Trypsinogen Activation Peptide Research Articles

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.

Robust autoactivation, chymotrypsin C independence and diminished secretion define a subset of hereditary pancreatitis‐associated cationic trypsinogen mutants

Mutations in human cationic trypsinogen cause hereditary pancreatitis by altering its proteolytic regulation of activation and degradation by chymotrypsin C (CTRC). CTRC stimulates trypsinogen autoactivation by processing the activation peptide to a shorter form, but also promotes degradation by cleaving the calcium-binding loop in trypsinogen. Mutations render trypsinogen resistant to CTRC-mediated degradation and/or increase processing of the activation peptide by CTRC. Here we demonstrate that the activation peptide mutations D19A, D22G, K23R and K23_I24insIDK robustly increased the rate of trypsinogen autoactivation, both in the presence and absence of CTRC. Degradation of the mutants by CTRC was unchanged, and processing of the activation peptide was increased fourfold in the D19A mutant only. Surprisingly, however, this increased processing had only a minimal effect on autoactivation. The tetra-aspartate motif in the trypsinogen activation peptide binds calcium (KD of ~ 1.6 mM), which stimulates autoactivation. Unexpectedly, calcium binding was not compromised by any of the activation peptide mutations. Despite normal binding, autoactivation of mutants D22G and K23_I24insIDK was not stimulated by calcium. Finally, the activation peptide mutants exhibited reduced secretion from transfected cells, and secreted trypsinogen levels were inversely proportional with autoactivation rates. We conclude that D19A, D22G, K23R and K23_I24insIDK form a mechanistically distinct subset of hereditary pancreatitis-associated mutations that exert their effect primarily through direct stimulation of autoactivation, independently of CTRC. The potentially severe clinical impact of the markedly increased autoactivation is offset by diminished secretion, resulting in a clinical phenotype that is indistinguishable from typical hereditary pancreatitis.

Mutations of the trypsinogen activation peptide in hereditary pancreatitis

Mutations of the trypsinogen activation peptide in hereditary pancreatitis

New Insights into Pathogenesis, Diagnosis, and Treatment of Pancreatic Disorders

New Insights into Pathogenesis, Diagnosis, and Treatment of Pancreatic Disorders

Prediction of the severity of acute pancreatitis on admission by urinary trypsinogen activation peptide: A meta-analysis

To undertake a meta-analysis on the value of urinary trypsinogen activation peptide (uTAP) in predicting severity of acute pancreatitis on admission. Major databases including Medline, Embase, Science Citation Index Expanded and the Cochrane Central Register of Controlled Trials in the Cochrane Library were searched to identify all relevant studies from January 1990 to January 2013. Pooled sensitivity, specificity and the diagnostic odds ratios (DORs) with 95%CI were calculated for each study and were compared to other systems/biomarkers if mentioned within the same study. Summary receiver-operating curves were conducted and the area under the curve (AUC) was evaluated. In total, six studies of uTAP with a cut-off value of 35 nmol/L were included in this meta-analysis. Overall, the pooled sensitivity and specificity of uTAP for predicting severity of acute pancreatitis, at time of admission, was 71% and 75%, respectively (AUC = 0.83, DOR = 8.67, 95%CI: 3.70-20.33). When uTAP was compared with plasma C-reactive protein, the pooled sensitivity, specificity, AUC and DOR were 0.64 vs 0.67, 0.77 vs 0.75, 0.82 vs 0.79 and 6.27 vs 6.32, respectively. Similarly, the pooled sensitivity, specificity, AUC and DOR of uTAP vs Acute Physiology and Chronic Health Evaluation II within the first 48 h of admission were found to be 0.64 vs 0.69, 0.77 vs 0.61, 0.82 vs 0.73 and 6.27 vs 4.61, respectively. uTAP has the potential to act as a stratification marker on admission for differentiating disease severity of acute pancreatitis.

