抗ABCA1自身抗体在SLE患者动脉粥样硬化发病机制中的意义

检测系统性红斑狼疮(SLE)患者血清中抗三磷酸腺苷结合盒转运子(ABCA1)抗体及探讨其致SLE早发动脉粥样硬化(AS)的机制。采用免疫印迹法检测75份SLE患者血清中抗ABCA1自身抗体。75份性别年龄匹配正常人血清为对照。观察抗ABCA1抗体阳性患者血清IgG对细胞胆固醇流出的影响。结果显示,SLE患者血清中存在抗ABCA1抗体,阳性率为29.3%,正常人中无一阳性(P<0.05)。SLE有斑块组的抗ABCA1抗体阳性率高于SLE无斑块组(43.8%和16.3%,P<0.05)。纯化的抗ABCA1抗体阳性SLE患者IgG体外可抑制THP-1细胞内胆固醇的流出,抑制率最高可达28.7%,正常人IgG抑制率相比有显著性差异。实验表明,SLE患者血清中抗ABCA1抗体阳性率明显高于正常人,且纯化的抗AB-CA1抗体阳性患者IgG在体外能抑制细胞内胆固醇的流出,从而促进SLE患者早发动脉粥样硬化的发生。     

Apo A-对LDL受体的表达作用

Brown等证明细胞LDL受体的表达是受细胞内外环境LDL胆固醇浓度的调控,当细胞在缺乏脂蛋白血清(LDS)中培养,LDL受体表达加强。因此,体外观察脂蛋白受体一直沿用LDS方法。作者曾报导国产人清蛋白(HSA)代替LDS,观察到LDL受体作用,并进一步比较了HSA、LDS、BSA(牛清蛋白)的作用,发现HSA>LDS>BSA,经鉴定发现HSA试剂中无脂质而含Apo A-I,BSA有脂质而无Apo A-I。提示在缺脂前提下,Apo A-I可能加强LDL受体表达作用,这种作用是否由于Apo A-I有接纳细胞内胆固醇外流的功能,为此,作者同时观察了不同量的Apo A-I对细胞内胆固醇外流及LDL受体表达作用。     

载脂蛋白A-Ⅰ对三磷酸腺苷结合盒转运体A1的影响

目的：以THP-1巨噬细胞源性泡沫细胞为研究对象，观察载脂蛋白A-I对THP-1巨噬细胞源性泡沫细胞胆固醇流出和三磷酸腺苷结合盒转运体A1(ABCA1)的影响,从而探讨载脂蛋白A-I对动脉粥样硬化(As)发生发展的影响。方法:用液体闪烁计数器检测细胞内胆固醇流出, 高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量, 运用逆转录-多聚酶链反应和Western 印迹分别检测ABCA1 mRNA 与ABCA1蛋白的表达,用流式细胞术检测细胞平均ABCA1荧光强度。 结果:载脂蛋白A-I引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯呈时间依赖性减少, 而ABCA1蛋白质水平、细胞平均ABCA1荧光强度以及细胞内胆固醇流出呈时间依赖性增加,但ABCA1 mRNA没有明显变化。巯基蛋白酶抑制剂(leupeptin 和ALLN)增加ABCA1蛋白质水平,而其它蛋白酶抑制剂(pepstatin A、aprotinin及phosphoramiddon)不增加ABCA1蛋白质水平,蛋白体抑制剂(lactacytin)和溶酶体抑制剂(NH4Cl)也不影响ABCA1蛋白质水平。 结论:巯基蛋白酶可降解THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质,而载脂蛋白A-I可阻碍巯基蛋白酶降解ABCA1蛋白质,从而提高THP-1巨噬细胞源性泡沫细胞ABCA1 蛋白质水平, 增加细胞内胆固醇流出, 降低细胞内胆固醇聚积。     

