Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression

BALB/c mice are susceptible to cutaneous lelshmanlasls uponinfection with Leishmania major while C57BL/6 are not. Thereis a major promastigote surface protease (PSP or gp63) whichis available in both native and recomblnant forms, and for whichthe primary amlno acid sequence is known. Immunization withPSP has been shown to offer some protection against challengewith the live organism. Therefore, we attempted to develop apeptide vaccine with PSP peptldes. In the first experiments,recall prollferatlve responses to PSP were measured using aset of 15mer peptldes spanning the entire PSP molecule whichallowed designation of major determinant regions in BALB/c,C57BL/6, and CBA mice. Several of these determinants were promiscuousand shared almost the identical core amlno acid residues inthe different strains. Immunization with major determinant peptldeswas recalled vigorously with L. major soluble antigen as wellas with PSP. The response to peptide was almost entirely T_h1as measured by a localized ELISA assay for single-cell productionof IFN-. A similar assay for IL-5, which overcomes problemsof sensitivity and inhibition by lymphoklnes produced by T_h1cells, Indicates very little production of T_h1 cells even byBALB/c. It was found that if a major responsive peak was examinedby recall with overlapping peptldes, the highest, central peptidegave a mainly Th1 response while the boundary, less efficientpeptldes gave more of a T_h2 response. Possible reasons for thiswere discussed. These results point to the importance of selectingthe exactly appropriate peptide in considering a vacclnogenthat might protect susceptible individuals. Even the choiceof a somewhat immunogenlc peptide within the determinant envelopemight actually exacerbate infection by steering the responsein a T_h2 direction.     

Antileismanial activity,mechanism of action study and molecular docking of 1,4‐bis(substituted benzalhydrazino)phthalazines

To identify new agents for the treatment of American cutaneous leishmaniasis, a series of eight 1,4‐bis(substituted benzalhydrazino)phthalazines was evaluated against Leishmania braziliensis and Leishmania mexicana parasites. These compounds represent a disubstituted version of the 1‐chloro‐4‐(monoaryl/heteroarylhydranizyl)phthalazine that exhibited a significant response against L. braziliensis according to our previous findings. Two disubstituted phthalazines 3b and 3f were identified as potential antileishmanial agents against L. braziliensis parasites, exhibiting a submicromolar IC₅₀ response of 2.37 and 7.90 µM on the promastigote form, and of 1.82 and 4.56 µM against intracellular amastigotes, respectively. In particular, compound 3b showed interesting responses against amastigote isolates from reference, glucantime‐resistant and clinical human strains, which were by far superior to the biological response found for the glucantime drug. With regard to the toxicity results, both 3b and 3f exhibited moderate LD₅₀ values against murine macrophages (BMDM), with good selectivity indexes on promastigotes and intracellular amastigotes of L. braziliensis. A comparison of biological response was established between the monosubstituted and disubstituted versions of these benzalhydrazino‐phthalazines. Easy synthetic procedure and significant response against amastigote strains including against resistant lines made compound 3b a potential candidate for further pharmacokinetic and in vivo experiments as antileishmanial agent, and as a platform for further structural optimization. Mechanism‐of‐action studies and molecular docking simulations discarded to inhibition of superoxide dismutase as possible mode of action.     

杜氏利什曼原虫表膜的制备及保护性McAb识别膜抗原的研究

本文用匀浆及超声波破碎杜氏利什曼原虫四川犬分离株前鞭毛体,蔗糖密度梯度离心分离其表膜,电镜观察见所得样品是单位膜和膜下微管组成的表膜,测得微管直径平均为23.3nm,微管间距巨平均为15.7nm,经SDS-PAGE分离显示1条主带,次带约15条,再转移到硝化纤维膜上,用单克隆抗体2H6-E3识别,仅见有一条区带,分子量为63kDa,该单克隆抗体是己经被证实定位于前鞭毛体表膜,并具有免疫保护性的。     

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-γ and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-β concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-γ, TGF-β and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.     

Study the effects of PLGA-PEG encapsulated Amphotericin B nanoparticle drug delivery system against Leishmania donovani

Abstract

Drug delivery systems are a promising technology to increase poor solubility and bioavailability of compounds. Therefore we have developed PLGA-PEG encapsulated amphotericin B nanoparticles (NPs) drug delivery technology to increase the solubility of amphotericin B and target the macrophage of infected tissues during visceral leishmaniasis. The structural characterization by transmission electron microscopy and dynamic light scattering revealed the nano-size of the particle (30–35 nanometers). Fourier transform infrared spectroscopy confirmed the PLGA-PEG encapsulation. The mean cytotoxic assay (0.0803?+?0.0253) of extracellular promastigote of PLGA-PEG encapsulated amphotericin B is significantly lower than that of amphotericin B (0.1134?+?0.0153) and inhibition of amastigotes in the splenic tissue was significantly more than with conventional amphotericin B (93.02?+?6.63 versus 74.42?+14.78). Amphotericin B encapsulated PLGA-PEG nanoparticles were found to be more effective than free amphotericin B in terms of therapeutic efficacy during in vitro and in vivo study.     

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。     

Paromomycin in the treatment of leishmaniasis

Background: The treatment options for leishmaniasis are limited. Most of the drugs available need parenteral administration, are toxic, require monitoring and have a prolonged treatment duration. All these factors increase the cost of the treatment. The development of resistance to pentavalent antimonials in patients with visceral leishmaniasis in North Bihar, India, has added another dimension to the problem. Objective: To summarise the pharmacological and clinical data on antileishmanial activity of paromomycin and discuss the impact this agent may have on present treatment regimens. Methods: A literature search on paromomycin and leishmaniasis was done on PubMed and through Google. Results: Paromomycin, with its excellent efficacy, low cost, shorter duration of administration and good safety profile, has the potential to be used as a first-line drug.     

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。     

In vitro activity of an essential oil against Leishmania donovani

The in vitro antileishmanial effect of the essential oil from Chenopodium ambrosioides against Leishmania donovani was investigated. The product showed significant activity against promastigotes and amastigotes, with a 50% effective concentration of 4.45 and 5.1 microg/mL, respectively. The essential oil caused an irreversible inhibition of the growth of promastigotes after a treatment with 100 or 10 microg/mL for 1 or 24 h, respectively. The phagocytic activity of the macrophages was preserved at a concentration toxic to the parasite. The essential oil from C. ambrosioides may be a potential candidate drug to development a new agent to combat this parasitic disease.