they asked for better characterization of the mouse models to be used to investigate the effects of Claudin-2 deficiency in acute pancreatitis/chronic pancreatitis; Also, the reviewers asked for more preliminary data on the potential regulation of Claudin-2 pancreatic expression by NFkB/cytokines. To generate the requested preliminary information, we will perform the following experiments: 1. We will investigate the expression of Claudin-2 and the effects of Claudin-2 deficiency using a published mouse model of alcohol-induced pancreatitis with i.p. injections of ethanol + palmitoleic acid (Huang W et al., Gut, 2014). This mouse model is clinically relevant because excessive alcohol consumption can cause pancreatitis and mutations in CLDN2 sensitize humans to alcohol-induced pancreatitis (Withcomb DC et al., Nat. Genet., 2012). For these experiments, C57Bl/6 (wildtype) mice will be injected twice weekly with EtHO+POA during 1 - 3 weeks to determine the experimental regimen for the induction of acute (AP) or chronic (CP) pancreatitis. Once these conditions are established, Cldn2-GFP transgenic mice will be injected with different doses of EtHO+POA to determine how Claudin-2 pancreatic expression changes during the course of AP/CP (anti-GFP antibodies will be used to stain the pancreatic tissue). Next, control and Cldn2-null mice will be administered with EtHO+POA to investigate the effects of Claudin-2 deficiency in alcohol-induced AP/CP. Histologic and immunostaining analyses will be used to quantify tissue damage, edema and immune infiltrates. These analyses should not be problematic since my laboratory has extensive expertise working with mouse models of pancreatitis. Based on our preliminary results and those from GWAS studies, I predict that these pilot experiments will demonstrate that Claudin-2 function is relevant in alcohol-induced pancreatitis. 2. Our preliminary results showed that Claudin-2 expression is upregulated in the pancreatic ducts under conditions of acute or sustained inflammation. Similarly, published studies demonstrated that NFkB, IL-6 and IL- 22 upregulate Claudin-2 expression in the intestine. To begin investigating the mechanisms that regulate Claudin-2 pancreatic expression, we will culture the immortalized, non-cancerous human pancreatic ductal cell line H6c7 in the presence of TNF-alpha (to stimulate NFkB signaling), IL-6 or IL-22. For these experiments, we will use culture conditions similar to those reported in studies of Claudin-2 regulation in intestinal cells and the colon cancer cell line Caco-2 as positive control (Wang Y et al., J. Immunol., 2017 and Suzuki T et al., JBC, 2011).