携带IL-10基因的双歧杆菌对溃疡性结肠炎模型小鼠血浆及结肠组织NO和VEGF-A表达影响

目的观察携带IL-10基因的双歧杆菌对溃疡性结肠炎(UC)小鼠血浆及结肠组织一氧化氮(NO)与血管内皮生长因子A(VEGF-A)表达的影响,进一步探讨携带IL-10基因的双歧杆菌治疗UC的相关机制。方法筛选出能稳定表达具有生物活性hIL-10蛋白的BL-hIL-10菌株。用5%DSS诱导UC小鼠模型,50只小鼠分为正常对照组、UC模型组、BL治疗组、BL0治疗组和BL-hIL-10治疗组,每组10只。计算小鼠DAI、HE染色评估结肠组织病理学变化;ELISA法测小鼠血浆及结肠组织IL-13、VEGF-A和NO的含量。结果 (1)BL-hIL-10可以降低UC小鼠DAI,减轻UC小鼠结肠组织炎症程度。(2)BL-hIL-10能降低UC小鼠血浆和结肠组织NO、VEGF-A和IL-13水平。结论口服BL-hIL-10对UC小鼠的治疗作用可能与抑制NO产生及下调VEGF-A表达有关。     

携带IL-10基因的双歧杆菌对 溃疡性结肠炎小鼠肠道屏障功能的影响

目的观察表达IL-10基因的双歧杆菌对溃疡性结肠炎(UC)小鼠肠道屏障功能的影响,进一步探讨转基因双歧杆菌治疗UC的相关机制。方法 25只小鼠分为空白对照组、结肠炎组(UC-blank组)、双歧杆菌治疗组(UC-bacteria组)、空质粒双歧杆菌治疗组(UC-pBBAD/X-bacteria组)和表达IL-10基因双歧杆菌治疗组(UC-BL-hIL-10-bacteria组),每组5只。采用5%DSS诱导建立UC小鼠模型,利用前期研究已成功筛选出的可稳定表达hIL-10蛋白的BL-hIL-10菌株对小鼠进行治疗。采用HE染色评估小鼠结肠炎症情况;采用荧光定量PCR检测小鼠结肠组织Claudin1、2、4、5、7和8的表达水平;计算小鼠DAI。结果 (1)BL-hIL-10菌株可以降低UC小鼠DAI,减轻UC小鼠结肠组织炎症程度。(2)BL-hIL-10菌株能下调UC小鼠结肠组织Claudin2表达水平,同时能够上调Claudin1、4、5、7和8的表达水平。(3)UC-BL-hIL-10-bacteria组UC小鼠的治疗效果明显优于UC-bacteria组和UC-pBBAD/Xbacteria组。结论 BL-hIL-10菌株能改善UC小鼠结肠黏膜的通透性,其机制可能与其能上调Claudin1、4、5、7和8的表达水平,同时降低Claudin-2的表达水平有关。     

地衣芽胞杆菌BL63516对抗生素诱导的 菌群紊乱小鼠肠黏膜屏障功能的影响

目的探索地衣芽胞杆菌BL63516对肠道菌群和肠黏膜屏障功能调节的效果和可能机制,为临床疾病的治疗提供新思路。方法采用健康清洁级雄性BALB/C小鼠30只构建动物模型,将小鼠分为3组(每组10只),分别为对照组(CON组)、头孢曲松钠灌胃组(CS组)和地衣芽胞杆菌BL63516治疗组(BL组),除CON组外其余两组小鼠给予300 mg/mL头孢曲松钠0.2 mL,2次/d灌服8 d,短期大量服用抗生素以建立肠道菌群紊乱模型。第9天起BL组小鼠给予地衣芽胞杆菌干预连续8 d。第17天摘眼球处死小鼠,取血、结肠、粪便样本。ELISA方法检测小鼠血清及肝脏组织中的细胞因子及LPS水平。PCR-变性梯度凝胶电泳方法及16S rRNA高通量测序测定小鼠肠道菌群结构。结果地衣芽胞杆菌干预后小鼠血清LPS水平较模型组显著降低,屏障功能改善。且地衣芽胞杆菌治疗后BL组小鼠肠道菌群丰富度和多样性显著升高,菌群结构有所改变。结论实验结果提示地衣芽胞杆菌BL63516可以有效改善抗生素所致的肠道菌群失调,对肠屏障功能有一定调节作用。     

