Decrease in the inflammatory cytokines of LPS-stimulated PBMCs of patients with atherosclerosis by a TLR-4 antagonist in the co-culture with HUVECs.

Toll-like receptors (TLRs) are among the players of inflammation during atherosclerosis. We assessed the effects of Eritoran, a TLR-4 antagonist, on lipopolysaccharide (LPS)-induced cytokines production by Peripheral Blood Mononuclear Cells (PBMCs) of patients with high-stenosis (HS) (n = 6) and healthy controls (HCs) (n = 6) co-cultured with Human Umbilical Vein Endothelial Cells (HUVECs). LPS stimulation significantly increased the levels of IL-6 (P = 0.007 and P = 0.005), TNF-α (P = 0.006 and P = 0.005), IL-2 (P = 0.007 and P = 0.002), IFN-γ (P = 0.006 and P = 0.003), IL-17A (P = 0.004 and P = 0.003), IL-17F (P = 0.005 and P = 0.003), IL-5 (P = 0.007 and P = 0.005), IL-13 (P = 0.006 and P = 0.005), IL-9 (P = 0.005 and P = 0.005) and IL-21 (P = 0.007 and P = 0.005) in HUVECs co-cultured with HC and HS PBMCs as compared with un-stimulated co-culture condition, respectively. Eritoran treatment (50 µg/mL and 100 µg/mL) significantly reduced the levels of LPS-induced IL-6 (P = 0.007 and P = 0.006; P = 0.007 and P = 0.007), TNF-α (P = 0.005 and P = 0.003; P = 0.007 and P = 0.005), IL-2 (P = 0.007 and P = 0.005; P = 0.005 and P = 0.004), IFN-γ (P = 0.007 and P = 0.005; P = 0.005 and P = 0.004), IL-17A (P = 0.005 and P = 0.002; P = 0.005 and P = 0.002), IL-17F (P = 0.006 and P = 0.006; P = 0.005 and P = 0.005), IL-5 (P = 0.007 and P = 0.006; P = 0.007 and P = 0.007), IL-9 (P = 0.005 and P = 0.005; P = 0.005 and P = 0.005) and IL-21 (P = 0.007 and P = 0.007; P = 0.005 and P = 0.005) in stimulated HUVECs co-cultured with HC and HS PBMCs, compared to un-treated condition, respectively. Our results demonstrate that attenuating effect of Eritoran on the inflammatory responses to LPS is higher in PBMCs of patients with high stenosis, suggesting its potential role in ameliorating inflammatory conditions in atherosclerosis.

S100A8 may govern hyper-inflammation in severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic threatens human species with mortality rate of roughly 2%. We can hardly predict the time of herd immunity against and end of COVID-19 with or without success of vaccine. One way to overcome the situation is to define what delineates disease severity and serves as a molecular target. The most successful analogy is found in BCR-ABL in chronic myeloid leukemia, which is the golden biomarker, and simultaneously, the most effective molecular target. We hypothesize that S100 calcium-binding protein A8 (S100A8) is one such molecule. The underlying evidence includes accumulating clinical information that S100A8 is upregulated in severe forms of COVID-19, pathological similarities of the affected lungs between COVID-19 and S100A8-induced acute respiratory distress syndrome (ARDS) model, homeostatic inflammation theory in which S100A8 is an endogenous ligand for endotoxin sensor Toll-like receptor 4/Myeloid differentiation protein-2 (TLR4/MD-2) and mediates hyper-inflammation even after elimination of endotoxin-producing extrinsic pathogens, analogous findings between COVID-19-associated ARDS and pre-metastatic lungs such as S100A8 upregulation, pulmonary recruitment of myeloid cells, increased vascular permeability, and activation coagulation cascade. A successful treatment in an animal COVID-19 model is given with a reagent capable of abrogating interaction between S100A8/S100A9 and TLR4. In this paper, we try to verify our hypothesis that S100A8 governs COVID-19-associated ARDS.

Eritoran Attenuates Hepatic Inflammation and Fibrosis in Mice with Chronic Liver Injury.

