Congenital toxoplasmosis--prenatal aspects of Toxoplasma gondii infection

Toxoplasma gondii (T. gondii) is the cause of toxoplasmosis. Primary infection in an immunocompetent person is usually asymptomatic. Serological surveys demonstrate that world-wide exposure to T. gondii is high (30% in US and 50–80% in Europe). Vertical transmission from a recently infected pregnant woman to her fetus may lead to congenital toxoplasmosis. The risk of such transmission increases as primary maternal infection occurs later in pregnancy. However, consequences for the fetus are more severe with transmission closer to conception. The timing of maternal primary infection is, therefore, critically linked to the clinical manifestations of the infection. Fetal infection may result in natural abortion. Often, no apparent symptoms are observed at birth and complications develop only later in life. The laboratory methods of assessing fetal risk of T. gondii infection are serology and direct tests.

Screening programs for women at childbearing age or of the newborn, as well as education of the public regarding infection prevention, proved to be cost-effective and reduce the rate of infection.

The impact of antiparasytic therapy on vertical transmission from mother to fetus is still controversial. However, specific therapy is recommended to be initiated as soon as infection is diagnosed.     

Screening for small molecule inhibitors of Toxoplasma gondii

Introduction: Toxoplasma gondii, the agent that causes toxoplasmosis, is an opportunistic parasite that infects many mammalian species. It is an obligate intracellular parasite that causes severe congenital neurological and ocular disease mostly in immunocompromised humans. The current regimen of therapy includes only a few medications that often lead to hypersensitivity and toxicity. In addition, there are no vaccines available to prevent the transmission of this agent. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed.

Areas covered: The author presents in silico and in vitro strategies that are currently used to screen for novel targets and unique chemotypes against T. gondii. Furthermore, this review highlights the screening technologies and characterization of some novel targets and new chemical entities that could be developed into highly efficacious treatments for toxoplasmosis.

Expert opinion: A number of diverse methods are being used to design inhibitors against T. gondii. These include ligand-based methods, in which drugs that have been shown to be efficacious against other Apicomplexa parasites can be repurposed to identify lead molecules against T. gondii. In addition, structure-based methods use currently available repertoire of structural information in various databases to rationally design small-molecule inhibitors of T. gondii. Whereas the screening methods have their advantages and limitations, a combination of methods is ideally suited to design small-molecule inhibitors of complex parasites such as T. gondii.     

弓形虫RH强毒株对大白鼠中枢神经系统影响的研究

目的 探讨弓形虫感染对中枢神经系统的影响.方法 对不同孕期的大鼠腹腔感染弓形虫,观察其产仔及仔鼠生长存活情况,用迷宫实验检测仔鼠学习分辨及记忆能力,并对死亡的仔鼠大脑进行病理切片. 结果 早期感染弓形虫者其仔鼠在迷宫测试中,学习分辨能力及记忆能力显著低于对照组(P＜0.001);不同孕期感染各组学习成绩超过对照组算术均数3.21天的仔鼠数中,早期感染组所产仔鼠与对照组差异有显著性(P＜0.05);早期、中期感染组所产仔鼠在学习中的电击次数超过对照组算术均数4.41次的仔鼠数与对照组差异呈显著性(P＜0.05);晚期感染组其仔鼠6～7周死亡数显著高于对照组(P＜0.01)且大脑病理切片结果显示:大脑皮质空洞,大量嗜酸性细胞增生、集聚,提示寄生虫感染.结论 不同孕期感染弓形虫均可导致胎儿的先天性感染,造成中枢神经系统的损害.     

