Staurosporine-Induced Cell Death in Trypanosoma brucei and the Role of Endonuclease G during Apoptosis

Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.

Evaluation of the TbgI_(2) and TbgI_(17) Tandem Repeat Antigens as Potential Antigens for the Diagnosis of Trypanosoma brucei rhodesiense

Human African trypanosomiasis (HAT) affects up to half a million people every year in sub-Saharan Africa. Interruption of transmission of the disease by early diagnosis and treatment is core to the control and eventual elimination of HAT. The routine diagnostic method for HAT is light microscopy of blood samples. The present study sought to evaluate the potential of TbgI2 and TbgI17 tandem repeat antigens as candidates for the diagnosis of Trypanosoma brucei rhodesiense. The expressed proteins were purified and the antigenic reactivity evaluation was done using multiplex assay using sera obtained from HAT patients. Receiver operating characteristic analysis showed that recombinant antigen, TbgI2 had high sensitivity for sera from patients infected with T. b. rhodesiense with the area under the curve being 0.577 and a sensitivity of 0.641 and specificity 0.650. The results suggest that TbgI2 is a potential biomarker for T. b. rhodesiense HAT serodiagnostic tests.

Anti-trypanosomal Activity of Bufonidae(Toad)Venom Crude Extract on Trypanosoma brucei brucei in Swiss Mice

Trypanosomiasis afflicts about 6~7 million people globally and to a large extent impedes livestock production in Africa.Naturally,trypanosomal parasites undergo genetic mutation and have developed resistance over a wide range of therapies.The utilization of animals and plants products has presented therapeutic potential for identifying novel anti-trypanosomal drugs.This study evaluated toad venom for anti-trypanosomal potency in-vivo in Swiss mice.Toads were collected from July to August 2019.The acute oral toxicity and biochemical characterization of the toad venom were determined.The experimental mice were administered various doses(130 mg/kg,173 mg/kg and 217 mg/kg)of the toad venom crude extract and 0.75 mg/mL of Diamizan Plus standard drug for the treatment of trypanosomiasis,once daily for 3 days.The in-vivo anti-trypanosomal activity was evaluated by a curative test,after infecting the mice with Trypanosoma brucei brucei.The pre-patent period was 72 hours before treatment commenced.The overall results showed that trypanosomal load was highest in the control group while the group treated with Diamizan drug had the least trypanosomal load.As such,the mean trypanosomal load in relation to treatments showed a very high significant difference(P<0.05).Also,the mean trypanosomal load in Swiss mice in relation to the highest dosage of toad venom versus Diamizan drug showed a very high significant difference(P<0.05).The mean change in relation to the haematological parameters across treatments groups varied significantly(P<0.05)with the exception of Hb which showed no significant difference(P>0.05)across treatment groups.The over 50%reduction in the trypanosomal load in the 130 mg/kg group in comparison with the control group brings to bare the anti-trypanosomal potency of the toad venom.The anti-trypanosomal activity demonstrated by the toad venom has provided basis for development of new therapeutic agents from different toad species.The study recommends further studies(both in-vivo and in-vitro)followed by the characterization of the active compounds present in the toad venom responsible for the anti-tyrpanosomal activity observed alongside the management and conservation of these species.

Effect of diminazene aceturate,levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei

Objective:To investigate the effect of diminazene aceturale(DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei.Methods:Thirty adult male albino rats,randomly assigned into 6 groups(A—F) of 5rats each were used.They were either infected with 1×10~a trypanosomes intraperitoneally(groups A-E) or uninfected(group F).The different groups were treated respectively as follows:group A-with 3.5 mg/kg DA;group B-3.5 mg/kg DA and 7.5 mg/kg levamisole;group C-3.S mg/kg DA and 100 mg/kg vitamin C;and group D-3.S mg/kg DA and 7.S mg/kg levamisole and 100 mg/kg vitamin C.Croup E was left untreated.Parameters assessed include:rectal temperature,body weight changes,packed cell volume(PCV),Haemoglobin concentration(Hb),total leucocyte count(TLC) differential leucocyte count(DLC),parasitaemia,clinical signs and survivability.Results:Average pre-patent period of 5 days was recorded.Parasites in the blood were cleared in all treated groups(A-D) within 48 hours post treatment(PT).Untreated rats in group E died between25 and 32 days post infection(PI).Relapse was not recorded in all the treated groups(A-D).The initial reduction in PCV,Hb,TLC and increases in rectal temperature following infection were reversed by the treatments.The rats that received drug combinations(groups B,C and D)showed faster and higher recovery rates than the uninfected control and group A.Conclusions:Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei.

