Identification of a 17 kDa protein that is a potentially novel antigen of lettuce-associated respiratory allergy in farmers.

BACKGROUND: We have identified and reported a novel antigen, nonprotein-specific secreted EP1-like glycoprotein (51 kDa), for lettuce-related respiratory allergy. OBJECTIVE: We aimed to identify a novel antigen for lettuce-related respiratory allergy that is different from epidermis-specific secreted EP1-like glycoprotein. METHODS: Immunoblotting was performed using an immunoglobulin E-specific antibody. The antigen-antibody reaction was confirmed by means of enzyme-linked immunosorbent assaying. LC-MS/MS analysis was carried out to detect a novel protein found in sera from 3 of 13 patients with lettuce-related respiratory allergy. Finally, we purified a novel protein from Escherichia coli. RESULTS: Immunoblotting assays showed common bands of 17 kDa in the sera of 3 of 13 patients. An enzyme-linked immunosorbent assay confirmed that the patient sera reacted with lettuce latex juice. A 17 kDa protein band that showed antigenic reactivity in 3 of 13 patient sera was identified as a kirola-like protein by LC-MS/MS. In addition, although we purified this protein, we failed to show the inhibitory effect. CONCLUSION: A 17 kDa protein that is a potentially novel antigen of lettuce-associated respiratory allergy was identified. In further studies, we will focus on purifying this novel protein to diagnose lettuce allergy.

N-Methyl-D-Aspartic Acid Receptor-Mediated Vasodilation Is Attenuated in the Retinas of Diabetic Rats.

PURPOSE: Activation of N-methyl-d-aspartic acid (NMDA) receptors enhances nitric oxide (NO) production in retinal neuronal cells, and in turn, NO released from neuronal cells induces glial cell-mediated dilation of retinal arterioles in rats. The purpose of this study was to examine how neuronal cell-dependent, glial cell-mediated vasodilation is impacted in diabetic rat retinas. METHODS: Diabetes was induced in 6-week-old male Wistar rats by combining streptozotocin injection and D-glucose feeding. Two weeks later, the dilator function of retinal arterioles was assessed. RESULTS: Compared with non-diabetic rats, the dilator responses of retinal arterioles induced by intravitreal injection of NMDA and NOR3, an NO donor, were reduced in diabetic rats. Following the blockade of large-conductance Ca2+-activated K+ (BKCa) channels with iberiotoxin, no significant difference in the retinal vasodilator response to NOR3 was observed between non-diabetic and diabetic rats. Intravitreal injection of 14,15-epoxyeicosatrienoic acid, a vasodilatory factor released from glial cells, dilated retinal arterioles, and the response was diminished by diabetes. CONCLUSION: These findings suggest that the impaired BKCa channel function in vascular cells is responsible for the diminished neuronal cell-dependent, glial cell-mediated dilation of retinal arterioles during the early stage of diabetes.

Hypersensitivity Pneumonitis Caused by Exposure to a Gray Parrot (Psittacus erithacus).

A 73-year-old woman complaining of cough and dyspnea was admitted to our hospital. High-resolution computed tomography chest revealed patchy ground-glass attenuation in the upper lung field. The patient suffered an asthma attack and was diagnosed with allergic pneumonitis; prednisolone was administered for treatment. Bird-related hypersensitivity pneumonitis was suspected, as she had a gray parrot (Psittacus erithacus) and a budgerigar (Melopsittacus undulatus) at home. An immunoblotting analysis with the patient's serum demonstrated IgG-binding fractions to the gray parrot's feathers only; no binding was noted with the budgerigar antigens. The patient was conclusively diagnosed with hypersensitivity pneumonitis related to exposure to a gray parrot.

Dietary syringic acid reduces fat mass in an ovariectomy-induced mouse model of obesity.

