CN1968965A - 磷酸胆碱偶联物和相应抗体 - Google Patents
磷酸胆碱偶联物和相应抗体 Download PDFInfo
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- CN1968965A CN1968965A CNA2005800197019A CN200580019701A CN1968965A CN 1968965 A CN1968965 A CN 1968965A CN A2005800197019 A CNA2005800197019 A CN A2005800197019A CN 200580019701 A CN200580019701 A CN 200580019701A CN 1968965 A CN1968965 A CN 1968965A
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Abstract
本发明测定了高血压患者体内抗磷酸胆碱的IgG和IgM自身抗体的基础水平,目的是确定抗体在动脉粥样硬化进展中的重要性。自基础水平开始随访四年的结果表明,内膜-中膜厚度的增加在具有高水平的抗磷酸胆碱自身抗体、尤其是具有高水平的抗磷酸胆碱的IgM自身抗体的患者体内明显少见。因此,本发明认为,抗磷酸胆碱自身抗体、尤其是抗磷酸胆碱的IgM自身抗体的有无,与缺血性心血管疾病的风险的增加或减少有关。本发明提供了一种测定抗磷酸胆碱自身抗体、尤其是抗磷酸胆碱的IgM自身抗体,从而鉴定有患缺血性心血管疾病风险的患者的方法。动物实验显示,用匙孔血蓝蛋白(KLH)-磷酸胆碱偶联物进行主动免疫之后,可以检测到血浆中的中-高水平抗磷酸胆碱自身抗体、尤其是抗磷酸胆碱的IgM自身抗体。本发明提供了一种药物组合物,其包含磷酸胆碱偶联物(主动免疫)或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体(被动免疫),本发明还提供了用这些组合物作为主动免疫或被动免疫的免疫原治疗或预防动脉粥样硬化的用途。
Description
技术领域
本发明涉及动脉粥样硬化和缺血性心血管疾病的治疗和风险评估领域。
背景技术
动脉粥样硬化是一种慢性疾病,引起大动脉和中等大小动脉的最内层(内膜)增厚。它使血流缓慢,并可以在由受感染血管供血的器官中引起缺血和组织破坏。动脉粥样硬化是导致心肌梗塞等心血管疾病、中风和外周动脉疾病的主要原因。它是西方国家的主要死因,预计不出二十年,将成为全球最主要的死亡原因。
这种疾病始于血管的细胞外基质中积累脂蛋白,主要是低密度脂蛋白(LDL)。这些LDL颗粒聚集并发生氧化。氧化后的LDL具有促炎特性和毒性,引起血管损伤。动脉粥样硬化在很多方面代表了对这种损伤的一种反应,包括炎症和纤维变性。
1989年,Palinski及其同事鉴定出人体内抗氧化型LDL的循环自身抗体。这一发现提示,动脉粥样硬化可能是由抗氧化型脂蛋白的免疫反应引起的自身免疫病。同一时期,多个实验室开始寻找抗氧化型LDL抗体滴度与心血管疾病之间的关系。但这些研究没有得出明确的结论。目前已经有针对氧化型LDL表面众多不同表位的多种抗体,但这些表位的结构尚不清楚。因此,术语″氧化型LDL抗体″是指多种不同抗体的未知混合物,而不是指一种特殊的抗体。
众所周知,动脉硬化病灶存在持续炎症,其特征是免疫活性细胞被活化,产生炎性细胞因子。已经确认的风险因素,例如高血压、血脂、糖尿病和吸烟都很可能促进这种炎症反应,但这种现象的发生机制尚不明确,存在多种互不相干的排他性可能性。已经有人提出多种可以引起该免疫反应的自身抗原,包括氧化型低密度脂蛋白(oxLDL)和热休克蛋白(HSP)2,3。有关免疫反应在动脉粥样硬化中的作用的现有信息提示一种复杂的关系。其中一种情况是,在动物模型中进行免疫接种,以便影响动脉粥样化的形成。当使用HSP时,动脉粥样硬化增加,当以oxLDL作为抗原时,动脉粥样硬化减少4,5。
aOxLDL在人类疾病中的作用似乎比较复杂。此前有人证实,aOxLDL在健康对照人群中的水平高于临界高血压(borderline hypertension,一种早期心血管疾病6)人群。近期研究与该发现是一致的7,8。另一方面,有多位研究者报道,aOxLDL在人类心血管疾病(CVD)中升高,尤其是在晚期疾病中2.3.9.10。实例之一是系统性红斑狼疮(SLE)以及与CVD极高风险有关的自身免疫病。有CVD病史的SLE-患者aOxLDL水平明显升高11。这些在某种程度上相互矛盾的结果可能主要是由于:LDL-氧化的不同方法和状态在抗原性方面产生差异。疾病阶段和风险系数变化谱都倾向于与抗体水平相关。
氧化型低密度脂蛋白(oxLDL)本身具有多种促炎特性,包括活化T细胞12.13,单核细胞/巨噬细胞和内皮细胞14-16。OxLDL还在来自动脉粥样硬化病灶的免疫活性细胞中促进炎症17。但要注意,oxLDL也可以缓解急性炎症反应,并替代促进更低级的慢性炎症,这在动脉粥样硬化中可见18。有趣的是,oxLDL的多种生物学活性是由oxLDL中的血小板活化因子(PAF)-样脂质引起的19-21。
磷酸胆碱(phosphorylcholine,PC)不仅是血小板活化因子PAF(它在与PAF-受体相互作用时是必需的)等炎症性磷脂和oxLDL中的主要成分,还可以作为肺炎链球菌(S.Pneumoniae)等多种细菌的免疫原成分22。而且,PC由凋亡细胞表达2,23。
在US5455032中,在用于提供抗感染(例如肺炎链球菌(Streptococcuspneumoniae))的免疫保护的疫苗中使用磷酸胆碱偶联物。Binder等人最近对小鼠中肺炎球菌疫苗的研究24显示,免疫接种减少了动脉粥样硬化灶的形成。有研究发现,由动脉粥样硬化小鼠产生的多种抗oxLDL自身抗体与提供抗常见感染性病原体(包括肺炎链球菌)的保护作用的抗体有相同的结构。在小鼠、而非人类中的研究并未提供任何关于特异性的信息,也未能提示,IgM抗-磷酸胆碱抗体在动脉粥样硬化中是比相应IgG抗体明显更为重要的保护因子。此外,磷酸胆碱偶联物未被用于肺炎球菌疫苗中。
另一项研究25显示,抗磷酸胆碱抗体水平在牙周病(periodontal diseases)患者体内升高。其结论是,磷酸胆碱是与牙周菌群中生物相关的重要的口腔抗原,而抗-PC抗体水平的升高是牙周患病的结果。至今尚无动脉粥样硬化的抗体和可能的保护作用或进展的任何信息。
目前已有多篇关于动脉粥样硬化的免疫接种治疗的文献被公开(如WO2002080954和WO0168119),但这些是基于使用脱脂载脂蛋白B的肽片段或抗T细胞受体alpha/beta链的抗体。有一篇文献(WO9908109)描述了用抗脂蛋白表面氧化作用特异性表位的单克隆抗体检测动脉粥样硬化斑的方法。与该方法不同的是,本发明的方法使用磷酸胆碱偶联物检测受试者样品中的IgM或IgG等抗体。
发明简述
