嗜酸乳杆菌对β-乳球蛋白过敏诱发Thl/Th2细胞平衡的影响

目的:观察嗜酸乳杆菌对牛乳β-乳球蛋白(BLG)致敏小鼠Th1/Th2细胞平衡及血清抗体水平的影响,以研究其缓解过敏反应的作用。方法:用牛乳BLG和弗氏佐剂的混合液腹腔注射诱发BALB/c小鼠致敏,建立动物过敏模型。将实验动物随机分为空白组、过敏组与不同剂量的嗜酸乳杆菌组。采用ELISA法测定各组小鼠血清总IgE、BLG特异性IgE和总IgG含量。体外分离培养各组小鼠脾淋巴细胞,采用ELISA法检测细胞上清液中Th1型细胞因子(IL-12、IFN-γ)和Th2型细胞因子(IL-4)的水平。结果:中、高剂量嗜酸乳杆菌组小鼠的IFN-γ/IL-4比值(代表Th1/Th2细胞平衡)显著高于过敏组(P<0.05);而其血清中的总IgE、BLG特异性IgE和总IgG水平显著低于过敏组(P<0.05),与空白组相比无差异性(P>0.05)。结论:嗜酸乳杆菌干预可改善小鼠的BLG过敏症状,其作用可能与促进Th1占优势的Th1/Th2细胞平衡,阻断IgE及IgG分泌相关。     

miRNA在牛乳过敏小鼠模型中的表达及嗜酸乳杆菌的干预作用

观察β-乳球蛋白(β-Lg)过敏小鼠模型中miRNA的异常表达和Th17相关细胞因子分泌的相关性,同时研究嗜酸乳杆菌的干预作用,探究益生菌缓解牛乳过敏的分子机制。BALB/c小鼠随机被分为正常对照组,β-Lg过敏模型组和低、中、高剂量嗜酸乳杆菌干预组,以ELISA法检测过敏小鼠血清中Th17相关细胞因子的分泌,RT-PCR法检测过敏小鼠脾脏组织CD4+T细胞中与炎症相关的miRNA基因表达,并对两者进行相关性分析。在此基础上研究嗜酸乳杆菌干预过敏模型对miRNA表达的影响。与正常对照组比较,过敏组小鼠血清IgE,ILI7,IL-6,TNF-α及IL-21水平显著增高(P0.05);其脾脏组织CD4+T细胞中miR-155,miR-21,miR-9和miR-146a mRNA表达量明显上调(P0.05),与Th17细胞的RORγt及IL-17mRNA表达水平呈正相关(P0.05)。嗜酸乳杆菌干预可显著下调过敏小鼠Th17细胞亢进,以及上述miRNA基因的异常表达(P0.05),具有剂量依赖性。以上结果显示,miRNA在β-Lg过敏中发挥重要作用,嗜酸乳杆菌缓解过敏的作用可能与其调节miRNA表达密切相关。     

具有缓解对虾原肌球蛋白致敏性乳酸菌的筛选及其免疫调节作用

目的:通过体外实验比较5种乳酸菌对致敏肥大细胞模型组胺释放量、脱颗粒的影响,筛选具有最佳抗过敏作用的乳酸菌;体内检测最佳抗过敏作用芽孢乳酸菌09.712对致敏小鼠血清中特异性抗体、脾脏T淋巴细胞亚群等的影响,探讨其缓解过敏的免疫调节作用机理。方法:体外构建P815肥大细胞致敏模型,与芽孢乳酸菌09.712共培养,采用ELISA测定细胞上清液中组胺含量,甲基红染色观察肥大细胞脱颗粒数,筛选最佳抗过敏乳酸菌。建立小鼠虾原肌球蛋白致敏模型,芽孢乳酸菌09.712灌胃处理,ELISA法测定小鼠血清中特异性抗体和细胞上清液细胞因子分泌水平,流式细胞术检测T细胞亚群,荧光定量PCR检测相关细胞因子和转录因子m RNA表达量。结果:比较5种乳酸菌,芽孢乳酸菌09.712可有效降低组胺含量,抑制肥大细胞脱颗粒,具有显著的抗过敏作用。体内试验表明,芽孢乳酸菌09.712处理组小鼠的过敏症状减弱,Th1/Th2、Treg/Th2比值显著高于致敏组小鼠(P0.05),且Th1型细胞因子IFN-γ分泌量增高,Th2型细胞因子IL-4、IL-13分泌量降低,血清中特异性抗体Ig E和Ig G1水平显著低于致敏组小鼠。此外,芽孢乳酸菌09.712还显著增加Foxp3、TGF-β和IL-10的m RNA表达水平(P0.001)。结论 :芽孢乳酸菌09.712能有效缓解小鼠过敏症状,其作用与促进初始T细胞向Th1和调节性T细胞(Treg)分化以及调控Treg平衡Th1/Th2有关。     

