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1.  Effects of autoclaving and high pressure on allergenicity of hazelnut proteins 
Background
Hazelnut is reported as a causative agent of allergic reactions. However it is also an edible nut with health benefits. The allergenic characteristics of hazelnut-samples after autoclaving (AC) and high-pressure (HHP) processing have been studied and are also presented here. Previous studies demonstrated that AC treatments were responsible for structural transformation of protein structure motifs. Thus, structural analyses of allergen proteins from hazelnut were carried out to observe what is occurring in relation to the specific-IgE recognition of the related allergenic proteins. The aims of this work are to evaluate the effect of AC and HHP processing on hazelnut in vitro allergenicity using human-sera and to analyse the complexity of hazelnut allergen-protein structures.
Methods
Hazelnut-samples were subjected to AC and HHP processing. The specific IgE- reactivity was studied in 15 allergic clinic-patients via western blotting analyses. A series of homology-based-bioinformatics 3D-models (Cora 1, Cora 8, Cora 9 and Cora 11) were generated for the antigens included in the study to analyse the co mplexity of their protein structure. This study is supported by the Declaration of Helsinki and subsequent ethical guidelines.
Results
A severe reduction in vitro in allergenicity to hazelnut after AC processing was observed in the allergic clinic-patients studied. The specific-IgE binding of some of the described immunoreactive hazelnut protein-bands: Cora 1 ~18KDa, Cora 8 ~9KDa, Cora 9 ~35-40KDa and Cora 11 ~47-48 KDa decreases. Furthermore a relevant glycosylation was assigned and visualized via structural analysis of proteins (3D-modelling) for the first time in the protein-allergen Cora 11 showing a new role which could open a new door for allergenicity-unravellings.
Conclusion
Hazelnut allergenicity-studies in vivo via Prick-Prick and other means using AC processing are crucial to verify the data we observed via in vitro analyses. Glycosylation studies provided us with clues to elucidate, in the near future, mechanisms of the structures that contribute to hazelnut allergenicity, which thus, in turn, help alleviate food allergens.
doi:10.1186/2043-9113-2-12
PMCID: PMC3467176  PMID: 22616776
Structural analysis of allergen-proteins and Glycosylation
2.  Oil body-associated hazelnut allergens including oleosins are underrepresented in diagnostic extracts but associated with severe symptoms 
Background
Oil body-associated allergens such as oleosins have been reported for important allergenic foods such as peanut, sesame and hazelnut. Here we investigate whether oil body associated proteins (OAPs) are linked with specific clinical phenotypes and whether they are represented in skin prick test (SPT) reagents.
Methods
A hazelnut OAP fraction was characterized by mass-spectrometry (MS) to identify its major constituents. Polyclonal rabbit antibodies were generated against hazelnut OAPs. The presence of OAPs in commercially available hazelnut SPTs was studied by immunoblot and spiking experiments. OAP-specific IgE antibodies were measured in sera from patients with a convincing history of hazelnut allergy by RAST (n = 91), immunoblot (n = 22) and basophil histamine release (BHR; n = 14).
Results
Hazelnut OAPs were analysed by MS and found to be dominated by oleosins at ~14 and ~17 kDa, and a 27 kDa band containing oleosin dimers and unidentified protein. In 36/91 sera specific IgE against hazelnut OAPs was detected, and confirmed to be biologically active by BHR (n = 14). The majority (21/22) recognized the oleosin bands at 17 kDa on immunoblot, of which 11 exclusively. These OAP-specific IgE responses dominated by oleosin were associated with systemic reactions to hazelnut (OR 4.24; p = 0.015) and negative SPT (χ2 6.3, p = 0.012). Immunoblot analysis using OAP-specific rabbit antiserum demonstrated that commercial SPT reagents are virtually devoid of OAPs, sometimes (3/9) resulting in false-negative SPT. Spiking of SPT reagents with OAP restored serum IgE binding of these false-negative patients on immunoblot at mainly 17 kDa.
Conclusion
Hazelnut allergens found in oil bodies dominated by oleosin are associated with more severe systemic reactions and negative SPT. Defatted diagnostic extracts are virtually devoid of these allergens, resulting in poor sensitivity for detection of IgE antibodies against these clinically relevant molecules.
doi:10.1186/2045-7022-4-4
PMCID: PMC4015814  PMID: 24484687
Food allergy; IgE-mediated; Oil bodies; Oleosins; Hazelnut
3.  High-Throughput NMR Assessment of the Tertiary Structure of Food Allergens 
PLoS ONE  2012;7(7):e39785.
