我国间日疟原虫混合流行区分离株Dufry抗原结合蛋白基因多态性分析

为了解我国季节混合流行区云南省间日疟原虫红内期候选抗原Dufly抗原结合蛋白（DBP）基因多态性特点,提取19株我国云南株疟原虫基因组DNA,PCR扩增DBPRⅡ区基因片段。以SalI标准株作为参照,对所获得的基因序列进行排序分析,检测基因突变位点,以中性检验分析评估序列多态性。结果表明,中国间日疟原虫混合流行区云南分离株PvDBPRII的基因多态性与南美、中非等流行突变位点一致,未发现地区特异性的突变位点,但与国内单一流行区湖北和浙江地区突变位点存在一定差异。原因可能为整体上受到自然选择的影响,呈正向选择趋势。上述结果可为以PvDBPRⅡ为基础建立有效传播阻断疫苗的研究提供一定的线索。     

Extense variant gene family repertoire overlap in Western Amazon Plasmodium falciparum isolates

In order to find a molecular basis for observations of relatively fast developing immunity to malarial infections in the Western Amazon region, the partial var, stevor and rif gene repertoires of nine different Plasmodium falciparum isolates collected in 1985 and 2000–2004 were evaluated. In contrast to previous results from South East Asia, the variant gene repertoire in Brazilian isolates is rather small and redundant. While the individual var repertoire sizes of Brazilian strains did not differ from Southeast Asian/African isolates, we found an over three times higher overlap of var sequence repertoires in Amazonian strains which was also conserved over time, suggesting the ongoing circulation of a similar var gene repertoire. Coincidently, almost 40% of the sequences identified herein showed the highest degree of similarity to var genes from either Brazilian or Venezuelan isolates, indicating a limited var repertoire of P. falciparum in the Amazon Basin as a whole. The intrastrain similarities of var genes were slightly but significantly lower than in Southeast Asian/African samples suggesting a higher selective pressure for diversification in Amazonian isolates. Despite of higher copy numbers per genome, rif genes also showed a significant repertoire overlap. stevor genes, which share the same predominant subtelomeric localization as var and rif genes, showed a still higher repertoire overlap and were highly similar to 3D7 stevor genes, indicating stronger functional conservation than var and rif genes. This is the first study that reveals that P. falciparum variant gene repertoires of certain areas can be limited. This has important implications for the strain-specific immunity against variant antigens occurring in these areas.     

恶性疟原虫FCC1/HN株环子孢子蛋白的原核表达、纯化及其对肝细胞靶向作用的观察

目的从恶性疟原虫FCC1／HN株基因组中扩增得到环子孢子蛋白(circumsporozoite protein,CSP)的全长编码基因,克隆至原核表达载体pET-32c中进行表达纯化,观察重组环子孢子蛋白对肝细胞的结合能力,探讨其作为原发性肝癌基因治疗的靶向分子的可行性。方法根据恶性疟原虫3D7株环子孢子蛋白的编码序列设计一对引物,利用聚合酶链反应(PCR)技术从恶性疟原虫FCC1／HN株基因组中扩增出CSP的全长编码基因,将其克隆到载体pGEM-T中,通过基因测序加以证实;进一步亚克隆至原核表达载体pET-32c中,在大肠杆菌BL21中用IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,采用免疫印迹技术(WB)对纯化的融合蛋白进行免疫反应性检测;采用免疫组化(IHC)技术观察重组环子孢子蛋白对不同组织细胞的结合能力。结果从恶性疟原虫FCC1／HN株基因组中成功扩增到1263 bp的全长CSP基因,该基因在原核系统中经诱导表达出一相对分子质量(Mr)约62×103大小的融合蛋白,表达产物以包涵体的形式存在;通过Ni2+亲和柱纯化获得重组CSP融合蛋白;WB表明,重组CSP融合蛋白能被疟原虫阳性血清特异性识别;免疫组化结果显示重组CSP融合蛋白能够与肝癌和正常肝细胞特异性地结合,与其他组织来源的细胞则未见反应。结论CSP是疟原虫子孢子表面主要的蛋白,重组环子孢子蛋白作为原发性肝癌基因治疗的靶向分子具有一定的潜在应用价值。     

青蒿提取物对伯氏疟原虫超微结构的影响

采用4天抑制试验法,观察青蒿提取物和青蒿素对伯氏疟原虫超微结构的影响。结果显示,青蒿提取物和青蒿素均使原虫滋养体膜结构损伤,表现为虫体表膜、食物泡膜、限制膜、线粒体膜、内质网、核膜呈现肿胀及膜间隙增宽。有些虫体表膜、食物泡膜呈现多层螺纹膜样改变。严重病变的原虫滋养体退变崩解,结构消失,残留退变的食物泡和吞噬泡散在红细胞内。此外,还观察到青蒿提取物和青蒿素对疟原虫纳虫泡内裂殖子发育有抑制作用。试验证明,青蒿提取物和青蒿素对伯氏疟原虫滋养体的超微结构损伤部位和病变特征基本相同;并提示对疟原虫纳虫泡内裂殖子发育有一定影响。     

