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Premature intrapancreatic trypsinogen activation is widely regarded as an initiating event for acute pancreatitis. Previous studies have alternatively implicated secretory vesicles, endosomes, lysosomes, or autophagosomes/autophagolysosomes as the primary site of trypsinogen activation, from which a cell-damaging proteolytic cascade originates. To identify the subcellular compartment of initial trypsinogen activation we performed a time-resolution analysis of the first 12 h of caerulein-induced pancreatitis in transgenic light chain 3 (LC3)-GFP autophagy reporter mice. Intrapancreatic trypsin activity increased within 60 min and serum amylase within 2 h, but fluorescent autophagosome formation only by 4 h of pancreatitis in parallel with a shift from cytosolic LC3-I to membranous LC3-II on Western blots. At 60 min, activated trypsin in heavier subcellular fractions was co-distributed with cathepsin B, but not with the autophagy markers LC3 or autophagy protein 16 (ATG16). Supramaximal caerulein stimulation of primary pancreatic acini derived from LC3-GFP mice revealed that trypsinogen activation is independent of autophagolysosome formation already during the first 15 min of exposure to caerulein. Co-localization studies (with GFP-LC3 autophagosomes versus Ile–Pro–Arg–AMC trypsin activity and immunogold-labelling of lysosomal-associated membrane protein 2 [LAMP-2] versus trypsinogen activation peptide [TAP]) indicated active trypsin in autophagolysosomes only at the later timepoints. In conclusion, during the initiating phase of caerulein-induced pancreatitis, premature protease activation develops independently of autophagolysosome formation and in vesicles arising from the secretory pathway. However, autophagy is likely to regulate overall intracellular trypsin activity during the later stages of this disease.

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific.

Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.

Hintergrund: Die akute Pankreatitis stellt aufgrund einer unkontrollierten Aktivierung der in der Bauchspeicheldruse synthetisierten Verdauungsenzyme eine Erkrankung mit hohem Komplikationspotenzial und stark variablem Verlaufsmuster dar. Da sich die pathophysiologischen Prozesse der Pankreatitis am Menschen ungenugend studieren lassen, haben sich eine Anzahl unterschiedlicher Tiermodelle etabliert, um die spezifischen Fragestellungen beantworten zu konnen. Die odematos-interstitielle Entzundung der Bauchspeicheldruse nach supramaximaler Caerulein-Injektion ermoglicht es, Einblicke in intrazellulare Ereignisse der fruhen Phase einer akuten Pankreatitis zu gewinnen. Fur dieses Pankreatitismodell werden ublicherweise uber die Nacht gefastete Tiere verwendet, um infolge einer verminderten CCK-(Cholezystokinin-)Sekretion eine maximale Zymogengranulaakkumulation sowie eine standardisierte Ausgangssituation zu erreichen. Die Rolle der Ernahrung fur Pathogenese und Verlauf der akuten Pankreatitis wird kontrovers diskutiert. Ziel der Studie war es, den Einfluss des Ernahrungszustands auf die lokale Auspragung der Pankreasschadigung bei experimenteller akuter Pankreatitis zu untersuchen. Methode: Mittels standardisierter supramaximaler Caerulein-Stimulation (Dosis: 50 µg/kg; Intervalle: 1/h, max. 7×) wurde eine akute odematos-interstitielle Pankreatitis an gefasteten und nicht gefasteten Mausen induziert. Die lokalen Veranderungen wurden durch Schadigungsmarker wie Pankreasodem und -histologie sowie die Pankreasenzymaktivitat im Serum und Pankreashomogenat und systemisch anhand von IL-6, CRP und pulmonaler MPO (Myeloperoxidase) objektiviert. Ergebnisse: 1) Die erhohten Aktivitaten der Pankreasenzyme im Serum wahrend der akuten Pankreatitis bei nicht gefasteten Tieren sind kein Ausdruck einer starkeren Pankreasaffektion, da sich die Aktivitaten der Verdauungsenzyme im Serum und im Pankreasgewebe proportional zueinander verhalten. Die basolaterale Enzymfreisetzung betrug ca. 1,3 % bzw. 0,7 % fur Amylase und Lipase unabhangig vom Ernahrungsregime. 2) Weder die lokalen noch die systemischen Schadigungsparameter zeigten Abhangigkeiten vom Ernahrungsregime. Histologisch zeigten sich neben einer Zunahme der Zymogengranula und Zellgrose bei den nicht gefasteten Tieren keine weiteren Unterschiede. 3) In einer 16-stundigen Erholungsphase (keine weitere Caerulein-Injektion) wurde eine weitgehende Normalisierung der lokalen Schadigungsparameter beobachtet. Diskussion: Im Caerulein-Modell konnte trotz hoherer intrapankreatischer Enzymakkumulation unter Nahrungszufuhr keine starkere lokale bzw. systemische Schadigung nachgewiesen werden, was auf ein Gleichgewicht zwischen potenziell schadigenden und protektiven Faktoren hindeutet.