Background
In vitro component-resolved diagnosis of food allergy requires purified allergens that have to meet high standards of quality. These include the authentication of their conformation, which is relevant for the recognition by specific IgE antibodies from allergic patients. Therefore, highly sensitive and reliable screening methods for the analysis of proteins/allergens are required to assess their structural integrity. In the present study one-dimensional 1H Nuclear Magnetic Resonance (1D 1H-NMR) analysis was adopted for the assessment of overall structural and dynamic properties and authentication of a set of relevant food allergens, including non-specific lipid transfer proteins from apple, peach and hazelnut, 7/8S seed storage globulins from hazelnut and peanut, 11S seed storage globulins from hazelnut and peanut, caseins from cows' and goats' milk and tropomyosin from shrimp.
Methodology/Principal Findings
Two sets of 1D 1H-NMR experiments, using 700 MHz and 600 MHz instruments at 298 K were carried out to determine the presence and the extent of tertiary structure. Structural similarity among members of the individual allergen families was also assessed and changes under thermal stress investigated. The nuclear magnetic resonance (NMR) results were compared with structural information available either from the literature, Protein Data Bank entries, or derived from molecular models.
Conclusions/Significance
1D 1H-NMR analysis of food allergens allowed their classification into molecules with rigid, extended and ordered tertiary structures, molecules without a rigid tertiary structure and molecules which displayed both features. Differences in thermal stability were also detected. In summary, 1D 1H-NMR gives insights into molecular fold of proteins and offers an independent method for assessing structural properties of proteins.
doi:10.1371/journal.pone.0039785
PMCID: PMC3388089  PMID: 22768312
4.  Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis 
Allergy  2013;69(2):208-215.
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.
doi:10.1111/all.12306
PMCID: PMC4041322  PMID: 24224690
apple allergy; birch pollen-associated food allergies; hazelnut allergy; pollen–food syndrome; protein remodeling
5.  Differences in the intrinsic immunogenicity and allergenicity of Bet v 1 and related food allergens revealed by site-directed mutagenesis 
Allergy  2013;69(2):208-215.
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.
doi:10.1111/all.12306
PMCID: PMC4041322  PMID: 24224690
apple allergy; birch pollen-associated food allergies; hazelnut allergy; pollen–food syndrome; protein remodeling
6.  IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome 
Allergy  2014;70(1):59-66.
Background
Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail.
Methods
Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA.
Results
Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased.
Conclusion
Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.
doi:10.1111/all.12534
PMCID: PMC4283702  PMID: 25327982
Bet v 1; food allergy; IgA; IgE; IgG
7.  Long-Term Characteristics of Hazelnut Allergy in an Adjuvant-Free Mouse Model 
Background
Clinically it is recognized that tree nut allergies such as hazelnut allergy are not usually outgrown. Specific mechanisms underlying the persistence of such food allergies are incompletely understood. Here we studied the natural history and the long-term immune and clinical characteristics of hazelnut allergy in an adjuvant-free mouse model.
Methods
BALB/c mice were sensitized to hazelnut protein using a transdermal sensitization protocol that does not use adjuvant. After establishing sensitization, exposure to hazelnut was withdrawn for 3, 5 or 8 months. The fate of circulating IgE antibodies was monitored. Subsequently, mice were given booster exposures and examined for memory IgE antibody and spleen cell IL-4 responses. Clinical characteristics and hypothermia responses upon oral allergen challenge were studied.
Results
Upon allergen withdrawal, circulating hazelnut-specific IgE antibody levels began to drop. Nevertheless, IgE responses once established remained at significantly high levels for up to 8 months (the last time point studied) despite withdrawal of allergen exposure. Memory IgE responses to booster exposures were robust after 3, 5 or 8 months of allergen withdrawal. Furthermore, significant clinical reactivity to oral hazelnut challenge, and hypothermia responses were demonstrable at each of these time points. Long-lasting spleen cell memory IL-4 responses to hazelnut were detectable in these mice explaining the mechanism of sustenance of IgE responses and clinical sensitization.