地衣芽胞杆菌BL63516对 DSS诱导的小鼠结肠炎的调节作用

目的探索地衣芽胞杆菌BL63516对DSS诱导的小鼠结肠炎的缓解作用和可能机制,为临床疾病的治疗提供新思路。方法用BALB/C雌性小鼠构建动物模型,将30只小鼠分为CON组、DSS组和BL组,每组10只,除CON组外,其余两组均给予3%(m/v)DSS自由饮用,并给予地衣芽胞杆菌BL63516干预。第8天摘眼球处死小鼠,取血、结肠、粪便样本。以ELISA法检测小鼠血清和结肠研磨液中的细胞因子。用PCR-变性梯度凝胶电泳(PCR-DGGE)方法分析小鼠肠道菌群结构。结果 DSS组小鼠血清IL-10及结肠MPO水平均与CON组有明显差异,地衣芽胞杆菌干预后有不同程度改变;使用非加权成对算术平均算法(UPGMA)对PCR-DGGE图谱中各泳道条带类型聚类分析结果显示各组小鼠结肠菌群结构具有显著差异,其中BL组增加双歧杆菌属Bifidobacterium saguini及疣微菌门的噬黏蛋白阿克曼菌(Akkermansia muciniphila)数量。结论地衣芽胞杆菌可以有效改善DSS诱导的结肠炎症状,其作用机制与肠屏障功能和肠道菌群的调节有密切关系。     

思连康对小鼠腹腔巨噬细胞吞噬率和吞噬指数的影响

目的研究双歧杆菌四联活菌片(商品名:思连康)对小鼠腹腔巨噬细胞吞噬鸡红细胞吞噬率及吞噬指数的影响。方法将SPF小鼠30只随机分成三组,每组10只,Ⅰ组灌胃生理盐水,Ⅱ组灌胃婴儿双歧杆菌菌悬液,Ⅲ组灌胃双歧杆菌四联活菌片菌悬液,每天给药0.5 mL,菌液浓度为1.0×10~8 CFU/mL,连续给药10 d后小鼠腹腔注入2%鸡红细胞悬液1 mL(红细胞数量为2×10~8个/mL),30 min后处死,取小鼠腹腔洗液,观察并记录吞噬鸡红细胞的巨噬细胞数及被吞噬的鸡红细胞数,计算吞噬率及吞噬指数。结果与Ⅰ组相比,Ⅱ组和Ⅲ组小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬率和吞噬指数均显著升高(Ps0.05),其中Ⅲ组高于Ⅱ组(P0.05)。结论双歧杆菌四联活菌片及其婴儿双歧杆菌通过提高小鼠腹腔巨噬细胞的吞噬率和吞噬指数提高机体的免疫力。     

益生菌对肠易激综合征小鼠肠道黏膜CRF-R1表达及肠道屏障功能的影响

目的通过观察肠易激综合征(IBS)小鼠模型结肠黏膜肥大细胞上CRF-R1及结肠黏膜PAR-2、Claudin1~4等的表达变化,探讨IBS中应激通过肥大细胞引起肠道屏障功能障碍的可能机制并观察婴儿双歧杆菌的治疗作用。方法 30只雄性Balb/c小鼠随机分为对照组、模型组及婴儿双歧杆菌组。以束缚应激法建立IBS小鼠模型。婴儿双歧杆菌组给予婴儿双歧杆菌灌胃,而对照组及模型组给予等体积生理盐水灌胃。观测腹肌收缩反射(AWR)后处死小鼠。ELISA检测外周血中类胰蛋白酶的表达变化。免疫组织化学分析结肠黏膜CRF、PAR-2、Claudin1、Claudin2、Claudin3、Claudin4的表达情况。免疫荧光双标分析结肠黏膜CRF-R1在肥大细胞的表达情况。RT-PCR检测结肠CRF-R1mRNA的表达情况。结果与对照组相比,模型组小鼠外周血类胰蛋白酶表达量增加;结肠黏膜中CRF、PAR-2、Claudin2表达量、CRF-R1+肥大细胞数目及CRF-R1 mRNA表达量增加,结肠黏膜Claudin1、Claudin3、Claudin4表达量降低,差异均有统计学意义(P0.05)。婴儿双歧杆菌干预后,该组小鼠外周血中类胰蛋白酶表达量降低;结肠黏膜中CRF、PAR-2、Claudin2表达量、CRF-R1+肥大细胞数目及CRF-R1 mRNA表达量降低,结肠黏膜Claudin1、Claudin3、Claudin4表达量增高,差异均有统计学意义(P0.05)。结论 IBS小鼠模型中,婴儿双歧杆菌可以减轻肠道屏障功能障碍,其机制可能与抑制结肠黏膜肥大细胞上CRF-R1的表达而抑制肥大细胞的激活及其免疫因子的释放,从而降低结肠黏膜PAR-2的表达有关。     

