利什曼原虫前鞭毛体体外生长动力学研究

目的观察不同种株利什曼原虫前鞭毛体在体外培养条件下的生长增殖情况，确定其最适体外培养条件。方法分别使用RPMI1640和M199复合培养液，观察温度、pH及新生小牛血清对硕大利什曼原虫、热带利什曼原虫、婴儿利什曼原虫、墨西哥利什曼原虫、杜氏利什曼原虫前鞭毛体体外增殖速度与增殖周期的影响。结果当培养温度为26℃时，杜氏利什曼原虫前鞭毛体在含和不含新生小牛血清、pH中性、偏碱和偏酸的PMI1640或M199复合培养基中，早期均能生长，且生长周期相对较长，其他种株利什曼原虫前鞭毛体在无血清或偏碱培养基中生长缓慢，增殖周期缩短；培养温度为37℃时，各种株利什曼原虫前鞭毛体均发生沉积，增殖停滞，不同程度地向无鞭毛体转化并发生死亡。结论使用复合培养液培养利什曼原虫前鞭毛体，温度、pH和新生小牛血清均可显著影响增殖速度和生长周期。各种株利什曼原虫前鞭毛体在相同培养条件下增殖速度和生长周期存在差异，可能与其遗传背景不同有关。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况，为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中，22 ℃温箱中无菌静置培养，显微镜下连续观察计数8 d，绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖，在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05），在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异，同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况,为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中,22 ℃温箱中无菌静置培养,显微镜下连续观察计数8 d,绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖,在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05）,在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异,同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

杜氏利什曼前鞭毛体与人单核细胞衍化的巨噬细胞的相互作用:寄生虫的侵袭、细胞内存活和繁殖

在体外,将杜氏利什曼原虫前鞭毛体用人单核细胞衍化的巨噬细胞(MDM)孵育,以估计巨噬细胞在早期内脏利什曼病中的作用。作者用健康献血员的血液分离MDM,在盖玻片上培养。然后在经热灭活的自体血清中,将MDM与无菌生长的前鞭毛体一起孵育。这时可见到前鞭毛体以其鞭毛端或无鞭毛端附着在MDM上,MDM可在前鞭毛体周围形成伪足。透射电镜证实,在MDM的空泡内含有未分裂的和正在分裂的无鞭毛体,其形态与仓鼠脾内的很相似。最初67±5%的巨噬细胞受到利什曼原虫的感染。平均每个受感染的巨噬细胞内有4.2±0.7个利什曼原虫。孵育6天后,感染率上升到     

墨西哥利什曼原虫无鞭毛体蛋囟的基因克隆化与序列分析

目的　克隆墨西哥利什曼原虫(L.mer)WR972株的无鞭毛体蛋白(amastin)的编码基因,并对其同源核苷酸序列进行分析。方法　根据已克隆的亚马逊利什曼原虫无鞭毛体蛋白的编码基因序列,设计并合成无鞭毛体蛋白基因特异性引物,以墨西哥利什曼原虫VR972株的基因组DNA作为模板,进行多聚酶链反应(PCR)扩增。将扩增的DNA片段克隆到pCR2.1T载体中,进行测序,并对同源的核苷酸序列分析、比较。结果　从体外培养的墨西哥利什曼原虫WR972株提取基因组DNA,以无鞭毛体蛋白基因特异性引物进行PCR扩增获得550bp的DNA片段。克隆到pCR2.1T载体片段进行的序列测定结果　表明,获得了墨西哥利什曼原虫的无鞭毛体蛋白编码基因片段,与亚马逊利什曼原虫的无鞭毛体蛋白基因之间具有高度的同源性。结论　实现了墨西哥利什曼原虫无鞭毛体蛋白基因的克隆化,为进一步研究其表达及作为疫苗研究的候选基因奠定了基础。     

克拉玛依地区的利什曼病ⅩⅢ.大沙鼠体内利什曼原虫的鞭毛体形态上的一种特征的记述

将克拉玛依大沙鼠体内的利什曼原虫接种BALB/c小鼠的足垫皮下后133～161天,用透射电镜观察了小鼠皮下组织内无鞭毛体的形态,发现在形态完整(鞭毛、鞭毛袋、动基体和细胞核位于同一切面上者)的81个无鞭毛体中有15个虫体(18.5％)的鞭毛从鞭毛袋内明显伸出体外,伸出部分的鞭毛长度平均为0.54μm。杜氏利什曼原虫的无鞭毛体则未见此种现象。     

