Persister cell phenotypes contribute to poor patient outcomes after neoadjuvant chemotherapy in PDAC.

Neoadjuvant chemotherapy can improve the survival of individuals with borderline and unresectable pancreatic ductal adenocarcinoma; however, heterogeneous responses to chemotherapy remain a significant clinical challenge. Here, we performed RNA sequencing (n = 97) and multiplexed immunofluorescence (n = 122) on chemo-naive and postchemotherapy (post-CTX) resected patient samples (chemoradiotherapy excluded) to define the impact of neoadjuvant chemotherapy. Transcriptome analysis combined with high-resolution mapping of whole-tissue sections identified GATA6 (classical), KRT17 (basal-like) and cytochrome P450 3A (CYP3A) coexpressing cells that were preferentially enriched in post-CTX resected samples. The persistence of GATA6hi and KRT17hi cells post-CTX was significantly associated with poor survival after mFOLFIRINOX (mFFX), but not gemcitabine (GEM), treatment. Analysis of organoid models derived from chemo-naive and post-CTX samples demonstrated that CYP3A expression is a predictor of chemotherapy response and that CYP3A-expressing drug detoxification pathways can metabolize the prodrug irinotecan, a constituent of mFFX. These findings identify CYP3A-expressing drug-tolerant cell phenotypes in residual disease that may ultimately inform adjuvant treatment selection.

Farnesoid X receptor activation inhibits pancreatic carcinogenesis.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists.

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma.

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

H2O2-mediated autophagy during ethanol metabolism.

BACKGROUND: Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes and ROS. METHODS: Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure by immunoblotting and immunohistochemistry. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and its major intermediator acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and knock down of NOX4 using siRNA. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls. RESULTS: Acute ethanol exposure of mice over 24 h significantly induced autophagy as measured by LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in huh7 cells in the presence of overexpressed CYP2E1. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA mediated knock down. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1-and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid. CONCLUSION: We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS. These data suggest that suppression of autophagy by ethanol is most likely due to longer alcohol exposure during chronic alcohol consumption with the accumulation of e.g. misfolded proteins.

Effects of neoadjuvant FOLFIRINOX and gemcitabine-based chemotherapy on cancer cell survival and death in patients with pancreatic ductal adenocarcinoma.

Background: The progression and response to systemic treatment of cancer is substantially dependent on the balance between cancer cell death (apoptosis and necroptosis) and cancer cell survival (autophagy). Although well characterized in experimental systems, the status of cancer cell survival and cell death in human pancreatic ductal adenocarcinoma (PDAC), especially in response to chemotherapy and different types of chemotherapy is poorly described. Results: The median (95% confidence interval) survival was 31.6 (24.5-44.5) months after FOLFIRINOX versus 15.8 (2.0-20.5) months after gemcitabine-based therapy (p = 0.039). PDAC tissue autophagy was reduced compared to normal pancreata based on reduced BECLIN-1 expression and LC3-Lamp-2 colocalization, whilst necroptosis (RIP-1) was increased. Neoadjuvant therapy was associated with further reduced autophagy based on p62/SQSTM-1 accumulation, and increased necroptosis (RIP3 and pMLKL) and apoptosis (BAX, cleaved CASPASE-9 and CASPASE-3) markers, increased nuclear p65 (NF-κB) and extracellular HMGB1 expression, with greater CD8+ lymphocyte infiltration. Survival was associated with reduced autophagy and increased apoptosis. Necroptosis (RIP-3, pMLKL) and apoptosis (BAX and cleaved CASPASE-9) markers were higher after FOLFIRINOX than gemcitabine-based treatment. Patients and methods: Cancer cell autophagy, apoptosis, and necroptosis marker expression was compared in pancreatic tissue samples from 51 subjects, comprising four groups: (1) surgical resection for PDAC after FOLFIRINOX (n = 11), or (2) after gemcitabine-based (n = 14) neoadjuvant therapy, (3) patients undergoing PDAC resection without prior chemotherapy (n = 13), and (4) normal pancreata from 13 organ donors. Marker expression was undertaken using semi-automated immunofluorescence-FACS-like analysis, defining PDAC cells by CK-7+ expression.

RIP3 attenuates the pancreatic damage induced by deletion of ATG7.