OBJECTIVE: Postmenopausal women are at increased risk of metabolic diseases such as obesity and diabetes. Therefore, the chemoprevention of postmenopausal changes in health via dietary supplements is important. Syringic acid (SA) is a phenolic compound present in the fruit of the assai palm, Euterpe oleracea, and in the mycelium of the shiitake mushroom, Lentinula edodes. This compound shows no affinity for estrogen receptors and may exert disease-preventive effects. Reportedly, dietary SA ameliorates high-fat diet-induced obesity in mice; however, its effects on estrogen deficiency-induced obesity are still unclear. Therefore, in this study, we investigated whether and how dietary SA affects these factors in ovariectomized (OVX) mice. METHODS: Ten-week-old OVX mice were fed SA-containing diets (100 mg/kg body weight/d) for 12 weeks. Their body weights, food intake, and uterus weights as well as other parameters were measured and comparisons were made with mice in the control group. RESULTS: Dietary SA did not affect the body weight, food intake, or uterus weight of OVX mice over the study period; however, the SA-fed group showed lower fat mass (ie, visceral, subcutaneous, and total fat) than the OVX-control group (11.1 ±â€Š3.3 vs. 8.3 ±â€Š2.4, P < 0.05; 7.9 ±â€Š1.1 vs. 5.9 ±â€Š1.6, P < 0.05; 19.0 ±â€Š4.2 vs. 14.1 ±â€Š3.8, P < 0.05, respectively). Furthermore, blood analysis revealed that SA-treatment resulted in a dose-dependent decrease and increase in serum triglyceride (59.2 ±â€Š8.3 vs. 43.9 ±â€Š12.2 mg/dL P < 0.05) and adiponectin (7.7 ±â€Š0.3 vs. 9.5 ±â€Š0.6 µg/mL, P < 0.05) levels, respectively. CONCLUSIONS: These results suggest that the SA diet improves lipid metabolism without affecting the uterus in OVX mice. Therefore, dietary SA has potential applicability for the prevention of postmenopausal obesity and type 2 diabetes.

Validation of Antiobesity Effects of Black Soybean Seed Coat Powder Suitable as a Food Material: Comparisons with Conventional Yellow Soybean Seed Coat Powder.

In this study, we fed obese model mice black soybean seed coat powder (BSCP) and evaluated the antiobesity effects. As a control, normal yellow soybean seed coat powder (YSCP) was used. C57BL/6J, a high-fat diet-induced obesity model mouse, was fed a high-fat diet containing BSCP or YSCP (20% fat) to induce obesity. The results showed that in the BSCP group, it caused significant suppression of body weight gain and suppression of white adipose tissue weight compared with the YSCP group. Moreover, it significantly decreased serum leptin levels, which correlated with visceral fat mass, and increased antidiabetic adipocytokine and adiponectin levels. Therefore, this suggests the pigmented components contained in BSCP have an antiobesity effect in obese model mice. It is suggested that this material, which can be prepared without extraction with an organic solvent and is suitable for use as a food material, could be a functional food material with a practicable antiobesity effect.

Thaumatin-Like Protein (Pru av 2) Is a Cherry Allergen That Triggers Percutaneous Sensitization in Mice.

Numerous recent studies have suggested that food allergens enter the skin and predispose individuals to food allergies through the production of IgE antibodies in the body. Cherries are a popular fruit eaten worldwide. However, cherries are an allergenic food and percutaneous sensitization with cherry allergens through the perioral region may occur while ingesting cherries. The identity of the cherry protein that triggers percutaneous sensitization in humans or animal models remains unknown. In this study, the backs of BALB/c mice were shaved and crude cherry extracts containing sodium dodecyl sulfate were applied to the skin. Thereafter, the cherry-specific IgE and IgG1 antibodies generated and secreted in response to the epidermal application were measured using an enzyme-linked immunosorbent assay or immunoblotting. Skin exposure to cherry extracts elevated cherry-specific IgG1 levels. Application of fractionated and purified cherry proteins (antigen candidates for percutaneous sensitization) that bound to the IgG1 antibodies led to the identification of a thaumatin-like protein (Pru av 2). This molecule is known as the major cherry allergen that affects humans. In conclusion, our study identified Pru av 2 as a cherry allergen that triggers percutaneous sensitization in mice for the first time.

Role of Epoxyeicosatrienoic Acids in Acetylcholine-Induced Dilation of Rat Retinal Arterioles in Vivo.

CYP epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent hyperpolarization (EDH)-related dilation in multiple vascular beds. The present study aimed to determine the role of EETs in the acetylcholine (ACh)-induced dilation of retinal arterioles in rats in vivo. The vasodilator responses were assessed by determining the change in diameter of the retinal arterioles on images of the ocular fundus. The intravitreal injection of 17-octadecynoic acid (1.4 nmol/eye), an inhibitor of CYP epoxygenase, and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E; 2 nmol/eye), an antagonist of EETs, reduced the ACh (0.3-10 µg/kg/min)-induced dilation of the retinal arterioles. The EET antagonist attenuated the vasodilator response to ACh under blockade of nitric oxide (NO) synthases and cyclooxygenases with NG-nitro-L-arginine methyl ester (30 mg/kg) plus indomethacin (5 mg/kg). Intravitreal injection of 14,15-EET (0.5 nmol/eye) dilated retinal arterioles and the response was prevented by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BKCa channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.

Development of Pretreatment Protocols for Determination of Soybean ß-Conglycinin in Processed Soybean Foods Using Commercial ELISA Kits.

