Regulation of inflammatory responses by oxidized phospholipids: structure-function relationships

Increasing evidence points to the role of oxidized phospholipids as modulators of inflammatory processes. These modified phospholipids are derived from lipoproteins or cellular membranes and accumulate at sites of inflammation such as atherosclerotic lesions. It has been shown that oxidized phospholipids influence a variety of cellular functions such as chemokine production and expression of adhesion molecules. Furthermore, recent reports indicate that oxidized phospholipids act as ligands for pattern-recognition receptors which detect conserved pathogen-associated molecular patterns during innate immune defense. Thus, the diversity of individual phospholipid oxidation products reflects the many aspects of the inflammatory process they influence. In this review, we focus on structural features used to classify different oxidized phospholipids and how they relate to specific biological responses. As the chemical identification of oxidized phospholipid products proceeds, distinctive structural motifs emerge that can help us to understand the mechanism of action of these unique compounds and how to intervene for therapeutic purposes.     

Cytochrome P450 gene induction in rats ex vivo assessed by quantitative real-time reverse transcriptase-polymerase chain reaction (TaqMan).

Drug-induced changes in expression of cytochrome P450 (P450) genes are a significant issue in the preclinical development of pharmaceuticals. For example, preclinically, P450 induction can affect safety studies by reducing the systemic exposure of a compound undergoing toxicological evaluation, thus limiting the exposure that can be safely investigated in patients. Therefore, the induction potential of candidate drugs has been studied as part of the drug development process, typically using protein and/or catalytic end points. However, measuring changes in the levels of mRNA using TaqMan technology offers the opportunity to investigate this issue with the advantages of better dynamic range and specific enzyme identification. Here, we describe the TaqMan application to study ex vivo the P450 gene induction in the rat. Initially, livers from rats dosed with the prototypic P450 inducers beta-napthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX), and clofibric acid (CLO) were analyzed for mRNA levels of CYP1A1, 1A2, 2B1, 2B2, 2E1, 3A2, 3A23, and 4A1 and compared with control animals. The maximum fold induction of mRNA varied: 2500-fold for CYP1A1 with BNF, 680-fold for CYP2B1 with PB, 59-fold for CYP3A23 with DEX, and 16-fold for CYP4A1 with CLO. This method was then applied to estimate the inductive potential of putative drug candidates undergoing rodent toxicological evaluation. We present a summary of these data that demonstrates the sensitivity and specificity of the TaqMan assay to distinguish between inducers and noninducers and that offers a highly specific alternative to the quantification of drug effects on P450 expression using immunodetection and substrate metabolism.     

用实时荧光定量RT-PCR检测单个小鼠种植前胚胎中特异性基因表达

目的用单细胞实时荧光定量RT-PCR,检测单个小鼠种植前胚胎中特异性基因表达。方法采集小鼠卵母细胞和原核期胚胎,后者经体外培养至囊胚。采用实时荧光定量RT-PCR对多个及单个卵母细胞和2-细胞期胚胎中Tcl1基因的表达进行定量检测;用同样方法对小鼠种植前各发育阶段的胚胎进行Tcl1基因的表达检测。结果在多个及单个卵母细胞和2-细胞期胚胎中,均检测出Tcl1的表达;多个卵母细胞、2-细胞期胚胎中Tcl1的表达量显著高于单个卵母细胞、2-细胞期胚胎;此方法也适用于胚胎早期发育的各个时期,Tcl1在单个卵母细胞、原核期胚胎和2-细胞期胚胎中表达量最高,在4-细胞期胚胎、8-细胞期胚胎、桑葚胚和囊胚中表达量极低。结论应用单细胞实时荧光定量RT-PCR可对单个小鼠种植前胚胎中基因的表达进行定量检测。     

Identification of genes induced by oxidized phospholipids in human aortic endothelial cells

