'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage.

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.

Developmental Accretion of Docosahexaenoic Acid Is Independent of Fatty Acid Transporter Expression in Brain and Lung Tissues of C57BL/6 and Fat1 Mice.

BACKGROUND: Developmental expression of fatty acid transporters and their role in polyunsaturated fatty acid concentrations in the postnatal period have not been evaluated. OBJECTIVE: We hypothesized that transporter expression is developmentally regulated, tissue-specific, and that expression can modulate fatty acid accretion independently of diet. METHODS: Brain and lung transporter expression were quantified in C57BL/6 wild-type (WT) and Fat1 mice. Pups were dam-fed until day 21. Dams were fed AIN-76A 10% corn oil to represent a typical North American/European diet. After weaning, mice were fed the same diet as dams. Gene expression of Fatp1, Fatp4, Fabp5, and Fat/cd36 was quantified by quantitative reverse transcriptase-polymerase chain reaction. Fatty acid concentrations were measured by GC-MS. RESULTS: Brain docosahexaenoic acid (DHA) concentrations increased from day 3 to day 28 in both genotypes, with higher concentrations at days 3 and 14 in Fat1 than in WT mice [median (IQR)]: 10.7 (10.6-11.2) mol% compared with 6.6 (6.4-7.2) mol% and 12.5 (12.4-12.9) mol% compared with 8.9 (8.7-9.1) mol%, respectively; P < 0.05). During DHA accrual, transporter expression decreased. Fold changes in brain Fatp4, Fabp5, and Fat/cd36 were inversely correlated with fold changes in brain DHA concentrations in Fat1 relative to WT mice (ρ = -0.85, -0.75, and -0.78, respectively; P ≤ 0.001). Lung DHA concentrations were unchanged across the 3 time points for both genotypes. Despite unchanging DHA concentrations, there was increased expression of Fatp1 at days 14 and 28 (5-fold), Fatp4 at day 14 (2.3-fold), and Fabp5 at day 14 (3.8-fold) relative to day 3 in Fat1 mice. In WT mice, Fatp1 increased almost 5-fold at day 28 relative to day 3. There was no correlation between lung transporters and DHA concentrations in Fat1 relative to WT mice. CONCLUSIONS: Development of fatty acid transporter expression in C57BL/6 WT and Fat1 mice is genotype and tissue specific. Further, postnatal accretion of brain DHA appears independent of transporter status, with tissue concentrations representing dietary contributions.

Effect of polyunsaturated fatty acids on postnatal ileum development using the fat-1 transgenic mouse model.

BACKGROUND: Long-chain polyunsaturated fatty acids (LCPUFAs) play a critical role in neonatal health. We hypothesized that LCPUFAs play an essential role in priming postnatal gut development. We studied the effect of LCPUFAs on postnatal gut development using fat-1 transgenic mice, which are capable of converting n-6 to n-3 LCPUFAs, and wild-type (WT) C57BL/6 mice. METHODS: Distal ileum sections were collected from fat-1 and WT mice on days 3, 14, and 28. Fatty acid analyses, histology, RT-qPCR and intestinal permeability were performed. RESULTS: Fat-1 mice, relative to WT mice, showed increased n-3 LCPUFAs levels (α-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid, p < 0.05) and decreased arachidonic acid levels (p < 0.05) in the ileum. Preweaning fat-1 mice, compared to WT, showed >50% reduced muc2, Tff3, TLR9, and Camp expression (p < 0.05), markers of the innate immune response. There was a >two-fold increased expression of Fzd5 and EphB2, markers of cell differentiation (p < 0.05), and Fabp2 and 6, regulators of fatty acid transport and metabolism (p < 0.05). Despite reduced expression of tight junction genes, intestinal permeability in fat-1 was comparable to WT mice. CONCLUSIONS: Our data support the hypothesis that fatty acid profiles early in development modulate intestinal gene expression in formative domains, such as cell differentiation, tight junctions, other innate host defenses, and lipid metabolism.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aß (CnAß) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Mechanically unloading the left ventricle before coronary reperfusion reduces left ventricular wall stress and myocardial infarct size.

BACKGROUND: Ischemia/reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular wall stress with a percutaneous left atrial-to-femoral artery centrifugal bypass system while delaying coronary reperfusion limits myocardial injury in a model of acute MI. METHODS AND RESULTS: MI was induced by balloon occlusion of the left anterior descending artery in adult male swine. In the MI group (n=4), 120 minutes of left anterior descending artery occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+unload; n=4), percutaneous left atrial-to-femoral artery bypass was initiated after 120 minutes of ischemia, and left anterior descending artery occlusion was prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were euthanized after reperfusion, and infarct size was quantified by triphenyltetrazolium chloride staining. Compared with baseline, mean left ventricular wall stress and stroke work were not changed at any point in the MI group but were decreased after reperfusion in the MI+unload group (mean left ventricular wall stress, 44 658 versus 22 963 dynes/cm(2); stroke work, 2823 versus 655 mm Hg·mL, MI versus MI+unload). Phosphorylation of reperfusion injury salvage kinase pathway proteins from noninfarcted left ventricular tissue was unchanged in the MI group but was increased in the MI+unload group. Compared with the MI group, total infarct size was reduced in the MI+unload group (49% versus 28%, MI versus MI+unload). CONCLUSIONS: These data support that first unloading the left ventricle despite delaying coronary reperfusion during an acute MI reduces myocardial injury.

