Murine Model Of Visceral Leishmaniasis Research Articles

Treatment of visceral leishmaniasis with sterically stabilized liposomes containing camptothecin.

The efficacy of 20(S)-camptothecin (CPT), free and incorporated into sterically stabilized liposomes, has been investigated in vitro against Leishmania donovani promastigotes and in vivo in a murine model of visceral leishmaniasis. Incubation of L. donovani promastigotes with free or liposomal CPT inhibited the growth of parasites in a dose-dependent manner. Tissue distribution studies revealed that the intraperitoneal administration of liposomal CPT was efficient for the delivery of high drug levels to the liver and spleen. Treatment of infected mice with intraperitoneal injections of free and liposomal CPT significantly reduced the parasite loads in the livers by 43 and 55%, respectively, compared with the loads for untreated controls. However, both treatments caused normochromic anemia and neutropenia.

Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events.

Leishmania donovani, an intracellular protozoan parasite, challenges host defense mechanisms by impairing the signal transduction of macrophages. In this study we investigated whether interleukin-10 (IL-10)-mediated alteration of signaling events in a murine model of visceral leishmaniasis is associated with macrophage deactivation. Primary in vitro cultures of macrophages infected with leishmanial parasites markedly elevated the endogenous release of IL-10. Treatment with either L. donovani or recombinant IL-10 (rIL-10) inhibited both the activity and expression of the Ca2+-dependent protein kinase C (PKC) isoform. However, preincubation with neutralizing anti-IL-10 monoclonal antibody (MAb) restored the PKC activity in the parasitized macrophage. Furthermore, we observed that coincubation of macrophages with rIL-10 and L. donovani increased the intracellular parasite burden, which was abrogated by anti-IL-10 MAb. Consistent with these observations, generation of superoxide (O2-) and nitric oxide and the release of murine tumor necrosis factor-alpha were attenuated in response to L. donovani or rIL-10 treatment. On the other hand, preincubation of the infected macrophages with neutralizing anti-IL-10 MAb significantly blocked the inhibition of nitric oxide and murine tumor necrosis factor-alpha release by the infected macrophages. These findings imply that infection with L. donovani induces endogenous secretion of murine IL-10, which in turn facilitates the intracellular survival of the protozoan and orchestrates several immunomodulatory roles via selective impairment of PKC-mediated signal transduction.

Identification of vaccine candidates for experimental visceral leishmaniasis by immunization with sequential fractions of a cDNA expression library.

Visceral leishmaniasis caused by the intracellular parasite Leishmania donovani is a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in an L. donovani amastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries ( approximately 2,000 cDNAs/sublibrary). Following systemic challenge with L. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encoded L. donovani histone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates.

Therapeutic evaluation of free and liposome-encapsulated atovaquone in the treatment of murine leishmaniasis

The use of drug delivery systems may reduce the toxicity and improve the activity of anti-leishmanial compounds. The activity of atovaquone (ATV)-loaded liposomes was compared by determination of median effective doses (ED 25 and ED 50), with that of free ATV in a murine model of visceral leishmaniasis induced by Leishmania infantum. On day 0, mice were infected intravenously with 4.10 7 promastigotes and treated via the tail vein on days 15, 17 and 19 by free drug in a DMSO/cremophor/water solution (0.2 to 1.6 mg/kg body weight) or by liposomal drug (0.04 to 0.32 mg/kg body weight). Mice were killed and livers and spleens were removed and weighed on day 21 p.i. and liver parasite burdens evaluated using the Stauber method. Effective doses were determined using the Hill representation relating the percentage of parasite suppression to the dose. Liposomal ATV was significantly more effective than the free drug in reducing liver parasites (61.6% of parasite suppression at a dose of 0.32 mg/kg vs 34.9% at a dose of 1.6mg/kg). Liposomal ATV was 23 times more active than the free drug (ED 25 value=0.02±0.01 mg/kg vs 0.46±0.15 mg/kg for free drug). It was not possible to obtain the ED 50 for free ATV because the dose-response curve reached a plateau around 33% of parasite suppression. Conversely, the ED 50 for liposomal ATV was 0.17±0.05 mg/kg. 100% efficacy of bound ATV could be obtained with a concentration of 1.77±0.35 mg/kg. A significant decrease in spleen weights was also observed reflecting a leishmanicidal activity of ATV. These results suggest that liposome loaded ATV is more efficacious than the free drug against Leishmania infantum in this murine model.