本发明涉及药物组合物及其在治疗、预防动脉粥样硬化,例如治疗、预防或缓解动脉粥样硬化的进一步进展方面的用途,所述药物组合物包含磷酸胆碱偶联物、或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体。本发明还涉及磷酸胆碱偶联物或所述抗体制剂,例如单克隆抗体在制备药物组合物中的用途,所述偶联物或抗体制剂可以与佐剂组合或者不与佐剂组合。本发明还涉及诊断有无抗体,例如IgM或IgG抗体的方法,所述抗体与患缺血性心血管病风险的增加或降低相关。
本发明第一方面提供了药物组合物在制备免疫接种和治疗哺乳动物动脉粥样硬化或动脉粥样硬化相关疾病的药物中的用途,所述药物组合物包含至少一种磷酸胆碱偶联物,或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体,所述哺乳动物包括人。所述药物是用于对动脉粥样硬化有致免疫特性或治疗特性的免疫接种。
本发明第二方面提供了免疫接种和治疗哺乳动物动脉粥样硬化或动脉粥样硬化相关疾病的方法,所述方法包括对所述哺乳动物施用药物组合物,所述药物组合物包含至少一种磷酸胆碱偶联物,或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体,所述哺乳动物包括人。所述药物组合物是用于对动脉粥样硬化有致免疫特性或治疗特性的免疫接种。
磷酸胆碱偶联物是指磷酸胆碱组分与载体相连,优选通过间隔臂(spacer)相连。作为结构性组件的磷酸胆碱可包括磷酸胆碱衍生物。合适的磷酸胆碱偶联物实例可参见US 5,455,032,见上文。例如,US 5,455,032提供了这样的磷酸胆碱偶联物,其中磷酸胆碱组分借助直链烷基和酰胺键与各种不同的免疫学载体相连。所述磷酸胆碱偶联物可以是,例如人血清白蛋白(HSA)-或匙孔血蓝蛋白(keyhole limpet hemocyanin,KLH)-磷酸胆碱偶联物或牛血清白蛋白(BSA)-磷酸胆碱偶联物(例如见实施例)。PC-BSA(磷酸胆碱-牛血清白蛋白)可以从Biosearch Technologies,INC(Ca,USA)购买。HSA-BSA可以通过化学方法偶联,所述方法举例如下:
可以按照Chesebro,B.在Biochemistry 11,(1972)766中所述方法,以含10%钯的活性碳(charcoal)作为催化剂,使O-(4-硝基苯基-磷酰基)-胆碱(Sigma N 5879)与氢气在1atm进行还原反应,大量产生O-(4-氨基苯基磷酰基)-胆碱(I)。
用EDC(1-乙基-3-(3-二甲基-氨基丙基)-碳二亚胺)的MES液(pH 4)使(I)与HAS偶联,方法基本如Padilla,N.D.等在J.Immun..Methods 293(2004)1-11中所述。偶联的HAS可以通过对经过缓冲的盐水(pH 7.4)透析而被分离。
载体可以是,例如,蛋白质、脂质或聚合物。载体可以是乳胶珠,例如实施例中所述。
所述药物可以用于注射给药。
本发明另一方面提供了在先任一方面定义的一或多种磷酸胆碱偶联物在制备免疫治疗或治疗缺血性心血管疾病的药物组合物中的用途,可以与佐剂组合或不与佐剂组合。
本发明又一方面提供了预防性治疗或治愈性治疗哺乳动物的方法,所述哺乳动物是指患有动脉粥样硬化或者有患缺血性心血管病的风险的哺乳动物,例如人,所述方法包括施用治疗有效量的至少一种磷酸胆碱偶联物、或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体。
本发明还涉及确定有无抗磷酸胆碱的抗体,例如抗磷酸胆碱的IgM或IgG抗体的方法,所述抗体与患缺血性心血管病风险的增加或降低相关。
本发明又一方面提供了用磷酸胆碱偶联物诊断有无抗体,例如IgM或IgG抗体的方法,所述抗体与患缺血性心血管病风险的增加或降低相关。
故而本发明还有一方面提供了磷酸胆碱偶联物在评估患者发生或进展缺血性心血管疾病的风险的方法中的用途,在所述方法中,评估所述患者的与磷酸胆碱偶联物反应的抗体,例如IgM或IgG抗体的水平。
磷酸胆碱偶联物已经在上文中描述。磷酸胆碱可以与载体通过间隔臂相连。所述载体可以是蛋白质,这可以是KLH(匙孔血蓝蛋白)或人血清白蛋白(HSA)。所述载体可以是乳胶珠。
所述患者的与磷酸胆碱偶联物反应的抗体,例如IgM或IgG抗体的水平可以用免疫分析方法评估。合适的免疫分析方法的实例在下丈中描述,并且是本领域技术人员在任何情况下都明显可以得知的。
可能需要测定与oxLDL或MD-LDL反应的抗体以及测定与磷酸胆碱偶联物反应的抗体,例如IgM或IgG抗体。或者,或进一步地,可能需要测定HSP70,HDL,TNF和/或HSP60的水平(见实施例)以及测定与磷酸胆碱偶联物反应的抗体,例如IgM或IgG抗体的水平。
发明详述
以下实施例旨在举例说明本发明,不应认为是对权利要求所概括的保护范围作任何限制。本文提到的文献都引入作为参考。
这里描述了一例测定与缺血性心血管疾病患病风险升高或降低相关的抗磷酸胆碱IgM抗体存在与否(或水平)的方法。也可以用本领域已知的其它方法。类似的方法可以用于测定抗磷酸胆碱IgG抗体的存在与否(或水平)。
测定抗磷酸胆碱的IgM抗体存在与否的方法
用酶联免疫吸附法测定抗PC-BSA的IgM抗体。
微量滴定板用PC-BSA(10μg/ml;例如可以购自Biosearch Technologies,INC(Ca,USA)的磷酸盐缓冲液(PBS)包被。用PBS洗板,然后将板用2%BSA溶液封闭。血清样品用0.2%BSA-PBS稀释(1∶30)。将微量滴定板40℃保温过夜,洗板。添加碱性磷酸酶偶联的山羊抗-人IgM(用样品缓冲液稀释成l∶7000),100ul/孔,40℃保温过夜。洗板后,添加碱性磷酸酶底物,将板于室温避光保温60min,进行显色。在分光光度计中读取405nm处的吸光度。
测试了磷酸胆碱的不同载体(carrier)和间隔臂(spacer)。载体不限于举例的这些。也可以用本领域已知的其它载体,如蛋白质、脂质或聚合物,例如乳胶珠。载体可以参见上文中提到的US 5,455,032。
由本发明方法检测到的IgM或IgG抗体还可以结合含有暴露在表面的磷酸胆碱(PC)的化合物中的PC,例如溶血磷脂酰胆碱(lysoPC;see,forexample,Kim et al,J Exp Med.2002 Sep 2;196(5):655-65)中的PC。因此,本发明的方法可以检测与溶血磷脂酰胆碱结合的IgM或IgG抗体。
合成磷酸胆碱偶联物和制备药物组合物
将乳胶珠(0.20μm或0.81μm)悬浮在PBS中,与10μg/ml磷酸胆碱-BSA溶液混合过夜。然后将这些珠离心,用缓冲液洗数次,用10μg/ml BSA溶液封闭。再洗一次,然后将这些珠重悬于适当的缓冲液中达到适当的浓度,冰箱保存备用。
带有连接臂的磷酸胆碱也可以借助重氮苯基(diazophenyl)与KLH(匙孔血蓝蛋白)偶联。更优选按照Chesebro,B.和Metzger,H.(1972)