a b s t r a c t Objectives: C-telopeptide crosslaps (CTX) and bone-specific alkaline phosphatase (BAP) do not provide sufficient sensitivity and specificity for diagnosis of osteoporosis. Cathepsin K (CatK), osteoprotegerin (OPG), and receptor activator of nuclear factor B ligand (total (t) and soluble (s) RANKL) play an important role in bone metabolism. Thus serum levels of biochemical markers, each or in combination, may be useful in diagnosis of osteoporosis. Study Design: In total, 121 healthy women, 27 premenopausal women aged between 20 and 45 years, and 94 postmenopausal women aged 59-81 years, all free of known skeletal disorders were included. They underwent bone density measurement and measurement of biochemical markers. Main Outcome Measures: Based on WHO criteria, women were stratified in four groups (premenopausal: healthy; postmenopausal: healthy, osteopenia, osteoporosis), and their levels of CatK, OPG, RANKL, CTX and BAP were analyzed. Results: Using WHO criteria 21 postmenopausal women had normal bone mineral density (BMD), 49 had osteopenia and 24 had osteoporosis. There were no significant correlations of CatK, OPG and RANKL with BMD (T-score) in age-adjusted analysis, but for BAP and CTX. ROC analyses resulted in poor diagnostic validity of all parameters. The best result - also confirmed by discriminant analysis - was yielded by BAP (AUC = 0.646 (0.510; 0.781)). A combination of variables did not significantly improve the diagnostic power. Conclusions: Baseline serum levels of BAP, CTX, CatK, OPG, sRANKL or tRANKL alone or in combination are not suitable to distinguish osteoporotic from non-osteoporotic postmenopausal women with sufficient accuracy.

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis–associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

Background & Aims The transcription factor nuclear factor-κB (NF-κB) (a heterodimer of NF-κB1p50 and RelA) is activated rapidly in acute pancreatitis (AP). However, it is not clear whether NF-κB promotes or protects against AP. We used the NF-κB inhibitor protein, inhibitor of κB (IκB)α, to study the roles of NF-κB in the development of AP in mice. Methods IκBα or the combination of IκBα and RelA selectively were deleted from pancreas of mice using the Cre/locus of cross-over P strategy; cerulein or L-arginine were used to induce AP. We performed microarray analyses of the IκBα- and RelA-deficient pancreata. DNA from healthy individuals and patients with acute or chronic pancreatitis were analyzed for variants in coding regions of alpha-1-antichymotrypsin . Results Mice with pancreas-specific deletion of IκBα had constitutive activation of RelA and a gene expression profile consistent with NF-κB activation; development of AP in these mice was attenuated and trypsin activation was impaired. However, AP was fully induced in mice with pancreas-specific deletion of IκBα and RelA. By using genome-wide expression analysis, we identified a cluster of NF-κB–regulated genes that might protect against the development of AP. The serine protease inhibitor 2A ( Spi2a ) was highly up-regulated in IκBα-deficient mice. Lentiviral-mediated expression of Spi2A reduced the development of AP in C57BL/6 and RelA-deficient mice. However, we did not correlate any variants of alpha-1-antichymotrypsin , the human homologue of Spi2a , with acute or chronic pancreatitis. Conclusions Pancreas-specific deletion of IκBα results in nuclear translocation of RelA and reduces AP induction and trypsin activation in mice after administration of cerulein or L-arginine. Constitutive activation of RelA up-regulates Spi2A, which protects mice against the development of AP.

Background:Chemoresistance is a main obstacle to effective esophageal cancer (EC) therapy. We hypothesize that altered expression of microRNAs (miRNAs) play a role in EC cancer progression and resistance to 5-fluorouracil (5-FU) based chemotherapeutic strategies.Methods:Four pairs of esophageal adenocarcinoma (EAC) cell lines and corresponding 5-FU resistant variants were established. The expression levels of miRNAs previously shown to be involved in the general regulation of stem cell pathways were analyzed by qRT-PCR. The effects of selected miRNAs on proliferation, apoptosis, and chemosensitivity were evaluated both in vitro and in vivo. We identified a particular miRNA and analyzed its putative target genes in 14 pairs of human EC tumor specimens with surrounding normal tissue by qRT-PCR as well as Wnt pathway associated genes by immunohistochemistry in another 45 EAC tumor samples.Results:MiR-221 was overexpressed in 5-FU resistant EC cell lines as well as in human EAC tissue. DKK2 was identified as a target gene for miR-221. Knockdown of miR-221 in 5-FU resistant cells resulted in reduced cell proliferation, increased apoptosis, restored chemosensitivity, and led to inactivation of the Wnt/-catenin pathway mediated by alteration in DKK2 expression. Moreover, miR-221 reduction resulted in alteration of EMT-associated genes such as E-cadherin and vimentin as well as significantly slower xenograft tumor growth in nude mice. RT2 profiler analysis identified a substantial dysregulation of 4 Wnt/-catenin signaling and chemoresistance target genes as a result of miR-221 modulation: CDH1, CD44, MYC, and ABCG2.Conclusion:MiR-221 controls 5-FU resistance of EC partly via modulation of Wnt/-catenin-EMT pathways by direct targeting of DKK2 expression. MiR-221 may serve as a prognostic marker and therapeutic target for patients with 5-FU resistant EAC.

