氯喹及其它红内期裂殖体杀灭剂的作用机理

随着恶性疟原虫对氯喹抗性的产生及扩散,药物作用机理的研究对寻找、设计新药极有意义。目前有两种解释氯喹作用机理的理论。Fitch指出:氯喹等是通过阻碍铁卟啉原Ⅸ(FP)形成疟色素而发挥抗疟作用的。疟色素中含有大量FP,红内期裂殖体杀灭剂的选择性抗疟作用常常与疟色素的形成有关。对氯喹有抗性的鼠疟原虫往往不产生疟色素,或当接触氯喹时便停止产生。迄今为止仅发现氯喹有间接抑制血红蛋白降解的作用。实验证明,FP和FP-氯喹复合物对纯化的恶性疟原虫酸性蛋白酶有抑制作用,推     

伯氏疟原虫氯喹敏感株和抗性株在疟色素形成和致病性上的差异

目的：研究伯氏疟原虫氯喹敏感株（Ｎ）和抗性株（ＲＣ）在疟色素形成和致病性上的差异。方法：用伯氏疟原虫氯喹敏感株和抗性株分别感染ＩＲＣ小鼠。实验分为５组：正常对照组（ＮＣ）,氯喹治疗对照组（ＣＣ）,Ｎ株感染组（Ｎ）,ＲＣ株感染组（ＲＣ）和ＲＣ株感染加氯喹治疗组（ＲＣＣ）,比较各组间末梢血中疟原虫的形态、原虫血症、肝脏组织学及超微结构的改变。结果：Ｎ组肝细胞损害较严重,细胞内线粒体肿胀融合,溶酶体增多,肝血窦内疟原虫少见。ＲＣ组炎症反应较突出,主要为单核细胞浸润以及枯否细胞活跃,大滋养体和裂殖体在肝血窦滞留;肝细胞损害较轻,表现为线粒体增生、肿胀及空泡化。Ｎ株疟原虫富含内食物泡,其内有疟色素颗粒,被寄生红细胞结构较完整;而ＲＣ株疟原虫外食物泡较多,位于疟原虫外围的红细胞胞质内,被寄生的红细胞呈蜂窝样改变,食物泡内无疟色素。结论：ＲＣ株疟原虫可能改变了原敏感株（Ｎ株）对血红蛋白的摄取和消化方式,从而阻碍了疟色素形成;Ｎ株和ＲＣ株疟原虫可能因诱导宿主免疫反应的明显差异,导致ＲＣ株较Ｎ株致病力为弱     

高尿酸血症及痛风患者尿液碱化治疗的利与弊

痛风是一种以高尿酸血症为特征的代谢性炎症疾病,当血清尿酸浓度(sUA)>420 μmol/L时,尿酸一钠(MSU)晶体可形成并沉积在关节和结缔组织,发生急性痛风性关节炎。除了尿酸盐浓度,pH值也是影响MSU沉积的影响因素之一,pH值降低促进MSU结晶,尿液碱化将pH提高至6.2～6.9,可增加尿酸盐溶解度,防止MSU沉...     

亚砷酸钠对人类黑色素瘤G361细胞系酪氨酸酶mRNA表达的影响

目的 研究亚砷酸钠(NaAsO2)对人类黑色素瘤G361细胞系酪氨酸酶(TYR)mRNA表达的影响.方法 将体外培养的G361细胞分别暴露于短时(12、24、48 h)低剂量(0.1、1.0μmol/L)、短时(3、6、12、24、48 h)高剂量(5.0、10.0μmol/L)及长时(8、16周)低剂量(0.1μmol/L)下,以荧光实时定量PCR方法测定细胞TYR mRNA表达水平.结果 染毒剂量较低(0.1、1.0μmol/L)、作用时间较短(12、24、48 h)时,黑色素瘤G361细胞TYRmRNA表达水平同一时段各剂量组问比较差异无统计学意义(F值分别为0.173、0.687、2.869,P＞0.05).染毒剂量较高(5.0、10.0μmol/L)、作用12、24、48 h时,细胞TYR mRNA表达水平同一时段各剂量组间比较差异有统计学意义(F值分别为81.056、7.616、24.132,P＜0.05或＜0.01);其中,与对照组比较,除5.0、10.0μmol/L组3、6 h及5.0μmol/L组12 h外,其余各组TYR mRNA相对表达水平比同一时段的对照组明显降低(P＜0.05),0.1μmol/L组染毒8周时细胞TYR mRNA表达水平(138.71±4.56)明显高于对照组(100.00±8.06),两组比较差异有统计学意义(t=-7.238,P＜0.01).结论 NaAsO2暴露可通过引起色素代谢关键酶TYR mRNA表达水平改变而影响色素代谢过程.     