CXCR3 determines strain susceptibility to murine cerebral malaria by mediating T lymphocyte migration toward IFN-gamma-induced chemokines

Cerebral malaria (CM) results from the binding of infected erythrocytes and leukocytes to brain endothelia. The precise mechanisms underlying lymphocyte recruitment and activation in CM remain unclear. Therefore, the expression of various chemokines was quantified in brains of mice infected with Plasmodium berghei ANKA (PbA). Several chemokines attracting monocytes and activated T-lymphocytes were expressed at high levels. Their expression was almost completely abrogated in IFN-gamma ligand and receptor KO mice, indicating that IFN-gamma is an essential chemokine inducer in vivo. Surprisingly, the expression levels of chemokines, IFN-gamma and also adhesion molecules in the brain were not lower in CM-resistant Balb/c and DBA/2 mice compared to CM-sensitive C57BL/6 and DBA/1 mice, although T lymphocyte sequestration in the brain was significantly less in CM-resistant than in CM-sensitive mice. This difference correlated with a higher up-regulation of the CXC chemokine receptor (CXCR)-3 on splenic T cells and a higher chemotactic response to IFN-gamma-inducible protein-10 (IP-10) in C57BL/6 compared to Balb/c mice. In conclusion, parasite-induced IFN-gamma in the brain results in high local expression levels of specific chemokines for monocytes and lymphocytes. The strain-dependent susceptibility to develop CM is more related to the expression of CXCR3 in circulating leukocytes than to the chemokine expression levels in the brain.     

Probing the roles of non-homologous insertions in the N-terminal domain of Plasmodium falciparum hydroxymethylpterin pyrophosphokinase–dihydropteroate synthase

Plasmodium falciparum bifunctional hydroxymethylpterin pyrophosphokinase–dihydropteroate synthase (pfHPPK–DHPS) is a crucial enzyme in the de novo folate biosynthesis pathway. The crystal structure is not yet available for this enzyme, however, homology model of the enzyme reported previously revealed the presence of parasite-specific insertions. Alignment of pfHPPK–DHPS with HPPK and DHPS sequences from other microorganisms reveals two insertions relative to the corresponding enzyme in other organisms, i.e. HPPK-1 and HPPK-2. The former encompasses amino acid residues 66–162, while the latter covers residues 213–311. In order to investigate the roles of the two insertions, we constructed a number of mutants in which parts of these two insertions were deleted. Characterization of the mutationally altered proteins revealed that deletions of residues 74–80 in the HPPK-1 sequence of the pfHPPK–DHPS, but not that of the monofunctional pfHPPK, decreased the HPPK activity. A longer deletion (residues 74–86) in the HPPK-1 sequence of the bifunctional pfHPPK–DHPS completely inactivated both HPPK and DHPS activities. However, deletion in the HPPK-2 sequence from residues 247–306 did not disrupt the activities of HPPK and DHPS, but the kinetic properties of the recombinant proteins were slightly changed. The importance of HPPK-1 sequence on the catalytic activities of HPPK and DHPS in the bifunctional pfHPPK–DHPS could have implications for development of inhibitors targeting the non-catalytic region of this chemotherapeutically important enzyme.     

Natural killer cells and malaria

Summary:  Malaria, caused by the infection with parasites of the germs Plasmodium , is one of the three most important infectious diseases worldwide, along with tuberculosis and infection with human immunodeficiency virus. Natural killer (NK) cells are lymphocytes classically involved in the early defense against viral infections and intracytoplasmic bacterial infections and are also implicated during the course of tumor development and allogeneic transplantation. These cells display important cytotoxic activity and produce high levels of proinflammatory cytokines. In both mouse and human models of malaria, NK cells appear to be a major source of interferon-γ during the early phase of infection. In humans, indirect signaling through monocytes/macrophages required to optimally stimulate NK cell activity. However, the in vivo functions of NK cells during malaria are still enigmatic, and many issues remain to be dissected, such as the molecular basis of the direct recognition of iRBCs by NK cells.     

Natural frequency of polymorphisms linked to the chondroitin 4-sulfotransferase genes and its association with placental malaria

Our understanding about the role of the maternal genetic factors on placental malaria is scarce. The general aim of this work was to examine whether common polymorphisms of genes involved in chondroitin sulphate A (CSA) synthesis influence susceptibility to and manifestation of malaria during pregnancy. To achieve this, 96 women with placental malaria and 180 healthy controls without malaria from the province of Luanda, Angola, were genotyped using six microsatellite loci. No associations were found between polymorphisms of genes involved in CSA synthesis and placental malaria. All these findings suggest that there is no genetic susceptibility or increased risk attributed to polymorphisms of the enzymes involved on the synthesis of CSA.