Background Osteopenia (OP) or osteoporosis (OST) was diagnosed by bone densitometry (DXA) in postmenopausal women free of known skeletal disorders and without acute fracture. DVO guidelines were applied to define therapeutic indication. Methods The study included 94 women aged 59-81 years. Fracture or operation ≤12 months, malignant tumor, ovariectomy, and drugs such as cortisone, strontium, fluorides, bisphosphonates, SERMs, estrogens, and steroids were exclusion criteria. The lowest T-score at the spine, femoral neck, or total hip was decisive. The indication for therapy was determined by evaluating age, BMD, and other risk factors. Results Using the WHO criteria 22.3% (n=21) had normal BMD, 52.1% (n=49) had OP, and 25.6% (n=24) had OST. According to "Dachverband Osteologie" (DVO) guidelines, 28 women (29.8%) of the whole group needed therapy. Of the 28 women receiving therapy, 9 had OP and 19 had OST. Therapy was indicated in 18.4% for OP and 79.2% for OST. Conclusion A preventive measurement of BMD with DXA provides a benefit for postmenopausal women. Combinatory assessment and consideration of other risk factors allows identification of women who might benefit from early treatment.

Objectives Cathepsin K (CatK) is expressed in high levels in osteoplasts and therefore plays an important role in bone resorption. Thus CatK serum levels may be useful in the diagnosis of chronic bone disorders such as osteopenia and osteoporosis. Therefore we aimed at studying CatK levels in women putatively free of known skeletal disorders. Study design In total, 121 voluntary women, 27 premenopausal women aged between 20 and 45 years, and 94 postmenopausal women aged 59–81 years, all free of known skeletal disorders were included. All women underwent bone density measurement, routine labor parameter and measurement of serum CatK levels. Main outcome measures Based on WHO criteria, women were stratified in four groups (premenopausal: healthy; postmenopausal: healthy, osteopenia, osteoporosis), and their CatK levels were statistically analyzed. Results Using WHO criteria 21 postmenopausal women had normal bone mineral density (BMD), 49 had osteopenia and 24 had osteoporosis. All 27 premenopausal women had normal BMD. There were no significant differences in CatK between these groups. ROC analysis resulted in poor diagnostic validity of CatK, where the area under curve was 0.544. There was no correlation neither between CatK and other biomarkers as C-telopeptide crosslaps (CTX) or bone-specific alkaline phosphatase (BAP) nor between CatK and age. Conclusions Serum levels of CatK are not suitable to differentiate women with osteoporosis from healthy subjects.

[Background and Aims]: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. [Methods]: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. [Results]: A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. [Conclusions]: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells., This study was supported in part by the Mildred-Scheel-Professur der Deutschen Krebshilfe 111464, DFG AL 1174/6-1 to H.A., DFG DI 2299/1-1 to K.N.D., DFG SFB1321 (S01) to K.S. and W.W., and the German Federal Ministry of Education and Research to the German Center for Diabetes Research (DZD e.V.) to J.A.

Background & Aims 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. Methods 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. Results 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. Conclusions 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.

Early events in the pathogenesis of experimental acute pancreatitis are intensively studied using isolated cells or animal models. However, the results and their interpretations are dependent on the complexity of biological structures. Therefore, we proposed that studies on isolated perfused pancreas can give additional information about processes leading to acinar cell injury. This hypothesis was examined adapting the well-established caerulein hyperstimulation model and the taurocholate model of acute pancreatitis to the extracorporeal perfused isolated rat pancreas. The pancreas was removed with the duodenum including the arterial supply. A continuous perfusion of the organ was performed with a modified Krebs–Ringer bicarbonate buffer. Intraarterial caerulein application or an intraductal taurocholate (3.5%) application were used to induce acinar cell injury which was determined as the release of amylase, lipase and lactate dehydrogenase into the portal outflow medium and into the transudation fluid and by examination of histological alterations. Trypsinogen release and activation was followed by analysis of trypsinogen activation peptide (TAP) in the transudation fluid and in pancreatic tissue. Perfusion of isolated rat pancreas with supramaximal concentrations of caerulein or retrograde injection of taurocholate (3.5%) resulted in acinar cell injury indicated by elevated levels of amylase and lipase into the perfusate and into the transudation fluid. TAP levels in the transudation fluid significantly increased after perfusion with caerulein or retrograde injection of taurocholate (3.5%). The histological alterations after taurocholate application include oedema and necrosis and show significant differences to the control perfusion. Extensive pancreatic necroses were not observed after caerulein hyperstimulation. The isolated perfused rat pancreas is a useful model to investigate pathophysiological mechanisms which are relevant for the early phase of acute pancreatitis. The caerulein and the taurocholate models are transferable to the isolated rat pancreas. Studies on isolated perfused rat pancreas enable pathophysiological investigations of the exocrine pancreas without influence of systemic components, but with preserved morphology.

