Amphipathic Liponecrosis Impairs Bacterial Clearance and Causes Infection During Sterile Inflammation.

BACKGROUND & AIMS: Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. METHODS: Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects-NEFA-membrane phospholipid (phosphatidylcholine) interactions-were studied on isothermal titration calorimetry. RESULTS: Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered ß-diversity; enrichment in Pseudomonadales; and increased annexin V-positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. In vivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. CONCLUSIONS: Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.

Adipose saturation reduces lipotoxic systemic inflammation and explains the obesity paradox.

Obesity sometimes seems protective in disease. This obesity paradox is predominantly described in reports from the Western Hemisphere during acute illnesses. Since adipose triglyceride composition corresponds to long-term dietary patterns, we performed a meta-analysis modeling the effect of obesity on severity of acute pancreatitis, in the context of dietary patterns of the countries from which the studies originated. Increased severity was noted in leaner populations with a higher proportion of unsaturated fat intake. In mice, greater hydrolysis of unsaturated visceral triglyceride caused worse organ failure during pancreatitis, even when the mice were leaner than those having saturated triglyceride. Saturation interfered with triglyceride's interaction and lipolysis by pancreatic triglyceride lipase, which mediates organ failure. Unsaturation increased fatty acid monomers in vivo and aqueous media, resulting in greater lipotoxic cellular responses and organ failure. Therefore, visceral triglyceride saturation reduces the ensuing lipotoxicity despite higher adiposity, thus explaining the obesity paradox.

Triptolide targets super-enhancer networks in pancreatic cancer cells and cancer-associated fibroblasts.

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is highly heterogeneous, fibrotic, and hypovascular, marked by extensive desmoplasia and maintained by the tumor cells, cancer-associated fibroblasts (CAFs) and other stromal cells. There is an urgent need to identify and develop treatment strategies that not only target the tumor cells but can also modulate the stromal cells. A growing number of studies implicate the role of regulatory DNA elements called super-enhancers (SE) in maintaining cell-type-specific gene expression networks in both normal and cancer cells. Using chromatin activation marks, we first mapped SE networks in pancreatic CAFs and epithelial tumor cells and found them to have distinct SE profiles. Next, we explored the role of triptolide (TPL), a natural compound with antitumor activity, in the context of modulating cell-type-specific SE signatures in PDAC. We found that TPL, cytotoxic to both pancreatic tumor cells and CAFs, disrupted SEs in a manner that resulted in the downregulation of SE-associated genes (e.g., BRD4, MYC, RNA Pol II, and Collagen 1) in both cell types at mRNA and protein levels. Our observations suggest that TPL acts as a SE interactive agent and may elicit its antitumor activity through SE disruption to re-program cellular cross talk and signaling in PDAC. Based on our findings, epigenetic reprogramming of transcriptional regulation using SE modulating compounds such as TPL may provide means for effective treatment options for pancreatic cancer patients.

Single-cell transcriptome analysis of tumor and stromal compartments of pancreatic ductal adenocarcinoma primary tumors and metastatic lesions.

BACKGROUND: Solid tumors such as pancreatic ductal adenocarcinoma (PDAC) comprise not just tumor cells but also a microenvironment with which the tumor cells constantly interact. Detailed characterization of the cellular composition of the tumor microenvironment is critical to the understanding of the disease and treatment of the patient. Single-cell transcriptomics has been used to study the cellular composition of different solid tumor types including PDAC. However, almost all of those studies used primary tumor tissues. METHODS: In this study, we employed a single-cell RNA sequencing technology to profile the transcriptomes of individual cells from dissociated primary tumors or metastatic biopsies obtained from patients with PDAC. Unsupervised clustering analysis as well as a new supervised classification algorithm, SuperCT, was used to identify the different cell types within the tumor tissues. The expression signatures of the different cell types were then compared between primary tumors and metastatic biopsies. The expressions of the cell type-specific signature genes were also correlated with patient survival using public datasets. RESULTS: Our single-cell RNA sequencing analysis revealed distinct cell types in primary and metastatic PDAC tissues including tumor cells, endothelial cells, cancer-associated fibroblasts (CAFs), and immune cells. The cancer cells showed high inter-patient heterogeneity, whereas the stromal cells were more homogenous across patients. Immune infiltration varies significantly from patient to patient with majority of the immune cells being macrophages and exhausted lymphocytes. We found that the tumor cellular composition was an important factor in defining the PDAC subtypes. Furthermore, the expression levels of cell type-specific markers for EMT+ cancer cells, activated CAFs, and endothelial cells significantly associated with patient survival. CONCLUSIONS: Taken together, our work identifies significant heterogeneity in cellular compositions of PDAC tumors and between primary tumors and metastatic lesions. Furthermore, the cellular composition was an important factor in defining PDAC subtypes and significantly correlated with patient outcome. These findings provide valuable insights on the PDAC microenvironment and could potentially inform the management of PDAC patients.

