[A CASE OF A GIRL WITH ANAPHYLAXIS INDUCED BY INHALATION OF SOYBEAN DUST].

The patient was a 3-year-old girl whose father was employed sorting and washing soybeans. She exhibited transient respiratory distress and loss of activity on the same day or the next day after her father came home wearing work clothes with soybean dust on them. One day, she developed anaphylaxis after being lifted into her father's arms while he was wearing his work clothes. Although a blood test was positive for soybean and Gly m 4-specific IgE antibodies, the girl was able to consume soy products (not including soy milk, which she had never consumed) without any issues. The father was instructed to change clothes before leaving work and bathe immediately upon returning home, and the girl has not had any further episodes of respiratory distress, loss of activity, or anaphylaxis. Though reports of anaphylaxis from soybean antigen inhalation are extremely rare, it is very likely that inhalation of soybean dust from the father's work clothes induced anaphylaxis in this case.

[TWO CASES OF ALLERGY WITH MUNG BEAN SPROUTS].

Patient 1 was a female patient in her teens who presented with swelling of the lips and oral discomfort after consuming mung bean sprouts. She had a history of this reaction since the age of 6 years and showed positive on a prick-to-prick test for mung bean sprouts. Patient 2 was a male patient in his twenties who also showed positive for mung bean sprouts as well as soybean sprout. Both patients were positive for IgE specific to birch, Gly m4, and Bet v1.Mung beans belong to the PR-10 family because they contain the allergenic component, Vig r1. A cross reaction to mung bean may occur in a patient with birch allergy as in the present cases. Mung bean sprouts are a cheap and common dietary item in Japan where, however, only a few cases of mung bean sprouts allergy have been reported. Mung bean sprouts allergy should be diagnosed with appropriate testing; if the patient has allergic reactions for this food item, an allergologist should provide detailed dietary guidance for avoiding pollen-food allergy syndrome.

Structural and Immunologic Properties of the Major Soybean Allergen Gly m 4 Causing Anaphylaxis.

Gly m 4 is the major soybean allergen, causing birch pollen cross allergic reactions. In some cases, Gly m 4-mediated anaphylaxis takes place, but the causative factors are still unknown. Here, we studied the structural and immunologic properties of Gly m 4 to shed light on this phenomenon. We showed that Gly m 4 retained its structure and IgE-binding capacity after heating. Gly m 4 was cleaved slowly under nonoptimal gastric conditions mimicking duodenal digestion, and IgE from the sera of allergic patients interacted with the intact allergen rather than with its proteolytic fragments. Similar peptide clusters of Bet v 1 and Gly m 4 were formed during allergen endolysosomal degradation in vitro, but their sequence identity was insignificant. Animal polyclonal anti-Gly m 4 and anti-Bet v 1 IgG weakly cross-reacted with Bet v 1 and Gly m 4, respectively. Thus, we supposed that not only conserved epitopes elicited cross-reactivity with Bet v 1, but also variable epitopes were present in the Gly m 4 structure. Our data suggests that consumption of moderately processed soybean-based drinks may lead to the neutralizing of gastric pH as a result of which intact Gly m 4 can reach the human intestine and cause IgE-mediated system allergic reactions.

Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max).

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30 K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.

Utility of the Basophil Activation Test Using Gly m 4, Gly m 5 and Gly m 6 Molecular Allergens for Characterizing Anaphylactic Reactions to Soy.

There are two major clinically described forms of IgE-dependent soy allergy: (i) a primary dietary form, linked to sensitization against soy storage proteins Gly m 5 and Glym 6, and (ii) a form included in birch-soy syndromes linked to Gly m 4, a PR-10-like allergen. This second form sometimes causes severe systemic reactions, even anaphylaxis, especially on consuming certain forms of soy such as soymilks or smoothies. Skin prick tests and specific IgE assays against soy whole extracts lack sensitivity. Assays of anti-Gly m 4, Gly m 5 and Gly m 6 specific IgEs have been developed to overcome this obstacle, but they unfortunately lack specificity, especially for anti-Gly m 4. We hypothesized that the basophil activation test (BAT) using molecular soy allergens Gly m 4, Gly m 5 and Gly m 6 would both remedy the lack of sensitivity of other tests and offer, through its mechanistic contribution, greater specificity than the assay of anti-Gly m 4 specific IgEs. This would enable the two types of soy allergy to be separately identified. In a characteristic clinical example of PR-10-induced anaphylactic reaction after consuming soymilk, we report preliminary results of Gly m 4-exclusive positivity of BAT supporting our hypothesis. It will be necessary to confirm these results on more patients in subsequent studies, and to specify the place of the BAT in an overall diagnostic strategy. Meanwhile, soy BAT using molecular allergens is a promising diagnostic tool for soy allergy and probably also for follow-up in specific immunotherapies.