Prior peritoneal lavage with hot 0.9 % saline induces HSP70 expression and protects against cerulein-induced acute pancreatitis in rats

Recent studies have indicated that pre-induction of heat shock protein 70 (HSP70) expression in the pancreas protects against secretagogue-induced pancreatitis. In those studies, the HSP70 was mostly induced by unfeasible conditions. The aim of this current study was to investigate the effect of peritoneal lavage with hot 0.9% saline (42°C) on the pancreatic expression of HSP70 and its protective effect on cerulein-induced acute pancreatitis in rats. Male Wistar rats were peritoneally lavaged with 0.9% saline at 42°C for 30min. HSP70 expression was evaluated by western blotting analysis. Prior peritoneal lavages with hot and warm saline were performed. Acute pancreatitis was induced by administration of intraperitoneal injection of cerulein (20μg/kg) four times, and its severity was assessed by measuring serum amylase, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and trypsinogen activation peptide (TAP) levels. Pancreatic sections were stained with hematoxylin and eosin for histological evaluation. Peritoneal lavage with hot 0.9% saline increased intrapancreatic HSP70 expression and ameliorated the cerulein-induced pancreatitis in rats, judged by the significantly reduced serum amylase, TNF-α, and IL-6 concentrations; histopathological scores, and serum TAP levels. Peritoneal lavage with hot 0.9% saline can induce HSP70 expression and prevent cerulein-induced acute pancreatitis in rats. The results suggest that HSP70 protects against cerulein-induced pancreatitis by preventing proinflammatory cytokine synthesis and trypsinogen activation during acute pancreatitis.

Increased Activation of Hereditary Pancreatitis-associated Human Cationic Trypsinogen Mutants in Presence of Chymotrypsin C

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP). Moreover, we have discovered that L-HEP hydrolyzed several nonspecific peptidic substrates. In this work, we aimed to further characterize species-specific enteropeptidase activities and to reveal their structural basis. First, we compared hydrolysis of peptides and proteins lacking DDDDK sequence by L-HEP and L-BEP. In each case human enzyme was more efficient, with the highest hydrolysis rate observed for substrates with a large hydrophobic residue in P2-position. Computer modeling suggested enzyme exosite residues 96 (Arg in L-HEP, Lys in L-BEP) and 219 (Lys in L-HEP, Gln in L-BEP) to be responsible for these differences in enteropeptidase catalytic activity. Indeed, human-to-bovine mutations Arg96Lys, Lys219Gln shifted catalytic properties of L-HEP toward those of L-BEP. This effect was amplified in case of the double mutation Arg96Lys/Lys219Gln, but still did not cover the full difference in catalytic activities of human and bovine enzymes. To find a missing link, we studied monopeptide benzyl-arginine-β-naphthylamide hydrolysis. L-HEP catalyzed it with an order lower K m than L-BEP, suggesting the monopeptide-binding S1 site input into catalytic distinction between two enteropeptidase species. Together, our findings suggest structural basis of the unique catalytic properties of human enteropeptidase and instigate further studies of its tentative physiological and pathological roles.

Autophagy Regulation by the Nuclear Factor κB Signal Axis in Acute Pancreatitis

The objective of the study was to investigate the significance of LC3 and beclin 1 in the pancreas of rat after acute necrotizing pancreatitis (ANP) and whether nuclear factor κB (NF-κB) signal regulates autophagy during sodium taurocholate-induced ANP. Acute necrotizing pancreatitis was induced in rat by sodium taurocholate injection in the pancreaticobiliary duct. Pyrrolidine dithiocarbamate was administrated before the injection of sodium taurocholate. Then, serum amylase activity, trypsinogen activation peptide and tumor necrosis factor α, and morphological signs of pancreatitis were measured. The formation of autophagosome and the activation of lysosome were observed in the pancreas after ANP by using electron microscope. Meanwhile, the pancreatic levels of NF-κB and essential proteins involved in formation of the autophagosome (LC3 and beclin 1) were assessed by Western blotting and immunohistochemistry. Sodium taurocholate increased serum amylase, trypsinogen activation peptide, tumor necrosis factor α, and pancreatic NF-κB levels compared with sham-operated rats and increased the levels of LC3-II and beclin 1. Inhibition of the NF-κB signal axis with pyrrolidine dithiocarbamate reduced serum amylase, blocked pancreatic NF-κB activation, and reduced the levels of LC3-II and beclin 1. Nuclear factor κB pathway activation stimulates autophagy during induction of ANP. Targeted inhibition of the NF-κB pathway may provide novel therapeutic strategies for reducing the severity of ANP.