粪菌移植对溃疡性结肠炎小鼠结肠黏膜Th1/Th2细胞及其细胞因子表达的影响

目的观察粪菌移植(FMT)对结肠炎小鼠结肠组织内Th1/Th2平衡及其细胞因子的影响。方法用葡聚糖硫酸钠(DSS)制备溃疡性结肠炎小鼠模型,共48只雄性C57BL/6小鼠,按3∶2∶1随机分为干预组、对照组和粪便供体组。其中干预组24只小鼠自由饮用2%DSS溶液,第6天时再次称重随机分为3组,每组6只:粪便滤液组(A组)、美沙拉嗪缓释颗粒组(B组)、生理盐水组(C组),并分别灌肠给予粪便滤液(500mg/mL,0.3mL/次)、美沙拉嗪缓释颗粒混悬液(25mg/mL,0.3mL/次)和生理盐水(0.3mL/次),每日2次,连续5d;对照组分为急性结肠炎组(D组)和正常对照组(E组),每组6只,分别自由饮用2%DSS溶液和蒸馏水7d后处死;粪便供体组(F组)正常进食饮水,采集新鲜粪便制作粪便滤液。评估疾病活动指数、脾脏指数、结肠长度和结肠组织病理评分。免疫组化法和酶联免疫吸附试验(ELISA)分别检测结肠组织Th1/Th2细胞及细胞因子(TNF-α、IFN-γ、IL-4和IL-10)的表达。结果 (1)粪菌移植治疗明显改善结肠挛缩和病理损伤(P0.05),而脾脏指数明显增加(P0.05);(2)粪菌移植使结肠炎小鼠结肠组织的T-bet阳性细胞和Gata-3阳性细胞表达升高,T-bet/Gata-3平衡右移;(3)粪菌移植使结肠炎小鼠结肠组织细胞因子(TNF-α、IFN-γ、IL-4、IL-10)水平下降。结论粪菌移植促进结肠炎小鼠的结肠黏膜修复,可能与调节结肠组织Th1/Th2细胞平衡,下调炎症因子,介导Th2免疫修复有关。     

双歧杆菌小鼠肠道定植的优化

目的评价自制的小鼠灌肠器灌肠效果,并探讨双歧杆菌定植的优化方式,更好地建立双歧杆菌在结肠的定植模型。方法选用SPF级C57BL小鼠40只,每组10只,随机分成4组：正常对照组,常规灌胃组,市售灌肠组,自制灌肠组。从市面不同的材料中筛选小鼠灌肠管,并设计小鼠专用灌肠器。记录不同灌注方式时隐血试验等一般指标检测。RT-PCR检测4组脾脏TLR2 mRNA表达水平。免疫组化观察颈动脉血管HGF蛋白水平。体外模拟不同pH值检测双歧杆菌定植HT-29细胞的粘附水平。结果自制的灌肠器在小鼠灌肠操作过程中明显优于市售灌肠器;自制灌肠组隐血试验明显低于市售灌肠组（P〈0.01）;RT-PCR结果显示,自制灌肠组效果最强,市售灌肠组次之,常规灌胃组一般,但均高于正常对照组;HGF的免疫组化显示,自制灌肠组表达最强;粘附试验显示双歧杆菌在pH 7.0环境中粘附能力优于pH 4.5。结论双歧杆菌在结肠定植采用灌肠方式优于灌胃方式;自制灌肠器优于市售灌肠器;优化灌肠液,采用自制的灌肠器灌肠同时优化灌肠操作,能够在小鼠结肠建立较好的双歧杆菌定植模型。     

