During a study aimed at generating a bispecific molecule between BN antagonist (D-Trp(6),Leu(13)-psi[CH(2)NH]-Phe(14))BN(6-14) (Antag1) and mAb22 (anti-FcgammaRI), we attempted to cross-link the two molecules by introducing a thiol group into Antag1 via 2-iminothiolane (2-IT, Traut's reagent). We found that reaction of Antag1 with 2-IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, D-Trp-Gln-Trp-NH(2) (TP1), the N-terminal tripeptide of Antag1. Reaction of TP1 with 2-IT for 5 min gave products 1a and 3a; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a, the expected Traut product, was identified by collecting it in a vial containing N-methylmaleimide and then isolating the resultant Michael addition product 2a, which was confirmed by mass spectrometry. Thiol 1a is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH3 to give N-TP1-2-iminothiolane (3a), ES-MS (m/z) [602.1 (M+H)(+)], as well as regenerating TP1. Repeat reaction with Antag1 and 2-IT allowed us to isolate N-Antag1-2-iminothiolane (3b), FAB-MS (m/z) [1212.8 (M+H)(+)] and trap the normal Traut product 1b as its N-methylmaleimide Michael addition product 2b, ES-MS (m/z) [1340.8 (M+H)(+)]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.

Introduction: People of South Asian descent carry a high burden of cardiovascular disease (CVD) and type 2 diabetes mellitus (DM2) both globally and in the United States. Although retrospective cohort studies indicate decreased medication adherence amongSouth Asian immigrants, data exploring contributing factors are limited.

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis-associated colon cancer (CACC). Despite the strong relationship between inflammation and cancer, the mechanistic events that contribute to the transition from IBD to CACC remain undefined. Changes in the glycosylation profile of the known oncoprotein MUC1 commonly occur in chronic inflammation and may facilitate progression to cancer. We used a transwell coculture model system to examine the effect of polarized macrophages, such as those contributing to inflammation, on the expression of glycosylation-related enzymes in colon cancer cells. We found that M2-like macrophages induce the expression of ST6GALNAC1 glycosyltransferase which, by adding sialic acid to O-linked GalNAc residues, promotes the formation of the tumor-associated MUC1-sTn glycoform. Cytokine antibody arrays and blocking antibody experiments revealed that high levels of IL-13 and CCL17, present in the conditioned medium of colon cells cocultured with M2 macrophages, activate ST6GALNAC1 expression in colon cancer cells. In silico and in vivo murine and human models of colitis and CACC showed that IL-13 induces the phosphorylation of STAT-6 that directly binds ST6GALNAC1 promoter resulting in its transcription activation and protein over-expression. On the other hand, CCL17 activates ST6GALNAC1 expression via NF-kB pathway signaling. Our findings revealed a novel cross-talk between M2-like macrophages and inflamed and malignant colon cells that contributes to the pathogenesis of colitis and progression to CACC.