by Leila M. Lopes Bezerra, and Scott G. Filler Interactions of Aspergillus fumigatus with endothelial cells: internalization, injury, and stimulation of tissue factor activity by Leila M. Lopes Bezerra, and Scott G. Filler Blood Volume 103(6):2143-2149 March 15, 2004 ©2004 by American Society of Hematology

Interactions of A fumigatus conidia and germ tubes with endothelial cells. Interactions of A fumigatus conidia and germ tubes with endothelial cells. Photomicrographs of endothelial cell monolayers infected with A fumigatus H237 conidia (A-C) and germ tubes (D-F) after 30 minutes (A,D), 3 hours (B,E), and 8 hours (C,F). Original magnification × 20. Arrows indicate the organisms. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Endocytosis of A fumigatus by endothelial cells. Endocytosis of A fumigatus by endothelial cells.A fumigatus H237 conidia and germ tubes were incubated with endothelial cells for 45 minutes in the presence (□) or absence (▪) of the microfilament inhibitor, cytochalasin D. The number of organisms (orgs) endocytosed by the endothelial cells was determined by a differential fluorescence assay. Results are mean ± standard deviation of 3 experiments. *P < .001 compared with cells without cytochalasin D; §P < .05 compared with conidia. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Confocal microscopy of actin filaments surrounding endocytosed organisms. Confocal microscopy of actin filaments surrounding endocytosed organisms. Endothelial cells were incubated for 45 minutes with A fumigatus H237 conidia (A-B) or germ tubes (C-D), and then stained for filamentous actin. (A,C) Confocal images of the filamentous actin. (B,D) Differential interference contrast images of the same microscopic fields shown in panels A and C, respectively. Arrows indicate endothelial cell actin polymerizing around the organisms (A,C) and the organisms themselves (B,D). Original magnification × 1000. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Endothelial cell injury induced by A fumigatus. Endothelial cell injury induced by A fumigatus. (A) Time course of injury. Endothelial cells were incubated with conidia of A fumigatus H237 for the indicated times, and the extent of endothelial cell injury was determined by the specific release of 51Cr as described in “Materials and methods.” (B) Extent of injury caused by different clinical isolates. Endothelial cells were infected with conidia of the indicated clinical isolates of A fumigatus for 8 hours, after which endothelial cell injury was measured. (C) Comparison of endothelial cell injury caused by live and thimerosal-killed conidia and hyphae of A fumigatus H237 after 8 hours. Results are means ± standard deviation of at least 3 independent experiments. Error bars indicate standard deviation. *P < .001 versus uninfected control wells; §P < .05 versus live organisms; †P < .001 versus conidia. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Effects of cell contact and endocytosis on endothelial cell injury caused by A fumigatus. Effects of cell contact and endocytosis on endothelial cell injury caused by A fumigatus. (A) Extent of endothelial cell injury caused by conidia of A fumigatus H237 that were added directly onto endothelial cell monolayers in a 24-well plate and/or into 0.4 μm cell culture inserts suspended above the monolayers. (B) Extent of endothelial cell injury caused by A fumigatus H237 conidia in the presence of the indicated concentrations of the microfilament inhibitor, cytochalasin D. Endothelial cell injury was measured after 8 hours of infection and the results are the mean ± standard deviation of at least 3 independent experiments. Error bars indicate standard deviation. *P < .001 compared with control. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Tissue factor activity of endothelial cells incubated with A fumigatus. Tissue factor activity of endothelial cells incubated with A fumigatus. Time course of tissue factor activity of endothelial cells incubated for 4, 8, and 16 hours with viable and nonviable conidia or germ tubes of A fumigatus. Endothelial cells were also stimulated with LPS as a positive control (insert). Tissue factor activity was expressed as fold increase (stimulation index) relative to the basal level of activity of unstimulated cells. The results are means ± standard deviation of at least 3 independent experiments. Error bars indicate standard deviation. *P < .05 compared with control. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology

Expression of tissue factor on the surface of infected endothelial cells. Expression of tissue factor on the surface of infected endothelial cells. Endothelial cell monolayers grown on glass coverslips were incubated for 8 hours with tissue culture medium (A), LPS (B), or germ tubes of A fumigatus (C-D). The expression of tissue factor on the endothelial cell surface was detected by confocal microscopy using an anti–tissue factor monoclonal antibody (A,C). Panel D shows the differential interference contrast image corresponding to the same field as panel C. Results are representative of 3 independent experiments. Original magnification × 400. Leila M. Lopes Bezerra, and Scott G. Filler Blood 2004;103:2143-2149 ©2004 by American Society of Hematology