pH值改变对大鼠离体冠状动脉静息张力的影响及机制

目的 研究在基础张力状态下,不同pH对大鼠冠状动脉血管静息张力的影响并探讨机制.方法 采用离体血管张力记录方法,大鼠冠状动脉环的张力舒缩状态采用PowerLab和DMT系统记录.观察pH值梯度改变对大鼠冠脉血管环张力的影响.观察内外钙、Na+/Ca2+交换体抑制剂KB - R7943(1×10-6 mol/L)、氯离子通道阻断剂NPPB(3×10-5 mol/L)、钙通道阻断剂维拉帕米(1×10-5 mol/L)、Na+/H+交换体抑制剂氧氯吡咪(AM,3×10-5 mol/L)、Na+ -K+- ATP酶抑制剂哇巴因(1×10-6mol/L)、NO合酶抑制剂L - NAME(1×10 4 mol/L)对浴液pH6.4时冠脉张力的影响.结果 随胞外pH值逐渐降低,大鼠离体冠脉血管环的静息张力逐渐增强.外钙内流和内钙释放均参与pH6.4时的冠脉收缩,钙通道阻断剂Verapamil可部分阻断冠脉收缩幅度的升高.与对照组相比,KB- R7943、哇巴因对pH6.4时冠脉收缩幅度无显著影响(P＞0.05);NPPB、氨氯吡咪均可抑制pH6.4时冠脉收缩幅度的升高(P＜0.05);L - NAME可增强pH6.4时冠脉收缩幅度(P＜0.05).结论 酸中毒时,随胞外pH值降低,大鼠冠脉的静息张力升高.其作用机制可能与内外钙、氯通道、Na+/H+交换有关.     

利多卡因和普罗帕酮对离体大鼠心脏功能的影响

目的 观察在非缺血及缺血状态下,不同浓度利多卡因和普罗帕酮对离体大鼠心脏功能的影响.方法 采用Langendorff离体心脏灌流的方法,观察不同浓度利多卡因和普罗帕酮对非缺血及缺血大鼠心脏左室功能的影响.结果 非缺血组含不同浓度利多卡因、普罗帕酮的灌流液灌注大鼠心脏5 min,与基础值相比,利多卡因0.0 μg/mL、2.5 μg/mL、5.0 μg/mL、10.0 μg/mL、20.0 μg/mL使左室功能分别降低(0.5±0.1)%、(34.1±5.7)%、(38.0±7.4)%、(44.2±5.1)%和(64.7±6.0)%,普罗帕酮0.000 μmol/L、0.001 μmol/L、0.010 μmol/L、0.100 μmol/L、1.000 μmol/L使左室功能分别降低(0.5±0.1)%、(13.1±2.1)%、(23.1±2.6)%、(36.3±5.5)%和(46.4±2.5)%.缺血组含不同浓度利多卡因、普罗帕酮的缺血灌流液灌注大鼠心脏5min,与基础值相比,利多卡因0.0 μg/mL、2.5 μg/mL、5.0 μg/mL、10.0 μg/mL、20.0 μg/mL使左室功能分别降低(86.3±4.4)%、(76.6±6.7)%、(85.6±4.4)%、(92.1±1.8)%和(94.2±3.0)%,普罗帕酮0.000 μmol/L、0.001 μmol/L、0.010 μmol/L、0.100 μmol/L、1.000 μmol/L)使左室功能分别降低(86.3±4.4)%、(73.2±2.4)%、(84.0±3.1)%、(88.4±4.8)%和(90.3±0.3)%.结论 利多卡因和普罗帕酮剂量依赖性抑制非缺血大鼠心脏功能.低浓度利多卡因和普罗帕酮保护缺血大鼠心脏功能.     

