IL-8 production was determined by ELISA assay.Cell death was assessed by measuring DNA fragmentation.Total RNA was isolated and IL-8, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression was determined by real time RT-PCR.NF-kB activation was determined by luciferase assay.Results: Exposure of LX2 cells to palmitate significantly induces IL-8 production and gene expression, as well as TLR4, CXCL1, CXCL2, CCL2, ICAM, VCAM mRNA expression.Palmitate induced IL-8 production can not be blocked by pan-caspase inhibitor.However, palmitate induced MIP-2 (rodent homolog of human IL-8) production was completely abrogated in HSCs from TLR4-mutant (C3H/HeJ) mice, but not from wildtype (C3H/HeOuJ) mice.Palmitate also induces NF-kB activation in a dose-dependent manner.Conclusions: Our data demonstrate that palmitate directly stimulates IL-8 production in HSCs via TLR4 signaling pathway, suggesting that HSCs play an important role in FFAs induced liver inflammation and injury.

Nucleotide-binding oligomerization domain-containing-2 (NOD2) acts as a bacterial sensor in dendritic cells (DCs), but it is not clear how bacterial recognition links with antigen presentation after NOD2 stimulation. NOD2 variants are associated with Crohn's disease, where breakdown in self-recognition of commensal bacteria leads to gastrointestinal inflammation. Here we show NOD2 triggering by muramyldipeptide induces autophagy in DCs. This effect requires receptor-interacting serine-threonine kinase-2 (RIPK-2), autophagy-related protein-5 (ATG5), ATG7 and ATG16L1 but not NLR family, pyrin domain containing-3 (NALP3).We show that NOD2-mediated autophagy is required for both bacterial handling and generation of major histocompatibility complex (MHC) class II antigen-specific CD4(+) T cell responses in DCs. DCs from individuals with Crohn's disease expressing Crohn's disease-associated NOD2 or ATG16L1 risk variants are defective in autophagy induction, bacterial trafficking and antigen presentation. Our findings link two Crohn's disease-associated susceptibility genes in a single functional pathway and reveal defects in this pathway in Crohn's disease DCs that could lead to bacterial persistence via impaired lysosomal destruction and immune mediated clearance.

Background Nucleotide-binding oligomerization domain containing 2 (NOD2) is an intracellular pattern-recognition receptor activated by muramyldipeptide, a component of peptidoglycan contained in bacterial cell walls. Mutations in the NOD2 gene that occur in the muramyldipeptde-recognition domain (such as 1007fsinsC NOD2) predispose to ileal Crohn's disease (CD). NOD2 is expressed in intestinal epithelial cells including Paneth cells, and also in antigen-presenting cells such as dendritic cells (DCs). DCs process information from innate immune receptors such as NOD2 and translate this into specific signals that govern generation of correctly targeted or aberrant immune responses. Stimulation of NOD2 may induce cytokines, defensins, TH responses and autophagy, these being attenuated in the presence of 1007fsinsC NOD2. It remains unclear how this loss of function leads to the pro-inflammatory changes of CD.We have investigated themolecular basis of NOD2 signaling in DCs focusing on 1) documenting large scale gene expression changes following NOD2 triggering in combination with toll-like receptors (TLRs) and 2) using proteomic analysis to delineate the protein complex in which NOD2 acts. A comparison with effects occurring on expression of 1007fsinsC NOD2 reveals further aspects of NOD2 function in DCs that may have implications for inflammation in CD. Methods Large scale gene expression profiling was undertaken using mRNA and microRNA micro arrays pre and post stimulation through NOD2 +/TLRs. Data was analyzed in Genespring and validated using luminex, FACS, Western blot or qPCR. Biological experiments are being undertaken to investigate the effect of differential regulation of individual genes on NOD2 function. For proteomic studies, green-fluorescence protein (GFP)-tagged WT and 1007fsinsC NOD2 expression vectors were generated for expression in HEK293 cells and DCs. Proteins associating with GFP NOD2 are being identified using LC/MS-MS and Q-TOF analysis. Results NOD2 stimulation results in a transcriptional program in DCs that acts synergistically with that of TLRs to induce maturation of DCs and upregulation of inflammatory mediators. Comparison with signaling through 1007fsinsC NOD2 largely demonstrates loss of this synergy, although there is also loss of expression of some TLR negative regulators, which may accentuate signaling through these particular TLRs in specific contexts. Conclusion NOD2 induces signaling in DCs in a largely synergistic manner with TLRs that is lost following expression of 1007fsinsC NOD2. Definition of the protein complex in which NOD2 acts will help define the mechanism of NOD2 signaling in immune cells.