Anti-trypanosomal effect of Peristrophe bicalyculata extract on Trypanosoma brucei brucei-infected rats

Objective:To investigate thein vitroandin vivoeffect of whole plant extracts ofPeristrophe bicalyculataonTrypanosoma brucei brucei-infected rats.Methods:The experiment wasdivided into two phases:In the first phase,the anti-trypanosomal activity of the hot water,cold water,methanol and butanol extracts of the whole plant were determined by incubatingwithTrypanosoma brucei brucei.The cold water extract was partially-purified and the anti-trypanosomal activity of the fractions determined.In the second phase,Trypanosoma brucei brucei-infected rats were treated with fraction 2c for nine days.Packed cell volume(PCV),highdensity lipoprotein(HDL),low density lipoprotein(LDL),total cholesterol(TC),triacylglycerol(TAG),aspartate aminotransferase,alanine aminotransferases(ALT),alkaline phosphatase(ALP),total and direct bilirubin levels were determined at the end of the experiment.Results:Cold water extract immobilized 90%of the parasites after 60 min of incubation,and fraction 2ccompletely immobilized the parasites after 35 min.It significantly increased PCV inTrypanosoma brucei brucei-infected rats.Decreased TC,TAG,HDL and LDL levels of infected rats increasedsignificantly when rats were treated with the fraction,while elevated levels of total bilirubinand ALT also decreased.The difference in urea,direct bilirubin and ALP was not significantwhen infected rats were compared to rats in other groups.Conclusions:The ability of the plantto ameliorate the infection-induced biochemical changes calls for detailed investigation of thepotentials of the plant for antitrypanosomiasis drug delivery.

Experimental Trypanosoma brucei infection at immediate post partum period:effects on dam and the offspring

Objective:To investigate the effects of immediate post-partum infection with Trypanosoma brucei（T.brucei） on dam and offspring.Methods:Sixty female Albino rats（Rattus norvegicus） weighing between 130-170 g were used as animal model.The animals were divided as follows: 25 infected between 1-5 days post partum;10 infected unbred as positive controls;and 25 uninfected as negative controls.The following parameters were evaluated:packed cell volume （PCV）,level of parasitaemia,survival time,litter size and litter weight at birth and on days 7, 14 and 21 post delivery,using conventional methods.Possible trans-mammary transmission of infection to litter through milk was also assessed.Results:The results showed a comparatively （P【0.05） higher mean PCV value for the uninfected negative control on the 8 day post infection compared with the infected groups which corresponded with the increasing level of parasitaemia in the two infected groups.Mean litter size and litter weights were higher（P【0.05） in the uninfected controls on the 21st day.Survival time in the infected groups were similar.No evidence of trans-mammary transfer of infection was recorded.Conclusion:T.brucei infection during immediate post partum period is detrimental to the dam and impairs growth of the offspring.

Trypanosoma brucei rhodesiense infection in a Chinese traveler returning from the Serengeti National Park in Tanzania

Background:Human African trypanosomiasis(HAT)is one of the most complex parasitic diseases known to humankind.It usually occurs in endemic areas in Africa,but is occasionally detected in returning travelers and migrants in non-endemic countries.Case presentation:In August 2017,a case of HAT was diagnosed in China in a traveler returning from the Masai Mara area in Kenya and the Serengeti area in Tanzania.The traveler visited Africa from 23 July to 5 August,2017.Upon return to China,she developed a fever(on 8 August),and Trypanosoma brucei rhodesiense infection was confirmed by laboratory tests(on 14 August)including observation of parasites in blood films and by polymerase chain reaction.She was treated with pentamidine followed by suramin,and recovered 1 month later.Conclusions:This is the first imported rhodesiense HAT case reported in China.This case alerts clinical and public health workers to be aware of HAT in travelers,and expatriates and migrants who have visited at-risk areas in Africa.

