TLR4和IL-1β在幽门螺杆菌感染的SPF级小鼠胃内的表达及其相关性

目的比较SPF级小鼠在幽门螺杆菌(Hp)感染时和用三联疗法根除Hp后胃黏膜内Toll样受体4(TLR4)和白细胞介素-1β(IL-1β)在基因和蛋白水平上的表达含量变化,分析TLR4和IL-1β之间的相关性,探讨TLR4在Hp致病机制中的作用。方法20只成功感染Hp的SPF级BALB/c小鼠随机分为两组,第1组不予任何治疗,第2组予三联疗法治疗,并将10只正常SPF级BALB/c小鼠设为第3组。取各组小鼠的胃黏膜标本,用逆转录聚合酶链反应(RT-PCR)和蛋白质印记(Western blotting)的方法半定量检测TLR4、IL-1β在基因和蛋白水平上的表达。结果①在基因和蛋白水平,第2组小鼠胃黏膜内TLR4和IL-1β的表达均较第1组明显降低(P<0.05)。②TLR4与IL-1β呈正相关。结论TLR4参与了Hp的致病机制,并且与IL-1β呈正相关,TLR4通路与下游的IL-1β炎症因子的激活相关。     

Toll样受体4通路在幽门螺杆菌致病机制中的作用

比较Toll样受体4(TLR4)及MyD88在幽门螺杆菌(Hp)感染的BALB/c小鼠胃黏膜内表达含量随时间的变化,探讨TLR4通路在Hp致病机制中的作用.方法:70只BALB/c小鼠随机分为7组,分别于感染Hp的第0天、第3天、第7天、第14天、抗生素三联疗法治疗的第3天、第7天、第14天取得各组小鼠的胃黏膜标本,用RT-PCR和Western blotting的方法半定量检测TLR4、MyD88在基因和蛋白水平上的表达.结果:(1)小鼠TLR4、MyD88在感染Hp后均迅速升高,在治疗后迅速回落,但仍高于正常水平.(2)小鼠TLR4、MyD88的表达含量与病理严重程度无相关性.结论:TLR4信号通路参与了HP的致病机制,但与炎症反应的严重程度无相关性.     

FoxP3、CD4~+CD25~+调节性T细胞、TLR2和TLR9在儿童传染性单核细胞增多症中的变化

本研究旨在探讨TLR2(Toll-like receptors,TLRs)、TLR9和CD4+CD25+调节性T细胞(Treg)及其转录因子FoxP3在儿童传染性单核细胞增多症(infectious mononucleosis,IM)发病中的作用。2010年4月至2011年1月我院儿科诊治的初次发病的急性期IM患儿35例(IM急性期组),IM恢复期组35例以及健康对照儿童35例(对照组)纳入研究。采用SYBR GreenⅠ实时荧光定量PCR方法检测外周血单个核细胞TLR2、TLR9、FoxP3mRNA的表达,运用流式细胞术检测外周血中T淋巴细胞亚群CD4+CD25+的表达。结果显示:IM急性期组TLR2mRNA(4.03±0.56)、TLR9 mRNA(8.88±1.56)相对表达水平显著高于对照组TLR2 mRNA(2.22±0.57)、TLR9mRNA(3.63±1.30)及恢复期组TLR2 mRNA(2.76±0.83)、TLR9 mRNA(5.34±1.60)相对表达水平(P<0.01)。IM急性期组FoxP3 mRNA(2.82±0.90)、CD4+CD25+(2.38±1.32%)表达水平显著低于对照组FoxP3mRNA(4.65±1.23)、CD4+CD25+(7.85±1.97%)及恢复期组FoxP3 mRNA(4.11±1.37)、CD4+CD25+(6.81±1.84%)(P<0.01),IM恢复期组FoxP3 mRNA、CD4+CD25+表达水平与对照组比较差异无统计学意义(P>0.05)。结论:IM急性期存在CD4+CD25+Treg数量降低及其特异性转录因子FoxP3表达下调,而TLR2和TLR9在IM急性期表达上调。     

