Glial cell-derived soluble factors increase the metastatic potential of pancreatic adenocarcinoma cells and induce epithelial-to-mesenchymal transition.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive types of cancer, characterized by the spreading of highly metastatic cancer cells, including invasion into surrounding nerves and perineural spaces. Nerves, in turn, can invade the tumor tissue and, through the secretion of neurotrophic factors, chemokines, and cytokines, contribute to PDAC progression. However, the contribution of the nerve-associated glial cells to PDAC progression is not well characterized. METHODS: Two murine PDAC cell lines were cultured with the conditioned media (CM) of primary enteric glial cells or IMS32 Schwann cells (SCs). Different properties of PDAC cells, such as invasiveness, migratory capacity, and resistance to gemcitabine, were measured by RT-qPCR, microscopy, and MTT assays. Using a neuronal cell line, the observed effects were confirmed to be specific to the glial lineage. RESULTS: Compared to the control medium, PDAC cells in the glial cell-conditioned medium showed increased invasiveness and migratory capacity. These cells showed reduced E-cadherin and increased N-cadherin and Vimentin levels, all markers of epithelial-mesenchymal transition (EMT). Primary enteric glial cell CM inhibited the proliferation of PDAC cells but preserved their viability, upregulated transcription factor Snail, and increased their resistance to gemcitabine. The conditioned medium generated from the IMS32 SCs produced comparable results. CONCLUSION: Our data suggest that glial cells can increase the metastatic potential of PDAC cells by increasing their migratory capacity and inducing epithelial-to-mesenchymal transition, a re-programming that many solid tumors use to undergo metastasis. Glial cell-conditioned medium also increased the chemoresistance of PDAC cells. These findings may have implications for future therapeutic strategies, such as targeting glial cell-derived factor signaling in PDAC.

Sympathetic Innervation Modulates Mucosal Immune Homeostasis and Epithelial Host Defense.

Intestinal mucosal cells, such as resident macrophages and epithelial cells, express adrenergic receptors and are receptive to norepinephrine, the primary neurotransmitter of the sympathetic nervous system (SNS). It has been suggested that the SNS affects intestinal immune activity in conditions, such as inflammatory bowel disease; however, the underlying mechanisms remain ambiguous. Here, we investigated the effect of SNS on mucosal immune and epithelial cell functions. We employed 6-OHDA-induced sympathetic denervation (cSTX) to characterize muscularis-free mucosal transcriptomes by RNA-seq and qPCR, and quantified mucosal immune cells by flow cytometry. The role of norepinephrine and cytokines on epithelial functions was studied using small intestinal organoids. cSTX increased the presence of activated CD68+CD86+ macrophages and monocytes in the mucosa. In addition, through transcriptional profiling, the proinflammatory cytokines IL-1ß, TNF-α, and IFN-γ were induced, while Arg-1 and CD163 expression was reduced. Further, cSTX increased intestinal permeability in vivo and induced genes involved in barrier integrity and antimicrobial defense. In intestinal organoids, similar alterations were observed after treatment with proinflammatory cytokines, but not norepinephrine. We conclude that a loss in sympathetic input induces a proinflammatory mucosal state, leading to reduced epithelial barrier functioning and enhanced antimicrobial defense. This implies that the SNS might be required to maintain intestinal immune functions during homeostasis.

Sympathetic Denervation Alters the Inflammatory Response of Resident Muscularis Macrophages upon Surgical Trauma and Ameliorates Postoperative Ileus in Mice.

Interactions between the peripheral nervous system and resident macrophages (MMs) modulate intestinal homeostatic functions. Activation of ß2-adrenergic receptors on MMs has been shown to reduce bacterial challenges. These MMs are also crucial for the development of bowel inflammation in postoperative ileus (POI), an iatrogenic, noninfectious inflammation-based motility disorder. However, the role of the sympathetic nervous system (SNS) in the immune modulation of these MMs during POI or other noninfectious diseases is largely unknown. By employing 6-OHDA-induced denervation, we investigated the changes in the muscularis externa by RNA-seq, quantitative PCR, and flow cytometry. Further, we performed transcriptional phenotyping of sorted CX3CR1+ MMs and ex vivo LPS/M-CSF stimulation on these MMs. By combining denervation with a mouse POI model, we explored distinct changes on CX3CR1+ MMs as well as in the muscularis externa and their functional outcome during POI. Our results identify SNS as an important mediator in noninfectious postoperative inflammation. Upon denervation, MMs anti-inflammatory genes were reduced, and the muscularis externa profile is shaped toward a proinflammatory status. Further, denervation reduced MMs anti-inflammatory genes also in the early phase of POI. Finally, reduced leukocyte infiltration into the muscularis led to a quicker recovery of bowel motility in the late phase of POI.

End-ischemic reconditioning of liver allografts: Controlling the rewarming.