Invalidation of pancreatic autophagy entails pancreatic atrophy, endocrine and exocrine insufficiency and pancreatitis. The aim of this study was to investigate whether depletion of Rip3, which is involved in necroptotic signaling, may attenuate the pancreatic atrophy and pancreatitis resulting from autophagy inhibition. Autophagy and necroptosis signaling were evaluated in mice lacking expression of Rip3 in all organs and Atg7 in the pancreas. Acinar cell death, inflammation and fibrosis were evaluated by using of a compendium of immunofluorescence methods and immunoblots. Mice deficient for pancreatic Atg7 developed acute pancreatitis, which progressed to chronic pancreatitis. This phenotype reduces autophagy, increase apoptosis and necroptosis, inflammation and fibrosis, as well as premature death of the animals. Knockout of Rip3 exacerbated the apoptotic death of acinar cells, increased tissue damage, reduced macrophage infiltration and further accelerated the death of the mice with Atg7-deficient pancreas. The pancreatic degeneration induced by autophagy inhibition was exacerbated by Rip3 deletion.

Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells.

BACKGROUND: Sulforaphane (SFN), an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT). METHODS: In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT. RESULTS: Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4. CONCLUSION: Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

The bile acid receptor FXR attenuates acinar cell autophagy in chronic pancreatitis.

The functional relationship between bile acid (BA) and autophagy has not been evaluated in the context of pancreatitis. Here we investigated whether BA and their nuclear farnesoid X receptor (FXR) modulate autophagy and the development of pancreatitis. FXR expression, autophagy, apoptosis and necroptosis were determined in human chronic pancreatitis (CP) tissue in vivo and in pancreatic cells lines in vitro by means of real-time PCR, immunoblots and immunofluorescence. Pancreatic cell lines exposed to the most abundant BAs glycochenodeoxycholate (GCDC) and taurocholic acid (TCA) increased the expression of nuclear FXR and diminished that of the essential autophagy-related protein ATG7. BA was also elevated in pancreatic tissues from CP patients, correlating with elevated FXR and curtailed ATG7 expression with locally reduced autophagic activity. This was accompanied by an increased manifestation of CP hallmarks including apoptosis, necroptosis, inflammation and fibrosis. The present results suggest a cascade of events in which local accumulation of BA signals via FXR to suppress autophagy in pancreatic acinar cells, thereby unleashing acinar cell apoptosis and necroptosis. Thus, BA may cause CP by suppressing autophagy and exacerbating acinar cell apoptosis and necroptosis.

Phytotherapeutics Oridonin and Ponicidin show Additive Effects Combined with Irradiation in Pancreatic Cancer in Vitro.

Background: Chemoradiation of locally advanced non-metastatic pancreatic cancer can lead to secondary operability by tumor mass reduction. Here, we analyzed radiomodulating effects of oridonin and ponicidin in pancreatic cancer in vitro. Both agents are ent-kaurane diterpenoids, extracted from Isodon rubescens, a plant that is well known in Traditional Chinese Medicine. Cytotoxic effects have recently been shown in different tumor entities for both agents. Materials and methods: Pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MIA PaCa-2 were pretreated with oridonin or ponicidin and irradiated with 2 Gy to 6 Gy. Long-term survival was determined by clonogenic assay. Cell cycle effects and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytometry. Western blotting was used to study the DNA double-strand break repair proteins Ku70, Ku80 and XRCC4. Results: Oridonin and ponicidin lead to a dose-dependent reduction of clonogenic survival and an increase in γH2AX. Combined with irradiation we observed additive effects and a prolonged G2/M-arrest. No relevant changes in the levels of the DNA double-strand break repair proteins were detected. Conclusions: Pretreatment with oridonin or ponicidin followed by irradiation lead to an additional reduction in survival of pancreatic cancer cells in vitro, presumably explained by an induced prolonged G2/M-arrest. Both agents seem to induce DNA double-strand breaks but do not interact with the non-homologous end joining (NHEJ) pathway.

Retrospective electron microscopy: Preservation of fine structure by freezing and aldehyde fixation.

For many years it has been believed that ultrastructural analysis by transmission electron microscopy (TEM) is not possible using frozen tissues. We have developed a TEM method that allows the evaluation of organelles using pancreatic tissue that was previously liquid nitrogen snap-frozen and stored long-term at -80°C. This method is suitable for the quantitative assessment of mitochondria, rough endoplasmic reticulum (RER), and Golgi structures, as well as organelles originating from autophagy signaling. Frozen pancreatic tissue exhibited no signs of freezing- or storage-related damage and was undistinguishable from fresh material subjected to standard glutaraldehyde fixation. Since pancreatic tissue is the most delicate tissue to work with due to the high expression of digestive enzymes, our method is also suitable for other tissue types such as liver. Thus, by applying proper tissue freezing and fixation techniques, retrospective TEM analysis can be performed on mammalian tissues in a time- and cost-saving manner.

Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

Alcohol exacerbates LPS-induced fibrosis in subclinical acute pancreatitis.

The role of pancreatic acinar cells in initiating fibrogenic responses during the early stages of alcoholic acute pancreatitis has not been evaluated. We investigated the ability of injured acinar cells to generate pancreatic fibrosis in acute pancreatitis. Rats were fed either an ethanol-containing or control diet over 14 weeks and euthanized 3 or 24 hours after a single lipopolysaccharide injection. Profibrotic transforming growth factor-ß of acinar cells and pancreatic fibrosis were assessed by immunofluorescence, histological characteristics, and electron microscopy. Human pancreatic tissues were also evaluated. Periacinar cell fibrosis and collagen were exacerbated 24 hours after endotoxemia in alcohol-fed rats. Alcohol exposure exacerbated acinar cell-specific production of transforming growth factor ß in response to lipopolysaccharide in vivo and in acinar cell-like AR42J cells in vitro. Although a morphological examination showed no visible signs of necrosis, early pancreatic fibrosis can be initiated by little or no pancreatic necrosis. Transforming growth factor ß was also significantly increased in human acinar cells from patients with acute/recurrent pancreatitis compared with chronic pancreatitis tissue. Alcohol exacerbates lipopolysaccharide-induced pancreatic fibrosis during the early onset of mild, subclinical, acute pancreatitis. We suggest that multiple, subclinical, acute pancreatitis episodes can accumulate in fibrosis during the development of chronic pancreatitis, even if there is no history of acute pancreatitis.

Autophagy and cell death signaling following dietary sulforaphane act independently of each other and require oxidative stress in pancreatic cancer.

The broccoli isothiocyanate, sulforaphane (SFN), was recently identified as being capable of eliminating highly therapy-resistant pancreatic carcinoma (PC) cells without inducing toxic side effects. While SFN has been shown to stimulate autophagy or 'self-eating', it is unclear whether this catabolic process is a pro- or anti-tumorigenic response. To investigate the role of autophagy in SFN-induced cell death, established PC cell lines were treated with SFN, and the induction of autophagy was evaluated by detecting the abundance of autophagic vesicles by electron microscopy, the increase in converted LC3-II by Western blot analysis and the autophagosome puncta of GFP-LC3 by immunofluorescence. SFN-induced autophagy was suppressed by the autophagy inhibitor chloroquine, while the autophagy inducer rapamycin did not further enhance autophagy in PC cells. Importantly, neither modulator altered SFN cytotoxicity, suggesting that SFN-induced autophagy and cell death act independently of each other. In contrast, the antioxidant N-acetyl-cysteine sustained cell viability and prevented autophagy induction after SFN exposure, indicating that both signaling pathways depend on reactive oxygen species (ROS). Our studies provide a valuable new mechanistic insight into the SFN-induced elimination of PC cells and suggest that an SFN-enriched diet potentially enhances ROS-releasing chemotherapeutic agents.

Effects of gadolinium chloride and glycine on hepatic and pancreatic tissue damage in alcoholic pancreatitis.

OBJECTIVE: Systemic complications in alcoholic pancreatitis are supposed to be aggravated by inflammatory liver damage. Resident macrophages including hepatic Kupffer cells play a pivotal role in mediating systemic complications in severe necrotizing pancreatitis (SNP). The aim of this study was to evaluate the effects of Kupffer cell inhibition on the inflammatory liver damage in experimental alcoholic pancreatitis. METHODS: Rats were fed with either alcohol or control diet for 6 weeks before induction of SNP. Animals were allocated into 4 groups: healthy controls, controls with SNP, SNP with gadolinium chloride or glycine (permanent vs temporary inhibition of hepatic Kupffer cells) prophylaxis. Hepatic microcirculation and morphologic damage of the liver and pancreas were assessed. RESULTS: Alcohol feeding and SNP increased hepatic and pancreatic injury compared with SNP alone. Gadolinium chloride and glycine improved hepatic microcirculation. In contrast, pancreatic and hepatic morphological damage was reduced by gadolinium chloride but not by glycine. CONCLUSIONS: Alcohol exposure aggravates hepatic and pancreatic injury in SNP. Gadolinium chloride reduces both microcirculatory and morphological damage, whereas glycine did not improve histological damage.