婴儿型双歧杆菌对哮喘小鼠气道炎症的作用研究

目的探讨婴儿型双歧杆菌对气道过敏小鼠的气道炎症作用及其机制。方法 6周龄BALB/c雄性小鼠40只,随机分为4组:阴性对照组、哮喘组、婴儿型双歧杆菌预防组和婴儿型双歧杆菌治疗组。哮喘组、预防组和治疗组用卵清蛋白(Ovalbvmin,OVA)进行致敏和激发诱发哮喘,阴性对照组用生理盐水进行致敏和激发。预防组第0至14天灌胃菌液;治疗组第15至28天灌胃菌液;阴性对照组和哮喘组全程灌胃生理盐水作对照。观察小鼠气道过敏表现;计数肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)中细胞总数评估气道炎症程度;肺组织行HE染色;ELISA法测定血清中IL-10、OVA特异性IgE和OVA特异性IgG1的浓度以及肺泡灌洗液中细胞因子IL-4、IL-5、IL-10、IL-13和IFN-γ的浓度。结果 (1)哮喘组小鼠在激发后出现搔抓口鼻、竖毛、打喷嚏、腹肌扇动、弓背直立、二便失禁以及体重下降等表现,预防组和治疗组的症状较轻。(2)预防组和治疗组BALF细胞数量明显低于哮喘组(P0.05)。(3)肺组织HE染色后,哮喘组小鼠肺组织出现明显的炎症细胞浸润,预防组和治疗组肺组织炎症明显减轻。(4)预防组和治疗组血清OVA特异性IgE浓度明显低于哮喘组(P0.05),并且预防组低于治疗组(P0.05);治疗组血清OVA特异性IgG1浓度明显低于哮喘组和预防组(P0.05);预防组和治疗组血清IL-10浓度明显高于哮喘组(P0.05),且治疗组高于预防组(P0.05);(5)预防组和治疗组肺泡灌洗液中IL-4、IL-13含量明显低于哮喘组(P0.05);IL-5、IL-10、IFN-γ各组浓度都较低,未测出有效浓度。结论口服婴儿型双歧杆菌可以减轻过敏症状,降低肺组织炎症浸润程度,抑制Th2免疫反应。     

微生态制剂通过下调小鼠肝脏γδT细胞内IL-17的分泌缓解胆汁淤积性肝病

目的探讨微生态制剂通过下调小鼠肝脏γδT细胞内IL-17的分泌缓解胆汁淤积性肝病的机制。方法随机选取10只正常环境饲养的小鼠作为对照组,每日上下午给予10 mL/kg的生理盐水灌胃共20 d;按相关文献中小鼠胆汁淤积性肝病模型制备方法,将其余用于造模的30只小鼠每日上下午均采用50%葡萄糖+100%猪油脂1︰1比例的混合液10 mL/kg灌胃20 d,第21天,将α-萘基异硫氰酸盐(ANIT)100 mg/kg一次性灌胃,灌胃前后12 h禁食不禁水;随后在完成造模的30只小鼠中,随机选取10只作为模型组,再选取10只作为给药低剂量组,剩余10只为给药高剂量组;自第21天开始,对照组及模型组小鼠每日下午给予10 mL/kg生理盐水灌胃7 d,给药低剂量组以含双歧杆菌活菌数为10~3 CFU/mL的稀释液10 mL/kg灌胃7 d,给药高剂量组以含双歧杆菌活菌数为10~7 CFU/mL的稀释液10 mL/kg灌胃7 d。将小鼠处死后,小鼠肝脏HE染色进行组织病理学观察,小鼠血清采用ELISA试验进行肝功能及炎症因子IL-17检测;分离纯化小鼠γδT细胞并进行培养,通过qRT-PCR试验检测IL-17信号通路相关因子IL-17a、IL-17f、CCR6和CD163的表达水平。结果小鼠肝脏组织病理学观察显示微生态制剂对胆汁淤积性肝病有明显缓解效果,并且高剂量比低剂量的效果更明显;血清肝功能检测结果显示模型组小鼠血清ALT、AST、ALP、TBA、TBIL和LP-X水平较对照组升高(t=15.670、13.470、14.170、7.376、7.839、10.430,均P0.001),血清LEC、CHOL水平降低(t=4.089、6.022,均P0.001);给药低剂量组及给药高剂量组小鼠血清ALT、AST、ALP、TBA、TBIL和LP-X水平较模型组降低(F=83.330、39.400、71.680、20.820、17.000、27.300,均P0.001),血清LEC、CHOL水平升高(F=6.419,P=0.520;F=8.486,P=0.014);小鼠血清IL-17检测结果显示模型组较对照组升高(t=42.010,P0.001),给药低剂量组及给药高剂量组小鼠血清IL-17水平较模型组降低(F=454.700,P0.001);qRT-PCR试验结果显示微生态制剂给药能明显下调小鼠肝脏γδT细胞内IL-17信号通路相关主要因子IL-17a、IL-17f、CCR6和CD163的表达水平。结论微生态制剂能通过下调小鼠肝脏γδT细胞内IL-17的分泌缓解胆汁淤积性肝病的发展。     