Early intraperitoneal metabolic changes and protease activation as indicators of pancreatic fistula after pancreaticoduodenectomy

Ischaemia and local protease activation close to the pancreaticojejunal anastomosis (PJA) are potential mechanisms of postoperative pancreatic fistula (POPF) formation. To provide information on the pathophysiology of POPF, intraperitoneal microdialysis was used to monitor metabolic changes and protease activation close to the PJA after pancreaticoduodenectomy (PD). In patients who underwent PD, intraperitoneal metabolites (glycerol, lactate, pyruvate and glucose) were measured by microdialysis, and lactate and glucose in blood were monitored, every 4 h for 5 days, starting at 12.00 hours on the day after surgery. Trypsinogen activation peptide (TAP) was measured in microdialysates as a marker of protease activation. Intraperitoneal glycerol levels and the ratio of lactate to pyruvate were higher after PD and glucose levels were lower in seven patients who later developed symptomatic POPF than in eight patients with other surgical complications (OSC) and 33 with no surgical complications (NSC) (all P < 0·050). TAP was detected at a concentration greater than 0·1 µg/l in six of seven patients with POPF, two of eight with OSC and two of 33 with NSC. Intraperitoneal lactate concentrations were higher than systemic levels in all patients on days 1 to 5 after surgery (P < 0·001). In patients with POPF, high intraperitoneal lactate concentrations were observed without systemic hyperlactataemia. Early in the postoperative phase, patients who later developed clinically significant POPF had higher intraperitoneal glycerol concentrations and lactate/pyruvate ratios, and lower glucose concentrations in combination with a TAP level exceeding 0·1 µg/l close to the PJA, than patients who did not develop POPF.

Polyamine Catabolism in Relation to Trypsin Activation and Apoptosis in Experimental Acute Pancreatitis

Background: Overinduced polyamine catabolism (PC) in a transgenic rat model has been suggested to be a mediator of trypsin activation which is important in acinar cell necrosis. PC has also been observed in experimental taurodeoxycholate pancreatitis. We hypothesized that PC may be a mediator of trypsin activation in taurodeoxycholate pancreatitis. Methods: Pancreatitis was induced in wild-type rats by 2 or 6% taurodeoxycholate infusion or in transgenic rats by overexpressing spermidine/spermine N1-acetyltransferase (SSAT). The time courses of necrosis, caspase-3 immunostaining, SSAT, polyamine levels, and trypsinogen activation peptide (TAP) were monitored. The effect of the polyamine analogue bismethylspermine (Me2Spm) was investigated. Results: In a transgenic pancreatitis model, TAP and acinar necrosis increased simultaneously after the activation of SSAT, depletion of spermidine, and development of apoptosis. In taurodeoxycholate pancreatitis, necrosis developed along with the accumulation of TAP. SSAT was activated simultaneously or after TAP accumulation and less than in the transgenic model, with less depletion of spermidine than in the transgenic model. Supplementation with Me2Spm ameliorated the extent of acinar necrosis at 24 h, but contrary to previous findings in the transgenic model, in the taurodeoxycholate model it did not affect trypsin activation. Compared with the transgenic model, no extensive apoptosis was found in taurodeoxycholate pancreatitis. Conclusions: Contrary to transgenic SSAT-overinduced pancreatitis, PC may not be a mediator of trypsin activation in taurodeoxycholate pancreatitis. The beneficial effect of polyamine supplementation on necrosis in taurodeoxycholate pancreatitis may rather be mediated by other mechanisms than amelioration of trypsin activation.

Strategy for improvement of enteropeptidase efficiency in tag removal processes

Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. It has also great biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occurred during cleavage of fusions when high amount of enzyme is required. In this study we have improved the efficiency of fusion proteins cleavage by enteropeptidase by substitution of the Lys residue by Arg in specific cleavage sequence (Asp)4-Lys. We have demonstrated that 3–6-fold lower amounts of the catalytic subunit of human and bovine enteropeptidase is required for 95% cleavage of Trx/TRAIL and Trx/FGF-2 fusions with (Asp)4-Arg cleavage sequence in comparison to native sequence (Asp)4-Lys. As a result, reduced amount of non-specifically cleaved peptide fragments were observed during cleavage of (Asp)4-Lys/Arg mutated fusions. These findings overcome limitations of enteropeptidase in tag removal processes during recombinant proteins purification and extend its commercial benefit in the biopharmaceutical industry.

Significance of trypsinogen activation peptides and interleukin-6 in experimental acute pancreatitis.