不同时段嗜酸乳杆菌干预对β-乳球蛋白过敏缓解的作用

目的:分析和比较嗜酸乳杆菌预防和治疗β-乳球蛋白过敏的作用和效果,为抗过敏性能益生菌的使用提供科学依据。方法:BALB/c小鼠随机分为空白组、β-乳球蛋白过敏组、嗜酸乳杆菌预防组和嗜酸乳杆菌治疗组。建模结束后,采用酶联免疫吸附法(ELISA)检测各组小鼠血清中IgE及Thl/Th2型细胞因子(IL-12、IFN-γ、IL-4)的含量;RT-PCR法检测脾脏中T-bet和GATA-3 mRNA的表达量。结果:嗜酸乳杆菌干预可有效缓解β-乳球蛋白过敏,与过敏组小鼠比较,其血清总IgE和特异性IgE水平明显降低(P0.05),IFN-γ、IL-12含量及T-bet mRNA表达量显著增加,IL-4含量及GATA-3 mRNA表达显著降低(P0.05)。特别是嗜酸乳杆菌预防组的抗过敏效果优于治疗组,且预防组调节IFN-γ/IL-4比值(代表Thl/Th2细胞平衡)的能力显著优于治疗组(P0.05)。结论:嗜酸乳杆菌预防β-乳球蛋白过敏的效果优于其治疗效果。     

EGC和EGCG降低αs1-酪蛋白致敏小鼠过敏反应

为研究茶多酚中的表没食子儿茶素(EGC)和表没食子儿茶素没食子酸酯(EGCG)的抗αs1-酪蛋白致敏机理,首先用αs1-酪蛋白致敏BALB/C小鼠,在激发阶段用EGC和EGCG给予营养干预,以降低αs1-酪蛋白致敏小鼠的过敏反应。以小鼠体内肥大细胞蛋白酶含量、组胺含量、特异性抗体IgE水平、细胞因子分泌水平以及组织病理学观察为主要指标,评价EGC和EGCG降低αs1-酪蛋白致敏小鼠过敏反应的能力。结果表明:EGC和EGCG均可显著降低过敏小鼠的肥大细胞蛋白酶、组胺、特异性IgE抗体和Th2型细胞因子水平。同时,组织病理学结果显示:EGC和EGCG显著降低肠、胸腺、脾脏和肺的病变程度。本研究结果可为茶多酚调控牛乳过敏原致敏反应发生的路径提供理论参考,未来可从信号转导和基因表达水平方面进一步探讨抗过敏机制。     

长双歧杆菌BBMN68诱导的树突状细胞对小鼠牛乳β-乳球蛋白过敏的缓解作用

为研究益生菌缓解食物过敏的作用机制,本实验以牛乳β-乳球蛋白(BLG)为过敏原构建小鼠食物过敏模型,灌服长双歧杆菌BBMN68(BBMN68),采用ELISA方法检测小鼠血清及细胞培养上清中抗体和细胞因子含量,流式细胞术分析小鼠体内树突状细胞(DCs)亚型及CD4+CD25+Foxp3+Treg细胞数量的变化。结果表明,BBMN68调节了BLG小鼠体内的Th1/Th2细胞失衡,缓解了过敏反应。与过敏组小鼠相比,BBMN68显著提高了派氏淋巴结DCs中CD103表达(p0.05),并降低了CD86和MHC-II表达(p0.05);提高了派氏淋巴结,肠系膜淋巴结和脾脏中CD4+CD25+Foxp3+Treg细胞数量,分别增加41.91%、71.16%和61.25%。分离BBMN68组小鼠派氏淋巴结的DCs与BLG过敏小鼠的CD4+T细胞共培养,发现Foxp3+Treg细胞比例显著增加,调节性细胞因子TGF-β和IL-10分泌显著增多(p0.05)。以上结果说明BBMN68缓解小鼠牛乳β-乳球蛋白过敏的机制与其调节DCs功能,促进其介导的免疫抑制有关。     

