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BACKGROUND & AIMS: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. METHODS: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. RESULTS: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. CONCLUSIONS: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk i

The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch), Pharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH 5.9 (64 mg/L at 0.02 1/h) compared to pH 5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.

An enigmatic and never described hyper-reactivity of most of the cysteines resident in the reduced, molten globule-like intermediate of a few proteins has been recently discovered. In particular, all ten cysteines of chymotrypsinogen showed hundred times increased reactivity against hydrophobic reagents. A single cysteine (Cys1) was also found thousand times more reactive toward GSSG, making speculate that a single glutathionylation could represent the primordial event of its oxidative folding. In the present study, we compare these kinetic properties with those present in trypsinogen taken in its reduced, molten globule-like intermediate and identify the origin of these unusual properties. Despite the divergent evolution of these two proteins, the different amount of disulfides and the very different 3D localization of three disulfides, their hyper-reactivity toward hydrophobic thiol reagents and disulfides is very similar. Mass spectrometry identifies two cysteines in trypsinogen, Cys148 and Cys197, 800 times more reactive toward GSSG than an unperturbed protein cysteine. These results point toward a stringent and accurate preservation of these peculiar kinetic properties during a divergent evolution suggesting some important role, which at the present can only be hypothesized. Similar extraordinary hyper-reactivity has been found also in albumin, ribonuclease, and lysozyme confirming that it cannot be considered a kinetic singularity of a single protein. Interestingly, the very flexible and fluctuating structures like those typical of the molten globule status prove capable of enabling sophisticated actions typical of enzymes such as binding to GSSG with relevant specificity and high affinity (K-D = 0.4 mm) and accelerating the reaction of its cysteines by thousands of times.

ObjectivesProteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity.DesignTrypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3.ResultsWe showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism.ConclusionsIn IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity.

ObjectivesProteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity.DesignTrypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3.ResultsWe showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism.ConclusionsIn IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity.

Zymogen secretory granules in pancreatic acinar cells express two vesicle-associated membrane proteins (VAMP), VAMP2 and -8, each controlling 50% of stimulated secretion. Analysis of secretion kinetics identified a first phase (0-2 min) mediated by VAMP2 and second (2-10 min) and third phases (10-30 min) mediated by VAMP8. Induction of acinar pancreatitis by supramaximal cholecystokinin (CCK-8) stimulation inhibits VAMP8-mediated mid- and late-phase but not VAMP2-mediated early-phase secretion. Elevation of cAMP during supramaximal CCK-8 mitigates third-phase secretory inhibition and acinar damage caused by the accumulation of prematurely activated trypsin. VAMP8-/- acini are resistant to secretory inhibition by supramaximal CCK-8, and despite a 4.5-fold increase in total cellular trypsinogen levels, are fully protected from intracellular trypsin accumulation and acinar damage. VAMP8-mediated secretion is dependent on expression of the early endosomal proteins Rab5, D52, and EEA1. Supramaximal CCK-8 (60 min) caused a 60% reduction in the expression of D52 followed by Rab5 and EEA1 in isolated acini and in in vivo The loss of D52 occurred as a consequence of its entry into autophagic vacuoles and was blocked by lysosomal cathepsin B and L inhibition. Accordingly, adenoviral overexpression of Rab5 or D52 enhanced secretion in response to supramaximal CCK-8 and prevented accumulation of activated trypsin. These data support that acute inhibition of VAMP8-mediated secretion during pancreatitis triggers intracellular trypsin accumulation and loss of the early endosomal compartment. Maintaining anterograde endosomal trafficking during pancreatitis maintains VAMP8-dependent secretion, thereby preventing accumulation of activated trypsin.

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N-terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N-terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.