Biochem.11:776的方法合成PC的p-硝基苯基-6-(O-磷酸胆碱)羟基己酸酯衍生物。先将p-硝基苯基-6-(O-磷酸胆碱)羟基己酸酯溶于干乙腈(100mg/ml),再迅速将其加入KLH中。使衍生物和KLH在4℃混合过夜,然后透析除去未结合的间隔臂和作为离去基团(leaving group)的p-硝基苯(nitrophenylate)。
将所得磷酸胆碱偶联物悬浮在合适的缓冲液中,制得注射液,其可以直接用于免疫接种。
用磷酸胆碱偶联物进行免疫接种
BALB/c小鼠腹腔注射(i.p.)200μg[p-硝基苯基-6-(O-磷酸胆碱)羟基己酸酯-KLH]进行免疫接种,按照建议的免疫分析方法,在所述小鼠血浆中检测到高滴度的识别磷酸胆碱的IgM抗体。
抗磷酸胆碱偶联物的单克隆抗体
单克隆抗体可以用本领域已知的任何标准方法制备。例如,可参见“Briles DE,Forman C,Hudak S,Claflin JL.Anti-phosphorylcholine antibodiesof the T15 idiotype are optimally protective against Streptococcus pneumoniae.
J Exp Med.1982;156:1177-85”或“T15 PC binding monoclonal antibodies retainspecificity when they switch from IgM to IgG.,Spira,Gad;Aguila,Hector L.;Scharff.Matthew D.Fac.Med.,Techniton-Israel Inst.Technol.,Haifa,Israel.
Journal of Immunology(1988),140(8),2675-80。
其它抗磷酸胆碱偶联物抗体可以用本领域已知的方法制备。例如,可以按照以下的描述,用磷酸胆碱偶联物进行亲和纯化,制得人免疫球蛋白制剂的具有aPC活性的部分(subfraction)。静脉免疫球蛋白制剂(如IGIV;Baxter等)是市面上可以买到的高纯度IgG制剂,可以用于治疗不具有抗体生成能力或者抗体生成能力极低的患者。免疫球蛋白制剂包括可以从以下厂家购买到的那些:Baxter(US)eg Gammagard,Isiven(Antimo Naples,ltaly),Omrix(Tel-Hashomer,Israel),Miles(Biological Products Division,WestHeaven,CT),Sclavo(Lucca,Italy),Sandoz(Novartis,Basel,Swizerland)egSandoglobulin,Biotest Diagnostic Corporation(Deville,NJ)。免疫球蛋白制剂的实例有Gammagard S/D,Gammar IV,Gammar-P IV,GammimuneN,Iveegam,Panglobulin,Polygam S/D,Sandoglobulin,Venoglobulin。免疫球蛋白制剂通常包含一些IgM,还含有IgG。Gammagard中有很少量IgM。Pentaglobin(Biotest)是富含IgM的制剂,可用于治疗SARS。具有aPC活性的部分可包含IgG和IgM二者,或者可以选择主要包含IgG(例如,用Gammagard等富含IgG的制剂开始,和/或选择IgG);或主要包含IgM(例如,用Pentaglobin等富含IgM的制剂开始,和/或选择IgM)。
具有磷酸胆碱偶联物特异性的抗体制剂结合未被偶联的磷酸胆碱,也可以结合含有暴露的磷酸胆碱(PC)的化合物中的PC、例如溶血磷脂酰胆碱(lysoPC;参见,例如,Kim et al,J Exp Med.2002 Sep 2;196(5):655-65)中的PC。因此,具有磷酸胆碱偶联物特异性的抗体制剂也可以结合溶血磷脂酰胆碱。
动脉粥样硬化受试者中的IgM免疫球蛋白水平
在动脉粥样硬化风险因子相关研究中,测定高血压患者(舒张压(diastolic pressure)>95mmHg)中抗磷酸胆碱的IgM自身抗体的基础水平和4年后水平。结果概述如下:
在刚加入试验时的77例受试者(35%)和4年随访后的84例受试者(38%)中检测到颈动脉斑(Carotid plaque)。该研究共包括218例受试人。中膜-内膜厚度(IMT)的增加在随访组中比在那些刚加入试验时具有高血清水平的抗PC IgM(75th或90th百分点)的患者中少见。IMT增加者与减少者之间抗-磷酸胆碱IgM抗体水平均值有显著性差异(638.8±219.6比(vs.)734.8±266.9,p=.004)。
抗PC的IgM自身抗体与IMT变化之间的关系与年龄、吸烟习惯、阿替洛尔(atenolol)或拉西地平(lacidipine)治疗以及血脂都无关。IgM自身抗体也与IgG水平无关。
因而本发明的一个实施方案是,用磷酸胆碱偶联物制备药物组合物,以便在动脉粥样硬化的治疗或预防中应用。所述偶联物可以是磷酸胆碱与蛋白质或聚合物相连。所述药物组合物优选经注射给药。
本发明建议的主动免疫方法将可以调节自身抗体的滴度,从而在对抗动脉粥样硬化发生方面起到积极有利的作用。
本发明另一实施方案是,用识别磷酸胆碱偶联物的抗体制剂(例如单克隆抗体)制备药物组合物,以便在动脉粥样硬化的治疗或预防中应用。所述单克隆抗体可以用本领域已知的方法制备。
本发明另一实施方案是,提供一种用磷酸胆碱偶联物检测抗磷酸胆碱抗体(例如IgM或IgG抗体)存在与否的方法,所述抗体与患缺血性心血管病风险的增加或降低相关。优选的方法是免疫分析方法。所述方法可以用于评估患者发生或进展缺血性心血管疾病的风险。
附图说明
图1a:用β2GPI,PS和CL抑制抗体(IgM)与经过PC白蛋白包被的ELISA-板的结合。通过不同抗原与PC白蛋白包被板的结合来抑制。为研究aPC的特异性,按照试验部分所述进行竞争试验。结果用均值±SD表示。
图1b:用β2GPI,PS和CL抑制抗体(IgG)与经过PC白蛋白包被的ELISA-板的结合。通过不同抗原与PC白蛋白包被板的结合来抑制。为研究aPC的特异性,按照试验部分所述进行竞争试验。结果用均值±SD表示。
图2a:用oxLDL和MDA-LDL抑制抗体(IgM)与经过PC白蛋白包被的ELISA-板的结合。通过不同抗原与PC白蛋白包被板的结合来抑制。为研究aPC的特异性,按照试验部分所述进行竞争试验。结果用均值±SD表示。
图2b:用oxLDL和MDA-LDL抑制抗体(IgG)与经过PC白蛋白包被的ELISA-板的结合。通过不同抗原与PC白蛋白包被板的结合来抑制。为研究aPC的特异性,按照试验部分所述进行竞争试验。结果用均值±SD表示。
图3:从IGIV提取的aPC对巨噬细胞中oxLDL摄取的影响
测试了两组:巨噬细胞加oxLDL,巨噬细胞加已经与从IVIG提取的aPC预保温的oxLDL。
巨噬细胞+-oxLDL(共107个细胞):
