伯氏疟原虫感染早期的根治性治疗对再感染细胞免疫应答的影响

目的探讨疟疾感染早期根治性治疗对再感染细胞免疫应答的影响。方法用伯氏疟原虫感染DBA/2小鼠,感染后3d进行根治性治疗,并于初次感染后90d进行再感染。通过吉姆萨薄血膜染色法计数红细胞感染率,流式细胞术检测再感染前（0d）和再感染后（1、3、5d）不同时间点脾T细胞中活化性T细胞百分含量,ELISA检测脾细胞培养上清中IFN-γ、TNF-α、IL-4和IL-10水平。结果同源疟原虫再感染后,根治性治疗小鼠仅出现短暂的低水平虫体血症;再感染后第1～5天活化性T细胞百分率持续升高。IFN-γ于再感染后第1天即出现有意义的升高,第3天达到峰值水平,与此同时,TNF-α和IL-10水平也开始出现有意义的升高,但IL-4的升高出现在再感染后的第5天。结论疟疾感染早期的根治性治疗并不影响宿主在再感染时产生有效的细胞免疫应答,CD4＋Th1应答反应也是抵御疟疾再感染的关键因素之一。     

伯氏疟原虫CSP片段与肝细胞特异结合的生物学特性

目的明确去除伯氏疟原虫CSP基因的中央重复序列不影响该蛋白的膜定位及与肝细胞特异结合的特性。方法将去除中央重复序列的伯氏疟原虫CSP基因片段（PbCSP’）克隆入原核表达质粒pGEX一4T一1,在大肠杆菌BL21／DE3中诱导表达,纯化重组蛋白,免疫大鼠获得相应抗体;用流式细胞仪筛选表达EGFP—PbCSP’的Hela细胞,应用Confocal照微镜及免疫组化方法观察表达的蛋白是否定位于细胞膜及能否与小鼠肝癌细胞（H22）特异结合。结果pGEX—PbCSP’的原核表达及纯化、免疫大鼠获得抗体,在Hela细胞表达的EGFP—PbCSP’蛋白定位于细胞膜,并能与小鼠肝癌细胞（H22）特异结合。结论去除中央重复序列的伯氏疟原虫CSP片段与肝细胞特异结合,为进一步研究其是否可作为肝细胞的靶向分子奠定了基础。     

柏氏疟原虫红内期体外培养的初步研究

按烛缸法对柏氏疟原虫(Plasmodium berghei)红内期进行了体外培养的初步研究。先后5次,每次连续培养7天。除培养的第2天,其原虫率稍有增长外,随后便逐日下降;与此同时,培养的第4天,环状体有增多外,其后也逐日减少,而未成熟裂殖体及成熟裂殖体相对地逐日增加。培养结果提示有1～2个周期的裂体增殖。另将第7天的培养物无菌注入健康正常小白鼠腹腔内,7天后血检,原虫率明显上升(>5％),各红内期形态均可见到,还偶见到配子体,鼠间继续人工传代,原虫活力不减,实验鼠均于1周左右自然致死,反映原虫经体外培养7天后对宿主仍具有很强的毒力。     

昆明小鼠对伯氏疟原虫的易感性和组织病理研究

目的探讨昆明小鼠对伯氏疟原虫(Plasmodium berghei)的易感性及其感染后的组织病理变化。方法 6周龄昆明小鼠腹腔接种1×106个感染伯氏疟原虫的红细胞,观察小鼠的生存时间及临床症状。感染后第2天开始每天采尾静脉血制作薄血膜片,Giemsa染色,计数血虫率。感染后第4天开始经肛门测小鼠体温。小鼠临死时取脑、肺、肝和脾组织,经10%中性福尔马林固定24h以上,脱水、包埋后制成4μm厚石蜡切片,苏木素-伊红染色,显微镜下观察各组织的病理变化。结果昆明小鼠腹腔接种感染伯氏疟原虫后的生存时间为8～21d;小鼠感染后第2天外周血可检出疟原虫,小鼠临死前血虫率达高峰;肉眼观察肝、脾体积增大,颜色变深,显微镜下见大量疟色素沉着,肝实质组织可见少量灶性炎性细胞聚集;脑和肺组织见感染疟原虫红细胞沉积在微血管壁,引起脑血管堵塞、大脑皮层出血灶和肺水肿、肺泡腔内炎性细胞渗出。结论昆明小鼠对伯氏疟原虫易感性较高,脑、肺、肝和脾组织均呈现出典型的疟疾病理特征,可作为研究疟原虫致病机制的动物模型。     

Comparative study on schizontocidal activity of recrystallized or crude daphnetin against malaria parasites

Objective To compare the schizontocidal activity of recrystallized or crude daphnetin against malaria parasites in vivo. Methods Schizontocidal activity of recrystallized or crude daphnetin at various dosages was assessed in mice infected with Plasmodium berghei ANKA using a “4-day suppress assay”. Results The comparison of the reduction rate of parasitemia caused by either recrystallized or crude dephnetin showed that ED50 of crude daphnetin was 18.36 mg/kg, with 95% confidence limit of 5.96-56.54 mg/kg while ED50 of recrystallized daphnetin was 11.46 mg/kg, with 95% confidence limit of 8.63-15.22 mg/kg. Conclution The results indicate that the efficacy of recrystallized daphnetin is 37.6% higher than that of crude daphnetin.     