慢性阻塞性肺病外周血单个核细胞表面TLR2、TLR4的表达与血清TNF-α、IL-6、IL-8相关性的研究

目的:探讨慢性阻塞性肺病(chronic obstructive pulmonary disease,COPD)患者外周血单个核细胞表面Toll样受体(Toll-like receptors)TLR2和TLR4的表达情况及其与血清肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)的关系。方法:本研究涉及的3组研究对象分别为:吸烟COPD组(n=68)、吸烟非COPD组(n=42)和不吸烟非COPD组(n=43)。将68例吸烟COPD患者进一步分为急性发作期亚组(n=32)和稳定期亚组(n=36)。采用实时荧光定量PCR法检测外周血单个核细胞表面TLR2、TLR4的表达水平,同时采用酶联免疫吸附试验(ELISA)试剂盒测定血清细胞因子的浓度。结果:吸烟COPD组外周血单个核细胞表面TLR2、TLR4的表达水平及血清TNF-α、IL-6、IL-8的浓度均显著高于吸烟非COPD组(TLR2:1.87±2.04比0.47±0.24;TLR4:4.08±3.84比0.41±0.18;TNF-α:68.20±56.26比26.20±5.43;IL-6:201.62±174.69比72.99±8.03;IL-8:235.42±158.22比77.50±8.38)。吸烟非COPD组外周血单个核细胞上TLR2、TLR4的表达水平均显著低于不吸烟非COPD组(TLR2:0.47±0.24比2.96±2.71;TLR4:0.41±0.18比2.67±1.74),而吸烟非COPD组血清TNF-α、IL-6、IL-8的浓度均显著高于不吸烟非COPD组(TNF-α:26.60±5.43比8.93±6.41;IL-6:72.99±8.03比25.61±8.63;IL-8:77.50±8.38比47.10±11.11)。COPD急性发作期单个核细胞表面TLR2、TLR4的表达水平及血清TNF-α、IL-6、IL-8的浓度均显著高于COPD稳定期(TLR2:3.56±1.80比0.368±0.222;TLR4:6.97±3.76比1.51±0.943;TNF-α:106.17±61.94比90.01±19.66;IL-6:327.18±183.56比105.36±13.45;IL-8:381.74±108.51比34.44±10.45)。吸烟COPD组外周血单个核细胞表面TLR2、TLR4的表达水平与血清TNF-α、IL-6、IL-8的浓度成正相关。结论:COPD患者尤其是COPD急性发作期患者外周血单个核细胞表面TLR2、TLR4的表达水平及血清TNF-α、IL-6、IL-8的浓度均显著升高;并且COPD患者外周血单个核细胞表面TLR2、TLR4的表达水平与血清TNF-α、IL-6、IL-8的浓度成正相关。提示COPD的发生、发展与机体天然免疫密切相关。     

Dectin-1和TLR2/TLR3/TLR4受体在免疫缺陷小鼠侵袭性肺曲霉菌病的表达

目的本研究利用小鼠动物模型,探讨烟曲霉感染免疫缺陷小鼠TLR2/TLR3/TLR4对树突状细胞植物血凝素-1(Dectin-1)表达的影响。通过检测TLRs和CLRs受体在不同免疫状态下的表达,揭示介导侵袭性肺曲霉菌病(IPA)肺损伤的关键受体。方法小鼠随机分成四组,A组为正常对照组;B组为环磷酰胺免疫抑制+未接种烟曲霉菌;C组正常状态小鼠+烟曲霉菌接种;D组为IPA感染组,免疫抑制+烟曲霉菌接种。采用real-time PCR方法进行小鼠肺组织各个时相点的TLR2、TLR3、TLR4、Dectin-1和β-actin m RNA的表达,检测受体间的相互表达调控。结果正常状态感染组(C组N+AF),TLR4/TLR2、Dectin-1 m RNA比正常对照组表达上调;IPA模型组(D组CP+AF),Dectin-1和TLR4、TLR2 m RNA比正常状态感染组表达下调;但TLR3 m RNA 48 h、72 h表达没有明显不同(P>0.05)。病理切片模型组小鼠72 h可见较严重的出血和充血;96 h时肺内有菌丝,肺泡间隔增宽,支气管壁破坏。正常状态感染烟曲霉小鼠72 h可见充血,肺泡弹性纤维破坏。结论成功建立了小鼠IPA动物模型,Dectin-1受体表达下调可能是环磷酰胺免疫抑制引起IPA的机制之一。Dectin-1的表达可能是建立在TLR2/TLR4对烟曲霉早期识别的基础上,TLR3可能起协同作用。     

