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1.  Necroptosis Is an Important Severity Determinant and Potential Therapeutic Target in Experimental Severe Pancreatitis 
Background and Aims
Severe acute pancreatitis is characterized by acinar cell death and inflammation. Necroptosis is an aggressive and pro-inflammatory mode of cell death that can be prevented by necrostatin-1 administration or RIP3 deletion.
Mouse pancreatic acinar cells were incubated with supramaximally stimulating concentrations of caerulein or sub-micellar concentrations of TLCS and necroptosis was inhibited by either addition of necrostatin or by RIP3 deletion. Cell death was quantitated using either LDH leakage from acini or PI staining of nuclei. Necrosome formation was tracked and quantitated using cell fractionation or immunoprecipitation. Pancreatitis was induced in mice by retrograde intraductal infusion of TLCS or by repetitive supramaximal stimulation with caerulein.
Necroptosis was found to be the most prevalent mode of acinar cell in vitro death and little or no apoptosis was observed. Acinar cell death was associated with necrosome formation and prevented by either necrostatin administration or RIP3 deletion. Both of these interventions reduced the severity of TLCS- or caerulein-induced pancreatitis. Delaying necrostatin administration until after pancreatitis had already been established could still reduce the severity of TLCS-induced pancreatitis.
Necroptosis is the predominant mode of acinar cell death in severe experimental mouse pancreatitis. The severity of pancreatitis can be reduced by administration of necrostatin and that necrostatin can still reduce the cell injury of pancreatitis even if it is administered after the disease has already been established. Inhibition of necroptosis may be an effective strategy for the treatment of severe clinical pancreatitis.
PMCID: PMC5020563  PMID: 27642624
acute pancreatitis; biliary pancreatitis; necroptosis; apoptosis; pancreatic cell death
2.  Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection 
The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.
PMCID: PMC3731432  PMID: 23851390
Cancer Biology; Issue 77; Cellular Biology; Molecular Biology; Medicine; Biochemistry; Biomedical Engineering; Acinar Cells; Pancreatitis; Transfection; Microscopy; Confocal; Calcium Signaling; Pancreatic Acinar Cells; Pancreatitis; Calcium Signaling; Cytotoxicity; LDH Leakage; cell injury; imaging
3.  ‘Role reversal’ for the receptor PAR1 in sepsis-induced vascular damage 
Nature immunology  2007;8(12):1303-1312.
Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1–PAR2 complexes may be beneficial in the treatment of sepsis.
PMCID: PMC3059149  PMID: 17965715
Gastroenterology  2009;138(2):715.
The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G protein-coupled, cell surface, bile acid receptor.
Acute pancreatitis was induced in wild type and Gpbar1−/− mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild type and Gpbar1−/− mice were exposed to either sub-micellar concentrations of TLCS (200–500 μM) or a supramaximally stimulating concentration of caerulein (10 nM).
Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS- but not in caerulein-induced pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS but not when they are induced by exposure to caerulein.
Gpbar1 may play a critical role in the evolution of bile acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a “receptor-mediated” disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.
PMCID: PMC2819588  PMID: 19900448
5.  A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na‐taurocholate 
Gut  2007;56(11):1590-1598.
Most mechanistic studies of pancreatitis in mice employ the secretagogue‐induced model. The currently reported studies were designed to develop an alternative, and possibly more clinically relevant, mouse model of pancreatitis.
Na‐taurocholate (10–50 μl, 1–5%) in saline, or saline alone, was retrogradely infused into the mouse pancreatic duct. The animals were killed 6–24 hours later and the severity of pancreatitis in the pancreatic head and tail was examined by quantitating hyperamylasemia, pancreatic edema, acinar cell necrosis, and pancreatic inflammation. In addition, intrapancreatic activation of trypsinogen, generation of IL‐6, intrapulmonary sequestration of neutrophils, and alterations in lung compliance were evaluated. The effects of Na‐taurocholate on in‐vitro acinar cell calcium transients, viability, and trypsinogen activation were examined.
