杜氏利什曼原虫自动免疫的初步研究

<正> 在探索黑热病自动免疫的研究中,曾使用活的前鞭毛体以及经 X 射线和甲醛处理的前鞭毛体与佐剂合用的免疫原,发现有很弱的保护力。近年来又报告了卡介苗对实验性皮肤利什曼病和内脏利什曼病以及一种葡聚糖(glucall)在黑热病自动免疫中作为佐剂的作用。Preston 等还报道了豚鼠利什曼原虫(Leishmania enriettii)核糖体的免疫保护作用。本文报告了用杜氏利什曼(Leishmania donovani)前鞭毛体及其核糖体制剂自动免疫以及短棒杆菌菌苗作为免疫佐剂的初步实验结果。     

雄性激素对鼠巨噬细胞杜氏利什曼原虫感染的影响

目的 :研究雄性激素对鼠巨噬细胞株 (ana 1)细胞杜氏利什曼原虫感染率及感染水平的影响。方法 :体外培养巨噬细胞 ,实验组用雄激素 (0 4μmol L)处理 2 4h ,按巨噬细胞和前鞭毛体 1∶10的比例感染杜氏利什曼原虫。Giemsa染色法观察不同时限 (感染初期 ,3h ,6h ,12h ,2 4h ,36h ,48h)的感染率及感染水平。结果 :雄性激素处理的巨噬细胞对杜氏利什曼原虫感染率及感染水平明显高于未加雄性激素的对照组 ,并且随着时间的延长这种差别愈趋明显 (感染后 12h ,2 4h ,P <0 0 5 ;36h ,48h ,P <0 0 1)。结论 :雄性激素增强杜氏利什曼原虫对巨噬细胞株细胞感染率及感染水平。     

我国厌氧棒菌菌苗在小鼠体内抗流感病毒、诱生干扰素及调节免疫的作用

我国分离的厌氧棒菌制成的菌苗北京7903和上海7503两批样品,静脉注入小鼠20mg/kg一次,对6～48小时后经静脉感染流感病毒所致死亡有保护作用,可减少小鼠死亡率。最佳保护时间为注射后24小时,保护率87.6～100％。静脉注射菌苗后6小时出现血清干扰素,高峰达320～640u/ml,18小时降低,持续24小时以上,其性质可能为γ-干扰素。静脉注射菌苗6～72小时间,激活巨噬细胞活性,高峰时间在注射后24小时。腹腔巨噬细胞吞噬鸡红细胞的％和吞噬指数,比对照组高,P<0.01。静脉注射菌苗对流感病毒抗体生成有促进作用,可使血清流感病毒血凝抑制抗体增高3～4倍。腹腔及皮下注射菌苗保护率低,腹腔注射后未测出血清干扰素。 小鼠静脉注射棒菌菌苗后抗流感病毒的最佳保护时间,与血清干扰素高峰时间不一致,但与腹腔巨噬细胞吞噬功能高峰一致。给小鼠腹腔注射石英粉尘,破坏巨噬细胞吞噬功能,也降低菌苗对流感病毒感染的保护作用,但对血清干扰素滴度无显著影响,提示厌氧棒菌菌苗抗流感病毒作用,与激活巨噬细胞的关系密切。     

低强度半导体激光照射对小鼠腹腔巨噬细胞功能的影响

目的：观察低强度650nm半导体激光对小鼠腹腔巨噬细胞吞噬功能的影响。方法：用不同功率和照射时间的650nm半导体激光辐照小鼠腹腔巨噬细胞，采用中性红吞噬实验测定巨噬细胞的吞噬能力。结果：在适当的激光功率和照射时间下巨噬细胞的吞噬能力有显著性增强。结论：低强度650nm半导体激光照射对小鼠腹腔巨噬细胞有激活作用，可增强其免疫活性。     