利用噬菌体肽库技术获得与人Fas结合的多肽基序

目的　利用噬菌体随机肽库技术获得与人Fas胞外区结合的多肽及其多肽基序 ,观察多肽基序的生物学功能。方法　以人Fas胞外区与IgGFc段的融合蛋白Fas .Fc为筛选配基 ,筛选噬菌体随机九肽库 ;微量淘洗与ELISA相结合鉴定阳性克隆 ,DNA测序和分析。化学合成多肽进行竞争性ELISA以及细胞增殖抑制实验。结果　经过 4轮亲和筛选 ,微量淘洗鉴定 ,获得 4 2个阳性克隆 ;固定ELISA实验显示筛选到的噬菌体短肽能与Fas .Fc特异性结合 ,并呈剂量依赖关系 ;随机选取 13个阳性克隆进行DNA测序 ,其序列及出现几率分别为 :PRKARVDTS(2 / 13)、YKKKSLQVQ (2 / 13)、YKKKSMLQA(2 / 13)、SRKKYDQYA(4/ 13)、YARKIKPTA(2 / 13)和ARKKTEGAG(1/ 13)。经多重序列分析 ,获得多肽基序 : R/KKK A。在ELISA和竞争性ELISA实验中 ,化学合成多肽EGEFYKKKSM LQADPAK (P3)可抑制Fas与抗人Fas单抗Apo 1的结合 ,且呈剂量效应关系 ;P3不能抑制Fas与FasL的结合。细胞增殖实验表明 ,多肽可抑制Jurkat细胞增殖 ,且随多肽剂量的增加而加强。多肽与单抗Apo 1联合作用对Jurkat细胞增殖的影响与单独使用P3没有明显差别。结论　通过噬菌体随机肽库技术获得与Fas结合的多肽及其多肽基序 ,它们可能模拟了抗Fas抗体Apo 1对Fas的结合位点 ,为基于Fas凋     

自身抗体联合检测对系统性红斑狼疮诊断的意义

为了评价抗核小体抗体(anti-nucleosome antibody,AnuA)、抗Sm抗体、抗双链DNA(double stranded-DNA,dsDNA)抗体、抗核糖体P蛋白(ribosomal P protein,rRNP)抗体联合检测对系统性红斑狼疮(SLE)诊断的价值,采用酶联免疫吸附法(ELISA)测定123例SLE患者、61例其他结缔组织病患者和30名健康对照者血清中An.uA、抗dsDNA抗体、抗Sm抗体、抗rRNP抗体含量.结果显示,AnuA、抗dsDNA抗体、抗Sm抗体和抗rRNP抗体在SLE患者中的阳性率明显高于疾病对照组和正常对照组;AnuA与抗dsDNA抗体的敏感性显著高于其他两种自身抗体;抗dsDNA抗体、抗sm抗体和抗rRNP抗体阴性的SLE患者中,AnuA阳性率为52.6%～68.0%;SLE活动期患者与非活动期患者AnuA、抗dsDNA抗体阳性率有显著性差别.四种自身抗体在SLE的诊断中有明显的互补作用,特别是AnuA和抗dsDNA抗体可以弥补其他抗体的不足;AnuA、抗dsDNA抗体与疾病活动性密切相关.AnuA与抗sm抗体或AnuA与抗dsDNA抗体的二联检测可明显提高其对SLE诊断的敏感性.AnuA、抗dsDNA抗体、抗Sm抗体三联检测的阳性率可达87%.自身抗体联合检测提高了诊断的敏感性,对SLE的诊断和治疗有重要意义.     