ß-Conglycinin is the major storage protein in soybeans. Pre-clinical animal models and human clinical studies have demonstrated the triglyceride-lowering effect of this protein, suggesting that it could be put into practical use as a functional food material. To date, however, there are no accurate and simple assays for quantification of ß-conglycinin. In this study, samples were pretreated by mixing them with rice flour powder prior to extraction of proteins. Then, we used commercially available ELISA kits for detection of allergens that could be present in any contaminating soybean residue. This enabled accurate and highly reproducible quantitation of ß-conglycinin content in several processed soybean foods.

Occupational respiratory allergy to lettuce in lettuce farmers.

BACKGROUND: Lettuce-associated respiratory allergy has never been reported before. The aim of this study was to clarify the clinical condition of lettuce-associated respiratory allergy and to identify the lettuce antigen which induces allergic symptoms. METHODS: We distributed questionnaires to 1168 lettuce farmers and performed medical examinations in those who exhibited respiratory symptoms related to occupational exposure to lettuce. We analysed specific IgE-binding proteins in the sera of patients through immunoblotting analysis and determined molecular characterization of the IgE-binding bands using liquid chromatography-mass spectrometry. RESULTS: A total of 932 farmers (80%) responded to the questionnaire. Of those, 7% exhibited lettuce-associated respiratory symptoms, during harvesting and packaging. Thirteen patients were diagnosed with allergy to lettuce and agreed to undergo further examinations. The percentage of activated basophils in these patients was significantly higher compared with that reported in negative controls (P < .05). Lettuce-specific IgE (ImmunoCAP® ) and skin prick testing was positive in 46% and 62% of patients, respectively. Notably, occupational lettuce-allergic asthma was detected in one patient through specific bronchial provocation testing. The IgE-binding bands recognized in the sera of >50% of patients were identified as epidermis-specific secreted glycoprotein EP1-like (51 kDa). CONCLUSION: The present analysis identified a novel lettuce allergen. This allergen may have clinically useful applications, such as specific IgE testing and allergen-specific immunotherapy.

Comparison of Various Soybean Allergen Levels in Genetically and Non-Genetically Modified Soybeans.

Several analyses of allergen levels have been reported as part of the safety assessment of genetically modified (GM) soybean; however, few comprehensive analyses have included new allergens. Thus, in this study the levels of eight major soybean allergens, including Gly m 7 (a newly reported soybean allergen), were semi-quantitatively detected in six GM soybeans and six non-GM soybeans using antigen-immobilized ELISA and immunoblotting. We also analyzed the IgE-reactivity to these soybeans through immunoblotting, using sera from three soybean-allergic patients. The results showed that there were no significant differences in the levels of the major soybean allergens in the GM and non-GM soybeans. Moreover, there were no significant differences in the serum IgE-reactive protein profiles of the patients, as analyzed using immunoblotting. These results indicate that, in general, CP4-EPSPS-transfected GM soybeans are not more allergenic than non-GM soybeans.

Group B Soyasaponin Aglycone Suppresses Body Weight Gain and Fat Levels in High Fat-Fed Mice.

Group B soyasaponins, found in soy, have various health-promoting properties, but it is unclear whether they have an anti-obesity effect. The aim of this study was to evaluate the anti-obesity effect of group B soyasaponin glycosides and aglycone in mice fed a high-fat diet. Six-week-old C57/BL6 mice were divided into three groups (each n=10) and orally administered a high-fat diet for 35 d; two of the groups also received group B soyasaponin glycosides or aglycone. Although there was no significant difference among the three groups in consumption, the weight of fat adipose tissue at autopsy was more than 30% lower in the group B soyasaponin aglycone group than in the control group, but X-ray computed tomography showed no significant difference in muscle weight between these two groups. The ratio of muscle to whole body weight was higher in the group B soyasaponin aglycone group than in the control group. These results suggest that group B soyasaponin aglycone has a stronger anti-obesity effect than group B soyasaponin glycosides, without a loss in muscle weight, and that it increases the ratio of muscle to whole body weight. To our knowledge, this is the first report showing the anti-obesity effect of soyasaponin aglycone in vivo using animal models.

Identification of the 7S and 11S globulins as percutaneously sensitizing soybean allergens as demonstrated through epidermal application of crude soybean extract.

Cutaneous exposure to food allergens can predispose individuals to food allergies. Soybean, a major allergenic food, is an ingredient in various cosmetic products. However, the types of soybean proteins that are percutaneously sensitizing in humans or animal models remain unknown. In this study, BALB/c mice were dorsally shaved and epicutaneously exposed to a crude soybean extract including sodium dodecyl sulfate or distilled water alone. Specific IgEs secreted in response to 7S globulin (Gly m 5), 11S globulin (Gly m 6), Gly m 3, and Gly m 4 were measured using enzyme-linked immunosorbent assays or immunoblots. Exposure to soybean extract elicited the secretion of soybean-specific IgEs. Of the soybean proteins, 7S and 11S globulins acted as percutaneous sensitizers in 6/9 mice (67%). Additionally, IgE bound specifically and preferentially to the 7S globulin ß subunit. In conclusion, this is the first report to identify percutaneously sensitizing soybean allergens in a mouse model.