阿齐霉素对弓形虫形态影响的研究

为评价阿齐霉素对弓形虫的抑制、杀伤效应,体外培养的猴肾细胞经弓形虫感染后,给予不同浓度阿齐霉素(0．1μg／ml、1μg／ml、10μg／m1),然后以电镜观察药物作用后的弓形虫形态、体积、超微结构的改变。结果经透射电镜观察发现：虫体在药物作用下发生崩解,细胞内留下空泡,有的呈团块,没有明显的细胞器可见。通过扫描电镜观察,由于阿齐霉素的影响,使附着于猴肾细胞表面的虫体与未加药组相比明显皱缩,从而充分反映阿齐霉素的抑制、杀伤虫体作用。     

阿齐霉素对弓形虫形态影响的研究

为评价阿齐霉素对弓形虫的抑制、杀伤效应,体外培养的猴肾细胞经弓形虫感染后,给予不同浓度阿齐霉素(0.1μg/ml、1μg/ml、10μg/ml),然后以电镜观察药物作用后的弓形虫形态、体积、超微结构的改变。结果经透射电镜观察发现:虫体在药物作用下发生崩解,细胞内留下空泡,有的呈团块,没有明显的细胞器可见。通过扫描电镜观察,由于阿齐霉素的影响,使附着于猴肾细胞表面的虫体与未加药组相比明显皱缩,从而充分反映阿齐霉素的抑制、杀伤虫体作用。     

一氧化氮在弓形虫致生精细胞损伤中的作用

目的：研究弓形虫感染后对雄性小鼠一氧化氮（nitric oxide,NO）水平的改变对睾丸生精细胞的影响。方法：选用9-10周龄雄性BALB,C小鼠60只,随机分为正常对照组、弓形虫速殖子感染组（0．5×10^3,1×10^3、2×10^3、4×10^3）。采用腹腔注射法,建立弓形虫急性感染模型,免疫组化SP法测定睾丸Bax、Bcl-2表达的变化,硝酸还原法测定睾丸NO含量。结果：感染小鼠睾丸组织的NO水平明显增高,睾丸细胞Bcl-2表达无明显改变,而生精细胞尤其是精母细胞的Bax表达明显增加。结论：弓形虫感染导致NO升高．并可能通过Bcl蛋白途径促使生精细胞,尤其是精母细胞的损伤。     

弓形虫SAG1基因在HeLa细胞表达的研究

目的 :研究弓形虫 SAG1基因在 He L a细胞中的表达 ,为研制弓形虫疫苗奠定基础。方法 :采用磷酸钙 -DNA共沉淀转化法将重组质粒 pc DNA3- SAG1导入 He L a细胞 ,用 G418进行选择培养 ,筛选出表达阳性的细胞克隆 ,再通过培养和加压 ,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株 ,分离表达产物 ,进行 SDS- PAGE、Western blot分析。结果 :采用钙沉淀法成功地将重组质粒 SAG1导入 He L a细胞 ,通过 G418选择和加压培养 ,获得稳定分泌 SAG1抗原的阳性 He L a细胞 ,表达的蛋白经 SDS- PAGE、Western blot分析 ,显示其分子量 30 k Da蛋白 ,与理论值相符。结论 :成功构建重组质粒 pc DNA3- SAG1,建立稳定分泌 SAG1抗原的阳性 He L a细胞克隆株     

珠海地区无偿献血者弓形虫感染的研究

目的了解珠海地区无偿献血者弓形虫（TOX）的血清学阳性率,探讨血清学与核酸检测的相关性。方法用ELISA捕获法对2008年珠海地区1000名无偿献血者血液标本进行抗TOX—IgM、TOX-IgG的检测,然后对血清学阳性者95名血样采用实时荧光定量聚合酶链反应（FQ—PCR）技术进行检测,分析两者的相关性。结果1000名无偿献血者中TOX—IgM阳性数1例,阳性率为0．1％、TOX—IgG阳性数为94例,阳性率为9．4％,而TOX—IgM和TOX—IgG阳性的95份标本用FQ—PCR检测结果均为阴性。结论珠海地区无偿献血者弓形虫血清学阳性率与同内其它地区相近,但经FQ—PCR检测均未发现TOX—DNA阳性,输血感染弓形虫病的可能性较小。     