免疫血清对同种和异种利什曼原虫的影响

利什曼原虫的种类颇多,它们的形态和结构等颇为相似,难于相互鉴别。因此我们用NNN培养基加抗血清对同种及异种利什曼原虫生长影响作了光学显微镜的观察。 一、材料和方法 1.利什曼前鞭毛体抗血清的制备: 抗原:将利什曼原虫置NNN培养基内培养10～12天,收集前鞭毛体用生理盐水离心洗涤4～5次,取有原虫部分。最后按前鞭毛体的压积量加9倍的pH7.2PBS制成15     

杜氏利什曼原虫DNA探针及其在流行病学和诊断中的应用

描述了杜氏利什曼原虫的一种特异而敏感的DNA探针的制备、序列分析及在诊断中的应用。从体外培养的杜氏利什曼原虫HU3株前鞭毛体提取DNA和RNA。取后循环前鞭毛体的poly(A)~+RNA用RNase H法在λgt10中构建cDNA库,经源于HU3株对数生长期和后循环期前鞭毛体的标记cDNA     

杜氏利什曼原虫无鞭毛体表达抗原的Western blot和MALDI-TOF/TOF分析

目的鉴定杜氏利什曼原虫无鞭毛体特异表达抗原。方法培养杜氏利什曼原虫前鞭毛体并体外转化无鞭毛体,其总蛋白经2-DE电泳后以小鼠抗杜氏利什曼原虫无鞭毛体血清进行Western blot,对前鞭毛体与无鞭毛体特异表达抗原蛋白进行MALDI-TOF/TOF串联质谱鉴定。重组表达无鞭毛体特异表达抗原编码基因,以Western blot法对重组蛋白进行鉴定。结果等量的杜氏利什曼原虫前鞭毛体与无鞭毛体蛋白经2-DE电泳均可呈现680~742个蛋白点,Western blot及MALDI-TOF/TOF-MS分析甘油醛3-磷酸脱氢酶与延伸因子2为杜氏利什曼原虫前鞭毛体特异表达抗原,核苷二磷酸激酶为无鞭毛体特异表达抗原。重组核苷二磷酸激酶编码基因表达产物经Western blot证实为杜氏利什曼原虫无鞭毛体特异表达强抗原。结论杜氏利什曼原虫前鞭毛体与无鞭毛体抗原表达存在差异,核苷二磷酸激酶为杜氏利什曼原虫无鞭毛体特异表达强抗原。     

克拉玛依地区的利什曼病XIV．硕大白蛉吴氏亚种自然感染前鞭毛体的鉴定

硕大白蛉吴氏亚种是新疆克拉玛依地区的主要蛉种之一，具有强的亲人性，在野外和居民点内常能查见该蛉有前鞭毛体的自然感染。本文结果表明，白蛉自然感染的前鞭毛体能使仓鼠及ＢＡＬＢ／ｃ小鼠发生内脏利什曼病；在感染仓鼠内脏涂片上的无鞭毛体，由蛉体而来的明显较由大沙鼠而来的都兰利什曼原虫为小；白蛉自然感染的前鞭毛体在ＮＮＮ培养基内生长不良；用￣（３２）Ｐ标记的ｇｐ￣（６３）基因为探针，与婴儿利什曼原虫、都兰利什曼原虫及白蛉自然感染的前鞭毛体的ＤＮＡ进行杂交，证实蚌体自然感染的原虫与婴儿利什曼原虫同源。克拉玛依无内脏利什曼病人，但人群中有皮肤利什曼病流行。关于硕大白蛉吴氏亚种自然感染的来源以及当地的皮肤利什曼病究竟是由都兰利什曼原虫抑或婴儿利什曼原虫所致，尚待阐明。     

应用单克隆抗体检测白蛉体内的前鞭毛体

以杜氏利什曼前鞭毛体为靶抗原的L12G9单克隆抗体(McAb),检测人工感染杜氏利什曼原虫的白蛉。当空腹雌蛉吸取病鼠血液后,分别饲养4、6、8和10d后解剖。吸血后4d,前鞭毛体较少。阳性率仅为15.9％;而10d,其感染程度较重,可获100％的阳性检出率。其阳性率与白蛉的感染程度呈正相关。另外又证明应用单克隆抗体检测时,原虫数不能低于11×10~7/ml,宜选择胃血完全消化后的白蛉,方可得到良好的效果。如捕获的白蛉,胃内前鞭毛体较少,则可经NNN基培养后,再进行检测,亦可获得同样效果。     