Little or no evidence of pancreatitis was observed in mice infused with saline alone or in the tail of pancreata removed from animals infused with Na‐taurocholate. In the head of the pancreas, evidence of pancreatitis was observed 12–24 hours after infusion of 20–50 μl 2–5% Na‐taurocholate and the earliest morphological changes involved terminal duct and acinar cells. Intrapancreatic trypsin activity was transiently elevated within 5 minutes of Na‐taurocholate infusion and pancreatic IL‐6 levels were elevated 24 hours later. Under in‐vitro conditions, Na‐taurocholate triggered pathological acinar cell calcium transients, cell death, and calcium‐dependent trypsinogen activation.
This clinically relevant model of acute biliary pancreatitis yields reproducible results and its severity can be easily manipulated. It is ideally suited for use in mechanistic studies employing genetically modified mouse strains.
PMCID: PMC2095649  PMID: 17591621
acute pancreatitis; animal model; bile acids
6.  Matrix Metalloproteinase 9 Plays a Key Role in Lyme Arthritis but Not in Dissemination of Borrelia burgdorferi▿  
Infection and Immunity  2009;77(7):2643-2649.
Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9−/− mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9−/− exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9−/− and wild-type mice. We examined whether decreased inflammation in MMP-9−/− mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.
PMCID: PMC2708580  PMID: 19364840
7.  Targeting of melanoma brain metastases using engineered neural stem/progenitor cells1 
Neuro-Oncology  2006;8(2):119-126.
Brain metastases are an increasingly frequent and serious clinical problem for cancer patients, especially those with advanced melanoma. Given the extensive tropism of neural stem/progenitor cells (NSPCs) for pathological areas in the central nervous system, we expanded investigations to determine whether NSPCs could also target multiple sites of brain metastases in a syngeneic experimental melanoma model. Using cytosine deaminase–expressing NSPCs (CD-NSPCs) and systemic 5-fluorocytosine (5-FC) pro-drug administration, we explored their potential as a cell-based targeted drug delivery system to disseminated brain metastases. Our results indicate a strong tropism of NSPCs for intracerebral melanoma metastases. Furthermore, in our therapeutic paradigm, animals with established melanoma brain metastasis received intracranial implantation of CD-NSPCs followed by systemic 5-FC treatment, resulting in a significant (71%) reduction in tumor burden. These data provide proof of principle for the use of NSPCs for targeted delivery of therapeutic gene products to melanoma brain metastases.
PMCID: PMC1871940  PMID: 16524944
brain metastases; cytosine deaminase; gene therapy; melanoma; neural progenitor cells; neural stem cells; tumor targeting
8.  MyD88 Deficiency Results in Tissue-Specific Changes in Cytokine Induction and Inflammation in Interleukin-18-Independent Mice Infected with Borrelia burgdorferi  
Infection and Immunity  2006;74(3):1462-1470.
Toll-like receptors (TLRs) play an important role in the control of infection with Borrelia burgdorferi. Deficiencies in TLR-2 or the shared TLR adapter molecule MyD88 have been shown to result in greatly increased bacterial burdens in mice. However, although in vitro studies have shown that the activation of TLR pathways by B. burgdorferi results in the release of inflammatory cytokines, studies in deficient mice have shown either no change or increased rather than decreased inflammation in infected animals. In this study, we looked at mechanisms to explain the increase in inflammation in the absence of MyD88. We found that MyD88-deficient mice infected with B. burgdorferi did not show increased inflammation at sites typically associated with Lyme disease (joints and heart). However, there was markedly increased inflammation in the muscles, kidneys, pancreas, and lungs of deficient animals. Muscle inflammation was typically seen perivascularly and perineuronally similar to that seen in infected humans. Chemotactic chemokines and cytokines were greatly increased in the muscle and kidneys of MyD88-deficient animals but not in the joints or heart tissue, suggesting that MyD88-independent pathways for recognizing B. burgdorferi and inducing these chemokines are present in the muscle and kidneys. Interleukin-18 signaling through MyD88 does not appear to play a role in either control of infection or inflammation.
