Buckwheat: an emerging allergen

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Real-time PCR Detection Methods for Food Allergens (Wheat, Buckwheat, and Peanuts) Using Reference Plasmids.

Specific and sensitive real-time qualitative polymerase chain reaction (PCR) methods for the detection of food allergens including wheat, buckwheat, and peanuts were developed that could cancel between instrument effects and avoid risks of false-positives and false-negatives. In these real-time PCR analysis, the cutoff for determination of positive samples was set in every PCR run by using reference plasmids containing known copies of the target sequences. The copy numbers of the plasmids were used to detect the allergenic ingredients corresponding to 10 ppm (w/w) protein in highly processed foods (cooked for more than 30 min at 122 °C). Reference plasmid analysis for each real-time PCR run helped to minimize variability between runs and instruments (7900HT Real-Time PCR systems and Light Cycler Nano). It also helped to avoid false positives due to trace levels of contaminants from the laboratory environment or agricultural products. The specificity of the real-time PCR method was verified using 79 commonly used food materials and some of their relatives. The method was sensitive enough to detect those allergenic ingredients corresponding to 10 ppm (w/w) in seven types of incurred samples. The current official Japanese method was not able to detect the allergens in some of the incurred samples. The developed method can avoid false negatives due to lack of sensitivity and is useful to confirm positive ELISA screening tests.

Oral Immunotherapy with a Phosphorylated Hypoallergenic Allergen Ameliorates Allergic Responses More Effectively Than Intact Allergen in a Murine Model of Buckwheat Allergy.

SCOPE: Buckwheat is a common food allergen frequently consumed in Asian countries, with Fag e 1 and Fag e 2 being the major buckwheat allergens. The purpose of this study is to prepare an oral immunotherapy agent by attenuating these allergens via phosphorylation. The immunomodulatory effects of phosphorylated Fag e 2 (P-Fag e 2) in a mouse model of buckwheat allergy are evaluated. METHODS AND RESULTS: Phosphorylated Fag e 1 (P-Fag e 1) and P-Fag e 2 are prepared by dry-heating in the presence of pyrophosphate. Subsequent dot-blot analysis using serum from food-allergic patient indicates that both proteins exhibit reduced allergenicity upon phosphorylation. Mice subjected to oral administration of P-Fag e 2 for 6 weeks exhibit decreased specific serum IgE and increased specific IgA after Fag e 2 sensitization compared to the sham-treated mice. Moreover, the Peyer's patches (PP) of phosphorylated antigen-fed mice show decreased IL-4 production and induction of T follicular helper (Tfh) cells. Increased production of IL-6 is observed in the CD11c+ cells isolated from the PPs of P-Fag e 2-fed mice. CONCLUSION: These results indicate that attenuated allergens can suppress Th2-induced allergic responses via induction of Tfh cells, which are regulated by IL-6 secreted from dendritic cells.

Specific IgE for Fag e 3 Predicts Oral Buckwheat Food Challenge Test Results and Anaphylaxis: A Pilot Study.

BACKGROUND: Buckwheat (BW) is the source of a life-threatening allergen. Fag e 3-specific serum IgE (sIgE) is more useful than BW-sIgE for diagnosis; however, it is unknown whether Fag e 3-sIgE can predict oral food challenge (OFC) results and anaphylaxis. This study aimed to clarify the efficacy of Fag e 3-sIgE in predicting OFC results and anaphylaxis. METHODS: We conducted a retrospective review of BW- and Fag e 3-sIgE data obtained using the ImmunoCAP® assay system and fluorescent enzyme-linked immunosorbent assay from children who underwent OFC using 3,072 mg of BW protein between July 2006 and March 2014 at Sagamihara National Hospital, Kanagawa, Japan. RESULTS: We analyzed 60 patients aged 1.9-13.4 years (median 6.0 years); 20 (33%) showed objective symptoms upon BW OFC. The patients without symptoms had significantly lower Fag e 3-sIgE than those with non-anaphylactic (p < 0.001) and anaphylactic reactions to BW (p = 0.004). Fag e 3-sIgE was the only tested factor that significantly predicted positive OFC results (odds ratio 8.93, 95% confidence interval 3.10-25.73, p < 0.001) and OFC-induced anaphylaxis (2.67, 1.12-6.35, p = 0.027). We suggest that a threshold Fag e 3-sIgE level of 18.0 kUE/L has 95% probability of provoking a positive reaction to BW. CONCLUSIONS: Fag e 3-sIgE predicted OFC results and OFC-induced anaphylaxis. We further emphasize paying careful attention to the risk of BW OFC-induced anaphylaxis.

Biphasic buckwheat anaphylaxis:Case report and systematic review.