弓形虫STAg免疫小鼠后血清抗体水平的动态检测

目的 观察弓形虫可溶性速殖子抗原(STAg)免疫小鼠后,小鼠血清IgG抗体水平的动态变化,探索STAg抗原的最佳免疫次数.方法 5～6周龄BALB/c小鼠20只,随机分成对照组和实验组,其中对照组头背部皮下注射生理盐水,实验组头背部皮下注射20 μg STAg,免疫5次,每次免疫1周后取5只小鼠,采血并分离血清,通过ELISA法测定IgG抗体;通过Western Blot方法检测血清中产生的IgG和IgM抗体条带.结果 第二次免疫后小鼠血清IgG值明显高于初次免疫小鼠的血清IgG值,差异有统计学意义;但是.Western Blot检测的IgG抗体条带颜色未明显增强.第四次免疫后小鼠血清IgG再次增高并达到峰值,同时第四次免疫后小鼠血清中的IgG抗体条带的反应颜色也明显变深.结论 利用STAg免疫小鼠,免疫2次就会产生明显的免疫效果,但是免疫4次可进一步有效提高小鼠的免疫力.     

Preparation of 131I-Pyrimethamine and evaluation for scintigraphy of experimentally Toxoplasma gondii-infected rats

We aimed to assess the ability of ¹³¹I-Pyrimethamine scintigraphy to detect the lesions of Toxoplasma gondii infection. An experimental model of toxoplasmosis was developed. The presence of toxoplasmosis was confirmed 60 days after implantation. Pyrimethamine was radioiodinated with I-131. The radioligand was validated by the requisite quality control tests to check its radiolabeling efficiency, in vitro stability and radiochemical purity etc. ¹³¹I-Pyrimethamine (specific activity: 7.08 MBq/µmol) was injected intravenously into the tail vein of the control and infected rats. Static whole body images of the rats were acquired under the gamma camera at 5?min, 45?min, 2?h, 6?h, and 24?h following the intravenous administration of the radioactivity (3.7 MBq/rat). Then the scintigraphic data were analyzed both visually and semiquantitatively. Regions of interest (ROIs) were drawn over the organs (thyroid, stomach, liver, bladder, and soft tissues) to calculate the ratios of the radiotracer in infected vs. control rats. The mean ratio of radiotracer in infected/control rats in the liver and diaphragm was over 1 at 45?min which persisted till 24?h. In conclusion, ¹³¹I-Pyrimethamine may be useful agent for diagnosis toxoplasmosis especially involving liver and diaphragm, needs further preclinical validation before being extended for use in clinical applications.     

Prevalence and genotyping of Toxoplasma gondii among Saudi pregnant women in Saudi Arabia

Introduction: Toxoplasma gondii (T. gondii) is an intracellular protozoan that can infect all mammals, who serve as intermediate host. It causes congenital, neurological, eyes complications and mild or asymptomatic infections in humans. Purpose of this study: To investigate not only the prevalence of T. gondii, but also to find out its genotyping using multiple sequential molecular methods to predict exactly the precise genotyping of T. gondii among Saudi pregnant women. Methods: A cross-sectional study was conducted using multi-stage methods. Initial stage involved enrolment of 250 Saudi pregnant women from multi-centre healthcare and community based settings in the capital of Saudi Arabia Riyadh. The second stage was embracement of the laboratory investigation that included Enzyme immunoassay (ELISA), DNA extraction, PCR, nested-PCR assay, and genotyping of the seropositive cases. Results: 203 women agreed to take part in our study with a response rate of 81.2% (203/250). Using ELISA, we found that the prevalence of Toxoplasma gondii IgG and IgM antibodies was 32.5% and 6.4%, respectively. We found that 29 samples (80.6%) were of genotype II; however 7 samples (19.4%) were of genotype III. Conclusion: Defining the population structure of T. gondii from Saudi Arabia has important implications for transmission, immunogenicity, pathogenesis, and in planning preventive strategies. Relationship between such variation in structure and disease manifestation in pregnant women is still difficult to assess due to the role of host immune status and genetic background on the control of infection, and of other parasitic features such as the infecting dose or parasite stage. Our finding of the genotyping of T. gondii might facilitate and inform future studies on comparative genomics and identification of genes that control important biological phenotypes including pathogenesis and transmission among Saudi women.     

