A new model of progressive visceral leishmaniasis in hamsters by natural transmission via bites of vector sand flies.

BACKGROUND: Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. METHODS: We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. RESULTS: Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)-10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%-82%) rather than total GMPL (2.0 × 10(4)-8.0 × 10(4)) per midgut. CONCLUSIONS: This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL.

Electrospray encapsulation of toll-like receptor agonist resiquimod in polymer microparticles for the treatment of visceral leishmaniasis.

Leishmaniasis is a disease caused by the intracellular protozoan, Leishmania. A current treatment for cutaneous leishmaniasis involves the delivery of imidazoquinolines via a topical cream. However, there are no parenteral formulations of imidazoquinolines for the most deadly version of the disease, visceral leishmaniasis. This work investigates the use of electrospray to encapsulate the imidazoquinoline adjuvant resiquimod in acid sensitive microparticles composed of acetalated dextran (Ac-DEX) or Ac-DEX/Tween blends. The particles were characterized and tested both in vitro and in vivo. Solutions of Ac-DEX and resiquimod in ethanol were electrosprayed to generate approximately 2 µm Ac-DEX particles containing resiquimod with an encapsulation efficiency of 85%. To prevent particle aggregation, blends of Ac-DEX with Tween 20 and Tween 80 were investigated. Tween 80 was then blended with the Ac-DEX at ∼10% (w/w) of total polymer and particles containing resiquimod were formed via electrospray with encapsulation efficiencies between 40% and 60%. In vitro release profiles of resiquimod from Ac-DEX/Tween 80 particles exhibited the acid-sensitive nature of Ac-DEX, with 100% drug release after 8 h at pH 5 (phagosomal pH) and after 48 h at pH 7.4 (physiological pH). Treatment with Ac-DEX/Tween 80 particles elicited significantly greater immune response in RAW macrophages over free drug. When injected intravenously into mice inoculated with Leishmania, parasite load reduced significantly in the bone marrow compared to blank particles and phosphate-buffered saline controls. Overall, electrospray appears to offer an elegant, scalable way to encapsulate adjuvant into an acid sensitive delivery vehicle for use in treating visceral leishmaniasis.

fMLP receptor stimulated activation of macrophage: its effect on killing of intracellular Leishmania donovani.

The fMLP receptor of peritoneal macrophages stimulated by fMLP grafted liposomes as ligand, was analysed and compared with respective controls for its ability to promote killing of intracellular Leishmania parasites. fMLP grafted liposomes show greater efficacy in killing intracellular L. donovani (MHOM/IN/1983/AG83) parasites in a time dependent manner than free fMLP. fMLP grafted liposomes also release more active oxygen intermediates and reactive nitrogen intermediates (O2-, H2O2, NO) than free fMLP. The key enzymes PKC and PTK for the respiratory burst and nitric oxide generation were found to be important in this fMLP receptor mediated signaling process as the enzyme specific inhibitors viz. staurosporine, genistein and AG126 suppressed the leishmanicidal effect of fMLP grafted liposomes. The above findings suggest that the fMLP receptor of macrophages activates PKC and PTK mediated signalling that is responsible for the intracellular parasite killing.

Potentiating effects of MPL on DSPC bearing cationic liposomes promote recombinant GP63 vaccine efficacy: high immunogenicity and protection.

BACKGROUND: Vaccines that activate strong specific Th1-predominant immune responses are critically needed for many intracellular pathogens, including Leishmania. The requirement for sustained and efficient vaccination against leishmaniasis is to formulate the best combination of immunopotentiating adjuvant with the stable antigen (Ag) delivery system. The aim of the present study is to evaluate the effectiveness of an immunomodulator on liposomal Ag through subcutaneous (s.c.) route of immunization, and its usefulness during prime/boost against visceral leishmaniasis (VL) in BALB/c mice. METHODOLOGY/PRINCIPAL FINDINGS: Towards this goal, we formulated recombinant GP63 (rGP63)-based vaccines either with monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) or entrapped within cationic liposomes or both. Combinatorial administration of liposomes with MPL-TDM during prime confers activation of dendritic cells, and induces an early robust T cell response. To investigate whether the combined formulation is required for optimum immune response during boost as well, we chose to evaluate the vaccine efficacy in mice primed with combined adjuvant system followed by boosting with either rGP63 alone, in association with MPL-TDM, liposomes or both. We provide evidences that the presence of either liposomal rGP63 or combined formulations during boost is necessary for effective Th1 immune responses (IFN-γ, IL-12, NO) before challenge infection. However, boosting with MPL-TDM in conjugation with liposomal rGP63 resulted in a greater number of IFN-γ producing effector T cells, significantly higher levels of splenocyte proliferation, and Th1 responses compared to mice boosted with liposomal rGP63, after virulent Leishmania donovani (L. donovani) challenge. Moreover, combined formulations offered superior protection against intracellular amastigote replication in macrophages in vitro, and hepatic and splenic parasite load in vivo. CONCLUSION: Our results define the immunopotentiating effect of MPL-TDM on protein Ag encapsulated in a controlled release system against experimental VL.