Pancreatitis is an inflammatory disease of the pancreas characterized by dysregulated activity of digestive enzymes, necrosis, immune infiltration, and pain. Repeated incidence of pancreatitis is an important risk factor for pancreatic cancer. Legumain, a lysosomal cysteine protease, has been linked to inflammatory diseases such as atherosclerosis, stroke, and cancer. Until now, legumain activation has not been studied during pancreatitis. We used a fluorescently quenched activity-based probe to assess legumain activation during caerulein-induced pancreatitis in mice. We detected activated legumain by ex vivo imaging, confocal microscopy, and gel electrophoresis. Compared with healthy controls, legumain activity in the pancreas of caerulein-treated mice was increased in a time-dependent manner. Legumain was localized to CD68+ macrophages and was not active in pancreatic acinar cells. Using a small-molecule inhibitor of legumain, we found that this protease is not essential for the initiation of pancreatitis. However, it may serve as a biomarker of disease, since patients with chronic pancreatitis show strongly increased legumain expression in macrophages. Moreover, the occurrence of legumain-expressing macrophages in regions of acinar-to-ductal metaplasia suggests that this protease may influence reprogramming events that lead to inflammation-induced pancreatic cancer.

Background & Aims Under conditions of inflammation in the absence of micro-organisms (sterile inflammation), necrotic cells release damage-associated molecular patterns that bind to Toll-like receptors on immune cells to activate a signaling pathway that involves activation of IκB kinase and nuclear factor κB (NF-κB). Little is known about the mechanisms that control NF-κB activity during sterile inflammation. We analyzed the contribution of B-cell CLL/lymphoma 3 (BCL3), a transcription factor that associates with NF-κB, in control of sterile inflammation in the pancreas and biliary system of mice. Methods Acute pancreatitis (AP) was induced in C57BL/6 (control) and Bcl3 −/− mice by intraperitoneal injection of cerulein or pancreatic infusion of sodium taurocholate. We also studied Mdr2 −/− mice, which develop spontaneous biliary inflammation, as well as Bcl3 −/− Mdr2 −/− mice. We performed immunohistochemical analyses of inflamed and noninflamed regions of pancreatic tissue from patients with AP or primary sclerosing cholangitis (PSC), as well as from mice. Immune cells were characterized by fluorescence-activated cell sorting analysis. Control or Bcl3 −/− mice were irradiated, injected with bone marrow from Bcl3 −/− or control mice, and AP was induced. Results Pancreatic or biliary tissues from patients with AP or PSC had higher levels of BCL3 and phosphorylated RelA and IκBα in inflamed vs noninflamed regions. Levels of BCL3 were higher in pancreata from control mice given cerulein than from mice without AP, and were higher in biliary tissues from Mdr2 −/− mice than from control mice. Bcl3 −/− mice developed more severe AP after administration of cerulein or sodium taurocholate than control mice; pancreata from the Bcl3 −/− mice with AP had greater numbers of macrophages, myeloid-derived suppressor cells, dendritic cells, and granulocytes than control mice with AP. Activation of NF-κB was significantly prolonged in Bcl3 −/− mice with AP, compared with control mice with AP. Bcl3 −/− Mdr2 −/− mice developed more severe cholestasis and had increased markers of liver injury and increased proliferation of biliary epithelial cells and hepatocytes than Mdr2 −/− mice. In experiments with bone marrow chimeras, expression of BCL3 by acinar cells, but not myeloid cells, was required for reduction of inflammation during development of AP. BCL3 inhibited ubiquitination and proteasome-mediated degradation of p50 homodimers, which prolonged binding of NF-κB heterodimers to DNA. Conclusions BCL3 is up-regulated in inflamed pancreatic or biliary tissues from mice and patients with AP or cholangitis. Its production appears to reduce the inflammatory response in these tissues via blocking ubiquitination and proteasome-mediated degradation of p50 homodimers.

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis. The initial experiments were supported by the DFG (Wi 2036/1-1). This work was supported by the Sonnenfeld-Stiftung, Berlin, Germany (to H.W.), the US National Institutes of Health (NIH) (grant DK058088 to M.S.-T.), INSERM (Institut National de la Santé et de la Recherche Médicale) and the Programme Hospitalier de Recherche Clinique (grant PHRC R 08-04 to C.F.)

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs. Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels ( 100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins. Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.

Excessive ethanol consumption is a common risk factor for acute and chronic pancreatitis. Ethanol could lead to the onset of pancreatitis in a number of ways; the most recently discovered is its effect on intrapancreatic digestive enzyme activation, by either sensitizing acinar cells to pathologic stimuli or stimulating the release of a secretagogue (cholecystokinin) from duodenal I cells. Recent advances in cell biologic and molecular techniques have permitted us to address the intracellular events involved in digestive enzyme activation in a manner that was previously considered impossible. Investigations that used these novel techniques found that (a) trypsin is, in contrast to its role in the small intestine, not necessarily involved in the premature intracellular activation of other digestive proteases such as proelastase; (b) trypsinogen does not autoactivate intracellularly but is instead largely activated by the lysosomal hydrolase cathepsin B; and (c) the role of trypsin in the intrapancreatic protease cascade is most likely one that involves the degradation, rather than the activation, of active digestive proteases including trypsin itself. These studies, as well as investigations that have addressed the role of mutant trypsin in the disease onset of hereditary pancreatitis, suggest that trypsin may not be critical for triggering pancreatitis but might have a protective role against the action of some of the other digestive proteases. While the specific role of different digestive enzymes in initiating pancreatitis is still a matter of debate and the topic of ongoing investigations, experimental evidence suggests that ethanol can directly interfere with the processes involved in digestive zymogen activation.