弱染色37/107=34.58% 强染色10/107=9.35% 总染色阳性47/107=43.93%
巨噬细胞+Dil-oxLDL+aPC-组(检查了156个细胞):
弱染色37/156=23.72% 强染色2/156=1.28% 总染色阳性39/156=25%
图3A示Dil-标记的oxLDL染色。图3B显示无标记的oxLDL的染色。
图4:将高血清滴度抗磷脂抗体(aPL)与人免疫球蛋白集合物Gammagard预保温对膜联蛋白V与人脐静脉内皮细胞(HUVEC)的结合的影响:培养24小时后进行流式细胞术分析
IVIG与以下浓度的血清预保温 | 膜联蛋白V结合的中值荧光强度(MFI) |
0mg/ml | 649 |
2.5mg/ml | 913 |
5mg/ml | 1269 |
10mg/ml | 1382 |
图5:aPC对内皮细胞中ICAM-诱导的影响
1ug/ml PAF与aPC IgM预保温或不经预保温而添加到内皮细胞培养物中。ICAM-1的表达用FACScan测定。绿线代表PAF的作用,红线是PAF+aPC IgM以及空白对照。数据清楚表明,添加aPC IgM后,柱状图左移。
试验
受试者
226例确认为高血压(舒张压>95mm Hg)的受试者在加入欧洲拉西地平对动脉粥样硬化影响的研究的瑞典分组(the Swedish component of theEuropean Lacidipine Study on Atherosclerosis(ELSA)25,26)之前取血样。在为期4周的清除期(washout period)内不使用任何药物,以便将治疗对测量值的影响减至最小,4周后采血。按照文献25,26所述测定血压、胆固醇和甘油三酯水平。115例受试者随后用β-阻断剂阿替洛尔治疗,111例受试者用钙拮抗剂拉西地平治疗。该研究经Ethics Committee of the Karolinska Hospital核准,符合the Helsinki Declaration的规定。所有受试者都是在知情的情况下表示同意加入研究。
颈动脉超声(Carotid ultrasound)
按照文献25,26所述进行颈动脉超声并分析。共有218例患者获得了有效的基础超声值和4年随访超声值。简言之,左右颈动脉用Biosound 2000IIA 型多普勒仪(duplex scanner)以8.0MHz环形振子转换器(annular arraytransducer)进行测定。测出内腔-内膜回声(lumen-intima echo)的最边缘(theleading edge)与中膜-外层回声(media-adventitious echo)的最边缘之间的距离,将其作为远壁(the far wall)的内膜-中膜(I-M)厚度。将测量结果作为动脉粥样硬化的替代指示物,它是,四年随访期间,颈总动脉远端(the distalcommon carotids)和双侧颈动脉分支(carotid bifurcations bilaterally,CBMmax)中4处远壁的平均最大内膜-中膜厚度(IMT)的改变。对加入本研究之时的抗PC抗体水平与4年随访期间IMT的增加或减少之间的关联性进行评估。
试剂
Polysorp F96微量免疫滴定板购自Nunc(Roskilde Denmark),PC-BSA(磷酸胆碱-牛血清白蛋白)购自Biosearch Technologies,INC(USA)。
牛血清白蛋白(BSA),碱性磷酸酶偶联的山羊抗人IgG(r-链特异性),碱性磷酸酶偶联的山羊抗人IgM(u-链特异性),PNPP(碱性磷酸酶底物)都购自Sigma(St.Louis,MO,USA)。心磷脂(CL)购自AVANTT(US,β2糖蛋白(β2GP1)得自Calbiochem(US)。
IgG和IgM总水平用文献6所述常规技术测定。
血清CRP用高敏感度颗粒增强型免疫浊度测定法(particle-enhancedimmunonephelometry,Behring Nephelometer Analyzer,BN II(Dade BehringGmBH,Marburg,Germany))进行分析,将试验误差(antir-assay variation)设定为<4%。
测定抗PC,oxLDL和MDA-LDL的自身抗体
用酶联免疫吸附试验(ELISA)测定抗PC-BSA的lgG和IgM抗体。从17例抗磷脂综合征患者收集血清作为内部标准,并在每个平板上都进行检测。用10μg/ml抗原浓度达到抗体结合的平台期。F96微量滴定polysorp板用每孔50μg PC-BSA(10μg/ml)的PBS液包被。将包被板在4℃保温过夜。用PBS洗5次,然后将板用2%BSA-PBS室温封闭2h,再如上述洗板。血清样品用0.2%BSA-PBS稀释(1∶30),按照50μl/孔进行加样。
按照文献所述6,用连续制备型超速离心法(sequential preparativeultra-centrifugation)从健康供血者血浆分离LDL,用铜离子氧化(OxLDL),或用MDA衍化(MDA-LDL)。
基本按照文献6所述,用ELISA测定OxLDL和MDA-LDL。将OxLDL或MDA-LDL用包被缓冲液(碳酸盐-碳酸氢盐缓冲液50mM pH9.7)稀释至2μg/ml,按照100μl/孔添加到包被的ELISA板(Costar 2581)中。将板在4℃静置过夜,用PBS洗4次,然后用20%成年牛血清的PBS溶液(20%ABS-PBS)室温封闭2小时。再与已用20%ABS-PBS稀释至1∶30的100μl血清一起4℃保温过夜。
将板在4℃保温过夜,如上述洗板。按照100μl/孔添加碱性磷酸酶偶联的山羊抗人IgG(用样品缓冲液稀释至1∶9000)和碱性磷酸酶偶联的山羊抗人IgM(用样品缓冲液稀释至1∶7000),4℃保温过夜。洗5次,然后按照100μl/孔添加碱性磷酸酶底物(PNPP),将板在室温避光保温60min以便显色。在ELISA Multiskan Plus spectrophotometer上用405nm读板。在一项实验中测定所有样品,偏差系数(coefficient of variation)低于10-15%。
抗-磷酸胆碱-BSA抗体的特异性
为了检查抗-磷酸胆碱-BSA的特异性,用收集汇总的高滴度血清进行吸附试验。将高滴度血清集合物在与PC-BSA有50%最大结合的稀释度与不同浓度的PC-BSA一起预保温。将试管涡旋后,40℃保温过夜,13000r.p.m.离心30min(40℃)。按照文献所述测定上清中抗体与PC-BSA的结合。抑制百分比如下计算:
抑制百分比=(不含竞争物时的OD-有竞争物时的OD)×100/不含竞争物时的OD
统计学分析