Pancreatic triglyceride lipase mediates lipotoxic systemic inflammation.

Visceral adipose tissue plays a critical role in numerous diseases. Although imaging studies often show adipose involvement in abdominal diseases, their outcomes may vary from being a mild self-limited illness to one with systemic inflammation and organ failure. We therefore compared the pattern of visceral adipose injury during acute pancreatitis and acute diverticulitis to determine its role in organ failure. Acute pancreatitis-associated adipose tissue had ongoing lipolysis in the absence of adipocyte triglyceride lipase (ATGL). Pancreatic lipase injected into mouse visceral adipose tissue hydrolyzed adipose triglyceride and generated excess nonesterified fatty acids (NEFAs), which caused organ failure in the absence of acute pancreatitis. Pancreatic triglyceride lipase (PNLIP) increased in adipose tissue during pancreatitis and entered adipocytes by multiple mechanisms, hydrolyzing adipose triglyceride and generating excess NEFAs. During pancreatitis, obese PNLIP-knockout mice, unlike obese adipocyte-specific ATGL knockouts, had lower visceral adipose tissue lipolysis, milder inflammation, less severe organ failure, and improved survival. PNLIP-knockout mice, unlike ATGL knockouts, were protected from adipocyte-induced pancreatic acinar injury without affecting NEFA signaling or acute pancreatitis induction. Therefore, during pancreatitis, unlike diverticulitis, PNLIP leaking into visceral adipose tissue can cause excessive visceral adipose tissue lipolysis independently of adipocyte-autonomous ATGL, and thereby worsen organ failure.

Single-Cell Sequencing in Precision Medicine.

The application of next-generation sequencing in cancer genomics allowed for a better understanding of the genetics and pathogenesis of cancer. Single-cell genomics is a relatively new field that has enhanced our current knowledge of the genetic diversity of cells involved in the complex biological systems of cancer. Single-cell genomics is a rapidly developing field, and current technologies can assay a single cell's gene expression, DNA variation, epigenetic state, and nuclear structure. Statistical and computational methods are central to single-cell genomics and allows for extraction of meaningful information. The translational application of single-cell sequencing in precision cancer therapy has the potential to improve cancer diagnostics, prognostics, targeted therapy, early detection, and noninvasive monitoring. Furthermore, single-cell genomics will transform cancer research as even initial experiments have revolutionized our current understanding of gene regulation and disease.

A Multiscale Map of the Stem Cell State in Pancreatic Adenocarcinoma.

Drug resistance and relapse remain key challenges in pancreatic cancer. Here, we have used RNA sequencing (RNA-seq), chromatin immunoprecipitation (ChIP)-seq, and genome-wide CRISPR analysis to map the molecular dependencies of pancreatic cancer stem cells, highly therapy-resistant cells that preferentially drive tumorigenesis and progression. This integrated genomic approach revealed an unexpected utilization of immuno-regulatory signals by pancreatic cancer epithelial cells. In particular, the nuclear hormone receptor retinoic-acid-receptor-related orphan receptor gamma (RORγ), known to drive inflammation and T cell differentiation, was upregulated during pancreatic cancer progression, and its genetic or pharmacologic inhibition led to a striking defect in pancreatic cancer growth and a marked improvement in survival. Further, a large-scale retrospective analysis in patients revealed that RORγ expression may predict pancreatic cancer aggressiveness, as it positively correlated with advanced disease and metastasis. Collectively, these data identify an orthogonal co-option of immuno-regulatory signals by pancreatic cancer stem cells, suggesting that autoimmune drugs should be evaluated as novel treatment strategies for pancreatic cancer patients.