Acid phosphatase activity of virulent and avirulent clones of Leishmania donovani promastigotes.

Virulent and avirulent clones of Leishmania donovani promastigotes were examined for their acid phosphatase activity. The acid phosphatase activity of whole-cell lysates of virulent clones was 1.5 to 2.0 times higher than that of avirulent clones. Pellet fractions (260,000 x g, 30 min) from sonicated promastigotes of a virulent clone and an avirulent clone contained 60 and 40% of the total enzyme activity, respectively. Membrane-bound acid phosphatase was extracted with Triton X-100 from the pellet. This membrane-bound phosphatase activity was 2.4-fold higher in virulent organisms than in avirulent organisms. The membrane acid phosphatase exhibited two distinct bands on polyacrylamide gels stained for enzyme activity. One diffuse, faster-migrating band showed identical electrophoretic mobility in both virulent and avirulent clones, although a higher enzymatic activity was observed with the extract from virulent cells. In contrast, a slower-migrating band was different between the two clones in the mobility. These results suggest that membrane-bound acid phosphatase was quantitatively and qualitatively different between virulent and avirulent promastigotes of L. donovani. In addition, virulent cells produced a relatively high level of acid phosphatase throughout the growth in culture.

Neoglycoprotein conjugated liposomes as macrophage specific drug carrier in the therapy of leishmaniasis.

The potential utility of neoglycoprotein conjugated multilamellar liposomes as macrophage specific drug delivery system was studied using hamycin as the model drug and visceral leishmaniasis as the model macrophage disease. Hamycin, a polyene antibiotic, was found to have a growth inhibitory effect on cultured Leishmania donovani promastigotes at a concentration of 0.05 microgram/ml. Hamycin entrapped in neoglycoprotein conjugated liposome (neohamysome) eliminated intracellular amastigotes of L. donovani in peritoneal macrophages 10 and 1.5 times more efficiently than did the free and liposome entrapped drug (hamysome), respectively. Moreover, neohamysome possibly could completely eliminate splenic intracellular parasites in a 45 day BALB/c mouse model of visceral leishmaniasis at a dose of 1.5 mg/Kg/day given for 4 consecutive days. Hamysome at a similar dose had 80% parasite suppressive effect whereas free drug could not be administered more than the dosage of 0.5 mg/Kg/day due to mortality problem. Neohamysome and hamysome were generally less toxic than the free drug as judged by erythrocyte lysis and several clinical parameters of liver toxicity. These results suggest a possible use of neoglycoprotein conjugated liposomes in macrophage-associated diseases.

Ligation of Fc receptor of macrophages stimulates protein kinase C and anti-leishmanial activity.

Fc receptors are known to express on the surface of mature monocytes, macrophages and lymphocytes. In this study a ligand e.g. liposomal IgG (human IgG coupled to PE-liposome via carbodimide reaction) was developed to ligate the Fc receptor of macrophages. When liposomal IgG was incubated with macrophages at 37 degrees C for 5 min, it induced the macrophage activation which suppress the parasite burden approximately to an extent of 60%, 50% and 45%, when macrophages were infected with UR6, AG83 and GE1 strains of L-donovani respectively. Superior efficacy of liposomal IgG were achieved compared to the treatment with free IgG and free liposomes. The activity of protein kinase C (PKC) has been found to be higher in the Fc receptor targeted macrophage membrane fraction, suggesting its translocation from the cytosol. Staurosporine, a potent inhibitor of the enzyme protein kinase C (PKC) has been found to protect the parasite inside the macrophage indicating the role of PKC in the signaling process. The liposomal IgG treatment has been found to induce the generation of significant amount of superoxide and hydrogen peroxide which helped to suppress the parasite burden. Further when liposomal IgG were incubated with IFN-gamma primed, LPS activated macrophages, a significant amount of NO release was also noticed, indicating its role in parasite killing. The above results suggest that Fc receptor mediated activation by liposomal IgG may be used as an alternative approach to kill parasites intracellularly.