我国不同地区的杜氏利什曼原虫对亚历山大白蛉感染性的观察

用白蛉人工感染利什曼原虫的方法,观察从新疆荒漠、甘肃山区及河南平原三地自人体成蛉体分离出来的杜氏利什曼原虫对新疆亚历山大白蛉的感染性。以白蛉的感染率、感染程度以及原虫在白蛉消化道内的进展等项指标,来衡量原虫对白蛉的适应性。结果发现,新疆荒漠的原虫对亚历山大白蛉有高度的感染性,甘肃的原虫居次,河南的原虫对白蛉的感染性很差。结合以往用单克隆抗体检测法和K-DNA杂交法对我国一些地区杜氏利什曼原虫的研究,以及我国荒漠、山区和平原地区的黑热病具有不同的流行病学特征等的结果分析,认为我国的杜氏利什曼原虫很可能存在不同的地域株。     

Infectivity of the subspecies of the Leishmania braziliensis complex in vivo and in vitro

The infectivity of Leishmania braziliensis ssp. in relation to their growth kinetics in Senekjie's medium was determined using the human macrophage cell line U937 and inbred hamsters. In both systems, infectivity was shown to be distinctive for each subspecies. While L. b. panamensis promastigotes from 6-day-old cultures (early stationary phase) were more infective than parasites from any other culture day, L. b. guyanensis and L. b. braziliensis reached maximum infectivity on days 8-10 and day 10 (late stationary phase of growth), respectively. Although maximum infectivity occurred during stationary growth, strict growth phase dependency was not observed. The populations of parasites on these culture days were composed mostly of small, highly motile promastigotes with flagella 2-3 times the length of their cell bodies. These promastigotes resembled the infective forms transmitted by the sand fly vector. A distinct pathological picture characterized the disease caused by the different WHO reference strains for these subspecies in hamsters: L. b. guyanensis developed the most severe lesions, while moderate and inconspicuous lesions were observed when L. b. panamensis and L. b. braziliensis, respectively, constituted the inocula.     

雄性激素对小鼠骨髓巨噬细胞杜氏利什曼原虫感染水平的影响

目的：研究雄性激素对杜氏利什曼原虫感染的C57BL／6J小鼠骨髓巨噬细胞（BMMs）水平的影响。方法：用雄性激素处理小鼠3wk后，收集BMMs，培养5d后，按BMMs前鞭毛体＝110的比例，接种杜氏利什曼原虫，用吉姬氏染色法观察不同时相的感染水平。结果：与植物油处理的对照组相比，雄性激素处理的小鼠BMMs对前鞭毛体较为易感，并随着感染时间的延长其感染水平增高（感染后3h，P＜0．05；24h，48h和72h，P＜0．01）。结论：雄性激素可增加杜氏利什曼原虫对BMMs 的感染水平, 这可能与雄性激素诱导的免疫抑制作用有关。     

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.     

Arginase activity of Leishmania isolated from patients with cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is one of the most important vector‐borne parasitic diseases, highly endemic in Iran, and its prevalence is increasing all over the country. Arginase (ARG) activity in isolated Leishmania parasites from CL patients is yet to be explored. This study aimed to compare the ARG activity of isolated Leishmania promastigotes from CL patients with a standard strain of Leishmania major and its influences on the disease pathogenesis. We recruited 16 confirmed CL patients from Qom Province, in central Iran; after detection of Leishmania species using PCR‐RFLP, we assessed the levels of ARG in the isolated promastigotes and determined the parasites’ growth rate. Only L. major was identified from CL patients. The level of ARG activity in the isolated Leishmania promastigotes from CL patients was significantly higher than that obtained from the standard strain of L. major. No significant correlations between ARG activity and lesion size, number or duration were observed; in contrast, a significant negative correlation was seen between ARG level and Leishmania’ growth rate. The obtained results suggest that increased ARG expression and activity in the isolated Leishmania promastigotes might contribute to the higher parasite infectivity and play a major role in the pathogenicity of the CL.     

Surface antigenic change during differentiation of a parasitic protozoan, Leishmania mexicana: Identification by monoclonal antibodies.

