前列腺酸性磷酸酶体外诱导结肠癌特异性CTLs的研究

目的探讨前列腺酸性磷酸酶（PAP）是否可以诱导结肠癌患者的外周血单核细胞（PBMCs）产生特异性细胞毒性T淋巴细胞（CTLs）。方法利用负载有PAP213-221（LYCESVHNF）多肽、Flu多肽（阳性对照）、EBV多肽（阳性对照）、HIV多肽（阴性对照）的C1R-A2402细胞与结肠癌患者（5例）及健康人（8例）的HLA-A24＋PBMCs在体外进行CTLs诱导,ELISA法检测PAP多肽特异性IFN-γ的分泌水平,51Cr释放法测定CTLs的细胞毒活性。结果3例结肠癌患者和1例健康人的PBMCs与负载有PAP213-221多肽的C1R-A2402细胞孵育后所分泌的IFN-γ,显著高于与负载有HIV多肽的C1R-A2402细胞孵育后分泌的INF-γ水平（P〈0.05）,说明本实验诱导出了PAP多肽特异性的CTLs。与对PAP-/HLA-A24＋淋巴母细胞的杀伤率比较,这种CTLs对PAP＋/HLA-A24＋的结肠癌细胞colo320的杀伤率显著增高,而对PAP＋/HLA-A24-的结肠癌细胞colo205的杀伤率无显著增加。而且这种CTLs的细胞毒活性可以被抗CD8和抗HLAclassⅠ的抗体所封闭,说明其细胞毒活性依赖于CD8＋的T淋巴细胞。结论PAP可能成为结肠癌特异性免疫治疗的新靶点。     

树突状细胞负载人工合成甲胎蛋白表位肽后诱导的细胞毒作用

目的 观察HLA-A2树突状细胞(dendritic cells,DCs)负载人甲胎蛋白(human α-fetoprotein, hAFP) HLA-A2限制性九肽(FMNKFIYEI,AFP158-166)后,激活特异的细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTLs),杀伤肝癌细胞株的能力.方法 用贴壁法分离HLA-A2成人外周血单核细胞,分次加入IL-4与GM-CSF共同诱导培养6 d,给予单核细胞调控培养液(monocyte conditioned medium,MCM)诱导成熟2 d,然后负载AFP158-166,激活特异性CTLs,用MTT法检测CTL杀伤肝细胞癌(hepatocellular carcinoma, HCC)细胞株HepG2和T2细胞株的能力.结果 采用贴壁法、多次加入细胞因子和MCM为成熟诱导剂的培养方法可以获得足量的成熟DCs,并且在去除细胞因子后,仍能保持成熟的DCs形态;负载AFP158-166的DCs激活的CTLs对HepG2和负载AFP158-166的T2具有较强的杀伤作用.结论 负载HLA-A2限制性表位肽AFP158-166的DCs对表达AFP的HLA-A2HCC有潜在的治疗作用,具有一定的临床运用前景.     

热休克蛋白65增强小鼠对HBV DNA 疫苗免疫反应的研究

目的:观察热休克蛋白65的真核表达载体(pHSP65)对HBV DNA疫苗诱导BALB/c小鼠(H-2d)免疫应答的调节作用。方法:肌内注射空载体pcDNA3,HBV DNA疫苗加HSP65佐剂(pHBVS2S pHSP65)或不加佐剂(pHBVS2S);ELISA法测定血清抗HBs抗体;ELISPOT检测分泌IFN-γ的脾淋巴细胞;4 h51Cr释放法检测小鼠脾细胞CTLs活性。结果:HBV DNA佐剂组免疫小鼠抗HBsAg抗体滴度虽高于不加佐剂组,但无显著性差异;其IgG1/IgG2 a的比例不同于多肽免疫组,二者分别为0.395与10。佐剂组小鼠分泌IFN-γ的脾淋巴细胞量是不加佐剂组的2~3倍。CTLs细胞杀伤活性(E∶T=100)佐剂组与不加佐剂组分别为:(53.68±7.5)%、(42.81±7.7)%,差异显著(P<0.05)。结论:HBV DNA疫苗具有较强的免疫原性,能够诱导机体产生特异性的抗体及CTLs反应;HSP65佐剂能够有效提高小鼠对DNA疫苗的细胞免疫应答,有望成为DNA疫苗的免疫佐剂。     

