Effects of veterinary drugs on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3)

Little is known about the effects of residual veterinary drugs on the allergic reaction, except for the antigenicity of antibiotics and synthetic antimicrobials. Therefore, 59 kinds of veterinary drugs were investigated for their effects on the IgE receptor-mediated beta-hexosaminidase release from RBL-2H3 cells as an index of immediate allergic reaction. We found that the antibiotics chlorotetracycline, doxycycline, monensin, the synthetic antimicrobial pyrimethamine and the steroid hormone testosterone inhibited beta-hexosaminidase release. Most of the veterinary drugs showed no action, though the ionophores lasalocid, salinomycin and the steroid hormone hexestrol promoted beta-hexosaminidase release from injured cells. Based on the residual levels of these drugs and the frequencies of detection in actual food samples, it seems unlikely that these drugs have any immediate allergic effect in practice.     

Anti-allergic activity of grapeseed extract (GSE) on RBL-2H3 mast cells

Grapeseed extract (GSE) is a rich source of natural phenolic compounds and possesses various pharmacological activities, including antioxidant, anticarcinogenic and anti-inflammatory properties. However, effects of GSE on immunoglobulin (Ig) E-mediated allergic responses still remain elusive. In the present study, the effects of GSE on activation and degranulation of RBL-2H3 mast cells were investigated. GSE pretreatments (20-100 μg/ml) reduced IgE-antigen mediated release of β-hexosaminidase and histamine in RBL-2H3 cells. Additionally, GSE reversibly inhibited expression of FcεRI on RBL-2H3 cells. GSE treatments caused a significant elevation of intracellular cAMP levels, whereas the Ca²⁺ influx upon antigen stimulation was inhibited. Suppression on FcεRI expression together with decreased calcium uptake and increased cAMP level might be involved in attenuated degranulation of mast cells by GSE treatment. Our results suggest a possible pharmaceutical application of GSE in treating type I allergic diseases.     

Effects of organonitrogen, carbamate pesticides and others on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3)

There have been very few reports of the effect of pesticides on immediate allergy. In the previous report, the effect of pyrethroid pesticides and organophosphorus pesticides on immediate allergic reaction was investigated. Subsequently, 12 organonitrogen pesticides, 14 carbamate pesticides and 4 other pesticides were investigated for their effects on the enzyme activity of beta-hexosaminidase as an index of chemical mediator release from rat basophilic leukemia cells (RBL-2H3). Two organonitrogen pesticides, bitertanol and pyridaben, and two organotin pesticides, cyhexatin and fenbutatin oxide, were found to promote beta-hexosaminidase release. Bitertanol non-specifically caused the promotion of chemical mediator release, while the release-promotive action of pyridaben was related to IgE antibody and those of cyhexatin and fenbutatin oxide to cell injury. On the other hand, two organonitrogen pesticides, propiconazole and triadimenol, and imazalil showed release-inhibitory action. These data suggested that some pesticides can affect immediate allergy.     

Allergenic characteristics of a modified peanut allergen

Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.     

Inhibitory effect of aqueous spinach extract on degranulation of RBL-2H3 cells

The inhibitory effect of an aqueous extract from spinach on degranulation of RBL-2H3 cells is herein reported. The extract significantly suppressed antigen-induced degranulation in a dose-dependent manner without affecting cell viability. Active substances in the extract were heat-stable and trypsin-resistant with molecular weights ranging from 500 Da to 14 kDa. The extract inhibited elevation of the intracellular Ca²⁺ concentration caused by stimulation by antigen, while not suppressing degranulation induced by a calcium ionophore A23187. Immunoblot analysis revealed that the inhibitory effect results from downregulation of phosphorylation of both Syk kinase and phosphatidylinositol 3-kinase in the signalling pathways involved in degranulation caused by the antigen–antibody interaction. Taken together, these findings suggest that aqueous spinach extract has an anti-allergic activity that controls degranulation.     

Anti-allergy effect of marc from silver vine Actinidia polygama prepared by supercritical fluid extraction

Anti-allergic effect of marc extract after supercritical fluid extraction SFE comparing the crude extract from silver vine was investigated to increase its potential for use as a new source of functional food that reduced non-favorable flavor. The results tested by thiazolyl blue tetrazolium bromide MTT assay indicated that the ethanol extract of silver vine ETOH and ethanol extract of SFE marc SFEM showed no cytotoxic effects on IgE-sensitized rat basophilic leukemia RBL-2H3 cells under the conditions. ETOH, as well as SFEM inhibited antigen-induced degranulation in RBL-2H3 mast cells dose dependently. The morphological change of the cells treated with compound 48/80 was prevented by the extracts. Production of tumor necrosis factor TNF-α on the activated RBL-2H3 cells was also reduced by ETOH and SFEM, similarly. Moreover, the total phenolic content of the extracts seems to correlate with the anti-allergic activity. In conclusion, the results suggest that the flavor-improved SFE marc extract suppressed the antigen-induced allergic reactions in RBL-2H3 mast cells.     