The fusion of SP2/0 myeloma cells with spleen cells from mice immunized with Leishmania mexicana amazonensis promastigotes produced hybridoma clones. Indirect immunofluorescent antibody assay with live leishmanias showed that the monoclonal antibody 6H12 recognized only the antigens bound to the surface of L. mexicana amazonensis promastigotes. It also showed that the antibody bound to neither amastigotes of this species nor to other Leishmania species--i.e., L. braziliensis braziliensis, L. tropica, and L. donovani. Monoclonal antibodies from three other clones (4D11, 4H9, and 6A11) were found to compete with 6H12 for binding to L. mexicana promastigotes. With lysates of [35S]methionine-labeled promastigotes, all four monoclonal antibodies precipitated the same triplet set of protein bands at the approximately equal to 68,000-dalton region, whereas another monoclonal antibody (6G5) precipitated a different band at approximately equal to 90,000 daltons. During differentiation of L. mexicana amazonensis from amastigotes to promastigotes, there was a 4- to 8-fold increase above the initial level in the binding of 6H12 monoclonal antibody to leishmanias, as detected by enzyme-linked immunosorbent assay and quantitative fluorometric assay, respectively. Thus, we have demonstrated the use of monoclonal antibodies as probes for antigens that change during leishmanial differentiation.     

Purine nucleotide metabolism in promastigotes of Leishmania tropica: inhibitory effect on allopurinol and analogues of purine nucleosides.

The catabolism of adenosine 5'-monophosphate in promastigotes of L. tropica suggests the presence of a parasite specific pathway. In continuing the investigation on enzymes of this pathway two purine nucleoside cleaving enzymes have been found, adenosine phosphorylase and inosine nucleosidase. Various purine nucleoside analogues inhibited the activity of both enzymes. A mode of action of the growth inhibition of L. tropica promastigotes by allopurinol has been suggested on the basis of Michaelis and inhibitor constants.     

Application of Specific Monoclonal Antibodies for Characterization of Leishmania spp. Promastigotes Using Indirect Immunofluorescent and Immunoperoxidase Tests

Background: Different methods have been used for characterization of Leishmania promastigotes. Monoclonal antibodies are useful in characterization of   Leishmania spp . both amastogotes and promastigotes. Objective: Comparing the characterization of   Leishmania spp. promastigotes with immunoperoxidase test (Avidin-Biotin) techniques and an indirect immunofluorescent assay (IFA).   Methods: Application of specific monoclonal antibodies for characterization of different   Leishmania species. Immunoperoxidase tests (Avidin-Biotin) and indirect immunofluorescent assay (IFA) were employed for characterization of different   Leishmnia promastigotes from culture. Five monoclonal antibodies including LXXVIII-2E5- A8 (D2) specific for   L. donovani:L. infantum , IS2-2B4 (A11) specific for L. tropica, XCIV-H2- AB (T10) specific for   L. tropica, XLVI-5B8- B3 (T1) specific for L. major, and T7 reactive to both   L. major and L. tropica as well as an anti GP63 mAb were used. Results: The best result was obtained with the dilution of 1:50 for both mAb and conjugate. One hundred percent sensitivity and specificity was achieved for characterization of Leishmania promastogotes with both methods. Conclusion: As immunoperoxidase method needs less equipments compared to IFA technique, it would be a preferred method for characterization of promastigotes.     

Development of metacyclic Leishmania promastigotes is associated with the increasing expression of GP65, the major surface antigen

Using immunofluorescence techniques and flow microfluorometry analysis, we have demonstrated that the binding of a monoclonal antibody (VD5/25) produced against GP65, the major surface antigen of Leishmania braziliensis, increased on the surface of stationary-phase promastigotes from all the New World Leishmania species causing mucocutaneous or cutaneous disease as compared with the log-phase parasites. In addition, a sequential development of Leishmania amazonensis promastigotes from a non-infective to an infective stage was demonstrated. Indeed, promastigotes in the stationary phase (days 6-7) were found to be far more infective than those in the logarithmic phase of growth (day 3) both in vitro for mouse peritoneal macrophages and in vivo for BALB/c mice. The intracellular survival and multiplication of L. amazonensis were significantly inhibited when infective promastigotes were treated with the VD5/25 monoclonal antibody. The increasing expression of GP65 on the promastigote surface may thus contribute to Leishmania infectivity. This seems to represent a characteristic mechanism applicable to all New World Leishmania species studied.