Serine proteases are involved in many fundamental physiological processes, and control of their activity mainly results from the fact that they are synthetized in an inactive form that becomes active upon cleavage. Three decades ago Martin Karplus's group performed the first molecular dynamics simulations of trypsin, the most studied member of the serine protease family, to address the transition from the zymogen to its active form. Based on the computational power available at the time, only high frequency fluctuations, but not the transition steps, could be observed. By performing accelerated molecular dynamics (aMD) simulations, an interesting approach that increases the configurational sampling of atomistic simulations, we were able to observe the N-terminal tail insertion, a crucial step of the transition mechanism. Our results also support the hypothesis that the hydrophobic effect is the main force guiding the insertion step, although substantial enthalpic contributions are important in the activation mechanism. As the N-terminal tail insertion is a conserved step in the activation of serine proteases, these results afford new perspective on the underlying thermodynamics of the transition from the zymogen to the active enzyme.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

Objective: To compare three cystic fibrosis (CF) newborn screening strategies used in Victoria since 1989. Design, setting and participants: Retrospective review of newborn screening and clinical records for people with CF born in Victoria between 1989 and 2008 to compare screening strategies: repeat immunoreactive trypsinogen (IRT) testing (IRT/IRT, 1989-1990), IRT and p.F508del mutation analysis (IRT/ p.F508del, 1991-2006) and IRT with analysis of 12 CFTR mutations (IRT/12 mutations, 2007-2008). Main Outcome Measure(s): Total number of infants screened, people identified with CF (by screening or clinical diagnosis), number of CF-affected terminations of pregnancy, and number of carriers detected. Result(s): There were 420 people born with CF (live-birth prevalence, 1/3139; 95% CI, 1/2853-1/3462) and 78 CF-affected pregnancy terminations (overall prevalence, 1/2647; 95% CI, 1/2425-1/2896). Of the babies born with CF, 283 (67.4%) were detected by newborn screening alone, 61 (14.5%) had meconium ileus, 33 (7.9%) had a family history of CF, nine (2.1%) were diagnosed antenatally, and 34 (8.1%) were missed by screening (17 missed because IRT level was < 99th percentile, two with repeat IRT level not elevated, 14 without a screened CFTR mutation, and one with missing data). The sensitivities of the protocols were 86.6% for IRT/IRT, 89.9% for IRT/p.F508del, and 95.8% for IRT/ 12 mutations. Including 12 mutations in the analysis detected one patient who would otherwise have been missed and, had this protocol been implemented from 1989, it would have detected four others. Conclusion(s): Most babies with CF without meconium ileus, a family history or antenatal diagnosis are detected by newborn screening. Despite improved sensitivity with the 12-mutation analysis, most infants detected would have been diagnosed using the IRT/p.F508del protocol.

Objective: To compare three cystic fibrosis (CF) newborn screening strategies used in Victoria since 1989. Design, setting and participants: Retrospective review of newborn screening and clinical records for people with CF born in Victoria between 1989 and 2008 to compare screening strategies: repeat immunoreactive trypsinogen (IRT) testing (IRT/IRT, 1989-1990), IRT and p.F508del mutation analysis (IRT/ p.F508del, 1991-2006) and IRT with analysis of 12 CFTR mutations (IRT/12 mutations, 2007-2008). Main Outcome Measure(s): Total number of infants screened, people identified with CF (by screening or clinical diagnosis), number of CF-affected terminations of pregnancy, and number of carriers detected. Result(s): There were 420 people born with CF (live-birth prevalence, 1/3139; 95% CI, 1/2853-1/3462) and 78 CF-affected pregnancy terminations (overall prevalence, 1/2647; 95% CI, 1/2425-1/2896). Of the babies born with CF, 283 (67.4%) were detected by newborn screening alone, 61 (14.5%) had meconium ileus, 33 (7.9%) had a family history of CF, nine (2.1%) were diagnosed antenatally, and 34 (8.1%) were missed by screening (17 missed because IRT level was < 99th percentile, two with repeat IRT level not elevated, 14 without a screened CFTR mutation, and one with missing data). The sensitivities of the protocols were 86.6% for IRT/IRT, 89.9% for IRT/p.F508del, and 95.8% for IRT/ 12 mutations. Including 12 mutations in the analysis detected one patient who would otherwise have been missed and, had this protocol been implemented from 1989, it would have detected four others. Conclusion(s): Most babies with CF without meconium ileus, a family history or antenatal diagnosis are detected by newborn screening. Despite improved sensitivity with the 12-mutation analysis, most infants detected would have been diagnosed using the IRT/p.F508del protocol.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

