雄性激素抑制感染杜氏利什曼原虫的巨噬细胞的凋亡

目的：研究雄性激素对Ｃ５７ＢＬ／６ｊ雌性小鼠骨髓巨噬细胞凋亡的影响。方法：溴化丙锭染色及透射电镜观察凋亡特征性形态学变化。来自雄性激素和油处理过的小鼠骨髓巨噬细胞分别用杜氏利什曼原虫（Ｌｅｉｓｈｍａｎｉａｄｏｎｏｖａｎｉ）攻击２４ｈ后,检测特征性的ＤＮＡ凋亡梯形。结果：在培养基中去掉Ｍ－ＣＳＦ后可以诱导骨髓巨噬细胞的凋亡。ＤＮＡ片段电泳提示：①在雄性激素和油处理组间凋亡细胞的量没有区别,然而②经杜氏利什曼原虫攻击后,雄性激素处理组的凋亡细胞数明显低于油处理组。结论：雄性激素可以抑制感染了杜氏利什曼原虫的巨噬细胞的凋亡,不感染利什曼原虫时,这种抑制作用并不出现。雄性激素对骨髓巨噬细胞凋亡的抑制作用可能在雄性激素诱导的免疫抑制作用中发挥着重要的作用     

TESTOSTERONE INHIBITS APOPTOSIS OF LEISHMANIA DONOVANI INFECTED MACROPHAGES*

目的：研究雄性激素对Ｃ５７ＢＬ／６ｊ雌性小鼠骨髓巨噬细胞凋亡的影响。方法：溴化丙锭染色及透射电镜观察凋亡特征性形态学变化。来自雄性激素和油处理过的小鼠骨髓巨噬细胞分别用杜氏利什曼原虫（Ｌｅｉｓｈｍａｎｉａｄｏｎｏｖａｎｉ）攻击２４ｈ后,检测特征性的ＤＮＡ凋亡梯形。结果：在培养基中去掉Ｍ－ＣＳＦ后可以诱导骨髓巨噬细胞的凋亡。ＤＮＡ片段电泳提示：①在雄性激素和油处理组间凋亡细胞的量没有区别,然而②经杜氏利什曼原虫攻击后,雄性激素处理组的凋亡细胞数明显低于油处理组。结论：雄性激素可以抑制感染了杜氏利什曼原虫的巨噬细胞的凋亡,不感染利什曼原虫时,这种抑制作用并不出现。雄性激素对骨髓巨噬细胞凋亡的抑制作用可能在雄性激素诱导的免疫抑制作用中发挥着重要的作用     

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。     

草原兔尾鼠（Lagurus lagurus）对利什曼原虫敏感性的初步观察

本文报道了用杜氏利什曼和都兰利什曼两种原虫，进行人工感染草原兔尾鼠的初步结果。杜氏利什曼９１１株腹腔接种动物三个月后剖检，发现以５×１０７个原虫／０．２ｍｌ／只接种的５只鼠，有３只感染；９０１株以５×１０７个原虫／０．２ｍｌ／只无鞭毛体接种４只中有３只感染，前鞭毛体接种７只中４只感染；都兰利什曼原虫前鞭毛体同样剂量接种７只中２只足垫出现皮肤损害，涂片上查到大量原虫，而内脏无感染。提示草原兔尾鼠可能是黑热病的一种敏感的实验动物。     

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。     

热带利什曼原虫的特异性单克隆抗体:期及种特异性抗原的特性

本文报道以热带利什曼大型亚种前鞭毛体膜的单克隆抗体对热带利什曼种团及杜氏利什曼种团各10株共20株原虫的膜或完整的前鞭毛期,进行了间接放射免疫,免疫荧光及免疫沉淀等方法的测定。各种原虫的前鞭毛体是用Schneider’s Drosophlia培养基保存的,无鞭毛体则从感染BALB/c雌性小鼠取得。原虫混悬于pH7.3含40mM NaCl,10mM乙二胺四乙     

杜氏利什曼原虫人工感染家犬血清中IgG动态观察

本文采用杜氏利什曼原虫四川人分离株前出毛体人工感染家犬１１只，按不同感染剂量（１×１０４～１×１０８／只）分组并设未感染对照犬２只，骨髓涂片法感染组均查见利什曼原虫无鞭毛体，感染成功。感染前犬血清ＩｇＧ含量均数为１２６．５９Ｉｕ／ｍｌ，１５周时达１９２．５４Ｉｕ／ｍｌ，（Ｐ＜０．０５）。说明本试验人工感染Ｌ．ｄ前毛体导致了犬ＩｇＧ的上升。     