小鼠感染肺炎衣原体后肺组织TLR2、TLR4基因表达的变化及意义

目的通过建立小鼠肺炎衣原体感染的模型,探讨小鼠感染肺炎衣原体后肺脏组织Toll样受体(TLR)2、4mRNA表达及意义。方法Icr雄性小鼠60只,随机分为实验组、对照组,实验组鼻内接种肺炎衣原体,对照组接种二磷酸蔗糖(2sp)缓冲液,再分别按预定的处死时间1、3、5、7、14d各分为5小组,取肺脏组织。应用逆转录-聚合酶链反应(RT-PCR)半定量方法检测肺组织TLR2mRNA、TLR4mRNA的表达变化。结果小鼠感染肺炎衣原体后,引起TLR表达明显变化。其中TLR2mRNA表达水平在3、5、7、14d其密度值(0.849±0.107、0.981±0.104、0.926±0.116、0.905±0.046)显著高于对照组(0.375±0.149、0.340±0.069、0.314±0.074、0.302±0.010,P<0.01),并在第5天达到高峰;而TLR4mRNA表达水平在第1、3、5、7、14天均低于对照组,但无明显的统计学意义。结论肺炎衣原体感染与TLR的变化有密切的联系,主要在TLR2上表达变化,TLR的变化增强了机体非特异免疫能力,并随着肺炎衣原体感染程度的加重与减轻,TLR2mRNA的表达变化上调或下降。     

库普弗细胞对急性肝衰竭模型鼠TLR4表达的影响

目的:探讨抑制库普弗细胞对急性肝衰竭模型鼠Toll样受体4(Toll-like receptor 4.TLR4)表达的影响.方法:80只SD雄性大鼠随机分为正常对照组、氯化钆(gadolinium chloride,GdCl3)组、内毒素(LPS)组、GdCl3+LPS组.免疫组化检测EDl表达,观察GdCl3对库普弗细胞的抑制程度,并检测血清ALT、TNFα、IL-1β的水平,Western blot分析肝组织TLR4的表达.结果:GdCl3组肝组织中EDl免疫反应阳性细胞与正常对照组相比明显减少(P＜0.05),LPS组EDl阳性细胞数量最多,GdCl3+LPS组EDl阳性细胞数少于LPS组(P＜0.05).GdCl3+LPS组AIJT、TNFct、IL-1β水平高于正常对照组,但低于LPS组(P＜0.05).GdCl3+LPS组大鼠肝脏TLR4表达与LPS组相比较弱(P＜0.05),与正常对照组相比差异无统计学意义(P＞0.05).结论:抑制库普弗细胞激活可减少肝组织11LR4的表达,库普弗细胞在急性肝衰竭中发挥重要作用.     

白癜风患者外周血单个核细胞中TLR7、TLR9、NF-κB的表达及意义研究

目的探究白癜风患者外周血单个核细胞(PBMC)中Toll样受体7(TLR7)、Toll样受体9(TLR9)、核因子-κB(NF-κB)的表达情况及其意义。方法前瞻性选取2017年4月至2018年4月沈阳市第七人民医院收治的33例白癜风患者作为观察组,33例同期健康体检者为对照组。使用流式细胞仪检测两组PBMC中的TLR7、TLR9表达情况,采用逆转录-聚合酶链反应(RT-PCR)检测两组PBMC中的NF-κB表达情况。结果流式细胞仪结果显示,观察组患者PBMC中的TLR7表达量为(94.65±3.931)%,高于对照组的(92.84±3.52)%,但差异无统计学意义(P0.05);而观察组患者PBMC中的TLR9表达量为(86.68±11.01)%,低于对照组的(90.75±5.74)%,但差异无统计学意义(P 0.05); PCR反应结果显示,观察组患者PBMC中的NF-κB mRNA表达量为(0.96±1.52),高于对照组的(0.24±0.35),差异具有统计学意义(P 0.05)。结论白癜风患者PBMC中TLR7表达过量,TLR9则低表达,但与正常健康者的表达情况未见较大差异,而该信号通路的信号分子NF-κB表达量远高于正常健康者,推测TLR7、TLR9、NF-κB的表达与白癜风可能存在一定的关系,但具体机制需进一步探寻。     