Background and aims: In spite of the fact that pancreatic cancer is a relatively infrequent disease, it ranks 8th in the worldwide ranking of cancer death due to the poor prognosis. The mortality rate is almost as high as the incidence with a M/I ratio of 98%, indicating an extremely dismal clinical course. This makes it imperative to try to develop new therapeutic strategies and to try to identify risk factors in order to intensify preventive efforts. The most important risk factor for pancreatic cancer is tobacco smoking, but there are other putative environmental risk factors and some pre-existing diseases that have been linked to pancreatic cancer. The aim of this thesis is to evaluate different epidemiological aspects in relation to pancreatic cancer; in more specific terms to investigate the relation between alcohol and pancreatic cancer, between trypsinogen, pancreatic secretory trypsin inhibitor (PSTI) and pancreatic cancer, between Helicobacter pylori infection and pancreatic cancer and to investigate if the metabolic syndrome is associated with the risk of pancreatic cancer. Results and conclusion: High alcohol intake, estimated using both a questionnaire on attitude towards alcohol and a laboratory marker in the form of γ-GT is associated with a subsequent high risk of developing pancreatic cancer. The previously established association between smoking and pancreatic cancer is confirmed. The hypothesis that pancreatic cancer is related to an imbalance between the trypsinogen isoforms is in line with the finding concerning the ratio of human anionic trypsinogen and human cationic trypsinogen (HAT/HCT). There is no overall association between H.pylori infection and the risk of pancreatic cancer, but H.pylori infection may increase the risk of pancreatic cancer in never smokers and in low alcohol consumers. High mid-blood pressure, high fasting glucose and the metabolic syndrome as an entity are associated with an increased risk of pancreatic cancer in women

Matrix metalloproteinases, MMPs, are a family of enzymes capable of modulating connective tissue components. The expression of several MMPs is increased in oral squamous cell carcinomas (OSCCs). They are assumed to have an important role in the development and progression of OSCCs. However, the exact role and mechanism of the regulation of MMPs in malignant transformation are still largely unknown. In this study, tumour-associated trypsin-2 (TAT-2) was detected in OSCC tissue sections, and its role in MMP-2 and -9 regulation in carcinoma cells was evaluated. The TAT-2 gene was transfected into two different OSCC cell lines and one immortalized oral epithelial cell line. In TAT-2-transfected cells, MMP-9 activation increased OSCC cell invasion in chicken chorionallantoic membrane assay. Increased intravasation was prevented by tumour-associated trypsin inhibitor or specific gelatinase-inhibiting CTT-peptide. TAT-2 also converted MMP-1, -8, -13 and -3 into smaller molecular weight forms in vitro. However, TAT-2-transfected OSCC cells showed no conversion. TAT-2 was demonstrated to degrade powerfully type I collagen into small fragments in vitro. The cell surface receptor αvβ6 integrin is strongly up-regulated in OSCCs. By using β6-transfected OSCC cells, it was demonstrated that αvβ6 integrin down-regulates MMP-13 expression. However, this integrin did not regulate other collagenases or TIMP-1. β6-transfected cells invaded more efficiently through the basement membrane matrix, but their migration through type I collagen remained unchanged. MMP-8 expression was detected for the first time in head and neck squamous cell carcinoma (HNSCC) cell lines and corresponding cultured dermal and tumour fibroblasts. The localization of MMP-8 in HNSCC was determined by immunohistochemical stainings and in situ hybridization. MMP-8 production levels in carcinoma cells were faint and sporadic in HNSCCs sections. Ninety-two primary mobile tongue SCCs were subjected to MMP-8 i

Cancer progression is a complex multi-step process. Two critical steps in tumor growth and invasion are the proteolytic processing of the extracellular matrix environment and the angiogenic switch enabling blood supply into the tumor. Matrix metalloproteases (MMPs) are a group of proteolytic enzymes involved in physiological and pathological extracellular matrix processing. Trypsinogen, a serine protease, is one of the first proteolytic enzymes characterized. The amount of one of its isoforms, tumor associated trypsinogen-2 (TAT-2) correlates with the malignant phenotype of several forms of cancers. Both of these protease groups are critically dependent on their activation from latent proforms to fully active enzymes. We found that the overproduction of TAT-2 in malignant oral squamous cell carcinoma cell line was associated with elevated proMMP-9 (but not proMMP-2) activation, as well as enhanced cancer cell intravasation in an in vivo model. This indicates that TAT-2 and MMP-9 activation play a role in the invasive growth of oral carcinomas. Proteases are involved in angiogenesis, the formation of new blood vessels, in several ways. One mechanism is the release of cryptic anti-angiogenic molecules from larger extracellular matrix components. Endostatin is one such cryptic endogenous inhibitor of angiogenesis. Certain MMPs were able to cleave endostatin from its parent molecule, collagen XVIII. The endostatin fragments generated by MMP-3, -7, -9, -13 and -20 inhibited angiogenesis in a similar fashion as the native endostatin. The regulation between MMPs and endostatin was shown to be reciprocal, as endostatin was able to block the activation and activities of MMP-2, -9 and -13. The inhibition of these tumor-associated MMPs explains at least in part the anti-tumor activity of endostatin. Endostatin not only affects endothelial cell growth as is usually thought, but it also inhibits the migration of oral carcinoma cells. In addition, cell density and proper

