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ment in areas with intact crypts and normal appearing surface epithelium (not shown). In the distal colon, however, some surface enterocytes were CA I negative during DSS administration (days 5 and 7) in areas with intact crypts and otherwise normally appearing surface epithelium. Moreover, in both proximal colon and distal colon CA I expression was almost completely absent on days 5 and 7 in areas with a flattened surface epithelium and crypt loss (Fig. 4B). During the regenerative phase (day 28) the CA I expression was restored in the entire proximal colon and also in those areas of the distal colon where the epithelial morphology appeared normal (not shown). Moreover, flattened surface epithelium adjacent to ulcers was frequently CA I positive (Fig. 4C). Biochemical analysis of CA I protein levels in the proximal colon revealed a slight but not significant decrease in CA I protein levels in the course of DSS treatment (Fig. 5). Remarkably, during the regenerative phase (day 28) an overshoot in CA I expression was seen in this segment, with CA I levels that were significantly higher than on days 5 and 7. In the distal colon more pronounced alterations in CA I protein levels were observed. CA I expression levels decreased significantly until day 7, the end of DSS treatment. During the regenerative phase (day 28) CA I expression returned to levels that were not significant different from control values. Goblet cell specific MUC2 and TFF3 expression To study goblet cell function MUC2 and TFF3 expression was studied. In controls MUC2 was expressed by goblet cells in the proximal and distal colon from crypt base to the surface epithelium (Fig. 6A, E). In contrast, TFF3 was expressed in the upper crypts in the proximal colon and upper two-thirds of the crypts in the distal colon (Fig. 6C, H). Alterations in the localization and numbers of goblet cells were seen during and after DSS treatment in both colonic segments. During the onset of disease (day 2) MUC2- and TFF3-positive goblet cells accumulated in the surface epithelium in both proximal and distal colon (not shown). As DSS treatment progressed (days 5 and 7) the accumulation of MUC2- and TFF3-positive goblet cells in the surface epithelium became more pronounced, particularly in the proximal colon (Fig. 6B, D). In the distal colon the area of TFF3 expression was extended from upper two-thirds of the crypt toward the crypt bottom in elongated crypts (Fig. 6J). In areas with flattened crypts TFF3 was expressed strongly in the upper crypts and surface epithelium, and, although very weakly, also in the deeper crypt region (Fig. 6I). MUC2 expression in the proximal and distal colon appeared unaltered, independent of crypt morphology, during active disease. Specifically, Muc2 was expressed in small goblet cells within damaged crypts and in small goblet cells in elongated crypts (Fig. 6F, G). Due to the massive crypt dam-

Fig. 7 MUC2 (A) and TFF3 expression levels (B) during the different phases of the DSS-induced disease. MUC2 and TFF3 levels in each colonic segment of control and DSS-treated rats were quantified. Thereafter the values of the control rats were averaged per segment and arbitrarily set at 1. The MUC2 and TFF3 levels in each segment of DSS-treated rats were expressed as relative values compared to control values (day 0). Finally, the mean Âąstandard error of the mean is presented. Statistical analysis was performed using analysis of variance followed by an unpaired t test. In the proximal colon MUC2 expression was significantly higher on day 7 than on day 0 (aP<0.01) and day 2 (bP<0.05). In the distal colon significant differences in MUC2 levels were seen between day 28 and days 5 and 7 (cP<0.05). Significantly lower TFF3 levels were observed in the distal colon on day 7 than in controls (aP<0.05), and on days 5 and 7 than on day 28 (bP<0.05)

age, crypt loss, and loss of surface epithelium in the distal colon (approx. 50%), the overall number of goblet cells decreased in the latter segment. During the regenerative phase (day 28) elevated numbers of MUC2- and TFF3positive goblet cells were still seen in the surface epithelium of distal colon (Fig. 6K, L) but not in the proximal colon. Especially in the distal colon crypts were elongated and primarily contained large goblet cells. Biochemical analysis of MUC2 protein levels demonstrated a progressive increase in MUC2 levels in the proximal colon in the course of DSS treatment (Fig. 7A). On day 7 this increase in MUC2 levels in the proximal colon was fourfold control values and was significantly different from control values and onset of disease. There-

Epithelial proliferation, cell death, and gene expression in experimental colitis  

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