Triptolide and Its Derivatives as Cancer Therapies.

Triptolide, a compound isolated from a Chinese medicinal herb, possesses potent antitumor, immunosuppressive, and anti-inflammatory properties, but is clinically limited due to its poor solubility, bioavailability, and toxicity. Recently, Minnelide, a water-soluble prodrug of triptolide, was shown to have potent antitumor activity in various preclinical cancer models. Minnelide is currently in Phase II clinical trials for treatment of advanced pancreatic cancer, which has fueled increased interest in this promising agent. Here, we review the recent advances in the biological activity of triptolide and its analogs, their mechanisms of actions, and their clinical developments. A special emphasis is given to proteins and pathways within the tumor and stromal compartments that are targeted by triptolide and its analogs as well as the ongoing clinical trials.

Carboxyl Ester Lipase May Not Mediate Lipotoxic Injury during Severe Acute Pancreatitis.

Acute lipolysis of visceral fat or circulating triglycerides may worsen acute pancreatitis (AP)-associated local and systemic injury. The pancreas expresses pancreatic triacylglycerol lipase (PNLIP), pancreatic lipase-related protein 2 (PNLIPRP2), and carboxyl ester lipase (CEL), which may leak into the visceral fat or systemic circulation during pancreatitis. We, thus, aimed to determine the pancreatic lipase(s) regulating lipotoxicity during AP. For this AP, associated fat necrosis was analyzed using Western blot analysis. Bile acid (using liquid chromatography-tandem mass spectrometry) and fatty acid (using gas chromatography) concentrations were measured in human fat necrosis. The fat necrosis milieu was simulated in vitro using glyceryl trilinoleate because linoleic acid is increased in fat necrosis. Bile acid requirements to effectively hydrolyze glyceryl trilinoleate were studied using exogenous or overexpressed lipases. The renal cell line (HEK 293) was used to study lipotoxic injury. Because dual pancreatic lipase knockouts are lethal, exocrine parotid acini lacking lipases were used to verify the results. PNLIP, PNLIPRP2, and CEL were increased in fat necrosis. Although PNLIP and PNLIPRP2 were equipotent in inducing lipolysis and lipotoxic injury, CEL required bile acid concentrations higher than in human fat necrosis. The high bile acid requirements for effective lipolysis make CEL an unlikely mediator of lipotoxic injury in AP. It remains to be explored whether PNLIP or PNLIPRP2 worsens AP severity in vivo.

SuperCT: a supervised-learning framework for enhanced characterization of single-cell transcriptomic profiles.

Characterization of individual cell types is fundamental to the study of multicellular samples. Single-cell RNAseq techniques, which allow high-throughput expression profiling of individual cells, have significantly advanced our ability of this task. Currently, most of the scRNA-seq data analyses are commenced with unsupervised clustering. Clusters are often assigned to different cell types based on the enriched canonical markers. However, this process is inefficient and arbitrary. In this study, we present a technical framework of training the expandable supervised-classifier in order to reveal the single-cell identities as soon as the single-cell expression profile is input. Using multiple scRNA-seq datasets we demonstrate the superior accuracy, robustness, compatibility and expandability of this new solution compared to the traditional methods. We use two examples of the model upgrade to demonstrate how the projected evolution of the cell-type classifier is realized.

The Effects of Synthetically Modified Natural Compounds on ABC Transporters.