溴氰菊酯药浴阳性鼠阻断内脏利什曼病传播的实验研究

用中华白蛉分别饲吸两组染有杜氏利什曼原虫的背纹仓鼠（Ｃｒｉｃｅｔｕｌｕｓｂａｒａｂｅｎｓｉｓ），一组系经澳氰菊酯药浴后９－４５ｄ的仓鼠，另一组系未经药浴的仓鼠。实验结果表明饲吸药浴鼠的白岭在２４ｈ内全部中毒致死，饲吸未经药浴鼠的白蛉存活率很高。在剖检的吸血白蛉中，有６９．１％的白蛉，在其消化道内发现有前鞭毛体的感染，这些前鞭毛体不仅在白蛉消化道内发育良好，且随饲吸时间的推移，前鞭毛体侵入食道、咽和喙部。背纹仓鼠是杜氏利什曼原虫理想的保虫宿主，经药浴的仓鼠进行人工感染，已失去感染能力。     

犬血清的免疫复合物去补体用作诊断内脏利什曼的补体结合试验

犬血清经56℃灭活后,常出现抗补体影响补体结合试验(CF)的效果。作者用抗羊红细胞抗体吸收犬血清中的补体后进行CF试验。病犬血清采自感染杜氏利什曼原虫无鞭毛体的雄性牧羊犬。感染后每周或隔周采血,直至12周。血清保存于-20℃。以杜氏利什曼原虫肯尼亚株和巴西利什曼巴拿马亚种新大陆株的前鞭毛体和无鞭毛体,经冻融、     

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     

Studies on the effect of the anti-phagocytic agent cytochalasin B on Leishmania-macrophage interaction.

Mouse peritoneal exudate cells were cultured on coverslips in Eagle's Basal Essential Medium. The adhering cells were infected with promastigotes of three different species of Leishmania. After 8 h incubation, the macrophages were fixed and stained, and a total of one hundred cells were counted. The rates of infection of macrophages were respectively 53.5 +/- 5% for L. enriettii, 52.3 +/- 5% for L. donovani and 11.7 +/- 2% for L. tropica. When cytochalasin B at concentrations of 2.5, 5 and 10 microgram/ml and Leishmania promastigotes were added to the adhering cells at the same time, the drug did not have any effect on the uptake of the organisms by the macrophages. However, when the cells were treated for a 2-h period with the drug and then were infected with the promastigotes, only 1-2% of the cells were infected. On the other hand, when cytochalasin B-treated cells which had lost their phagocytic ability were washed and then were infected with the promastigotes, some degree of cellular infection was observed. It was concluded that infection of mouse p.e.c. by three different species of Leishmania which were used in our study was by phagocytosis rather than active penetration of the organisms into the cells. It was also of interest to note that although our outbred strain of mice gets infected easily with L. tropica, the p.e.c. of these animals phagocytosed L. tropica with least efficiency in comparison with L. donovani and L. enriettii.     

Cross-protection against Leishmania donovani but not L. Braziliensis caused by vaccination with L. Major soluble promastigote exogenous antigens in BALB/c mice

Vaccinating with soluble Leishmania major promastigote exogenous antigens (LmSEAgs) protects mice against challenge with L. major. To explore the potential of LmSEAgs to cross-protect against infection with other species of Leishmania, BALB/c mice were immunized with LmSEAgs prior to challenge with either L. donovani or L. braziliensis promastigotes. Such mice were protected against L. donovani but not L. braziliensis infection. Leishmania braziliensis-infected mice developed lesions that were not significantly different from those of controls and that contained 13-fold more parasites. In contrast, immunized mice infected with L. donovani were protected as illustrated by low splenic parasite loads (as much as 4,913-fold fewer parasites). This protection corresponded to significant increases in gamma interferon and low production of interleukin-4 (IL-4) IL-4 or IL-10, which suggested an enhanced type 1 response.     

