Satoh K, Kawakami A, Shirabe S, Tamai M, Sato A, Tsujihata M, Nagasato K, Eguchi K

Satoh K, Kawakami A, Shirabe S, Tamai M, Sato A, Tsujihata M, Nagasato K, Eguchi K. disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets Brivudine for the treatment of IBD and PEPCK-C further suggest Cl-amidine as a candidate therapy for this disease. [for oral gavage/treatment experiment (see Supplemental Fig. S1 in Supplemental Material for this article, available at the Journal website)] or [for intraperitoneal/prevention experiment (see Supplemental Fig. S1)]. Cl-amidine dissolved in 1 PBS or vehicle control only (1 PBS) was administrated by two methods: (see Supplemental Fig. S1) at 75 mg/kg body wt, which is a human equivalent dose of 6.1 mg/kg daily (29), or (see Supplemental Fig. S1). On 0.001). = 5) and normal colon tissues from patients without colitis (= 5)] by automated cellular imaging system. *Significantly increased PAD levels in colitis ( 0.01). (see Supplemental Fig. S1). On for 30 min at 4C. The supernatant was separated, and the protein concentration was determined using the Lowry method. PAD activity in samples was measured using a previously described PAD activity assay (13). Briefly, 20 l of each sample were added to a reaction buffer containing 50 mM NaCl, 10 mM CaCl2, 2 mM DTT, 100 mM Tris (pH 7.6), and 10 mM = 0.05 was chosen for significance. RESULTS PAD levels are elevated in mouse and human colitis. Given that Brivudine recent studies have demonstrated that increased protein citrullination is apparent in human IBD (23), we wished to confirm that PAD levels were also increased in the colonic inflammatory lesions present in DSS-induced colitis. For these experiments, we used tissues archived from previous studies using the DSS mouse model of colitis (11, 15). The relative amount of PAD enzyme in this tissue was detected using a polyclonal anti-pan-PAD antibody that recognizes all five PAD isozymes (Fig. 1 0.001). Given that PAD2 and PAD4 are known to be expressed in immune cells, we next used immunohistochemistry to determine whether the levels of one, or both, of these isozymes were elevated in these tissues. The results indicate that the levels of both of these enzymes are increased in mouse colon colitis tissues (Fig. 1and = 0.89, 0.05) between the percentage of cells positive for PAD (anti-pan-PAD slides were evaluated) and the total estimated number of inflammatory Brivudine cells (in MLN and LP) in a serial H&E-stained section (see Supplemental Fig. S2). In total, these studies demonstrate that PAD levels are increased in human UC patients and DSS-induced mouse colitis, thereby helping validate the use of PAD inhibitors, such as Cl-amidine, as a potential treatment for this disease. Cl-amidine suppresses and treats DSS-induced colitis. Brivudine To explore whether PAD inhibition represents a viable approach to the treatment of IBD, we set out to determine whether Cl-amidine, a pan-PAD inhibitor, could inhibit colon inflammation in DSS-induced colitis. Initial experiments used injections of Cl-amidine (75 mgkg?1day?1 ip), beginning concomitantly with the initiation of 2% DSS in the drinking water. This dose was chosen based on results in a RA model that used 100 mgkg?1day?1 without overt side effects and without immunosuppressive outcomes. In our DSS model, 50 mice in 4 groups were examined, and inflammation scores were recorded as described in materials and methods. Figure 2shows significantly higher levels of colon inflammation in the 2% DSS than the 2% DSS.