Rapamycin和cyclosporin A对同种排斥过程中TLR5及Foxp3表达的影响

目的:研究雷帕霉素(Rapamycin,RAPA)和环孢素A(cyclosperin A,CsA)体内处理对同种移植小鼠急性排斥过程中TLR5及Foxp3表达的影响.方法:建立同种皮肤移植模型,术后给予RAPA或CsA,1次/d,连续14 d,同时设生理盐水对照组.每日观察移植物存活状况.术后选取1、7、10、14、21 d共5个时间点,提取受体小鼠脾细胞,RT-PCR检测TLR5及Foxp3 mRNA表达,探讨不同处理组基因表达与移植物生存的相关性.体外实验利用鞭毛蛋白(flagellin)联合RAPA和CsA处理正常小鼠T细胞6 h,RT-PCR检测TLR5表达.结果:RAPA和CsA可明显延长小鼠同种移植皮片存活期.RAPA可促进脾细胞TLR5 mRNA表达升高,停药后的第21天仍明显高于CsA组和对照组(P＜0.05);CsA组在第7、10天有显著升高,第21天降低(P＜0.05);3组中最高表达的时间点为移植后第10天,此时正值排斥高峰期.RAPA可引起Foxp3 mRNA的表达升高(P＜0.05),停药后仍维持较高水平;CsA组仅在移植后第7、10天短暂升高,第14天低于对照组(P＜0.05).移植后1、7、10、21 d 3组的TLR5与Foxp3 mRNA变化趋势正相关.体外实验中,RAPA或CsA与flagellin联合使用,可促进TLR5的表达,以前者的作用更为明显.结论:RAPA和CsA在同种移植急性排斥高峰期可引起TLR5和Foxp3表达的协同升高,且RAPA的作用更加明显和持久,鞭毛蛋白体外可以增强这种作用效果.     

γδT17/Th17/Tc17细胞在H1N1重症感染小鼠肺脏中的分布及其与肺脏免疫损伤的关系

目的:探讨γδT17/Th17/Tc17细胞在H1N1重症感染小鼠肺脏中的分布及其与肺组织炎性损伤的关系。方法:通过滴鼻感染建立H1N1重症感染小鼠模型;采用流式细胞术检测小鼠肺组织中γδT17/Th17/Tc17细胞的比例和数目;采用ELISA和Luminex多因子试剂盒检测肺泡灌洗液(BALF)中IL-17A、IL-1β、IL-23的浓度和血清中IL-17A的浓度。结果:(1)建立了H1N1重症感染小鼠模型;(2)感染后第3天小鼠肺脏中γδT细胞占总淋巴细胞的比例较对照组显著升高(P0.01),而Th和Tc细胞的比例较对照组无明显差异;(3)感染后第1天肺脏中γδT17细胞占总γδT细胞的比例和数目较对照组显著升高(P0.05);Th17和Tc17细胞占Th和Tc细胞的比例和数目较对照组也升高;其中γδT17细胞的数目显著高于Th17和Tc17细胞(P0.05);(4)感染后小鼠BALF中IL-17A的浓度逐渐升高,与对照组相比有显著性差异(P0.05);感染后第3天血清中IL-17A也显著升高(P0.05);BALF中可能参与γδT17细胞活化的IL-1β和IL-23的浓度较对照组显著升高。结论:γδT17细胞有可能以γδTCR非依赖的作用方式活化,并通过释放IL-17A参与H1N1重症感染小鼠早期肺组织炎性损伤过程。     

IL-1β和IL-18在鼠巨细胞病毒播散性感染中的表达及意义

目的:在整体水平观察鼠巨细胞病毒(Murine cytomegalovirus,MCMV)播散性感染时脏器病毒载量、caspase-1的活化及其下游因子IL-1β和IL-18的表达状况。方法:建立MCMV播散性感染模型,MCMV Smith株接种后第0、3、7和14天各处死4只小鼠;同时设模拟感染小鼠作为对照。标准空斑试验检测唾液腺、肺和肝组织病毒滴度;Western blot法检测脾细胞中procaspase-1及其活化形式caspase-1的表达强度;双抗体夹心ELISA法检测血清IL-1β和IL-18水平;免疫组化法检测唾液腺、肺和肝组织中IL-1β和IL-18表达状况。结果:肝组织病毒滴度于MCMV感染后3天升高,其后迅速减低,感染2周内肺组织中未检测到病毒,而唾液腺组织病毒滴度呈逐渐增高趋势;与模拟感染对照组比较,播散性感染组感染后3天脾细胞中procaspase-1和caspase-1的表达明显升高(相对吸光值均P<0.01);同时,血清IL-1β和IL-18水平升高达峰值(均P<0.01)。结论:MCMV感染后炎性体活化,caspase-1表达升高;其下游信号IL-1β和IL-18成熟释放增加,并呈组织差异性表达。     