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。     

Efficacy of Trypan: a diminazene based drug as antileishmanial agent

Trypan, a diamidine based drug, was tested as an antileishmanial agent. Duplicate cultures of both Leishmania major and Leishmania donovani promastigotes in M199 medium and Trypan at various concentrations were tested. The cultures were incubated at 25 degrees C and parasites counted at 48 h interval, and the data generated was used to establish growth inhibition curves. Drug-free cultures were included to serve as control. In the in vivo study, a total of 40 BALB/c mice were divided into five groups of 8 mice each. They were infected with 2 x 10(6) promastogotes on the left footpad. Two groups were treated with 70 microg/ml of Trypan, a total of 500 microl used immediately after infection, one group by topical application and the other administered intraperitoneally. The treatments were repeated for the two other groups 10 weeks post infection, one by topical application and the other administered intraperitoneally. One group was not treated and thus served as control. Footpad sizes were measured using Vernier calliper every 2 weeks for 21 weeks. In the in vitro studies, Trypan inhibited growth of either L. major or L. donovani promastigotes in all the concentrations tested with more dramatic inhibition in high concentrations. Based on the in vivo studies, it was evident that Trypan had effect on L. major infected lesions when applied topically immediately after infection. However, there was no effect when treatment commenced after the lesions were established. The data is discussed.     

Leishmania donovani: immunization against infection as a function of parasite growth phase

C57BL/6 mice were immunized against Leishmania donovani infection with a subcutaneous vaccination protocol. Groups received 3 injections at 4-day intervals combining glucan and killed promastigotes harvested from either logarithmic or stationary phase cultures. Controls were immunized with glucan alone, stationary or log phase promastigotes alone, or were untreated. All groups were challenged intravenously with stationary phase promastigotes at day 45 post-immunization. Results revealed that animals immunized with the glucan-killed parasite vaccine, utilizing promastigotes derived from either log (GPL) or stationary phase cultures (GPS), demonstrated significant resistance against infection as compared to controls or untreated mice. Additionally, the reduction in hepatic amastigote proliferation in mice immunized with GPS was significantly greater than in mice immunized with GPL.     

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。     

Experimental infections of the multimammate rat (Mastomys natalensis) with Leishmania donovani and Leishmania major

The susceptibility of the multimammate rat, Mastomys natalensis, to experimental infections with Leishmania donovani and L. major was examined. Inoculations of L. major promastigotes into the skin resulted in nonulcerating lesions in which parasites could be detected for more than 30 weeks later. Intravenous inoculations of L. donovani promastigotes produced visceral infections characterized by a continuing increase in splenic parasite burdens and liver parasite burdens which peaked during the first few weeks of infection and gradually decreased as the disease became chronic. L. donovani could be isolated from the blood throughout the infection, and promastigotes were cultured from the spleens of rats inoculated intradermally. Thus, the multimammate rat appears to be a good reservoir host for these parasites.     

Eicosanoid metabolism by Leishmania donovani-infected macrophages: mouse strain responses in prostanoid synthesis

Arachidonic acid (20:4) conversion to prostanoids was examined in murine peritoneal macrophages infected in vitro with Leishmania donovani. Four strains of mice differing in resistance to in vivo L. donovani infection were studied. Normal macrophages from all strains converted 20:4 to prostanoids and this was augmented by L. donovani infection. Although cells from each strain synthesized elevated levels of prostaglandin-E2 (PGE2), there were differences with respect to relative increases of this product, with infected macrophages from the C3H/HeJ strain showing the smallest increase above basal levels. These results indicate that macrophages from mouse strains with distinct levels of in vivo resistance to L. donovani all respond to infection with augmented prostanoid synthesis. Although some heterogeneity in strain-specific PGE2 responses to in vitro infection were observed (with respect to increases in PGE2 synthesis above basal levels), it is unlikely that differential resistance of these strains to in vivo infection is strictly related to these relative differences. It seems likely that other genetically controlled factors may have a major impact on disease expression.     

Leishmania-induced cellular recruitment during the early inflammatory response: modulation of proinflammatory mediators

This study investigated whether Leishmania species, the etiologic agent of cutaneous (Leishmania major) and visceral (Leishmania donovani) leishmaniasis, could differentially elicit early inflammatory events in vivo correlating with the subsequent development of their reciprocal pathogenesis. By use of the murine air pouch system, injection of Leishmania led to a rapid and transient accumulation of a mixed population of leukocytes, and L. major recruited 31-fold more leukocytes than did controls, compared with 7-fold more leukocytes for L. donovani. L. major promastigotes were better than L. donovani promastigotes at inducing proinflammatory cytokine secretion and chemokine gene expression in pouch exudates. L. major infection elicited significantly increased chemokine receptor gene expression, compared with L. donovani infection. Collectively, the data reveal that L. major is a strong inducer of the early inflammatory response, compared with L. donovani, and suggest that such an immunologic event potentially could restrain this parasite to the inoculation site, favoring the development of local swelling and cutaneous lesions.