ABSTRACT The main objective of this grant is to use genetically engineered mouse models to determine how genetic changes in human chymotrypsin increase risk for chronic pancreatitis. Previous studies demonstrated the protective role of chymotrypsin against pancreatitis in humans and in mouse models. In this proposal, the overarching hypothesis is that loss of protective chymotrypsin function leads to pancreatitis in the context of additional genetic or environmental risk factors that facilitate intrapancreatic trypsin activation. To prove this notion, a CTRB1-deleted mouse strain (Ctrb1-del) will be crossed with mice carrying various pancreatitis risk alleles or will be exposed to an alcohol diet. Although CTRB1-deleted mice show heightened pancreatitis responses when disease is induced experimentally by cerulein stimulation; they do not develop pancreatitis naturally. Introduction of additional risk factors in the setting of chymotrypsin deficiency is predicted to result in the spontaneous onset and progression of pancreatitis. Alternatively, mice will exhibit additive susceptibility to cerulein- induced pancreatitis. The following specific aims will be studied. (1) Chymotrypsin deficiency synergizes with trypsinogen mutations to cause pancreatitis. In this aim, the hypothesis is that mice deficient in chymotrypsin and carrying mutations in mouse cationic trypsinogen (isoform T7) will develop spontaneous acute pancreatitis that will progress to chronic pancreatitis. Alternatively, mice will exhibit increased susceptibility to cerulein-induced pancreatitis. Adjusting gene dosage through combinations of heterozygous and homozygous alleles should result in variations in age of onset and/or severity. (2) Combined insufficiency of chymotrypsin and trypsin inhibitor in pancreatitis. In this aim, Ctrb1-del mice will be crossed either with a SPINK1-deleted strain (Spink1-KO) or with mice carrying a T7 trypsinogen allele with the p.G199R mutation, which conveys inhibitor-degrading properties (functional ortholog of human mesotrypsin). The hypothesis is that mice with combined deficiency in chymotrypsin and trypsin inhibitor will develop either spontaneous pancreatitis or will exhibit increased severity of cerulein- induced pancreatitis. (3) Alcoholic pancreatitis in the setting of chymotrypsin deficiency. In this aim, chymotrypsin-deficient mice will be fed the Lieber-DeCarli alcohol liquid diet. The hypothesis is that in the background of chymotrypsin-deficiency alcohol will cause spontaneous pancreatitis through selective upregulation of trypsinogen. Successful completion of these aims will offer conclusive evidence that alterations in chymotrypsin genes in humans promote the development of pancreatitis by interacting with other genetic and environmental factors that increase intrapancreatic trypsin activity.