分析(dichotomize)第75和第90百分点的抗-磷酸胆碱水平。抗-磷酸胆碱抗体(或其它抗体)与动脉粥样硬化进展之间的4年期间的关联性如下确定:用逻辑回归分析来估计IMT增长(有增长或无增长)并计算差异率(oddsratios,ORs)和95%置信区间(CI),或用Spearman correlation如所述进行比较。针对有可能造成混淆的因素,如年龄、吸烟习惯、血清胆固醇、血清甘油三酯以及抗-高血压治疗(拉西地平,阿替洛尔)模式等,作出适当调整。双侧p-值<0.05时认为具有显著性。
结果
受试者刚加入研究时的基本情况另有详述(Pockley et al(2003)Hypertension 42,235-238),可参见本文表I。
竞争性研究表明,IgM和IgG类的aPC与PC-BSA预保温时有竞争力(competed out),心磷脂竞争能力弱,磷脂酰丝氨酸没有竞争能力(图1a,1b)。β2-糖蛋白I可在一定程度上与IgG竞争对PC-BSA的结合,但不如与IgM竞争的程度(图1a,1b)。PC-BSA对结合其它受试抗原的竞争力弱(数据未显示)。OxLDL和MDA-LDL可以竞争IgM aPC对PC-BSA的结合,也可以竞争IgG aPC的结合,但后一种情况的程度弱(图2a,2b)。
随访组的IMT增长在那些刚加入研究时具有抗PC(第75或第90百分点),oxLDL和MDA-LDL(第90百分点)的IgM高血清水平的患者中少见,CRP与IMT变化无关(表2)。
逻辑回归分析表明,抗PC,oxLDL和MDA-LDL的IgM自身抗体与IMT变化之间的关联性与年龄、吸烟习惯、阿替洛尔或拉西地平治疗以及血脂无关。aPC IgM在第75百分点和第90百分点都与IMT变化显著相关,IgM类aOxLDL和aMDA-LDL仅在第90百分点显著相关(表3a-d)。IgM自身抗体也与IgG值无关(数据未显示)。IgG总水平和IgM总水平与IMT测量值或变化无关(数据未显示)。
抗PC的IgG自身抗体在有IMT增长的受试者中倾向于更低水平,但这种差异无统计学显著性(表2)。
男性和女性之间存在显著差异。IgM类的aPC,aMDA-LDL和aOxLDL在女性中明显高于男性(p’s<0.05)。但这些自身抗体的IgG水平在男女之间无差别。此外,女性在刚加入研究时以及随访中,动脉斑发生率明显更低(p<0.05)。
aPC IgM水平与IMT增长呈负相关(Rho 0.18,p=0.006),这与另两种保护因子,HDL和HSP70的表现相反,后二者在连续测量中与IMT变化无关(数据未显示)。与aPC IgM不同,aOXLDL和aMDA-LDL在这些测定中未达到显著性(数据未显示)。
aPC IgM水平与aOCLDL IgM(Rho 0.74,p<0.001)和aMDA-LDL IgM(rho 0.51,p<0.001)之间有显著相关性。类似地,aPC与HSP60(Rho 0.28,p<0.001),HSP70(Rho 0.35,p<0.001)和HDL(Rho 0.23,p<0.01)都相关,其中HSP70在最近的文献中被描述为该群体(cohort)中新的人动脉粥样硬化保护因子(Pockley et al(2003),出处同上)。aPC IgM,aOxLDL IgM或aOxLDL,MDA-LDL和LDL,CRP或甘油三酯之间无关联(数据未显示)。
在控制年龄、总胆固醇、甘油三酯、吸烟和治疗等因素的情况下,分别对男性和女性进行逻辑回归分析,结果表明,IgM aPC在女性中仅在第90百分点时显示明显的保护效应(EXP(B)=.17,95%CI=0.05-.68;p=0.01),在男性中仅在第70百分点时显示明显的保护效应(EXP(B)=.18,95%CI=0.04-.74;p=0.01)。
抗MDA-LDL和oxLDL的IgM在这方面有不同,仅有女性的检测值具有统计学显著性。因此,在对男性和女性分别进行逻辑回归分析时,在控制年龄、总胆固醇、甘油三酯、吸烟和治疗这些因素的情况下,抗MDA-LDL的IgM以及抗OxLDL的IgM在女性中有明显保护作用(分别为EXP(B)=.17,95%CI=.05-.68,p=0.01和EXP(B)=.18,95%CI=.04-.74,p=0.01),男性中这种效应不具有显著性(分别为EXP(B)=.60,95%CI=.15-2.2,p=0.44和EXP(B)=.39,95%CI=.10-1.5,p=0.17),这表明,高滴度的抗OxLDL IgM和抗MDA-LDL IgM在女性中有特异性保护作用。
表1.刚加入研究时的基本情况。结果表示为平均值(SD)或百分比(%),脂质结果表示为mg/dL。
总水平(N=226) | 阿替洛尔(N=115) | 拉西地平(N=111) | |
年龄(岁) | 57.7(7.8) | 57.6(7.6) | 57.7(7.9) |
性别(%男性) | 50 | 46 | 53 |
BMI | 26.7(3.7) | 26.3(3.3) | 27.1(3.9) |
总胆固醇 | 232.4(37.8) | 233.5(38.1) | 231.4(37.4) |
HDL | 55.6(27.6) | 56.5(25.8) | 54.7(27.6) |
LDL | 149.4(37.8) | 149.7(37.1) | 149.2(38.6) |
甘油三酯 | 131.6(58.2) | 128.6(57.0) | 134.7(59.5) |
表2.抗磷酸胆碱(PC)的IgG和IgM自身抗体为基础水平时对IMT变化的直接预测。
变量 | 差异率第75百分点 | (95%CI)下限(Lower) | 上限(Upper) | P |
aPC(IgG) | .60 | .32 | 1.1 | .10 |
aPC(IgM) | .46 | .25 | .85 | .01 |
aOxLDL(IgG) | 1.2 | .64 | 2.3 | .57 |
aOxLDL(IgM) | .77 | .41 | 1.4 | .40 |
aMDA-LDL(IgG) | .80 | .43 | 1.5 | .48 |
aMDA-LDL(IgM) | .67 | .36 | 1.2 | .18 |
C反应蛋白 | .80 | .43 | 1.5 | .46 |
第90百分点 | ||||
aPC(IgG) | .60 | .25 | 1.4 | .24 |
aPC(IgM) | .36 | .15 | 0.87 | .024 |
aOxLDL(IgG) | .94 | .38 | 2.31 | .90 |
aOxLDL(IgM) | .27 | .11 | .69 | .006 |
aMDA-LDL(IgG) | .63 | .26 | 1.5 | .30 |
aMDA-LDL(IgM) | .27 | .11 | .69 | .006 |
C反应蛋白 | .60 | .24 | 1.4 | .24 |
表3a:用高血压受试者aPC IgM自身抗体基础水平的第75百分点预测4年期间的IMT变化。
模型中变量 | 系数(B) | 差异率估计值Exp(B) | P | 95%CI | |
下限 | 上限 | ||||
吸烟 | -.01 | .99 | .95 | .66 | 1.5 |