鼠巨细胞病毒感染对小鼠脾细胞IL-12 p35和p40基因转录和表达的影响

目的 探讨鼠巨细胞病毒（MCMV）感染对小鼠脾细胞内TH-1细胞调控因子白细胞介素-12（IL-12）p35和p40基因转录和表达影响的时序性变化特点。方法 制备全身播散型MCMV感染小鼠模型，于感染后3d,5d、7d、10d和14d分离小鼠脾细胞，在PHA刺激后用RT-PCR法检测脾细胞内IL-12 p35和p40 mRNA水平时序性变化，ELISA法测定脾细胞培养上清中IL-12 p70和IFN-γ蛋白水平变化。结果 模型鼠在感染后第3天IL-12 p70表达显著增高（P〈0．05），但第5天后急剧下降，显著低于正常鼠（P〈0．05）；IFN-γ在感染后第3天增高达峰值（P〈0．01），随后逐渐下降，在感染后第10～14天降至正常水平；IL-12 p35mRNA水平变化与IL-12 p70完全一致；而p40mRNA则从感染第3天开始持续高水平表达（P〈0．01）。结论 IL-12 p35mRNA和IL-12 p70在感染5d后持续明显下降是感染后期TH1类细胞因子持续低表达、TH1反应受抑制的主要原因之一；感染后IL-12 p40mRNA持续高水平可能导致拮抗剂（p40）2表达增加，后者可进一步抑制IL-12功能。     

HSV-1感染单核巨噬细胞及其影响因素的研究

用HSV-1接种人单核细胞传代系U937细胞和小鼠腹腔巨噬细胞,并在接种前后以细菌脂多糖(LPS)或/和卡介苗(BCG)处理细胞,通过病毒滴定、间接荧光抗体试验检测病毒抗原及PCR技术检测病毒基因,初步研究了HSV-1感染单核巨噬细胞的特点及其影响因素。结果如下。 U937细胞接种HSV-1的剂量为1.0 TCID_(50)/细胞,在2周内降至测不出水平。病毒抗原阳性细胞于感染后第4天达高峰,为47.6％,随即迅速下降,2周内降至10％以下,3周后几乎全部消失。病毒DNA在感染2周后即不能从细胞中检出。感染过程中始终未见典型的CPE,但MTT比色分析法显示感染后1～2周细胞增殖受到明显抑制。2周后细胞增殖开始恢复。将细胞     

AIM2炎性体识别胞浆鼠巨细胞病毒DNA的实验研究

目的 观察AIM2( absent in melanoma 2)在感染早期识别胞浆小鼠巨细胞病毒(MCMV) DNA的变化状况.方法 建立MCMV全身播散型感染模型,接种MCMV Smith株后第1、3、5和7天各处死3只小鼠;同时设模拟感染小鼠作为正常对照.Western blot检测脾脏巨噬细胞中AIM2、衔接子凋亡相关点样蛋白(ASC)和caspase-1蛋白的表达状况;同时应用ELISA法检测血清中IL-1β和IL-18的水平;空斑法测定感染小鼠唾液腺感染性病毒滴度.结果 MCMV感染后第3、5、7天,小鼠唾液腺组织中感染性病毒滴度逐渐增加;MCMV感染鼠脾脏巨噬细胞中AIM2、ASC和caspase-1蛋白的表达呈现一致的变化,与模拟感染对照鼠比较,AIM2、ASC和caspase-1蛋白相对吸光值在感染后第1天开始升高(P＞0.05),第3天明显升高并达峰值[分别为(1.121±0.243) vs(0.240±0.046),(1.318±0.333) vs (0.248±0.090),(1.085±0.243) vs (0.247±0.064); P＜0.01],其后接近正常;MCMV感染鼠血清IL-1β和IL-18水平在感染后第3天也明显高于模拟感染对照鼠[分别为(112.72±5.20) pg/ml vs (47.86±4.35) pg/ml,(42.74±4.23) pg/ml vs( 22.60±2.82)pg/ml;P＜0.01],其后均逐渐下降接近正常.结论 MCMC感染早期巨噬细胞通过AIM2炎性体识别胞浆MCMV DNA,可能成为CMV感染及感染后所引起的疾病的治疗靶点.     