Inhibitory effect of chlorophyll c2 from brown algae,Sargassum horneri,on degranulation of RBL-2H3 cells

The anti-allergic effect of alcohol extracts from edible seaweeds was evaluated by the degranulation of a rat basophilic leukemia cell line (RBL-2H3). Extracts prepared from the brown algae Sargassum horneri, Ecklonia cava and Ecklonia kurome, inhibited the degranulation of RBL-2H3 cells, whereas those from green algae and red algae did not. The active substance in S. horneri was purified and identified as chlorophyll c2, a major pigment in brown algae. The purified chlorophyll c2 preparation also suppressed the degranulation of RBL-2H3 cells. In contrast, chlorophyll a and b did not affect cell degranulation. Therefore, chlorophyll c2 from brown algae might be useful for the prevention or mitigation of allergic symptoms.     

Inhibitory effects of water extracts of fermented rice bran on allergic response

In this study, the effects of water extracts of fermented rice bran (FRBE) on immediate-type allergic reactions in vitro (RBL-2H3 cell line) and in vivo [ovalbumin (OVA) sensitized mice model] were investigated. FRBE inhibited β-hexosaminidase release from RBL-2H3 cells activated by anti-dinitrophenyl (DNP) IgE while FRBE pretreatment profoundly inhibited the systemic allergic reaction induced by compound 48/80. Moreover, FRBE had a significant inhibitory effect on the mRNA expression of anti-DNP IgE-induced tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-4, IL-6, and IL-10, all of which mediate inflammatory reactions triggered by allergic reaction. To determine the mRNA levels of cytokines in an OVA-sensitized mice model after FRBE treatment, spleen cells were harvested with several concentrations of FRBE. The FRBE-treated group showed decreased levels of TNF-α, IFN-γ, IL-6, and IL-10. These results suggest that FRBE suppressed allergic and inflammatory reactions through inhibition of degranulation, histamine release, and pro-inflammatory cytokine production from mast cells.     

Effect of the structure and potential allergenicity of glycated tropomyosin,the shrimp allergen

The aim is to elucidate whether the structural changes of shrimp tropomyosin (TM) following ribose treatment affected its potential allergenicity. The structural changes of glycated TM were analysed by fluorescence, ultraviolet spectrum (UV), circular dichroism (CD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the changes in allergenicity were investigated by enzyme-linked immunosorbent assay (ELISA) and RBL-2H3 cell model. The result indicated that ribose could cause the conformational structural changes of TM. Moreover, the phenylalanine, isoleucine and DL-methionine residues were altered by ribose by LC-MS/MS analysis. These modified amino acids also appeared in the shrimp allergic epitopes region. Furthermore, the glycated TM (4000 mmol L⁻¹ ribose) could significantly decrease the IgE-binding capacity and inhibit the release of cytokines and mediators from RBL-2H3 cells. These findings suggest that ribose-induced glycation could damage the allergic epitope of TM, decreasing the potential allergenicity and could be considered for alleviating TM-induced food allergy.     

Inhibitory effects of flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu) on antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells

We found that two distinct flavonoid glycosides isolated from the peel of Japanese persimmon (Diospyros kaki Fuyu), isoquercitrin (Isq) and hyperin (Hyp), are capable of inhibiting antigen-stimulated degranulation in rat basophilic leukaemia RBL-2H3 cells. In order to elucidate the underlying mechanisms, we examined effects of Isq and Hyp on cellular responses induced by antigen stimulation. Treatment with both Isq and Hyp markedly inhibited antigen-stimulated elevation of intracellular free Ca²⁺ concentration and reactive oxygen species (ROS). Isq and Hyp did not affect NADPH oxidase (NOX) activity, but they possessed DPPH radical-scavenging activity similar to that of epigallocatechin gallate, a potent anti-oxidant, Finally, Isq and Hyp showed little or no effects on Ag-stimulated Syk activation or phosphorylation of signalling molecules. These results indicate that inhibition of antigen-stimulated degranulation by Isq and Hyp is mainly due to suppression of intracellular Ca²⁺ elevation, which is caused by direct scavenging of ROS that are generated by NOX. Our findings suggest that Isq and Hyp, isolated from the peel of persimmon, would be beneficial for alleviating symptoms of type I allergy.     