Background and aims: Activation of pancreatic proteases is considered to be a crucial event in the early phase of acute pancreatitis but the cause of this activation is not known. Most cases of acute pancreatitis can be attributed to either gallstone disease or alcohol abuse. However, little is known about other risk factors. The aim of this thesis is to investigate the mechanisms involved in the initiation of acute pancreatitis, trends in the incidence, and risk factors for the disease. The potential role of smoking as a risk factor was given special attention and the effect of nicotine on exocrine pancreas was studied in a rat model. Results and conclusions: Cathepsin B activated trypsinogen but not proelastase or procarboxypeptidase B. Hence, if cathepsin B is to play a role in the activation of digestive enzymes in acute pancreatitis, this probably occurs through activation of trypsinogen. The incidence of gallstone-related acute pancreatitis increased by 7.6% per year (95% confidence interval (CI), 4.0 to 11.4) in Malmö 1985?1999. The incidence of alcohol-related acute pancreatitis decreased by ?5.1% per year (95 % CI, ?7.4 to ?2.8). The risk for acute pancreatitis was increased in smokers (relative risk 2.14, (95% CI, 1.48 to 3.09)), after adjustment for age, sex, body mass index and alcohol consumption. There was a weak association between body mass index and the risk for acute pancreatitis (p=0.02). Nicotine induced increased concentrations of pancreatic proenzymes in pancreatic extract but had no impact on the production of the same enzymes. These findings suggest that nicotine impairs acinar cell secretion. We propose that this might be a contributory mechanism behind the association between smoking and pancreatic disease.

Endoscopic Retrograde Cholangiopancreatography (ERCP) is a widely performed gastroenterological procedure often accompanied by the development of acute pancreatitis post-ERCP - the current literature quoting an incidence between 0% and 39.5%. Because acute pancreatitis can be a very serious outcome, sometimes leading to death, a method for predicting poor outcomes post-ERCP would be an advantage to the clinicians and patients. Described below is the development of two algorithms with a strong capacity to predict poor outcomes post-ERCP. In addition to assessing a battery of common laboratory blood tests, this study also took the opportunity to study a recently described but still research-based analyte, the enzymatic activity of the trypsin bound to the serine protease inhibitor alpha- 2- macroglobulin ( a2M) complex called Macroglobulin-Trypsin complex-Like Substance (MTLS), high levels of MTLS have been identified in patients who developed acute pancreatitis post-ERCP. It was hypothesized that MTLS might be a useful, novel indicator of patients at risk of development of post-ERCP pancreatitis. Three separate studies were carried out. The first was a retrospective study of 72 patients presenting for ERCP to the collaborating gastroenterologist, and the second was a prospective study involving 29 more patients. The retrospective study involved the analysis of previously collected blood specimens pre-ERCP and post-ERCP. On the pre-ERCP specimens 22 laboratory tests were performed to test the haematological, biochemical and coagulation status of the patients. Lipase, a classical diagnostic marker for acute pancreatitis, was measured on the patient's post-ERCP samples. From this study sophisticated statistical analysis was based on a 2-step process - Factor Analysis with Principal Component Extraction followed by Discriminant Analysis on the key components identified by Factor Analysis - in order to develop a predictive algorithm for poor outcomes post-ERCP. The object

Objective: To explore relationships between malnutrition and pancreatic damage in hospitalised aboriginal children. Methods: Immunoreactive trypsinogen (IRT) concentrations were measured in two populations of hospitalised aboriginal children in Australia; 472 children aged 0-3 years, in Alice Springs (Northern Territory); and 187 children aged 0-16 years in Mount Isa (Queensland). Correlation of whole blood IRT with height and weight z-scores, four-site skinfold thickness and upper arm circumference was sought. Results: In Mount Isa, the geometric mean IRT concentration rose with decreasing weight z-score. The IRT concentration was otherwise unrelated to nutritional indices. Sixty percent of the 39 Mount Isa patients with gastroenteritis and 24.5% of the 358 Alice Springs patients with gastroenteritis had an IRT concentration in the upper quartile for their population, compared with 16% for patients with other diagnoses in both populations. Conclusions: A high IRT concentration in patients with low weight z-scores is a confounding effect of gastroenteritis, and may result from subclinical pancreatic disease in gastroenteritis.