Understandable controversy surrounds the use of antiscale magnetic treatment (AMT) though considerable published evidence suggests it can often be effective. Common factors in reported successful applications include continuous recirculation of a hot (and often supersaturated) process feed water usually including a high ionic and suspended load. Much less success has been claimed at preventing scaling in once through, cooler systems or in membrane desalination. AMT for prevention of RO scaling could represent an ideal solution, specifically in those cases where the membrane is liable to foul predominantly with crystalline material. If it could be proven to be both effective and reliable, the need for chemical antiscalants could be reduced and considerable cost benefits realised. The effects of magnetic treatment on low temperature crystallisation of CaCO3 in a flow through system has been investigated. Magnetically induced changes were most apparent in sedimented scale provided that a significant magnetic field contact velocity (Vmf) had been obtained. This deposit, forming mostly as calcite under non magnetised conditions was modified to spherical grains displaying a characteristic form. Scale precipitating onto PVC pipe was also visibly altered, but only when a high strength magnetic field was applied to strongly supersaturated solution flowing with a sufficient velocity. Preliminary experiments were conducted using a magnetically treated feed in once through and recirculating RO systems operated under accelerated scaling conditions. The efficacy of the treatment was ascertained by analyses of flux and salt rejection decline during formation of the scale deposit with reference to a control system operating under the same physicochemical conditions. Destructive autopsy was also carried out on the membrane elements to deduce the nature and extent of the deposit. No change was evident in a once through system with a single magnetic exposure of the water. However under recirculation (and repeated exposure) the grain size of the deposit (precipitating exclusively as aragonite) was reproducibly increased. Improvements in salt rejection decline were occasionally evident due to this more porous deposit but no repeatable substantial improvement in flux decline was found. It was found that magnetic treatment was promoting enhanced precipitation (up to 10× more; depending upon experimental conditions) in the prefilter units. SEM analysis of the prefilter deposit indicated this had grown in situ rather than having been trapped by virtue of having increased particle size. Further work is needed to deduce if these effects can be applicable to RO engineering situations.

Process industry remains sceptical of antiscale magnetic treatment (AMT) despite its long history. Manufacturer's claims concerning AMT comprise: (a) a reduction in the amount of scale formed, (b) production of a less tenacious scale due to a change in its crystal morphology, (c) removal of existing scale, and (d) a retention of the antiscaling properties of the treated water for hours following treatment. Scientific research has both substantiated and refuted these claims, creating widespread controversy as to the credibility of this type of water conditioning. Positive results indicate effects on: (a) colloidal systems where aggregation is generally enhanced and (b) crystallisation where larger hydrophilic crystals, usually with modified crystal growth, are generated. Investigations have incorporated scaling kinetics, scale morphology, scale solubility, particle coagulation and corrosion. Effects have been reported for different scale-forming compounds and for various microscopic and macroscopic parameters in single-phase systems. AMT appears to be enhanced by prolonged or repeated magnetic exposure, and is more effective above a threshold magnetic field contact time and in flowing systems. Effects have been reported in treated waters up to 130 h after exposure has ceased. Industrial case studies indicate that the most successful implementations are in hot recirculating systems. Mechanisms presented to account for the observed effects comprise (a) intramolecular/intraionic interaction, (b) Lorentz force effects, (c) dissolution of contaminants, and (d) interfacial effects. The most plausible of these is the latter, in which the interaction of the magnetic field with the charged species present (ion clusters and crystallites) affects crystal nucleation and subsequent growth. The reported scale inhibition (and descaling) then occurs as a result of magnetically-produced hydrophilic discrete scale particles of substantially different size and crystal morphology to untreated systems, in which more adherent crystals are generated.