Table 1.
Characteristics of the subjects and biopsy specimens.
Table 2.
Patients inclusion and exclusion criteria.
Fig 1.
Immunocytochemical characterization of fibroblasts.
The cells from both SM-exposed patients and controls were negative for pancytokeratin staining (A and B; 10×) and positive for vimentin (C and D; 10×).
Fig 2.
Immunocytochemical detection of α-SMA and fibronectin expression.
Both SM-exposed patients and controls expressed α-SMA (A and B, 10×) and fibronectin (C and D, 20×). The number of positive cells was determined by enumeration of 100 cells/sample. Figures E and F present the count of positive cells as mean ± SEM for patients (n = 5) and controls (n = 4). *p < 0.0001. Arrows show positive cells; Abbreviations: α-SMA alpha smooth muscle actin, SM sulfur mustard.
Fig 3.
Morphologic characterization of fibroblast.
Fibroblasts from both SM-exposed patients and controls (A and B; 40×) were stained with Crystal Violet. Twenty cells/sample were measured for each patient. The results are presented for both length (C) and width (D) as mean ± SEM for patients (n = 5) and controls (n = 4). *p < 0.05. Abbreviations: SM sulfur mustard.
Fig 4.
Electron microscopic photomicrographs of fibroblasts.
SM-exposed patient and control (A and B). Filopodia are seen in SM-exposed fibroblasts. Abbreviations: SM sulfur mustard, ER endoplasmic reticulum, F filopodium, N nucleus.
Fig 5.
Population doubling levels (PDLs).
PDLs of SM-exposed patients and controls were compared at each passage performed at different time points after the start of the primary culture. Each individual culture's PDL was presented by a red dot. PDL of the SM-exposed patients were significantly higher than controls from the day 7 through 28, but did not show any significant difference on the day 35. * p = 0.0025, ** p = 0.0001, *** p < 0.05.
Fig 6.
The migration distance of fibroblasts isolated from SM-exposed patients (n = 5) was significantly higher than the distance migrated by controls (n = 4); *p < 0.05. Abbreviations: SM sulfur mustard.
Fig 7.
Flow cytometric analysis for apoptosis of fibroblasts.
Representative dot plots of the flow cytometric analysis of the Annexin V–FITC/propidium iodide (PI) following induction of apoptosis with H2O2. Fibroblasts derived from SM-exposed patients and controls were assessed for spontaneous apoptosis and apoptosis following treatment with 500, 700 and 900 μM of H2O2. Spontaneous apoptosis was not significantly different in SM-exposed patients and controls. However, a higher dose-dependent sensitivity to H2O2 is observed in patients as compared with controls. Q1: necrotic cells, Q2: late apoptotic cells, Q3: viable cells, and Q4: early apoptotic cells.