国外利什曼原虫病免疫学及免疫诊断研究近况

利什曼原虫病免疫学一利什曼原虫的多种型性利什曼原虫的特征是在其动基体的细胞器中有‘核外’的脱氧核糖核酸。这种原虫种型繁多,均寄生在不同脊椎动物宿主的细胞内部,而这些细胞主要是属于单核吞唁细胞系统。这种原虫在动物宿主细胞内繁殖为无鞭毛的圆形小体,称无鞭毛体(amastigoles)并在媒介昆虫——白蛉的肠壁内发育为单鞭毛体,称前鞭毛体(Promastigatas)。     

内蒙古额济纳旗大沙鼠和蜥蜴体内的利什曼原虫及其媒介的调查研究

现场调查结果表明:内蒙古额济纳旗大沙鼠和蜥蜴均自然感染了利什曼原虫;感染原虫的大沙鼠耳组织无溃疡;该原虫虫体比其它利什曼原虫大,对小鼠不致病。但能在黑线仓鼠睾丸的塞氏细胞内繁殖;蒙古白蛉和安氏白蛉是大沙鼠利什曼原虫的主要传播媒介,微小白蛉新疆亚种为蜥蜴利什曼原虫的媒介;未发现当地居民有皮肤利什曼病,提示大沙鼠利什曼原虫对人类无致病作用。     

利什曼原虫的感染及免疫

<正> 利什曼原虫无鞭毛体寄生于人体巨噬细胞中,有效地逃避了机体免疫系统对它的作用,引起人的利什曼病。长期以来,学者们对利什曼原虫侵入人体后,原虫与人体的相互作用,特别是与机体免疫系统的相互作用,进行了大量的研究和观察,1975年Zuckerman 曾对此做过详细的综述。然而,到目前为止,对利什曼原虫寄生后,引起的一系列免疫系统的特殊反应,以及利什曼原虫免疫逃避机理,还没有一个十分完     

杜氏利什曼原虫39KD抗原基因同源性分析

目的：确定杜氏利什曼原虫39KD抗原基因的基因分析与同源性。方法：以杜氏利什曼原虫39KD抗原基因为探针,与不同种株的利什曼原虫进行Southem杂交,确定不同种株的利什曼原虫该基因的变化,并将该基因序列测定后,进行DNA序列数据库检索,分析其同源性,并进行数据库登记。结果：杜氏利什曼原虫不同地域株及婴儿利什曼原虫基因组中均含有该基因,并且无明显定位变化,整个基因在DNA数据库中无完全DNA同源序列,在GenBanK中的序列号为3280。结论：该抗原基因是利什曼原虫保守基因。     

深圳市1例黑热病病例实验室检测与分子鉴定

目的 对深圳市1例输入性黑热病病例进行实验室检测和分子溯源分析以确定感染虫株。方法 收集2023年3月15日深圳市确诊1例黑热病病例的骨髓穿刺液和血液进行实验室检测。对患者骨髓穿刺液涂片姬姆萨染色后进行显微镜检查,对血液样品采用内脏利什曼原虫快速诊断试剂（rk39）进行血清抗体检查,并提取全血DNA,PCR扩增内转录间隔区Ⅰ(internal transcribed spacer-1, ITS-1)序列并测序比对,同时基于ITS-1序列构建系统进化树。结果 对患者骨髓涂片显微镜检查查见大量利什曼原虫无鞭毛体,确诊为黑热病,患者血液采用rk39快速诊断试剂检测结果呈阳性,PCR扩增出ITS-1基因产物序列与预期大小一致,经NCBI数据库中比对,与婴儿利什曼原虫ITS-1基因序列相似度为100%,确定感染虫株为婴儿利什曼原虫。对扩增的ITS-1序列进行系统发育树构建发现与婴儿利什曼原虫聚到一个分支,且与所选的参比序列中的KC347299距离较近。结论 深圳市1例黑热病病例是由婴儿利什曼原虫引起的,黑热病在我国仍时有发生,应加强非疫区医务人员诊断技术,积极配合使用新的诊断技术进行辅助诊断,同...     

杜氏利什曼原虫前鞭毛体的扫描电镜观察

杜氏利什曼原虫前鞭毛体的透射电镜观察较多,而用扫描电镜观察其表面结构及分裂过程的报道甚少,为了研究前鞭毛体表面结构与取食、活动的关系及其分裂繁殖特点,本文用扫描电镜对离体培养的前鞭毛体进行初步观察。 材料与方法 一、虫种:选用体外培养的杜氏利什曼原虫前鞭毛体,培养基为MEM(pH 7.4),培养前加15％小牛     

复染杜氏利什曼原虫前鞭毛体简便法

复染杜氏利什曼原虫前鞭毛体简便法栾希英，周吉礼，刘春会（人体寄生虫学教研室，２５６６０３）杜氏利什曼原虫前鞭毛体标本通常以吉氏染液染色，长期使用虫体往往褪色，失去可辨认的形态特点。近年来，笔者对部分褪色待弃的前鞭毛体玻片标本，改换染色方法，进行复染处...     