Treg细胞参与LIGHT缺失小鼠对利什曼原虫感染的敏感性

目的探讨LIGHT对利什曼原虫(L.major)感染模型中CD4+T细胞亚群免疫应答的影响。方法采用LIGHT KO和野生型(WT)小鼠,经小鼠脚掌注射L.major建立感染模型。从感染第2周至24周连续观察被感染脚掌的病损程度,并测定感染24周小鼠体内寄生虫载量。其次,取感染第7天小鼠引流淋巴结(d LN)细胞,运用3H-Td R掺入方法检测T细胞体外刺激的增殖情况,并且利用ELSIA检测体外刺激后d LN细胞IL-12和IFN-γ的表达水平。最后,通过流式细胞术检测感染第7天小鼠脾脏和d LN中Treg水平。结果 LIGHT KO小鼠被感染脚掌和d LN的寄生虫载量均显著高于WT对照小鼠。感染早期LIGHTKO小鼠d LN中T细胞增殖水平,以及IL-12和IFN-γ的表达水平均显著低于WT对照小鼠。感染早期LIGHT KO小鼠中脾脏和d LN中的Treg细胞百分率相对WT小鼠均显著升高。结论 LIGHT可能通过抑制Treg细胞的产生,促进Th1细胞的分化,从而增强小鼠抗L.major感染的能力。     

小鼠MD-1表达的影响因素及其在移植排斥反应中的作用

目的：探讨影响小鼠MD-1表达的因素以及MD-1在移植排斥反应中的作用。方法：实验采用C57BL／6-BALB／c小鼠同种皮肤移植模型，以非特异性免疫抑制剂CsA、FK506、MMF和SRL，以及特异性MD-1反义脱氧寡核苷酸(AS-ODNs)为干预手段，于术后第11天留取标本检测脾细胞MD-1表达水平和增殖反应强度、血清IL-2和IL-10水平以及皮肤移植物组织学改变，并记录移植物存活时间。结果：CsA、MMF和AS-ODNs处理能减少脾脏MD-1表达阳性细胞数、降低脾细胞增殖反应强度、下调血清IL-2水平和上调IL-10水平，并显著延长皮肤移植物平均存活时间(MST)；FK506与CsA和MMF处理结果的差异在于其对血清IL-10水平无明显影响；SRL处理仅能减低脾细胞增殖反应强度和延长皮肤移植物MST，对MD-1表达及IL-2与IL-10分泌无明显影响。MD-1表达水平与脾细胞增殖反应强度和血清IL-2水平呈极显著正相关，和IL-10水平呈极显著负相关。结论：CsA、FK506、MMF和AS-ODNs处理均能有效抑制小鼠MD-1的表达。免疫抑制剂干预MD-1表达的作用与血清IL-2水平下调有关。MD-1通过影响淋巴细胞增殖反应和血清IL-2、IL-10水平，在移植排斥反应中发挥重要作用。     

宿主的遗传背景对呼吸道感染沙眼衣原体后Treg产生的影响

目的 探讨宿主的遗传背景对呼吸道感染沙眼衣原体后调节性T细胞(Treg)产生的影响.方法 对衣原体感染具有明显易感性差异的C57 BL/6(C57)和C3H/HeN(C3H)小鼠鼻腔吸入1×103 IFU沙眼衣原体小鼠肺炎菌株(Chlamydia muridarum,Cm),于感染后不同天数处死小鼠.利用细胞内细胞因子染色技术检测小鼠脾脏单个核细胞CD4+ CD25+T细胞、Foxp3+ CD4+ CD25+T细胞百分率,利用RT-PCR技术检测小鼠肺组织Treg细胞分泌的相关细胞因子IL-10和IL-2的mRNA表达水平,并比较Cm呼吸道感染不同时期C57和C3H小鼠Treg免疫应答水平的差异.结果 Cm感染在两组小鼠均诱导较高水平的CD4+ CD25+T细胞、Foxp3+ CD4+ CD25+T细胞产生及IL-10、IL-2mRNA表达.感染后第3天和第7天,高易感性的C3H小鼠脾脏CD4+ CD25+T细胞、Foxp3+ CD4+CD25+T细胞扩增水平,以及肺组织细胞因子IL-2 mRNA的表达水平均高于C57小鼠,感染后第14天,C3H小鼠IL-10 mRNA表达水平明显高于C57小鼠.结论 衣原体呼吸道感染在高易感性的C3H小鼠诱导高水平的Treg的增殖及Treg相关细胞因子IL-10、IL-2的表达,从而对衣原体特异的Th1免疫应答抑制作用增强,在小鼠衣原体呼吸道感染易感性差异中发挥重要作用.     