Toll-like receptor 4 (TLR4) signaling plays a key role in liver inflammation and fibrosis. The therapeutic effects of eritoran, a TLR4 antagonist, in mice with chronic liver injury remained unclear. C57BL/6 mice were fed a fast-food diet (FFD) or treated with carbon tetrachloride (CCl4) to induce chronic liver injury. Eritoran (10 mg/kg) or a vehicle was randomly intraperitoneally administered to the FFD-fed mice and the CCl4-injured mice. Primary mouse liver cells were cultured with lipopolysaccharide (LPS) or eritoran. In both FFD and CCl4 mouse models, eritoran significantly reduced serum ALT levels and decreased hepatic inflammatory cell infiltration without altering hepatic steatosis. Additionally, eritoran attenuated liver fibrosis by decreasing hepatic stellate cells (HSCs) activation and the abundance of α-smooth muscle actin and transforming growth factor-ß1. Hepatic TLR4 downstream signaling including MyD88 expression, NF-κB p65 nuclear translocation, p38 and JNK phosphorylation were successfully inhibited by eritoran. In the in vitro study, LPS-induced nuclear translocation of NF-κB in primary HSCs and Kupffer cells was significantly suppressed by eritoran. In conclusion, eritoran attenuated hepatic inflammation and fibrosis by inhibition of the TLR4 signaling pathway in mice with chronic liver injury. Eritoran may serve as a potential drug for chronic liver disease.

Characterization of P. aeruginosa Glucose 6- Phosphate Isomerase: A Functional Insight via In-Vitro Activity Study.

BACKGROUND: Glucose-6-phosphate isomerase (G6PI) catalyses the second step in glycolysis in the reversible interconversion of an aldohexose glucose 6-phosphate, a six membered ring moiety to a ketohexose, fructose 6-phosphate five membered ring moiety. This enzyme is of utmost importance due to its multifunctional role like neuroleukin, autocrine motility factor, etc. in various species. G6PI from Pseudomonas aeruginosa is less explored for its moonlighting properties. These properties can be predicted by studying the active site conservation of residues and their interaction with the specific ligand. METHODS: Here, we study the G6PI in a self-inducible construct in bacterial expression system with its purification using Ni-NTA chromatography. The secondary structure of pure G6PI is estimated using circular dichroism to further predict the proper folding form of the protein. The bioactivity of the purified enzyme is quantified using phosphoglucose isomerase colorimetric kit with a value of 12.5 mU/mL. Differential scanning fluorimetry and isothermal titration calorimetry were employed to monitor the interaction of G6PI with its competitive inhibitor, erythrose 4-phosphate and calculated the Tm, Kd and IC50 values. Further, the homology model for the protein was prepared to study the interaction with the erythrose 4-phosphate. MD simulation of the complex was performed at 100 ns to identify the binding interactions. RESULTS: We identified hydrogen bonds and water bridges dominating the interactions in the active site holding the protein and ligand with strong affinity. CONCLUSION: G6PI was successfully crystallized and data has been collected at 6Å. We are focused on improving the crystal quality for obtaining higher resolution data.

Glucose 6-Phosphate Accumulates via Phosphoglucose Isomerase Inhibition in Heart Muscle.

RATIONALE: Metabolic and structural remodeling is a hallmark of heart failure. This remodeling involves activation of the mTOR (mammalian target of rapamycin) signaling pathway, but little is known on how intermediary metabolites are integrated as metabolic signals. OBJECTIVE: We investigated the metabolic control of cardiac glycolysis and explored the potential of glucose 6-phosphate (G6P) to regulate glycolytic flux and mTOR activation. METHODS AND RESULTS: We developed a kinetic model of cardiomyocyte carbohydrate metabolism, CardioGlyco, to study the metabolic control of myocardial glycolysis and G6P levels. Metabolic control analysis revealed that G6P concentration is dependent on phosphoglucose isomerase (PGI) activity. Next, we integrated ex vivo tracer studies with mathematical simulations to test how changes in glucose supply and glycolytic flux affect mTOR activation. Nutrient deprivation promoted a tight coupling between glucose uptake and oxidation, G6P reduction, and increased protein-protein interaction between hexokinase II and mTOR. We validated the in silico modeling in cultured adult mouse ventricular cardiomyocytes by modulating PGI activity using erythrose 4-phosphate. Inhibition of glycolytic flux at the level of PGI caused G6P accumulation, which correlated with increased mTOR activation. Using click chemistry, we labeled newly synthesized proteins and confirmed that inhibition of PGI increases protein synthesis. CONCLUSIONS: The reduction of PGI activity directly affects myocyte growth by regulating mTOR activation.

