PMC

Isolated surface and crypt epithelium from control, active, and inactive ulcerative colitis (UC) colonic biopsy specimens. Photomicrographs of isolated sheet of colonic epithelium (×20) (A, C, E) and histological sections of isolated colonic epithelium stained with haematoxylin and eosin (original magnification ×50) (B, D, F). The isolated epithelia from control (A, B), active (C, D), and inactive UC (E, F) colonic biopsy specimens were free of stromal elements that surround each crypt in vivo.

Immunohistochemical detection of Bcl-2 and Bax proteins in control and active ulcerative colitis (UC) colonic epithelium. Normal (A and B) and active UC (C and D) colonic mucosa samples were stained for Bcl-2 (A and C) or Bax (B and D). Note expression of Bcl-2 in the lower half of the crypt epithelium in normal (A) and active UC (C) colonic mucosa. Bax expression is confined to the surface epithelium in the normal colonic mucosa (B). Bax staining is obscure in colonic epithelium in active UC (D) (original magnification ×100).

Detection of Bax, Bcl-2, Bcl-xL, CD95, and β-actin proteins in epithelial tissues isolated from control, active, and inactive ulcerative colitis (UC) colonic biopsy specimens by western blotting. Results are representative of duplicate experiments with similar results. Lysates were prepared from epithelium isolated from control, active, and inactive UC colonic biopsy specimens. Immunoblotting was performed using anti-Bax antibody, anti-Bcl-2 antibody, anti-Bcl-xL antibody, anti-CD95 antibody, and anti-β-actin antibody. No bands of Bax protein were evident in lysates of colonic epithelium isolated from active UC colonic biopsy specimens while one major band of Bax was observed in lysates of all samples from control and inactive UC colonic biopsy specimens. In contrast with Bax, the bands of Bcl-2, Bcl-xL, CD95, and β-actin were detected in lysates of all subjects at about the same intensity.

Immunoblot analysis of lysates of HT29 cells cultured for 24 hours with or without various inflammatory mediators. HT29 cells (5×10⁵ cells) were cultured with or without interleukin (IL)-1β (0.5, 2, or 10 ng/ml), IL-4 (5, 50, or 500 U/ml), IL-6 (1,10, or 100 ng/ml), tumour necrosis factor α (TNF-α) (1,10, or 100 ng/ml), interferon γ (IFN-γ) (0.1, 1, or 10 µg/ml) or 3-morpholinosydnonimine (SIN-1) (1, 10, or 100 mM) in 2 ml of RPMI 1640 with 10% fetal calf serum for 24 hours. Cells were harvested and whole cell lysates were obtained for comparison of protein levels of Bax and β-actin in lysates (20 µg) of cultured HT29 cells by immunoblot analysis. No up or downregulation of Bax was noted under any culture condition studied. Results are from one of two experiments with similar results.

(A, B) Detection of Bax mRNA in colonic epithelium obtained from ulcerative colitis (UC) and control colonic biopsy specimens. Total RNA was extracted from colonic epithelium isolated from control, active, and inactive UC colonic biopsy specimens. Bax and β-actin cDNA was amplified by reverse transcription-polymerase chain (RT-PCR) and detected by Southern hybridisation using mouse Bax cDNA or human β-actin cDNA fragment as a probe. Bands of Bax transcripts were not detected or markedly reduced in extracted RNA of colonic epithelium from active UC colonic mucosa, while intense bands of Bax transcripts were observed in all samples from normal subjects and in all colonic epithelial samples isolated from inactive UC colonic mucosa. These results are representative of duplicate experiments with similar results. (C) Relative quantitation of Bax cDNA by real time PCR. Total RNA was extracted from colonic epithelium isolated from control, inactive, and active UC colonic biopsy specimens. After synthesis of cDNA by RT reaction, real time PCR was performed using the Taq-Man PCR kit as described in patients and methods. The mean threshold cycle (C_T ) value for GAPDH gene transcripts of the standard sample was 25.6 and this value was used for standardisation of clinical specimens. Each sample was tested in duplicate, and the average of the two values was used for calculation. After standardisation, the amount of Bax gene transcript (fg) was expressed relative to that of GAPDH (C_T=25.6). Horizontal bars represent the mean values of each group. Results are representative of duplicate experiments with similar results.