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1.
FIG. 2.

FIG. 2. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

P. aeruginosa traversal of multilayered human corneal epithelial cells in vitro. PAO1 (106 CFU) was added to the apical compartment, and viable counts were taken from the apical (white bar) and basal (black bar) compartments at 2, 4, 6, and 8 h postinoculation. P. aeruginosa traversal was detected at each time point. Data from one of three independent experiments are shown.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
2.
FIG. 8.

FIG. 8. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

Viable P. aeruginosa strain PAO1 in the tear fluid of mBD-3−/− versus wild-type C57BL/6 mice at 3 h (A) or 6 h (B) postinoculation with ∼109 CFU bacteria in vivo using the null infection clearance model (n = 6 to 8 mice per group) (43). Data are expressed as a median (lower quartile:upper quartile). *, P < 0.03 by a Mann-Whitney test. Data from one of three independent experiments are shown.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
3.
FIG. 6.

FIG. 6. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

(A and B) Confocal reflection microscopy of mBD-3−/− (A) or wild-type (B) C57BL/6 mouse eyes at 4.5 h after inoculation ex vivo with ∼109 CFU P. aeruginosa strain PAO1-GFP (green). Eyeballs were carefully enucleated, rinsed with PBS, and then tissue paper blotted before bacterial challenge (see Materials and Methods). z-stack images were split into an orthogonal view to show x, y, and z planes of the corneal epithelium. Bacteria showed greater adherence to mBD-3−/− corneas (red) but did not traverse the epithelium. (C) These differences were clearly shown by quantifying the bacterial distribution over the corneal epithelium (red indicates the reflection of corneal epithelial cells at 633 nm). Magnification, ×∼1,000.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
4.
FIG. 1.

FIG. 1. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

Schematic diagram (A) and immunofluorescence microscopy pictures (B) illustrating the in vitro traversal model. Human corneal epithelial cells were grown air lifted on Transwell tissue culture inserts (3-μm pore size) under high-calcium conditions (1.15 mM) to induce a polarized multilayered epithelium. To measure bacterial traversal, bacteria were added only to the apical compartment, and bacteria traversing to the basal compartment were enumerated at various times postinoculation. Fluorescence microscopy using rhodamine-conjugated phalloidin (red) to label the actin cytoskeleton and DAPI (blue) to label cell nuclei showed that the cells formed a multilayered epithelium to which P. aeruginosa (FITC labeled using antibody to PAO1 [green]) can adhere and traverse (B). Magnification, ∼×1,000. Bar, 10 μm.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
5.
FIG. 3.

FIG. 3. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

P. aeruginosa in vitro traversal assay using multilayered human corneal epithelia that were preexposed to PAO1 culture supernatant (1:5 dilution in KGM-2 medium) for 4, 8, or 12 h before inoculation with 106 CFU of strain PAO1 for 8 h in the continued presence of the supernatant. Controls were treated with KGM-2 medium only. Viable counts were taken from the apical compartment (A), internalized bacteria were assessed by gentamicin survival (B), and viable traversed bacteria (basal compartment) were counted (C). *, P < 0.001 in each instance versus untreated controls (ANOVA with Fisher PLSD post hoc analysis). Data representative of three experiments are shown. Note that the x axis represents different supernatant pretreatment times prior to PAO1 inoculation.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
6.
FIG. 7.

FIG. 7. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

(A and B) Confocal reflection microscopy of mBD-3−/− (A) or wild-type (B) C57BL/6 mouse eyes at 7.5 h after inoculation ex vivo with ∼109 CFU P. aeruginosa strain PAO1-GFP (green). Eyeballs were carefully enucleated, rinsed with PBS, and then tissue paper blotted before bacterial challenge (see Materials and Methods). z-stack images were split into an orthogonal view to show x, y, and z planes of the intact corneal epithelium. After 7.5 h, bacteria showed equal levels of adherence to mBD-3−/− and wild-type corneas but still did not traverse the epithelium. (C) These data were confirmed by quantifying the bacterial distribution over the corneal epithelium (red indicates the reflection of corneal epithelial cells at 633 nm). Magnification, ×∼1,000.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
7.
FIG. 4.

FIG. 4. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

(A) Immunoblot analysis of secreted hBD-1 to hBD-3 or LL-37 peptide expression by human corneal epithelial cells after siRNA treatment versus scrambled siRNA controls, followed by a 4-h pretreatment with PAO1 culture supernatant (1:5 dilution in KGM-2 medium) and an 8-h exposure to bacteria in a traversal assay in the continued presence of the supernatant. (Left) Two micrograms of recombinant (r) positive-control peptide was included in each experiment. (Right) Relative knockdown (K.D) of each peptide (i.e., the integrated density of immunoblot bands normalized to the scrambled siRNA control for each peptide). (B) Gene expression levels of hBD-1 to hBD-3 or LL-37 in siRNA-treated human corneal epithelial cells (under the same experimental conditions), as measured by RT-qPCR and normalized to β-actin, show successful siRNA knockdown. Data are representative of data from three similar experiments, each using six pooled culture inserts.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.
8.
FIG. 5.

FIG. 5. From: Role of Defensins in Corneal Epithelial Barrier Function against Pseudomonas aeruginosa Traversal .

P. aeruginosa traversal of human corneal epithelia in vitro after siRNA knockdown of AMPs. Cells were treated with siRNA specific to hBD-1 to hBD-3 or LL-37 or with a scrambled siRNA control (SC) stimulated with bacterial culture supernatant (1:5 in KGM-2 medium) for 4 h prior to inoculation with 106 CFU of PAO1 in the continued presence of the supernatant. Viable counts of the apical (A) and basal (B) compartments showed significant increases in bacterial survival in the apical compartment, and in bacterial traversal to the basal compartment, in siRNA-treated cells versus the scrambled control at 8 h postinoculation. Effects on bacterial survival and traversal were greater when AMPs were knocked down in combination. *, P < 0.001 (by a Student's t test) in each instance, compared to scrambled siRNA controls. Results represent data from one of three independent experiments.

Danielle K. Augustin, et al. Infect Immun. 2011 February;79(2):595-605.

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