Volume 117, Issue 5, Pages 1089-1097 (November 1999) The intestinal mucus layer from patients with inflammatory bowel disease harbors high numbers of bacteria compared with controls  Constance Schultsz*, Frank M. van den Berg‡, Fiebo W. ten Kate‡, Guido N.J. Tytgat§, Jacob Dankert*  Gastroenterology  Volume 117, Issue 5, Pages 1089-1097 (November 1999) DOI: 10.1016/S0016-5085(99)70393-8 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 Results of RISH of sheep collagen sections containing HT-29 cells and E. coli, K. pneumoniae, and S. typhi in equal amounts. (A) Hybridization with probe Eub338M. (B) Pretreatment of section with ribonuclease before hybridization abolishes the signal of the bacteria. (C) Omission of pretreatment with proteinase K decreases antibody penetration, resulting in diminished staining of bacteria. (D) Hybridization with probe dT35f results in staining of eukaryotic mRNA. Gastroenterology 1999 117, 1089-1097DOI: (10.1016/S0016-5085(99)70393-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Results of RISH hybridization with probe Eub338M of rectal biopsy specimens from patients with IBD and healthy controls. ■, UC; ▴, CD; ●, indeterminate colitis. Each symbol represents 1 patient or control. Median lengths of total mucosal surface of sections examined from patients and controls were not statistically different. Specimens from IBD patients contained significantly more bacteria than control specimens (P = 0.004, Fisher exact test). Gastroenterology 1999 117, 1089-1097DOI: (10.1016/S0016-5085(99)70393-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Hybridization of rectal biopsy sections from a patient with UC and a healthy control with probe Eub33M (counterstained with hematoxylin). (A–D) Patient 1; original magnifications: A, 100×; B, 400×; C, 1000×; D, 100×; staining with alcian blue. (E–F) Control; E, 400×; F, 400×; staining with alcian blue. Brackets designate an area containing bacteria. Gastroenterology 1999 117, 1089-1097DOI: (10.1016/S0016-5085(99)70393-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Results of RISH with probe Eub338M of rectal biopsy specimens from patients with IBD and relationship with inflammation. ■, UC; ▴, CD; & <!--sexagon-->;, indeterminate colitis. Each symbol represents 1 patient or control. In the inflammation score, the presence of ulceration, erosion, crypt abcesses, and cryptitis was separately assessed semiquantitatively by assigning scores from 0 (absent) to 3 (severe). A final score was calculated for each specimen by addition of the individual scores, with a maximum score of 12. Inflammation was considered to be absent if final scores were 0 or 1 due to minimal cryptitis (only 1 crypt affected). Active inflammation or relapse was considered to be present if final scores were higher than 0, except in case of a score of 1, due to minimal cryptitis. The number of bacteria per total mucosal surface length examined and the degree of inflammation are not correlated (P = 0.4, Pearson correlation test). Gastroenterology 1999 117, 1089-1097DOI: (10.1016/S0016-5085(99)70393-8) Copyright © 1999 American Gastroenterological Association Terms and Conditions