers to the role of Mal d 1 in apples, to health aspects and possible conclusions for apple allergy sufferers, and to open questions and future research fields with regard to research on birch-apple syndrome.

Apple-related allergy is increasing among European population. Avoidance of apple fruit consumption is the only strategy for sensitized people, with a subsequent shortage of healthy nutrients in the diet. Four different protein families, Mal d 1 to 4, determine the allergy to apple causing syndromes known as oral allergy syndrome (OAS) or gastro-intestinal/generalized syndrome (GS) in sensitized patients. With the aim to highlight the dissimilarities in the immunological response inside the Mal d 1 family, recognized as main determinant for OAS, single purified allergens were tested against sera of paediatric and adult patients suffering for both syndromes. Here, we found that Mal d 1 isoallergens showed a remarkable different allergenicity potential, thus indicating an individual pattern in each patient and a diverse sensitization between age groups. Moreover, two different allergens, Mald 2.01 and Mal d 4.01, were analyzed with the above sera. In addition to patients and allergens variability, differences in amount and localization of the allergens, detected by patient’s sera, were revealed among hypo- and hyper allergenic apple varieties, confirming the complexity of this food allergy.

BackgroundGenetic 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.MethodsWe 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).ResultsGene 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 3.0mm vs 0.0mm for e-DR1, P=0.017 and 0.0mm for e-DR2, P=0.043), as well as significantly less intense mean symptoms per dose (meanV/d) than with Elstar (meanV/d Elstar 2.2mm vs 0.2mm for e-DR1, P=0.017 and 0.0mm 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.ConclusionThese 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.

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, PLA2and 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.

Patients with Oral Allergy Syndrome (OAS) to fresh apple may tolerate low allergenic apple cultivars. We aimed to investigate if the low allergenic properties of Elise and Santana, as previously identified in a Dutch population, could be generalised within North West Europe within the birch pollen region with regard to both the prevalence and degree of sensitization. Prick-to-prick tests (PTP) were performed in eighty-five adult patients with OAS to fresh apple in Great Britain, Switzerland and Northern Italy, before the birch pollen season, using the putatively low allergenic apple cultivars Elise, Santana, Granny Smith, Modi and Mcintosh, as well as the putatively high allergenic apple cultivars Golden Delicious and Kanzi. No significant differences in percentages of negative responses of PTPs were found between the three countries. Negative responses did not differ from negative responses to the different apple cultivars we previously found in 2006/2007 in the Netherlands. The size of the PTPs of all apple cultivars tested were correlated to the size of the skin prick tests with birch pollen. These results add to the indications for the low allergenic properties of the low allergenic apple cultivars Santana and Elise, as the number of negative responses were reproducible in three countries within the birch pollen region and were similar to previous results in the Netherlands. These results justify oral challenge studies with Elise and Santana within the birch pollen region, to establish the low allergenic properties for the benefit for apple allergic consumers for definite conclusions.

Background Birch pollen allergies are frequently associated with adverse reactions to various fruits, nuts, or vegetables, described as pollen–food syndrome (PFS) and caused by cross-reactive IgE antibodies primarily directed against Bet v 1. Specific immunotherapy (SIT) represents an effective treatment for inhalant allergies; however, successful birch pollen SIT does not correlate well with the amelioration of concomitant food allergies. Methods As vaccine candidates, apple Mal d 1 as well as hazelnut Cor a 1 derivatives were designed by in silico backbone analyses of the respective allergens. The proteins were produced by site-directed mutagenesis as fold variants of their parental allergens. Because Mal d 1 and Cor a 1 form cysteine-mediated aggregates, nonaggregative cysteine to serine mutants were also generated. The proteins were characterized physicochemically, immunologically, and in in vivo models with or without adjuvant. Results The structurally modified proteins showed significantly decreased IgE binding capacity. Notably, both in vivo models revealed reduced immunogenicity of the hypoallergenic fold variants. When formulated with alum, the monomeric cysteine mutants induced a similar immune response as the aggregated parental allergens, which is in contrast with data published on Bet v 1. Conclusion These findings lead to the suggestion that the Bet v 1 structure has unique intrinsic properties, which could account for its high allergenicity. Obviously, these characteristics are not entirely shared with its food homologues from apple and hazelnut. Thus, it is important to tackle pollen-related food allergies from different angles for the generation of effective vaccine candidates to treat birch PFS.

The apple (Malus ×domestica) cultivar Santana was shown to have a low allergenicity in a previously reported clinical trial. A protocol was developed to validate these results outside a clinical setting and to market the Santana as a hypoallergenic apple for large-scale consumption. This protocol was designed to ensure safe consumption of the Santana by consumers suffering from apple allergy. We tested the protocol in a pilot in 100 supermarket stores and evaluated in an internet-based survey with 295 respondents. Forty-one percent of the consumers with an apple allergy did not have an allergic reaction to the Santana. Only five percent of the allergic consumers that tried the Santana reported a strong allergic reaction. In concordance with this, the majority (78%) of the mild apple allergic consumers appreciated the Santana. The developed protocol is considered suitable for safe consumption of the Santana providing that information about the possibility of having an allergic reaction among consumers with a mild apple allergy is added to the label.

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 2cm 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.

Background - A considerable number of individuals suffer from oral allergy syndrome (OAS) to apple, resulting in the avoidance of apple consumption. Apple cultivars differ greatly in their allergenic properties, but knowledge of the causes for such differences is incomplete. Mal d 1 is considered the major apple allergen. For Mal d 1, a wide range of isoallergens and variants exist, and they are encoded by a large gene family. To identify the specific proteins/genes that are potentially involved in the allergy, we developed a PCR assay to monitor the expression of each individual Mal d 1 gene. Gene-specific primer pairs were designed for the exploitation of sequence differences among Mal d 1 genes. The specificity of these primers was validated using both in silico and in vitro techniques. Subsequently, this assay was applied to the peel and flesh of fruits from the two cultivars 'Florina' and 'Gala'. Results - We successfully developed gene-specific primer pairs for each of the 31 Mal d 1 genes and incorporated them into a qRT-PCR assay. The results from the application of the assay showed that 11 genes were not expressed in fruit. In addition, differential expression was observed among the Mal d 1 genes that were expressed in the fruit. Moreover, the expression levels were tissue and cultivar dependent. Conclusion - The assay developed in this study facilitated the first characterisation of the expression levels of all known Mal d 1 genes in a gene-specific manner. Using this assay on different fruit tissues and cultivars, we obtained knowledge concerning gene relevance in allergenicity. This study provides new perspectives for research on both plant breeding and immunotherapy.