Protective effects and anti-inflammatory pathways of exogenous calcitonin gene-related peptide in severe necrotizing pancreatitis.

BACKGROUND: Microcirculatory disturbances are known to play a pivotal role in the pathogenesis of severe necrotizing pancreatitis (SNP). Calcitonin gene-related peptide (CGRP) is a vasodilatatory neuropeptide with potential anti-inflammatory effects. This study characterizes the protective effects and the anti-inflammatory pathway of exogenous CGRP in SNP. METHODS: SNP was induced in rats using the glycodeoxycholic acid model. CGRP was injected prophylactically before or therapeutically after initiation of the disease. Pancreatic damage was assessed using intravital microscopy, histology, NF-kappaB p50/p65 electrophoretic mobility shift assay, serum cytokine assay and ICAM-1 immunohistochemistry at 6 or 12 h after the onset of disease. RESULTS: Pancreatic microcirculatory disturbances, nuclear NF-kappaB levels and pancreatic ICAM-1 expression were increased in SNP compared to controls. After CGRP application, microcirculatory disturbances, NF-kappaB levels and pancreatic ICAM-1 expression were attenuated compared to pancreatitis alone. Moreover, pancreatic morphologic damage was significantly reduced by both prophylactic and therapeutic application of CGRP. CONCLUSIONS: CGRP is a neuropeptide that ameliorates the development of SNP in rats and may provide new treatment options. Its anti-inflammatory effects appear to be mediated by the modulation of pancreatic microcirculation and the inflammatory cascade.

Impaired autophagosome-lysosome fusion in the pathogenesis of pancreatitis.

In contrast to apoptosis, necrosis is generally viewed as a pro-inflammatory cell death mechanism. Accumulation of autophagosomes and massive acinar cell necrosis is observed in human acute pancreatitis, a severe and potentially lethal inflammatory condition. We have investigated the incidence of apoptosis, autophagy and necrosis affecting acinar cells in a rat model of acute pancreatitis induced by chronic alcohol intake and acute endotoxemia. We have observed that the combination of alcohol exposure and endotoxemia results in substantial accumulation of autophagosomes without an increase in autolysosomes, coupled to the depletion of LAMP-2, a lysosomal protein required for the proper fusion of autophagosomes with lysosomes. Alcohol plus endotoxemia favors the switch from apoptotic to necrotic cell death, as indicated by histopathological examination, reduced ATP levels, suppressed caspase activation, as well as the nuclear release of the proinflammatory factor HMGB1. Importantly, patients with alcoholic pancreatitis also exhibit local LAMP-2 depletion, recapitulating the results obtained in the animal model. We suggest that acinar cell vacuolization in pancreatitis is mediated by an endotoxemia-induced depletion of LAMP-2, which in turn facilitates the accumulation of autophagosomes due to the deficient formation of autolysosomes. Hence, we postulate that the depletion of lysosomal proteins may play a critical role in the pathogenesis of acute pancreatitis.

Impaired autolysosome formation correlates with Lamp-2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis.

BACKGROUND & AIMS: Acute pancreatitis constitutes a life-threatening condition in which pancreatic acinar cells undergo massive cell death. We investigated the incidence of apoptosis, autophagy, and necrosis affecting acinar cells in the early onset of acute pancreatitis induced by chronic alcohol feeding and acute endotoxemia. METHODS: Rats were fed either an ethanol-containing or a control diet over 14 weeks and killed 3 or 24 hours after a single lipopolysaccharide injection. Apoptosis, necrosis, and autophagy of pancreatic acinar cells were assessed by histology, electron microscopy, immunofluorescence, and biochemical methods. RESULTS: The combination of alcohol exposure and endotoxemia resulted in the depletion of several lysosomal proteins including lysosomal-associated membrane protein-2 (Lamp-2), a protein that is required for the proper fusion of autophagosomes with lysosomes. Accordingly, Lamp-2 depletion correlated with the accumulation of autophagosomes and a relative paucity of autolysosomes, reduced adenosine-5'-triphosphate levels, and a switch from apoptotic to necrotic cell death. This switch to necrosis was accompanied by reduced caspase activation and the nuclear release of the proinflammatory factor high mobility group box 1. Importantly, human patients with alcoholic pancreatitis also exhibited local Lamp-2 depletion, which points to a crucial role for Lamp-2 and autophagy in pancreatic acinar cell death. CONCLUSIONS: Our data suggest that acinar cell vacuolization in pancreatitis is mediated by an endotoxemia-induced inhibition of the late stage of autophagy. The combination of alcohol and endotoxemia attenuated apoptosis response yet enhanced acinar cell necrosis. The depletion of lysosomal proteins plays a critical role in the early onset of acute pancreatitis.