果胶与双歧杆菌对Balb/c小鼠机体敏感性的影响

目的研究果胶是否能够降低Balb/c小鼠对OVA(鸡卵清白蛋白)的过敏反应水平,以及同时经口给予双歧杆菌是否能够调整小鼠肠道菌群,从而影响过敏反应的发生和反应强度。方法连续40 d经口给予小鼠20μg/只(低剂量)和200μg/只(高剂量)的果胶溶液或溶有双歧杆菌(2×106 CFU/只)的果胶溶液,每10 d腹腔注射OVA和铝佐剂致敏小鼠,10 d后进行大刺激,采血测定组胺、肥大细胞蛋白酶-1、免疫球蛋白E水平,并对脾脏T细胞进行体外刺激,检测IFN-γ和IL-4的水平,同时取小鼠粪便筛选双歧杆菌培养。结果果胶处理组小鼠血浆中组胺、肥大细胞蛋白酶-1水平与OVA致敏组相比无显著差异(P0.05);高剂量处理组小鼠血清总IgE水平显著低于OVA致敏组(P0.05),特异性IgE及细胞因子水平无显著差异(P0.05);双歧杆菌筛选培养可以看出,果胶有增加双歧杆菌数量的作用,但经口给予双歧杆菌活菌无显著作用。结论补充果胶不能降低Balb/c小鼠的肥大细胞脱颗粒情况、抗体水平以及细胞因子产生水平;果胶有增加肠道内双歧杆菌的作用,但经口给予活菌不能增加肠道内双歧杆菌水平。     

果胶与双歧杆菌对Balb/c小鼠机体敏感性的影响

目的：研究果胶是否能够降低Balb/c小鼠对OVA(鸡卵清白蛋白)的过敏反应水平,以及同时经口给予双歧杆菌是否能够调整小鼠肠道菌群,从而影响过敏反应的发生和反应强度。方法：连续40天经口给予小鼠20μg/只(低剂量)和200μg/只(高剂量)的果胶溶液或溶有双歧杆菌(2*10₆CFU/只)的果胶溶液,每10天腹腔注射OVA和铝佐剂致敏小鼠,10天后进行大刺激,采血测定组胺、肥大细胞蛋白酶-1、免疫球蛋白E水平,并对脾脏T细胞进行体外刺激,检测IFN-γ和IL-4的水平,同时取小鼠粪便筛选双歧杆菌培养。结果：果胶处理组小鼠血浆中组胺、肥大细胞蛋白酶-1水平与OVA致敏组相比无显著差异(P>0.05);高剂量处理组小鼠血清总IgE水平显著低于OVA致敏组(P<0.05),特异性IgE及细胞因子水平无显著差异(P>0.05);双歧杆菌筛选培养可以看出,果胶有增加双歧杆菌数量的作用,但经口给予双歧杆菌活菌无显著作用。结论：补充果胶不能降低Balb/c小鼠的肥大细胞脱颗粒情况、抗体水平以及细胞因子产生水平;果胶有增加肠道内双歧杆菌的作用,但经口给予活菌不能增加肠道内双歧杆菌水平。     

植物乳杆菌调节抗体和细胞因子水平缓解食物过敏的机制

食物过敏危害人体健康,利用益生菌特有的抗过敏作用来缓解食物过敏是治疗食物过敏的新方向,然而目前关于益生菌缓解过敏的机制研究还不够深入。本文以前期筛选出的具有抗过敏功能的4株植物乳杆菌为研究对象,利用Balb/c小鼠建立食物过敏模型,采用酶联免疫吸附实验(ELISA)、荧光定量PCR(RT-PCR)等方法研究植物乳杆菌对Th1/Th2相关细胞因子、抗体分泌的影响。ELISA结果表明,将4株植物乳杆菌与致敏小鼠脾细胞共培养可降低IL-4的分泌水平,增加IFN-γ的分泌水平,而荧光定量结果证实IL-4及其转录因子GATA-3 m RNA表达水平显著降低,IFN-γ及其转录因子T-bet mRNA表达水平显著升高,这一结果与细胞因子分泌水平结果一致。此外,4株植物乳杆菌均可下调致敏小鼠血清中IgE分泌水平,缓解由食物过敏造成的肠道黏膜及肺泡组织损伤。这些结果说明植物乳杆菌通过调节Th1/Th2平衡来缓解食物过敏。     