抗中性粒细胞胞浆抗体与抗核抗体谱联合检测对系统性红斑狼疮的临床意义

目的探讨系统性红斑狼疮(systemic lupus erythematosus,SLE)患者抗核抗体谱(antinuclear antibody spectrum,ANAs)与抗中性粒细胞胞浆抗体(antineutrophil cytoplasmic antibody,ANCA)联合检测的临床意义。方法采用间接免疫荧光法(IIF)和免疫印迹法(Western blot)检测120例SLE组患者、60例疾病对照组患者和20位健康人的ANCA和ANAs。用ELISA法检测验证ANCA阳性率,用SLE活动指数(SLEDAI)判断SLE活动性,分析ANCA、SLEDAI和ANAS之间的关系。结果在120例SLE患者中IIF法检测ANCA的阳性率为27.5(33/120),且均为核周型(perinuclear ANCA,pANCA)。用ELISA法验证全部ANCA阳性患者血清,阳性率为81.8(27/33),且均为髓过氧化物酶(myeloperoxidase,MPO)即为pANCA。ANCA阳性组SLEDAI评分>10分者(即SLE活动期)占81.4%与ANCA阴性组(36.5%)相比差异有显著性意义(P<0.01)。ANCA阳性组在血沉升高,补体C3、C4降低,肾脏损害、肺损害、关节肿痛等方面与ANCA阴性组比较差异具有显著性(P<0.01)。ANCA阳性率与抗ds-DNA抗体、抗Sm抗体、抗核小体抗体(AnuA)及抗核糖体P蛋白抗体(ARPA)阳性率呈正相关。结论联合检测ANCA和SLE的各项特异性自身抗体可能对SLE患者的早期发现、治疗、病情活动判断具有重要的意义。     

抗β2GPI抗体对系统性红斑狼疮患者血栓形成的影响

目的:探讨抗β2GPI抗体对系统性红斑狼疮患者血栓形成的作用及对疾病诊断的临床意义.方法:选取系统性红斑狼疮(SLE)并发血栓患者20例,单纯SLE患者30例,正常对照23例,采用AggRAM-四通道血小板聚集仪检测血小板聚集率,用酶联免疫吸附试验(ELISA)检测抗β2GPI抗体.结果:SLE血栓组患者和SLE非血栓组患者抗β2GPI抗体滴度和抗体阳性率都显著高于正常对照组(P＜0.05),并且SLE血栓组患者抗β2GPI抗体滴度和抗体阳性率高于SLE非血栓组患者;SLE血栓组患者血小板聚集率比SLE非血栓组和正常对照组显著升高(P＜0.05),SLE非血栓组患者血小板聚集率和正常对照组没有统计学差异;所有SLE患者中抗β2GPI抗体阳性组比抗体阴性组血小板聚集率升高,差异有统计学意义(P＜0.05).结论:抗β2GPI抗体和血小板聚集率升高可为SLE患者继发血栓性疾病提供辅助诊断;抗β2GPI抗体促进SLE患者血小板聚集率上升,加速SLE患者血栓形成.     

血清抗C1q抗体在系统性红斑狼疮疾病活动性判断及狼疮肾炎诊断中的价值

目的探讨抗C1q抗体(C1qAb)在系统性红斑狼疮(SLE)活动性及狼疮肾炎(LN)诊断和疾病活动性判断中的价值。方法采用酶联免疫吸附法检测SLE患者(n=89)、疾病对照组(n=56)和正常对照组(n=42)血清中的抗C1q抗体阳性率,并与SLE患者临床实验室指标﹑活动性评分进行分析。结果 C1qAb的阳性率在SLE患者中显著高于疾病对照组和正常对照组患者(P<0.05);C1qAb阳性的SLE患者肾损发生率、活动性狼疮发生率及抗dsDNA抗体的阳性率均高于C1qAb阴性患者(P<0.05);C1qAb与SLEDAI活动性评分、抗核小体抗体(anti-nucleosome antibody,AnuA)及抗dsDNA抗体呈正相关(P<0.05)。结论抗C1q抗体对SLE的诊断和疾病活动性判断有重要价值;抗C1q抗体参与了SLE肾脏损害的发病机制。     

自身抗体检测在儿童系统性红斑狼疮诊断中的意义

目的探讨抗核抗体(ANA)、抗双链DNA(ds-DNA)、抗ENA抗体检测在儿童系统性红斑狼疮(SLE)诊断中的价值.方法利用酶免疫斑点技术对56例SLE患儿的抗核抗体、抗ds-DNA、抗ENA抗体进行检测.结果56例SLE患儿ANA阳性率最高94.6%,抗ds-DNA阳性率为55.4%,抗Sm/RNP阳性率64.8%,抗SSA/SSB阳性率60.7%.结论SLE患者血清中存在多种自身抗体,自身抗体的联合检测对SLE的诊断和病情的监测有着重要的意义.     