杜氏利什曼原虫amastin基因重组真核表达质粒的构建与表达

目的：构建杜氏利什曼原虫无鞭毛体蛋白（alllastin）编码基因的真核表达重组质粒pcDNA3．1-amastin，并研究其在NIH3T3细胞中的表达。方法：提取杜氏利什曼原虫基因组DNA进行PCR扩增。将扩增的无鞭毛体蛋白基因片段导入质粒pcDNA3．1（＋）中，构建真核表达重组质粒pcDNA3．1-amastin。以pcDNA3．1-amastin转染NIH3T3细胞，采用免疫荧光染色法和RT-PCR分别鉴定pcDNA3．1-amastin的瞬时表达和稳定表达。结果：在细胞膜和细胞内均观察到较强的绿色荧光，表明pcDNA3．1-amastin成功地转入NIH3T3细胞，并在细胞膜和细胞内获得短暂表达。稳定转染的NIH3T3细胞的总RNA经反转录后，用PCR扩增出无鞭毛体蛋白基因，表明获得了稳定表达。结论：成功地构建杜氏利什曼原虫无鞭毛体蛋白基因的真核表达重组质粒，并且该基因在NIH3T3细胞中获得了稳定表达。     

精制厌氧棒状杆菌细胞壁的佐剂作用及其对巨噬细胞的激活性能

本文主要观察经冷冻高压破碎菌体、SDS处理以及蛋白酶消化提纯的精制厌氧棒菌细胞壁的佐剂作用;用溶血空斑法测定抗体形成细胞数以及间接血凝法测定免疫小鼠血清抗体,证明其对抗原SRBC及BSA具有明显佐剂作用;此外亦能恢复带瘤小鼠(艾氏腹水癌)的吞噬功能。     

小鼠腹腔巨噬细胞Fc受体的观察

本文对不同免疫水平的小鼠腹腔巨噬细胞作了 EA-花环试验。发现Fc受体功能在昆明杂交系小鼠比近交系C_(57)BL 的同年龄小鼠活跃,而在C_(57)BL系小鼠中,年青动物的Fc受体功能又较老年鼠强,从而证明小鼠腹腔巨噬细胞的Fc受体功能状态可随机体的免疫水平变化而发生改变,因此,对巨噬细胞 Fc受体的观察可作为衡量机体免疫水平的实验指标之一。 当小鼠腹腔巨噬细胞被厌氧棒状杆菌菌苗激活后,发现其 EA-花环形成百分率明显上升,而且吞噬活性也明显增强,尤其是在抗体调理吞噬反应中,还表现出单位细胞吞噬能力的显著提高,这说明菌苗激活的巨噬细胞可促进其表面 Fc 受体的功能,进而使巨噬细胞在调理吞噬反应中变得更为活跃。由此可见,提高巨噬细胞 Fc 受体的功能,对其功能的发挥具有积极意义。     

SAP基因注射干预小鼠SLE样综合征的发生

目的：研究SAP基因注射对SLE发病的干预作用，并进一步探讨SAP发挥作用的可能机制。方法：通过RT-PCR方法克隆SAP基因，构建其真核表达质粒pcDNA3-SAP，观察SAP基因注射对活化淋巴细胞免疫诱导小鼠产生的SLE样综合征的干预作用，以ELISA方法检测抗dsDNA抗体的产生情况，以免疫荧光法检测肾脏免疫复合物沉积；通过巨噬细胞吞噬实验观察SAP与DNA结合后对DNA清除的影响；用增殖实验检测巨噬细胞吞噬SAP结合的DNA后对预致敏淋巴细胞活化的影响。结果：SAP基因注射可有效干预小鼠SLE样综合征的发生，SAP与活化淋巴细胞DNA结合后可明显促进巨噬细胞对DNA的吞噬，并且巨噬细胞吞噬SAP结合的DNA后不引起预致敏淋巴细胞的增殖。结论：提示SAP通过促进DNA等自身抗原的有效清除干预SLE疾病的发生。     