Classical clinical signs in rats experimemtally infected with Trypanosoma brucei

Objective:To investigate clinical signs in Trypanosoma brucei infection in albino rats.Methods:Fourteen rats grouped into 2 with 7 rats in each group were used to determine classical clinical manifestation of Trypanosoma brucei infection in rats.Group A rats were uninfected control and Group B rats were infected with Trypanosoma brucei.Results:Parasitaemia was recorded in Group B by(3.86±0.34)d and the peak of parasitaemia was observed at Day 5 post infection.Classical signs observed included squint eyes,raised whiskers,lethargy,no weight loss,pyrexia,isolation from the other rats,and starry hair coat.Conclusions:These signs could be diagnostic or aid in diagnosis of Trypanosoma brucei infection in rats.

Changes in blood sugar levels of rats experimentally infected with Trypanosoma brucei and treated with imidocarb dipropionate and diminazene aceturate

Objective:To determine the effect of Trypanosoma brucei(T.brucei)on blood sugar level of infected rats.Methods:The experiment was done with 42 albino rats grouped into 3 groups of 14 members each.Group A was uninfected(control group),Group B was infected with T.brucei and treated with diminazene aceturate,and Group C was infected with T.brucei and treated with imidocarb dipropionate.Blood samples were collected from the media canthus of the experimental rats on Days 0,1,2,3,4,5,6,7,8,9,10 for the assessment of change in blood sugar levels.The blood sugar levels were determined with a glucometer(Accu-chek active serial No.GN:10023338).Results:By 4 to 5 days post infection,there was a significant increase(P<0.05)in the blood sugar of Group B and Group C.By Day 6 post infection(Day 2 post treatment),no significant difference(P>0.05)was observed in the groups when compared with the control group till Day 12 of the experiment.Conclusions:T.brucei caused a significant increase in blood sugar of infected rats.

MRI Signal Abnormalities of the Optic Tracts, a Marker of Meningoencephalitis Caused by Trypanosoma Gambiense (TG)—A Delayed Patho-Radlological Correlation

Parasitic meningoencephalitis presents several etiologies which sometimes depend on their geographical location. They require thorough blood and cerebrospinal fluid check-up for directing an efficient treatment. Clinicians and radiologists are constantly looking for specific signs that could point to a particular etiology. The meningoencephalitis caused by Human African Trypanosomiasis (HAT) due to Trypanosoma brucei gambiense (TG) is a rare disease characterized by a slow progression, over years sometimes. Its non-specific presentation either clinically or in imaging can lead to misdiagnosis and thus, delay the treatment. However, involvement of the optic tracts seems to be characteristic of this condition, on old data from animal experimentation and recent high-field MRI data. MRI is the best current technique to explore the brain, cranial nerves, and visual pathways. In this article, we are going to present two observations of meningoencephalitis caused by HAT and then discuss some specific aspects of this neglected and re-emerging disease.

伊氏锥虫、马媾疫锥虫、布氏锥虫、刚果锥虫的18S rDNA部分序列测定与系统发育关系

对伊氏锥虫(Trypanosomaevansi):新疆株(XJCA)、湖北株(HBM)、云南株(YNB)、广东株(GDB2);马媾疫锥虫(Trypanosomaequiperdum)、布氏锥虫(Trypanosomabrucei)、刚果锥虫(Trypanosomacongolense)提取基因组DNA,根据已报道的伊氏锥虫株18SrDNA基因序列设计合成引物,用PCR扩增了锥虫虫株基因组DNA,伊氏锥虫新疆株、湖北株、云南株、广东株、布氏锥虫、刚果锥虫均为373bp的片段;马媾疫锥虫为372bp的片段,PCR产物经电泳鉴定后用试剂盒回收纯化,纯化后PCR产物经连接、转化后测序,将测得的序列用DNAMAN软件分析并与国外已发表的相应序列进行了同源性比较,并绘制了系统发育进化树。结果与国外AJ009153、AJ223564、D89527株同源性达到99%～100%,与另外11株同源性75%。本研究为锥虫分子流行病学研究及分类研究打下基础。

