Distal colons were selected as this is the site of migration of protective appendiceal lymphocytes (Ng et al., submitted). Our approach merges data from groups of gene-sets described previously in the literature to detect significant expression differences.
These gene-set groups were Kegg pathways (150 gene-sets), micro-RNAs (200 gene-sets), transcription factors (579 gene-sets), biological processes (536 gene-sets) and others (1387 gene-sets). We used stringent statistical cut-offs: false discovery rates (FDR) values < 1% and P value < 0·001. Expression of 266 gene-sets was up-regulated significantly in AA group samples; distributed across Kegg pathways (9 gene-sets), transcription factors (41 gene-sets), biological processes (seven gene-sets) and others Selleck Kinase Inhibitor Library (209 gene-sets) as depicted in Table 1. The 266 gene-sets up-regulated in the AA group (Table S1) included immunity-related and unrelated gene-sets. No gene-sets were up-regulated in the SS group when compared to the AA group. The tnfsf10 gene was up-regulated 1·46-fold, the SLC22A5 gene (OCTN2) 1·31-fold, the C3 gene 1·74-fold, the ccr5 gene 1·5-fold, the irgm gene 1·66-fold and
the ptger4 gene 1·43-fold in the AA mice 3 days after surgery. Conversely, the ccl20 gene was decreased 0·6-fold in the AA mice 3 days after surgery. We selected 14 genes for confirmation of our gene expression studies. These genes were immunological genes of interest which were find more up-regulated in the AA group in this study.
They broadly belonged to four major groups: innate immunity (slpi, s100A8, lbp, CD68), immune mediators (IL18R1, IL33), cell migration-chemokines (ccl8, cxcl10, ccl12 or mcp5, pf4, ccl5, ccl7 or mcp3) and cell migration-receptors (fpr1, ccr5). The RT–PCR results (Fig. 1) indicate that eight of Tryptophan synthase the total 14 genes tested were up-regulated significantly in the AA group; three of these genes just missed statistical significance, and three genes showed no difference between the SS and AA groups. These RT–PCR results validate our microarray data. Distal colonic samples from 3 days, 14 days and 28 days after the last (second) surgery from SS and AA mice were assessed. SS and AA expression levels of all 14 genes analysed (except for the pf4 gene) either decreased or remained level. Pertaining to the four innate immunity genes that were quantified (slpi, s100A8, lbp, CD68), slpi was reduced significantly in the AA group when compared to the SS group at the 28 day post-surgery time-point, in contrast to the 3-day post-surgery time-point (Fig. 2). CD68 was relatively up-regulated in the SS group, although being expressed to a relatively lesser extent in the AA group (Fig. 2).
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