人参皂甙对小鼠腹腔巨噬细胞活性的影响及其作用机制初步探讨

目的:观察人参皂甙(GS)对巨噬细胞的免疫激活作用,探讨GS活化巨噬细胞及免疫调节机制。方法:在体外培养的小鼠腹腔巨噬细胞中加入不同浓度的GS后,观察巨噬细胞一氧化氮(NO)合成及MTT比色法检测活化后的巨噬细胞对肿瘤细胞杀伤活性的影响;扫描电子显微镜(SEM)观察巨噬细胞的超微结构改变;激光扫描共聚焦显微镜(LSCM)观察巨噬细胞表面组织相容性复合体Ⅱ(MHCⅡ)的变化;以特异性荧光探针Fluo-3/AM负载细胞,应用LSCM检测巨噬细胞内Ca2+浓度。结果:小鼠腹腔巨噬细胞经GS作用后,细胞形态发生活化性改变,杀瘤活性增强,细胞表面MHCⅡ表达增加并增强对H22细胞杀伤活性;GS作用细胞4小时后,25~200 mg/L GS细胞内Ca2+浓度升高,并与药物浓度呈正相关。结论:GS在体外能激活小鼠巨噬细胞,促进其发挥免疫防御功能,其免疫调节机制可能与细胞内Ca2+浓度升高有关。     

约氏疟原虫保护性抗原对鼠疟红内期保护性免疫调节作用的研究──Ⅱ．53kD抗原细胞增殖反应及血清抗体与保护作用的关系

用约氏疟原虫（Ｐ．ｙ）裂殖子抗原免疫ＥＡＬＢ／ｃ小鼠，再用约氏疟原虫非致死株１０５ＰＲＢＣ／只攻击。免疫组小鼠血中原虫推迟出现，提早消失，原虫率明显低于未免疫小鼠和佐剂对照小鼠；佐剂对照组仅原虫高峰推迟出现，与易感小鼠无明显差异。抗原免疫小鼠脾脏Ｔ淋巴细胞对抗原特异性和非特异性增殖反应明显高于其它两组，原虫攻击后增殖反应普遍受到抑制，但免疫小鼠比其它两组恢复快，感染后１８天即恢复至正常水平。     

Impairment of Leishmania mexicana phagocytosis in peritoneal macrophages induced by Amaranthus leucocarpus lectin

The lectin from Amaranthus leucocarpus (ALL) is specific for N-acetyl-D-galactosamine and inhibits phagocytosis of Leishmania mexicana promastigotes in Balb/c mice peritoneal macrophages by 38%. The lipophosphoglycan (LPG) purified from L. mexicana inhibits penetration of promastigotes into peritoneal macrophages by 31%; interestingly, treatment of macrophages with both, ALL and LPG, inhibits phagocytosis of promastigotes by 72%, confirming that ALL induces modification of the macrophage's phagocytic activity by a different route than mannose or C3b receptors. The Inhibitory effect of ALL was time-dependent. N-acetyl-D-galactosamine (GalNAc) or O-glycosidically linked glycoproteins modified macrophage phagocytosis of Leishmania. These results suggest that macrophage membrane glycoproteins, possessing constitutive GalNAc, can influence the signaling pathways used by this intracellular parasite to infect.     

Leishmania braziliensis promastigotes and amastigotes interact differently with host macrophages

In vitro and in vivo ultrastructral studies reveal that the parasite entrance into the macrophage occurs by phagocytosis. The early stage of phagocytosis exhibited different ultrastructural characteristics in both forms of the parasite. Long and prominent projections from peritoneal exudate macrophages made focal contacts with the promastigote surface. The amastigotes, in turn, laid on cup-shaped extensions of the macrophage membrane. Later stages of the phagocytosis are characterized by progressive and complete engulfment of both promastigotes and amastigotes.     

Cyclosporin A enhances elimination of intracellular L. major parasites by murine macrophages.

In this study we analyzed the influence of cyclosporin A (CyA) on the process of phagocytosis of L. major promastigotes and amastigotes by inflammatory peritoneal macrophages (MP) from BALB/c mice. Our data clearly demonstrate that CyA profoundly enhanced the degradation by peritoneal MP of both intracellular L. major promastigotes and amastigotes. This effect was T cell-independent and specifically associated with CyA, since the similarly structured cyclosporin F (CyF) was ineffective. CyA did not alter the replication and infectivity of extracellular parasites. From these results we conclude that the inhibition of intracellular parasite replication in the presence of CyA substantially contributes to the previously described suppressive effect of CyA on the development of L. major-induced lesions in BALB/c mice.     