Background. Oxidative stress is a relevant event in the pathogenesis of acute pancreatitis. Investigations in vivo are limited because of the complexity of the organism and the short half-life of free radicals. The isolated perfused rat pancreas could be useful for investigations in the early phase of acute pancreatitis especially under conditions of oxidative stress. Methods. External perfusions of the pancreatic glands of Wistar rats were carried out using a modified Krebs-Ringer buffer including an additive of the detergents Triton X-100 and a perfusion including hydrogen peroxide (0.0012%) or tert-butylhydroperoxide (0.0042%) or xanthine oxidase (0.1 U/ml). Changes in amylase, lipase, LDH in the portal outflow fluid and for histological alterations were analyzed. Results. Damage to pancreatic parenchyma using Triton X-100 was indicated by increased levels of pancreatic enzymes in the perfusion medium. During perfusion with hydrogen peroxide or tert-butylhydroperoxide we found no changes in pancreatic enzymes in the portal outflow. In contrast, perfusion with xanthine oxidase induced a significant elevation in lipase and amylase in the effusion fluid after 30 min. We found a significant increase in edema in the hydrogen peroxide and in the xanthine oxidase group. Focal necroses of the pancreatic parenchyma were detected in all groups of oxidative stress. Conclusions. The isolated perfused rat pancreas is a valuable experimental model for investigating the early phase of pathophysiology in acute pancreatitis, for instance, the effect of oxidative stress as an early event in acute pancreatitis. Using hydrogen peroxide tert-butylhydroperoxide or xanthine oxidase, only xanthine oxidase was able to induce a typical elevation in the pancreas enzymes in the effusion fluid.

The lysosomal cysteine protease cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of experimental pancreatitis. Recent in vitro studies have suggested that this mechanism might be of pathophysiological relevance in hereditary pancreatitis, a human inborn disorder associated with mutations in the cationic trypsinogen gene. In the present study evidence is presented that cathepsin B is abundantly present in the secretory compartment of the human exocrine pancreas, as judged by immunogold electron microscopy. Moreover, pro-cathepsin B and mature cathepsin B are both secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with sporadic pancreatitis or hereditary pancreatitis. Finally, cathepsin B- catalyzed activation of recombinant human cationic trypsinogen with hereditary pancreatitis-associated mutations N29I, N29T, or R122H were characterized. In contrast to a previous report, cathepsin B-mediated activation of wild type and all three mutant trypsinogen forms was essentially identical under a wide range of experimental conditions. These observations confirm the presence of active cathepsin B in the human pancreatic secretory pathway and are consistent with the notion that cathepsin B-mediated trypsinogen activation might play a pathogenic role in human pancreatitis. On the other hand, the results clearly demonstrate that hereditary pancreatitis-associated mutations do not lead to increased or decreased trypsinogen activation by cathepsin B. Therefore, mutation-dependent alterations in cathepsin B-induced trypsinogen activation are not the cause of hereditary pancreatitis.

La laparoscopie est utilisee de plus en plus souvent pour traiter les infections abdominales. Les effets du pneumoperitoine n'ont pas encore ete completement elucides. Dans ce modele experimental de peritonite chez le rat, nous avons analyse l'influence du lavage sous laparoscopie compare aux effets de la technique de lavage conventionnelle. On a installe dans la cavite abdominale de 80 rats, un echantillon multibacterien defini. Ces animaux ont ensuite ete randomises en trois groupes: groupe 1 (n = 32), pas d'intervention; groupe 2 (n = 24), lavage conventionnel; et groupe 3 (n = 24) lavage laparoscopique. Les animaux ont ete sacrifies et autopsies 1, 2 et 8 heures apres intervention chirurgicale. Les criteres de jugement principaux ont ete la bacteriemie, le taux d'interleukine-6 dans le plasma et dans l'ascite, les modifications de la numeration globulaire et l'activite de la myeloperoxidase (MPO) dans le poumon, le foie, le rein et le pancreas. On n'a note aucune difference en ce qui concerne la bacteriemie. Dans l'ascite on a note une elevation marquee de l'interleukine-6 8 heures apres, avec un taux plus bas dans les deux groupes de traitement, compare aux taux des controles (p < 0.025). L'activite MPO, temoin des granulocytes presents dans les tissus, a change de facon significative uniquement dans le poumon. Deux heures apres l'intervention chirurgicale, l'activite MPO etait significativement plus basse dans le poumon du groupe laparoscopique que dans les controles et le groupe laparotomie. En conclusion, le lavage conventionnel et laparoscopique reduisent l'inflammation. Dans ce modele, le lavage laparoscopique en meme temps que le pneumoperitoine au CO 2 apparaisse n'avoir aucune influence negative sur la reaction inflammatoire dans la phase postoperatoire precoce. Une sequestration reduite de neutrophiles dans les tissu pulmonaire apres lavage laparoscopique reflete le degre de traumatisme peu eleve lors du lavage laparoscopique.