To explore the feasibility of using plasma trypsinogen activation peptides (TAP) and serum interleukin-6(IL-6) as early markers for predicting the severity of experimental acute pancreatitis. Ninety male adult Sprague-Dawley rats were equally randomized into five groups: edema pancreatitis group, treated with retrograde ductal infusion of 3% sodium taurocholate solution; necrosis pancreatitis group, treated with retrograde ductal infusion of 5% sodium taurocholate solution; treatment pancreatitis group, treated with retrograde ductal infusion of 3% sodium taurocholate solution and ulinastatin intravenous infusion half an hour later; control pancreatitis group, treated with 0.9% normal saline retrograde ductal infusion; and sham operation group, treated with sham operation. Rats in each group were equally randomized into three subgroups, which were killed by exsanguination 3, 6, or 24 hours after infusion, and blood specimens were obtained. Serum amylase, plasma TAP, and serum IL-6 were determined. The severity of pancreatitis was scored by two blinded pathologists under microscope. At 3 and 6 hours after infusion, plasma TAP concentration of necrosis pancreatitis group [(4.798±0.169) and (3.999±0.299)nmol/L, respectively]were significantly higher than those of edema pancreatitis group [(2.416±0.148) and (3.356±0.211)nmol/L, respectively] (P<0.01); at 6 hours after infusion, serum IL-6 level of necrosis pancreatitis group [(1339.51±56.43)pg/ml]was significantly higher than that of edema pancreatitis group [(619.07±42.25)pg/ml] (P<0.01). In this acute pancreatitis model, the peak levels of plasma TAP and serum IL-6 may appear earlier in rats with severer disease. Serum TAP level may be used as a marker for the accurate early prediction of the severity of acute pancreatitis.

Rho‐kinase signalling regulates trypsinogen activation and tissue damage in severe acute pancreatitis

Severe acute pancreatitis (SAP) is characterized by trypsinogen activation, infiltration of leucocytes and tissue necrosis but the intracellular signalling mechanisms regulating organ injury in the pancreas remain elusive. Rho-kinase is a potent regulator of specific cellular processes effecting several pro-inflammatory activities. Herein, we examined the role of Rho-kinase signalling in acute pancreatitis. Pancreatitis was induced by infusion of taurocholate into the pancreatic duct in C57BL/6 mice. Animals were treated with a Rho-kinase inhibitor Y-27632 (0.5-5 mg·kg⁻¹) before induction of pancreatitis. Taurocholate infusion caused a clear-cut increase in blood amylase, pancreatic neutrophil infiltration, acinar cell necrosis and oedema formation in the pancreas. Levels of pancreatic myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), trypsinogen activation peptide (TAP) and lung MPO were significantly increased, indicating local and systemic disease. Inhibition of Rho-kinase activity dose-dependently protected against pancreatitis. For example, 5 mg·kg⁻¹ Y-27632 reduced acinar cell necrosis, leucocyte infiltration and pancreatic oedema by 90%, 89% and 58%, respectively, as well as tissue levels of MPO by 75% and MIP-2 by 84%. Moreover, Rho-kinase inhibition decreased lung MPO by 75% and blood amylase by 83%. Pancreatitis-induced TAP levels were reduced by 61% in Y-27632-treated mice. Inhibition of Rho-kinase abolished secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro. Our novel data suggest that Rho-kinase signalling plays an important role in acute pancreatitis by regulating trypsinogen activation and subsequent CXC chemokine formation, neutrophil infiltration and tissue injury. Thus, these results indicate that Rho-kinase may constitute a novel target in the management of SAP.

Mechanisms of the Beneficial Effect of Hypertonic Saline Solution in Acute Pancreatitis

Administration of hypertonic saline (HS) solution to rats with acute pancreatitis (AP) decreases mortality and systemic inflammation. We hypothesized that these effects are related not only to systemic inflammatory reduction, but also to a reduction of the pancreatic lesion. Acute pancreatitis was induced in Wistar rats by injection of 2.5% sodium taurocholate. Animals were divided in groups: without AP, not treated AP, AP treated with NaCl 0.9%, and AP treated with NaCl 7.5%. Trypsinogen activation peptides and amylase activity were increased in ascitic fluid and serum and were not affected by treatment with HS. Pancreatic inflammation was evaluated by increased myeloperoxidase activity, malondialdehyde formation, and histopathology for severity of pancreatic lesions. The HS did not affect these parameters. Expression of cyclooxygenase 2 and inducible nitric oxide synthase was markedly increased in the pancreas of the AP group and was reduced by treatment with HS. This treatment also reduced the levels of TNF-α and IL-6 but not of IL-10 in the pancreatic tissue. These results show that HS modulates cytokine production and expression of enzymes responsible for inflammatory mediator production in the pancreas without affecting the severity of the pancreatic lesions.