Isotretinoin-associated exercise-induced anaphylaxis in a patient with birch pollinosis and soybean sensitization: case presentation and literature review.

BACKGROUND: Peanut and soybean allergies are listed as contraindication in the package leaflet of isotretinoin, a widely used treatment of acne vulgaris. Cross-reactivity between PR10-proteins in peanut, tree nuts, and soybean is particularly common in patients with birch pollinosis and may lead to anaphylactic reactions in sensitized patients after intake of soybean oil containing isotretinoin capsules. CASE PRESENTATION: Here, we describe a young man with hazelnut and birch pollen allergy, who experienced exercise-induced anaphylaxis after isotretinoin intake on the third day of treatment. A complete allergy work-up was carried out, and sensitization to both peanut and soybean PR10-proteins was confirmed. However, oral provocation with isotretinoin remained negative in the absence of intense physical activity and longterm treatment was well tolerated. CONCLUSION: To our knowledge, this is the first report of an exercise-induced anaphylaxis due to isotretinoin therapy. Our literature review to assess tolerability of isotretinoin in patients allergic to peanut, tree nuts or soybean revealed only one other case of anaphylaxis in a cashew-nut allergic patient sensitized to soybean PR10-protein Gly m 4. While there are no reports on soybean allergic patients treated with isotretinoin, the vast majority of peanut or tree nut allergic patients tolerated isotretinoin. Therefore, we conclude that sensitization to soybean, peanut or tree nuts should not preclude isotretinoin therapy. Particular caution is however warranted in patients with soybean sensitization. Pre-treatment oral challenges with isotretinoin may be recommended and physicians should be aware of the potential role of cofactors.

Investigation of Sensitization Potential of the Soybean Allergen Gly m 4 by Using Caco-2/Immune Cells Co-Culture Model.

The soybean allergen Gly m 4 is known to cause severe allergic reactions including anaphylaxis, unlike other Bet v 1 homologues, which induce mainly local allergic reactions. In the present study, we aimed to investigate whether the food Bet v 1 homologue Gly m 4 can be a sensitizer of the immune system. Susceptibility to gastrointestinal digestion was assessed in vitro. Transport through intestinal epithelium was estimated using the Caco-2 monolayer. Cytokine response of different immunocompetent cells was evaluated by using Caco-2/Immune cells co-culture model. Absolute levels of 48 cytokines were measured by multiplex xMAP technology. It was shown that Gly m 4 can cross the epithelial barrier with a moderate rate and then induce production of IL-4 by mature dendritic cells in vitro. Although Gly m 4 was shown to be susceptible to gastrointestinal enzymes, some of its proteolytic fragments can selectively cross the epithelial barrier and induce production of Th2-polarizing IL-5, IL-10, and IL-13, which may point at the presence of the T-cell epitope among the crossed fragments. Our current data indicate that Gly m 4 can potentially be a sensitizer of the immune system, and intercommunication between immunocompetent and epithelial cells may play a key role in the sensitization process.

Do Lipids Influence Gastrointestinal Processing: A Case Study of Major Soybean Allergen Gly m 4.

Previously, we have demonstrated that Gly m 4, one of the major soybean allergens, could pass through the Caco-2 epithelial barrier and have proposed a mechanism of sensitization. However, it is not known yet whether Gly m 4 can reach the intestine in its intact form after digestion in stomach. In the present work, we studied an influence of various factors including lipids (fatty acids and lysolipids) on digestibility of Gly m 4. Using fluorescent and CD spectroscopies, we showed that Gly m 4 interacted with oleic acid and LPPG (lyso-palmitoyl phosphatidylglycerol), but its binding affinity greatly decreased under acidic conditions, probably due to the protein denaturation. The mimicking of gastric digestion revealed that Gly m 4 digestibility could be significantly reduced with the change of pH value and pepsin-to-allergen ratio, as well as by the presence of LPPG. We suggested that the protective effect of LPPG was unlikely associated with the allergen binding, but rather connected to the pepsin inhibition due to the lipid interaction with its catalytic site. As a result, we assumed that, under certain conditions, the intact Gly m 4 might be able to reach the human intestine and thereby could be responsible for allergic sensitization.

Food-dependent exercise-induced anaphylaxis to soybean: Gly m 5 and Gly m 6 as causative allergen components.

Food-dependent exercise-induced anaphylaxis (FDEIA) is a life-threatening but relatively rare disorder which occurs mainly in older children and young adults and manifests with symptoms of anaphylaxis upon exercise following ingestion of certain kinds of food. We herewith report 3 cases of soybean-induced FDEIA. We also highlight 2 types of soybean-induced FDEIA, one caused by storage protein components Gly m 5 and Gly m 6 and the other caused by pollen-related allergen components.