Oxidative stress is considered to be a pathogenic factor for multisystem organ failure during acute pancreatitis. Infusion of 3% and 5% sodium taurocholate into the pancreatic duct of rats resulted in a 24-hr lethality of 8% and 82%, respectively. Kidney tissue showed a long-lasting significant elevation of malondialdehyde (lipid peroxidation). Only small amounts of this aldehyde were formed in the liver. In the lung malondialdehyde was increased during the first 6 hr after pancreatitis induction. Malondialdehyde levels were not different for pancreatitis initiated by 3% or 5% taurocholate. Protein-bound carbonyls (protein oxidation) in the tissues were not significantly changed at any time point. However, after infusion of 5% taurocholate, lung proteins were oxidatively modified by the product of lipid peroxidation, 4-hydroxynonenal. Another parameter characteristic for pancreatitis with high lethality was the high number of neutrophils in the lungs. We conclude that oxidative stress is important for the injury of extrapancreatic tissues during pancreatitis, but survival is determined by the degree of systemic inflammation.

METHODS The pancreas of 24 male Wistar rats was perfused extracoporally by modified Krebs-Ringer-buffer for 80 minutes (including a 20 minutes equilibration period). To verify any organ damage we measured the activity of pancreatic enzymes like amylase, lipase and lactatdehydrogenase in the portal effluent. Furthermore histological changes were analysed after perfusion. Organ damage was induced by adding cerulein in a physiological dose (10(-10) M, n = 6) and in a supramaximal dose (10(-8) M, n = 6) and by intraductal injection of taurocholate (3.5 %, n = 6). RESULTS Already 10 minutes after stimulation with cerulein (10(-8) M) and after intraductal injection of taurocholate increased activities (p < 0.01) of amylase and lipase were measured in the portal effluent compared to the group without any treatment. Lactatdehydrogenase levels did not changed. Apart from marked oedema in both groups considerable zones of necrosis could be noticed especially in the taurocholate group. CONCLUSION Our data suggest that the isolated perfused rat pancreas (IPRP) is a valuable experimental tool to verify pathophysiological changes in the early phase of acute pancreatitis (AP). Various established models of AP such as by cerulein hyperstimulation or intraductal injection of taurocholate, could be applied to the IPRP. We conclude that this method enlarges the spectrum of established experimental models of acute pancreatitis.

Clinical trials have found that the pneumoperitoneum has potentially hazardous side effects. The biochemical basis of organ injury induced by pneumoperitoneum is, however, not well defined. Since oxidative stress is believed to play an important role in many pathological conditions, we set out to examine oxidative stress markers in the lung, liver, kidney, and pancreas by using a rat model of laparoscopy with CO(2) pneumoperitoneum and comparing it to a group with gasless laparoscopy.Malondialdehyde (for lipid peroxidation), protein-bound carbonyls (for protein oxidation), reduced and oxidized glutathione, and the neutrophil marker myeloperoxidase were evaluated in tissue homogenates at 2 h, 6 h, and 18 h after laparoscopy. Immunoblotting was used to analyze the modification of lung proteins by 4-hydroxynonenal at 6 h.Significant lipid peroxidation was found selectively in lungs at 2 h and 6 h after CO(2) pneumoperitoneum. This was accompanied by a loss of glutathione but only minor protein oxidation. Further, lung proteins were clearly modified by the aldehydic product of lipid peroxidation 4-hydroxynonenal. Myeloperoxidase in lungs increased continuously up to 18 h in both experimental groups, but there were higher levels in the group with pneumoperitoneum.Oxidative stress is likely to contribute to the impairment of pulmonary function after laparoscopic operations using a CO(2) pneumoperitoneum.

The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Derivatization of protein carbonyls with 2,4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS–PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS–PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls.

The exocrine pancreas synthesises more protein per gram of tissue than any other mammalian organ (with the exception of the lactating breast) and produces 6–20 g of digestive enzyme per day. This enormous metabolic activity requires an equally high energy supply for protein synthesis, transport and storage, as well as regulated secretion of active digestive enzymes and protease zymogens. Pancreatic acinar cells have therefore a high rate of mitochondrial energy metabolism. Pancreatic mitochondria effectively use nicotine adenine dinucleotide (NAD+)-dependent substrates to maintain the mitochondrial membrane potential (Δψm). Energy stored as Δψm is mainly used for two energy-requiring processes: ATP synthesis and Ca2+ accumulation. ATP is further needed for protein synthesis, signal transduction, intracellular ion homeostasis, vesicular transport to the apical pole of the acinar cell, and exocytosis of digestive enzymes into the duct lumen. In pancreatic acinar cells a rise in intracellular free Ca2+ is the key signal transduction mechanism regulating stimulus–secretion coupling. Following exposure of the cell to cholecystokinin (CCK) or other hormones Ca2+ is released from intracellular stores via either Inositol triphosphate (IP3), nicotinic acid adenine dinucleotide phosphate (NAADP) or cyclic ADP ribose (cADPR) signalling pathways.1 2 The endoplasmatic reticulum (ER) operates as the main Ca2+ storing compartment, and this organelle is mostly located in the basal portion of the cell surrounding the nucleus.3 Additional Ca2+ storing organelles have been identified in the apical area of acinar cells and may be composed of ER protrusions into that region.2 3 Under physiological conditions hormonal stimulation results in an oscillating wave of free Ca2+ that is initiated in the apical portion of acinar cells and enables exocytosis of zymogen granule content from the luminal plasma membrane. Recently, …

Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model.

