Toll like receptor 2 and CC chemokine receptor 5 cluster in the lipid raft enhances the susceptibility of Leishmania donovani infection in macrophages.

In experimental visceral leishmaniasis the causative obligate protozoan parasite, L. donovani invades and multiplies inside of macrophages, one of the sentries of the mammalian immune system. The initial host-parasite interaction between the Leishmania promastigote and the macrophage takes place at the plasma membrane interface. To trace any possible interaction between Toll-like receptor 2 (TLR2) and CC chemokine receptor 5 (CCR5) during early Leishmania-macrophage interactions, it was observed that the expression of both TLR2 and CCR5 were significantly increased, along with their recruitment to the lipid raft. TLR2 silencing attenuates CCR5 expression and restricts L. donovani infection, indicating a regulatory role of TLR2 and CCR5 during infection. Silencing of CCR5 and TLR2 markedly reduced the number of intracellular parasites in macrophages by host protective cytokine responses, while raft disruption using β-MCD affected TLR2/CCR5 cross-talk and resulted in a significant reduction in parasite invasion. In vivo RNA interference of TLR2 and CCR5 using shRNA plasmids rendered protection in Leishmania donovani-infected mice. Thus, this study for the first time demonstrates the importance of TLR2/CCR5 crosstalk as a significant determinant of Leishmania donovani entry in host macrophages.

Functionality of drug efflux pumps in antimonial resistant Leishmania donovani field isolates.

The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.

Drug resistance mechanisms in clinical isolates of Leishmania donovani.

Leishmania are protozoan parasites distributed worldwide. About 1.5-2.0 million cases are reported in the world annually from this disease and the death toll is estimated to be 57,000. Along with Brazil, Sudan and Bangladesh, India contributes to 90 per cent of the global burden of visceral leishmaniasis (VL). The absence of effective vaccines and vector control programmes, makes chemotherapy the most widely used tool against leishmaniasis. Chemotherapy based on pentavalent antimonials has been used for more than 50 years and remains the mainstay for treatment of leishmaniasis. Clinical resistance to pentavalent antimonials, in the form of sodium antimony gluconate (SAG), has become a major problem in the treatment of kala-azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates although a lot of work has been carried out with Leishmania mutants selected in vitro by step-wise increasing drug concentration using the antimony related metal arsenic and more recently sodium antimony gluconate. We for the first time, investigated the molecular aspect of drug resistance in clinically confirmed sodium antimony gluconate resistant field isolates and found that the parasite evaded cytotoxic effects of therapy by enhanced efflux of drugs through overexpressed membrane proteins belonging to the superfamily of ABC (ATP-binding cassette) transporters. Additionally, our study also points towards cell surface changes in resistant isolates.

Childhood visceral leishmaniasis.

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and transmitted by the bite of infected sandfly Phlebotomus argentipes. Nearly half of the VL cases occur in children (childhood or paediatric VL). The clinical manifestations of childhood VL are more or less same as in the adults. Prolonged fever with anorexia and loss of appetite are the major presenting features. Marked enlargement of the spleen and liver (spleen larger than liver) with moderate to severe anaemia and changes in hair take place. Bacterial infection is a common coinfection and intestinal parasitic infestations are very common in children with VL. Liver function tests, blood, urine and stool may show abnormalities. Confirmation of diagnosis is made by demonstration of parasite by microscopic examination and culture of materials obtained by bone marrow aspiration or splenic puncture. Sodium antimony gluconate (stibogluconate) has been the drug of choice for over past 50 yr. Pentamidine isothionate, though effective is relatively toxic. Amphotericin B is the most effective drug for the treatment of VL. Miltefosine is the first-ever oral drug, is highly effective. Post kala-azar dermal leishmaniasis (PKDL) in children poses a therapeutic challenge. In the absence of an ideal vaccine for VL, control measures would essentially include prevention of transmission through vector control and community awareness.

Visceral leishmaniasis in the New World & Africa.

Visceral leishmaniasis in the New World, primarily found in northeastern Brazil, is caused by Leishmania chagasi. Compared to India, unusual features of Brazilian disease are the large number of asymptomatic infections versus symptomatic infections, and the apparent change from a zoonotic disease to a partially anthroponotic one. Visceral disease in Africa is caused by L. donovani as in India, but disease differs from that in India in being zoonotic rather than anthroponotic, and in the large numbers of patients who acquire post-kala-azar dermal leishmaniasis.

Immune response to Leishmania infection.

Antileishmanial immune response is shown to be host genotype dependent so that some inbred strains of mouse are susceptible while others are resistant. The resistance is conferred by T-helper type-1 (Th1) cells while the susceptibility is conferred by Th2 cells. Th1 cells secrete IL-2 and IFN-gamma but Th2 cells secrete IL-4, IL-5 and IL-10. It has been shown that IFN-gamma activates macrophages to express iNOS2, the enzyme catalyzing the formation of nitric oxide. Nitric oxide kills the intracellular amastigotes. In contrast, Th2 immune response limits the action of Th1 functions via IL-10 and IL-4, which deactivate macrophages helping intracellular parasite growth and disease progression. Being a parasite, Leishmania ensures its own survival by modulating host immune system either by inducing immunosuppression or by promoting pro-parasitic host functions. A detailed knowledge of this host-parasite interaction would help in designing prophylactic and therapeutic strategies against this infection.

Monoclonal antibody affinity purification of a 78 kDa membrane protein of Leishmania donovani of Indian origin and its role in host-parasite interaction.

Monoclonal antibodies were raised against pathogenic promastigotes of Leishmania donovani of Indian origin. Among these, one was used for immuno-affinity purification of a 78 kDa membrane protein present in both the amastigote and promastigote forms of the parasite. Results of immunoblot experiments with the anti-78 kDa antibody revealed that the protein was present only in parasites belonging to the L. donovani complex. The expression of the protein was observed to be the same during different phases of growth of the promastigotes. Therefore, the 78 kDa protein is neither stage-specific nor differentially regulated. Surface iodination and subcellular fractionation of the promastigotes indicated that the protein was localized on the cell surface. The 78 kDa protein was found to inhibit the binding of promastigotes to macrophages significantly, suggesting that it may play a role in the process of infection. Thus, here we report the purification of a surface protein of L. donovani of Indian origin, which may play an important role in the process of infection.