Dendritic Cell (DC)-Based Anti-Infective Strategies: DCs Engineered to Secrete IL-12 Are a Potent Vaccine in a Murine Model of an Intracellular Infection

Infections with intracellular pathogens such as Leishmania donovani and Mycobacterium tuberculosis pose serious health problems worldwide. Effective vaccines for these pathogens are not available. Furthermore, despite optimal therapy, disease progression is often seen with several intracellular infections. For these reasons, we initiated studies to develop novel anti-infective vaccine and treatment strategies that couple the potent Ag-presenting capacity of dendritic cells (DC) with paracrine delivery of potent anti-infective cytokines such as IL-12 to local immune response sites. We tested this strategy in a murine model of visceral leishmaniasis. Adoptive transfer of DCs pulsed ex vivo with soluble L. donovani Ags (SLDA) to naive mice induced the Ag-specific production of IFN-gamma, and increased the percentage of activation markers on spleen lymphocytes. SLDA-pulsed DCs engineered by retroviral gene transfer techniques to secrete high levels of biologically active murine IL-12 augmented this immune response further. In several different vaccination and immunotherapy protocols, compared with sham-treated mice, animals receiving SLDA-pulsed DCs either before or following infection had 1-3 log lower parasite burdens, and this protection was associated with a pronounced enhancement in the parasite-specific IFN-gamma response. The augmentation of this protection by IL-12-engineered DCs was striking. First, live parasites were not detected in the liver of mice vaccinated with IL-12-transduced, SLDA-pulsed DCs. Second, this parasitological response was associated with a nearly normal liver histology. In contrast, parasites and granulomas were found in mice vaccinated with SLDA-pulsed, nontransduced DCs. Collectively, these studies provide the rationale for the development of potent DC-based immunotherapies.

Vaccination of Swiss Albino mice against experimental visceral leishmaniasis with the FML antigen of Leishmaniadonovani

The FML antigen of Leishmania donovani in combination with saponin, aluminum hydroxide (Al(OH) 3) and Freund's incomplete adjuvant (FIA) was used in vaccines tested in an outbred murine model of visceral leishmaniasis, either through intraperitoneal or subcutaneous routes. The humoral response was significantly higher in the groups treated with FML+saponin or FML+Al(OH) 3 than in controls, both before and after the infection. Animals immunized by the i.p. route developed higher antibody titres. A significant and specific reduction of parasitic load in relation to saline (85%, p<0.01) and saponin ( p<0.025) controls, was seen in animals treated with FML+saponin by the i.p. Coincidentally with this reduction, an increase in antibodies of the IgG2a subtype was detected only in animals treated with FML+saponin i.p. A reduction of 88% in parasitic load was achieved by the combination of FML+Al(OH) 3 (s.c.), but the Al(OH) 3 treatment itself accounted for 68% of this protection. In our conditions, vaccination with FML+saponin i.p. was superior to other treatments and had no toxic effect due to saponin.

Influence of the host and parasite strain in a mouse model of visceral Leishmania infantum infection

We investigated the respective roles of the host and parasite strain in a murine model of visceral leishmaniasis. Balb/c and C57Bl/6 mice were selected for their respective `non cure' and `cure' haplotypes vis-a-vis Leishmania major. Mice were infected with 10 7 stationary-phase promastigotes of four strains of Leishmania infantum with different infection profiles in mice: visceralization or regulation, as established by Sulahian et al. (Sulahian et al. (1998) FEMS Immunol. Med. Microbiol. 17, 131–138). The infection was monitored by measuring parasite load in the liver and spleen on days 9, 22, 44 and 87 post-infection, using a sensitive microtitration technique. Similar profiles (visceralizing or regulating) were observed in the two mouse strains, suggesting a predominant role of the Leishmania strain in the visceralization process. The host response was assessed by analyzing the granulomatous response in the liver and by quantifying specific IgG, IgG1 and IgG2a as a marker of the Th1/Th2 immune response. A granulomatous response was observed in both strains of mice but was more pronounced with visceralizing strains of L. infantum and in C57Bl/6 mice compared to Balb/c mice. The kinetics of anti- Leishmania IgG antibody production was similar in all the groups, but the distribution of IgG1 and IgG2a isotypes was different between the two mouse strains: Balb/c mice had a predominantly Th2-like response whereas C57Bl/6 had a mixed Th1/Th2-like response. This study demonstrates the determining role of both the parasite and mouse strain in the outcome of L. infantum infection. The Th1/Th2 concept does not seem to explain susceptibility and resistance to infection in our model of visceral L. infantum infection, contrary to the L. major model.