人ⅡA型磷脂酶A2的cDNA顺序与婴儿利什曼原虫DNA相似性分析

目的：了解人ⅡA型磷脂酶A2cDNA顺序和婴儿利什曼原虫DNA相似性的情况和原因。方法：利用BLAST工具,以人ⅡA型磷脂酶cDNA顺序中的片段对核酸数据库进行搜寻和比对,将获得的一致性片段进行收集、整理并分析、汇总利什曼原虫DNA的相关信息。结果：发现人ⅡA型磷脂酶A2cDNA与婴儿利什曼原虫DNA之间具有9个完全一致序列（≥16bp）,这些序列集中分布于利什曼原虫36条染色体的29、30号染色体上。结论：人ⅡA型磷脂酶cDNA顺序与婴儿利什曼原虫DNA片段之间具有一致性,可能与基因由原虫向人的横向传递有关。     

墨西哥利什曼原虫无鞭毛体超微结构研究

从感染墨西哥利什曼原虫的仓鼠的肿大趾跖部位,切取病变组织,电镜观察原虫形态。无鞭毛体平均长度3.64μm。表膜分内外两层。膜下微管中心间距55.2nm。膜下微管总数约100～110根。鞭毛袋内藏鞭毛。鞭毛从基体发出。基体后部鞭毛公式为“9×2+O”,前部为“9×2+2”。动基体腊肠状。内腔中有高度弯曲的DNA纤丝。动基体有时与线粒体相连,表明前者具线粒体功能。墨西哥利什曼原虫的超微结构,与其他利什曼原虫相似。但原虫长度、膜下微管中心间距及膜下微管数有一定区别,可能为利什曼原虫的虫种鉴定提供线索。     

抗Mac-1单克隆抗体和细胞因子对利什曼原虫入侵和细胞内生存的抑制作用

目的：探索细胞内利什曼原虫的入侵、生存机制,为研制阻断利什曼原虫与巨噬细胞结合的新型抗利什曼药物以及利什曼病免疫预防和免疫治疗的研究提供理论基础。方法：采用抗Ｍａｃ １单克隆抗体（Ｍ１／７０和Ｍ１８／２）和重组的ＩＦＮ γ,ＴＮＦ α和ＩＬ ３处理巨噬细胞,观察经上述因子处理后的巨噬细胞对杜氏利什曼原虫前鞭毛体入侵和无鞭毛体在细胞内生存的影响。结果：经Ｍ１／７０和Ｍ１８／２单抗处理后巨噬细胞对杜氏利什曼原虫的易感性明显降低,其原虫感染率和受染巨噬细胞内入侵的原虫数量减低,原虫对巨噬细胞的入侵过程和速度也减慢。Ｍ１／７０和Ｍ１８／２两种单抗同时应用,则对原虫入侵巨噬细胞的抑制作用更为显著。通过对实验中所用的三种细胞因子活化巨噬细胞能力的观察发现,重组的ＩＦＮ γ或ＴＮＦ α均可激活巨噬细胞,提高巨噬细胞的杀原虫活性,且上述两种细胞因子联合对巨噬细胞的活化具有协同增强作用。但重组的ＩＬ ３不仅不能激活巨噬细胞而且尚抑制ＩＦＮ γ对巨噬细胞的活化作用。通过测定活化巨噬细胞内氧化氮产物（ＮＯ２）的生成量,观察ＮＯ２生成与巨噬细胞对原虫杀伤活性的关系,进一步探索了活化巨噬细胞对细胞内原虫的杀伤机制。结论：作用于巨噬细?     