Anthocyanins,but not Anthocyanidins,from Bilberry (Vaccinium myrtillus L.) Alleviate Pruritus via Inhibition of Mast Cell Degranulation

Abstract: We have previously reported that bilberry anthocyanins exhibit an anti‐pruritic effect in a mouse model of allergic contact dermatitis. It has been reported that anthocyanins are particularly sensitive to thermal treatment and are easily hydrolyzed to anthocyanidins when exposed to high temperatures. The objective of this study was to compare the anti‐pruritic effect of anthocyanin‐rich quality‐controlled bilberry extract and anthocyanidin‐rich degraded extract using a mouse model of allergic contact dermatitis. BALB/c mice with allergic contact dermatitis induced by 4 weeks of repeated application of 2,4,6‐trinitro‐1‐chlorobenzene (TNCB) were administered Bilberon‐25 orally for 4 weeks after sensitization with TNCB. The effect of Bilberon‐25 on pruritus was evaluated by measurement of scratching behavior. RBL‐2H3 mast cells were used to investigate the effect of Bilberon‐25 on degranulation in 48/80‐stimulated mast cells. Compared with nonheated Bilberon‐25, the proportion of anthocyanins in heated Bilberon‐25 decreased, and the proportion of anthocyanidins was increased in heated‐time dependent manner. Treatment with non‐heated Bilberon‐25 significantly attenuated the TNCB‐induced increase in scratching behavior, whereas treatment with 2 h‐heated Bilberon‐25 did not. Moreover, 300 μg/mL nonheated Bilberon‐25 showed significant inhibition of degranulation in RBL‐2H3 mast cells, whereas 2 h‐heated Bilberon‐25 had no effect at any concentration studied. It is assumed that the inhibitory effect of bilberry anthocyanins on pruritus might be mediated, at least in part, by its inhibitory effect on mast cell degranulation. In conclusion, the anthocyanin‐rich but not anthocyanidin‐rich bilberry extract may be a useful dietary supplement for skin diseases involving pruritic symptoms, such as chronic allergic contact dermatitis, atopic dermatitis, and rhinitis.     

基于RBL-2H3模型分析过敏反应效应细胞激活的差异基因

构建IgE介导的大鼠嗜碱性粒细胞（rat basophilic leukemia，RBL）-2H3作为过敏反应细胞模型，并利用转录组测序分析比较RBL-2H3细胞激活前后的转录组基因差异，富集相关信号通路。IgE介导的RBL-2H3细胞激活后，过敏介质和细胞因子分泌显著提高；转录组测序得到RBL-2H3细胞激活前后的232 个差异表达基因，其中有127 个（54.74%）可进行基因功能注释；RBL-2H3细胞激活主要影响肿瘤坏死因子、丝裂原活化蛋白激酶、Janus激酶-信号转导与转录激活子、Toll样受体等信号通路，涉及的转录因子有MAP3K8、Nfkbia、Junb、Jun、Fos等。本研究建立了IgE介导的过敏反应体外细胞模型，并且通过对效应细胞转录组信息的全面分析和潜在靶点及信号通路的富集为过敏性疾病的防控治疗提供参考。     

Antibacterial activities of semipurified fractions of Quercus infectoria against enterohemorrhagic Escherichia coli O157:H7 and its verocytotoxin production

Escherichia coli O157:H7 is one of the most important foodborne pathogens, causing nonbloody and bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Use of antibiotics has been demonstrated to result in increased levels of verocytotoxin (VT) production as well as antibiotic resistance. Quercus infectoria was investigated for its antibacterial activity against E. coli O157:H7 and other VT-producing enterohemorrhagic E. coli (VTEC). The MIC was determined by a broth microdilution method, and the MBC was assessed by subculturing the bacteria from the wells that showed no apparent growth onto Mueller-Hinton agar. The fractions Qi2, Qi3, and Qi4 of Q. infectoria were demonstrated to possess good antibacterial activity, with MICs and MBCs ranging from 250 to 500 microg/ml. The effect of the effective fraction, Qi4, on the production of VT was determined using a reversed passive latex agglutination. The results indicate that at 20 h, fraction Qi4 markedly inhibits the release of VT1 and VT2 from VTEC cells at both inhibitory and subinhibitory concentrations. Furthermore, verotoxicity assay demonstrated that bacterial cultures treated with fraction Qi4 exerted less toxic effect on Vero cells. These in vitro results clearly indicate that the fraction Qi4 might constitute a promising natural food additive for the control of food poisoning by E. coli O157:H7 as well as other VTEC strains.     