副干酪乳杆菌L9对过敏小鼠淋巴细胞Th1/Th2平衡的调节作用

目的:研究副干酪乳杆菌L9对牛乳β-乳球蛋白(BLG)过敏小鼠淋巴细胞Th1/Th2平衡的影响,探讨L9缓解机体过敏反应的机制。方法 :构建BLG过敏小鼠模型,通过ELISA方法和流式细胞术分析L9对过敏小鼠原代淋巴细胞和骨髓源树突状细胞(BM-DCs)细胞因子分泌及调节性T细胞(Foxp3+Treg)数量的影响。结果:不同剂量的活/热致死L9均可显著提高过敏小鼠淋巴细胞上清中IFN-γ水平,显著降低IL-4含量,减少BLG特异性抗体的产生(P0.05);显著促进BM-DCs细胞上清中调节性细胞因子IL-10、TGF-β的分泌(P0.05),提高过敏小鼠CD4+T淋巴细胞中Foxp3+Treg细胞比例,并且活菌与热致死菌的调节效果相似。结论 :L9能够调节过敏小鼠淋巴细胞的Thl/Th2失衡,这可能与树突状细胞和Foxp3+Treg介导的免疫抑制有关。     

Hesperidin inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice by suppressing Th17 activity

Hesperidin (previously called vitamin P) is a predominant flavanone present in citrus fruits, and is presumed to have a role in their beneficial effect for human health because it possesses various physiological activities. In this study, we investigated the anti-allergic and anti-inflammatory effects of hesperidin and α-glucopyranosyl (αG)-hesperidin, its derivative with enhanced water-solubility, in NC/Nga mice, a human-like mouse model of atopic dermatitis. NC/Nga mice were fed a 0.1% αG-hesperidin or hesperidin diet for 8 weeks. αG-hesperidin and hesperidin feeding effectively inhibited skin lesions and immunoglobulin E (IgE) elevation. At the end of the 8-week-experimental period, the production of inflammatory cytokine interleukin (IL)-17 and interferon-gamma (IFN-γ) from splenocytes was lower in the αG-hesperidin/hesperidin-fed group than in the control group. Changes in mRNA expression in splenocytes are also examined using DNA microarray and real-time RT-PCR. It was revealed that cytotoxic T-lymphocyte antigen 4 (CTLA4), a regulatory T-cell (Treg) marker, was markedly upregulated in splenocytes, particularly by αG-hesperidin feeding. These results suggest that αG-hesperidin attenuated exacerbation of AD-like symptoms, decreased systemic immune hyper-responsiveness in part through the reduction of IgE, IL-17 and IFN-γ, and also modulated Th17/Treg balance in NC/Nga mice. Therefore, αG-hesperidin may be useful in the management of Th17-mediated allergic disorders.     

Partially hydrolyzed whey proteins prevent clinical symptoms in a cow's milk allergy mouse model and enhance regulatory T and B cell frequencies

1 Scope

Partially hydrolyzed cow's milk proteins are used to prevent cow's milk allergy in children. Here we studied the immunomodulatory mechanisms of partial cow's milk hydrolysates in vivo.

2 Methods and results

Mice were sensitized with whey or partially hydrolyzed whey using cholera toxin. Whey‐specific IgE levels were measured to determine sensitization and immune cell populations from spleen, mesenteric lymph nodes and Peyer's patches after oral whey administration were measured by flowcytometry. Whey‐specific IgE and IgG1 levels in partial whey hydrolysate sensitized animals were enhanced, but challenge did not induce clinical symptoms. This immunomodulatory effect of partial whey hydrolysate was associated with increased regulatory B and T cells in the spleen, together with a prevention of IgM‐IgA class switching in the mesenteric lymph nodes and an increased Th1 and activated Th17 in the Peyer's patches.

