Figure 5

In vivo efficacy of mahanine in mice. (a) Female Balb/c mice were infected with stationary phase virulent AG83 promastigotes. Infection was established for 15 days and fed with vehicle control (DMSO) or mahanine or miltefosine for five consecutive days. Mice were sacrificed after four-day post feeding and splenic parasite burden was measured by stamp-smear method after Giemsa staining. (b) Giemsa-stained infected and treated splenic smears bearing amastigotes as indicated by white arrow (100 X under oil immersion lens). The slides were viewed and the image captured on an inverted bright-field microscope (IX73 inverted microscope; Olympus). (c) Splenocytes isolated from infected untreated and treated groups of mice were incubated for 3 days in presence of SLA and supernatant was collected. Nitrite was quantified by Griess reaction. (d) Splenocytes isolated from infected and treated groups were plated (1 × 10⁶/ml) in a six-well plate and incubated for 48 hr in presence of SLA. ROS generation was measured by adding H₂DCFDA in FACS. (e) Splenocytes from control and fed mice were plated in a 96 well plate and incubated for 3 days in presence of SLA. Cell proliferation was measured by MTT. (f) Total RNA was isolated from infected untreated and treated mice splenocytes and cDNA were prepared. cDNA was amplified by real-time PCR by using specific primers from IL-12 and iNOS and represented as fold changes compared to control. HGPRT was used for normalization.