Oxidized-L-alpha-1-Palmitoyl-2-Arachidonoyl-sn-glycero-3-Phosphorylcholine (Ox-PAPC), a component of mildly oxidized/minimally modified low-density lipoprotein (MM-LDL), accounts for many of the biological activities of MM-LDL. Having hypothesized that Ox-PAPC initiates gene expression changes in endothelial cells that result in enhanced endothelial/monocyte interactions and the subsequent development of atherosclerotic lesions, we used the suppression subtractive hybridization (SSH) procedure to compare mRNA isolated from PAPC-treated human aortic endothelial cells (HAEC) with mRNA isolated from Ox-PAPC-treated cells. Genes induced by Ox-PAPC but not by PAPC in HAEC included genes involved in signal transduction, extracellular matrix, growth factors, chemokines and several genes with unknown functions. The observed pattern of gene induction suggests that Ox-PAPC may play multiple roles in angiogenesis, atherosclerosis, and inflammation and wound healing.     

实时荧光定量RT-PCR检测内皮细胞t-PA mRNA方法的建立

目的建立实时RT-PCR检测人微血管内皮细胞组织型纤溶酶原激活物(t-PA)mRNA的表达。方法提取人微血管内皮细胞总RNA,经RT-PCR获得靶基因(t-PA)及管家基因(β-actin)的PCR产物。纯化后,作为标准品梯度稀释,采用SYBR G reen I定量PCR检测,建立标准曲线。方法学考核参数为特异性、线性范围、精密度和重复性。分析全反式维甲酸对内皮细胞表达t-PA mRNA的干预效果。结果定量方法特异性好,检测的灵敏度达103拷贝,线性范围为103~1010拷贝,循环阈值与PCR体系中起始模板量的对数值之间有着良好的线性关系(r2>0.990),批内变异≤3.10%,批间变异≤4.93%。1.25~20.00μmol.L-1的全反式维甲酸能明显上调内皮细胞t-PA mRNA的表达(P<0.01),且呈剂量依赖性。结论实时荧光定量RT-PCR的方法可对t-PA mRNA的表达进行准确定量,有助于溶栓药物药理学研究和新药筛选。     

In vitro and in vivo immunomodulatory effects of microdispersed oxidized cellulose

The immune system can be manipulated specifically by vaccination or nonspecifically by immunomodulation. Many of biological response modifiers (BRM) have polysaccharidic structure similar to that of microdispersed oxidized cellulose (MDOC). We have investigated the immunomodulatory activity of different inorganic MDOC salts (H, Na, Ca, Mg, Zn, Al, Co, Ca/Na) and organic MDOC derivatives (urea, gelatine, arginine) both in vitro and in vivo. A dose-dependent stimulation by a number of MDOC derivatives was observed with spontaneous and mitogen-induced proliferation of human peripheral blood leukocytes (PBLs) and mouse splenocytes in vitro. In both primary cultures, the most intensive proliferation was induced by a Ca/Na salt at a concentration of 1 mg/ml. We have also demonstrated stimulatory effects of MDOC Ca/Na salt on the mouse mixed leukocyte reaction (MLR). The stimulatory activity of MDOC towards the immune system was further supported by the fact that in vitro the product stimulates the release of Th1 cytokine TNF-alpha, but not IFN-gamma, IL-4 or IL-6. In vivo MDOC application increases more than 50% the number of colony-forming units spleen (CFU-s), i.e., stimulates the stem cells in bone marrow, and increases relative percentage of monocytes and B lymphocytes in the mouse peripheral blood.     