γ射线诱导癌细胞凋亡致敏树突状细胞后的免疫应答

目的 观察树突状细胞（DCs）从γ射线诱导的凋亡胆管癌细胞获取抗原后，抗肿瘤免疫应答及对胆管癌细胞的特异性免疫杀伤效果。方法 用粒－巨噬细胞集落刺激因子（GM－CSF）加白介素－4（IL－4）从人外周血分化、诱导DCs,γ射线在体外诱导培养的人胆管癌细胞凋亡，将DCs、T淋巴细胞和凋亡胆管癌细胞共培养，同时设计不同类型肿瘤细胞（坏死胆管癌细胞及培养胆管癌细胞）作对照，7d后，分离、富集DCs、T淋巴细胞进行免疫应答及肿瘤细胞杀伤试验。结果 与凋亡胆管癌细胞共培养之DCs可以有效提呈胆管癌细胞抗原，有强烈的免疫应答，刺激的细胞毒T淋巴细胞（CTLs)特异性地杀伤胆管癌细胞。结论 γ射线诱导癌细胞凋亡可以致敏rhGM-CSF加rhIL-4从人外周血单核细胞诱导、扩增出的DCs并产生显著的杀伤胆管癌细胞的免疫反应，可望成为特异性免疫治疗肿瘤的一条新途径。     

结核分枝杆菌抗原Ag85A(Rv3840c)限制性CD8+CTL表位的预测及鉴定

目的预测及鉴定新的HLA-A*0201结核分枝杆菌(Mycobacterium tuberculosis,Mtb)抗原Ag85A中的CD8+CTL优势表位。方法应用数据库SYFPEITHI预测可能存在的HLA-A*0201的限制性CD8+CTL表位,并经流式细胞术分析抗原肽与HLA-A*0201的亲和力、经时间荧光分辨法检测结核(TB)患者外周血单个核细胞(PBMCs)对抗原肽的增殖反应,再通过细胞毒实验研究抗原肽诱导的T细胞毒杀伤活性,逐步鉴定Ag85A的HLA-A*0201限制性CD8+CTL表位。结果位于Ag85A氨基酸序列(48-56)的肽表位与HLA-A*0201分子具有较高的亲和力,并能刺激HLA-A*0201阳性结核患者PBMC增殖,诱导产生具有特异性杀伤活性的CTL。结论肽6(GLPVEYLQV,48-56aa)是Mtb抗原Ag85A的HLA-A*0201限制性CD8+CTL的优势表位,这为今后进一步研究结核杆菌,作为设计结核疫苗的候选表位奠定了一些基础。     

HBV表面抗原和核心抗原联合负载的HBV相关性肝癌患者树突状细胞功能研究

目的 研究HBV相关性肝癌(HCC)患者单核细胞来源的树突状细胞(MoDC)联合负载HBV表面抗原(HBsAg)和HBV核心抗原(HBcAg)后表型和功能的变化.方法 从20例HBV感染相关性HCC患者外周血单个核细胞(PBMC)中诱导出未成熟的MoDC(imaDC),HBcAg与HBsAg联合负载后用"细胞因子鸡尾酒"(IL-1β、ID-6、TNF-α和PGE2)促成熟,流式细胞仪检测成熟MoDC的表型,酶联免疫吸附试验(ELISA)检测MoDC分泌的细胞因子,酶联免疫斑点(Elispot)实验检测MoDC诱导自身T细胞分泌IFN-γ的频率,五聚体染色法检测MoDC诱导自身T细胞产生HBV特异性CD8 T细胞的能力.结果 HBsAg和HBcAg联合负载的成熟MoDC(scDC)表面分子表达水平比imaDC显著增加,其中HBsAg、抗-HBe、抗-HBc三项阳性(简称为1-4-5阳性组)的患者MoDC表面分子明显高于HBsAg和抗-HBc两项阳性(简称为1-5阳性组)的患者;scDC分泌IL-12和IL-10水平明显高于用无关蛋白负载的对照组(conDC),1-4-5阳性组患者MoDC分泌的IL-12水平明显高于1-5阳性组;1-4-5阳性组的scDC诱导自体T细胞分泌IFN-γ的频率明显高于conDC;有4例HLA-A2 的1-4-5阳性组患者的scDC诱导自体T细胞产生HBVcorel8-27特异性CD8 T细胞.结论 HBsAg和HBcAg联合负载可以逆转HBV相关性HCC患者MoDC受损的功能,增强1-4-5阳性组MoDC;诱导自体T细胞特异性应答的能力.1-4-5阳性组患者的MoDC功能强于1-5阳性组患者.     