A study of the differential respiratory burst activity elicited by promastigotes and amastigotes of Leishmania donovani in murine resident peritoneal macrophages.

Acridine orange and ethidium bromide and a combination of fluorescent and transmitted light microscopy used in conjunction with the qualitative nitroblue tetrazolium assay for superoxide anion (O2-) release demonstrated dramatic differences in the binding of and respiratory burst (RB) activity elicited by promastigotes and amastigotes of Leishmania donovani in resident peritoneal macrophages (M phi) from C57BL/10ScSn mice. When amastigotes were incubated with M phi for 30 min the number of parasites per 100 M phi was 2-4-fold higher, a higher proportion of M phi became infected and the mean number of parasites per infected M phi was higher than in promastigote infections. RB activity was higher for promastigotes than amastigotes both in terms of the percentage of infected M phi containing formazan positive parasites and the percentage of individual formazan positive parasites. In an attempt to explain the differential response to promastigotes and amastigotes, RB activity was examined for sodium azide-treated, glutaraldehyde-fixed and heat-killed parasites and for various transformation intermediates between amastigotes and promastigotes. Binding and RB activity were also examined in conjunction with competitive binding assays designed to determine the specific receptors involved in ligand binding of both forms of the parasite to the M phi. The results indicate that, while amastigotes may possess an azide-sensitive mechanism which either competes for O2- produced or causes localized inactivation of RB activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. The transformation and competitive binding studies suggest that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the M phi plasma membrane and that the well characterized mannose/fucose receptor may be important in promastigote, but not amastigote, binding and RB activity.     

Expression of Leishmania antigen on the surface membrane of infected human macrophages in vitro.

Resolution of leishmaniasis is associated with host immunological responsiveness to parasite antigens. In clinical disease, leishmania are found as amastigotes contained with macrophages. We investigated the possibility that Leishmania antigens are expressed on the infected macrophage surface by reacting infected macrophages with antibody to Leishmania. In vitro-infected human monocyte-derived macrophages were labelled with antibody to amastigotes when examined with immunofluorescent or immunoelectron microscopic techniques. Infected macrophages were poorly labelled by antibody to promastigotes (insect forms of Leishmania). Certain antisera that reacted with the surface membranes of amastigotes did not label the infected macrophage surface. These results indicate that human macrophages infected in vitro express Leishmania amastigote antigen(s) on their surface membranes, that such antigen(s) may not be present in large quantities in promastigotes, and that certain antigen(s) on the amastigote surface are not expressed on the surface membranes of infected macrophages.     

Growth of Leishmania amastigotes in macrophages from normal and immune mice

Summary An in vitro system of prolonged culture of Leishmania tropica amastigotes in mouse macrophages is presented. The division rate of parasites was monitored by microscopic observations and by ³H-thymidine incorporation. The dynamics of macrophage infection and parasite division are influenced by the initial rate of promastigotes per cells in culture. Parasites multiply, gradually infect and finally destroy all available macrophages from normal mice releasing large numbers of viable amastigotes. Macrophages from immune donors were inferior in their ability to support parasite multiplication and did not survive long periods in culture.     

Correlation of Increased Metabolic Activity, Resistance to Infection, Enhanced Phagocytosis, and Inhibition of Bacterial Growth by Macrophages from Listeria- and BCG-Infected Mice

Macrophages from mice infected with facultative intracellular organisms such as Listeria monocytogenes and BCG have been shown to resist infection by antigenically unrelated intracellular bacterial parasites. This study compares phagocytosis, bacterial growth inhibition, and oxidation of glucose by macrophages from normal mice, mice infected with listeria or BCG, or mice immunized with killed listeria in incomplete Freund's adjuvant. Macrophages from listeria- and BCG-infected mice ingested more listeria; 67 and 57%, respectively, had three or more cell-associated bacteria versus 22% of controls (P < 0.001). Peritoneal macrophages from listeria- and BCG-infected animals significantly (P < 0.001 covariance analysis) inhibited growth of listeria in suspension, whereas control macrophages had no such inhibitory effect. The rate of oxidation of glucose-1-(14)C was higher in macrophages from listeria- and BCG-infected mice than from either uninfected animals or those immunized with killed listeria. During phagocytosis of killed or live bacteria, or latex particles, the rate of glucose oxidation was increased (P < 0.01). These data suggest that the cellular immunity after infection by an intracellular organism is associated with an increase in metabolic activity of macrophages, namely, an increase in the rate of glucose oxidation resulting in enhancement of phagocytosis and killing.     