Targeting GNE Myopathy: A Dual Prodrug Approach for the Delivery of N-Acetylmannosamine 6-Phosphate.

ProTides comprise an important class of prodrugs currently marketed and developed as antiviral and anticancer therapies. The ProTide technology employs phosphate masking groups capable of providing more favorable druglike properties and an intracellular activation mechanism for enzyme-mediated release of a nucleoside monophosphate. Herein, we describe the application of phosphoramidate chemistry to 1,3,4-O-acetylated N-acetylmannosamine (Ac3ManNAc) to deliver ManNAc-6-phosphate (ManNAc-6-P), a critical intermediate in sialic acid biosynthesis. Sialic acid deficiency is a hallmark of GNE myopathy, a rare congenital disorder of glycosylation (CDG) caused by mutations in GNE that limit the production of ManNAc-6-P. Synthetic methods were developed to provide a library of Ac3ManNAc-6-phosphoramidates that were evaluated in a series of studies for their potential as a treatment for GNE myopathy. Prodrug 12b showed rapid activation in a carboxylesterase (CPY) enzymatic assay and favorable ADME properties, while also being more effective than ManNAc at increasing sialic acid levels in GNE-deficient cell lines. These results provide a potential platform to address substrate deficiencies in GNE myopathy and other CDGs.

Human Toll-Like Receptor 4 (hTLR4): Structural and functional dynamics in cancer.

Toll-Like Receptors (TLRs) recognize a wide variety of pathogen-associated molecular patterns and are centrally involved in the initiation of the innate and adaptive immune responses. Extensive analysis of TLRs has shown specificity in terms of ligand recognition, expression and cellular localization in different cell types and tissues, and most importantly, its role in the pathogenesis of multiple chronic inflammatory diseases. In recent past extensive investigations showed that many TLRs are profoundly expressed in various types of cancers. This review is emphasized on human TLR4 structural and functional dynamics in cancer. The review was intended to explore the present understanding of the involvement of hTLR4 in different types of cancer and different danger signals that can affect the expression and function of TLR4 in both normal and cancer cells. Dual role of TLR4 in cancer has also been discussed along with therapeutic targeting, cellular response via signaling and the possible conformational changes that occur in response to agonist and antagonist. This review provides a comprehensive resource for designing and discovery of novel TLR4 ligands for therapeutic intervention.

Disaccharide-Based Anionic Amphiphiles as Potent Inhibitors of Lipopolysaccharide-Induced Inflammation.

Despite significant advances made in the last decade in the understanding of molecular mechanisms of sepsis and in the development of clinically relevant therapies, sepsis remains the leading cause of mortality in intensive care units with increasing incidence worldwide. Toll-like receptor 4 (TLR4)-a transmembrane pattern-recognition receptor responsible for propagating the immediate immune response to Gram-negative bacterial infection-plays a central role in the pathogenesis of sepsis and chronic inflammation-related disorders. TLR4 is complexed with the lipopolysaccharide (LPS)-sensing protein myeloid differentiation-2 (MD-2) which represents a preferred target for establishing new anti-inflammatory treatment strategies. Herein we report the development, facile synthesis, and biological evaluation of novel disaccharide-based TLR4⋅MD-2 antagonists with potent anti-endotoxic activity at micromolar concentrations. A series of synthetic anionic glycolipids entailing amide-linked ß-ketoacyl lipid residues was prepared in a straightforward manner by using a single orthogonally protected nonreducing diglucosamine scaffold. Suppression of the LPS-induced release of interleukin-6 and tumor necrosis factor was monitored and confirmed in human immune cells (MNC and THP1) and mouse macrophages. Structure-activity relationship studies and molecular dynamics simulations revealed the structural basis for the high-affinity interaction between anionic glycolipids and MD-2, and highlighted two compounds as leads for the development of potential anti-inflammatory therapeutics.

Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1.