Conclusions
Hazelnut allergy once established persists for long periods, despite withdrawal of allergen exposure, due to long-lasting, memory IgE and IL-4 responses.
doi:10.1159/000283028
PMCID: PMC2895740  PMID: 20145410
Hazelnut allergy, natural history; Systemic anaphylaxis; Immunoglobulin E; T helper 2 lymphocyte profile; Mouse model, adjuvant-free; Food allergy
8.  Serum IgE Antibodies against Hazelnut in Hazelnut Processing Workers 
The Scientific World Journal  2012;2012:108953.
Aim. Previous studies have shown a higher sensitization rate to hazelnut in processing workers but no relation was found between the respiratory symptoms in workplace and hazelnut sensitization. Material and Method. To evaluate the association between the hazelnut sensitization and workplace-related respiratory complaints, hazelnut processing workers had undergone a questionnaire included work-related respiratory symptoms, smoking history, pulmonary function testing, and measurement of serum IgE antibodies against hazelnut. Results. This study consisted of 88 hazelnut processing workers (79 females and 9 males), aged 14–59 years (Mean ± SD: 33.8 ± 10.5 years). The mean working duration was 38.8 ± 36.6 months (min: 1–max: 180). Specific IgE against hazelnut allergens was positive in 14 of cases (17.1%). There was no significant difference between the cases with and without specific IgE against hazelnut allergens regarding respiratory symptoms, history of allergy, smoking status and spirometric values. Conclusion. 17.1% of the hazelnut processing workers were seropositive against hazelnut. Being sensitized to hazelnut was not found to be associated with work-related respiratory symptoms in this study. Further studies are needed in hazelnut workers respiratory health to search topics other than asthma.
doi:10.1100/2012/108953
PMCID: PMC3541565  PMID: 23326210
9.  The 11S globulin Sin a 2 from yellow mustard seeds shows IgE cross-reactivity with homologous counterparts from tree nuts and peanut 
Background
The 11S globulin Sin a 2 is a marker to predict severity of symptoms in mustard allergic patients. The potential implication of Sin a 2 in cross-reactivity with tree nuts and peanut has not been investigated so far. In this work, we studied at the IgG and IgE level the involvement of the 11S globulin Sin a 2 in cross-reactivity among mustard, tree nuts and peanut.
Methods
Eleven well-characterized mustard-allergic patients sensitized to Sin a 2 were included in the study. A specific anti-Sin a 2 serum was obtained in rabbit. Skin prick tests (SPT), enzyme-linked immunosorbent assay (ELISA), immunoblotting and IgG or IgE-inhibition immunoblotting experiments using purified Sin a 2, Sin a 1, Sin a 3, mustard, almond, hazelnut, pistachio, walnut or peanut extracts were performed.
Results
The rabbit anti-Sin a 2 serum showed high affinity and specificity to Sin a 2, which allowed us to demonstrate that Sin a 2 shares IgG epitopes with allergenic 11S globulins from tree nuts (almond, hazelnut, pistachio and walnut) but not from peanut. All the patients included in the study had positive skin prick test to tree nuts and/or peanut and we subdivided them into two different groups according to their clinical symptoms after ingestion of such allergenic sources. We showed that 11S globulins contain conserved IgE epitopes involved in cross-reactivity among mustard, tree nuts and peanut as well as species-specific IgE epitopes.
Conclusions
The allergenic 11S globulin Sin a 2 from mustard is involved in cross-reactivity at the IgE level with tree nuts and peanut. Although the clinical relevance of the cross-reactive IgE epitopes present in 11S globulins needs to be investigated in further detail, our results contribute to improve the diagnosis and management of mustard allergic patients sensitized to Sin a 2.