婴儿利什曼原虫细胞免疫优势抗原预测

目的:预测婴儿利什曼原虫全基因组编码蛋白中刺激细胞免疫应答的优势抗原。方法:以计算机软件Net CTLpan对婴儿利什曼原虫基因组编码蛋白进行分析,确定与各类人HLA I类分子超型强结合抗原蛋白,综合预测细胞免疫优势抗原。结果:84个婴儿利什曼原虫基因组编码蛋白可与人HLA A2超型强结合,其中13个蛋白质还可与人HLA A1、A3、A26、B44、B7、B8、B58、B62、B39及B27超型强结合,它们大多数为膜蛋白,具有种属特异性。结论:预测婴儿利什曼原虫优势抗原可为发展抗内脏利什曼病疫苗提供新的视角。     

Progress towards a Leishmania vaccine

Leishmaniasis is a vector-born protozoan disease. Approximately 12 million individuals are affected worldwide with an estimated annual incidence of 1.5-2 million. Two clinical manifestations are recognized, cutaneous, and visceral, both of which are common in the Middle East. In both forms, infection is chronic, with potential deformities, persistence following cure, and lifelong risk of reactivation. Attempts to develop an effective human Leishmania vaccine have not yet succeeded. Leishmanization, a crude form of live vaccination historically originated in this part of the world. Experimental vaccination has been extensively studied in model animals in the past 2 decades. In this review, major human killed vaccine trials are surveyed, and modern trends in Leishmania vaccine development, including subunit vaccines, naked DNA vaccines, and transmission blocking vaccines are explored. Recent findings of a link between persistence of live parasites, and maintenance of long-term immunity suggest live vaccination with attenuated strains, as a future vaccination strategy.     

Atypical mucocutaneous involvement with Leishmania donovani

Mucocutaneous leishmaniasis has rarely been reported from India. The usual causative organisms of this infection are Leishmania braziliensis and L. tropica. Another species, L. donovani, which usually causes visceral leishmaniasis, has recently been reported to cause mucocutaneous disease in a few patients from Sri Lanka. We report two patients who had undiagnosed chronic skin lesions for several years. Skin biopsies revealed Leishmania and the species was characterized as L. donovani in both patients. There was considerable improvement in the skin lesions following treatment with liposomal amphotericin B.     

A review of adjuvants for Leishmania vaccine candidates

Over the last decade, there has been a flurry of research on adjuvants for vaccines, and several novel adjuvants are now licensed products or in late stage clinical development. The success of adjuvants in enhancing the immune response to antigens has led many researchers to re-focus their vaccine development programs. Although several vaccine candidates have been tested against leishmaniasis, there is yet no effective vaccine against this parasitic disease. Recent research has documented that efforts to develop effective Leishmania vaccine have been limited due to lack of an appropriate adjuvant. In view of this, this review paper outlines some of the adjuvants that have been used in Leishmania vaccine candidates and cites a few of the responses obtained from these studies. The aim of the present review is to consolidate these findings to facilitate the application of these adjuvants in general and experimental vaccinology.     

婴儿利什曼原虫磷酸烯醇丙酮酸羧基酶优势表位基因的分子克隆与表达

目的 基于前期分析并选取的婴儿利什曼原虫PEPCK的优势表位基因,构建相应重组原核表达载体以获得重组蛋白,构建相应重组真核表达载体并验证真核载体在NIH3T3细胞中的表达,为后续动物的免疫和感染实验备下基础。方法 根据PEPCK优势表位基因序列,经PCR反应及酶切连接构建重组原核与真核表达质粒pET32a-PEPCK和pVAX1-EPEPCK并分别转染至E. coli和NIH3T3细胞中进行表达。采用SDS-PAGE电泳和Western Blot鉴定原核重组质粒在大肠杆菌中的表达和原核表达蛋白被镍柱纯化后的情况,采用免疫荧光实验验证真核重组质粒在细胞中的表达。结果 重组原核与真核载体的正确测序结果表明重组载体构建成功。SDS-PAGE电泳和Western Blot的结果显示,大肠杆菌中表达的原核重组蛋白以及纯化后的蛋白在48.08 kD处存在条带,转染了真核重组载体的NIH3T3细胞的免疫荧光结果呈阳性。结论 成功构建了PEPCK优势表位基因的重组原核和真核表达载体:pET32a-PEPCK和pVAX1-EPEPCK,成功表达相应的重组蛋白并纯化并验证了pVAX1-EPEPCK在NIH3T3细胞中的表达,为后续DNA疫苗初次免疫和蛋白疫苗加强免疫的动物实验备下基础。     

抗杜氏利什曼原虫保护性单克隆抗体研究

Kala-azar is increasing in incidence in some endemic areas of China, such as provinces of Sichuan and Gansu. Monoclonal antibodies have been shown to interfere with the cutaneous leishmaniasis infection in the experimental animals. We have now raised McAb against membrane antigen of promastigotes of L. donovani canine isolate (the pathogen of visceral leishmaniasis). In this paper we report 3 McAb which inhibit the growth of promastigotes in vitro culture by incorporation of 3H-thymine deoxyriboside, such as McAb 2B12-A8, 2H6-E3, in the presence of complement, showing strong inhibiting effects with a decrease in incorporation of 3H-TdR, 94-99.3% rate of inhibition, whereas the inhibition rate induced by McAb 1B1-C2 fluctuated in a range of 68-95%, which is identical with the results of macrophage infection inhibition test by the same McAb, 61-87%. The results of ultrastructural localization of L. donovani antigen by protective McAb 2H6-E3 with immunogold technique showed the gold particles, in clumps, widely distributed on the outer side of promastigotes membrane. According to the high density of localized gold particles, it is suggested that this antigen is abundant on the plasma membrane.     