Down-regulation of HSP60 expression by RNAi increases lipopolysaccharide- and cerulein-induced damages on isolated rat pancreatic tissues

The objective of this study was to investigate the function of heat shock protein 60 (HSP60) on pancreatic tissues by applying HSP60 small interfering RNA (siRNA) to reduce HSP60 expression. Rat pancreas was isolated and pancreatic tissue snips were prepared, cultured, and stimulated with low and high concentrations of cerulein (10(-11) and 10(-5) mol/L) or lipopolysaccharide (LPS, 10 and 20μg/mL). Before the stimulation and 1 and 4h after the stimulation, the viability and the level of trypsinogen activation peptide (TAP) in the tissue fragments were determined and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) in the culture supernatants were measured. Real-time PCR and Western blotting were used to evaluate the HSP60 mRNA and protein expression. After the administration of siRNA to inhibit HSP60 expression in the isolated tissues, these injury parameters were measured and compared. The pancreatic tissues in the control (mock-interfering) group showed a decreased viability to varying degrees after being stimulated with cerulein or LPS, and the levels of TAP, TNF-α, and IL-6 increased significantly (p < 0.05) in the tissues and/or in the culture supernatant. The expressions of HSP60 mRNA and protein were raised moderately after stimulating 1h with low concentrations of cerulein or LPS, but decreased with high concentrations of the toxicants. In particular, the expression of HSP60 protein was reduced significantly (p < 0.05) when the tissues were stimulated by the two toxicants for 4h. In contrast, the tissue fragments in which HSP60 siRNA was applied showed much lower tissue viability (p < 0.01) and higher levels of TNF-a, IL-6, and TAP (p < 0.01) in the tissues or culture supernatant after stimulating with the toxicants at the same dose and for the same time duration as compared with those of the control groups (p < 0.05). The results indicated that both cerulein and LPS can induce injuries on isolated pancreatic tissues, but the induction effects are dependent on the duration of the stimulation and on the concentrations of the toxicants. HSP60 siRNA reduces HSP60 expression and worsens the cerulein- or LPS-induced injuries on isolated pancreatic tissues, suggesting that HSP60 has a protective effect on pancreatic tissues against these toxicants.

Pancreatic injury in hepatic alcohol dehydrogenase-deficient deer mice after subchronic exposure to ethanol

Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH−) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH− and hepatic ADH-normal (ADH+) deer mice fed 1%, 2% or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was ∼1.5-fold greater in ADH− vs. ADH+ deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH− deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.

Serum and Urine Trypsinogen Activation Peptide in Assessing Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis

Pezzilli, Raffaele MD1; Mariani, Alberto MD2; Gabbrielli, Armando MD3; Morselli-Labate, Antonio M. PhD1; Barassi, Alessandra MD4 Author Information

Cathepsin L Inactivates Human Trypsinogen, Whereas Cathepsin L-Deletion Reduces the Severity of Pancreatitis in Mice

Acute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL) can also process trypsinogen to active trypsin and has a role in pancreatitis. In CTSL-deficient (Ctsl(-/-)) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl(-/-) and Ctsb(-/-) mice were studied. CTSL was expressed in human and mouse pancreas, colocalized with trypsinogen in secretory vesicles and lysosomes, and secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl(-/-) mice, whereas apoptosis and intrapancreatic trypsin activity were increased. CTSL-induced cleavage of trypsinogen occurred 3 amino acids toward the C-terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (trypsinogen activation peptide [TAP]). This elongated TAP was not detected by enzyme-linked immunosorbent assay (ELISA) but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic. CTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity.

Recent Developments in Acute Pancreatitis

The incidence of acute pancreatitis (AP) has been increasing worldwide, but the major etiologies remain gallstones and alcohol. Several studies have reported that smoking is an independent risk factor for developing AP. Classification of AP has traditionally used the categories of mild and severe disease. However, a new intermediate category of moderately severe AP has been described with intermediate characteristics including a high incidence of local complications but a low mortality. Assessment criteria that can serve as early predictors of AP severity are often complex and not sufficiently accurate. However, several recently described criteria that rely on criteria such as the body mass index, physical findings, and simple laboratory measurements could prove useful if validated in large prospective studies. Many issues related to the therapy of AP are still unresolved. Although preliminary studies support the importance of early volume expansion for the treatment of acute pancreatitis, optimization of the amount and type of fluids will require further studies. Similarly, preliminary studies suggest that enteral nutrition might benefit patients with AP and could even be useful early in the course of disease. However, the timing and type of fluids as well as the intestinal infusion site require further study. Finally, issues related to the prophylactic use of antibiotics in patients with severe AP have not been resolved. While the process of clinical investigation moves slowly, progress has been made in clinical studies of AP.