实时荧光定量RT-PCR检测大肠腺癌中WT1基因表达

目的探讨大肠腺癌标本中WT1基因的表达水平及其与临床病理特征的关系。方法建立实时荧光定量反转录-聚合酶链反应(RT-PCR)方法,检测了27例大肠腺癌标本和11份正常大肠黏膜组织中WT1基因及内参照β-actin基因的表达水平。结果大肠腺癌标本中WT1基因表达水平的范围5.4×10-5～8.9×10-1,在11份正常肠黏膜中有5份存在WT1的低表达,范围从7.6×10-5～7.2×10-4,其余6份未检出表达,81%(22/27)的患者癌组织中WT1 mRNA表达水平高于正常肠黏膜组织,另外,研究表明WT1 mRNA表达水平与病理分级,淋巴结转移,Duke′s分期无明显相关性。结论WT1基因在大肠腺癌中高表达,可作为诊断和治疗大肠癌新的分子指标。     

In vitro and in vivo effects of piroxicam on rat polymorphonuclear responsiveness after induction of two acute non-specific inflammatory reactions

The effect of piroxicam on rat polymorphonuclear leucocytes (PMN) has been studied in vitro and in vivo after the induction of two acute, non specific inflammatory reactions (pleurisies induced by calcium pyrophosphate crystals (CaPP) or isologous serum). An inhibition of chemotaxis by piroxicam has been demonstrated by two techniques, the filter and agarose assays in vivo and in vitro. An inhibition of random cell migration has been observed only at the higher drug concentration using agarose assay with CaPP-elicited cells. Piroxicam also inhibited superoxide anion generation and O2 consumption of CaPP- and serum-elicited cells. These findings suggest that piroxicam may have a direct effect on PMN responses and that this activity could, at least in part, contribute to its anti-inflammatory properties.     

Protective effects of aspirin against oxidized LDL-induced inflammatory protein expression in human endothelial cells

Atherosclerosis is a complex vascular inflammatory disease. Oxidized low-density lipoprotein (ox-LDL) is directly associated with chronic vascular inflammation. In this study, we hypothesized that nonselective cyclooxygenase inhibitor aspirin might affect the ox-LDL-induced inflammatory responses on human endothelial cells. To test this assumption, cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) expression, IkappaB and p38 mitogen-activated protein kinase (MAPK) phosphorylation were determined in endothelial cells exposed to ox-LDL in the presence of aspirin. The results showed that aspirin significantly suppressed COX-2 and ICAM-1 expression induced by ox-LDL and also inhibited IkappaB phosphorylation in human umbilical vein endothelial cells (HUVECs). Moreover, aspirin reduced the level of p38 MAPK phosphorylation. Our findings suggest that aspirin can decrease inflammatory responses induced by ox-LDL, and the mechanism might be associated with NF-kappaB activation pathway and inhibition of p38 MAPK phosphorylation.     

抗感染联合激素雾化对小儿肺炎体内炎性介质的影响

目的观察抗感染联合激素雾化对肺炎患儿血液中白细胞介素6(IL-6)、白细胞介素10(IL-10)及C反应蛋白(CRP)的影响。方法 51例临床诊断为肺炎患儿分别在抗感染及布地奈德雾化治疗前后抽血检查血液中IL-6、IL-10、CRP,对比三者前后变化。结果治疗后三种介质均较前改变,P<0.05,有统计学意义。结论抗染感联合布地奈德雾化治疗对肺炎患儿有治疗作用,可明显降低患儿炎症程度,有一定疗效。     

In vivo versus in vitro comparison of swine cardiac performance: induction of cardiodepression with halothane

An in situ versus in vitro comparison of relative dose-dependent effects of halothane on cardiac performance was investigated, including ventricular systolic/diastolic function. Such comparative studies may be of interest to individuals working on heart failure models, cardiac device testing, or xenotransplantation. Normal swine (n=9) received halothane at levels of 0.25, 0.5 and 1 MAC (minimum alveolar concentration) for 30 min each. Parameters assessed included: 1) heart rate; 2) arterial blood pressure; 3) pulmonary artery, central venous, left and right ventricular pressures; 4) cardiac output; 5) end-expiratory CO(2) and halothane levels; 6) cardiac temperature; and 7) arterial blood gases. Hearts were removed using standard cardioplegic procedures and reperfused in four-chamber working mode (n=8); again the effects of increasing halothane concentrations on cardiac performance were analyzed. When comparing biventricular depressive effects (negative inotropic, negative lusitropic) of halothane in vivo and in vitro, there were distinct quantitative differences. The negative lusitropic effects were less pronounced in vitro; this was especially true for the right ventricle. Yet, in vitro, halothane at all doses induced more pronounced decreases in left heart output compared to the right. The large mammalian isolated four-chamber working heart model allows for novel assessment of pharmacodynamics and/or evaluation of cardiac devices under a range of hemodynamic performances. Halothane, a cardiodepressive agent, induced direct myocardial depressive effects in vitro similar to those recorded in vivo; hence additional systemic effects are considered to play a minor role in ultimate performances, e.g., compensatory responses due to autonomic controls.     