首例输入性布氏罗得西亚锥虫病病例分析

目的分析和总结首例输入性布氏罗得西亚锥虫病病例的诊断、治疗过程,为输入性锥虫病的防治提供科学依据。方法应用流行病学方法调查病例的流行病学史,吉氏染色显微镜下进行虫体观察,结合临床表现和其他辅助检查,进行诊断和治疗。结果发热病人的外周血涂片检出锥虫,经基因扩增和测序,为布氏罗得西亚锥虫,结合流行病学史和临床表现,诊断为布氏罗得西亚非洲锥虫病;脑脊液检查未检出锥虫,且白细胞≤5mm3,判定为病程第一阶段,采用苏拉明抗锥虫治疗;疗程结束病人症状体征消失,多次复查血液,均未检出锥虫,痊愈出院。结论该例病例为我国首例输入性布氏罗得西亚锥虫病,应加强输入性寄生虫病的防控。

伊氏锥虫同工酶、蛋白质和抗原组分的比较研究

本文采用生化技术对八个中国伊氏锥虫株及一个布氏锥虫株的同工酶、蛋白质和抗原组分进行比较研究。根据同工酶电泳结果,可将伊氏锥虫和与其形态上不能区分的布氏锥虫区别开来,亦可将伊氏锥虫分为两个酶株群(Z1、Z2)。根据SDS-聚丙烯酰胺凝胶电泳、等电聚焦电泳和免疫印迹试验的结果,可将酶株群Z1分成五个不同多肽群(株)。本研究结果表明,中国伊氏锥虫遗传变异程度较低,是一个相对稳定的种群。

应用质粒PTK探针鉴定锥虫的初步研究

用^(32)P标记质粒探针PTK_1、PTK_(1.1)和PTK_(1·2),对12株中国伊氏锥虫的斑点杂交试验显示,3个探针均能与8株具有正常动基体的伊氏锥虫杂交,而不与其余4株异常动基体伊氏锥虫杂交,对正常动基体株的敏感度为10~2虫体。探针PTK_1亦能与马媾疫锥虫杂交,敏感度为10~2个虫体。但PTK_1与布氏锥虫仅发生微弱的杂交反应,敏感度为10~5个虫体。试验表明伊氏锥虫株之间的kDNA微环是同源的,伊氏锥虫与马媾疫锥虫和布氏锥虫的kDNA微环存在着共同序列。

锥虫长期超低温保藏方法的研究

作者观测了不同保护剂、稀释液、pH值、降温方法、复苏温度、冻融次数、液相气相交替以及解冻后在普通冰瓶中存放时间对伊氏锥虫浙江虫株的感染性及致病力的影响。在此试验基础上,又对伊氏锥虫的6个不同虫株、媾疫锥虫、刚果锥虫、布氏锥虫等4个种的9个虫株,进行了超低温保藏试验和长期保藏效果观察。已测定的有效保藏期伊氏锥虫达574～3200天、媾疫锥虫达2866天、刚果锥虫达763天、布氏锥虫达783天。通过反复试验,提出了锥虫的常规保藏方法。

布氏锥虫C型凝集素类似蛋白的鉴定

C型凝集素在机体免疫系统中具有重要的作用。为了解布氏锥虫(T.brucei)基因组编码的C型凝集素类似蛋白(CLLPs),本研究在GeneDB数据库中通过软件鉴定了13种T.brucei基因组编码的CLLPs家族基因序列,并分析它们之间的遗传进化关系。同时,采用RT-PCR方法检测了其中3种CLLPs基因在血液阶段(BSF)T.brucei虫体内的转录。利用PCR扩增并在大肠杆菌中对所选3种基因进行表达,以表达的重组蛋白免疫小鼠制备多克隆抗体。实验结果表明,本研究选择的3种CLLPs基因在BSF T.brucei虫体内均能够进行转录;而通过间接免疫荧光检测表明所选的3种CLLPs基因中,只有一种CLLP(Tb5290)在T.brucei的BSF阶段和昆虫阶段(PCF)表达。本研究首次在T.brucei中鉴定了C型凝集素家族的新成员,为进一步了解CLLPs在T.brucei中的生物学功能的研究奠定了基础。

