Figure 1.
Rationale for selection of the time for establishing the ex vivo splenic explant culture.
Hamsters infected with 106 Luc-transfected L. donovani were evaluated from 7 to 21 days post infection (n = 6 per time point). (A) Spleen weight. Shown is the mean ± standard deviation (SD) of the spleen to body weight ratio (spleen weight divided the body weight). (B) Splenic parasite burden. The number of amastigotes (mean ± SD) was determined by luminometry in 500,000 splenocytes by extrapolating the counts (photons/sec) to a standard curve of microscopy-enumerated spleen-derived amastigotes. (C) Total splenocyte number. Splenocyte number (mean ± SD) was determined by counting the cells by microscopy. (D) Splenocyte lymphoproliferative response. The splenocyte stimulation index (shown as the mean ± SD) was determined by dividing the cpm of concanavalin A-stimulated and non-stimulated splenocytes. (E) Splenic soluble collagen content. The soluble collagen content (shown as the mean ± SD) was determined in spleens from uninfected and infected hamsters by the Sircol assay (Biocolor). (F) Splenic Arginase activity. Tissue arginase activity was determined by measurement of urea catalysis and is shown as the mean ± SD. Statistical analysis for all panels was performed by one-way analysis of variance (ANOVA).
Table 1.
Cellular composition of the splenic explant culture from hamsters infected with L. donovani.
Figure 2.
Characterization of the splenic explant cultures.
(A) Representative amastigote standard curve. Correlation between the number of L. donovani amastigotes counted by microscopy and the luciferase activity determined by luminometry. (B) Amastigote replication in splenic explant cultures. Number of amastigotes in ex vivo explants cultures determined by luminometry and interpolation from the standard curve over 0 to 72 hours of incubation (100,000 splenocytes per well). (C) Percent of infected macrophages in splenic ex vivo cultures. Splenocytes harvested from infected hamsters (21 days p.i.) were plated and the proportion of infected macrophages (shown as the mean ± SD) was determined by microscopy at 0, 24, 48, and 72 hours of ex vivo culture. (D) Number amastigotes per 100 macrophages. Amastigotes enumerated by direct microscopy in Giemsa stained cytospin slides (mean ± SD in 4 different samples per time point). (E, F) Infected splenocytes in ex vivo culture. Representative Giemsa-stained photomicrograph of splenocytes from hamsters infected with L. donovani at pre-culture (E) and after 48h of ex vivo culture (F).
Table 2.
Determination of splenocyte numbers required for the ex vivo assay.
Table 3.
Hit and lead compounds identified from chemical libraries using the ex vivo splenic explant system.
Table 4.
Lead compounds newly identified or previously known to have anti-leishmanial activity.
Table 5.
Therapeutic category of anti-leishmanial compounds identified in the ex vivo splenic explant system.
Table 6.
Comparative anti-Leishmania donovani activity of lead compounds in the ex vivo splenic explant model and in vitro infected peritoneal macrophages.