Discovery of potential Toxoplasma gondii CDPK1 inhibitors with new scaffolds based on the combination of QSAR and scaffold‐hopping method with in vitro validation

To discover drugs for toxoplasmosis with less side‐effects and less probability to get drug resistance is eagerly appealed for pregnant women, infant or immunocompromised patients. In this work, using TgCDPK1 as drug target, we design a method to discover new inhibitors for CDPK1 as potential drug lead for toxoplasmosis with novel scaffolds based on the combination of 2D/3D‐QSAR and scaffold‐hopping methods. All the binding sites of the potential inhibitors were checked by docking method, and only the ones that docked to the most conserved sites of TgCDPK1, which make them have less probability to get drug resistance, were remained. As a result, 10 potential inhibitors within two new scaffolds were discovered for TgCDPK1 with experimentally verified inhibitory activities in micromole level. The discovery of these inhibitors may contribute to the drug development for toxoplasmosis. Besides, the pipeline which is composed in this work as the combination of QSAR and scaffold‐hopping is simple, easy to repeat for researchers without need of in‐depth knowledge of pharmacology to get inhibitors with novel scaffolds, which will accelerate the procedure of drug discovery and contribute to the drug repurposing study.     

香菇多糖促BALB/c小鼠腹腔巨噬细胞抗弓形虫感染的研究

目的:研究香菇多糖预处理对弓形虫感染后BALB/c小鼠巨噬细胞免疫功能的影响.方法:将45只周龄、体重相近的雌性BALB/c小鼠随机均分为3组,即空白对照(无菌生理盐水)、模型(无菌生理盐水)和香菇多糖组(0.5g·L-1).各组小鼠以每次1mL、每天1次腹腔注射给药,连续给药5 d后,第6天模型与香菇多糖组各用3×1...     

致弱RH株弓形虫抗小鼠B16黑色素瘤的实验研究

目的观察紫外线辐照致弱RH株弓形虫对小鼠体内黑色素瘤生长的抑制作用。方法1×10^7紫外线致弱RH株弓形虫速殖子经腹腔注射免疫C57BL/6J小鼠,7d后再次接种相同数量弓形虫,并在接种当天给小鼠荷瘤,在荷瘤后21d处死小鼠,测定小鼠肿瘤体积与质量,并检测脾细胞T细胞亚群和淋巴细胞杀伤活性。结果致弱弓形虫能够显著抑制肿瘤生长,实验组小鼠肿瘤体积与质量显著小于对照组（P〈0．05）,抑瘤率为52．09％。实验组小鼠脾细胞CD3^＋、CD4^＋、CD8^＋、CD4^＋／CD8^＋、NK细胞杀伤活性显著高于对照组（P〈0．05）。结论致弱免疫弓形虫可以抑制小鼠体内黑色素瘤生长。     

Chinese 1基因型弓形虫棒状体蛋白 ROP16的基因克隆表达及生物信息学分析

目的：构建弓形虫Chinese 1基因型TgCtWh3（以后均简称Wh3）株棒状体蛋白（ rhoptry protein ,ROPs）16的原核和真核重组表达质粒及其3D结构。方法参照ROP16序列分别设计引物,采用PCR从弓形虫Chinese 1基因型Wh3株基因组DNA中扩增出编码ROP16的基因片段,克隆至pMD18-T载体;经PCR及测序分析鉴定;阳性克隆的质粒分别亚克隆至原核表达载体pET-28a和真核表达载体pEGFP-C2,分别转化大肠杆菌BL21和DH5α,PCR和酶切鉴定转化菌落插入的序列;将构建的原核表达菌株经IPTG诱导,SDS-PAGE和免疫印迹分析融合蛋白的表达;将构建的真核重组质粒经脂质体转染293T细胞,观察其在细胞中表达;采用生物信息学方法分析构建了蛋白的3 D结构。结果各组均PCR扩增出约2．1 kb ROP16基因的特异片段,序列检测结果均正确;分别亚克隆到原核表达载体pET-28a和真核表达载体pEGFP-C2中,成功构建了Chinese 1基因型弓形虫棒状体蛋白ROP16的原核表达质粒和真核表达质粒;原核表达质粒在大肠杆菌中表达了ROP16的融合蛋白;真核表达质粒在293T细胞中成功表达,成功构建出ROP16基因的3D结构图。结论以pET-28a和pEGFP-C2为载体,分别成功构建并表达了ROP16的原核和真核重组质粒,并构建出其3D结构图。     