TLR4基因的shRNA对内毒素刺激下RAW264.7细胞TLR2表达的影响及机制

目的 观察针对Toll样受体4的发夹结构RNA(short hairpin RNA,shRNA)对内毒素刺激后小鼠巨噬细胞系RAW264.7细胞Toll样受体2表达的影响,并探讨其机制.方法 将携带增强型绿色荧光蛋白(EGFP)及shRNA克隆位点的质粒pEGFP/TLR4-siRNA,运用脂质体Lipofectamine2000转染至小鼠巨噬细胞系RAW264.7.转染24 h后予新霉素(G418)筛选获取稳定转染细胞株.然后将细胞分为三组:空白组(Blank group),未转染细胞LPS刺激组(LPS group)和稳定转染细胞LPS刺激组(LPS-TLR4-siRNA group),采用荧光定量PCR及流式细胞学方法检测各组细胞TLR2 mRNA和蛋白的表达,Western blot检测NF-κB p65的表达,同时用ELISA方法检测细胞分泌的TNF-α水平的变化.结果 将pEGFP/TLR4-siRNA转染至RAW264.7细胞后,增强型荧光蛋白的表达为(45.25±9.23)%.LPS-TLR4-siRNA组细胞的TLR2mRNA及蛋白的表达均明显低于LPS组(P＜0.05),同时NF-κB p65表达下调及分泌TNF-α的水平下降(P＜0.01).结论 针对TLR4 mRNA的shRNA能降低LPS刺激下的RAW204.7细胞TLB2的表达.     

TLR4与COX-2在不同宫颈病变组织中的表达及临床意义

目的检测TLR4(Toll like receptor-4)与COX-2(Cyclooxygenase-2)在不同宫颈病变组织中的表达及差异,探讨TLR4、COX-2在宫颈癌发生发展过程中的作用。方法采用免疫组织化学方法对正常宫颈组织、CIN及宫颈癌组织中TLR4、COX-2基因的表达与差异性进行检测。结果 (1)在正常宫颈组织中TLR4的表达程度偏低,但伴随宫颈病变程度的不断增强,其阳性表达率也会随之提升,且宫颈癌组织中TLR4阳性表达率比CIN组、正常宫颈组都高,差异存在统计学差异(P0.05)。(2)在正常宫颈组织中COX-2不表达,但伴随宫颈病变程度的不断增强,COX-2阳性表达率也会随之提升,且宫颈癌组织中COX-2阳性表达率比CIN组、正常宫颈组都高,组间比较差异均有统计学意义(P0.05)。结论 TLR4与COX-2参与了宫颈癌的发生发展。     

BALB/C小鼠Hp感染胃炎动物模型的建立和免疫治疗实验研究

目的　通过人类Hp的接种 ,建立BALB/C小鼠感染胃炎动物模型 ,研究纯化尿素酶在治疗H .pylori(Hp) ,感染中的作用。方法　取小鼠胃粘膜组织 ,通过Giemsa染色和HE染色评价SS1在BALB/C小鼠的定植及病理组织学改变。结果　 4、8周时小鼠胃窦、胃体可见较少量的Hp定植 ,16周后B、C、D组Hp定植量明显增多 ;4周时动物未发现有明显炎症反应 ,第 8周时小鼠腺胃出现一定程度的炎症反应 ,16周时B、C、D组小鼠胃窦及胃体的炎症明显加重。经免疫治疗各组Hp根除情况如下 ,尿素酶 +霍乱毒素 (CT) +羟磷石灰 (HAP)为 61.5 3 % (8/ 13 ) ,而单纯尿素酶、CT +HAP根除率为零。结论　Hp可感染BALB/C小鼠导致胃炎发生 ,其病理改变与人类类似 ,另外由尿素酶加免疫佐剂组成的口服免疫治疗 ,有根除已感染的Hp的作用。     

TLR4、MyD88在泡状棘球蚴感染小鼠中的表达水平及机制

目的 研究泡状棘球蚴感染小鼠中TLR4、MyD88的表达水平,相关性及其作用机制.方法 选取北京维通利华实验动物技术有限公司供给的18只C57BL/6小鼠,将小鼠从门静脉注射约含2000个原头蚴的生理盐水,分别于感染后3个月、6个月各处死9只小鼠,采用免疫组织化学法检测不同感染时间段小鼠肝病变组织中TLR4、MyD88...     