性别 | -.05 | .95 | .87 | .54 | 1.4 |
总胆固醇 | .003 | 1.0 | .45 | .99 | 1.0 |
血浆甘油三酯 | -.001 | .99 | .63 | .99 | 1.0 |
年龄(岁) | .01 | 1.0 | .59 | .97 | 1.0 |
治疗(A/L) | -.23 | .79 | .40 | .45 | 1.4 |
APC IgM | -1.0 | .37 | .0027 | .15 | .89 |
表3b:用高血压受试者aPC IgM自身抗体的第90百分点预测4年期间的IMT变化。
模型中变量 | 系数(B) | 差异率估计值Exp(B) | P | 95%CI | |
下限 | 上限 | ||||
吸烟 | -.02 | .97 | .90 | .65 | 1.5 |
性别 | -.005 | 1.0 | .98 | .56 | 1.8 |
总胆固醇 | .003 | 1.0 | .42 | .99 | 1.0 |
血桨甘油三酯 | -.001 | 0.99 | .67 | .99 | 1.0 |
年龄(岁) | .003 | 1.0 | .87 | .97 | 1.0 |
治疗(A/L) | -.22 | .80 | .43 | .46 | 1.4 |
aPC IgM | -.77 | .46 | .017 | .24 | .87 |
表3c:用高血压受试者抗OxLDL IgM自身抗体和其它风险因子的第90百分点预测4年期间的IMT变化。
模型中变量 | 系数(B) | 差异率估计值Exp(B) | P | 95%CI | |
下限 | 上限 | ||||
吸烟 | -.01 | .99 | .95 | .66 | 1.5 |
性别 | .001 | 1.1 | .98 | .56 | 1.8 |
总胆固醇 | .001 | 1.0 | .72 | .99 | 1.1 |
血桨甘油三酯 | -.001 | 1.0 | .71 | .99 | 1.0 |
年龄(岁) | .01 | 1.0 | .60 | .97 | 1.1 |
治疗(A/L) | -.28 | .77 | .35 | .44 | 1.3 |
aOxLDL IgM | -1.3 | .26 | .008 | .11 | .72 |
表3d:用高血压受试者抗MDA-LDL IgM自身抗体和其它风险因子的第90百分点预测4年期间的IMT变化。
模型中变量 | 系数(B) | 差异率估计值Exp(B) | P | 95%CI下限上限 | |
吸烟 | -.07 | 0.93 | .73 | .62 | 1.4 |
性别 | -.001 | .99 | .99 | .56 | 1.7 |
总胆固醇 | 0.001 | 1.0 | .78 | .99 | 1.0 |
血浆甘油三酯 | -.001 | .99 | .74 | .99 | 1.1 |
年龄(岁) | 0.01 | 1.0 | .54 | .97 | 1.0 |
治疗(A/L) | -.27 | .76 | .34 | .44 | 1.3 |
aMDM-LDL IgM | -1.1 | .31 | .01 | .12 | .79 |
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显示aPC的保护作用的研究
在Malm的观察研究(the Malm Diet and Cancer Study)中,30000例受试者有约6000例接受了进一步的心血管调查,包括用颈动脉超声测量来对亚临床动脉粥样硬化进行非侵入性评估。还测定了其它心血管风险因子的基础水平。对这些受试者随访10年,考查他们的心血管疾病新事件(心肌梗塞、慢性冠状动脉心脏疾病、动脉粥样血栓性中风(atherothrombotic stroke))发生率。为了评估95%置信区间内的相对风险(计算为相对危险(relativehazard)),对低水平的抗磷酸胆碱抗体(aPC-IgM)进行嵌套式-病例-控制分析(nested-case-control analyse)(每种情况3个对照)。共有145例心血管疾病(CVD)病例(主要是心肌梗塞(MI)和缺血性中风)和400例在年龄和性别上相匹配的对照。aPC水平第10百分点的临界值是307。共有20例CVD病例的aPC水平低于第10百分点(14%),对照有34例低于该百分点(9%),相对危险为1,9(95%CI 1,1-4,3)。在男性病例中,共有16例CVD病例的aPC水平低于第10百分点(19%),25例对照低于该水平(11%),相对危险为1,9(95%CI 1,1-3,5)。女性病例数太少,无法获得有效的相对风险信息(见表1和2)。
这些结果提示,aPC低水平预示健康受试者心血管疾病的发生,其可以作为心血管疾病的标记物。
表1.aPC统计结果(<第10百分点)
性别 | 总计 | ||||||
男性 | 女性 | ||||||
病例 | 对照 | 病例 | 对照 | 病例 | 对照 | ||
低于第10百分点 | 68 | 206 | 57 | 160 | 125 | 366 | |
无 | n | ||||||
% | 81 | 89 | 93 | 95 | 86 | 92 | |
有 | N | 16 | 25 | 4 | 9 | 20 | 34 |
% | 19 | 11 | 7 | 5 | 14 | 9 |
表2.对所有患者、男性患者、以及女性患者分别进行传统的逻辑回归单变量分析(Univariate analysis),考查aPC(<第10百分点)对CVD的影响。
变量 | P值 | 危险比(hazard ratio) | 95%危险比置信限制(Confidence Limits) | ||
所有患者 | aPC | 0.0308 | 1.939 | 1.063 | 3.536 |
男性 | aPC | 0.0262 | 2.181 | 1.097 | 4.338 |
女性 | aPC | 0.6556 | 1.331 | 0.379 | 4.676 |
aPC的作用
简介
利用预先吸附了PC-BSA,PC-KLH或肺炎球菌疫苗(Statens SerumInstitute,Denmark)的柱,提取可与这些化合物反应的抗体。至少前两种化合物的aPC IgG水平升高。从IVIG提取出少量IgM,将其加载至已经预先吸附了上述抗原的柱上。我们通过这种方法获得了IgG和IgM类的多克隆人aPC。蛋白测定表明,可提取出0.5mg/ml aPC IgM。用体外模型测试这些抗体的功能:
1.在单核细胞/巨噬细胞系THP1中,浓度递增的aPC IgM与氧化型LDL预先保温是否可以减少结合和摄取?可以使用配合共焦显微术和/或FACS的测试系统。
