Monitoring of Intracellular Nitric Oxide in Leishmaniasis: Its Applicability in Patients with Visceral Leishmaniasis

Sarkar, Avijit and Saha , Piu and Mandal, Goutam and Mukhopadhyay, Debanjan and Roy , Susmita and Singh, Sunny Kumar and Das, Sukhen and Goswami, Rama Prosad and Saha, Bibhuti and Kumar, Deepak and Das, Padma and Chatterjee, Mitali (2011) Monitoring of Intracellular Nitric Oxide in Leishmaniasis: Its Applicability in Patients with Visceral Leishmaniasis. Cytometry Part A, 79A. pp. 35-45.

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    Abstract

    Nitric oxide (NO) has been demonstrated to be a principal effector molecule responsible for mediating intracellular killing of Leishmania parasites, the causative organism of leishmaniasis. As measurement of intracellular NO remains a challenge to biologists, we have developed a flow cytometric approach to perform real time biological detection of NO within Leishmania parasites and parasitized macrophages using a membrane permeable derivative of diaminofluorescein [4,5- diaminofluorescein diacetate (DAF-2DA)]. Initially, assay optimization was performed in Leishmania donovani promastigotes, assay specificity being confirmed using both a NO donor [S-nitroso-N-acetyl-penicillamine (SNAP)] and a NO scavenger [2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, C-PTIO]. Using 40 lM DAF- 2DA, basal levels of intracellular NO were measured which varied in different Leishmania species; addition of conventional anti-leishmanial drugs, antimony and miltefosine translated into a dramatic increase in DAF-2T fluorescence. Furthermore, the assay also measured levels of NO in macrophages, but needed a 20 fold lower concentration of DAF-2DA, being 2 lM. Following parasitization, levels of NO decreased which was normalized following treatment with anti-leishmanial drugs. Similarlymonocytes of patients with visceral leishmaniasis at disease presentation showeddecreased levels of NO which too reverted on completion of treatment. Taken together,this study opens new perspectives of research regarding monocyte function and provides a real time approach for monitoring the effect of anti-leishmanial compounds.

    Item Type: Article
    URI: http://www.eprints.iicb.res.in/id/eprint/55
    Subjects: Cell Biology & Physiology
    Divisions: Indian Institute of Chemical Biology
    Depositing User: Mr Shyamal Nath
    Date Deposited: 28 Sep 2011 19:41
    Last Modified: 13 Dec 2012 11:41
    Official URL: http://dx.doi.org/10.1002/cyto.a.21001
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