doi:10.1186/2045-7022-2-23
PMCID: PMC3583068  PMID: 23231956
Food allergy; Mustard allergy; Tree nut allergy; Peanut allergy; Cross-reactivity; 11S globulins; Sin a 2; lgG/IgE epitopes
10.  Biosensor immunoassay for traces of hazelnut protein in olive oil 
The fraudulent addition of hazelnut oil to more expensive olive oil not only causes economical loss but may also result in problems for allergic individuals as they may inadvertently be exposed to potentially allergenic hazelnut proteins. To improve consumer safety, a rapid and sensitive direct biosensor immunoassay, based on a highly specific monoclonal antibody, was developed to detect the presence of hazelnut proteins in olive oils. The sample preparation was easy (extraction with buffer); the assay time was fast (4.5 min only) and the limit of detection was low (0.08 μg/g of hazelnut proteins in olive oil). Recoveries obtained with an olive oil mixed with different amounts of a hazelnut protein containing hazelnut oil varied between 93% and 109%.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-009-2720-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s00216-009-2720-1
PMCID: PMC2724632  PMID: 19263041
Biosensor; Immunoassay; Monoclonal antibody; Olive oil; Hazelnut oil; Hidden allergens
11.  Allergenic Lipid Transfer Proteins from Plant-Derived Foods Do Not Immunologically and Clinically Behave Homogeneously: The Kiwifruit LTP as a Model 
PLoS ONE  2011;6(11):e27856.
Background
Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet.
Objective
Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins.
Methods
Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out.
Results
Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients' diet.
Conclusion
The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention.
doi:10.1371/journal.pone.0027856
PMCID: PMC3219694  PMID: 22114713
12.  Food allergies in developing and emerging economies: need for comprehensive data on prevalence rates 
Although much is known today about the prevalence of food allergy in the developed world, there are serious knowledge gaps about the prevalence rates of food allergy in developing countries. Food allergy affects up to 6% of children and 4% of adults. Symptoms include urticaria, gastrointestinal distress, failure to thrive, anaphylaxis and even death. There are over 170 foods known to provoke allergic reactions. Of these, the most common foods responsible for inducing 90% of reported allergic reactions are peanuts, milk, eggs, wheat, nuts (e.g., hazelnuts, walnuts, almonds, cashews, pecans, etc.), soybeans, fish, crustaceans and shellfish. Current assumptions are that prevalence rates are lower in developing countries and emerging economies such as China, Brazil and India which raises questions about potential health impacts should the assumptions not be supported by evidence. As the health and social burden of food allergy can be significant, national and international efforts focusing on food security, food safety, food quality and dietary diversity need to pay special attention to the role of food allergy in order to avoid marginalization of sub-populations in the community. More importantly, as the major food sources used in international food aid programs are frequently priority allergens (e.g., peanut, milk, eggs, soybean, fish, wheat), and due to the similarities between food allergy and some malnutrition symptoms, it will be increasingly important to understand and assess the interplay between food allergy and nutrition in order to protect and identify appropriate sources of foods for sensitized sub-populations especially in economically disadvantaged countries and communities.
doi:10.1186/2045-7022-2-25
PMCID: PMC3551706  PMID: 23256652
Food allergy; Food hypersensitivity; Nutrition; Developing countries
13.  435 Increasing Incidence of Food Allergy in Zimbabwe 
The World Allergy Organization Journal  2012;5(Suppl 2):S155-S156.
Background
Data relating to allergic diseases in general and food allergies in particular in the Central African region is scant. Despite observations by the ISAAC studies that airborne allergen sources were common, little has been reported about food allergens. We reviewed data from our laboratory and outpatient records of patients consulted to estimate the magnitude of the disease in our population.
Methods
Patients attending the only specialist allergy diagnostic facility in the country (Asthma, Allergy and Immune Dysfunction Clinic) were offered semi-quantitative allergen specific IgE antibody determination as part of their diagnostic work-up. Alongside skin-prick testing, the Euroimmun immunoblots were used to establish IgE reactivity to a variety of allergen sources.
Results
Six hundred thirty five patients were enrolled between January 2009 and April 2011. These were born between 1931 and 2010. IgE reactivity to egg, codfish, cows milk, wheat flour, rice, soya bean, peanut, hazelnut, carrot, potato and apple was investigated using the immunoblot technique. Results were scored negative or positive. The grades of positive were weak (±), low (+), moderate (++) and high (+++). Overall, 47% of the patients reacted to one or multiple allergen sources. Across the age spectrum, allergen specific IgE reactivity was most frequent against potato (16%) and peanut (15%) and lowest against milk (2.7%) and codfish (2.7%), others were intermediate. Egg white reactivity was highest in those below the age of 5 years (7%). IgE reactivity in patients born before 1959 was less than 1%. This increased to 3.4%, 4.8% and 64% respectively in those born before 1969, 1979 and 1989. Nineteen (19%) of patients born in 1990 to 1999 were reactive to a variety of food allergen sources. Likewise, 12% of those born between 2000 and 2011 were reactive. Food allergen reactivity paralleled inhalant allergen source sensitisation in all age groups.