2.以正常IgM为对照,浓度递增的aPC IgM与PAF、溶血磷脂酰胆碱(LPC)一起预先保温,是否可以抑制这些脂质对内皮细胞表面粘附分子ICAM的诱导?还可以用购买的试剂盒(多种不同细胞因子;BioSource)测试其它细胞因子。测试可以用FACScan进行。
细胞培养
从Cascade Biologics,Inc.(Portland,OR,USA)购买冻存的第2代HUVEC集合物。将这些培养物在不含酚红、但含有2%胎牛血清和添加剂的EGMTM培养基(Clonetics,San Diego,CA,USA)中维持。细胞在75cm2烧瓶(TPP,AG,Trasadingen,瑞士)中,于37℃、5%CO2湿境中孵育。
所有实验在第3-4代进行。将细胞以2×104细胞/ml的密度接种在12孔板(NUNC,Inc,Naperville,IL,USA)中,准备用于流式细胞分析。经过12-24小时,使细胞贴壁,然后使细胞在SFM中静置至少12小时,再进行处理。
单核细胞系THP-1来自AT&T(USA)。细胞在含10%FCS的RPMI中维持。
制备aPC
用HiTrap IgM或IgG分离柱(Amersham Biosciences)从购买的人免疫球蛋白集合物(Gammagard)中分离出总IgM或IgG级分,其浓度为50mg/ml。洗脱出抗磷酸胆碱(PC)抗体,方法是,将PC与匙孔血蓝蛋白(KLH)(1或5mg/ml)或牛血清白蛋白(BSA)(1mg/ml)偶联,使该偶联的PC结合NHS-Sepharose柱,将IgM或IgG级分加载到该柱中,随后加载到仅结合BSA的柱中。PC-BSA(磷酸胆碱-牛血清白蛋白)和PC-KLH从BiosearchTechnologies,INC(Ca,USA)购买。洗脱的级分在PD-10柱上更换缓冲液,用Millipore Centricone装置浓缩。方法见说明书。所得IgM aPC浓度主要为50μg/ml,IgG aPC浓度主要为30μg/ml。
THP-1衍生的巨噬细胞对oxLDLS的清除结合(cavenger binding)和摄取
按文献所述,通过与铜离子保温来制备氧化型LDL(oxLDL)。首先,将oxLDL用Dil(Dil-(1,1′-双十八烷基-3,3,3′,3′-四甲基吲哚碳菁高氯酸酯(Dil-(1,1′-dioctadecy1-3,3,3′,3′-tetramethylindocarbocyanine perchlorate);Molecular Probes,Inc))标记,在盐水-EDTA缓冲液中稀释至1mg/ml。每1mgoxLDL添加2ml不含脂蛋白的血清,然后过滤(0.45um)。每1mg oxLDL添加50ul Dil(3mg/ml)的DMSO溶液,将该混合物37℃保温15h,然后对盐水-EDTA透析6h,期间换几次透析液。之后,将该混合物再用0.45um滤膜过滤。
oxLDL的摄取用荧光/共焦显微镜检来研究。THP-1细胞作为单核细胞/巨噬细胞的模型,在载玻片小室(slide chamber)中过夜培养(培养基是DMEM/10%FBS/Glu/PEST)
用不含胎牛血清(FBS)的DMEM培养基洗3次
与oxLDL-DiO 5ug/ml(SFM培养基)一起保温6小时
细胞用0.2%BSA-PBS洗5次,用PBS洗1次
巨噬细胞细胞核染色:将细胞与1ug/ml二苯甲亚胺(bisbenzimide)一起保温10’,用PBS洗3次。
固定和封片(mount):细胞用4%低聚甲醛(paraformaldehyde)的PBS溶液固定30’,用PBS洗3次,加1滴封片胶,最后将载玻片用盖玻片覆盖。
膜联蛋白V与内皮细胞的结合
将已添加肝素进行保护、并且具有高度的抑制膜联蛋白V结合的能力的血浆置于SFM中,使其浓度为10%,将该血浆液添加到HUVEC单层培养物中。24小时后用细胞分离液(Cell Dissociation Solution,CDS;Sigma-Aldrich,St.Louis,MO,USA)收获细胞,小心地收集上清,避免丢失已脱壁的悬浮细胞,然后在1200rpm离心7分钟。将沉淀在100μl膜联蛋白V-结合缓冲液(Molecular Probes Inc,Eugene,OR,USA)中重新悬浮,用2μl 5mg/ml膜联蛋白V-FITC(Molecular Probes)染色,在冰上保持15分钟。添加1mg/ml碘化丙锭(propidium iodide)(PI;活体染料;R&DSystems Europe Ltd,Abingdon,UK),然后迅速采样(acquisition)。如上文所述进行分析。
统计学分析
用Stat View software,SAS Institute AB,Gteborg,Sweden进行统计学分析。将偏离的连续变量(Skewed continuous variables)转换为对数形式。用方差分析(ANOVA)比较各个研究小组的连续变量(continuous variable),用卡方(Chi square)比较各个研究小组的绝对变量(categorical variable)。用Fischer′sPLSD作为post hoc检验(test)。相关系数用简单回归计算,或者对于非正态分布的变量,用Spearman的秩相关(rank correlation)计算。将显著性水平设定在p<0.05。
结果
测量膜联蛋白V对内皮细胞的结合
测定膜联蛋白V染色阳性的HUVEC的频率,表示为双变量点图(bivariate dot plot)上的膜联蛋白V+/PI-细胞百分比或柱状图(histogram)上的膜联蛋白V+细胞百分比。在有已知可减少结合的血清存在、并且已与IVIG预保温的情况下,测定膜联蛋白V与HUVEC的结合。与IVIG预保温可以恢复(restore)膜联蛋白的结合,这表明,IVIG中的抗体可以中和结合作用(图4)。
在有CVD病史的SLE患者中,APC-BSA和aPC-KHL都与膜联蛋白V对EC的结合显著相关(分别为r=0.45;p=0.02和r=0.42和p=0.03)。aPC按照上丈的描述进行测定。
aPC对巨噬细胞中oxLDL摄取的影响
将如上所述从IVIG提取的IgM和IgG类aPC与oxLDL按照上丈所述进行预保温(图3)。用总IgM作为aPC IgM(巨噬细胞+Dil-oxLDL+IgM)的对照,和对巨噬细胞摄取的影响。阳性染色的细胞的总百分数为46.62%,表明,IgM本身不具有aPC那样的抑制效应。IgM从SIGMA购买,是用正常人血清作为起始材料,通过沉淀和凝胶过滤技术产生的纯化的人IgM。这种免疫球蛋白经测定纯度至少为95%。
aPC对内皮细胞中ICAM诱导作用的影响
将指定浓度的PAF与EC一起保温。如图5所示,这种脂质可以诱导ICAM表达的显著增加。将按照上文所述从IVIG提取的IgM类aPC与这些脂质按照上文所述一起保温(图5)。
ELSA研究中aPC与其它风险标记物的相关性(226例高血压个体,如前述
aPC IgM与另两种保护因子HSP 70和HDL相关,见表4所示。与TNF也有弱但显著的相关性,TNF是炎症标记物,也是促动脉粥样硬化的细胞因子(proatherogenic cytokine)。