Repeated intratracheal challenge with particulate antigen modulates murine lung cytokines

When lungs of experimental animals are repeatedly challenged with Ag, pulmonary inflammation wanes via unknown mechanisms. We hypothesized that changes in the balance of lung cytokines are responsible for immune down-regulation to repeated Ag challenge. We used intratracheal (IT) challenge of primed C57BL/6 mice with SRBC and on various days after single (1IT) or triple (3IT) challenge counted lung inflammatory cells and measured whole-lung cytokine mRNA and protein concentrations using RT-PCR and ELISA. We found that lung lymphocyte numbers and parenchymal lung inflammation decreased significantly at days 6 and 9 after final Ag challenge in 3IT mice compared with 1IT mice. Lungs of 3IT mice showed the following changes in relative mRNA expression: an earlier peak in IL-10, decreased IL-1beta, and a change from a Th2 response in 1IT mice to a Th1 response in 3IT mice (with pronounced increases in IL-12, IL-18, and IFN-gamma and decreased IL-4, IL-13, and IL-5). Similar types of changes were seen in whole-lung protein concentrations for TNF-alpha, IL-10, IL-12 p40, IFN-gamma, and IL-4. Additionally, mRNA expression of the endothelial selectins CD62E and CD62P decreased and lung lymphocyte apoptosis increased in the 3IT group. Thus, physiologic down-regulation of the pulmonary immune response to repeated Ag exposure is characterized by increased anti- and decreased proinflammatory cytokines that accompanies Th1 polarization. Similar mechanisms may act to minimize chronic lung inflammation in the majority of normal humans who do not develop progressive lung pathology when repeatedly exposed to inhaled or aspirated environmental Ags.     

Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-gamma-/- and IL-4-/-) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-gamma, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-gamma, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-gamma in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4-/- animals had more severe gastritis and higher levels of IFN-gamma production by urease-stimulated splenocytes (p < 0.01), whereas IFN-gamma-/- mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-gamma, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.     

Preventative Effect of an Herbal Preparation (HemoHIM) on Development of Airway Inflammation in Mice via Modulation of Th1/2 Cells Differentiation

HemoHIM, an herbal preparation of three edible herbs (Angelica gigas Nakai, Cnidium officinale Makino, Paeonia japonica Miyabe) is known to increase the Th1 immune response as well as reduce the allergic response in human mast cells. Here, our goal was to determine whether or not HemoHIM could induce Th1 cell differentiation as well as inhibit the development of airway inflammation. To study Th1/Th2 cell differentiation, naive CD4⁺ T cells isolated from C57BL/6 mouse spleens were cultured with or without HemoHIM. To examine airway inflammation, C57BL/6 mice were fed HemoHIM for 4 weeks before sensitization and provocation with ovalbumin (OVA). In an in vitro experiment, naive CD4⁺ T cells displayed increased Th1 (IFN-γ⁺ cell) as well as decreased Th2 (IL-4⁺ cell) differentiation in a HemoHIM concentration-dependent manner. Furthermore, in an airway inflammation mice model, eosinophil numbers in BALF, serum levels of OVA-specific IgE and IgG1, and cytokine (IL-4, IL-5, and IL-13) levels in BALF and the supernatant of splenocytes all decreased upon HemoHIM (100 mg/kg body weight) pretreatment (4 weeks). These results show that HemoHIM attenuated allergic airway inflammation in the mouse model through regulation of the Th1/Th2 balance.     