Different nonhypothermic preservation modalities have shown beneficial effects in liver transplantation models. This study compares controlled oxygenated rewarming (COR) to normothermic machine perfusion (NMP) to resuscitate liver grafts following cold storage (CS). Porcine livers were preserved for 18 hours by CS. Before reperfusion, the grafts were put on a machine perfusion device (Liver Assist) for 3 hours and were randomly assigned to COR (n = 6) or NMP (n = 5) and compared to standard CS. COR was carried out with the new Custodiol-N solution, slowly increasing temperature from 8 °C to 20 °C during the first 90 minutes. NMP was carried out with diluted autologous blood at 37 °C for 3 hours. In both cases, the perfusate was oxygenated to partial pressure of oxygen > 500 mm Hg. Then liver viability was tested for 180 minutes during in vitro isolated sanguineous reperfusion. Activity of the mitochondrial caspase 9 was lower after COR. Measurement of tissue adenosine triphosphate and total adenine nucleotides at the end of the reconditioning period showed better energetic recovery after COR. COR also resulted in significantly lower enzyme leakage and higher bile production (P < 0.05) during reperfusion. This first comparison of COR and NMP as end-ischemic reconditioning modalities demonstrates superior results in terms of mitochondrial integrity resulting in better energetic recovery, less hepatocellular injury, and ultimately superior function in favor of COR. Liver Transplantation 22 1223-1230 2016 AASLD.

One or 4 h of "in-house" reconditioning by machine perfusion after cold storage improve reperfusion parameters in porcine kidneys.

In-house machine perfusion after cold storage (hypothermic reconditioning) has been proposed as convenient tool to improve kidney graft function. This study investigated the role of machine perfusion duration for early reperfusion parameters in porcine kidneys. Kidney function after cold preservation (4 °C, 18 h) and subsequent reconditioning by one or 4 h of pulsatile, nonoxygenated hypothermic machine perfusion (HMP) was studied in an isolated kidney perfusion model in pigs (n = 6, respectively) and compared with simply cold-stored grafts (CS). Compared with CS alone, one or 4 h of subsequent HMP similarly and significantly improved renal flow and kidney function (clearance and sodium reabsorption) upon warm reperfusion, along with reduced perfusate concentrations of endothelin-1 and increased vascular release of nitric oxide. Molecular effects of HMP comprised a significant (vs CS) mRNA increase in the endothelial transcription factor KLF2 and lower expression of endothelin that were observed already at the end of one-hour HMP after CS. Reconditioning of cold-stored kidneys is possible, even if clinical logistics only permit one hour of therapy, while limited extension of the overall storage time by in-house machine perfusion might also allow for postponing of transplantation from night to early day work.

Energetic recovery in porcine grafts by minimally invasive liver oxygenation.

BACKGROUND: Gaseous insufflation of oxygen via the venous vascular system has proven to be an effective tool for preventing anoxic tissue injury after extended time periods of ischemic liver preservation. Most experimental studies so far have been undertaken in rat models and include a series of pinpricks into postsinusoidal venules as an outlet for the insufflated gas. Here, we describe a simplified technique for minimally invasive liver oxygenation in porcine grafts, representing a hassle-free access to organ oxygenation without vascular lesions. METHODS: We retrieved livers from Landrace pigs and cold-stored them in histidine-tryptophan-ketoglutarate solution. Subsequent to 18 h preservation, we treated some livers for an additional 2 h with gaseous oxygen, insufflated via silicone tubing inserted into the suprahepatic caval vein. Gas pressure was limited to 18 mm Hg. We occluded the infrahepatic caval vein with a bulldog clamp. Gas bubbles left the graft via the portal vein. We assessed liver integrity by energetic tissue status and by controlled in vitro reperfusion with autologous blood. RESULTS: Magnetic resonance imaging demonstrated homogeneous gas distribution in the persufflated tissue without major shunting. Biochemical analyses revealed effective and homogeneous restoration of energetic homeostasis in the ischemic graft before reperfusion. Sinusoidal endothelial clearance of hyaluronic acid was significantly improved upon reperfusion, as was hepatic arterial flow. Parenchymal enzyme loss was concordantly mitigated after minimally invasive liver oxygenation. CONCLUSIONS: Our results indicate that gaseous oxygen persufflation of the porcine liver is possible without tissue trauma, and significantly enhances post-preservation recovery of the graft.

Optimal time for hypothermic reconditioning of liver grafts by venous systemic oxygen persufflation in a large animal model.

BACKGROUND: Quality of cold-stored livers declines beyond 12 hr of ischemia, increasing the risk of primary dysfunction. Here we evaluate the potential and optimal treatment interval of gaseous oxygen persufflation for grafts reconditioning after long storage times in an experimental pig liver model. METHOD: Porcine livers (n=6/group) were cold stored at 4°C for 18 hr in histidine-tryptophan-ketoglutarate solution. Hypothermic reconditioning (HR) was performed in some livers, by insufflation of gaseous oxygen through the caval vein for 1, 2, or 3 hr subsequent to cold storage. Liver integrity was assessed by controlled in vitro reperfusion with autologous blood. RESULTS: HR resulted in a 40% to 50% reduction of serum levels of aspartate aminotransferase, lactate dehydrogenase, and tumor necrosis factor-α with a maximal effect after 2 hr of HR (P<0.05). Functional parameters (bile production, cholinesterase and energetic recovery) were likewise enhanced (P<0.05). Two hours of HR also improved hepatic arterial flow and abrogated the postischemic increase in portal venous perfusion resistance compared with untreated (P<0.05). Gene expression of Toll-like receptor-4 was reduced by 2 hr of HR as was platelet adherence in the reperfused graft (P<0.05), in line with a trend toward lower expression of von Willebrand factor. CONCLUSION: HR effectively ameliorated graft dysfunction after extended preservation of porcine livers. Two hours of "a posteriori" treatment provide the maximal effect and are recommended for further application.