荧光定量PCR技术在基因治疗药物组织分布研究中的应用

介绍应用荧光定量PCR技术对基因治疗药物组织分布研究的实验方法。从以下几个方面,即定量PCR技术原理、实验设计思路、各种检测方法的比较来说明这一问题。其中,可以使用的检测方法包括紫外定量法和引入内参基因法,后者又可以分为外标法和内标法。充分对比了这些方法的优缺点,并结合本实验室的研究现状,介绍了本监测中心在选用外标法进行研究时所取得的一些成果和实验经验。     

地高辛掺入RT-PCR半定量检测WT1基因在AML中的表达及其意义

目的 建立客观的半定量检测ＷＴ１基因表达的方法，探讨ＷＴ１基因表达水平与急性髓细胞性白血病（ＡＭＬ）预后的关系。方法 用地高辛标记的脱氧三磷酸尿苷（ＤＩＧ－１１－ｄＵＴＰ）掺入逆转录聚合酶链反应（ＲＴ－ＰＣＲ）的方法检测，用ＫＧ－１细胞建立ＷＴ１表达的半定量稀释曲线，分析５３例ＡＭＬ及２１世纪非恶性疾患病人骨髓中ＷＴ１基因的表达。结果 倍比稀释的ＫＧ－１细胞ＷＴ１基因表达水平与稀释倍数有良好的线性     

Liposomes from soya phospholipids as percutaneous drug carriers. 2nd communication: quantitative in vivo investigations with radioactively labelled liposomes

The percutaneous absorption of liposomes (prepared from NAT 106) was investigated with radioactively labelled substances in comparison with percutaneous absorption without liposomes (controls) in vivo on the skin of young pigs. Radioactivity was monitored as a function of time in skin tissue, plasma or blood and in urine. Elevated tissue levels, increased absorption and renal elimination of the various liposomal preparations in comparison to the controls were measured.     

LPS potentiates nucleotide-induced inflammatory gene expression in macrophages via the upregulation of P2Y2 receptor

Sepsis is a severe systemic inflammatory response that is associated with high morbidity and mortality. A previous study using an animal model of sepsis showed that survival was significantly lower in WT mice than in P2Y₂ receptor (P2Y₂R)-deficient mice, suggesting that P2Y₂R plays a role in septic death. We therefore investigated the role of P2Y₂R in the inflammatory responses of RAW264.7 murine macrophages to LPS. LPS time-dependently upregulated P2Y₂R mRNA levels, with a prominent increase observed at 4 h. In addition, LPS increased ATP release in a time dependent manner (5–120 min post LPS treatment). Accordingly, we pretreated cells with LPS for 4 h to induce P2Y₂R expression and then stimulated the cells with UTP or ATP for 16 h. Interestingly, ATP- or UTP-dependent P2Y₂R activation in LPS-pretreated cells resulted in dramatically enhanced HMGB1 secretion, COX-2 and iNOS expression, and furthermore PGE₂ and NO production compared to LPS treatment alone (4 h) or ATP or UTP treatment alone (16 h), an effect that was inhibited by P2Y₂R silencing. In addition, these increases in HMGB1 secretion, COX-2 and iNOS expression and PGE₂ and NO production commonly involved the JNK, PKC and PDK pathways. Taken together, these data demonstrate that LPS-dependent upregulation of P2Y₂R plays a critical role in facilitating the inflammatory responses induced by LPS.