抗人血清的伊氏锥虫选育

初步研究了伊氏锥虫（Ｔ．ｂ．ｅｖａｎｓｉ）对人血清敏感性的变异。以不断递增的人血清处理在免疫抑制鼠体内连续传代的伊氏锥虫，经２０代的压力选择，获得了能耐受０．５ｍＬ正常人血清的抗性变异克隆。该变异克隆在无人血清压力的正常鼠体内经１０次连续传代，又恢复了对人血清的敏感性。结果表明，伊氏锥虫对人血清的敏感性或抗性均不稳定。

布氏锥虫亮氨酰-tRNA合成酶的克隆表达纯化及活性测定

目的克隆布氏锥虫亮氨酸-tRNA合成酶基因,并进行表达纯化及活性测定。方法通过PCR方法从布氏锥虫细胞基因组中扩增出亮氨酰-tRNA合成酶(LeuRS)基因,依次克隆入pBS-T克隆载体及pET21a(+)表达载体,在大肠杆菌BL21(DE3)RIPL中通过条件优化后表达。表达产物用His-Bind亲和色谱纯化,并用免疫印迹进行鉴定。采用放射性同位素方法进行酶活性测定。结果PCR扩增得到3.2 kb的DNA片段,重组质粒pET21a(+)/LeuRS经酶切鉴定和测序分析,表明构建正确。表达的重组蛋白占菌体可溶性蛋白的20%,纯化后蛋白纯度达85%,免疫印迹进行鉴定蛋白正确,酶活性测定计算出提纯物酶活性约为72 U/mL。结论已成功克隆表达纯化出布氏锥虫亮氨酸-tRNA合成酶,并用放射性同位素方法进行了酶活性测定,这为下一步进行布氏锥虫亮氨酰-tRNA合成酶抑制物的设计和体外筛选奠定了良好的基础。

锥虫早老素蛋白亲水区的克隆和表达纯化

目的体外表达锥虫早老素蛋白亲水区肽段,以制备抗血清用于功能研究。方法根据锥虫早老素蛋白二级结构特性,设计引物分别扩增N端及C端片段的亲水区肽段基因,装入原核表达载体进行表达,并通过变性纯化方法获得足够量表达蛋白,制备兔抗血清。结果成功扩增并克隆锥虫早老素蛋白亲水区片段L2及L7,并分别采用变性磁珠法和变性树脂法进行大量蛋白产物纯化,浓缩纯化产物制备兔抗血清经Western blot杂交验证,出现目的蛋白大小阳性条带。结论成功表达锥虫早老素蛋推测N端及C端亲水肽段,并成功制备抗血清,可用于锥虫早老素蛋白功能分析。

利用MGE-PCR技术研究锥虫亚属种间与亚种间的分子特性

目的分析锥虫亚属布氏锥虫、伊氏锥虫和马媾疫锥虫3个种以及布氏锥虫:指名亚种、冈比亚亚种、罗得西亚3个亚种间的遗传变异与进化关系。方法利用移动遗传因子(MGE)-PCR技术对26个锥虫虫株基因组进行扩增,同时利用邻位相连(NJ)法对扩增产物进行聚类分析。结果锥虫亚属的种间及亚种间的遗传差异明显,遗传距离在0到100%间均有分布(平均遗传距离为41.2%);布氏锥虫种内遗传变异度最大,遗传相似系数只有41.15%,伊氏锥虫和布氏锥虫的同源性最高,相似系数为62.94%;布氏锥虫指名亚种和罗得西亚亚种明显高于冈比亚亚种的遗传变异程度。结论锥虫亚属的种及亚种间的遗传变异程度较高;中国与南美洲的伊氏锥虫及中国与南美的马媾疫锥虫均应为同一起源。