PMCID: PMC1418660  PMID: 16495516
9.  Co-localization hypothesis: A mechanism for the intrapancreatic activation of digestive enzymes during the early phases of acute pancreatitis 
Acute pancreatitis is generally believed to be a disease in which the pancreas is injured by digestive enzymes that it normally produces. Most of the potentially harmful digestive enzymes produced by pancreatic acinar cells are synthesized and secreted as inactive zymogens which are normally activated only upon entry into the duodenum but, during the early stages of acute pancreatitis, those zymogens become prematurely activated within the pancreas and, presumably, that activation occurs within pancreatic acinar cells. The mechanisms responsible for intracellular activation of digestive enzyme zymogens have not been elucidated with certainty but, according to one widely recognized theory (the “co-localization hypothesis"), digestive enzyme zymogens are activated by lysosomal hydrolases when the two types of enzymes become co-localized within the same intracellular compartment. This review focuses on the evidence supporting the validity of the co-localization hypothesis as an explanation for digestive enzyme activation during the early stages of pancreatitis. The findings, summarized in this review, support the conclusion that co-localization of lysosomal hydrolases with digestive enzyme zymogens plays a critical role in permitting the intracellular activation of digestive enzymes that leads to acinar cell injury and pancreatitis.
PMCID: PMC4087673  PMID: 16610045
Acute pancreatitis; Digestive enzymes; Pancreatic acinar cells
10.  The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis 
BMC Cancer  2006;6:56.
Patients with metastatic tumors to the brain have a very poor prognosis. Increased metastatic potential has been associated with the fibrinolytic system. We investigated the role of the fibrinolytic enzyme plasmin in tumor cell migration across brain endothelial cells and growth of brain metastases in an experimental metastatic melanoma model.
Metastatic tumors to the brain were established by direct injection into the striatum or by intracarotid injection of B16F10 mouse melanoma cells in C57Bl mice. The role of plasminogen in the ability of human melanoma cells to cross a human blood-brain barrier model was studied on a transwell system.
Wild type mice treated with the plasmin inhibitor epsilon-aminocaproic acid (EACA) and plg-/- mice developed smaller tumors and survived longer than untreated wild type mice. Tumors metastasized to the brain of wild type mice treated with EACA and plg-/- less efficiently than in untreated wild type mice. No difference was observed in the tumor growth in any of the three groups of mice. Human melanoma cells were able to cross the human blood-brain barrier model in a plasmin dependent manner.
Plasmin facilitates the development of tumor metastasis to the brain. Inhibition of the fibrinolytic system could be considered as means to prevent tumor metastasis to the brain.
PMCID: PMC1421425  PMID: 16524486
12.  Borrelia burgdorferi, Host-Derived Proteases, and the Blood-Brain Barrier  
Infection and Immunity  2005;73(2):1014-1022.
Neurological manifestations of Lyme disease in humans are attributed in part to penetration of the blood-brain barrier (BBB) and invasion of the central nervous system (CNS) by Borrelia burgdorferi. However, how the spirochetes cross the BBB remains an unresolved issue. We examined the traversal of B. burgdorferi across the human BBB and systemic endothelial cell barriers using in vitro model systems constructed of human brain microvascular endothelial cells (BMEC) and EA.hy 926, a human umbilical vein endothelial cell (HUVEC) line grown on Costar Transwell inserts. These studies showed that B. burgdorferi differentially crosses human BMEC and HUVEC and that the human BMEC form a barrier to traversal. During the transmigration by the spirochetes, it was found that the integrity of the endothelial cell monolayers was maintained, as assessed by transendothelial electrical resistance measurements at the end of the experimental period, and that B. burgdorferi appeared to bind human BMEC by their tips near or at cell borders, suggesting a paracellular route of transmigration. Importantly, traversal of B. burgdorferi across human BMEC induces the expression of plasminogen activators, plasminogen activator receptors, and matrix metalloproteinases. Thus, the fibrinolytic system linked by an activation cascade may lead to focal and transient degradation of tight junction proteins that allows B. burgdorferi to invade the CNS.
PMCID: PMC546937  PMID: 15664945

Results 1-12 (12)