Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and In Vivo Activity.

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.

Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages.

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-ß and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-ß pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.

A novel small molecule TLR4 antagonist (IAXO-102) negatively regulates non-hematopoietic toll like receptor 4 signalling and inhibits aortic aneurysms development.

OBJECTIVES: The toll-like receptors (TLRs), including TLR4, have been shown to play a crucial role in vascular inflammatory diseases, such as atherosclerosis and aneurysm. The main goal of this study was to determine the potential of IAXO-102 (Innaxon, Tewkesbury), a novel small molecule TLR4 antagonist, to modulate non-hematopoietic TLR4 proinflammatory signalling and inhibit experimental abdominal aortic aneurysm (AAA) development. METHODS: Human umbilical vein endothelial cells (HUVEC) and Angiotensin II-induced experimental AAA development were our in vitro and in vivo models respectively. Western blotting, antibody array and ELISA approaches were used to explore the effect of IAXO-102 on TLR4 functional activity on two levels: modulation of TLR4-induced mitogen activated protein kinases (MAPK) and p65 NF-kB phosphorylation and expression of TLR4 dependent proinflammatory proteins. RESULTS: Following activation of TLR4, in vitro/in vivo data revealed that IAXO-102 inhibited MAPK and p65 NF-kB phosphorylation associated with down regulation of the expression of TLR4 and TLR4 dependent proinflammatory proteins. Furthermore, IAXO-102 decreased Angiotensin II-induced aortic expansion, rupture and incidence of AAA. CONCLUSIONS: These results demonstrate the ability of IAXO-102 to negatively regulate TLR4 signalling and to inhibit experimental AAA development, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of AAA.

Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm.

Peroxisome proliferator-activated receptor x03B3; agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor x03B3; agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.

Increased Expression of Lamin A/C Correlate with Regions of High Wall Stress in Abdominal Aortic Aneurysms.

BACKGROUND: Since aortic diameter is the most -significant risk factor for rupture, we sought to identify stress-dependent changes in gene expression to illuminate novel molecular processes in aneurysm rupture. MATERIALS AND METHODS: We constructed finite element maps of abdominal computerized tomography scans (CTs) of seven abdominal aortic aneurysm (AAA) patients to map wall stress. Paired biopsies from high- and low-stress areas were collected at surgery using vascular landmarks as coordinates. Differential gene expression was evaluated by Illumina Array analysis, using the whole genome DNA-mediated, annealing, selection, extension, and ligation (DASL) gene chip (n = 3 paired samples). RESULTS: The sole significant candidate from this analysis, Lamin A/C, was validated at the protein level, using western blotting. Lamin A/C expression in the inferior mesenteric vein (IMV) of AAA patients was compared to a control group and in aortic smooth muscle cells in culture in response to physiological pulsatile stretch. -Areas of high wall stress (n = 7) correlate to those -regions which have the thinnest walls [778 µm (585-1120 µm)] in comparison to areas of lowest wall stress [1620 µm (962-2919 µm)]. Induced expression of Lamin A/C -correlated with areas of high wall stress from AAAs but was not significantly induced in the IMV from AAA patients compared to controls (n = 16). Stress-induced expression of Lamin A/C was mimicked by exposing aortic smooth muscle cells to prolonged pulsatile stretch. CONCLUSION: Lamin A/C protein is specifically increased in areas of high wall stress in AAA from patients, but is not increased on other vascular beds of aneurysm patients, suggesting that its elevation may be a compensatory response to the pathobiology leading to aneurysms.

Differential role of apoptosis and autophagy associated with anticancer effect of lupulone (hop ß-acid) derivatives on prostate cancer cells.

Lupulone, a ß-acid derived from hop extracts has been shown to exhibit cytotoxic activity against cancer cells. In this study we investigated the functional role of different modes of cell death that mediate anticancer effect of lupulone derivatives in prostate cancer cells. ELISA, immunoblotting and siRNA approaches were utilised to study cell death, expression of proteins of interest and their functional activities. We found that the anticancer effect of lupulone derivatives on prostate cancer cells is associated with induction of apoptosis and autophagy as determined by increases of DNA fragmentation and LC3I/ LC3II conversion respectively. Inhibition of apoptosis using a pan-caspase inhibitor resulted in increased levels of autophagy. Following screening of proteins associated with autophagy we found that Atg4ß expression was increased in prostate cancer cells after treatment with lupulone. Transfection of cells with siRNA against Atg4ß resulted in increased levels of apoptosis in prostate cancer cells. Treatment of prostate cancer cells with lupulone derivatives initiated two modes of cell death: apoptosis as a killing pathway and autophagy as a protection against cell death. Further studies are required to investigate the regulation of Atg4ß activity in lupulone derivatives-induced negative crosstalk between apoptosis and autophagy.

An investigation into the anticancer effects and mechanism of action of hop ß-acid lupulone and its natural and synthetic derivatives in prostate cancer cells.

Lupulone, a ß-acid derived from hop extracts has been shown to exhibit antibacterial and anticancer activity. In this study we investigated the anticancer potency of lupulone and its novel derivatives and their mechanism of action on prostate cancer cells. Cell viability was determined using the MTT assay, and the ELISA approach was used to investigate induction of apoptosis. Immunoblot analysis was carried out to determine activation and regulation of proteins associated with cell death. Screening of natural and new lupulone derivatives for their anticancer activity demonstrated that one (lupulone derivative 1h) displayed stronger anticancer activity than lupulone itself on PC3 and DU145 prostate cancer cells. We further found that lupulone derivatives induced caspase-dependent apoptosis that is associated with activation of caspases 8, 9, and 3. Furthermore, caspase 8 inhibitor Z-IETD-fmk reduced cell death induced by lupulone derivatives, suggesting that apoptosis is mediated by caspase 8. Finally, we found that lupulone and its synthetic derivatives also increased formation of LC3II suggesting that autophagy is also implicated in prostate cancer cell death. The new lupulone derivatives induce caspase-dependent apoptosis and autophagy in prostate cancer cells and appear to be good candidates for further preclinical studies of prostate cancer treatment.

