Gene expression changes induced by the sitagliptin treatment were assessed from whole blood samples taken at days 0 and 28. Paired analysis was performed to identify changes

within individuals. In the sitagliptin group, a group of 86 transcripts was identified as significantly changed between days 0 and 28 (paired t-test, P < 0·001). Sixteen transcripts changed in the placebo GSK1120212 group (P < 0·001) and none overlapped with those changed in the sitagliptin group, indicating the specificity of the genes identified in the treatment group. Although these changes were statistically significant, with a stringent P-value cut-off, the magnitude of these observed changes was small (most with a fold change < 1·2), indicating that the changes observed might not be biologically relevant. Shown in Supporting information, Table S2, are transcripts changed significantly with either sitagliptin or placebo treatment (P < 0·001) that had a fold change > 1·2. One of the transcripts with the highest significance and fold change was matrix metallopeptidase 9, a protein important for leucocyte trafficking that is up-regulated in many autoimmune diseases [26]. Another gene changed significantly BGJ398 mw in the sitagliptin group was small ubiquitin-related modifier (SUMO-1), that can modify other proteins via sumoylation. SUMO-1 interacts with dipeptidyl peptidase 9 (DPP-9), a protein with structural and functional

similarity to DPP-4, yet sitagliptin is specific for DPP-4 and does not inhibit DPP-9 [27]. Some alterations in immune function may not be directly Uroporphyrinogen III synthase observable ex vivo, and may require an immune stimulus to reveal differences. Therefore, we treated PBMCs with LPS as an innate immune stimulus, and measured

cytokine and chemokine levels. TGF-β levels were measured by ELISA, and did not differ before and after drug treatment or between the sitagliptin and placebo groups (data not shown). The same 27 cytokines and chemokines measured in plasma were also measured in supernatants with and without LPS treatment, and no significant differences were observed between placebo and sitagliptin groups (Fig. 4 and data not shown). Shown in Fig. 4 are the expression levels of proteins from this panel that were induced with LPS treatment of day 3 samples. Although individuals from the sitagliptin group exhibit moderately higher levels of certain cytokines in PBMCs cultured without LPS [for example, IL-6 and macrophage inflammatory protein (MIP)-1α], this difference was not statistically significant. In order to elicit an adaptive immune response and activate T cells, PBMCs from participants were stimulated with anti-CD3 for 4 days. Samples were obtained from 11 individuals who received sitagliptin (this part of the study was not blinded). T cell activation was measured by up-regulation of CD25 and T cell proliferation was measured via CFSE dilution (Fig. 5). Both parameters were measured in CD4+ and CD8+ T cells.