TLR5激动剂鞭毛蛋白预防小鼠异基因造血干细胞移植后急性移植物抗宿主病的初步研究

本研究旨在探讨Toll样受体(TLR5)激动剂鞭毛蛋白(flagellin)对小鼠异基因造血干细胞移植(allo-HSCT)后急性移植物抗宿主病(aGVHD)的预防作用和可能作用机制。用主要组织相容性抗原完全不合的纯种近交系小鼠〔供体鼠:雄性C57BL∕6鼠;受体鼠:雌性BALB∕c鼠〕建立allo-HSCT的aGVHD动物模型。受鼠随机分为3组:鞭毛蛋白组,于移植前后2次尾静脉注射高纯度(纯度≥95%)的鞭毛蛋白50μl〔5μg∕(只.次)〕预防aGVHD;单纯移植组,移植后仅给予等容量PBS;单纯照射组,仅照射不移植,亦给予等容量PBS。观察比较移植后aGVHD的表现。结果表明,移植小鼠均出现典型的aGVHD症状,单纯移植组小鼠死亡高峰在移植后第4-5天。用鞭毛蛋白作为aGVHD预防方案小鼠的aGVHD症状明显减轻,平均生存时间较单纯移植组显著延长(P<0.05)。三组小鼠移植前外周血白细胞数比较均无显著性差异,但在照射后第14、21天,鞭毛蛋白组较单纯移植组外周血白细胞数显著性升高(P<0.05)。鞭毛蛋白预防组移植小鼠aGVHD病理表现较单纯移植组明显减轻。流式细胞仪检测鞭毛蛋白组与单纯移植组移植前后不同时间点Treg细胞/CD4+T细胞含量结果也表明,移植后2-4周鞭毛蛋白组小鼠的Treg细胞数较单纯移植组明显增加(P<0.05)。结论:鞭毛蛋白对小鼠allo-HSCT后发生aGVHD有预防作用,能减轻其症状和病理损害程度,显著延长其平均生存时间,其机制有可能通过增加移植后小鼠的Treg细胞含量,从而有效改善并减轻移植后小鼠aGVHD。     

幽门螺杆菌感染对胃黏膜三叶因子2表达的影响

目的探讨幽门螺杆菌感染(Hp)对慢性胃炎胃黏膜三叶因子2(TFF2)表达的影响。方法采用免疫组化及半定量的分析方法检测Hp阳性组与Hp阴性组,以及Hp根除前后胃窦黏膜TFF2表达量的变化。结果TFF2阳性物质表达于胃黏膜上皮近基底部的胃腺细胞的细胞质内,呈棕黄色颗粒;与Hp阴性组比较,Hp阳性组的TFF2染色评分显著减少(P<0.05);根除Hp后的胃黏膜较根除前TFF2的表达量显著增加(P<0.05)。结论Hp感染导致胃黏膜TFF2表达减少,提示TFF2在Hp相关性胃炎的发病机制中起一定作用。     

幽门螺杆菌感染使胃上皮8-羟-2-脱氧鸟苷表达水平增加

目的通过检测胃上皮组织DNA氧化损伤标记物8-羟-2-脱氧鸟苷(oh8dG)表达水平,探讨Hp的致癌性。方法对Hp阳性患者予以PPI三联治疗,以ELISA法分别检测Hp根治前后及Hp阴性胃上皮组织oh8dG含量。结果Hp阳性组胃上皮oh8dG为(2.017±1.134)ng/mg蛋白,明显高于Hp阴性组的(1.289±0.942)ng/mg蛋白(P<0.01),Hp感染患者在抗Hp根治治疗后胃上皮oh8dG较治疗前显著下降(P<0.05)。结论Hp感染增加了胃粘膜组织oh8dG水平并与胃癌发生有关,抗Hp治疗有助于改善胃上皮DNA氧化损伤的程度。     