Approaches for Designing new Potent Inhibitors of Farnesyl Pyrophosphate Synthase

Abstract

Introduction: Farnesyl pyrophosphate synthase (FPPS) catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give rise to one molecule of geranyl diphosphate, which on a further reaction with another molecule of isopentenyl diphosphate forms the 15-carbon isoprenoid farnesyl diphosphate. This molecule is the obliged precursor for the biosynthesis of sterols, ubiquinones, dolichols, heme A, and prenylated proteins. The blockade of FPPS prevents the synthesis of farnesyl diphosphate and the downstream essential products. Due to its crucial role in isoprenoid biosynthesis, this enzyme has been winnowed as a molecular target for the treatment of different bone disorders and to control parasitic diseases, particularly, those produced by trypanosomatids and Apicomplexan parasites.

Areas covered: This article discusses some relevant structural features of farnesyl pyrophosphate synthase. It also discusses the precise mode of action of relevant modulators, including both bisphosphonate and non-bisphosphonate inhibitors and the recent advances made in the development of effective inhibitors of the enzymatic activity of this target enzyme.

Expert opinion: Notwithstanding their lack of drug-like character, bisphosphonates are still the most advantageous class of inhibitors of the enzymatic activity of farnesyl pyrophosphate synthase. The poor drug-like character is largely compensated by the high affinity of the bisphosphonate moiety by bone mineral hydroxyapatite in humans. Several bisphosphonates are currently in use for the treatment of a variety of bone disorders. Currently, the great prospects that bisphosphonates behave as antiparasitic agents is due to their accumulation in acidocalcisomes, organelles with equivalent composition to bone mineral, hence facilitating their antiparasitic action.     

SAR Studies of 5-Aminopyrazole-4-carboxamide Analogues as Potent and Selective Inhibitors of Toxoplasma gondii CDPK1

相似文献   

利用弓形虫病人血清筛选弓形虫速殖子特异性抗原

目的筛选能引起人体免疫反应的弓形虫抗原标记。方法利用实验室保存的9份弓形虫病人血清标准品、6份阴性血清标准品,以及2份弓形虫病人血清国际标准品作为一抗,分别与刚地弓形虫（Toxoplasma gondii）RH株的可溶性速殖子抗原（soluble tachyzoite antigen,STAg）和排泄-分泌抗原（excreted/secreted antigens,ESA）进行免疫印迹反应。结果发现STAg中有10条蛋白带与所用阳性血清均发生免疫反应;ESA中有4条蛋白带能与所有阳性血清发生免疫反应,这些蛋白与阴性血清均不发生免疫反应。其中分子量为30、35、49、50、54kDa的STAg抗原以及分子量为23、24、28kDa的ESA抗原能与所用阳性血清发生较强免疫反应。结论本实验结果将有助于寻找在弓形虫病人体内能够引起免疫反应的弓形虫诊断抗原和免疫保护性抗原,为弓形虫诊断试剂以及疫苗开发奠定初步基础。     

Selective anti-Toxoplasma gondii activities of azasterols

We report potent and selective inhibitory effects of 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, known inhibitors of Δ²⁴⁽²⁵⁾-sterol methyltranferase (SMT) in fungi and protozoa, on the proliferation of Toxoplasma gondii in LLCMK2 cells. These compounds produced a dose-dependent reduction in parasite proliferation. 22,26-azasterol had an IC₅₀ of 5.3 μM after 24 h and 4.5 μM after 48 h, while for 24,25-(R,S)-epiminolanosterol the IC₅₀ values were 1 μM after 24 h and 0.12 μM after 48 h. The rapid reduction of parasite load suggested these compounds have selective cytotoxic effects against T. gondii. However, we were unable to detect 24-alkyl sterols in purified T. gondii tachyzoites using highly sensitive gas–liquid chromatography/mass spectrometry methods, a fact which indicated that the anti-proliferative effects of these azasterols were not mediated by inhibition of SMT. Transmission electron microscopy showed that the mitochondrion was the major target of drugs. Ultrastructural effects on plasma membrane, apicoplast and the formation of autophagosomal structures were also observed.