Conclusions
In this sample of symptomatic patients we have shown that allergen specific IgE reactivity to dietary sources was high. An exponential increase in IgE reactivity in patients born between 1990 and 2011 was a surprising observation. Possible explanations include urbanisation, life-style and dietary changes in this predominantly urban population. The results call for a systematic investigation of the predisposing factors.
doi:10.1097/01.WOX.0000412198.86370.67
PMCID: PMC3512812
14.  Purification, crystallization and initial crystallographic characterization of brazil-nut allergen Ber e 2 
The crystallization of the brazil nut allergen Ber e 2 is reported.
Peanut and tree-nut allergies have attracted considerable attention because of their frequency and their lifelong persistence. Brazil-nut (Bertholletia excelsa) allergies have been well documented and the 11S legumin-like seed storage protein Ber e 2 (excelsin) is one of the two known brazil-nut allergens. In this study, Ber e 2 was extracted from brazil-nut kernels and purified to high purity by crystalline precipitation and gel-filtration chromatography. Well diffracting single crystals were obtained using the hanging-drop vapour-diffusion method. A molecular-replacement structural solution has been obtained. Refinement of the structure is currently under way.
doi:10.1107/S1744309107051445
PMCID: PMC2339761  PMID: 18007055
brazil nut allergy; Ber e 2; cupin superfamily; allergenicity
15.  430 Comparative Analysis of Patients with Birch Pollinosis and Patients with Associated Plant Food Allergy 
Background
Even 70% patients allergic to pollens of plants are developing undesirable symptoms after eating foods of the plant origin. It is most often a result of the cross-allergy between these allergens. The aim of the study was to compare the group of patients with pollinosis with patients with pollinosis and food allergy.
Methods
Fifty eight patients at the age above 16 were included in the study. Patients were divided into 2 groups. Patients included in the first group were birch allergic without any symptoms after eating food (23 persons). Patients in the other group had birch pollen allergy and they had reported clinical symptoms after eating foods such as: apple, celery, carrot, tomato, banana, peach, peanut and hazelnut (35 persons). The skin prick tests with pollen and food allergens (commercial and native) and serum IgE concentration (total and specific) were determined for all individuals. The immunoblotting was performed for the patients with the positive value of birch, apple, celery and/or carrot specific IgE to confirm the cross-reactivity.
Results
Patients with pollinosis and symptoms after eating plant foods were characterized by a significantly larger percentage of positive skin tests with the hazel allergen. In the first group patients revealed positive results of skin tests with food allergens, although they didn't reported the problem after consumption of them. No difference in total IgE levels was found between the 2 groups (271.5 ± 403.8 IU/mL vs 242.5 ± 340.9 IU/mL). Patients with birch allergy and hypersensitivity to food allergens showed significantly higher birch pollen specific IgE levels (11.8 ± 14.1 IU/mL vs 4.1 ± 6.6 IU/mL).
Conclusions
Sixty percent of all the patients with birch pollinosis reported manifestations symptoms after eating certain kind of food. These patients had most often clinical symptoms after eating apples, hazelnuts and of peaches, and less frequently symptoms after eating carrots, celery, peanuts, tomatoes and bananas. Although it seems that false positive results of skin tests with food allergens in the control group and the high level of the birch specific IgE might be the predictive factor of the allergy which may develop later; they require further studies.
doi:10.1097/01.WOX.0000412193.93994.62
PMCID: PMC3513115
16.  Severe Allergic Reactions to Food in Norway: A Ten Year Survey of Cases Reported to the Food Allergy Register 
The Norwegian Food Allergy Register was established at the Norwegian Institute of Public Health in 2000. The purpose of the register is to gain information about severe allergic reactions to food in Norway and to survey food products in relation to allergen labelling and contamination. Cases are reported on a voluntary basis by first line doctors, and submitted together with a serum sample for specific IgE analysis. The register has received a total of 877 reports from 1 July, 2000 to 31 December, 2010. Two age groups, small children and young adults are over-represented, and the overall gender distribution is 40:60 males-females. The legumes lupine and fenugreek have been identified as two “new” allergens in processed foods and cases of contamination and faults in production of processed foods have been revealed. The highest frequency of food specific IgE is to hazelnuts and peanuts, with a marked increase in reactions to hazelnuts during the last three years. The Food Allergy Register has improved our knowledge about causes and severity of food allergic reactions in Norway. The results show the usefulness of population based national food allergy registers in providing information for health authorities and to secure safe food for individuals with food allergies.