Rats received up to four subcutaneous injections of caerulein (20 µg/kg) in hourly intervals to induce a mild edematous pancreatitis. A single dose of caerulein resulted in significant oxidative modification of proteins in pancreatic homogenates as compared to saline-injected controls (p < 0.01). Repeated injections of the secretagogue were unable to induce a further increase in modified proteins. Protein oxidation preceded the formation of the lipid peroxidation product malondialdehyde as well as edema and the increase in serum amylase by 1–2.5 h. Western blotting for oxidatively modified proteins confirmed the results of the quantitative measurements and did not reveal individual, selectively modified proteins. These findings indicate that oxidative stress and oxidative protein modification are very early events in the initial period of caerulein pancreatitis. This may explain the poor success rate of most studies that administered antioxidants therapeutically during or after initiation of pancreatitis.

Free radical-mediated injury is believed to play a key role in the pathogenesis of acute pancreatitis (AP). Therefore, oxidative damage of proteins may be an important event in the development of AP. The present study was performed to investigate oxidative protein modification, quantified as 2,4-dinitrophenylhydrazine-reactive protein-carbonyls, during the time course of taurocholate-induced pancreatitis of the rat and to analyze oxidatively modified proteins by Western blotting. Protein modification in pancreatic homogenates was found as early as 30 min after induction of severe AP with 3% taurocholate preceding the elevation of serum amylase activity and the increase of malondialdehyde in the tissue. A correlation of protein-carbonyl contents to a score of pancreatic macroscopic alterations (r = .69) and to the wet weight/dry weight ratio (r = .65) was found. Infusion of 5% taurocholate resulted in fulminant AP with high lethality during the 24 h of the experiment. However, rats surviving showed significantly lower level of protein-carbonyls than animals that died between 20–24 h after AP induction. The quantitative data were confirmed by the intensity of immunostained protein-carbonyls. The present data show a rather uniform increase in the staining pattern not revealing single, selectively damaged proteins. The aldehydic product of lipid peroxidation 4-hydroxynonenal (HNE) is known for its reactivity towards proteins. Interestingly, an antibody raised against protein-bound HNE did not indicate an increased protein modification by this aldehyde. In conclusion, experimental AP is characterized by an early oxidative protein modification, possibly contributing to functional impairment of the pancreas. This protein alteration may not be mediated by HNE.

Hyperstimulation with the cholecystokinin analogue cerulein induces mild edematous pancreatitis in rats. It is believed that an impaired energy metabolism diminishes the cellular defense capacity in the inflamed pancreatic tissue and, therefore, contributes to the injuries in acinar cells. In the present study, changes in the capacity of oxidative phosphorylation were quantified within the first 24 h after subcutaneous cerulein injections. Serum amylase level and pancreatic water content were maximally elevated 5 h after the first injection. The capacity of mitochondrial respiration was reduced in isolated acinar cells to 69 and 44% at 5 and 24 h, respectively, compared to that in saline controls. Simultaneously, glutamate dehydrogenase (GLDH) activity dropped to 70 and 46%. The respiration rates of acinar cells and of isolated mitochondria related to GLDH activities were not different from controls. This suggests that the major portion of the mitochondrial population within the acinar cell is inactivated in the course of cerulein treatment. After 24 h, the reduced population of functionally intact mitochondria restricted the rate of phosphorylating respiration in acinar cells (52%), which resulted in a diminution of cellular ATP to 57%. It is concluded that cerulein hyperstimulation induces a drastic and long-lasting reduction of the capacity for mitochondrial ATP production which may adversely affect energy-requiring reactions of the gland during regeneration.

Inappropriate trypsinogen activation is discussed as an early intracellular event in the secretagogue-induced model of acute pancreatitis. However, the mechanisms by which trypsinogen is activated are not well characterized. In the present work, trypsinogen activation was studied in intact acinar cells using bis-(CBZ-arginyl)-Rhodamine 110 [(CBZ-Arg)2-Rho 110] as a cell-permeant substrate for trypsin and also independently via the formation of trypsinogen activation peptide (TAP). Preincubation with 10nM caerulein increased the Rho 110-substrate cleavage more than threefold. This proteolytic activity was fully sensitive to a benzamidine (BA)-type serine protease inhibitor. The appearance of enzymatic activity was paralleled by the formation of TAP. The lack of effect of the high-molecular soybean trypsin inhibitor indicates an intracellular substrate cleavage. The cathepsin B inhibitor CA-074 prevented neither the caerulein-induced formation of TAP nor the (CBZ-Arg)2-Rho 110-cleaving activity. BA inhibited the Rho 110-substrate cleavage and significantly reduced the TAP formation. These results show that trypsinogen activation in caerulein-hyperstimulated acinar cells may occur independently of the activity of cathepsin B. On the contrary, the effect of BA suggests the role of a serine protease in trypsinogen activation.