SLE新的抗SmD1抗体检测的临床意义

目的：探讨新的抗SmD1抗体检测在系统性红斑狼疮（SLE）诊断中的价值。方法：用放射免疫分析检测SLE患者血清中的抗dsDNA抗体,ELISA定量法检测抗SmD1抗体、抗Sm抗体,同时用其它风湿病患者及正常健康人血清作对照。结果：80例SLE患者抗SmD1抗体阳性率为52.5,抗Sm抗体阳性率12.5,抗dsDNA抗体阳性率33.8,80例对照组抗SmD1抗体、抗Sm抗体阳性率分别为4.0、0。结论：新的抗SmD1抗体在SLE中敏感性和特异性均较高,抗SmD1抗体可作为诊断SLE的参考指标。     

Presence of autoantibodies to apolipoprotein A-1 in patients with acute coronary syndrome further links autoimmunity to cardiovascular disease

Anti-apolipoprotein A-1 (Apo A-1) autoantibodies were described in autoimmune disorders such as systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) and might be involved in the genesis of arterial and venous thrombotic events. To investigate the presence of these autoantibodies in patients with acute coronary syndrome (ACS) without other features of autoimmunity, we set up an enzyme-linked immunosorbent assay (ELISA) for anti-Apo A-1 antibodies. We used it to investigate their prevalence in ACS as compared to SLE and APS and correlated them to plasma Apo A-1 and serum amyloid A protein (SAA) concentrations. The prevalence of anti-Apo A-1 autoantibodies in the healthy control group was 1% (1/92), but was significantly higher in other groups: 21% (11/53) in ACS group (P=0.001), 13% (12/92) in SLE and/or APS group (P=0.005). Multiple linear regression revealed a significant correlation between plasma Apo A-1 (r=-0.72, P=0.013), plasma SAA concentration (r=0.76, P=0.0066) and anti-Apo A-1 IgG titre in ACS patients. The presence of anti-Apo A-1 autoantibodies in patients with ACS highlights an additional link between autoimmunity, inflammation and atherosclerosis.     

Receptors and molecular heterogeneity of lipoprotein particles containing apolipoprotein A-I

High density lipoproteins (HDL) are heterogeneous, with respect to their hydrated density (HDL2, HDL3), and to their apolipoprotein composition (Lp A-I : A-II contains both apolipoprotein A-I and A-II, Lp A-I contains apolipoprotein A-I but not apolipoprotein A-II). Lp A-I and Lp A-I and Lp A-I : A-II particles have different metabolic functions. Only Lp A-I particles seem to be involved in the antiatherogenic role of HDL. Alcohol consumption raises Lp A-I : A-II level but not Lp A-I. Different tissue possess specific binding sites for HDL: steroidogenic tissue, hepatocytes peripheral cells. Apolipoprotein A-I and/or A-II are possible ligands. HLD, after binding to the receptor, can provide the cells with cholesterol, or promote an efflux of cholesterol from the cells and the "reverse cholesterol transport" from the peripheral cells to the liver. The HDL subfractions possess different metabolic roles: binding of Lp A-I to mouse adipose cell receptors promotes cholesterol efflux. Apo A-II are antagonists for this effect.     

The study of serum apoprotein levels as indicators for the severity of angiographically assessed coronary artery disease

Serum apoprotein A-I (Apo A-I) and B (Apo-B) concentrations were determined in 40 subjects undergoing coronary angiography for past myocardial infarction and angina pectoris, and the authors studied the relationship between the apoprotein concentrations and the severity of coronary artery disease (CAD). During this study, serum total cholesterol, triglyceride, and high-density lipoprotein, low-density lipoprotein, and very low density lipoprotein cholesterol concentrations were determined to control analysis. The results showed that the decrease in serum Apo A-I levels was the best indicator distinguishing CAD from non-coronary artery disease; the Apo B/Apo A-I ratio had the most consistent association with the severity of CAD as assessed by angiography; Apo B/Apo A-I values ranging from 0.98 to 1.00 might be considered critical values for early CAD.     