Pancreatic exocrine dysfunction has been frequently recorded in protein-energy malnutrition in underdeveloped countries. In addition, the pancreas requires optimal nutrition for enzyme synthesis and potentially correctable pancreatic enzyme insufficiency may play a role in the continuation of protein-energy malnutrition. This problem has not been previously evaluated in Australian Aborigines. We have applied a screening test for pancreatic dysfunction (human immunoreactive trypsinogen [IRT] assay) to the study of 398 infants (6-36 months) admitted to the Alice Springs Hospital over a 20-month period. All infants were assessed by anthropometric measures and were assigned to to three nutritional groups (normal, moderate or severely malnourished) and two growth groups (stunted or not stunted). Of the 198 infants who had at least a single serum cationic trypsinogen measurement taken, normal values for serum IRT (with confidence limits) were obtained from 57 children, who were normally nourished. IRT levels were significantly correlated with the degree of underweight but there was no correlation with the degree of stunting or age. Mean IRT levels for the moderate and severely underweight groups were significantly greater than the mean for the normal group (P < 0.01). Seventeen children (8.6%) had trypsinogen levels in excess of the 95th percentile for the normally nourished group, reflecting acinar cell damage or ductal obstruction. We conclude that pancreatic dysfunction may be a common and important overlooked factor contributing to ongoing malnutrition and diseases in malnourished Australian Aboriginal children.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.

Indirect and qualitative tests of pancreatic function are commonly used to screen patients with cystic fibrosis for pancreatic insufficiency. In an attempt to develop a more quantitative assessment, we compared the usefulness of measuring serum pancreatic lipase using a newly developed enzyme-linked immunosorbent immunoassay with that of cationic trypsinogen using a radioimmunoassay in the assessment of exocrine pancreatic function in patients with cystic fibrosis. Previously, we have shown neither lipase nor trypsinogen to be of use in assessing pancreatic function prior to 5 years of age because the majority of patients with cystic fibrosis in early infancy have elevated serum levels regardless of pancreatic function. Therefore, we studied 77 patients with cystic fibrosis older than 5 years of age, 41 with steatorrhea and 36 without steatorrhea. In addition, 28 of 77 patients consented to undergo a quantitative pancreatic stimulation test. There was a significant difference between the steatorrheic and nonsteatorrheic patients with the steatorrheic group having lower lipase and trypsinogen values than the nonsteatorrheic group (P < .001). Sensitivities and specificities in detecting steatorrhea were 95% and 86%, respectively, for lipase and 93% and 92%, respectively, for trypsinogen. No correlations were found between the serum levels of lipase and trypsinogen and their respective duodenal concentrations because of abnormally high serum levels of both enzymes found in some nonsteatorrheic patients. We conclude from this study that both serum lipase and trypsinogen levels accurately detect steatorrhea in patients with cystic fibrosis who are older than 5 years but are imprecise indicators of specific pancreatic exocrine function above the level needed for normal fat absorption.

Serum immunoreactive pancreatic lipase and cationic trypsinogen are elevated in young infants with cystic fibrosis (CF) and may be useful neonatal screening tests for CF. We compared lipase measured by a recently developed ELISA immunoassay with trypsinogen measured by radioimmunoassay in 70 children (ages 0.1 to 9.9 years) with CF who had various degrees of pancreatic dysfunction and in 79 similarly aged children without CF (controls). In the control children, lipase activity increased with advancing age, whereas trypsinogen showed no age-related trend. Lipase and trypsinogen were significantly elevated in the infants with CF who were younger than 1 year, irrespective of pancreatic function (trypsinogen, P<0.001; lipase, P<0.05). Sensitivities in detecting CF were 76% and 90% for lipase and trypsinogen, respectively. After the first year of life, lipase and trypsinogen values declined toward normal, the rate of decline of lipase being greater than that of trypsinogen; 67% of lipase values were within or below the normal range by 3 years, whereas 67% of trypsinogen values continued to be elevated. We conclude that trypsinogen is an excellent screening test for CF in young infants regardless of pancreatic function, and that the addition of a serum pancreatic lipase determination does not improve the accuracy of trypsinogen as a screening test for cystic fibrosis.