Phagocytic and bactericidal properties of macrophages of mice immunized with outer membrane proteins (OMP) of Shigella

Macrophages obtained from peritoneal exudates of mice immunized with the single doses (1 and 5 micrograms) of OMP were shown to have stronger phagocytic as well as bactericidal properties in relation to Shigella flexneri bacilli than nonactivated macrophages. Macrophages from the animals immunized with 10, 20 and 30 micrograms OMP doses showed phagocytic and bactericidal properties similar to those of nonactivated macrophages while the immunization with the dose 50 micrograms resulted in their suppression. Likewise, activity of macrophages from mice immunized twice or three times with various doses of OMP did not differ much from that obtained after immunization with single OMP doses. On the other hand, immunization of mice with a sublethal dose of live Shigella flexneri did not activate either phagocytic or bactericidal properties of macrophages. Besides, phagocytic and bactericidal activity of macrophages of mice immunized with OMP of Shigella was determined in relation to Salmonella typhimurium. The doses 1 and 5 micrograms of OMP resulted in slight activation of macrophages which manifested itself by a little increase in their phagocytic and bactericidal ability. When used in the dose 10 micrograms, OMP remained without any effect on the above activity of macrophages. Only the dose 50 micrograms, slightly suppressed their phagocytic properties.     

Leishmania amazonensis Amastigotes Trigger Neutrophil Activation but Resist Neutrophil Microbicidal Mechanisms

Neutrophils are the first cells to infiltrate to the site of Leishmania promastigote infection, and these cells help to reduce parasite burden shortly after infection is initiated. Several clinical reports indicate that neutrophil recruitment is sustained over the course of leishmaniasis, and amastigote-laden neutrophils have been isolated from chronically infected patients and experimentally infected animals. The goal of this study was to compare how thioglycolate-elicited murine neutrophils respond to L. amazonensis metacyclic promastigotes and amastigotes derived from axenic cultures or from the lesions of infected mice. Neutrophils efficiently internalized both amastigote and promastigote forms of the parasite, and phagocytosis was enhanced in lipopolysaccharide (LPS)-activated neutrophils or when parasites were opsonized in serum from infected mice. Parasite uptake resulted in neutrophil activation, oxidative burst, and accelerated neutrophil death. While promastigotes triggered the release of tumor necrosis factor alpha (TNF-α), uptake of amastigotes preferentially resulted in the secretion of interleukin-10 (IL-10) from neutrophils. Finally, the majority of promastigotes were killed by neutrophils, while axenic culture- and lesion-derived amastigotes were highly resistant to neutrophil microbicidal mechanisms. This study indicates that neutrophils exhibit distinct responses to promastigote and amastigote infection. Our findings have important implications for determining the impact of sustained neutrophil recruitment and amastigote-neutrophil interactions during the late phase of cutaneous leishmaniasis.     

Leishmania donovani singly deficient in HGPRT, APRT or XPRT are viable in vitro and within mammalian macrophages

Leishmania species express three phosphoribosyltransferase enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and xanthine phosphoribosyltransferase (XPRT), which enable this genus to acquire purine nutrients from their hosts. To test whether any of these enzymes is essential for viability, transformation into amastigotes, and infectivity and proliferation within mammalian macrophages, Deltahgprt, Deltaaprt, and Deltaxprt null mutants were created by targeted gene replacement within a virulent background of Leishmania donovani. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in bone marrow-derived murine macrophages. These data support the hypothesis that none of the three phosphoribosyltransferases is essential for purine salvage or viability by itself and that purine salvage occurs through multiple anabolic routes in both parasite life cycle stages. In addition these studies revealed the presence of an adenine aminohydrolase enzyme in L. donovani axenic amastigotes, an activity previously thought to be restricted to promastigotes.