Plasma lipoprotein abnormalities in a case of primary high-density-lipoprotein (HDL) deficiency

A 53-year-old patient with primary HDL-deficiency is reported. About 2% of the normal concentration of alpha1 HDL was present in his plasma. The alpha1-high-density-lipoproteins separated into two fast-moving components in polyacrylamide gel electrophoresis. The Apo HDL contained both the main apolipoproteins, Apo A-I and Apo A-II, but in disproportionally reduced amounts, the concentration of Apo A-I being reduced about 360-fold, and that of Apo A-II about 14-fold. Concomitantly, the amount of the Apo C polypeptides in the HDL-fractions was decreased to about 5.5% and the activity of the enzyme lecithin cholesterol acyltransferase (EC 2.3.1.4.3) in plasma was found to be only 40% of normal. Apoprotein D was present in the LDL in association with Apo B, forming an abnormal, fast-moving LDL-complex. Apo A-I and Apo A-II were both of normal size as determined by SDS-PAGE, and reduction with thiols resulted in the shift of the M.W. of Apo A-II from 17,000 daltons to about 8,500 daltons. Both proteins were found in the same position as their normal counterparts in analytical isoelectric focusing. The most likely explanation for the multiple lipoprotein abnormalities seems to be that a defect in the regulation or structure of Apo A-I is the basis of the HDL-deficeincy.     

Coronary artery disease, lipid disorders and genetic polymorphisms

Coronary artery disease (CAD) is the leading cause of morbidity and mortality in most industrialized countries, accounting for one out of every two deaths in the United States. Disorders of the lipid transport system resulting from complex interactions among nutritional, environmental and genetic factors, play a very important role in the development of this disease. It has been proposed that low density lipoproteins (LDL) cause cholesterol deposition in the arterial wall, whereas high density lipoproteins (HDL) promote efflux of cholesterol from this site. Thus, low levels of HDL and/or high levels of LDL, have been associated with increased risk of CAD. Apolipoprotein A-I (Apo A-I) is the major protein component of HDL, and it has been proposed that the levels of this protein are a better predictor of risk of CAD than the level of cholesterol in HDL. The human Apo A-I gene has been characterized, and it has been found to be adjacent to the genes for apolipoproteins C-lll and A-lV on the long arm of chromosome 11. The cloning of these genes provides the appropriate tools to apply molecular genetic techniques to find differences between individuals at the gene level (restriction fragment length polymorphisms, RFLP) and to identify specific alleles at this particular gene locus which may be associated with a clinical phenotype, more specifically, premature CAD and familial hypoalphalipoproteinemia. In a preliminary study we have identified a Pst I restriction-endonuclease site flanking the human apolipoprotein A-I gene at its 3' end that is polymorphic.(ABSTRACT TRUNCATED AT 250 WORDS)     

A peptide DNA surrogate that binds and inhibits anti-dsDNA antibodies

Autoantibodies to dsDNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Although the anti-dsDNA antibodies present in SLE are indicative of an antigen-driven response, the antigen has not been conclusively identified. The specific SLE anti-dsDNA antibodies were obtained by affinity purification using a dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide display library, we demonstrated that purified polyclonal anti-dsDNA antibodies and a monoclonal anti-dsDNA antibody specifically bind a 15 mer peptide ASPVTARVLWKASHV. This chemically synthesized peptide could be recognized by anti-dsDNA antibodies in ELISA and Dot blot. This 15 mer peptide can inhibit anti-dsDNA antibodies binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay while a control peptide did not inhibit anti-dsDNA antibodies. This study demonstrates the potential usefulness of the peptide DNA surrogate in diagnostic tests of SLE and in the investigation of the origin of anti-dsDNA antibodies. It may also be used in studies of the DNA-anti-DNA antibody interaction.     