Buckwheat anaphylaxis is commonly recognized in Europe and Asia, and there is only one case reported in Taiwan so far. Here, we report a case of biphasic buckwheat anaphylaxis in a 57 year-old male patient who lost consciousness twice in the same day after having buckwheat noodles. The serum test shows that Dermatophagoides pteronyssinus (Dp)immunoglobulin E (IgE) (42.4 kU/L) and buckwheat-specific IgE (81.5 kU/L) are unusually high. Although biphasic buckwheat anaphylaxis is rare, we should still be aware the second episode could be life-threatening and happen within a day after the exposure to the buckwheat antigen.

Reactions of Buckwheat-Hypersensitive Patients during Oral Food Challenge Are Rare, but Often Anaphylactic.

BACKGROUND: Buckwheat (BW) is a common cause of life-threatening allergy in Asia. Few have examined oral food challenges (OFCs) using BW. We here describe the OFC outcomes for the diagnosis or confirmation of tolerance acquisition and clarify risk factors for positive OFCs. METHODS: Between July 2005 and March 2014, we retrospectively reviewed data from children who underwent OFCs using 3,072 mg of BW protein at Sagamihara National Hospital. Children were suspected of having BW allergy because of positive results for BW-specific IgE or because they had been previously diagnosed with BW allergy owing to immediate reactions to BW. RESULTS: Of 476 such patients, we analyzed 419 aged 1-17 years (median age 6.7 years). Forty-four (10.5%) reacted to the BW OFC and 24 (54.5%) experienced anaphylaxis. Among patients with suspected BW allergies (n = 369), 30 (8.1%) reacted to OFC. However, among patients with definitive BW allergies (n = 50) who underwent OFCs a median of 7.0 years after their last immediate reaction, 14 (28.0%) reacted to OFC. Among 12 patients with past anaphylactic reactions to BW, 8 exhibited tolerance to BW. A history of immediate reaction to BW and high BW-specific IgE levels were significant risk factors for a positive OFC. CONCLUSIONS: BW allergies are rare among children suspected of having BW allergies due to positivity for BW-specific IgE. Most children with definitive BW allergies can tolerate BW, even after anaphylactic reactions. Nevertheless, careful observation is needed when performing BW OFCs, considering the high incidence of anaphylactic reactions.

Effect of Proteolysis with Alkaline Protease Following High Hydrostatic Pressure Treatment on IgE Binding of Buckwheat Protein.

Buckwheat is a popular food material in many Asian countries and it contains major allergenic proteins. This study was performed to analyze the effects of hydrolysis with alkaline protease following high hydrostatic pressure (HHP) treatment on the IgE binding of buckwheat protein. Extracted buckwheat protein was treated with HHP at 600 MPa for 30 min and hydrolyzed with alkaline protease for 240 min. IgE binding was examined using an enzyme-linked immunosorbent assay (ELISA) with serum samples from 14 patients who were allergic to buckwheat. Depending on the serum samples, HHP treatment of buckwheat protein without enzymatic hydrolysis decreased the IgE binding by 8.9% to 73.2% or increased by 31% to 78%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease decreased by 73.8% to 100%. The IgE binding of buckwheat protein hydrolyzed with alkaline protease following HHP treatment decreased by 83.8% to 100%. This suggested that hydrolysis with alkaline protease following HHP treatment could be applied to reduce the IgE binding of buckwheat protein.

Development of a simple IgE-independent anaphylactic model using buckwheat antigen and B-type CpG oligodeoxynucleotide from Streptococcus thermophilus.

We developed a severe anaphylactic model in mice using buckwheat antigen and B-type CpG-oligodeoxynucleotides (CpG-ODNs) from Streptococcus thermophilus genome. In typical systemic anaphylaxis models, animals are challenged with large quantity of antigens via an intravenous (i.v.) route. Here, we showed a simple anaphylactic shock after challenge via intraperitoneal (i.p.) route. The i.p. method is simpler than i.v. administration and has a lower risk for failure. To generate this anaphylactic model, 5-week-old female BALB/c mice were first i.p. sensitized with buckwheat antigen mixed with B-type CpG-ODN. After 2 weeks, mice were challenged with antigen to induce anaphylactic shock, which was evaluated by scoring the severity symptoms and measuring serum levels of various proteins and splenic cell producing cytokines. Immunoglobulin (Ig)G2a production and interferon-γ positive cells were markedly increased in mice immunized with antigen mixed with B-type CpG-ODN, whereas serum IgE levels were decreased by B-type CpG-ODN. We also examined the effects of various ODNs (A, B and C-type CpG-ODNs) and antigens (buckwheat, α-casein, ß-lactoglobulin and ovalbumin) on anaphylactic severity, and found that the combination of buckwheat and B-type CpG-ODN induced the most intense anaphylactic shock. This model is expected to contribute to the study of the prevention of anaphylactic shock.