Activity of pentamidine-loaded poly (D,L-lactide) nanoparticles against Leishmania infantum in a murine model.

The activity of pentamidine-loaded poly(D,L-lactide) nanoparticles was compared, by determination of median effective doses (ED50), to that of free pentamidine in a murine model of visceral leishmaniasis induced by Leishmania infantum. BALB/c mice were infected intravenously on day O with promastigotes and then treated on days 14, 16, and 18. Groups of 5 mice received either 0.57, 1.14 and 2.28 mg/kg of free pentamidine (expressed in pentamidine base) or 0.055, 0.11, 0.22 and 0.44 mg/kg of pentamidine-loaded nanoparticles. In the control group, 12 mice received normal saline. The liver parasite burden was evaluated using the Stauber method 72 h after the last injection and drug levels in livers and spleens were determined. Bound pentamidine was 3.3 times more active than free drug (ED50 value = 0.32 mg/kg versus 1.05 mg/kg for free drug). Drug levels showed a weak accumulation in hepatic and splenic tissues following bound pentamidine administration. A lack of acute toxicity was noted in all groups treated by bound pentamidine. Results obtained with this biodegradable carrier may be of particular interest as no new major antileishmanial compound is today available.

Activity of a new liposomal formulation of amphotericin B against two strains of Leishmania infantum in a murine model

The efficacy of a new liposomal formulation of amphotericin B was compared to that of amphotericin B deoxycholate (Fungizone) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Median effective doses (ED50) were determined with two different strains: strain 1 was obtained from an untreated patient, and strain 2 was obtained from a patient who had received 12.5 g of amphotericin B over 3 years. BALB/c mice were infected intravenously on day 0 with promastigotes and then treated on days 14, 16, and 18 (strain 1) or on days 21, 23, and 25 (strain 2) with the liposomal formulation of amphotericin B (five doses were tested for each strain: 0.05, 0.1, 0.5, 0.8, and 3 mg/kg of body weight) or with conventional amphotericin B (four doses were tested for each strain: 0.05, 0.1, 0.5, and 0.8 mg/kg). Mice in the control group received normal saline solution. The liposomal amphotericin B formulation was about three times more active than the conventional drug against both strains. ED50 of the liposomal formulation were 0.054 (strain 1) and 0.194 (strain 2) mg/kg. ED50 of conventional amphotericin B were 0.171 (strain 1) and 0.406 (strain 2) mg/kg. Determination of drug tissular levels, 3 days after the last drug administration, showed a drug accumulation in hepatic and splenic tissues much higher after administration of liposomal amphotericin B than after conventional amphotericin B. A lack of toxicity was noted in all groups treated with the liposomal formulation.

Protective effect of neoglycoprotein-conjugated muramyl dipeptide against Leishmania donovani infection: the role of cytokines.

Active targeting of muramyl dipeptide (MDP) to macrophages was studied by conjugation with the neoglycoprotein, mannosyl human serum albumin (mannose-HSA) using visceral leishmaniasis as the model macrophage disease. Conjugation did not decrease the affinity of the neoglycoprotein for macrophage mannose receptor. Mannose-HSA-MDP was 50 times more efficient than free MDP in inhibiting the growth of Leishmania donovani inside peritoneal macrophages. Moreover, in a 60-day murine model of visceral leishmaniasis, 95% of the spleen parasite burden was reduced by mannose-HSA-MDP at a dose of 0.5 mg/kg/day given for 4 days. Free MDP at a similar dose had very little effect. In vitro exposure of MDP caused enhanced generation of O2- by macrophages, whereas generation of nitric oxide (NO) was not induced. The elevated antileishmanial activity of MDP-treated macrophages in culture was abrogated by O2- scavengers. In contrast, considerably enhanced amounts of NO and O2- were generated from macrophages of mannose-HSA-MDP-treated animals, and their splenocytes secreted soluble factors providing all the signals required for the induction of NO biosynthesis. The increase in NO production was paralleled by a concomitant increase in antileishmanial activity, which was reversed by NO synthesis inhibitors. Splenocyte supernatants treated with anti-IFN-gamma or anti-TNF-alpha Abs suppressed inducible NO generation by macrophages. Moreover, i.v. administration of anti-IFN-gamma and anti-TNF-alpha along with mannose-HSA-MDP greatly reduced protection against L. donovani infection. Neoglycoprotein-conjugated MDP, therefore, activated mouse macrophages in vivo to kill L. donovani, and this may depend on the physiologic generation of NO induced by IFN-gamma and TNF-alpha.