The BASALIT multicenter trial: Gly m 4 quantification for consistency control of challenge meal batches and toward Gly m 4 threshold data.

SCOPE: The BASALIT clinical trial (EudraCT 2009-011737-27) investigated efficacy of birch allergen immunotherapy on lowest observed adverse effect levels after soy food challenge in patients with birch-associated and Gly m 4 allergen mediated soy allergy. Thus, consistently stable Gly m 4 levels were required in standardized challenge meals. METHODS AND RESULTS: Soy meal included soy protein isolate (SPI, 88% total protein). A Gly m 4 specific ELISA was developed and validated. Six SPIs and 24 meal batches were analyzed for Gly m 4. (Repeated-measures) analyses of variance were done to identify potential changes between batches and time intervals. Gly m 4 was below the ELISA detection limit (2 ng/mL) in placebo batches. With <20% mean coefficient of variation, Gly m 4 levels were consistent in 24 soy meal batches and within individual 12-wk shelf-life. CONCLUSION: The novel Gly m 4 specific ELISA proved consistency of challenge meal batches over a 56-month study period. With an average of 178 µg/g Gly m 4 in SPI, Gly m 4 lowest observed adverse effect level can be calculated once clinical lowest observed adverse effect level data based on SPI are available. Hence, sensitivity of patients can be correlated to the relevant allergen content instead of total protein of the allergenic source.

Comparison of Allergenicity at Gly m 4 and Gly m Bd 30K of Soybean after Genetic Modification.

Despite rapid growth of genetically modified (GM) crops, effective evaluations of genetic modification on allergenicity are still lacking. Gly m Bd 30K is cross-reactive with cow's milk protein casein, Gly m 4, and with birch pollen allergen Bet v 1. Here we compared the allergenicity between GM and non-GM soybeans with respect to the foci Gly m 4 and Gly m Bd 30K. Recombinant allergens of Gly m Bd 30K and Gly m 4 were generated and polyclonal antibodies raised to identify these two allergenic components in soybeans. GM soybean was first PCR-confirmed using 35S promoter. A total of 20 soybeans (half GM, half non-GM) obtained from a food market were used to assess their allergenicity based on IgE-binding and histamine release. The concentrations of Gly m Bd 30K and Gly m 4 in soybeans were then determined. Most soybean-allergic patients (9 of 10) showed IgE-positive reactions to the allergen of 30 kDa in molecular weight. That allergen turned out to be Glycine max Gly m Bd 30K based on LC-MS/MS analyses. Gly m Bd 30K is therefore the major allergen in the soybean. An increase in the transcription of both the Gly m 4 (stress-induced protein SAM22) and Gly m Bd 28K (soybean allergen precursor) was found after genetic modification. The protein concentrations of Gly m 4 and Gly m Bd 30K were not statistically significant different between non-GM and GM soybeans. There were also no statistical significances between them in the tests of IgE binding and histamine release. In conclusion, soybeans showed similar concentrations of Gly m Bd 30K and Gly m 4 regardless of genetic modification or absence thereof. The allergenicity of both Gly m Bd 30K and Gly m 4 was therefore not altered after genetic modification. Patients showing hypersensitivity to soybeans and who had pre-existing allergy to birch pollen and cow's milk casein might not further increase their allergic reactions following exposures to the GM soybeans.

Development of a Sandwich ELISA for Quantification of Gly m 4, a Soybean Allergen.

Gly m 4 is a key soybean allergen that causes allergic symptoms in the skin, gastrointestinal tract, or respiratory tract of sensitive individuals. To understand naturally variable levels of Gly m 4 among conventional soybean varieties, a sandwich ELISA was developed and validated using a mouse anti-Gly m 4 monoclonal antibody and a goat anti-Gly m 4 polyclonal antibody as capture and detection antibodies, respectively. The ELISA shows high specificity to Gly m 4 without any cross-reactivity to other soybean proteins and has a quantification range of 7.8-250 ng/mL using an Escherichia coli-produced recombinant Gly m 4, with 2.1 ng/mL being the limit of detection. Within the quantification range, the coefficients of variation of the intra-assay and interassay precision are less than 5 and 12%, respectively. Moreover, extraction efficiency and dilutional parallelism experiments were completed to demonstrate the assay is accurate. The validated assay was used to quantify Gly m 4 levels in 128 soybean samples from 24 conventional soybean varieties grown at 8 distinct geographical locations. There was a 13-fold difference between the least and greatest amounts of Gly m 4 concentrations among the samples, and the results demonstrate that the most significant sources of variability in Gly m 4 levels in the conventional varieties were related to location and variety.