甘肃省文县、迭部县家犬感染利什曼原虫危险因素研究

目的 了解甘肃省黑热病流行区犬感染利什曼原虫的危险因素,为探索犬源型黑热病防控新方法提供依据。方法 采用分层整群抽样的方法 确定文县兴隆村和迭部县洛大村为研究点。入户调查家犬的基本状况和症状体征,采集犬血并用PCR方法开展检测。运用IBM SPSS 23.0、Pearson χ²检验和多元Logistic回归方法评估犬的基本情况、临床症状和阳性感染犬之间的影响关系,筛选犬感染利什曼原虫的危险因素。结果 共调查2县家犬537只,利什曼原虫PCR 检测阳性率41.15%（221/537）。单因素回归显示豢养方式（χ²=12.357,P<0.05）、精神是/否活跃（χ²=11.883,P<0.05）、眼睑有/无分泌物（χ²=4.314,P<0.05）和趾甲是/否增长（χ²=37.292,P<0.05）4个变量与犬感染利什曼原虫差异有统计学意义。犬的性别、犬龄、身高、体重、品种、犬毛长短6个变量之间与犬感染利什曼原虫差异无统计学意义。多因素Logistic回归显示：犬的豢养方式[OR=3.051,CI=（0.965~9.645）]与犬感染利什曼原虫存在显著的相关性。结论 在甘肃省文县、迭部县存在大量利什曼原虫感染犬,犬放养是当地家犬感染利什曼原虫的危险因素,应加大群众防治黑热病意识,改善管理犬只方式,疾控部门加强犬监测,对有症状犬采取淘汰措施,减少黑热病传染源。     

Identification of Leishmania donovani isolates from different kala-azar foci in China by kDNA hybridization.

kDNA sequence homology of Leishmania donovani isolates from three types of kala-azar foci in China were analyzed by using dot and Southern hybridization with biotin- and 32P-labelled probes. The results revealed kDNA sequence heterogeneity among Leishmania donovani isolates from the three kala-azar foci: sequence homology between isolates of hill and desert foci was higher than that between hill and plain foci isolates. The kDNA hybridization technique was also found to be specific and sensitive for direct identification of Leishmania in animal tissues. In a preliminary survey, kDNA hybridization of cutaneous tissue blots of 71 dogs from endemic regions showed a positive rate of 40.8%, and the rate of double positive cases (touch blot hybridization and bone marrow smear) reached 91.3%. The direct identification of Leishmania in tissues by kDNA hybridization seems to be a useful and convenient method for epidemiological study and clinical diagnosis, especially for species/strain characterization.     

Nitric oxide synthase (NOS) characterization in Leishmania amazonensis axenic amastigotes

BACKGROUND: Although Leishmania virulence may be modulated by environmental and genetic factors of their mammalian hosts and sand fly vectors, molecular determinants of Leishmania sp. are the key elements. This work evidences that Leishmania amazonensis axenic amastigotes produce comparatively more NO than infective promastigotes. METHODS: A soluble NOS was purified from L. amazonensis axenic amastigotes by affinity chromatography (2',5'-ADP-agarose), and on SDS-PAGE the enzyme migrates as a single protein band. RESULTS: The presence of a constitutive NOS was detected through immunofluorescence using antibody against neuronal NOS (nNOS) and in NADPH consumption assays. CONCLUSIONS: The present data show that NOS is prominent in axenic amastigote preparations, suggesting an association with the infectivity and/or an escaping mechanism of the parasite. The relationship between the NO-generating systems in the parasite and in their host cell warrants further investigation.     

杜氏利什曼原虫无鞭毛体蛋白基因原核表达重组质粒的构建

目的:构建杜氏利什曼原虫无鞭毛体蛋白(amastin)编码基因的原核表达重组质粒pET-amastin.方法:提取杜氏利什曼原虫基因组DNA进行PCR扩增,将扩增出的无鞭毛体蛋白基因片段导入质粒载体pET-32a(+),构建原核表达重组质粒pET-amastin.结果:扩增出大小约550 bp的无鞭毛体蛋白基因,重组质...