TNF是重要的促炎细胞因子,TNF水平与aPC IgM水平负相关。这种相关性较弱,但却是显著的。
HSP 70是最近由发明人及其他人描述的一种新的保护因子。存在明显的正相关性。HSP60是一种较弱的保护因子,也有相关性。
HDL是众所周知的“良好(good)”胆固醇,具有抗炎特性。它与aPC IgM显著相关。
ANTPCIGG | ANTpCIGM | |||
Spearman′s rho | ANTPCIGG | 相关系数Sig.(双侧)N | 1,000,220 | ,245,000220 |
ANTPCIGM | 相关系数Sig.(双侧)N | ,245,000220 | 1,000,220 | |
HDL | 相关系数Sig.(双侧)N | ,008,906206 | ,233,001206 | |
TNFA | 相关系数Sig.(双侧)N | -,012,863220 | -,136,044220 | |
HSP60 | 相关系数Sig.(双侧)N | ,138,047209 | ,279,000209 | |
HSP70 | 相关系数Sig.(双侧)N | ,157,022213 | ,356,000213 |
**在.01水平具有显著相关性(双侧)。
*在.05水平具有显著相关性(双侧)。
Claims (16)
1.药物组合物在制备免疫接种和治疗哺乳动物动脉粥样硬化或动脉粥样硬化相关疾病的药物中的用途,所述药物组合物包含至少一种磷酸胆碱偶联物,或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体,所述哺乳动物包括人。
2.免疫接种和治疗哺乳动物动脉粥样硬化或动脉粥样硬化相关疾病的方法,所述方法包括对所述哺乳动物施用药物组合物,所述药物组合物包含至少一种磷酸胆碱偶联物,或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体,所述哺乳动物包括人。
3.权利要求1的用途或权利要求2的方法,其中所述药物是用于通过注射来施用的药物或者所述组合物通过注射来施用。
4.在先任一权利要求的用途或方法,其中磷酸胆碱与载体经由间隔臂连接。
5.权利要求4的用途或方法,其中所述载体是蛋白质。
6.权利要求5的用途或方法,其中所述蛋白质是KLH(匙孔血蓝蛋白)或人血清白蛋白(HSA)。
7.权利要求4的用途或方法,其中所述载体是乳胶珠。
8.在先任一权利要求定义的一或多种磷酸胆碱偶联物在制备免疫治疗或治疗缺血性心血管疾病的药物组合物中的用途,所述偶联物可以与佐剂组合或不与佐剂组合。
9.预防性治疗或治愈性治疗哺乳动物的方法,所述哺乳动物是指患有动脉粥样硬化或者有患缺血性心血管病的风险的哺乳动物,例如人,所述方法包括施用治疗有效量的至少一种磷酸胆碱偶联物、或特异性针对磷酸胆碱偶联物的抗体制剂,例如单克隆抗体。
10.用磷酸胆碱偶联物诊断有无IgM或IgG抗体的方法,所述抗体与患缺血性心血管病风险的增加或降低相关。
11.权利要求10的方法,其中磷酸胆碱与载体经由间隔臂连接。
12.权利要求11的方法,其中所述载体是蛋白质。
13.权利要求12的方法,其中所述蛋白质是KLH(匙孔血蓝蛋白)或人血清白蛋白(HSA)。
14.权利要求11的方法,其中所述载体是乳胶珠。
15.权利要求10-14之一的方法,其中所述试验是免疫分析。
16.磷酸胆碱偶联物在评估患者发生或进展缺血性心血管疾病的风险的方法中的用途,在所述方法中,评估所述患者的与磷酸胆碱偶联物反应的IgM或IgG抗体的水平。
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- 2011-02-07 JP JP2011024073A patent/JP5366987B2/ja not_active Expired - Fee Related
- 2011-05-19 AU AU2011202334A patent/AU2011202334B2/en not_active Ceased
- 2011-08-11 US US13/208,138 patent/US20120039895A1/en not_active Abandoned
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2014
- 2014-01-15 US US14/155,903 patent/US10222382B2/en not_active Expired - Fee Related
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2015
- 2015-11-06 US US14/935,044 patent/US20160131661A1/en not_active Abandoned
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US5455032A (en) * | 1993-07-29 | 1995-10-03 | The United States Of America As Represented By The Department Of Health And Human Services | Use of phosphocholine hapten conjugates in vaccines |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103906532A (zh) * | 2011-08-09 | 2014-07-02 | 阿瑟拉生物技术公司 | 结合磷酰胆碱(pc)和/或pc结合物的抗体 |
US9796786B2 (en) | 2011-08-09 | 2017-10-24 | Athera Biotechnologies Ab | Antibodies binding to phosphorylcholine (PC) and/or PC conjugates |
US9803028B2 (en) | 2011-08-09 | 2017-10-31 | Athera Biotechnologies Ab | Antibodies against phosphorylcholine |
CN103906532B (zh) * | 2011-08-09 | 2018-01-26 | 阿瑟拉生物技术公司 | 结合磷酰胆碱(pc)和/或pc结合物的抗体 |
CN105175504A (zh) * | 2015-09-08 | 2015-12-23 | 苏州普罗达生物科技有限公司 | 血管性血友病因子抑制多肽及其应用 |
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