硼酸锌/氢氧化铝对聚氯乙烯纺织结构材料阻燃及稳定性的影响

为改善聚氯乙烯（PVC）纺织结构材料的阻燃、抑烟、防熔滴等性能,采用无机阻燃剂硼酸锌和氢氧化铝协同作用制备阻燃PVC纺织结构材料。利用极限氧指数仪、烟密度仪、热重分析仪、扫描电子显微镜分析了硼酸锌和氢氧化铝复配对PVC纺织结构材料性能的影响。结果表明：硼酸锌和氢氧化铝复配阻燃PVC纺织结构材料具有优异的阻燃性、抑烟性、防熔滴性,随着硼酸锌含量的增加,抑烟效果更佳;老化处理对PVC稳定性存在一定程度的影响,其极限氧指数值随热氧老化时间的延长先增加再降低,且随光氧老化时间的延长而增加。     

Free fatty acids promote degranulation of azurophil granules in neutrophils by inducing production of NADPH oxidase–derived reactive oxygen species in cows with subclinical ketosis

Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2–4) and days in milk (median = 6, range = 3–15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A⁺/CD63^high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.     

主要坚果类过敏原致敏机理的初步探讨

本文以坚果过敏蛋白为抗原,构建小鼠动物致敏免疫模型,探讨坚果过敏原的致敏机理,为坚果类食物安全评价和安全开发提供有效的实验依据。选取六种坚果为原料,以昆明小鼠为受试小鼠,坚果蛋白液以0.01 mg/g进行灌胃致敏,第33 d和34 d以0.02 mg/g激发致敏,通过测定小鼠血清IgE、IgG、腹腔肥大细胞及类胰蛋白酶和血象指标,研究其病理特征。结果表明,致敏激发后小鼠IgE与IgG最高水平为0.91和0.93,组胺释放率阳性组为58%,中剂量组为40%,高剂量组为55%;小鼠腹腔肥大细胞体外激发后脱颗粒高剂量组最多以及类胰蛋白酶活性也最高;中和高剂量组、阳性组白细胞数较高(9.7±6.1、9.6±2.4、9.2±2)×109/L,低剂量组、中剂量组和高剂量组的血小板数依次降低。说明致敏后小鼠凝血功能显著减弱,免疫系统受到侵袭,体内诱发产生IgE抗体,并启动过敏介质的释放机制,导致过敏反应Ⅰ型变态反应的发生。     

Rocket immunoelectrophoresis (RIE) for determination of potentially allergenic peanut proteins in processed foods as a simple means for quality assurance and food safety

 Peanuts are one of the most allergenic foods known. The presence of hidden allergens in processed food for reasons of mislabelling or cross-contamination expose allergic individuals to unpredictable risks, especially since highly sensitized subjects may experience severe anaphylactic reactions. The protection of consumers requires specific and sensitive methods for the detection of trace amounts of potentially allergenic peanut components. A rocket immunoelectrophoresis (RIE) procedure was developed allowing the detection of even spurious contaminations with peanut protein. For precipitation of peanut protein a commercially available antiserum was used. By amplifying precipitates with a secondary immunodetection step, 20 ng/ml peanut protein in chocolate extract, equivalent to 0.0002% peanut in chocolate, could still be detected. Model chocolate spiked with various amounts of peanut was investigated down to 0.001% peanut (10 ppm), the limit of quantitative determination. The method was optimized for detection of peanut in chocolate samples. Non-chocolate samples had to be standardized with a chocolate matrix prior to analysis in order to obtain a uniform response. Cross-reactivities with other food proteins did not occur. The method showed high recoveries of 85–101% for chocolate samples down to 10 ppm peanut and good reproducibility with coefficients of variation of ≤ 5 % for samples of ≥ 15 ppm peanut protein. The applicability of this method in the detection of peanut protein in various food commodities was demonstrated: two unlabelled products and two products which did not have peanut listed as an ingredient were identified as containing peanut protein. In all cases where peanut was listed, peanut protein could be determined. The results of RIE were always confirmed by those of a new cell-mediator-release assay that is based on a rat basophil leukaemia (RBL) cell-line (RBL-2H3), cells that are a functional equivalent to mucosal mast cells. Measuring the release of β-hexosaminidase resulting from cross-linking of basophil-bound peanut-specific immunoglobulin E, the assay mimics a main event of the allergic type-I reaction. The cell assay was adapted for food matrices and peanut could be detected down to 0.01% which additionally demonstrated in vitro that even trace amounts of peanut protein could elicit allergic reactions. Received: 28 May 1997 / Revised version: 11 July 1997