氯喹作为嵌入剂:一个假说的再次崛起

氯喹的抗疟机制曾有三种假说:1.嵌入DNA;2.溶酶体聚积;3.与铁原卟啉IX结合。认为氯喹的作用机制仍是其嵌入疟原虫DNA起作用。嵌入作用的依据五十及六十年代许多学者指出氯喹嵌入DNA。但是如何结合,由于使用的方法、材料不同认识不一。证据1.最近对氯喹结合到DNA的亲和性重新评价后发现,此结合具高度盐依赖性。在50mmol/L磷酸盐缓冲液中加入0.50或100mmol/L氯化钠,解离常数(K_D)的变动范围是27μmol/L～2.6mmol/L。DNA的K_D值是表示每个结合点,而一个核可有数百万个重要的结合点,即使细胞内药物浓度低于K_D值几倍时,仍将有数千个位点被占据。估计在治疗剂量时,疟原虫总嵌入点的0.03％～1.0％被氯喹所占据。证据2.氯喹并非以同等强度结合DNA     

胃内pH值对肝硬化伴幽门螺杆菌感染患者血氨的影响

目的研究胃内pH值对肝硬化伴幽门螺杆菌(Hp)感染患者血氨的影响.方法对37例Hp阳性的肝硬化患者及40例Hp阴性的肝硬化患者,测定基础血氨后,静脉注奥美拉唑针40 mg,同时动态监测胃内pH值的变化,待胃内pH＞6 且持续1 h后,复测血氨.结果Hp阴性与阳性肝硬化患者的基础血氨水平差异无显著性(P＞0.05).胃内pH升高后,发现Hp阴性的肝硬化患者及Hp阳性的肝硬化Child-Pugh A级患者,血氨水平与实验前差异无显著性(P＞0.05).Hp阳性的肝硬化Child-Pugh B、Child-Pugh C级患者血氨水平较前明显升高,分别由 26.33±6.49 μmol/L、35.30±10.25 μmol/L升高至35.80±6.25 μmol/L、52.20±17.01 μmol/L,差异有非常显著性意义(P＜0.01).结论肝功能状况和胃内pH值是影响Hp感染肝硬化患者血氨水平的两个重要因素.     

曲古菌素A对人胰腺癌PaTu-8988细胞增殖和细胞周期的影响

目的 观察曲古菌素A(tfichostatin A,TSA)对人胰腺癌PaTu-8988细胞增殖及细胞周期的影响.方法 不同浓度(0.1、0.5、2.0 μmol/L)TSA处理人胰腺癌PaTu-8988细胞,并设空白对照组.采用WST-8法检测细胞存活率;流式细胞仪检测细胞周期的变化;实时定量PCR检测细胞周期相关基因p21和cyclin DI mRNA的表达.结果 对照组、TSA 0.1μmol/L、0.5μmol/L和2.0μmol/L组的细胞存活率分别为(100.0±4.2)%、(88.5±4.2)%、(79.7±5.0)%和(64.3±7.2)%,各TSA组均显著低于对照组(P<0.01).TSA 0.1μmoL/L、0.5μmol/L组的细胞以G1期居多,2.0μmol/L组(50.29±7.53)%细胞阻滞在G2/M期.TSA各组p21 mRNA表达值分别为5.29±1.16、7.79±0.41、8.61±0.73,较对照组l,00±0.08显著升高(P<0.01);cyclin DI mRNA表达值分别为1.13±0.12、0.42±0.06、0.19±0.06,较对照组1.00±0.07表达降低(P<0.05).结论 TSA通过上调p21及下调cyclin D1基因的表达,导致细胞周期阻滞.     

间日疟原虫疟色素对树突状细胞成熟的影响

目的观察间日疟原虫代谢产物疟色素(HZ)对树突状细胞(DC)成熟分化的影响。方法以间日疟患者感染红细胞获得间日疟原虫制备纯化HZ,体外刺激人单核来源的未成熟DC。采用流式细胞术分析0.1、1.0、10.0μmol/L不同浓度的HZ作用下DC成熟相关分子CD83、CD86、HLA-DR的表达变化;同时观察DC在HZ刺激后再经脂多糖(LPS)诱导其上述分子的表达变化。结果 0.1、1.0、10.0μmol/L的HZ刺激的DC表达CD83、CD86和HLA-DR阳性百分率均低于LPS诱导组(P均0.05);1.0、10.0μmol/L的HZ刺激组的CD83、CD86和HLA-DR明显低于未刺激组(P均0.05);HZ1.0、10.0μmol/L组HLA-DR的表达低于HZ0.1μmol/L组(P均0.05)。与未刺激组DC相比,HZ0.1μmol/L+LPS组DC的CD83表达明显升高(P0.01),CD86表达明显升高(P0.05),HZ1.0μmol/L+LPS组的CD83明显升高(P0.01);HZ10.0μmol/L+LPS组CD86表达与HZ0.1μmol/L+LPS和HZ1.0μmol/L+LPS组相比明显降低(P均0.05)。结论间日疟原虫来源的HZ能导致DC的CD83、CD86和HLA-DR表达下调,但负载HZ的DC仍可以在LPS等诱导剂作用下部分上调这些成熟相关分子的表达。HZ对DC的成熟性分化具有剂量依赖性抑制的特征。DC对HZ的过度吞噬而导致成熟抑制可能是疟原虫逃逸免疫攻击的重要方式之一。     

