Browsing by Author "Nasib Singh"

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Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persist in in vitro and in vivo experimental models
(2005) Anuradha Dube; Nasib Singh; Shyam Sundar; Neeloo Singh
Ever since their discovery about 60 years ago as therapeutic agent for visceral leishmaniasis (VL) or kala-azar, pentavalent antimonials (Sb v) have remained the first line treatment of choice all over the world including India. But recently, the number of kala-azar patients unresponsive to sodium stibogluconate (SSG) therapy, is steadily increasing in India. In this study, three clinical isolates, of which two were from SSG unresponsive and one from SSG responsive patients were evaluated for their infectivity and for their chemotherapeutic responses in vitro (macrophage-amastigote system) and in vivo (in hamsters). Persistence of SSG resistance was also checked by repeated passages in vitro as well as in vivo. The drug resistant strains (2039 and 2041) did not respond to SSG therapy both in vitro as well as in vivo but strains 2001 and Dd8 showed full sensitivity to SSG treatment. All the four strains responded well to amphotericin B and miltefosine treatment both in macrophages and in hamsters. The specific chemotherapeutic responses of all the strains to SSG were consistently persistent after repeated passages in cultures and in vivo, which indicates that these isolates are truly refractory to SSG treatment in field conditions. Two isolates were also transfected with green fluorescent protein (GFP) for the development of in vitro assay for studying antileishmanial activities of new and reference drugs in macrophages by flow cytometry. © Springer-Verlag 2005.
Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening
(2009) Nasib Singh; Reema Gupta; Anil K. Jaiswal; Shyam Sundar; Anuradha Dube
Objectives: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. Methods: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites - both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates - were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. Results: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for ∼12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. Conclusions: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.