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.

Percutaneous Sensitization to Soybean Proteins Is Attenuated by Oral Tolerance.

Oral tolerance prevents allergic responses, but cutaneous exposure to food allergens predisposes individuals to food allergies. Soybean, a major allergenic food, is also an ingredient in various cosmetic products. However, it remains to be determined whether oral tolerance prevents percutaneous sensitization to soybean proteins in humans or animal models. In this study, BALB/c mice were divided into three groups; the SS group fed a soybean-containing diet, and the CS and control (C) groups fed a soybean-free diet. After being dorsally shaved, the CS and SS groups were epicutaneously exposed to a soybean extract while the control group was exposed to only the carrier. Specific IgE and IgG1 immunoglobulins secreted in response to the soybean proteins were measured using enzyme-linked immunosorbent assays. Exposure to the soybean extract elicited the secretion of IgE and IgG1 specific for Gly m 5 and Gly m 6, and trypsin inhibitor. Oral soybean consumption attenuated the secretion of all the soybean-specific IgEs and IgG1s, suggesting that percutaneous sensitization to soybean proteins is attenuated by oral tolerance.

Effect of gamma irradiation on soybean allergen levels.

The levels of food allergens in gamma-irradiated soybean (0, 2.5, 5, 7.5, 10, 20, and 30 kGy) were investigated by immunoblotting and ELISA, using allergen-specific antibodies and patient serum. After 3 months of storage, Coomassie brilliant blue (CBB) staining indicated similar total protein profiles among the treatments, but that some proteins were degraded by irradiation at high doses. Immunoblotting with specific antibodies for major soybean allergens (ß-conglycinin, Gly m Bd 30 K, soybean trypsin inhibitor, and Gly m 4) resulted in apparent band profiles and intensities that were not significantly changed by irradiation. Competitive inhibition ELISA analyses suggested that there were no significant changes in the allergen contents, except for a decrease in the soybean trypsin inhibitor. The patient IgE binding allergenic protein patterns were not changed by irradiation up to 30 kGy. ELISA using patient serum also revealed that the IgE reactivity to the irradiated soybean extract did not increase from the level of the control, but that the reactivity to some patient serum IgE was significantly decreased by irradiation.

Hypoallergenicity and immunological characterization of enzyme-treated royal jelly from Apis mellifera.

Royal jelly (RJ), the exclusive food for queen bees, is taken as a dietary supplement because it is highly rich in nutrients. However, RJ is known to induce an anaphylactic response in some individuals. We evaluated in the present study the hypoallergenicity of alkaline protease-treated RJ in vitro and in vivo. We first confirmed that this treated RJ contained the same levels of vitamins, minerals and specific fatty acid as in untreated RJ. We then showed that the IgE-binding capacity of the treated RJ was very significantly reduced by conducting in vitro assays of the blood from RJ-sensitive patients. An in vivo skin-prick test on the RJ-sensitive patients also showed that, in the majority of the patients (3 out of 4 tested), the treated RJ did not evoke any allergenic response. It is thus advantageous to prepare hypoallergenic RJ by a protease enzyme treatment for its safe consumption.

Hypoallergenicity of various miso pastes manufactured in Japan.

Miso paste (miso), a fermented soybean food, is popular in Japan and other Asian countries. However, the soybean is known to induce an allergenic response in some individuals. In the present study, we evaluated the allergenicity of various kinds of miso available in Japan. Total proteins were extracted from Amakuti-kome miso, Karakuti-kome miso, Mugi-miso and Mame-miso, and the protein profiles were analyzed. The major protein bands detected in the intact soybean extract were not present in any of the miso samples, which instead showed various low molecular weight protein bands of approximately 10-25 kDa. The existence levels of six major soybean allergens were determined by Western blotting using specific antibodies. We found that the allergen levels varied among miso and allergen types; however, allergen levels were consistently lower in miso than in the soybean extract. We obtained similar results for IgE-ELISA experiments using serum IgE from soybean allergy patients. Taken together, these results indicate that compared to soybean extract, various types of miso contain small quantities of intact soybean allergens. Additionally, several lines of evidence indicated that the allergen levels were exceptionally low in the dark-colored Karakuti-kome miso and Mame-miso, which are produced with relatively long fermentation periods, suggesting that the duration of fermentation might be a key factor in the hypoallergenicity of miso.