For T cell transfer colitis, tissue damage was quantified by combining the scores for each of the following parameters for a maximum score of 17: degree of inflammation in the lamina propria (score 0C3); goblet cell loss (score 0C3); abnormal crypts (score 0C3); presence of crypt abscesses (score 0C1); mucosal erosion and ulceration (score 0C1); transmural involvement (score 0C3); and number of neutrophils counted at 40x magnification (score 0C3)
For T cell transfer colitis, tissue damage was quantified by combining the scores for each of the following parameters for a maximum score of 17: degree of inflammation in the lamina propria (score 0C3); goblet cell loss (score 0C3); abnormal crypts (score 0C3); presence of crypt abscesses (score 0C1); mucosal erosion and ulceration (score 0C1); transmural involvement (score 0C3); and number of neutrophils counted at 40x magnification (score 0C3). and PC2 was strongly weighted toward TNF and MIP-2, while the contributions of other biomarkers varied depending on the etiology of inflammation. Disease severity was correlated with elevated PC2 scores in DSS colitis and reduced PC1 scores in T cell transfer colitis. Down-regulation of pIgR was a common feature observed in both colitis models and was associated with altered cellular localization of pIgR and failure to transport IgA. Conclusions A multifactorial analysis of epithelial gene expression may be more informative than examining single gene Tilorone dihydrochloride responses in IBD. These results provide insight into the homeostatic and pro-inflammatory functions of CEC in IBD pathogenesis and suggest that biomarker analysis could be useful for evaluating therapeutic options for IBD patients. C57BL/6 mice were purchased from the Jackson Laboratories (Bar Harbor, ME). Mice were housed in microisolator cages with sterile bedding, food and water in AAALAC-accredited facility. All procedures were conducted in accordance with guidelines set forth by the University of Kentucky Institutional Animal Care and Use Committee. DSS Model of Acute Colitis Acute colitis was induced by oral administration of dextran sulfate sodium (DSS), as described 22. DSS (molecular weight 36,000 C50,000; MP Biomedicals, Aurora, OH) was dissolved in water at indicated concentration and given Tilorone dihydrochloride to mice for 8 days in place of normal drinking water. Water intake was measured daily to ensure consistency in DSS intake among mice. A disease activity index (DAI) was calculated daily for each mouse during DSS treatment, on a 0C4 scale that assessed weight loss, stool consistency, and fecal blood (visible and occult), as described 23. ColoScreen-ES testing kits (Helena Laboratories, Beaumont, TX) were used to detect occult blood. T cell Transfer Model of Chronic Colitis Chronic colitis was induced by adoptive transfer of na?ve T cells from wild-type mice into recipients, which lack B and T lymphocytes, as described19. To purify T-effector (Teff) and T-regulatory (Treg) cell populations for adoptive transfer, single cell suspensions from spleens of 10 week-old wild-type C57BL/6 mice were prepared in phosphate-buffered saline with 4% fetal bovine serum. In two of the three impartial experiments, CD4+CD45RBhi and CD4+CD45RBlo cell populations were isolated by fluorescence-activated cell sorting. Spleen cells were labeled with anti-CD4 antibodies (eBiosciences, San Diego CA) and anti-CD45RB (Biolegend, San Diego CA) and sorted into CD4+CD45RBhi (as a source of Teff) Rabbit Polyclonal to CRHR2 and CD4+CD45RBlo fractions (as a source of natural Treg), defined as the uppermost 30% of cells with the brightest CD45RB staining and the lowest 15% of cells with the dimmest CD45RB expression, respectively (Supporting Fig. 1A). The Tilorone dihydrochloride Teff and Treg fractions were subsequently demonstrated to differ dramatically in cell surface expression of CD25, a marker for natural Treg (Supporting Fig. 1B). In one of the three impartial experiments, CD4+ splenic T cells were separated into CD25? (Teff) andCD25+ (Treg) populations by magnetic bead sorting using the Regulatory T cells kit (Miltenyi, Auburn CA). Nine-week-old C57BL/6 mice received a single intraperitoneal injection of 4 105 purified Teff, with or without 1 105 purified Treg. Following adoptive transfer, body weight was monitored every 3 days and mice were sacrificed at 10 weeks post-transfer. No significant differences were observed in clinical parameters, tissue histology or colonic EC gene expression that could be attributed to the different methods of purification of Teff and Treg populations (immunofluorescence vs. magnetic beads). Tissue Histology Colons were dissected from euthanized mice and the length of the colon from the anus to the cecal junction was measured. For T cell transfer colitis, colons were weighed and the weight/length ratio was calculated. Colons were cut Tilorone dihydrochloride lengthwise, and half of each colon was prepared in a Swiss roll24 and fixed in 10% buffered formalin (Fisher Scientific, Fair Lawn, NJ). Fixed tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and evaluated in a blinded fashion for.