WRINKLED1 (WRI1), the transcriptional activator of fatty acid synthesis, was recently identified as a target of KIN10, a catalytic α-subunit of the SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1). We tested the hypothesis that trehalose 6-phosphate (T6P), a signal of cellular sucrose status, can regulate fatty acid synthesis by inhibiting SnRK1. Incubation of Brassica napus suspension cells in medium containing T6P, or overexpression of the Escherichia coli T6P synthase, OtsA, in Nicotiana benthamiana, significantly increased T6P levels, WRI1 levels, and fatty acid synthesis rates. T6P directly bound to purified recombinant KIN10 with an equilibrium dissociation constant (K d) of 32 ± 6 µM based on microscale thermophoresis. GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1) bound to KIN10 (K d 19 ± 3 µM) and activated it by phosphorylation. In the presence of T6P, the GRIK1-KIN10 association was weakened by more than 3-fold (K d 68 ± 9.8 µM), which reduced both the phosphorylation of KIN10 and its activity. T6P-dependent inhibition of SnRK1 activity was reduced in extracts of individual Arabidopsis thaliana grik1 and grik2 mutants relative to the wild type, while SnRK1 activity in grik1 grik2 extracts was enhanced by T6P. These results indicate that the T6P sensitivity of SnRK1 in vivo is GRIK1/GRIK2 dependent. Based on our findings, we propose a mechanistic model that links sugar signaling and fatty acid homeostasis.

Lifestyle of the biotroph Agrobacterium tumefaciens in the ecological niche constructed on its host plant.

Agrobacterium tumefaciens constructs an ecological niche in its host plant by transferring the T-DNA from its Ti plasmid into the host genome and by diverting the host metabolism. We combined transcriptomics and genetics for understanding the A. tumefaciens lifestyle when it colonizes Arabidopsis thaliana tumors. Transcriptomics highlighted: a transition from a motile to sessile behavior that mobilizes some master regulators (Hfq, CtrA, DivK and PleD); a remodeling of some cell surface components (O-antigen, succinoglucan, curdlan, att genes, putative fasciclin) and functions associated with plant defense (Ef-Tu and flagellin pathogen-associated molecular pattern-response and glycerol-3-phosphate and nitric oxide signaling); and an exploitation of a wide variety of host resources, including opines, amino acids, sugars, organic acids, phosphate, phosphorylated compounds, and iron. In addition, construction of transgenic A. thaliana lines expressing a lactonase enzyme showed that Ti plasmid transfer could escape host-mediated quorum-quenching. Finally, construction of knock-out mutants in A. tumefaciens showed that expression of some At plasmid genes seemed more costly than the selective advantage they would have conferred in tumor colonization. We provide the first overview of A. tumefaciens lifestyle in a plant tumor and reveal novel signaling and trophic interplays for investigating host-pathogen interactions.

Eritoran Suppresses Colon Cancer by Altering a Functional Balance in Toll-like Receptors That Bind Lipopolysaccharide.

Colorectal carcinogenesis is affected by overexpression of the lipopolysaccharide (LPS) receptors CD14 and TLR4, which antagonize each other by affecting epithelial cell proliferation and apoptosis. Eritoran is an investigational drug for sepsis treatment that resembles the lipid A moiety of LPS and therefore acts as a TLR4 inhibitor. In the present study, we explored the potential therapeutic uses and mechanisms of action of eritoran in reducing colon cancer progression. Eritoran administration via intracolonic, intragastric, or intravenous routes significantly reduced tumor burden in a chemically induced mouse model of colorectal carcinoma. Decreased proliferation and increased apoptosis were observed in mouse tumor cells after eritoran treatment. In vitro cultures of mouse primary tumor spheroids and human cancer cell lines displayed increased cell proliferation and cell-cycle progression following LPS challenge. This effect was inhibited by eritoran and by silencing CD14 or TLR4. In contrast, apoptosis induced by eritoran was eliminated by silencing CD14 or protein kinase Cζ (PKCζ) but not TLR4. Lastly, LPS and eritoran caused hyperphosphorylation of PKCζ in a CD14-dependent and TLR4-independent manner. Blocking PKCζ activation by a Src kinase inhibitor and a PKCζ-pseudosubstrate prevented eritoran-induced apoptosis. In summary, our work offers a preclinical proof of concept for the exploration of eritoran as a clinical treatment, with a mechanistic rationale to reposition this drug to improve the management of colorectal cancer. Cancer Res; 76(16); 4684-95. ©2016 AACR.

Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2-89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2-89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8(+) T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages.

Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen's ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses.

Toll-like receptor 4 signaling in neurons of trigeminal ganglion contributes to nociception induced by acute pulpitis in rats.

Pain caused by acute pulpitis (AP) is a common symptom in clinical settings. However, its underlying mechanisms have largely remained unknown. Using AP model, we demonstrated that dental injury caused severe pulp inflammation with up-regulated serum IL-1ß. Assessment from head-withdrawal reflex thresholds (HWTs) and open-field test demonstrated nociceptive response at 1 day post injury. A consistent up-regulation of Toll-like receptor 4 (TLR4) in the trigeminal ganglion (TG) ipsilateral to the injured pulp was found; and downstream signaling components of TLR4, including MyD88, TRIF and NF-κB, and cytokines such as TNF-α and IL-1ß, were also increased. Retrograde labeling indicated that most TLR4 positve neuron in the TG innnervated the pulp and TLR4 immunoreactivity was mainly in the medium and small neurons. Double labeling showed that the TLR4 expressing neurons in the ipsilateral TG were TRPV1 and CGRP positive, but IB4 negative. Furthermore, blocking TLR4 by eritoran (TLR4 antagonist) in TGs of the AP model significantly down-regulated MyD88, TRIF, NF-κB, TNF-α and IL-1ß production and behavior of nociceptive response. Our findings suggest that TLR4 signaling in TG cells, particularly the peptidergic TRPV1 neurons, plays a key role in AP-induced nociception, and indicate that TLR4 signaling could be a potential therapeutic target for orofacial pain.

CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance.

Dimerization of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD2) heterodimers is critical for both MyD88- and TIR-domain-containing adapter-inducing IFN-ß (TRIF)-mediated signaling pathways. Recently, Zanoni et al. [(2011) Cell 147(4):868-880] reported that cluster of differentiation 14 (CD14) is required for LPS-/Escherichia coli- induced TLR4 internalization into endosomes and activation of TRIF-mediated signaling in macrophages. We confirmed their findings with LPS but report here that CD14 is not required for receptor endocytosis and downstream signaling mediated by TLR4/MD2 agonistic antibody (UT12) and synthetic small-molecule TLR4 ligands (1Z105) in murine macrophages. CD14 deficiency completely ablated the LPS-induced TBK1/IRF3 signaling axis that mediates production of IFN-ß in murine macrophages without affecting MyD88-mediated signaling, including NF-κB, MAPK activation, and TNF-α and IL-6 production. However, neither the MyD88- nor TRIF-signaling pathways and their associated cytokine profiles were altered in the absence of CD14 in UT12- or 1Z105-treated murine macrophages. Eritoran (E5564), a lipid A antagonist that binds the MD2 "pocket," completely blocked LPS- and 1Z105-driven, but not UT12-induced, TLR4 dimerization and endocytosis. Furthermore, TLR4 endocytosis is induced in macrophages tolerized by exposure to either LPS or UT12 and is independent of CD14. These data indicate that TLR4 receptor endocytosis and the TRIF-signaling pathway are dissociable and that TLR4 internalization in macrophages can be induced by UT12, 1Z105, and during endotoxin tolerance in the absence of CD14.

Toll-like receptor 4 (TLR4) antagonist eritoran tetrasodium attenuates liver ischemia and reperfusion injury through inhibition of high-mobility group box protein B1 (HMGB1) signaling.

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1-TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1's release from hypoxic hepatocytes in vitro and thereby weakened HMGB1's activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.

GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids.

Epithelial-mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2-15 times) of EMT (ß-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (>80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.

Expression of toll-like receptor 4 contributes to corneal inflammation in experimental dry eye disease.