Multidrug resistance (MDR) is a major hurdle which must be overcome to effectively treat cancer. ATP-binding cassette transporters (ABC transporters) play pivotal roles in drug absorption and disposition, and overexpression of ABC transporters has been shown to attenuate cellular/tissue drug accumulation and thus increase MDR across a variety of cancers. Overcoming MDR is one desired approach to improving the survival rate of patients. To date, a number of modulators have been identified which block the function and/or decrease the expression of ABC transporters, thereby restoring the efficacy of a range of anticancer drugs. However, clinical MDR reversal agents have thus far proven ineffective and/or toxic. The need for new, effective, well-tolerated and nontoxic compounds has led to the development of natural compounds and their derivatives to ameliorate MDR. This review evaluates whether synthetically modifying natural compounds is a viable strategy to generate potent, nontoxic, ABC transporter inhibitors which may potentially reverse MDR.

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 µm within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis.

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.

Characterization and Predictive Value of Near Infrared 2-Deoxyglucose Optical Imaging in Severe Acute Pancreatitis.

BACKGROUND: Studying the uptake of 2-deoxy glucose (2-DG) analogs such as 2-Deoxy-2-[18F] fluoroglucose (FDG) is a common approach to identify and monitor malignancies and more recently chronic inflammation. While pancreatitis is a common cause for false positive results in human studies on pancreatic cancer using FDG, the relevance of these findings to acute pancreatitis (AP) is unknown. FDG has a short half-life. Thus, with an aim to accurately characterize the metabolic demand of the pancreas during AP in real-time, we studied the uptake of the non-radioactive, near infrared fluorescence labelled 2-deoxyglucose analog, IRDye® 800CW 2-DG probe (NIR 2-DG; Li-Cor) during mild and severe biliary AP. METHODS: Wistar rats (300 g; 8-12/group) were administered NIR 2-DG (10 nM; I.V.). Mild and severe biliary AP were respectively induced by biliopancreatic duct ligation (DL) alone or along with infusing glyceryl trilinoleate (GTL; 50 µL/100 g) within 10 minutes of giving NIR 2-DG. Controls (CON) only received NIR 2-DG. Imaging was done every 5-10 minutes over 3 hrs. Average Radiant Efficiency [p/s/cm²/sr]/[µW/cm²] was measured over the pancreas using the IVIS 200 in-vivo imaging system (PerkinElmer) using the Living Image® software and verified in ex vivo pancreata. Blood amylase, lipase and pancreatic edema, necrosis were measured over the course of AP. RESULTS: NIR 2-DG uptake over the first hour was not influenced by AP induction. However, while the signal declined in controls and rats with mild AP, there was significantly higher retention of NIR 2-DG in the pancreas after 1 hour in those with GTL pancreatitis. The increase was > 3 fold over controls in the GTL group and was verified to be in the pancreas ex vivo. In vitro, pancreatic acini exposed to GTL had a similar increase in NIR 2-DG uptake which was followed by progressively worse acinar necrosis. Greater retention of NIR 2-DG in vivo was associated with worse pancreatic necrosis, reduced ATP concentrations and mortality, which were not predicted by the blood parameters. CONCLUSION: In-vivo fluorescent imaging of a non-radioactive near infrared 2-DG optical probe can predict the AP severity early during the disease.

Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections.

BACKGROUND AND AIMS: Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators. METHODS: We measured acinar and PBMC injury induced by agents increased in NCs and PCs. Outcomes of caerulein pancreatitis were studied in lean rats coadministered interleukin (IL)-1ß and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat. RESULTS: NCs had higher fatty acids, IL-8 and IL-1ß versus other fluids. Lipolysis of unsaturated triglyceride and resulting unsaturated fatty acids (UFA) oleic and linoleic acids induced necro-apoptosis at less than half the concentration in NCs but other agents did not do so at more than two times these concentrations. Cytokine coadministration resulted in higher pancreatic and lung inflammation than caerulein alone, but only triolein coadministration caused peripancreatic fat stranding, higher cytokines, UFAs, multisystem organ failure (MSOF) and mortality in 97% animals, which were prevented by orlistat. CONCLUSIONS: UFAs, IL-1ß and IL-8 are elevated in NCs. However, UFAs generated via peripancreatic fat lipolysis causes worse inflammation and MSOF, converting mild AP to SAP.

