Dynamic aspects of granulocyte activation in rat severe acute pancreatitis.

We demonstrated dynamic aspects of granulocyte activation in rat severe acute pancreatitis, which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. This decrease occurred simultaneous to induction of systemic hypercytokinemia and granulocyte migration into the lung. Overall results imply that: (1) circulating granulocytes are activated by induction of mild pancreatitis; (2) activation of granulocytes is mediated by factors other than systemic cytokinemia, such as locally produced cytokines; (3) those priming granulocytes immediately and significantly migrate from the circulation into the extravascular space by induction of endotoxemia; and (4) migration of granulocytes, in turn, may be mediated by systemic cytokinemia.

Mutational analysis of the pancreatic secretory trypsin inhibitor gene in familial and juvenile pancreatitis in Japan.

BACKGROUND: Mutations in the pancreatic secretory trypsin inhibitor ( PSTI) gene have been reported in patients suffering from chronic pancreatitis. The aim of the present study was to investigate whether similar gene mutations are present in familial and juvenile pancreatitis patients in Japan. METHODS: All four exons of the PSTI gene and their flanking intronic sequences were amplified by polymerase chain reaction and sequenced for 37 familial pancreatitis patients (24 families) and 15 juvenile pancreatitis patients, distributed throughout Japan. RESULTS: Three types of exonic mutation in the PSTI gene were observed. The N34S mutation was found in six familial pancreatitis patients (three families) and in one juvenile pancreatitis patient, and the R67C mutation was found in one familial pancreatitis patient and one juvenile pancreatitis patient. We also found a 272C>T mutation in the 3' untranslated region of exon 4 in one familial pancreatitis patient and four juvenile pancreatitis patients. It should be noted that the N34S mutation was cosegregated with two intronic mutations, specifically, IVS1-37T>C and IVS3-69insTTTT. CONCLUSIONS: The same set of N34S mutations (N34S + IVS1-37T>C + IVS3-69insTTTT) that exists in other countries was found in the PSTI gene in Japanese familial and juvenile pancreatitis patients. Another unique mutation (R67C) was also observed in two patients; 272C>T was suggested to be a normal polymorphism.

Further evidence for endothelin as an important mediator of pancreatic and intestinal ischemia in severe acute pancreatitis.

INTRODUCTION: Severe acute pancreatitis is occasionally associated with pancreatic and intestinal necrosis. Mesenteric vasoconstriction is one of the most probable types of pathogenesis of these complications. AIM: To investigate the involvement of endothelin-1 (ET-1), a potent vasoconstrictor. METHODOLOGY AND RESULTS: Plasma ET-1 concentrations were extremely high in patients with pancreatic and/or diffuse intestinal necrosis. ET-1 mRNA was demonstrated in the rat pancreas, and the production of ET-1 protein by human umbilical vein endothelial cells was enhanced by tumor necrosis factor-alpha, thrombin, and protease-activated receptor-2-activating peptide. Administration of ET-1 in vivo induced mesenteric arterial spasm and decreased pancreatic and intestinal blood flow. CONCLUSION: These results suggest the following: ET-1 is produced in and around the pancreas, mainly by endothelial cells, in severe acute pancreatitis; in the inflammatory setting, cytokines, activated thrombin and trypsin, may stimulate ET-1 production in a paracrine fashion; produced ET-1 may exaggerate the splanchnic microcirculation; and progressive ischemia may lead to necrosis of the pancreas and intestine.

From acute to chronic pancreatitis: the role of mutations in the pancreatic secretory trypsin inhibitor gene.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent natural inhibitor of trypsin. We proposed the hypothesis that, if the function of the PSTI is impaired by its genetic mutation, trypsin may easily promote autodigestion causing pancreatitis and we performed a mutational analysis of the PSTI gene in patients with pancreatitis. Two exonic mutations (N34S and R67C) were thought to be associated with a predisposition to pancreatitis. The N34S mutation was co-segregated with two intronic mutations, IVS1-37T>C and IVS3-69insTTTT. Although we analyzed the function of the recombinant N34S protein, we could not demonstrate the loss of function of this protein. Intronic mutations, rather than N34S itself (IVS1-37T>C + N34S + IVS3-69insTTTT complex), may be associated with the decreased function of the PSTI. Alternatively, increased digestion of N34S in vivo may be applicable. As for R67C, the conformational alteration of the protein by forming intra-molecular or inter-molecular disulfide bonds with 67Cys was strongly suggested. These results, along with the brand-new findings in PSTI knockout mice, suggest that the genetic mutation of the PSTI is one of the important mechanisms for predisposition to pancreatitis by lowering the trypsin inhibitory function.

Functional analysis of recombinant pancreatic secretory trypsin inhibitor protein with amino-acid substitution.

BACKGROUND: We hypothesized that mutation of the pancreatic secretory trypsin inhibitor ( PSTI) gene may promote a predisposition to pancreatitis, possibly by reducing the inhibition of trypsin activity. Based on this hypothesis, we performed a biochemical analysis of recombinant PSTI protein. METHODS: The trypsin inhibitory activity of the recombinant protein was analyzed. The activity of PSTI protein with a point mutation of the most common type: (34)Asn (AAT)-to-Ser (AGT)(101A>G N34S: N34S) in exon 3, was compared with that of the wild type. RESULTS: The function of N34S PSTI remained unchanged under both the usual alkaline and acidic conditions compared with the wild-type PSTI. The calcium concentration did not affect the activity of recombinant PSTI. The trypsin susceptibility of the N34S protein was not increased either. CONCLUSIONS: Mechanisms other than the conformational change of PSTI associated with amino-acid substitution, such as abnormal splicing, may underlie the predisposition to pancreatitis in patients with the N34S mutation.