Feasibility study on eradication of helicobacter pylori北大核心CSCD

Objective To explore the relationship between oral Helicobacter pylori (Hp) infection and gastric Hp infection and examine the possibility of complete eradication of Hp by control of oral Hp infection. Methods Oral Hp was tested in saliva to identify cases of oral Hp infection. A total of 180 patients with gastritis or gastric or duodenal ulcer were randomly assigned to two groups, 90 patients in treatment group and 90 in control group. The diagnosis of gastritis or gastric or duodenal ulcer was made according to positive urease test and pathological examination. The patients in treatment group received triple therapy and brushing teeth with toothpaste containing caries-preventing fluorine material. The patients in control group received triple therapy alone. Hp eradication was detected and compared between groups at various time points to analyze the effect of oral hygiene on eradication of oral and gastric Hp. Results Four weeks after the end of 2-week treatment, positive-to-negative conversion rate of both oral and gastric Hp in treatment group was significantly higher than that in control group (80.0% vs 64.4%, P<0.05). After one-year oral hygiene treatment, the positive-to-negative conversion rate and eradication rate of Hp were significantly higher than those in control group (74.4% vs 40.0%, 93.5% vs 62.1%, all P<0.05). Hp re-infection rate in both oral cavity and stomach was significantly reduced in control group. Conclusions Eradication of Hp by drug therapy is associated with control of oral Hp and oral hygiene. Brushing teeth with toothpaste containing caries-preventing fluorine material in addition to triple drug therapy is beneficial for eradication of Hp and reduced use of antibiotics. © by Editorial Department of Chinese Journal of Infection and Chemotherapy.     

Hp感染与胃炎性病变,不典型增生及胃癌关系的研究

目的　通过检测 392例不同胃部疾病 :慢性胃炎 (2 13例 )、消化性溃疡 (77例 )、不典型增生 (5 3例 )及胃癌(4 9例 )中幽门螺杆菌 (Hp)感染的不同阳性率 ,探讨幽门螺杆菌感染与胃部疾病的关系。方法　应用快速尿素酶法 ,Giemsa染色法及C1 4呼气实验等 3种不同方法检测胃粘膜Hp感染。应用免疫组织化学ABC法检测人胃癌组织中caspase 3和bcl xl的表达情况。并对比不同胃粘膜病变中Hp阳性率及caspase 3和bcl xl表达的差异。结果　Hp感染与胃炎性病变及不典型增生的严重程度密切相关 ,但在胃癌中的检出率较前二者低 (P <0 0 5 )。胃炎性病变、不典型增生及胃癌中caspase 3的表达依次降低 (P <0 0 5 ) ,bcl xl的表达依次升高 (P <0 0 5 )。Hp阳性组较Hp阴性组caspase 3表达降低 (P <0 0 5 ) ,而bcl xl表达升高 (P <0 0 5 )。结论　Hp感染与胃粘膜炎性病变及不典型增生程度正相关 ,Hp在胃癌的发生发展过程中可能是早期事件 ,可能与改变细胞凋亡有关     

胃黏膜病变组织中RASAL_1基因的表达及其与幽门螺杆菌感染的关系

目的探讨RASAL1基因在幽门螺杆菌(Hp)感染胃黏膜组织中的变化及其在胃癌发生发展中的意义。方法内镜及活检病理证实的慢性胃炎58例(慢性胃炎组)、肠化生及异型增生58例(肠化生及异型增生组)和胃癌58例(胃癌组),分别用13C-呼气试验及Giemsa染色检测Hp,链霉菌亲生物素蛋白-过氧化物酶连接(SP)免疫组化检测RASAL1,比较各组及组内Hp阳性亚组和Hp阴性亚组中RASAL1的表达。结果慢性胃炎组、肠化生及异型增生组和胃癌组中RASAL1的阳性率分别为89.66%、56.90%和13.79%,呈递减趋势。各组Hp阳性亚组与Hp阴性亚组中RASAL1的阳性率分别为,慢性胃炎组:73.33%和96.43%,肠化生及异型增生组:40.63%和76.92%,胃癌组:14.29%与13.51%,两两比较均有显著差异。结论 Hp感染可降低胃黏膜RASAL1表达,削弱其对胃黏膜损伤的保护作用,促进了胃癌的发生发展。     

Reciprocal expression of interferon gamma or interleukin 4 during the resolution or progression of murine leishmaniasis. Evidence for expansion of distinct helper T cell subsets