Lipolysis of visceral adipocyte triglyceride by pancreatic lipases converts mild acute pancreatitis to severe pancreatitis independent of necrosis and inflammation.

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.

Hypothermia slows sequential and parallel steps initiated during caerulein pancreatitis.

BACKGROUND AND OBJECTIVES: Multiple deleterious signaling cascades are simultaneously activated in acute pancreatitis (AP), which may limit the success of pharmacologic approaches targeting a single step. We explored whether cooling acinar cells slows distinct steps initiated from a stimulus causing pancreatitis simultaneously, and the temperature range over which inhibition of such deleterious signaling occurs. METHODS: Caerulein (100 nM) induced trypsinogen activation (TGA), CXCL1, CXCL2 mRNA levels, cell injury were studied at 37 °C, 34 °C, 31 °C, 29 °C and 25 °C in acinar cells. Trypsin, cathepsin B activities and cathepsin B mediated TGA were studied at 37 °C, 23 °C and 4 °C. RESULTS: There was >80% reduction in TGA, CXCL1 and CXCL2 mRNA levels at 29 °C, and in cell injury at 34 °C, compared to those at 37 °C. Trypsin activity, cathepsin B activity and cathepsin B mediated TGA at 23 °C were respectively, 53%, 64% and 26% of that at 37 °C. Acinar cooling to 31 °C reduced LDH leakage even when cooling was initiated an hour after caerulein stimulation at 37 °C. CONCLUSIONS: Hypothermia synergistically and simultaneously slows parallel and distinct signaling steps initiated by caerulein, thereby reducing TGA, upregulation of inflammatory mediators and acinar injury.

Acute lipotoxicity regulates severity of biliary acute pancreatitis without affecting its initiation.

Obese patients have worse outcomes during acute pancreatitis (AP). Previous animal models of AP have found worse outcomes in obese rodents who may have a baseline proinflammatory state. Our aim was to study the role of acute lipolytic generation of fatty acids on local severity and systemic complications of AP. Human postpancreatitis necrotic collections were analyzed for unsaturated fatty acids (UFAs) and saturated fatty acids. A model of biliary AP was designed to replicate the human variables by intraductal injection of the triglyceride glyceryl trilinoleate alone or with the chemically distinct lipase inhibitors orlistat or cetilistat. Parameters of AP etiology and outcomes of local and systemic severity were measured. Patients with postpancreatitis necrotic collections were obese, and 13 of 15 had biliary AP. Postpancreatitis necrotic collections were enriched in UFAs. Intraductal glyceryl trilinoleate with or without the lipase inhibitors resulted in oil red O-positive areas, resembling intrapancreatic fat. Both lipase inhibitors reduced the glyceryl trilinoleate-induced increase in serum lipase, UFAs, pancreatic necrosis, serum inflammatory markers, systemic injury, and mortality but not serum alanine aminotransferase, bilirubin, or amylase. We conclude that UFAs are enriched in human necrotic collections and acute UFA generation via lipolysis worsens pancreatic necrosis, systemic inflammation, and injury associated with severe AP. Inhibition of lipolysis reduces UFA generation and improves these outcomes of AP without interfering with its induction.

Potential influence of intravenous lipids on the outcomes of acute pancreatitis.

Parenteral nutrition (PN) has been associated with a higher rate of adverse outcomes compared with enteral feeding in patients with acute pancreatitis (AP). However, PN may be necessary when feeding via the enteral route is poorly tolerated or impossible, and PN is recommended as a second-line nutrition therapy in AP. Intravenous (IV) lipids are commonly used as a part of PN in patients with AP. While the adverse outcomes related to the use of PN in AP have commonly been attributed to infectious complications, data suggest that the unsaturated fatty acids in the triglycerides used in IV lipids may contribute to the development of organ failure. We discuss the clinical and experimental data on this issue and the alternative lipid emulsions that are being studied.

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.