BALB/c小鼠感染弓形虫后Thl/Th2免疫漂移的实验研究

目的:动态分析BALB/c小鼠感染弓形虫后Thl/Th2免疫失衡及免疫漂移特点,并探讨转录因子T-bet和GA.TA-3在此过程中的改变及其意义.方法:90只BALB/c小鼠随机分为正常对照组30只,弓形虫感染组60只.于感染后奇数天每天处死感染组小鼠2只,对照组小鼠1只,采用ELISA法动态检测各组小鼠血清中IFN-γ和IL-4的水平,同时应用荧光定量PCR方法检测小鼠脾细胞中T-bet和GATA-3 mRNA的表达情况.结果:感染组小鼠中,血清IFN-γ于感染后第4天开始显著升高,第5~7天维持在高峰值,从第8天开始下降,第9天降至正常水平;IL-4于感染后第8天开始显著升高,第9天升至峰值,从第14天开始下降,第15天降至正常水平;脾细胞T-bet mRNA的表达在感染后第3天升高,第5天达高峰后于第9天降至正常水平;脾细胞GATA-3 mRNA的表达在感染后第7天升高,第11天达高峰,于第13天降至正常水平.正常对照组小鼠在实验期内IFN-γ、IL-4水平没有明显变化,维持在正常的较低水平.结论:BALB/c小鼠感染弓形虫后诱导的免疫应答在感染急性期(第1-8天)以Th1应答为主,第9至13天,宿主免疫应答以Th2细胞应答为主,之后Thl/Th2应答基本恢复平衡.Thl应答向Th2应答的漂移与T-bet和GATA-3 mRNA的表达相关并受其调控,Thl/Th2型免疫应答的发生时相和效应强度可能影响弓形虫感染的最终结局.     

Napsin A基因转染干预A549细胞的上皮-间质转化

目的 探讨Napsin A基因转染至A549细胞对其上皮-间质转化(EMT)的作用和机制.方法 采用慢病毒载体质粒PLJM1构建重组质粒PLJM1-Napsin A,将Napsin A基因转染至A549细胞染色体中并鉴定.用转化生长因子-β1(TGF-β1)刺激A549细胞构建体外EMT模型,倒置显微镜下动态观察细胞形...     

Lack of complement-dependent cytolytic antibodies in hepatitis A virus infection

Sera collected from patients with acute hepatitis A virus (HAV) infection and convalescent sera were examined for cytolytic activity against HAV-infected human-embryo lung fibroblasts (HAV carrier fibroblasts). Using the 51chromium release assay, no complement dependent antibody mediated cytolytic activity against HAV carrier cells could be detected. In control experiments with identical cell strains, anti-herpes simplex virus (HSV) positive sera and complement caused specific lysis of HSV type 1 infected target cells. The data presented here do not support the hypothesis that in the possible immunopathogenesis of HAV infection, complement-dependent cytolytic antibodies play an essential role.     

Psychological correlates of the Type A behavior pattern

Psychological characteristics of 384 adult males classified as Type A or Type B by the structured interview were examined. Subjects classified Type A differed significantly from subjects classified Type B on a number of psychological scales including measures of aggression, autonomy, extroversion, and impulsiveness but not on measures of psychological distress. The extent to which pencil and paper questionnaire assessments of Type A differ from structured interview ratings was also studied. Correlations between the various Type A questionnaire scales and the structured interview were found to be notably low. The use of Type A questionnaires and implications for Type A interventions are discussed.The preparation of this article was supported by NIOSH Contract CDC-99-7442, NIMH Grant MH 31269, and NHLBI Grant HL 03429.     

Three new HLA-A alleles determined by sequence-based typing: A*2493, A*2918 and A*0342

In this study, we report the identification of three new human leukocyte antigen class I alleles: A*2493, A*2918 and A*0342 found by routine typing using commercial kits. The names A*2493 (HWS 10005702), A*2918 (HWS 10005703) and A*0342 (HWS 10005705) have been officially assigned by the World Health Organization Nomenclature Committee in August 2008.     