A novel animal model of severe pancreatitis in mice and its differences to the rat.

BACKGROUND: A noninvasive model of necrohemorrhagic pancreatitis induced by simultaneous intravenous cerulein/enterokinase (EK) infusion has recently been established in rats. The aim of the present study was to establish this new model in mice and to compare it with the rat model. METHODS: Male Balb/C mice (20 to 25 g) were used for the experiments. Pancreatitis was induced by simultaneous intravenous infusion of cerulein and EK. Controls were infused with either 0.9% NaCl, cerulein, or EK. Animals were humanely killed 6 hours after start of infusions. Pancreatic and pulmonary injury was assessed by histology, wet-to-dry weight ratio, and myeloperoxidase activity. Systemic cytokine, amylase, and lactate dehydrogenase (LDH) levels in blood were measured to assess pancreatic and systemic inflammatory response. To evaluate the role of protease activity in this model, trypsin, cathepsin B, and elastase activity were measured in pancreatic tissue. Survival experiments were performed to determine survival time and tissue injury in the later course of the disease. RESULTS: Mice with simultaneous cerulein/EK infusion developed marked local and systemic organ injury compared with those animals who received cerulein or EK alone. Pancreatic and pulmonary injury increased with high concentrations of cerulein/EK infusions. Survival decreased in these animals. Whereas acinar cell apoptosis was an early finding, pancreatic necrosis was observed later in the course of the disease. Serum levels of LDH, interleukin (IL)-1 alpha, and IL-1 beta reflected cell damage and the systemic inflammatory response. Protease activity in pancreatic tissue was greatest in animals with simultaneous cerulein/EK infusion. CONCLUSIONS: Using intravenous cerulein/EK infusions, a model of lethal acute pancreatitis has been established in mice. Major pancreatic edema, acinar cell apoptosis and necrosis, and pulmonary leukocyte sequestration are characteristic findings in this model. Although pancreatic injury was not as strong as in the rat model, this model may prove useful for future studies in transgenic mice.

Enterokinase induces severe necrosis and rapid mortality in cerulein pancreatitis: characterization of a novel noninvasive rat model of necro-hemorrhagic pancreatitis.

BACKGROUND: Unlike edematous pancreatitis, induction of severe necrotizing pancreatitis in rats generally requires an invasive laparotomy with infusion and/or ligation of the pancreatic duct or duodenal or arterial occlusion. The aim of this study was to establish and characterize a noninvasive model of severe acute pancreatitis in rats. METHODS: Wistar rats were infused intravenously with cerulein or a combination of cerulein and enterokinase. Saline (154-mmol/L NaCl) or enterokinase only was infused in controls. In a first set of experiments, intrapancreatic protease activation and the release of cytokines were correlated with the severity of organ injury. Pancreatic and pulmonary injuries were determined at 6 h. In a second set of experiments, we assessed 24-h survival, serum parameters possibly reflecting the course of the disease, and morphologic changes later in the course of the disease. RESULTS: The severity of pancreatic injury and survival were correlated strongly with the amount of enterokinase infused simultaneously with cerulein. Trypsin as well as elastase and cathepsin B activity in pancreatic tissue samples were increased markedly in these animals. Marked pancreatic hemorrhage, necrosis, and leukocyte infiltration were present in animals with the greatest amounts of enterokinase infused. IL-6 and LDH, but not IL-1beta, CRP, and amylase, in serum correlated with the severity of pancreatitis. CONCLUSIONS: This noninvasive rat model of acute pancreatitis is characterized by major pancreatic necrosis, hemorrhage, and fatality. The simple and noninvasive induction technique may have advantages for future studies on inflammatory changes and sepsis in necrotizing pancreatitis compared with other currently available invasive models.