QLFATINSTL(93-102aa)是隐球菌抗原MP98 CTL优势表位

目的鉴定隐球菌抗原MP98中的HLA-A*0201限制性CD8+CTL表位。方法应用数据库SYFPEITHI预测隐球菌MP98中可能存在的HLA-A*0201限制性CD8+CTL表位,经流式细胞术分析各抗原肽与HLA-A*0201的亲合力,经时间分辨荧光法检测外周血单个核细胞(PBMCs)对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定MP98的HLA-A*0201限制性CD8+CTL表位。结果位于MP98氨基酸序列肽3(93-102aa)与HLA-A*0201分子具有较高的亲合力,并都能刺激HLA-A*0201阳性个体PBMC增殖,并诱导产生具有特异性杀伤活性的CTL。结论肽3 QLFATINSTL(93-102aa)是抗原MP98上HLA-A*0201限制性CD8+CTL的优势表位,可作为隐球菌疫苗设计的候选表位。     

治疗型双质粒HBV DNA疫苗对食蟹猴免疫功能的影响

目的评价治疗型双质粒HBV DNA疫苗(简称HBV DNA疫苗)诱导食蟹猴HBV特异性体液免疫和细胞免疫的效果。方法30只食蟹猴随机均分为5组(雌雄各半),分别重复肌注高剂量2000μg/kg、中剂量660μg/kg、低剂量200μg/kgEP介导HBV DNA疫苗,330μg/kg EP介导的佐剂质粒和PBS对照。在不同时间点以酶联免疫吸附法(ELISA)及酶联免疫斑点法(ELIS-POT)检测其血清抗-HBs水平并计数外周血单个核细胞(PBMCs)HBsAg特异性分泌IFN-γ的T细胞频数。结果食蟹猴接种不同剂量HBV DNA疫苗后第10周起均产生不同滴度的特异性抗体。16周时EP介导的HBV DNA疫苗高、中、低剂量组各3只食蟹猴中分别有3、2、3只HBsAg特异性IFN-γT细胞计数阳性;29周时高、中、低剂量组全部动物应答呈阳性,其计数结果分别为30.0±13.5SFCs/3×105PBMCs,30.7±26.3SFCs/3×105PBMCs及17.7±6.4SFCs/3×105 PBMCs;而相同时间点佐剂组和PBS对照组3只食蟹猴均为阴性。结论EP介导的HBV DNA疫苗能有效诱导食蟹猴产生HBsAg特异性体液和细胞免疫应答。     

运动与γ-干扰素反应及其影响因素

适量运动可提高机体抗病能力,而过度训练则使机体免疫功能下降。γ-干扰素(IFN-γ)在机体免疫防御机制中发挥相当重要的作用。IFN-γ可诱导单核细胞、巨噬细胞、树突状细胞等MHCⅡ类抗原的表达,使其参与抗原提呈和特异性免疫的识别过程;促进巨噬细胞杀伤病原微生物;协同IL-2诱导LAK活性,促进T细胞IL-2R表达;诱导急性期蛋白合成,诱导髓样细胞分化;增强NK细胞活性等。运动和IFN-γ的关系正日益受到体育科学工作者的重视。本文对这一领域的研究现状做一综述。1运动诱导的IFN-γ反应较为早期的研究中,Haahr等[1]令10名年轻的健康志愿…     

HBsAg致敏的树突状细胞体外抗乙型肝炎病毒的效应

目的 探讨用HBsAg致敏的树突状细胞(DCs)体外诱导自体T淋巴细胞特异性抗乙型肝炎病毒的免疫效应.方法 从乙型肝炎患者外周血单个核细胞(PBMC)中诱导DCs,在DCs成熟前加入纯的HBsAg刺激,将成熟后的DCs与自身T淋巴细胞体外共培养,5天后收集T细胞,分组加入2.2.15细胞培养液中,分别收集第1、3、5和7天的培养上清液,检测其HBeAg和HBsAg的分泌情况以及其与T淋巴细胞共培养产生的IFN-γ和IL-12的含量.结果 经抗原刺激后的DCs刺激自身T淋巴细胞增殖可明显加强其细胞毒作用,同时其产生的IFN-γ和IL-12的浓度明显高于未经抗原刺激的DCs组(P＜0.05)和对照组PBMC(P＜0.01).经抗原激活的T细胞可有效地抑制HBeAg的表达,但未发现对HBsAg有明显的抑制作用.结论 体外经HBsAg刺激后的DCs激活的自身T细胞可明显地增强细胞毒作用并可提高产生IFN-γ和IL-12的含量,从而显著地抑制2.2.15细胞上清中HBeAg的表达,致敏后的DCs疫苗体外可有效地起到抗乙型肝炎病毒的效应.     