Description of three new HLA-A*02 subtypes in Caucasians: A*0281, A*0289 and A*9224

Three new HLA-A*02 alleles were completely characterized by sequencing-based typing (SBT). A*0281 and A*9224 showed eight clustered amino acid differences into the Bw4/Bw6 epitope at positions 74-83, regarding A*02010101. Both disclosed the same Bw4 motif described for A*25 and A*32 alleles. A*9224 has an additional mismatch at residue 265 (G>V) in the alpha3 domain, which has not been reported for any other human leukocyte antigen (HLA) class I molecule. HLA-A*0289 differed from A*02010101 in the conserved amino acid residue 192 (H>Q).     

A1 and A2A receptor activation by endogenous adenosine is required for VIP enhancement of K+ -evoked [3H]-GABA release from rat hippocampal nerve terminals

Vasoactive intestinal peptide (VIP) modulates GABA release from hippocampal nerve terminals and enhances hippocampal synaptic transmission through a pathway dependent on GABAergic transmission. Since VIP modulation of hippocampal synaptic transmission is dependent on the tonic actions of adenosine we investigated if endogenous adenosine could influence VIP enhancement of GABA release from isolated hippocampal nerve endings, and which adenosine receptors could be mediating this influence. When extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1 U/ml), the enhancement (57.2 ± 3.7%) caused by VIP on GABA release was prevented. Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A_2A receptors with ZM241385 (50 nM) abolished the effect of VIP. In the presence of ADA, selective A_2A receptor-activation with CGS21680 (10 nM) readmitted most of the enhancement caused by VIP on GABA release (50.7 ± 5.3%). Also in the presence of ADA, A₁ receptor activation with N⁶-cyclopentyladenosine (CPA, 50 nM) partially readmitted that effect of VIP (32.6 ± 3.8%). In conclusion, the enhancement of GABA release caused by VIP in hippocampal nerve terminals is dependent on the tonic actions of adenosine on both A₁ and A_2A receptors, and this action of adenosine is essential to VIP modulation of GABA release.     

The effects of A1 and A2A adenosine receptor agonists on kainic acid excitotoxicity in the guinea pig cochlea

The present study aimed to clarify the protective effect of adenosine receptors against the excitotoxicity of cochlear afferent dendrites. The effects of 2-chloro-N6-cyclopentyladenosine (CCPA), an A1 adenosine receptor agonist, and 5′-N-cyclopropyl-carboxamidoadenosine (CPCA), an A2A adenosine receptor agonist, on cochlear excitotoxicity induced by kainic acid (KA) were examined using guinea pigs. KA was applied to the round window membrane at a concentration of 10 mM for 30 min. CCPA or CPCA was given at the onset of KA application. KA morphologically induced the swelling of cochlear afferent dendrites and significantly elevated the threshold of the compound action potential (CAP) of the cochlea. CCPA inhibited the KA-induced CAP threshold shift and swelling of the cochlear afferent dendrites. However, CPCA did not affect cochlear excitotoxicity induced by KA. The results suggest that adenosine A1 receptor activation could prevent the excitotoxicity of cochlear afferent dendrites.     

Three newly identified A*24 alleles: A*2406, A*2413 and A*2414

Three previously unknown A24-related alleles were identified by PCR-SSO typing and confirmed by DNA sequencing in Australian Aboriginal populations (A*2406, 2413) and in individuals of South American descent (A*2414). A*2406 and A*2413 both have two adjacent (but different) nucleotide substitutions in codon 156 in exon 3 compared to A*2402, resulting in a single amino acid replacement in each allele. The South American A*2414 is apparently a hybrid between A2 and A24 with a segment of the A*24 sequence between codons 95 and 107 in exon 3 replaced with the A*02 sequence. Interallelic sequence exchange is the most likely mechanism in the generation of all three novel alleles. Compared to A*2402, the four amino acid substitutions in the A*2414 molecule would be expected to significantly change the shape of the peptide binding cleft, leading to selection of different peptide ligands. The single amino acid replacements in position 156 of the two Australian Aboriginal A*24 alleles may also have significant functional effects. In particular, Trp replacing Gin in position 156 (A*2406) is predicted to markedly reduce the volume of the peptide binding cleft, influence the interaction of HLA pockets with peptide side chains, and therefore, cause major changes in peptide presentation. These newly defined alleles may reflect the adaptive process of HLA genes to local environments.