3 Conclusion

Partial hydrolysate sensitization did not induce whey‐induced clinical symptoms, even though sensitization was established. Increased regulatory cell populations in the systemic immune system and a prevention of increased total Th1 and activated Th17 in the intestinal immune organs could contribute to the suppression of allergic symptoms. This knowledge is important for a better understanding of the beneficial effects of hydrolysates.     

Induction of mud crab (Scylla paramamosain) tropomyosin and arginine kinase specific hypersensitivity in BALB/c mice

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. The allergens tropomyosin (TM) and arginine kinase (AK) from mud crab (Scylla paramamosain) were purified to homogeneity. BALB/c female mice were sensitized with TM and AK by intragastric administration. Mice treated with normal saline served as the negative control (NC) group. RESULTS: Compared with NC group, mice that were treated with TM and AK developed reduced activity; meanwhile, their scratching behavior and specific‐IgE level were increased. Specific‐CD4 + T cells were significantly elevated in the splenocyte cultures of the mice upon TM and AK stimulation. However, compared with the positive control group (ovalbumin, OVA), there was no significant difference. The expression of IL‐4 in culture cells stimulated by TM, AK, and OVA group showed significant differences from the NC group, respectively. CONCLUSION: These results indicated that a BALB/c mouse model for sensitization to TM and AK from mud crab was successfully established, and the Th2 response was observed, displaying increased immunoglobulin E levels, together with the production of interleukin 4 and allergic symptoms. Copyright © 2011 Society of Chemical Industry     

淡水鲈鱼小清蛋白致敏性及其对小鼠肠道菌群的影响

目的研究淡水鲈鱼小清蛋白(parvalbumin,PV)的致敏性,同时探索其对小鼠肠道菌群的影响。方法 36只C3H/HeJ小鼠按体重随机分为3组,分别为空白对照组(CK组)、卵清白蛋白(ovalbumin, OVA)致敏对照组(OVA组)和小清蛋白致敏组(PV组)。使用生理盐水、OVA溶液和PV溶液通过经口灌胃在6周时间内进行致敏,实验最后一天进行大剂量刺激,测定小鼠的体温变化、血清抗体水平和相关细胞因子水平,取腹腔灌洗液测定其中蛋白的含量以评价小鼠血管渗透性的变化,利用流式细胞术测定各组小鼠脾脏(SP)和肠系膜淋巴结(MLN)中T细胞亚群的水平。取小鼠粪便,对肠道菌群进行16S rRNA高通量测序,探索小清蛋白对小鼠肠道菌群的影响。结果与空白对照组相比,OVA小鼠血清抗体、细胞因子和免疫组织Th2相比水平均显著升高(P0.05),且表现出明显过敏症状。PV处理也使小鼠表现出明显的食物过敏症状,但仅显著升高了小鼠血清免疫球蛋白G1(Immunoglobulin G1, IgG1)和白细胞介素10(Interleukin 10, IL-10)的水平(P0.05)。16S rRNA高通量测序结果显示, OVA处理升高了小鼠肠道中属于拟杆菌门和变形菌门群落的水平,而PV处理升高了属于厚壁菌门菌群的丰度。结论淡水鲈鱼小清蛋白对小鼠具有一定的致敏性,但其致敏机理及对肠道菌群的影响作用与OVA不同。     

枸杞果多糖对哮喘小鼠炎症损伤影响及作用机制

目的 探究枸杞果多糖对Ⅴ级卵清蛋白(OVA)诱导的哮喘小鼠模型炎症损伤影响及其作用机制。方法 以OVA诱导的哮喘小鼠为模型,并进行枸杞果多糖干预。肺组织苏木素-伊红(HE)染色、流式细胞术检测肺组织嗜酸性粒细胞水平反映炎症细胞浸润程度；肺组织马松(masson)染色检测肺组织纤维化程度,过碘酸-雪夫(PAS)染色检测支气管杯状细胞化生程度；流式细胞术检测小鼠肺组织辅助性T细胞(Th1、Th2、Th17细胞)和调节性T细胞(Treg细胞)水平。结果 与对照组比较,枸杞果多糖干预的哮喘小鼠肺组织嗜酸性粒细胞水平明显降低,差异有统计学意义(P<0.05)；纤维化程度明显减轻,支气管化生的杯状细胞明显减少；Th1和Treg细胞水平上调,Th2和Th17细胞水平下调,差异均有统计学意义(P<0.05)。结论 枸杞果多糖可通过调节哮喘小鼠体内的Th细胞失衡缓解气道炎症。     