Knee injury and Osteoarthritis Outcome Score (KOOS) - validation of a Swedish version

The Knee injury and Osteoarthritis Outcome Score (KOOS) is a self-administered instrument measuring outcome after knee injury at impairment, disability, and handicap level in five subscales. Reliability, validity, and responsiveness of a Swedish version was assessed in 142 patients who underwent arthroscopy because of injury to the menisci, anterior cruciate ligament, or cartilage of the knee. The clinimetric properties were found to be good and comparable to the American version of the KOOS. Comparison to the Short Form-36 and the Lysholm knee scoring scale revealed expected correlations and construct validity. Item by item, symptoms and functional limitations were compared between diagnostic groups. High responsiveness was found three months after arthroscopic partial meniscectomy for all subscales but Activities of Daily Living.     

Endovascular management of carotid-cavernous fistulas

Objective To investigate endovascular treatment of traumatic direct carotid-cavernous fistulas （CCF） and their complications such as pseudoaneurysms. Methods： Over a five-year period, 22 patients with traumatic direct CCFs were treated endovascularly in our institution. Thirteen patients were treated once with the result of CCF occluded, 8 twice and 1 three times. Treatment modalities included balloon occlusion of the CCF, sacrifice of the ipsilateral internal carotid artery with detachable balloon, coll embolization of the cavernous sinus and secondary pseudoaneurysms, and covered-stem management of the pseudoaneurysms. Results All the direct CCFs were successfully managed endovascularly. Four patients developed a pseudoaneurysm after the occlusion of the CCF with an incidence of pseudoaneurysm formation of 18.2% （4/22）. A total number of 8 patients experienced permanent occlusion of the ICA with a rate of ICA occlusion reaching 36.4% （8/22）. Followed up through telephone consultation from 6 months to 5 years, all did well with no recurrence of CCF symptoms and signs. Conclusion Traumatic direct CCFs can be successfully managed with endovascular means. The pseudoaneurysms secondary to the occlusion of the CCFs can be occluded with stent-assisted coiling and implantation of covered stents.     

The acutely limping child

Acute limping may be the result of multiple pathologies in children. The differential diagnosis varies based on the age of the child. Irrespective of age, the initial imaging work-up includes AP and frog leg radiographs of the pelvis and ultrasound; MRI may sometimes be helpful. In children less than 3 years, infections and trauma are most frequent. MRI is the imaging modality of choice when osteomyelitis is clinically suspected. Between the ages of 3 and 10 years, transient synovitis of the hip and Legg-Calvé-Perthes disease are main considerations but infection, inflammation and focal bony lesions are also considered. In children over 10 years, slipped capital femoral epiphysis also is considered.     

Do Balance Board Training Programs Reduce the Risk of Ankle Sprains in Athletes?

Introduction Ankle sprains are the most common musculo-skeletal injury that occurs in athletes,particularly in sports that require jumping and landing on one foot such as soccer,and basketball(1-4).These injuries often result in significant time loss from participation,long-term disability,and have a major impact on health care costs and resources(5-8).     

High Intensity Interval Training:New Insights

KEY POINTS ·High-intensity interval training(HIT)is characterized by repeated sessions of relatively brief，intermittent exercise.often performed with an“a11 out”effort or at an intensity close to that which elicits peak oxygen uptake(i.e.，≥90%of VO2 peak).     

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard

In response to the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe, Promega^® developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex^® ESI 16 (European Standard Investigator 16) and the PowerPlex^® ESI 17 Systems. The PowerPlex^® ESI 16 System combines the 11 loci compatible with the UK National DNA Database^®, contained within the AmpFlSTR^® SGM Plus^® PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR^® SGM Plus^® kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex^® ESI 17 System amplifies the same loci as the PowerPlex^® ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR^® SGM Plus^® kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex^® ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54–86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.