Leishmania donovani secretory serine protease alters macrophage inflammatory response via COX-2 mediated PGE-2 production.

Leishmania parasites determine the outcome of the infection by inducing inflammatory response that suppresses macrophage’s activation. Defense against Leishmania is dependent on Th1 inflammatory response by turning off macrophages’ microbicidal property by upregulation of COX-2, as well as immunosuppressive PGE-2 production. To understand the role of L. donovani secretory serine protease (pSP) in these phenomena, pSP was inhibited by its antibody and serine protease inhibitor, aprotinin. Western blot and TAME assay demonstrated that pSP antibody and aprotinin significantly inhibited protease activity in the live Leishmania cells and reduced infection index of L. donovani-infected macrophages. Additionally, ELISA and RT-PCR analysis showed that treatment with pSP antibody or aprotinin hold back COX-2-mediated immunosuppressive PGE-2 secretion with enhancement of Th1 cytokine like IL-12 expression. This was also supported in Griess test and NBT assay, where inhibition of pSP with its inhibitors elevated ROS and NO production. Overall, our study implies the pSP is involved in down-regulation of macrophage microbicidal activity by inducing host inflammatory responses in terms of COX-2-mediated PGE-2 release with diminished reactive oxygen species generation and thus suggests its importance as a novel drug target of visceral leishmaniasis.

TLR4-mediated activation of MyD88 signaling induces protective immune response and IL-10 down-regulation in Leishmania donovani infection.

In visceral leishmaniasis, a fragmentary IL-12 driven type 1 immune response along with the expansion of IL-10 producing T-cells correlates with parasite burden and pathogenesis. Successful immunotherapy involves both suppression of IL-10 production and enhancement of IL-12 and nitric oxide (NO) production. As custodians of the innate immunity, the toll-like receptors (TLRs) constitute the first line of defense against invading pathogens. The TLR-signaling cascade initiated following innate recognition of microbes shapes the adaptive immune response. Whereas numerous studies have correlated parasite control to the adaptive response in Leishmania infection, growing body of evidence suggests that the activation of the innate immune response also plays a pivotal role in disease pathogenicity. In this study, using a TLR4 agonist, a Leishmania donovani (LD) derived 29 kDa β 1,4 galactose terminal glycoprotein (GP29), we demonstrated that the TLR adaptor myeloid differentiation primary response protein-88 (MyD88) was essential for optimal immunity following LD infection. Treatment of LD-infected cells with GP29 stimulated the production of IL-12 and NO while suppressing IL-10 production. Treatment of LD-infected cells with GP29 also induced the degradation of IKB and the nuclear translocation of NF-kB, as well as rapid phosphorylation of p38 MAPK and p54/56 JNK. Knockdown of TLR4 or MYD88 using siRNA showed reduced inflammatory response to GP29 in LD-infected cells. Biochemical inhibition of p38 MAPK, JNK or NF-kB, but not p42/44 ERK, reduced GP29-induced IL-12 and NO production in LD-infected cells. These results suggested a potential role for the TLR4-MyD88–IL-12 pathway to induce adaptive immune responses to LD infection that culminated in an effective control of intracellular parasite replication.

Topoisomerase research of kinetoplastid parasite Leishmania, with special reference to development of therapeutics.

Protozoan parasites of the order Kinetoplastida cause severe diseases primarily in the tropical and subtropical areas. The enormous development of molecular and cellular biology in recent times have provided opportunities for discovering newer molecular targets for drug designing, which now form a rational basis for the development of improved anti-parasitic therapy. DNA topoisomerases play a key role in cellular processes affecting the topology and organization of intracellular DNA. Recently, emergence of the bi-subunit topoisomerase I in the kinetoplastid family has brought a new twist in topoisomerase research related to evolution, functional conservation and as a potential target that can be exploited in drug designing and development of new intervention strategies. This review summarizes the biology of kinetoplastid topoisomerases, which are the key molecular targets in antileishmanial chemotherapy.

Diagnostic value of 5' Nucleotidase on kala - Azar patients

5'-Nucleotidase activity was measured in the sera of [67] Kala-azar patients before treatment and at different stages of treatment with pentostam as well as in [30] age matcher normal children. The changes in 5'-NT isoenzyme profile were also followed among the above cases. A change of the activity of the enzyme with the progress of the disease was observed. Our result suggest also that there are changes in the 5'-NT isoenzyme profile with the severity of the disease

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20 percent of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population

Biochemical changes in heat stressed promastigotes of Leishmania donovani.

On exposing promastigotes of L. donovani (Dd-8) to 34 degrees C for 30 hr, the flagella were shed, and size was decreased with 10% viability loss. The in vitro and in vivo infectivity of two forms was more or less similar. The 45Ca2+ uptake by the transformed cells was increased as compared to normal cells. Activity of 5'-nucleotidase was increased while activity of Mg(2+)-ATPase remained same. Parasite antioxidant enzymes were also significantly altered by heat shock. There was significant increase in superoxide dismutase, glutathione reductase and glutathione peroxidase. It was accompanied by decrease in ratio of reduced glutathione to oxidized glutathione.

A type 1 DNA topoisomerase from the kinetoplast hemoflagellate Leishmania donovani.

A type 1 DNA topoisomerase has been purified from the nuclei of the kinetoplast hemoflagellate Leishmania donovani using polyethylene glycol fractionation and chromatography on hydroxylapatite, phosphocellulose and phenylsepharose column. The relaxation activity is ATP independent. Mg2+ is an essential cofactor for the reaction with an optimum at 10 mM. Mg2+ can be substituted by Mn2+ at 5 mM concentration. The relaxation reaction exhibits a salt optimum at 100 mM KCl. The enzyme can not remove supercoils from positive superhelical DNAs nor can induce supercoiling of relaxed DNAs. The topoisomerase activity is associated with a polypeptide of molecular weight about 67 kDa as shown by sephacryl-S200 gel filtration and by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels.

Effect of heat-shock & nutritional stress on the expression of a neutral thiol protease in Leishmania donovani promastigotes.

The soluble intracellular protease was partially purified from L. donovani promastigotes. The activity of this enzyme increased with increase in temperature from 25 degrees C to 37 degrees C and was active optimally at 70 degrees C. This protease activity appeared to be decreased due to heat-shock of the promastigotes for 4 h at 37 degrees C and increased due to nutrient starvation. Inhibition of the protease by p-chloromercuribenzoate and iodoacetamide suggested that this enzyme could be a thiol protease.