[Allergic reaction after ingestion of pancakes]. / Allergisk reaktion efter indtag af pandekager.

Buckwheat is increasingly used in food and pillows in Europe and is a potent potential allergen when ingested or inhaled. A 74-year-old Danish woman experienced anaphylaxis after ingesting industrially manufactured pancakes. She was IgE-sensitized and was persistently asymptomatic after allergen elimination. A thorough medical history is pivotal to identify untraditional allergens that can evoke severe allergic reactions.

[Buckwheat allergy can cause live-threatening anaphylaxia].

Alongside with the globalization of the Danish kitchen and the introduction of a larger number of commercial food items containing buckwheat, buckwheat allergy no longer seems to be a rare disease in Denmark. Since buckwheat allergy can cause life-threathening reactions, it is important for medical doctors to be aware of this allergy. Reliable serological tests showing specific IgE to buckwheat protein 2S albumin (Fag e2) are available in most countries. Three patients with anaphylactic reactions due to buckwheat allergy are presented.

Ribonuclease activity of buckwheat plant (Fagopyrum esculentum) cultivars with different sensitivities to buckwheat burn virus.

Ribonucleases (RNases) are present in base-level amounts in intact plants, but this level is able to increase greatly under stress conditions. The possible cause for such an increase is protection against plant RNA-virus attack. Buckwheat burn virus (BBV) is a highly virulent pathogen that belongs to Rhabdoviridae family. In our study, we have analyzed the correlation between RNase activity and resistance of different buckwheat cultivars to BBV infection. Two cultivars, Kara-Dag and Roksolana, with different sensitivities to BBV have been used. Kara-Dag is a cultivar with medium sensitivity to virus and Roksolana is a tolerant cultivar. It has been shown that the base level of RNase activity in Roksolana cultivar was in most cases higher than the corresponding parameter in Kara-Dag cultivar. Both infected and uninfected plants of Roksolana cultivar demonstrated high RNase activity during two weeks. Whereas infected plants of Kara-Dag cultivar demonstrated unstable levels of RNase activity. Significant decline in RNase activity was detected on the 7th day post infection with subsequent gradual increase in RNase activity. Decline of the RNase activity during the first week could promote the virus replication and therefore more successful infection of upper leaves of plants. Unstable levels of RNase activity in infected buckwheat plants may be explained by insufficiency of virus-resistant mechanisms that determines the medium sensitivity of the cultivar to BBV. Thus, plants of buckwheat cultivar having less sensitivity to virus, displayed in general higher RNase activity.

Diversification of 13S globulins, allergenic seed storage proteins, of common buckwheat.

The α polypeptide of the 13S globulin subunit of common buckwheat is the counterpart of the major allergenic ß polypeptide. Trypsin digestibility varies between variants of the α polypeptide with and without a tandem repeat insert. To evaluate the intra-species diversity of 13S globulin, the comprehensive screening of a genomic DNA library was performed, resulting in the isolation of 14 and 3 genes for Met-poor and Met-rich subunits, respectively. Although most tandem repeat units were 45 bp in length, the two-repeat gene Glb2B and all one-repeat genes contained an additional 3 bp. Secondary structure predictions and polyacrylamide gel electrophoresis demonstrated that the sense strand of Glb2B-CCG, the additional 3 bp-deletion clone of Glb2B, formed a more rigid secondary structure than that of the wild-type. Thus, the large intra-species variation of 13S globulin revealed in this study and its diversification might be attributable to the unique nature of the tandem repeat sequences.

Effects of Maillard reaction on allergenicity of buckwheat allergen Fag t 3 during thermal processing.

BACKGROUND: Fag t 3 is a major allergenic protein in tartary buckwheat. The Maillard reaction commonly occurs in food processing, but few studies have been conducted on the influence of thermal processing on the allergenic potential of buckwheat allergen. The aim of the present study was to investigate the effects of autologous plant polysaccharides on the immunoreactivity of buckwheat Fag t 3 (11S globulin) following the Maillard reaction. RESULTS: Fag t 3 and crude polysaccharides were prepared from tartary buckwheat (Fagopyrum tataricum) flour. After heating, the polysaccharides were covalently linked to Fag t 3 via a Maillard reaction, and the IgE/IgG-binding properties of Fag t 3 decreased dramatically, with significant changes also being observed in the electrophoretic mobility, secondary structure and solubility of the glycated Fag t 3. The great influence of glycation on IgE/IgG binding to Fag t 3 was correlated with a significant change in the structure and epitopes of the allergenic protein. These data indicated that conjugation of polysaccharides to Fag t 3 markedly reduced the allergen's immunoreactivity. CONCLUSION: Glycation that occurs via the Maillard reaction during the processing of buckwheat food may be an efficient method to reduce Fag t 3 allergenicity.