The role of pancreatic ductal secretion in protection against acute pancreatitis in mice*.

OBJECTIVES: A common potentially fatal disease of the pancreas is acute pancreatitis, for which there is no treatment. Most studies of this disorder focus on the damage to acinar cells since they are assumed to be the primary target of multiple stressors affecting the pancreas. However, increasing evidence suggests that the ducts may also have a crucial role in induction of the disease. To test this hypothesis, we sought to determine the specific role of the duct in the induction of acute pancreatitis using well-established disease models and mice with deletion of the Na/H exchanger regulatory factor-1 that have selectively impaired ductal function. DESIGN: Randomized animal study. SETTING: Animal research laboratory. SUBJECTS: Wild-type and Na/H exchanger regulatory factor-1 knockout mice. INTERVENTIONS: Acute necrotizing pancreatitis was induced by i.p. administration of cerulein or by intraductal administration of sodium taurocholate. The pancreatic expression of Na/H exchanger regulatory factor-1 and cystic fibrosis transmembrane conductance regulator (a key player in the control of ductal secretion) was analyzed by immunohistochemistry. In vivo pancreatic ductal secretion was studied in anesthetized mice. Functions of pancreatic acinar and ductal cells as well as inflammatory cells were analyzed in vitro. MEASUREMENTS AND MAIN RESULTS: Deletion of Na/H exchanger regulatory factor-1 resulted in gross mislocalization of cystic fibrosis transmembrane conductance regulator, causing marked reduction in pancreatic ductal fluid and bicarbonate secretion. Importantly, deletion of Na/H exchanger regulatory factor-1 had no deleterious effect on functions of acinar and inflammatory cells. Deletion of Na/H exchanger regulatory factor-1, which specifically impaired ductal function, increased the severity of acute pancreatitis in the two mouse models tested. CONCLUSIONS: Our findings provide the first direct evidence for the crucial role of ductal secretion in protecting the pancreas from acute pancreatitis and strongly suggest that improved ductal function should be an important modality in prevention and treatment of the disease.

Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins.

Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).

TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice.

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-α. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-α(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-α(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-α by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-α expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 µM and a peak-plateau signal at 500 µM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule.

Molecular profiling of human biopsies and surgical specimens is frequently complicated by their inherent biological heterogeneity and by the need to conserve tissue for clinical diagnosis. We have developed a set of novel 'tissue print' and 'print-phoresis' technologies to facilitate tissue and tumor-marker profiling under these circumstances. Tissue printing transfers cells and extracellular matrix components from a tissue surface onto nitrocellulose membranes, generating a two-dimensional anatomical image on which molecular markers can be visualized by specific protein and RNA- and DNA-detection techniques. Print-phoresis is a complementary new electrophoresis method in which thin strips from the print are subjected to polyacrylamide gel electrophoresis, providing a straightforward interface between the tissue-print image and gel-based proteomic techniques. Here we have utilized these technologies to identify and characterize markers of tumor invasion of the prostate capsule, an event generally not apparent to the naked eye that may result in tumor at the surgical margins ('positive margins'). We have also shown that tissue-print technologies can provide a general platform for the generation of marker maps that can be superimposed directly onto histopathological and radiological images, permitting molecular identification and classification of individual malignant lesions.

Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1.

BACKGROUND & AIMS: 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. METHODS: 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 submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM). RESULTS: 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-induced, but not 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. CONCLUSIONS: 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.

Maltodextrin-induced intestinal injury in a neonatal mouse model.