doi:10.3390/ijerph8083144
PMCID: PMC3166732  PMID: 21909296
severe reactions to food; IgE-mediated reactions to food; food allergy register; food allergens
17.  Bacteria Associated with Hazelnut (Corylus avellana L.) Decline Are of Two Groups: Pseudomonas avellanae and Strains Resembling P. syringae pv. syringae 
A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the syrB gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with HinfI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the syrB gene, are pathogenically restricted to Corylus avellana, and belong to Pseudomonas avellanae. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to Pseudomonas syringae pv. syringae.
doi:10.1128/AEM.68.2.476-484.2002
PMCID: PMC126672  PMID: 11823181
18.  Food Allergy in Lebanon: Is Sesame Seed the "Middle Eastern" Peanut 
A randomly sampled, cross-sectional serology test-based survey was conducted in Lebanon to describe the pattern of food allergy among Lebanese population. The prevalence of specific Immunoglobulin E (IgE) to food allergens was investigated in 20 laboratories in different regions of Lebanon by an immunoblot assay over a 1 year period. Clinical correlation was determined in two university hospitals. There were 1842 patients with suspected IgE-mediated food allergic reactions tested for specific IgE upon their physician's request. Clinical correlation was done in 93 patients. We identified 386 out of 1842 (20.95%) patients with positive specific IgE to food allergens. The clinical presentations were cutaneous, digestive, and anaphylaxis. The major cause of allergy was cow's milk in infants and young children, hazelnut and wheat flour in adults. Although specific IgE to peanut in infants, children, and adults were higher than for sesame, peanut-induced allergic reactions were mild, in contrary to sesame where anaphylaxis was the only clinical manifestation. Recently, sesame has been recognized as an increasingly frequent and potentially severe allergen. Further studies with double-blind, placebo-controlled food challenge are needed to establish the real prevalence of food allergy in Lebanon, and to determine the most common allergens taking in consideration the nutritional habits of our population.
doi:10.1097/WOX.0b013e318204b745
PMCID: PMC3651063  PMID: 23283020
food allergy; sesame; peanut
19.  Clinical study of peanut and nut allergy in 62 consecutive patients: new features and associations. 
BMJ : British Medical Journal  1996;312(7038):1074-1078.
OBJECTIVE--To investigate clinical features of acute allergic reactions to peanuts and other nuts. DESIGN--Analysis of data from consecutive patients seen by one doctor over one year in an allergy clinic at a regional referral centre. SUBJECTS--62 patients aged 11 months to 53 years seen between October 1993 and September 1994. MAIN OUTCOME MEASURES--Type and severity of allergic reactions, age at onset of symptoms, type of nut causing allergy, results of skin prick tests, and incidence of other allergic diseases and associated allergies. RESULTS--Peanuts were the commonest cause of allergy (47) followed by Brazil nut (18), almond (14), and hazelnut (13). Onset of allergic symptoms occurred by the age of 2 years in 33/60 and by the age of 7 in 55/60. Peanuts accounted for all allergies in children sensitised in the first year of life and for 82% (27/33) of allergies in children sensitised by the third year of life. Multiple allergies appeared progressively with age. The commonest symptom was facial angioedema, and the major feature accounting for life threatening reactions was laryngeal oedema. Hypotension was uncommon. Of 55 patients, 53 were atopic--that is, had positive skin results of tests to common inhaled allergens--and all 53 had other allergic disorders (asthma, rhinitis, eczema) due to several inhaled allergens and other foods. CONCLUSIONS--Sensitisation, mainly to peanuts, is occurring in very young children, and multiple peanut/nut allergies appear progressively. Peanut and nut allergy is becoming common and can cause life threatening reactions. The main danger is laryngeal oedema. Young atopic children should avoid peanuts and nuts to prevent the development of this allergy.