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.

A one-step perifusion technique is described for studying the regulation of energy metabolism in intact hepatocytes and in mitochondria of the same cells after their permeabilization by digitonin. Cell count and activities of glutamate dehydrogenase, the latter being used as an indicator of mitochondrial integrity, were found to be nearly unchanged after permeabilization and perifusion for at least 40 min at 37°C. The residual activity of lactate dehydrogenase after permeabilization indicated that permeabilized cells were almost depleted of soluble cytosolic components. The composition of the perifusion medium was chosen so that various metabolic states could be adjusted of both intact and permeabilized hepatocytes without the need to change the perifusion medium. Oxidative phosphorylation of mitochondria within permeabilized hepatocytes remained intact throughout the perifusion as indicated by the response of respiration to the addition of ADP, carboxyatractyloside and uncoupler. The application of the perifusion technique allows us to sample indicator metabolites in the effluent medium like acetoacetate (AcAc) and 3-hydroxybutyrate (HB) for calculating the mitochondrial redox ratios and rates of ketogenesis. In the presence of octanoate and ADP, an improvement of substrate supply by glutamate and malate led to increases in the intramitochondrial HB/AcAc ratio and the respiration rate. Glutamate/malate concentrations of 1 mM resulted in maximal respiration rates, whereas concentrations of 5 mM further enhanced the HB/AcAc ratio. Mitochondria responded to increasing ATP/ADP ratios in the perifusion medium by decreased respiration rates at higher HB/AcAc ratios. By comparing respiration rates and redox ratios of mitochondria in permeabilized cells with those before permeabilization (gluconeogenic conditions of hepatocytes), it is concluded that in the intact cell oxidative phosphorylation is limited with respect to substrate supply as well as by the ATP demand.

OBJECTIVE To determine the value of pancreatic stone protein in predicting sepsis related postoperative complications and death in the ICU. DESIGN A prospective cohort study of postoperative patients admitted to the ICU. Blood samples for analysis were taken within 3 hours from admission to the ICU including pancreatic stone protein white blood cell counts C reactive protein interleukin 6 and procalcitonin. The Mannheim Peritonitis Index and Acute Physiology and Chronic Health Evaluation II clinical scores were also determined. Univariate and multivariate analyses were performed to determine the diagnostic accuracy and independent predictors of death in the ICU [Clinicaltrials.gov NCT01465711]. SETTING An adult medical surgical ICU in a teaching hospital in Germany. PATIENTS Ninety one consecutive postoperative patients with proven diagnosis of secondary peritonitis admitted to the ICU were included in the study from August 17 2007 to February 8 2010. INTERVENTIONS Peripheral vein blood sampling. MEASUREMENTS AND MAIN RESULTS Univariate analysis demonstrated that pancreatic stone protein has the highest diagnostic accuracy for complications and is the best predictor for death in the ICU. Pancreatic stone protein had the highest overall efficacy in predicting death with an odds ratio of 4.0 vs. procalcitonin (odds ratio 3.2) interleukin 6 (odds ratio 2.8) C reactive protein (odds ratio 1.3) and WBCs (odds ratio 1.4). By multivariate analysis pancreatic stone protein was the only independent predictor of death. CONCLUSIONS In a population of patients with sepsis related complications serum pancreatic stone protein levels demonstrate a high diagnostic accuracy to discriminate the severity of peritonitis and to predict death in the ICU. This test could be of value in the clinical diagnosis and therapeutic decision making in the ICU.

Background Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases. Design Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 μg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies. Results Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini. Conclusions The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings from the present work further suggest that targeting TNFα, for which pharmaceutical agents are readily available, could be an effective treatment strategy that directly addresses the cellular causes of pancreatitis.

In acinar cells of rat pancreas, plasma membrane permeability was investigated after electropulsing (EP). Adenosine 5′-triphosphate (ATP) level was determined to characterize the energy state of the cells. EP ( E = 3 kV cm −1 , τ =20 μs) reduced the proportion of trypan blue excluding cells from more than 90% to less than 20%. Thirty minutes after EP, 85% of the cells were found to exclude trypan blue, once again. Immediately after EP, ATP decreased by 15% and oxygen consumption increased by 30%, both indicating an enhanced energy demand. The dependence on energy supply for membrane resealing is demonstrated by the effect of the uncoupler 2,4-dinitrophenol (DNP). DNP added before EP inhibited the membrane resealing in a concentration-dependent manner. In EP-treated cells, DNP led to a reversal of the resealing process when added in the restoration period. The results demonstrate the energy demand of pancreatic acinar cells to maintain the intact state of the plasma membrane. Therefore, the capacity for reversible membrane perforation after EP may be used to evaluate cell viability.

Perinatal development of cytochrome-c oxidase (complex IV) and ubiquinol–cytochrome-c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densito-metrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schagger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223–231]. A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.