Prematurity and enteral feedings are major risk factors for intestinal injury leading to necrotizing enterocolitis (NEC). An immature digestive system can lead to maldigestion of macronutrients and increased vulnerability to intestinal injury. The aim of this study was to test in neonatal mice the effect of maltodextrin, a complex carbohydrate, on the risk of intestinal injury. The goal was to develop a robust and highly reproducible murine model of intestinal injury that allows insight into the pathogenesis and therapeutic interventions of nutrient-driven intestinal injury. Five- to 6-day-old C57BL/6 mice were assigned to the following groups: dam fed (D); D+hypoxia+Klebsiella pneumoniae; maltodextrin-dominant human infant formula (M) only; M+hypoxia; and M+hypoxia+K. pneumoniae. The mice in all M groups were gavage fed five times a day for 4 days. Mice were exposed to hypoxia twice a day for 10 min prior to the first and last feedings, and K. pneumoniae was added to feedings as per group assignment. Mice in all M groups demonstrated reduced body weight, increased small intestinal dilatation and increased intestinal injury scores. Maltodextrin-dominant infant formula with hypoxia led to intestinal injury in neonatal mice accompanied by loss of villi, increased MUC2 production, altered expression of tight junction proteins, enhanced intestinal permeability, increased cell death and higher levels of intestinal inflammatory mediators. This robust and highly reproducible model allows for further interrogation of the effects of nutrients on pathogenic factors leading to intestinal injury and NEC in preterm infants.This article has an associated First Person interview with the first author of the paper.

Early Enteral Administration of a Complex Lipid Emulsion Supplement Prevents Postnatal Deficits in Docosahexaenoic and Arachidonic Acids and Increases Tissue Accretion of Lipophilic Nutrients in Preterm Piglets.

BACKGROUND: Preterm delivery and current nutrition strategies result in deficiencies of critical long-chain fatty acids (FAs) and lipophilic nutrients, increasing the risk of preterm morbidities. We sought to determine the efficacy of preventing postnatal deficits in FAs and lipophilic nutrients using an enteral concentrated lipid supplement in preterm piglets. METHODS: Preterm piglets were fed a baseline diet devoid of arachidonic acid (AA) and docosahexaenoic acid (DHA) and randomized to enteral supplementation as follows: (1) Intralipid (IL), (2) complex lipid supplement 1 (CLS1) with an AA:DHA ratio of 0.25, or (3) CLS2 with an AA:DHA ratio of 1.2. On day 8, plasma and tissue levels of FAs and lipophilic nutrients were measured and ileum histology performed. RESULTS: Plasma DHA levels decreased in the IL group by day 2. In contrast, DHA increased by day 2 compared with birth levels in both CLS1 and CLS2 groups. The IL and CLS1 groups demonstrated a continued decline in AA levels during the 8-day protocol, whereas AA levels in the CLS2 group on day 8 were comparable to birth levels. Preserving AA levels in the CLS2 group was associated with greater ileal villus height and muscular layer thickness. Lipophilic nutrients were effectively absorbed in plasma and tissues. CONCLUSIONS: Enteral administration of CLS1 and CLS2 demonstrated similar increases in DHA levels compared with birth levels. Only CLS2 maintained AA birth levels. Providing a concentrated complex lipid emulsion with an AA:DHA ratio > 1 is important in preventing postnatal AA deficits.

Matrix metalloproteinase 9 plays a key role in lyme arthritis but not in dissemination of Borrelia burgdorferi.

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.

A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate.

OBJECTIVE: 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. DESIGN: Na-taurocholate (10-50 microl, 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. RESULTS: 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 microl 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. CONCLUSION: 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.

Targeting of melanoma brain metastases using engineered neural stem/progenitor cells.

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.

The fibrinolytic system facilitates tumor cell migration across the blood-brain barrier in experimental melanoma brain metastasis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

ADAMTS4 (aggrecanase-1) cleaves human brain versican V2 at Glu405-Gln406 to generate glial hyaluronate binding protein.

Human brain tissue from cerebellum and hippocampus was obtained between 2 h and 24 h post mortem and, after extraction in the presence of proteinase inhibitors, proteoglycans were purified by anion-exchange chromatography. The versican component was characterized by Western analysis with antibodies to the N-terminal peptide (LF99), the N-terminal globular domain (12C5) and the two GAG (glycosaminoglycan) attachment regions (anti-GAG-alpha and anti-GAG-beta). The results indicated that versican V2 is the major variant in all brain samples, and that it exists as the full-length form and also as at least six C-terminally truncated forms. The major immunoreactive species present is a 64 kDa product, which we identified by biochemical and immunological analysis as the brain protein previously termed GHAP (glial hyaluronate binding protein) [Perides, Lane, Andrews, Dahl and Bignami (1989) J. Biol. Chem. 264, 5981-5987]. Immunological analysis of purified human GHAP using a new anti-neoepitope antiserum (JSCNIV) showed that its C-terminal sequence is NIVSFE(405), and digestion of human cerebellum proteoglycans with ADAMTS4 (aggrecanase-1, where ADAMTS, a disintegrin and metalloproteinase with thrombospondin-1-like motifs) indicated that GHAP is a product of cleavage of versican V0 or V2 at the Glu(405)-Gln(406) bond. Since human cerebellum extracts contained multiple forms of ADAMTS4 protein on Western analysis, these data suggest that one or more members of the 'aggrecanase' group of the ADAMTS family (ADAMTS 1, 4, 5 and 9) are responsible for turnover of versican V2 in the adult human brain.