PMCID: PMC2350892  PMID: 8616415
20.  Crystallization and preliminary X-ray diffraction analysis of the magnesium transporter CorA 
Full-length and soluble domains of the integral membrane protein CorA have been expressed, purified and crystallized. X-ray diffraction data have been collected and analyzed.
The full-length integral membrane protein CorA from Thermotoga maritima (TmCorA1–351) has been expressed in Escherichia coli and purified without membrane isolation. TmCorA1–351 crystallized in the monoclinic space group C2, with unit-cell parameters a = 214.25, b = 86.30, c = 181.53 Å, β = 112.23°. Native crystals diffracted to 3.7 Å using synchrotron radiation, but selenomethionine-substituted crystals rarely diffracted to better than 5.0 Å. All full-length protein crystals were highly mosaic and produced anisotropic diffraction patterns. To aid in crystallographic phasing, soluble domain constructs were screened and the periplasmic domain of CorA from Archaeoglobus fulgidus (AfCorA1–263) was crystallized in the hexagonal space group P6122, with unit-cell parameters a = b = 101.17, c = 142.87 Å. Native and SeMet-substituted AfCorA1–263 crystals diffracted to ∼3.0 Å using synchrotron radiation.
doi:10.1107/S1744309106000996
PMCID: PMC2150960  PMID: 16511288
membrane proteins; transporter; magnesium
21.  Crystallization and preliminary X-ray analysis of a bifunctional catalase-phenol oxidase from Scytalidium thermophilum  
The bifunctional enzyme catalase-phenol oxidase from S. thermophilum was crystallized by the hanging-drop vapour-diffusion method in space group P21 and diffraction data were collected to 2.8 Å resolution.
Catalase-phenol oxidase from Scytalidium thermophilum is a bifunctional enzyme: its major activity is the catalase-mediated decomposition of hydrogen peroxide, but it also catalyzes phenol oxidation. To understand the structural basis of this dual functionality, the enzyme, which has been shown to be a tetramer in solution, has been purified by anion-exchange and gel-filtration chromatography and has been crystallized using the hanging-drop vapour-diffusion technique. Streak-seeding was used to obtain larger crystals suitable for X-ray analysis. Diffraction data were collected to 2.8 Å resolution at the Daresbury Synchrotron Radiation Source. The crystals belonged to space group P21 and contained one tetramer per asymmetric unit.
doi:10.1107/S1744309109012007
PMCID: PMC2675591  PMID: 19407383
Scytalidium thermophilum; Humicola insolens; catalases; phenol oxidases; catechol oxidases; CATPO
22.  Purification, crystallization and initial crystallographic characterization of peanut major allergen Ara h 3 
The crystallization of peanut allergen Ara h 3 is reported.
The peanut is a significant food source, but is responsible for many cases of anaphylaxis. The peanut 11S legumin-like seed storage protein Ara h 3 is one of the best characterized allergens. In this study, Ara h 3 was extracted from peanut kernels and purified by sequential anion-exchange, hydrophobic interaction and gel-filtration chromatography to very high purity to facilitate crystallization and structural studies. Well diffracting single crystals were obtained by the vapor-diffusion method. A molecular-replacement structural solution has been obtained and refinement of the structure is currently under way.
doi:10.1107/S1744309107041176
PMCID: PMC2339721  PMID: 17909286
food safety; allergy; cupin; glycinin
23.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae  
The major group 7 allergen, Der f 7, from the dust mite Dermatophagoides farinae has been crystallized and diffracted X-rays to a resolution of 2.24 Å.
Der f 7 is a major group 7 allergen from the dust mite Dermatophagoides farinae that shows 86% sequence identity to the homologous allergen Der p 7 from D. pteronyssinus. Der f 7 was successfully overexpressed in an Escherichia coli expression system and purified to homogeneity using Ni–NTA affinity and size-exclusion column chromatography. SeMet-labelled Der f 7 was crystallized by the hanging-drop vapour-diffusion method using a reservoir solution consisting of 0.1 M bis-tris pH 7.4 and 28% polyethylene glycol monomethyl ether 2000 at 293 K. X-ray diffraction data were collected to 2.24 Å resolution using synchrotron radiation. The crystals belonged to the orthorhombic system, space group P212121, with unit-cell parameters a = 50.19, b = 58.67, c = 123.81 Å. Based on the estimated Matthews coefficient (2.16 Å3 Da−1), two molecules of Der f 7 could be present in the asymmetric unit of the crystal lattice.
