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.     

Studies on mitochondrial ATPase of Leishmania donovani using digitonin-permeabilized promastigotes

Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated mitochondria. Maximum mitochondrial ATPase activity was obtained in promastigotes permeabilized with digitonin at a final concentration of 20 μM and the specific activity of the enzyme was 46% and 57% higher than that of homogenized and sonicated promastigotes, respectively. At concentrations above 20 μM digitonin inhibited ATPase activity and the degree of inhibition increased with increasing concentrations of the detergent. The ATPase activity of promastigotes remained DCCD-sensitive when permeabilized with digitonin at concentrations up to 120 μM but the enzyme became increasingly resistant to this inhibitor as digitonin concentrations were increased to 140 μM and more, indicating the loss of functional activity of the enzyme. The pH and temperature optima for mitochondrial ATPase were determined to be 7.5 and 30°C, respectively. Mg²⁺ ions were essential for ATPase activity but free Mg²⁺ ions were found to be inhibitory. A Mg²⁺/ATP ratio of 1:3 supported the optimum ATPase activity. Sulfite and hexanol activated the enzyme but failed to prevent the inhibition by free Mg²⁺ ions. The results indicate that digitonin-permeabilized promastigotes provide an ideal system for studying the mitochondrial ATPase of L. donovani.     

Paromomycin‐loaded albumin microspheres: Efficacy and stability studies

In the present work, paromomycin‐loaded albumin microspheres (PM‐MS) have been formulated for passive targeting of paromomycin (PM) to macrophages, for the treatment of visceral leishmaniasis (VL). PM‐MS were prepared by spray‐drying method with a mean particle size of ≈ 3 µm. Thermal and chemical cross‐linking methods were used for controlling drug release from the prepared microspheres (MS). PM‐MS were then tested for efficacy and stability studies. In efficacy study, in vitro promastigote assay was carried out to assess the susceptibility of promastigote to PM in the concentration range of 5.0–150 µg/ml; cytotoxicity assay was performed to determine possible toxicity of PM for the host cells (peritoneal macrophages) and intracellular amastigote assay was carried out to determine the efficacy of free PM (PM solution) and encapsulated PM (PM‐MS). Results obtained indicated a significant increase in efficacy of PM‐MS in comparison to PM solution at equivalent concentration. Subsequently, stability studies of prepared formulation was carried out at various temperature and humidity conditions, these studies provided stability of formulation at all tested conditions including accelerated conditions. Thus, it can be concluded that present work provides an optimized formulation with stability and enhanced efficacy. Copyright © 2012 John Wiley & Sons, Ltd.     

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。     

抗杜氏利什曼原虫单克隆抗体——蓖麻蛋白A链结合物的制备及对前鞭毛体的毒性初试

本文采用两种方法纯化抗体。方法一,经DE-52阴离子层析纯化获IgG;方法二,采用Sepharose-4B-Protein A亲和层析抗体,用纯化的McAb 2H_6-E_3与蓖麻毒蛋白A链偶连形成免疫毒素。体外将免疫毒素加入培养的杜氏利什曼原虫前鞭毛体内培养,经计数测定,可见免疫毒素对前鞭毛体有一定程度的抑制杀伤作用。     

Investigations on the in vitro metacyclogenesis of a visceral and a cutaneous human strain of Leishmania infantum

The in vitro metacyclogenesis of a visceral (VL) and cutaneous (CL) human strain of Leishmania infantum was monitored in order to find out the kinetics of this process and the in vitro infective capacity for macrophages of the metacyclic promastigotes developed. To identify, enumerate, and separate the metacyclic population, the complement-dependent lysis by normal serum and the agglutination by peanut agglutinin (PNA) were used, as they were shown to be useful for the purpose of this study. Maximum percentage of metacyclics was detected by both techniques on the 4th day of growth for VL and the 6th day for CL, and was higher for the VL strain. The in vitro infectivity for macrophages of two strains was assayed, and the high parasitization data obtained were transformed in order to determine the increase of the parasite burden for macrophages throughout the incubation time of the experiments (2–72 h post-infection (p.i.)). This parameter is denominated the infectivity ratio (%I) and calculated as follows: (number of intracellular parasites per infected macrophage at ‘x' time p.i./number of intracellular parasites per infected macrophage at 2 h p.i.)×100. When %I was calculated for promastigotes unagglutinated by PNA (PNA−)—metacyclic or infective promastigotes—at any time of culture, the %I at 72 h p.i. was always much higher than for agglutinated promastigotes (2.1–12.5 times)—non-infective promastigotes—and unfractionated promastigotes from culture (1.7–9.5 times), especially with VL parasites. Likewise, the %I for VL PNA− promastigotes from the 4th day of culture was 1.9 times higher than for CL PNA− promastigotes from the 6th day of culture. The higher resistance to lysis by serum, percentage of metacyclics (PNA−), and infectivity ratio of VL than CL could be related to a higher spreading capability into the host body associated with higher pathogenic effects of the visceral strain than the cutaneous one.     

In‐vitro antileishmanial potential of peptide drug hirudin

Hirudin is clinically an important drug used for the treatment of cardiac diseases, but has never been elucidated for antileishmanial potential. This study was designed to determine the therapeutic utility of hirudin against leishmaniasis. Binding affinities of 28 potent proteinase inhibitors were screened computationally against leishmanolysin (GP63), out of which hirudin exhibited higher binding affinity with GP63 and good expected IC₅₀ values. Experimentally, hirudin showed most promising activity against promastigote and axenic amastigote forms of leishmanial parasites with IC₅₀ values of 0.60 ± 0.36 μg/mL and 0.43 ± 0.23 μg/mL, respectively, in a dose‐ and time‐dependent assay. The cytotoxicity assay revealed no adverse effects on human macrophages with LD₅₀ value of 860.11 ± 53.44 μg/mL. Hirudin caused leishmanial cell death mainly by apoptosis and membrane permeability. In spite of the basic knowledge obtained, hirudin mechanism is considerably less prone to the induction of resistance than classical drugs. Collectively, this study fosters further studies for the hirudin as new antileishmania lead with a new mode of action.