Pentavalent antimony-mannan conjugate therapy of experimental visceral leishmaniasis.

Pentavalent antimony-mannan (Sb[V]-mannan) was 10-fold more potent than sodium stibogluconate in a murine model of visceral leishmaniasis. Liver antimony concentrations were six-fold higher after Sb[V]-mannan therapy compared with a dose of sodium stibogluconate that was equipotent in reducing liver parasite burdens. Murine toxicity of Sb[V]-mannan was variable, with a 50% lethal dose (LD50) for one preparation that was well above the concentration that killed 90% of the parasites, and for another preparation was only modestly higher than the concentration that killed 90% of the parasites.

Lipid formulations of amphotericin B in the treatment of experimental visceral leishmaniasis due to Leishmania infantum

Despite significant antileishmanial activity of amphotericin B (AmB) in vitro, the use of the deoxycholate formulation (Fungizone®) is limited because of serious side effects. Lipid formulations of AmB have been proposed to reduce this toxicity. We compared the tolerance and efficacy of the conventional AmB prepared with deoxycholate, AmB emulsified in Intralipid® 20%, amphotericin B lipid complex (Abelcet®), and liposomal AmB (AmBisome®) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Control groups included untreated mice and mice treated with the pentavalent antimonial (Glucantime®). Balb/C mice were infected intravenously on day 0 with 10 7 promastigotes of L. infantum, then treated from days 7 to 17 (early treatment group) or from days 60 to 70 (delayed treatment group). Glucantime® was administered daily by intraperitoneal injection, whereas AmB formulations were administered intravenously on alternate days. On days 20, 60 and 120 in the early treatment group and 72 and 125 in the delayed treatment group, parasite burdens were determined in liver, spleen, and lungs by subculturing using a microtitration method. Abelcet® (12 mg/kg) and AmBisome® (12 mg/kg) completely eradicated the parasites from the tissues. Both of these lipid formulations enabled higher dosages to be tolerated, and were remarkably more effective than Fungizone® (0·8 mg/kg) and AmB diluted in Intralipid 20% (1.2 mg/kg) in the treatment of murine visceral leishmaniasis due to L. infantum.

Therapy of visceral leishmaniasis due to Leishmania infantum: experimental assessment of efficacy of AmBisome.

The tolerance and efficacy of amphotericin B (AmB) deoxycholate (Fungizone) were compared with those of liposomal AmB (AmBisome) in a murine model of visceral leishmaniasis induced by Leishmania infantum. Control groups consisted of untreated mice and mice treated with a pentavalent antimonial (Glucantime). BALB/c mice were infected intravenously on day 0 with 10(7) promastigotes of L. infantum and then treated from day 7 to 17 (early treatment group) or from day 60 to 70 (delayed treatment group). The pentavalent antimonial was administered daily by intraperitoneal injection, whereas AmB formulations were administered intravenously on alternate days. On days 20, 60, and 120 (early treatment group) and on days 72 and 125 (delayed treatment group), parasite burdens in the liver, spleen, and lungs were determined by subculturings using a microtitration method. A dose range study showed that administration of AmBisome at the well-tolerated doses of 5 or 50 mg/kg of body weight completely eradicated the parasites from the tissues. At 0.8 mg/kg, AmBisome proved more efficacious than AmB deoxycholate administered at the same dose. We also compared the levels of AmB deoxycholate and AmBisome in plasma and tissue. Mice treated with AmBisome had levels of AmB in tissue much higher than did AmB deoxycholate-treated mice with persistent detectable levels 14 weeks after treatment. These results seem to account for the remarkable efficacy of the liposomal formulation of AmB in the treatment of visceral leishmaniasis due to L. infantum.