Infection patient Age Sex IBD status* Control patient 59 71 23 46 19 47 27 F F F M M F M No CD CD UC Different patient: matched for age, S. St Paul sex and biopsy site Same patient and biopsy site.12 months from acute infection Infectious agent C. jejuni C. jejuni C. jejuni C. difficile C. difficile S. enteritidis identical to a peptide from the human MUC3 mucin has been identified using proteomics on mouse mucins, suggesting the presence of the mouse orthologue to this mucin [35]). In a murine C. rodentium infection model, we demonstrate substantial changes in the amount of virtually all intestinal mucins after 12 days of infection. We further showed mucin binding to C. rodentium, demonstrating that the release of mucins has the capacity to remove bacteria from the epithelial surface. Materials and Methods Animals 8–16 weeks old male129/SvJxC57BL/6 mice were housed under clean conventional conditions, and allowed free access to sterilized food and water. All experiments were approved by the University of Queensland Animal Experimentation Ethics Com- mittee (approval no 059/07). Colonies routinely tested negative for murine viral and bacterial pathogens. Infection of mice Citrobactum rodentium strain ICC169 was grown on Lauria- Bertani agar for 20 h at 37uC. Bacteria harvested from plate cultures were suspended in warmed Lauria-Bertani broth. 8 mice were orally inoculated with 107 colony forming units (CFU) and sacrificed after 12 days by cervical dislocation. Duplicate samples of duodenum, caecum, small intestine and proximal, mid and distal colon were dissected and collected in either broth or 10% formalin. To assess the number of CFU/g tissue, tissue and fecal samples (collected as defecated samples) were homogenized in broth, serially diluted, plated onto McConkey’s selective agar, and grown for 20 h at 37uC. CFU were enumerated by counting C. rodentium ,1 mm diameter fuschia-coloured colonies. The mice were weighed and diarrhea was scored every second day. Histological assessment For analysis of colitis, formalin fixed tissue sections of the small intestine, caecum, proximal and distal colon stained with hematoxylin/eosin were coded to blind the analysis, and the entire section was systematically scored: aberrant crypt architec- ture (0–5), increased crypt length (0–3), goblet cell depletion (0–3), general leukocyte infiltration (0–3), lamina propria neutrophil counts (0–3), crypt abscesses (0–3), and epithelial damage and ulceration (0–3) were scored (full details in Table S1). Human biopsies Individuals with acute diarrhoea are not usually subjected to colonoscopic examination and biopsy, but archival biopsy material was available from seven patients with acute gastroenteritis. These patients included four previously healthy individuals and three patients with underlying inflammatory bowel disease, as described in Table 1. For controls, biopsies were examined from the same IBD patients when there was no detectable bacterial infection or from the same distal colonic site from age and sex matched healthy individuals (Table 1). The archived biopsies were collected and treated in accordance with the Australian National Statement on Ethical Conduct in Human Research (2007) (http://www.nhmrc.gov.au/ publications/synopses/e72syn.htm) guidelines for archived patho- logical material. There is no consent process required for this type of use. The biopsies were collected between 1/06 and 1/08 for diagnostic histopathology as requested by the attending doctor. The samples were processed by the Mater Health Services Pathology Service as requested by the attending doctor. The paraffin blocks are kept on level 6 of the Mater Adult Hospital, which is where the Mater Health Services Pathology Service is housed. PLoS ONE | www.plosone.org 2 December 2008 | Volume 3 | Issue 12 | e3952 Mucins in Infectious Colitis Table 1. Human biopsies used for investigating MUC1 induction in infectious colitis. *CD = Crohn’s disease, UC = ulcerative colitis. doi:10.1371/journal.pone.0003952.t001 Antibodies The MUC1 antibody (CT2[36]) and the Muc17/Muc3-S2 antibody (raised against the peptide KYTPGFENTLDTVVKN- LETKIKNAT [35] were kind gifts from Prof. S. Gendler, Scottsdale, USA, and Prof. G. Hansson, Gothenburg, Sweden. The polyclonal anti sera against the C-terminal sequence of MUC4 (hHA1-B-1) was a kind gift from Prof. K. Carraway, Miami, USA. The MUC5AC antibody (45MI) was purchased from Sigma [37]. Polyclonal antisera (rM13.C and MM2-2) against the cytoplasmic tail of the human MUC13 and murine Muc2 was raised in rabbit using bovine serum albumin conjugated peptides CMQNPYSRHSSMPRPDY and CPEDRPIY- DEDLKK, respectively [38,39], and then purified on a HiTrap NHS-activated HP column (Amersham Biosciences, Uppsala, Sweden) coupled to the same peptides according to manufacturers instructions. The monospecificity of the CT2 and rM13.C antibodies were demonstrated by that gastric and intestinal specimens from Muc1 and Muc13 knockout mice were negative for staining with the respective antibodies. The staining pattern of the hHA1-B-1 antisera was similar to that previously described for MUC4 in human tissue [40,41], and the pre-immune sera from the hHA1-B-1 rabbit did not stain any tissue. The 45M1 antibody recognizes MUC5AC (and no other molecules in gastric tissue extracts) in human and rhesus monkey as demonstrated by that only fractions from density gradient centrifugation that has a density appropriate for MUC5AC (1.4 g/ml) are recognized with this antibody [37], and the mouse gastric tissue used here as a positive control demonstrated a strong surface epithelial cell staining, similar to that of the human and rhesus monkey gastric tissue[37], whereas the intestinal tissue was negative. Similarly, the MM2-2 antibody recognize material from intestinal tissue extracts in fractions of appropriate density from density gradient centrifugation of MUC2/Muc2, show a distinct goblet cell staining pattern in the intestine of mice similar to that demonstrated in human [39], and did not stain the gastric negative control tissue. The specificity of the Muc3-S2 antibody has been confirmed using mass spectrometry [35]. Immunohistochemistry Mouse samples: Formalin-fixed, paraffin-embedded tissue sections (4 mm) of archived biopsies from mouse stomach, duodenum, caecum, small intestine and proximal-, mid- and distal colon were dewaxed and rehydrated. Antigen retrieval used