Camel nanobodies: Promising molecular tools against leishmaniasis.

AIM: To characterize several anti-Leishmania tropica nanobodies and to investigate their effect on Leishmania infection. METHODS: Several immunological tests were implied to characterize five different (as confirmed by sequencing) anti-L tropica nanobodies (NbLt05, NbLt06, NbLt14, NbLt24 and NbLt36) against parasite lysates or intact cells from different stages, promastigotes and amastigotes. Direct inhibitory effect of these nanobodies on parasite infection cycle on macrophages was tested in cell culture. RESULTS: All the five nanobodies (with distinguished characteristics) were more specific to L tropica than to L major, but could equally recognize the lysate and the outer surface of the intact cells from the two main stages of the parasite. Nanobodies recognized several leishmania antigens (majorly between 75 and 63 kDa), and their proteinaceous nature was confirmed. Because of its role in leishmania life cycle, gp63 was considered a potential antigen candidate for nanobodies, and bioinformatics predicted such interaction. All nanobodies have a negative effect on the infectivity of L tropica, as they decreased the number of infected macrophages and the amastigotes inside those macrophages. CONCLUSION: Such anti-leishmania nanobodies, with outstanding characteristics and important target, can be of great use in the development of promising treatment strategies against leishmaniasis.

Sequencing and Gene Expression Analysis of Leishmania tropica LACK Gene.

BACKGROUND: Leishmania Homologue of receptors for Activated C Kinase (LACK) antigen is a 36-kDa protein, which provokes a very early immune response against Leishmania infection. There are several reports on the expression of LACK through different life-cycle stages of genus Leishmania, but only a few of them have focused on L.tropica. METHODS: The present study provides details of the cloning, DNA sequencing and gene expression of LACK in this parasite species. First, several local isolates of Leishmania parasites were typed in our laboratory using PCR technique to verify of Leishmania parasite species. After that, LACK gene was amplified and cloned into a vector for sequencing. Finally, the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, was evaluated by Reverse Transcription-PCR (RT-PCR) technique. RESULTS: The typing result confirmed that all our local isolates belong to L.tropica. LACK gene sequence was determined and high similarity was observed with the sequences of other Leishmania species. Furthermore, the expression of LACK gene in both promastigotes and amastigotes forms was confirmed. CONCLUSION: Overall, the data set the stage for future studies of the properties and immune role of LACK gene products.