Modulation of lung allergic response by renal ischemia and reperfusion injury

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors.     

Transgenic expression of IL-10 in T cells facilitates development of experimental myasthenia gravis

Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.     

The effect of chloroquine on the production of interferon-gamma, interleukin (IL)-4, IL-6, and IL-10 in Plasmodium chabaudi chabaudi in infected C57BL6 mice

The effect of chloroquine (CQ) on the production pattern of interferon (IFN)-gamma, interleukin (IL)-4, IL-6, and IL-10 in female C57BL6 mice infected with Plasmodium chabaudi chabaudi AS was evaluated during a period of 35 days. Our data confirm that there is a switch from a T helper cell (Th)1 to a Th2 response during malaria infection in this model. Proliferation assays showed a decreased stimulation index in infected mice that was further reduced in infected mice treated with CQ. Noninfected control mice treated with CQ showed an increase production of IFN-gamma. However, no detectable changes in IL-4, IL-6, and IL-10 production were observed in this group. CQ treatment of infected mice resulted in parasite clearance that was associated with an earlier production of IL-4, IL-6, and IL-10 when compared with nontreated infected mice. We suggest that this earlier switch to a Th2 response is a consequence of parasite killing rather than CQ interference with cytokine production.     

IFN-gamma is necessary but not sufficient for anti-CD40 antibody-mediated inhibition of the Th2 response to Schistosoma mansoni eggs

The injection of Schistosoma mansoni eggs into the footpads of mice results in a localized Th2 cytokine response and tissue eosinophilia. We examined whether treatment with CD40-activating Abs would block the development of Th2 cytokine responses and eosinophilic tissue pathology in this model. Seven days after C57BL/6 mice were injected with eggs and the FGK45 anti-CD40 Ab, Ag-specific synthesis of IL-4, IL-5, and IL-13 in lymph node culture was reduced (>10-fold) relative to control mice treated with eggs and rat IgG. In contrast, IFN-gamma and IL-12 were increased in both culture supernatants and in the serum. Similar changes in lymph node cytokine mRNA were observed in vivo, and tissue eosinophilia was reduced nearly 20-fold. Th2 cytokine responses in anti-CD40-treated IFN-gamma-/- and IL-12 p40-/- C57BL/6 mice were unaffected, although anti-CD40 induced high levels of systemic and local IFN-gamma production in both wild-type and IL-12 p40-/- mice. We conclude that CD40-activating treatments strongly reverse the immune phenotype generated in response to a classic, Th2-biasing stimulus and stimulate IFN-gamma through a novel IL-12-independent pathway. This model for Th1-deviating immune therapy may have relevance to the treatment of Th2-dependent diseases in general.     

Bulleyaconitine A inhibits the lung inflammation and airway remodeling through restoring Th1/Th2 balance in asthmatic model mice

ABSTRACT

The current study aimed to study the effects of Bulleyaconitine A (BLA) on asthma. Asthmatic mice model was established by ovalbumin (OVA) stimulation, and the model mice were treated by BLA. After BLA treatment, the changes in lung and airway resistances, total and differential leukocytes in the bronchoalveolar lavage fluid (BALF) were detected, and the changes in lung inflammation and airway remodeling were observed. Moreover, the secretion of IgE, Th1/Th2-type and IL-17A cytokines in BALF and serum of the asthmatic mice were determined. The resuts showed that BLA attenuated OVA-induced lung and airway resistances, inhibited the inflammatory cell recruitment in BALF and the inflammation and airway remodeling of the asthmatic mice. In addition, BLA suppressed the secretion of IgE, Th2-type cytokines, and IL-17A, but enhanced secretions of Th1-type cytokines in BALF and serum. The current study discovered that BLA inhibited the lung inflammation and airway remodeling via restoring the Th1/Th2 balance in asthmatic mice.     

Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.