Allergenic responses of red kidney bean (Phaseolus vulgaris cv chitra) polypeptides in BALB/c mice recognized by bronchial asthma and allergic rhinitis patients

Allergenicity potential of red kidney beans (Phaseolus vulgaris cv chitra) was assessed and attempts were made to identify the responsible proteins by pepsin digestibility assay and IgE immunoblotting. To evaluate allergenic potential, BALB/c mice were sensitized with red kidney bean proteins and levels of specific immunoglobulin, histamine, mast cell protease-1, cytokines and CCL-2 were measured. To confirm our findings in BALB/c, the studies were also extended to human subjects. Human sera collected from control subjects and allergic patients after skin prick test were used for IgE immunoblotting, measuring the levels of total and specific IgE and determining cross reactivity of red kidney bean with other legumes. Red kidney bean allergenic potential was evident by significant increase in specific IgE, IgG1, histamine, mast cell protease-1 and Th2 cytokine levels in comparison to control. Enhanced level of eosinophils in jejunum, prominent anaphylactic symptoms, and eruptive histopathological changes give indication towards red kidney beans allergenicity. IgE immunoblotting detected five protein components with molecular weights of approximately 170, 100, 43, 34 and 20 kDa. Red kidney bean proteins showed cross reactivity with peanut, soybean, chickpea and black gram. Finally, this work demonstrated that red kidney beans may induce allergic response in mice similar to human subjects, with identification of five clinically relevant allergenic protein components.     

The antiallergic effect of kefir Lactobacilli

This study demonstrated that oral feeding of heat-inactivated Lactobacillus (L b.) kefiranofaciens M1 from kefir grains effectively inhibited immunoglobulin (Ig) E production in response to ovalbumin (OVA) in vivo. The pattern of cytokine production by splenocyte cells revealed that the levels of cytokines produced by T helper (Th) 1 cells increased, and those of cytokines produced by Th2 cells decreased in the heat-inactivated M1 feeding group. These findings indicated that Lactobacillus kefiranofaciens M1 in the kefir played an important role in antiallergic activities. By additional analysis using flow cytometry and microarray, the mechanism of suppression of IgE production by oral feeding of the heat-inactivated M1 probably occurs because of upregulation of the expression of Cd2, Stat4, and Ifnr leading to skewing the Th1/Th2 balance toward Th1 dominance, elevation of the CD4(+)CD25(+) regulatory T (Treg) percentage, and reduction of activated CD19(+) B cells. Downregulation of complement system and components was also involved in suppression of IgE production. Practical Application: Kefir has long been considered good for health. Its health benefits include immunoregulatory effects. However, there is a lack of knowledge concerning the immunoregulatory effects induced by kefir lactic acid bacteria (LAB). Our data clearly demonstrated the antiallergic activity of kefir LAB, Lactobacillus (L b.) kefiranofaciens M1. By additional analysis using flow cytometry and microarray, the possible mechanism of suppression of IgE production by oral feeding of the heat-inactivated M1 was also elucidated. Our findings indicated that Lactobacillus kefiranofaciens M1 may have a great potential for utilization in functional food products.     

Anti-allergic effect of low molecular weight β-glucan prepared by λ-irradiation

This study evaluated the anti-allergic effect of low molecular weight β-glucan (25 kDa) degraded by λ-irradiation. Allergy was induced in mouse models administrated with λ-irradiated low molecular weight and non-irradiated high molecular weight β-glucan, respectively, for 7 days. However, only the mice treated with low molecular weight β-glucan had a significantly decreased the total serum IgE and ovalbumin specific-IgE levels, and significantly increased Th 1 cytokine expression compared to only allergy induced mice. The allergic responses could be reduced by low molecular weight β-glucan treated λ-irradiation through the increased expression of Th 1 response and decreased expression of total IgE and antigen specific IgE.