Lipid profile of body builders with and without self-administration of anabolic steroids

Summary Twenty-four top-level body builders [13 anabolic steroid users (A); 11 non-users (N)] and 11 performance-matched controls (C) were examined to determine the effect on lipids, lipoproteins and apolipoproteins of many years of body building with and without simultaneous intake of anabolic steroids and testosterone. After an overnight fast, triglycerides (TG), total cholesterol (TOTC), high density lipoprotein cholesterol (HDLC), low density lipoprotein cholesterol (LDLC), the HDLC subfractions HDL₂C and HDL₃C, as well as apolipoprotein A-I (Apo A-I), apolipoprotein A-II (Apo A-II) and apolipoprotein B (Apo B) were determined. Both A and N, compared to C, showed significantly lower HDLC and higher LDLC concentrations, with the differences between A and C clearly pronounced. In a subgroup of 6 body builders taking anabolic steroids at the time of the study, HDLC, HDL₂C, HDL₃C, Apo A-I and Apo A-II were all significantly lower and LDLC was significantly higher than in a second subgroup of 7 body builders who had discontinued their intake of anabolic steroids at least 4 weeks prior to the study. In some single cases HDLC was barely detectable (2–7 mg·dl⁻¹). The TG and TOTC remained unchanged. The present findings suggest that many years of body building among top-level athletes have no beneficial effect on lipoproteins and apolipoproteins. Simultaneous use of anabolic steroids results in part in extreme alterations in lipoproteins and apolipoproteins, representing an atherogenic profile. After discontinuing the use of anabolic steroids, the changes in lipid metabolism appear to be reversible.     

Apolipoprotein A-I-derived amyloid in atherosclerosis. Its association with plasma levels of apolipoprotein A-I and cholesterol

Wild-type apolipoprotein A-I (apo A-I)-derived amyloid commonly occurs in atherosclerotic plaques. To clarify apo A-I amyloid formation, plasma levels of apo A-I and cholesterol were related to the presence of amyloid in atherosclerotic plaques in 15 patients with peripheral atherosclerosis, subjected to arterial reconstruction. Plasma levels of apo A-I and high-density lipoprotein (HDL) cholesterol were slightly higher in patients with apo A-I-derived amyloid than in those without, but the difference was not significant. Levels of low-density lipoprotein cholesterol and total cholesterol were significantly higher in the group with amyloid. High concentrations of apo A-I in the arterial intima are probably of greater importance to amyloid formation than high plasma levels of the protein. During atherosclerosis, the acute phase reactant serum amyloid A may displace apo A-I from HDL, leading to increased concentration of lipid-free apo A-I in the intima and conformational changes of apo A-I, which make it more fibrillogenic. Some forms of amyloid fibrils have been shown to be cytotoxic. Apo A-I-derived amyloid is possibly a pathogenically important factor in atherosclerosis.     

Human immunodeficiency virus protease inhibitor ritonavir inhibits cholesterol efflux from human macrophage-derived foam cells

Clinical use of human immunodeficiency virus protease inhibitors such as ritonavir may be associated with cardiovascular disease. The objective of this study was to determine the effects and molecular mechanisms of ritonavir on cholesterol efflux from human macrophage-derived foam cells, which is a critical factor of atherogenesis. Human THP-1 monocytes and peripheral blood mononuclear cells were preincubated with acetylated low-density lipoprotein and [(3)H]cholesterol to form foam cells, which were then treated with apolipoprotein A-I for cholesterol efflux assay. A clinically relevant concentration of ritonavir (15 mumol/L) significantly reduced cholesterol efflux from THP-1 and peripheral blood mononuclear cells to apolipoprotein A-I by 30 and 29%, respectively, as compared with controls. In addition, ritonavir significantly decreased the expression of scavenger receptor B1 and caveolin-1, whereas it significantly increased superoxide anion production and activated extracellular signal-regulated kinase (ERK) 1/2 in macrophages. Mitochondrial membrane potential was significantly reduced, whereas NADPH oxidase subunits were increased in ritonavir-treated macrophages. Consequently, the antioxidant seleno-l-methionine, the specific ERK1/2 inhibitor PD98059, or infection of a recombinant adenovirus encoding the dominant-negative form of ERK2 effectively blocked ritonavir-induced decrease of cholesterol efflux. Therefore, human immunodeficiency virus protease inhibitor ritonavir significantly inhibits cholesterol efflux from macrophages, which may be mediated by mitochondrial dysfunction, oxidative stress, ERK1/2 activation, and down-regulation of scavenger receptor B1 and caveolin-1.