Aligned hemozoin crystals in curved clusters in malarial red blood cells revealed by nanoprobe X-ray Fe fluorescence and diffraction

The human malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicron-sized hemozoin crystals. The crystal is composed of heme units interlinked to form cyclic dimers via reciprocal Fe─O (propionate) bonds. Templated hemozoin nucleation was envisaged to explain a classic observation by electron microscopy of a cluster of aligned hemozoin crystals within the parasite digestive vacuole. This dovetails with evidence that acylglycerol lipids are involved in hemozoin nucleation in vivo, and nucleation of β-hematin, the synthetic analogue of hemozoin, was consistently induced at an acylglycerol-water interface via their {100} crystal faces. In order to ascertain the nature of hemozoin nucleation in vivo, we probed the mutual orientations of hemozoin crystals in situ within RBCs using synchrotron-based X-ray nanoprobe Fe fluorescence and diffraction. The X-ray patterns indicated the presence of hemozoin clusters, each comprising several crystals aligned along their needle c axes and exposing {100} side faces to an approximately cylindrical surface, suggestive of nucleation via a common lipid layer. This experimental finding, and the associated nucleation model, are difficult to reconcile with recent reports of hemozoin formation within lipid droplets in the digestive vacuole. The diffraction results are verified by a study of the nucleation process using emerging tools of three-dimensional cellular microscopy, described in the companion paper.     

Hemozoin Enhances Maturation of Murine Bone Marrow Derived Macrophages and Myeloid Dendritic Cells

Background: Falciparum malaria is a severe health burden worldwide. Antigen presenting cells are reported to be affected by erythrocytic stage of the parasite. Malarial hemozoin (HZ), a metabolite of malaria parasite, has adjuvant properties and may play a role in the induction of immune response against the parasite. Objective: To determine the immunological impact of hemozoin on the capacity of innate immune cells maturation. Methods: Plasmodium falciparum (F32 strain) was cultured in O+ blood group up to 18% parasitemia. Natural hemozoin was extracted from infected red blood cells. Murine bone marrow derived macrophages and myeloid dendritic cells were stimulated with 4 ߤg/mL or 40 ߤg/mL of synthetic hemozoin (β-hematin) or natural hemozoin. We assessed the immunomodulatory role of synthetic or natural hemozoin in vitro by flowcytometric analysis. Results: The maturation markers MHCII, CD80 and CD86 were significantly upregulated (p<0.05) on the surface of murine bone marrow derived macrophages or myeloid dendritic cells. Data confirmed the potential of macrophages or myeloid dendritic cells, through hemozoin activation, to establish an innate immune response against malaria parasites. Conclusion: Both synthetic and natural hemozoin are potent inducers of cellular immunity against malaria infection. However, natural hemozoin is a stronger inducer as compared to synthetic hemozoin.     

Clinical efficacy of chloroquine followed by primaquine for Plasmodium vivax treatment in Azerbaijan

Efficacy of chloroquine followed by primaquine has been monitored in 153 patients in seven districts of Azerbaijan. Chloroquine is fully effective over the first 14 days and the combination of chloroquine and primaquine is 100% effective over 28 days.     

Effect of Inducers, Incubation Time and Heme Concentration on IC(50) Value Variation in Anti-heme Crystallization Assay

Heme detoxification through crystallization into hemozoin has been suggested as a good target for the development of screening assays for new antimalarials. However, comparisons among the data obtained from different experiments are difficult, and the IC(50) values (the concentrations of drug that are required to inhibit 50% of hemozoin formation) for the same drug vary widely. We studied the effects of changes in heme concentration (precursor of β-hematin), incubation time and three inducers (SDS, Tween 20 and linoleic acid) on the IC(50) of some antimalarials (chloroquine, quinine, amodiaquine, and clotrimazole). The results showed that increasing both inducer concentration and incubation time raised the IC(50) of selected antimalarials. Any change in those factors caused the IC(50) value to vary. Standardization of assay conditions is, therefore, necessary to increase reproducibility and reduce discrepancies in assay performance. Considering all of the variables, the best choice of inducers is in the order of SDS > Tween 20 > linoleic acid.     

