Article Figures & Data
Figures
- FIG. 1.
Representative in vitro phagocytosis of C. neoformans by BMDM. BMDM were incubated with FITC-labeled C. neoformans strain H99 and monoclonal antibody E1 (1.5 μg/106 yeasts) for 90 min at a BMDM/yeast ratio of 3:1. (A) Epifluorescence microscopy. The bright FITC staining is partially quenched after phagocytosis, allowing the distinction between free yeasts (thin arrow), intracellular yeasts (arrowheads), and adherent yeasts (large arrow). Uninfected BMDM (black stars) are also visible. Magnification, ×1,000. (B to D) Flow cytometry analysis of CD11b-APC-labeled BMDM (B), FITC-labeled C. neoformans H99 (C), and BMDM-H99 inoculum prepared as described above (D). CD11b-APC-labeled BMDM are seen in gate R1, free FITC-labeled yeasts (FITChigh APC− cells) are seen in R2, phagocytosed yeasts (FITClow APC+ cells) are seen in R3, and BMDM-adhering yeasts or just internalized yeasts (FITChigh/intermediate APC+ cells) are seen in R4.
- FIG. 2.
Effect of inoculation with yeasts internalized in monocytes on C. neoformans tissue burden. Mice were inoculated with 2 × 104 BMDM-H99 or 2 × 104 free H99 (free-H99) and sacrificed 1, 6, and 24 h after inoculation. Results are expressed as ratios of the mean CFU/g of organ for the experimental group (BMDM-H99) to that for the control group (free-H99) (data are means ± SD for three mice in each group). The dotted line denotes a ratio of 1. Two independent experiments included the three time points, and the last one included only the 1-h and 24-h study points. *, P < 0.05.
- FIG. 3.
Effect of established cryptococcal infection on brain invasion by a second isolate of C. neoformans. Inoculation with 2 × 104 free KN99α NAT or BMDM-KN99α NAT cells was done on day 0 to naïve mice (free-d0 or BMDM-d0 group) or mice injected 2 days before with 2 × 106 KN99α cells (free-d2 or BMDM-d2 group), as summarized in the table. Mice were sacrificed 1, 6, and 24 h thereafter. Results are expressed as CFU/g of brain (data are means ± SD for three mice). Only CFU data resulting from KN99α NAT inoculation are presented.
- FIG. 4.
Effects of delayed and sustained monocyte depletion on the course of cryptococcal infection. (A) Mice were infected with 105 H99 yeasts and treated by daily i.v. infection with clodronate liposomes (depleted group) or with PBS (control group) for 4 days. Macroscopic examination of representative organs from mice in the control group showed multiple abscesses in the spleen (B) and hemorrhages in the brain (D), but mice in the depleted group showed no visible lesions in the spleen (C) or the brain (E). (F) Ratios of fungal loads measured in the spleens, lungs, and brains of depleted and control mice (data are means ± SD for three animals per group from three independent experiments).
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Table S1. Comparison of fungal burdens in mice infected with C. neoformans strain H99 internalized in monocytes or free.
Table S2. Comparison of fungal burdens in clodronate-treated and control mice after inoculation with C. neoformans strain H99.
MS Word document, 72K. - Supplemental file 2
-
Fig. S1. Effect of inoculation with a mixture of yeasts internalized in monocytes and free C. neoformans on brain tissue burden.
Zipped TIF file, 24K. - Supplemental file 3
-
Legend for Fig. S1.
MS Word document, 25K.
- Supplemental file 1
-
Table S1. Comparison of fungal burdens in mice infected with C. neoformans strain H99 internalized in monocytes or free.