转基因伯氏疟原虫的建立及报告基因表达的初步研究

目的:通过转染含有绿色荧光蛋白(GFP)和PbfMSP1片段的重组质粒,建立伯氏疟原虫转染技术和表达恶性疟原虫MSP-1 19 000片段(PfMSP1-19)的转基因伯氏疟原虫.方法:构建重组转染质粒PyrFlu/PbfMSP1.伯氏疟原虫ANKA株经培养和分离后用电转化方法转入重组转染质粒,药物筛选转化原虫后进行PCR检测,并于荧光显微镜下检测报告基因GFP的表达.结果:构建了重组转染质粒PyrFlu/PbfMSP1.荧光显微镜下观察到呈绿色荧光的伯氏疟原虫.PCR检测显示转染伯氏疟原虫存在GFP和PbfMSP1基因.结论:重组质粒已成功转染伯氏疟原虫并表达了GFP报告基因,建立了伯氏疟原虫转染技术.     

Antimalarial and antiplasmodial activity of husk extract and fractions of Zea mays

Context: Zea mays L. (Poacae) husk decoctions are traditionally used in the treatment of malaria by various tribes in Nigeria.

Objective: To assess the antimalarial and antiplasmodial potentials of the husk extract and fractions on malaria parasites using in vivo and in vitro models.

Materials and methods: The ethanol husk extract and fractions (187–748?mg/kg, p.o.) of Zea mays were investigated for antimalarial activity against Plasmodium berghei using rodent (mice) malaria models and in vitro activity against chloroquine sensitive (Pf 3D7) and resistant (Pf INDO) strains of Plasmodium falciparum using the SRBR green assay method. Median lethal dose and cytotoxic activities against HeLa and HEKS cells were also carried out. The GCMS analysis of the most active fraction was carried out.

Results: The husk extract (187–748?mg/kg, p.o.) with LD₅₀ of 1874.83?mg/kg was found to exert significant (p?P. berghei infection in suppressive, prophylactive and curative tests. The crude extract and fractions also exerted prominent activity against both chloroquine sensitive (Pf 3D7) and resistant (Pf INDO) strains of P. falciparum with the ethyl acetate fraction exerting the highest activity with IC₅₀ values of 9.31?±?0.46?μg/mL (Pf 3D7) and 3.69?±?0.66?μg/mL (Pf INDO). The crude extract and fractions were not cytotoxic to the two cell lines tested with IC₅₀ values of?>100?μg/mL against both HeLa and HEKS cell lines.

Discussion and conclusion: These results suggest that the husk extract/fractions of Zea mays possesses antimalarial and antiplasmodial activities and these justify its use in ethnomedicine to treat malaria infections.     

Brine shrimp cytotoxicity and antimalarial activity of plants traditionally used in treatment of malaria in Msambweni district

Abstract

Context: In Kenya, most people use traditional medicine and medicinal plants to treat many diseases including malaria. To manage malaria, new knowledge and products are needed. Traditional herbal medicine has constituted a good basis for antimalarial lead discovery and drug development.

Objectives: To determine in vivo antimalarial activity and brine shrimp toxicity of five medicinal plants traditionally used to treat malaria in Msambweni district, Kenya.

Materials and methods: A 0.2?ml saline solution of 100?mg/kg aqueous crude extracts from five different plant parts were administered orally once a day and evaluated for their in vivo chemosuppressive effect using Plasmodium berghei berghei-infected Swiss mice for four consecutive days. Their safety was also determined using Brine shrimp lethality test: Grewia trichocarpa Hochst ex A. Rich (Tiliaceae) root, Dicrostachys cinerea (L) Wight et Am (Mimosaceae) root, Tamarindus indica L. (Caesalpiniaceae) stem bark, Azadirachta indica (L) Burn. (Meliaceae) root bark, and Acacia seyal Del. (Mimosaceae) root.

Results: Parasitaemia was as follows: A. indica, 3.1%; D. cinerea, 6.3%; T. indica, 25.1%; A. seyal, 27.8%; and G. trichocarpa, 35.8%. In terms of toxicity, A. indica root bark extract had an LC₅₀ of 285.8?µg/ml and was considered moderately toxic. T. indica stem bark extract and G. trichocarpa root extract had an LC₅₀ of 516.4 and 545.8?µg/ml, respectively, and were considered to be weakly toxic while A. seyal and D. cinerea root extracts had a LC₅₀ >1000?µg/ml and were, therefore, considered to be non-toxic.

Discussion and conclusion: All extracts had antimalarial activity that was not significant compared to chloroquine (p?≥?0.05). No extract was toxic to the arthropod invertebrate, Artemia salina L. (Artemiidae) larvae, justifying the continued use of the plant parts to treat malaria.     

Pharmacological assessment of the role of nitric oxide in mice infected with lethal and nonlethal species of malaria

This pharmacological investigation sought to determine whether nitric oxide (NO) had an antiparasitic effect and/or mediated pathology in mice infected with nonlethal P. chabaudi or lethal P. berghei. Nitric oxide synthase (NOS) inhibitors were evaluated for their ability to inhibit the rise in reactive nitrogen intermediates (RNI) induced by bacterial lipopolysaccharide (LPS) in mice. The more effective compound, aminoguanidine (AG) inhibited the rise in RNI induced by P. chabaudi and increased mortality, but had no effect on parasitaemia. Inducers and donors of NO were screened for their ability to increase RNI and the most effective agents evaluated for their ability to modify P. berghei infection. S-Nitrosoglutathione had little effect, but LPS decreased parasitaemia and mortality. An inconsistent relationship is evident between the abilities of these agents to modify NO activity and their effects on malaria in mice. Increased mortality in mice with P. chabaudi treated with AG indicates a reduction in resistance. The absence of an effect on parasitaemia by a NOS inhibitor or NO donor indicates either RNI have insignificant antimalarial action in vivo or the efficacy of the compounds is inadequade. Resistance to P. berghei in LPS-treated mice demonstrates an antiparasitic effect, but this may be attributable to factors other than NO.