Enhancement of the antimalarial effect of chloroquine by chloropheniramine in vivo

Summary The efficacy of chloroquine and chloroquine plus chloropheniramine, a histamine H_{1 receptor blocker which reverses chloroquine insensitivity in Plasmodium falciparum in vitro , was studied in 96 children with acute symptomatic uncomplicated falciparum malaria. The chloroquine/chloropheniramine combination produced a significantly higher cure rate than chloroquine alone and cured 77% of children with chloroquine treatment failures. Children with chloroquine treatment failure had mean plasma chloroquine concentrations above the minimum therapeutic concentration for the area. Chloroquine concentrations in plasma and red blood cells and ratio of red cell to plasma chloroquine concentrations on days 3 and 7 after initiation of therapy were not significantly different in the two groups. Chloroquine/chloropheniramine produces a higher cure rate than chloroquine alone and reverses chloroquine insensitivity in Plasmodium falciparum in vivo . It may be a useful way of optimising the antimalarial effect of chloroquine.}     

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ?-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ?-hematin synthesis (I?HS) and hemoglobin proteolysis (IHbP) in vitro. The ?-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5%, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 μM and 14.4 ± 1.4 μM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0% and 55.3 ± 3.6%, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia (%P) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0% compared to 26.6 ± 3.7%, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.     

中缅边境地区恶性疟原虫对氯喹、哌喹、咯萘啶敏感性的体外测定

目的了解中缅边境地区恶性疟原虫（Plasmodium falciparum）对氯喹、哌喹和咯萘啶敏感性的变化。方法 2009年9～12月在中缅边境的缅甸拉咱市采集了51份恶性疟血样,采用Rieckmann体外微量测定法进行药物敏感性测定。结果敏感性测定结果有效的42份血样中、其恶性疟原虫对氯喹、哌喹和咯萘啶的抗性率分别为95.2%、7.1%和54.8%,半数抑制量(ID₅₀)分别为320.5、128.2和96.0 nmol/L。在抗咯萘啶的23份血样中,对氯喹和哌喹的交叉抗性率分别为91.3%（21/23）和13.0%（3/23）;抗氯喹的40份血样中,对哌喹和咯萘啶的交叉抗性率分别为7.5%（3/40）和52.5%（21/40）;抗哌喹的3份血样中,对氯喹和咯萘啶的交叉抗性率均为100%。结论在缅甸拉咱市调查点流行的恶性疟原虫对氯喹已普遍产生抗性,约半数对咯萘啶具有抗性,多数对哌喹则敏感。     

Polarisation microscopy increases the sensitivity of hemozoin and Plasmodium detection in the histological assessment of placental malaria

The histological study of the placenta is useful in the diagnosis of malaria during pregnancy. However, the scarcity of parasites and pigment in many malarial infections renders their identification difficult. We have tested the accuracy of standard and polarisation microscopy in the evaluation of 500 placental specimens from an area of high malarial endemicity in Tanzania. Standard microscopy showed a low sensitivity (50.3% for parasites, 40.5% for pigment), due to poor detection rates in cases with scant parasites (12.7% for <1%; 97.8% for >5% parasitised erythrocytes, P<0.001) or minimal pigment deposition (42.4% versus 84.5% when severe, P<0.001). The use of polarisation microscopy significantly increased the sensitivity of detection of pigment to 100% and parasites to 98.1% because of the marked birefringence of hemozoin present in mature stage parasites which accumulate in the placenta. Formalin pigment shares many properties with hemozoin, but the use of neutral buffered formalin prevented the formation of formalin pigment in placentas even after long periods of fixation. In conclusion, polarisation microscopy is a simple tool that markedly increases the sensitivity of the detection of malaria infection in the placenta and has good specificity when used on tissues fixed in neutral formalin. This method can be useful to investigators working in the malaria field.     

缅甸拉咱市氯喹治疗间日疟的敏感性评价

缅甸拉咱市单纯间日疟原虫感染者48例分为氯喹A方案组（26例）和B方案组（22例）,分别采用成人总剂量1 200 mg （第1天顿服600 mg,第2、3天300 mg/d）和1 500 mg（第1天顿服750 mg,第2、3天375 mg/d）的三天疗法治疗。于服药当天、服药后第1、2、3、7、14、21和28天采集指血或耳垂血制作厚、薄血涂片检查疟原虫,测量体温和观察药物不良反应。氯喹治疗后,两组病例血内无性体原虫3 d内全部阴转。第28天随访治愈率为100%。结果表明,缅甸拉咱市的间日疟病例对氯喹治疗均敏感,该边境地区间日疟病例可采用氯喹进行临床治疗。