Purpose. To investigate the corneal expression of toll-like receptor (TLR) 4 and determine its contribution to the immunopathogenesis of dry eye disease (DED). Methods. Seven to 8-week-old female C57BL/6 mice were housed in a controlled environment chamber and administered scopolamine to induce experimental DED. Mice received intravenous TLR4 inhibitor (Eritoran) to block systemic TLR4-mediated activity. The expression of TLR4 by the corneal epithelium and stroma was evaluated using real-time polymerase chain reaction and flow cytometry. Corneal fluorescein staining (CFS) was performed to evaluate clinical disease severity. The corneal expression of proinflammatory cytokines (IL-1ß, IL-6, TNF, and CCL2), corneal infiltration of CD11b(+) antigen-presenting cells, and lymph node frequency of mature MHC-II(hi) CD11b(+) cells were assessed. Results. The epithelial cells of normal corneas expressed TLR4 intracellularly; however, DED significantly increased the cell surface expression of TLR4. Similarly, flow cytometric analysis of stromal cells revealed a significant increase in the expression of TLR4 proteins by DED-induced corneas as compared with normal corneas. DED increased the mRNA expression of TLR4 in corneal stromal cells, but not epithelial cells. TLR4 inhibition decreased the severity of CFS and significantly reduced the mRNA expression of IL-1ß, IL-6, and TNF. Furthermore, TLR4 inhibition significantly reduced the corneal infiltration of CD11b(+) cells and the lymph node frequency of MHC-II(hi) CD11b(+) cells. Conclusions. These results suggest that DED increases the corneal expression of TLR4 and that TLR4 participates in the inflammatory response to ocular surface desiccating stress.

Deactivation of the lipopolysaccharide antagonist eritoran (E5564) by high-density lipoprotein-associated apolipoproteins.

Lipid A, the active moiety of LPS, exerts its effects through interaction with TLR4, triggering a signalling cascade that results in the release of pro-inflammatory cytokines. Eritoran is a lipid A analogue that competes with LPS for binding to TLR4; however, after intravenous administration, it undergoes a time-dependent deactivation as a consequence of binding to high-density lipoproteins (HDLs). The site of eritoran association with HDL remains unknown. Therefore the aim of this study was to determine if HDL-associated apolipoproteins A1, A2, serum amyloid A (SAA) and C1, inhibit the ability of eritoran to block LPS-induced TNF-α release from whole blood. Eritoran activity after LPS stimulation in human whole blood was assessed in the presence of reconstituted HDL (rHDL) containing different apos. In rHDL, the major apolipoproteins in both the healthy and septic state, A1 and SAA, caused a significant reduction in eritoran antagonistic activity and had a greater effect than minor apolipoproteins A2 and C1. Apolipoproteins associated with HDL are likely to facilitate eritoran deactivation. Apolipoproteins A1 and SAA should be of particular focus as they are the major apos found on HDL in both the healthy and septic state. Further evaluation of the physical association between apolipoproteins and eritoran should be explored.

Toll-like receptor 4/nuclear factor-kappa B pathway is involved in myocardial injury in a rat chronic stress model.

Chronic stress is considered to predispose to various cardiovascular events such as coronary artery disease, hypertension, and even heart failure. In this study, rats were exposed to stress for 1 day, 1, 2, 3, and 4 weeks to establish a chronic stress model. A specific toll-like receptor 4 (TLR4) antagonist eritoran was used to block the activity of TLR4. On the second day after the last stress exposure, the animals were killed. The expression of TLR4 mRNA and nuclear factor-kappa B (NF-κB) DNA-binding activity in the myocardium were measured using reverse transcriptase polymerase chain reaction and electrophoretic mobility shift assay. The proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL-6) in myocardium were assayed by enzyme-linked immunosorbent assay. Myocardial injury was evident after chronic stress for 2 weeks. The TLR4 mRNA expression reached a peak after stress for 1 week. It was sustained at a stable level after stress exposure for 3 weeks and was restored to a nearly normal level in the fourth week. NF-κB DNA-binding activity was significantly enhanced after the stress for 1 day and markedly enhanced again after a 2-week stress exposure. It was weakened and reached a normal level after stress exposure for 4 weeks. The levels of TNF-α and IL-6 gradually increased and reached peaks after stress for 4 weeks. Meanwhile, eritoran significantly decreased the TLR4 mRNA expression and NF-κB activity in rats from the 2-week stress group. However, it did not downregulate the levels of TNF-α and IL-6. Importantly, it significantly improved the myocardial injury induced by the chronic stress. In conclusion, TLR4/NF-κB participates in myocardial injury during chronic stress.