Fig 4.

HCMV infection triggered apoptosis in epithelial and mesenchymal cells in human fetal lung tissue. Apoptotic cells were labeled with FITC by a TUNEL assay (A to C and E) or identified by laser confocal microscopy and immunostaining for Fas (D). HCMV-infected cells were identified by the expression of HCMV IE. Nuclei were counterstained with DAPI. In panel A, the dotted gray line separates human lung tissue from mouse kidney. The gray boxes outline areas 1 and 2 that are magnified in panels B and C, respectively. The yellow squares in panels B and C show the areas of the insets. Insets represent the merged and single images of triple labeling. The arrowheads show TUNEL-positive uninfected bystander cells in panel C and Fas-positive uninfected bystander cells in panel D. In panel E, lung implants (cohort F; 8 mice) were inoculated with 5.8 log₁₀ IU of VR1814 per implant, and mice were treated intraperitoneally once daily with ganciclovir (50 mg/kg) beginning the day before HCMV inoculation. Apoptotic cells were assessed by a TUNEL assay 14 days after virus inoculation. Data for cohort A (A to D and F), cohort B (F), and cohort F (E) are shown. In panel F, the fluorescence intensity of TUNEL labeling is expressed as an image mean gray value. For cohort A, 10 images of viral lesions from one infected implant and 10 images from three mock-infected implants were quantified. For cohort B, 17 images from three infected implants and 6 images from two mock-infected implants were quantified. Error bars represent standard errors of the means. As indicated by the fluorescence intensity of TUNEL labeling, the number of apoptotic cells in the area of the viral lesion was significantly higher than in mock-infected implants (P < 0.0001 [cohort A]; P < 0.05 [cohort B] [by Mann-Whitney U test]). These results are representative of those observed for 4 infected implants and 5 mock-infected implants obtained from two different lungs (cohorts A and B).