1. Volume 131, Issue 3, Pages 809-817 (September 2006)
Effects of Mouse and Human Lipocalin Homologues 24p3/lcn2 and Neutrophil Gelatinase–Associated Lipocalin on Gastrointestinal Mucosal Integrity and Repair 
Raymond J. Playford, Angelica Belo, Richard Poulsom, Anthony J. Fitzgerald, Kevin Harris, Isabella Pawluczyk, Joel Ryon, Thameen Darby, Marit Nilsen–Hamilton, Subrata Ghosh, Tania Marchbank 
Gastroenterology 
Volume 131, Issue 3, Pages (September 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

2. Figure 1
Changes in 24p3/lcn2 immunostaining in response to indomethacin. Under nondamaged circumstances, 24p3/lcn2 immunostaining was weak or absent in the stomach (A), small intestine (C), and liver (E). Following administration of indomethacin, a marked increased expression of 24p3/lcn2 (brown staining) was seen in the superficial epithelial cells and overlying mucus layer (arrow) of the stomach (B). The small intestine of the same indomethacin-treated animals showed increased 24p3/lcn2 immunostaining in the epithelial cells of the villi and also in the infiltrating inflammatory cells. As expected, the villi of treated animals also show some shrinkage and irregularity (D). Liver of indomethacin-treated animals also showed a marked increase in the 24p3/lcn2 immunostaining within the hepatocytes (F). Original magnifications were as follows: stomach, 40×; intestine, 10×; and liver, 20×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

3. Figure 2
Changes in colonic 24p3/lcn2 immunostaining and in situ hybridization in response to DSS. By using immunohistochemistry, (A) normal colonic tissue showed minimal 24p3 expression. (B) In contrast, colonic tissue from DSS-treated animals had a marked increase in 24p3/lcn2 immunoreactivity (brown staining) in the regenerating surface epithelial cells and underlying crypts. The same tissues were analyzed for 24p3/lcn2 mRNA using in situ hybridization. In keeping with the immunohistochemical studies, colonic tissue from DSS-treated animals (same tissue as used for B) showed marked up-regulation of 24p3/lcn2 mRNA in regenerating colonic crypts and superficial epithelial cells showed using bright field microscopy where RNA signals show as (C) black spots and dark field whereas (D) RNA shows as white areas. Original magnification, 20×.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

4. Figure 3
Changes in 24p3/lcn2 mRNA in response to injurious agents. In both the small intestine and colon, administration of the noxious agents indomethacin or DSS resulted in a marked increase in 24p3/lcn2 mRNA levels (in keeping with the results of immunohistochemistry). Data are the SEM of n = 3 per group and is expressed as a ratio to the housekeeper gene glyceraldehyde-3-phosphate dehydrogenase. Data are expressed as the mean ± SEM. **P < .01 vs relevant control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

5. Figure 4
Effect of 24p3/lcn2 and NGAL on wound healing of HT29 cells as assessed by cell migration. The addition of (A) 24p3/lcn2 (0–15 μg/mL) or (B) NGAL to wounded monolayers of the human colonic cell lines HT29 stimulated a dose-dependent increase in the rate of migration of the cells. □, control (no 24p3/lcn2 or NGAL); ▵, 5 μg/mL; ○, 15 μg/mL. P < .01 vs control for times after 4 hours for both peptides. (C) The promigratory effect of 24p3/lcn2 (○, 15 μg/mL) could be blocked by adding a 24p3/lcn2-neutralizing antibody (▵, 100 μg/mL), but not by a TGFβ-neutralizing antibody (X, 100 μg/mL). (D) Comparison of the promigratory effects of wild-type cys98-24p3/lcn2 (○), and mutant ala98-24p3/lcn2 (▵), each added at 15 μg/mL, showed them to be similar in activity. Data are expressed as the mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

6. Figure 5
Effect of 24p3/lcn2 on wound healing of HCT116 cells as assessed by cell migration. The human colonic cancer cell line, HCT116, does not produce trefoil peptides, which may be involved in promigratory effects. Despite this, compared with controls (□, medium alone), addition of 24p3/lcn2 (○, 15 μg/mL) caused an increase in the rate of cell migration. Similarly, co-incubation with the prostaglandin inhibitor indomethacin did not affect the promigratory effect of 24p3/lcn2 (*). Data are expressed as the mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

7. Figure 6
Effect of systemic 24p3/lcn2 administration on amount of indomethacin-induced gastric damage. Rats (n = 6–8 per group) were placed in restraint cages and subcutaneously infused with saline (control, X), EGF (positive control, □), wild-type cys98-24p3/lcn2 (○), and mutant ala98-24p3/lcn2 (▵) for 3.5 hours. Thirty minutes after the start of the study all animals also received indomethacin 20 mg/kg (subcutaneously). At the end of the study, animals were killed, and the amount of (A, mm2/stomach) macroscopic and (B) microscopic damage was determined. Microscopic assessment was graded as 0–4. Data are expressed as the mean ± SEM. **P < .01 vs control.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions

8. Figure 7
Effect of oral 24p3/lcn2 administration on amount of indomethacin-induced gastric damage. Rats (n = 6 per group) were gavaged with 1 mL of saline or 24p3/lcn2 (50 or 250 μg/mL). Thirty minutes later animals were treated with indomethacin and restraint as for Figure 5. When administered via this route, 24p3/lcn2 showed no protective effects either macroscopically (see Figure) or microscopically (data not shown). Data are expressed as the mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association (AGA) Institute Terms and Conditions