doi:10.1107/S174430911103836X
PMCID: PMC3232152  PMID: 22139179
group 7 allergens; Der f 7; Dermatophagoides farinae
24.  219 The Revised Edition of Korean Calendar for Allergenic Pollens 
Background
The old version of pollen calendar was used until this year in South Korea. That calendar did not reflect current pollen distribution and concentrations that can be influenced by changes in weather and environment. A new pollen calendar of allergenic pollens was made based on the data on pollen concentrations obtained in 8 regions nationwide between 1997 and 2009 in South Korea.
Methods
The distribution of pollen grains was assessed every day at 8 areas (Seoul, Guri, Busan, Daegu, Jeonju, Kwangju, Kangneung, and Jeju) nationwide for 12 years between July 1, 1997 and June 30, 2009. Pollen were collected by using Burkard 7-day sampler (Burkard manufacturing Co Ltd, Hertfordshire, UK), and the collected pollens were sent every week to Hanyang Guri Hospital. Then pollens were strained with Calberla's fuchsin staining solution and were identified. The number of pollen grains per m3 was calculated.
Results
Alder, birch and Japanese cedar started to appear in February. Japanese cedar showed a highest pollen concentration in Jeju. Pine became the highest pollen in May, and the pollen concentrations of oak and birch also became high. Common ragweed appeared in the middle of August and showed the highest pollen concentration in the middles of September. Japanese hop showed a high concentration between the middle of August and the end of September, and mugwort appeared in the middles of August and its concentration increased up until early September. Birch appeared earlier in Kangneung, and pine showed a higher pollen concentration than in the other areas. In Daegu, Oriental thuja, alder and juniper produced a large concentration of pollens. Pine produced a large concentration of pollens between the middle of April and the end of May. Weeds showed higher concentrations in September and mugwort appeared earlier than common ragweed. In Busan where is the southeast city, the time of flowering is relatively early, and alder and Oriental thuja appeared earliest among all areas. In Kwangju, Oriental thuja and hazelnut appeared in early February. In Jeju which is the end of southern island, Japanese cedar showed a higher pollen concentration than the other areas
Conclusions
New information on pollen distributions and concentrations should be provided for the general publics or allergic patients through the website in order to prevent the occurrence of pollinosis.
doi:10.1097/01.WOX.0000411976.47900.cf
PMCID: PMC3513006
25.  The Revised Edition of Korean Calendar for Allergenic Pollens 
The old calendar of pollens did not reflect current pollen distribution and concentrations that can be influenced by changes of weather and environment of each region in South Korea. A new pollen calendar of allergenic pollens was made based on the data on pollen concentrations obtained in eight regions nationwide between 1997 and 2009. The distribution of pollen was assessed every day at 8 areas (Seoul, Guri, Busan, Daegu, Jeonju, Kwangju, Kangneung, and Jeju) for 12 years between July 1, 1997 and June 30, 2009. Pollens were collected by using Burkard 7-day sampler (Burkard Manufacturing Co Ltd, UK). Pollens which were stained with Calberla's fuchsin staining solution were identified and counted. Pine became the highest pollen in May, and the pollen concentrations of oak and birch also became high. Ragweed appeared in the middle of August and showed the highest pollen concentration in the middles of September. Japanese hop showed a high concentration between the middle of August and the end of September, and mugwort appeared in the middles of August and its concentration increased up until early September. In Kangneung, birch appeared earlier, pine showed a higher pollen concentration than in the other areas. In Daegu, Oriental thuja and alder produced a large concentration of pollens. Pine produced a large concentration of pollens between the middle of April and the end of May. Weeds showed higher concentrations in September and mugwort appeared earlier than ragweed. In Busan the time of flowering is relatively early, and alder and Oriental thuja appeared earliest among all areas. In Kwangju, Oriental thuja and hazelnut appeared in early February. Japanese cedar showed the highest pollen concentration in March in Jeju. In conclusion, update information on pollen calendar in South Korea should be provided for allergic patients through the website to manage and prevent the pollinosis.
doi:10.4168/aair.2012.4.1.5
PMCID: PMC3242062  PMID: 22211164
Allergens; pollen

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