Serum immunoreactive cationic trypsinogen levels were determined in 99 control subjects and 381 cystic fibrosis (CF) patients. To evaluate the status of the exocrine pancreas all CF patients had previously undergone fecal fat balance studies and/or pancreatic stimulation tests. Three hundred fourteen CF patients had fat malabsorption and/or had inadequate pancreatic enzyme secretion (pancreatic insufficiency) requiring oral pancreatic enzyme supplements with meals. Sixty-seven CF patients did not have fat malabsorption and/or had adequate enzyme secretion (pancreatic sufficiency) and were not receiving pancreatic enzyme supplements with meals. Mean serum trypsinogen in 99 control subjects was 31.4 ± 14.8 /µg/hter (± 2 SD) and levels did not vary with age or sex. In CF infants (< 2 yr) with pancreatic insufficiency, mean serum trypsinogen was significantly above the non-CF values (p < 0.001). Ninety-one percent of the CF infants had elevated levels. Serum trypsinogen values in the pancreatic insuffi ient group declined steeply up to 5 years, reaching subnormal values by age 6. An equation was developed which described these age-related changes very accurately. Only six CF patients with pancreatic insufficiency had serum trypsinogen levels above the 95% confidence limits of this equation. In contrast, there was no age related decline in serum trypsinogen among the CF group with pancreatic sufficiency. Under 7 yr, serum trypsinogen failed to distinguish the two groups. In those over 7 yr of age, however, serum trypsinogen was significantly higher than the CF group with pancreatic insufficiency (p < 0.001), and 93% had values within or above the control range. In conclusion, serum trypsinogen appears to be a useful screening test for CF in infancy. Between 2 and 7 yr of age this test is of little diagnostic value. After 7 yr of age, serum trypsinogen can reliably distinguish between CF patients with and without pancreatic insufficiency.

A 17-year-old white adolescent had a history of chronic diarrhea, delayed puberty, and growth failure. Investigations excluded cystic fibrosis, Shwachman syndrome, and endocrine causes of growth failure. Severe steatorrhea was diagnosed from fecal fat studies, and a jejunal suction biopsy showed total villus atrophy, consistent with a diagnosis of celiac diseases. Following introduction of a gluten-free diet, his appetite and growth improved, but he continued to have abdominal discomfort and loose offensive bowel motions. One year later, severe steatorrhea was present. A repeat jejunal biopsy showed partial recovery of villus architecture. Serum immunoreactive trypsinogen level was low, which was highly suggestive of exocrine pancreatic failure. Results of quantitative pancreatic stimulation test confirmed the presence of primary pancreatic insufficiency. After introduction of oral pancreatic enzyme supplements with meals, his gastrointestinal symptoms resolved and growth velocity accelerated. Previously, primary pancreatic insufficiency has only been described in elderly patients with long-standing untreated celiac disease. This case, however, emphasizes that pancreatic failure can occur with celiac disease at any age. Determination of a serum immunoreactive trypsinogen level should be considered a useful screening tool for pancreatic insufficiency in patients with celiac disease who have not responded to a gluten-free diet.

The exocrine protein rat anionic trypsinogen has been expressed and is secreted from the murine anterior pituitary tumor cell line AtT-20. We examined which secretory pathway trypsinogen takes to the surface of this endocrine-derived cell line. The "constitutive" pathway externalizes proteins rapidly and in the absence of an external stimulus. In the alternate, "regulated" pathway, proteins are stored in secretory granules until the cells are stimulated to secrete with 8-Br-cAMP. On the basis of indirect immunofluorescence localization, stimulation of release, and subcellular fractionation, we find that trypsinogen is targeted into the regulated secretory pathway in AtT-20 cells. In contrast, laminin, an endogenous secretory glycoprotein, is shown to be secreted constitutively. Thus it appears that the transport apparatus for the regulated secretory pathway in endocrine cells can recognize not only endocrine prohormones, but also the exocrine protein trypsinogen, which suggests that a similar sorting mechanism is used by endocrine and exocrine cells.