We purified poly(A)+ mRNA from the spleen and lymph nodes at designated times after infection with Leishmania major in genetically susceptible BALB/c and resistant C57BL/6 mice. The steady-state levels of IL-2, IFN-gamma, IL-4, and IL-1 beta mRNA were determined using Northern hybridizations. IL-2 mRNA levels in the infected organs of BALB/c and C57BL/6 mice were comparable after infection, but IFN-gamma and IL-4 mRNA levels were reciprocally expressed. Levels of IFN-gamma mRNA in C57BL/6 draining nodes and spleen were significantly greater than in BALB/c mice except at 4 and 6 wk of infection, when splenic IFN-gamma mRNA levels were transiently comparable. In contrast, IL-4 mRNA was apparent only in BALB/c and not in C57BL/6 nodes and spleen. Tissue levels of IL-1 beta mRNA were 10-20-fold greater in BALB/c mice. BALB/c mice were pretreated with GK1.5 mAb, a manipulation that promotes healing of subsequent infection by transiently depleting L3T4+ cells. At 8 wk of infection, by which time lymphoid organs were repopulated with L3T4+ cells, GK1.5-pretreated BALB/c mice produced IFN-gamma, but not IL-4 message. Serum levels of IgE were markedly elevated in infected BALB/c, but not in infected C57BL/6 or GK1.5-pretreated BALB/c mice, consistent with in vivo biologic activity of IL-4 in nonhealing mice. Treatment of infected BALB/c mice with neutralizing anti-IL-4 antibody abolished the elevation of serum IgE and significantly attenuated the progression of disease as assessed by size and ulceration of the lesion, and by reduction in the number of tissue parasites. Both protective and deleterious responses to Leishmania infection have previously been shown to be L3T4+ cell dependent. Our findings are consistent with the differential expansion of protective, IFN-gamma-producing Th1 cells in healing mice, and the expansion of deleterious, IL-4-producing Th2 cells in nonhealing mice. The inverse relationship of IFN-gamma and IL-4 gene expression during leishmaniasis may underlie the divergence of cellular and humoral immunity that occurs during chronic infection with Leishmania and possibly other intracellular parasites.     

TLR ligand decreases mesenteric ischemia and reperfusion injury-induced gut damage through TNF-alpha signaling

Ischemic gut contributes to the development of sepsis and organ failure in critically ill patients. Toll-like receptors (TLRs) have been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We hypothesize that LPS, a ligand for TLR4, decreases mesenteric I/R injury-induced gut damage through tumor necrosis factor alpha (TNF-alpha) signaling. First, wild-type (WT) mice were fed with oral antibiotics for 4 weeks to deplete the intestinal commensal microflora. At week 3, drinking water was supplemented with LPS (10 microg/microL) to trigger TLRs. The intestinal mucosa was harvested for TLR4 protein, caspase 3 activity, and terminal deoxynucleotide transferase labeling assay. Second, WT and Tnfrsf1a mice received 30-min ischemia and 30-min reperfusion (30I-30R) or 30I-180R of the intestine; intestinal permeability and lipid peroxidation of the intestine were examined. Third, WT and Tnfrsf1a mice were fed with oral antibiotics with or without LPS and received 30I-180R of the intestine. The intestinal mucosa was harvested for lipid peroxidation; glutathione (GSH) level; nuclear factor kappaB (NF-kappaB) and AP-1 DNA-binding activity; Bcl-w, TNF-alpha, and CXCR2 mRNA expression; and HSP70 protein assay. Commensal depletion increased caspase 3 activity as well as villi apoptosis and decreased TLR4 expression of the intestinal mucosa. LPS increased TLR4 expression and decreased villi apoptosis. Commensal depletion augmented 30I-180R-induced intestine permeability as well as lipid peroxidation and decreased GSH level in WT mice but not in Tnfrsf1a mice. LPS decreased 30I-180R-induced intestinal permeability as well as lipid peroxidation and increased GSH level of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Commensal depletion with 30I-180R increased NF-kappaB and AP-1 DNA-binding activity, HSP70 protein expression, and decreased Bcl-w and TNF-alpha mRNA expression of the intestinal mucosa in WT mice but not in Tnfrsf1a mice. Collectively, commensal microflora induces TLR4 expression and decreases apoptosis of the intestinal mucosa. Commensal depletion enhances I/R-induced gut damage. LPS prevents I/R-induced intestinal permeability, lipid peroxidation, and decrease in GSH level. Given that the preventive effect of LPS on I/R-induced gut damage and NF-kappaB activity of the intestine is abolished in Tnfrsf1a mice, we conclude that TLR ligand decreases mesenteric I/R injury-induced gut damage through TNF-alpha signaling.