Role of the eIF4E binding protein 4E-BP1 in regulation of the sensitivity of human pancreatic cancer cells to TRAIL and celastrol-induced apoptosis.

BACKGROUND INFORMATION: Tumour cells can be induced to undergo apoptosis after treatment with the tumour necrosis factor α-related death-inducing ligand (TRAIL). Although human pancreatic cancer cells show varying degrees of response they can be sensitised to the pro-apoptotic effects of TRAIL in the presence of celastrol, a natural compound extracted from the plant Tripterygium wilfordii Hook F. One important aspect of the cellular response to TRAIL is the control of protein synthesis, a key regulator of which is the eukaryotic initiation factor 4E-binding protein, 4E-BP1. RESULTS: We examined the effects of celastrol and TRAIL in several pancreatic cancer cell lines. In cells that are normally resistant to TRAIL, synergistic effects of TRAIL plus celastrol on commitment to apoptosis and inhibition of protein synthesis were observed. These were associated with a strong up-regulation and dephosphorylation of 4E-BP1. The enhancement of 4E-BP1 expression, which correlated with a threefold increase in the level of the 4E-BP1 transcript, was blocked by inhibitors of reactive oxygen species and the JNK protein kinase. When the expression of 4E-BP1 was reduced by an inducible micro-RNA, TRAIL-mediated apoptosis was inhibited. CONCLUSION: These results suggest that 4E-BP1 plays a critical role in the mechanism by which TRAIL and celastrol together cause apoptotic cell death in human pancreatic tumour cells.

Urocortin is a novel regulator of osteoclast differentiation and function through inhibition of a canonical transient receptor potential 1-like cation channel.

This study investigated the role of urocortin (UCN), a member of the corticotrophin-releasing factor (CRF) family of peptides, in osteoclast maturation and function. We found that 10(-7) M UCN significantly (P<0.05) suppressed osteoclast differentiation from bone marrow precursor cells in culture and reduced the expression of several osteoclastic markers. Furthermore, UCN potently suppressed osteoclast bone resorption, by significantly inhibiting both the plan area of bone resorbed by osteoclasts and actin ring formation within osteoclasts at 10(-9) M (P<0.05), with complete inhibition at 10(-7) M (P<0.001). UCN also inhibited osteoclast motility (10(-7) M) but had no effect on osteoclast survival. Osteoclasts expressed mRNA encoding both UCN and the CRF receptor 2ß subtype. Pre-osteoclasts however, expressed CRF receptor 2ß alone. Unstimulated osteoclasts contained constitutively active cation channel currents with a unitary conductance of 3-4 pS, which were inhibited by over 70% with UCN (10(-7) M). Compounds that regulate calcium signalling and energy status of the cell, both crucial for osteoclast activity were investigated. The non-selective cation channel blockers, lanthanum (La(3)(+)) and gadolinium (Gd(3)(+)), inhibited actin ring formation in osteoclasts, whereas modulators of voltage-dependent Ca(2)(+) channels and K(ATP) channels had no effect. These findings show for the first time that UCN is a novel anti-resorptive molecule that acts through a direct effect on osteoclasts and their precursor cells.

Comparative proteomics reveals a systemic vulnerability in the vasculature of patients with abdominal aortic aneurysms.

INTRODUCTION: Abdominal aortic aneurysms (AAA) are associated with inflammation, apoptosis, and matrix degradation. AAA tissue represents the end stage of disease, limiting its utility in identification of factors culpable for initiation of aneurysm development. Recent evidence suggests that AAAs are a local representation of a systemic disease of the vasculature. Morphologic and molecular changes, comparable to those found in the aneurysm wall, have been demonstrated in veins from patients with AAAs. Changes in the vascular tissue proteome of patients with AAAs were investigated, using inferior mesenteric vein (IMV), to gain insight into early molecular changes contributing to AAA development. METHODS: IMV was harvested from 16 patients with AAA and 16 matched controls. Whole IMV lysates were subjected to 2-D difference in gel electrophoresis (2D-DIGE) with quantitative densitometry. Protein spots differentially expressed in AAA were identified using mass spectrometry. Differential protein expression was validated by Western blotting and localized to cell type by immunohistochemistry (IHC). RESULTS: Decreased levels of prohibitin (AAA, 2.00 ± 1.37; controls, 3.81 ± 1.39; 1.9-fold change; P = .02) AAA (7.33 ± 3.9; controls, 14.5 ± 5.6; 2-fold change; P = .001), along with relative increases in a cleaved fragment of vimentin (AAA, 12.9 ± 9; controls, 6.9 ± 4.7; 2-fold change; P = .11) were identified in AAA patients. All proteins were localized to the vascular smooth muscle cells. CONCLUSIONS: Proteins important in combating the injurious effects of oxidative stress and modulating the response to inflammation appear reduced in the vasculature of patients with AAA. These changes may represent early events in AAA formation. Enhancing expression of these proteins might offer a novel therapeutic avenue to inhibit AAA development.

Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia.

Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype.

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by preexposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.

Deletion of the c-Jun N-terminal kinase 3 gene protects neonatal mice against cerebral hypoxic-ischaemic injury.

c-Jun N-terminal kinase 3 (JNK3) is a member of the stress-activated group of mitogen-activated protein kinases. c-Jun N-terminal kinase 3 is a potent mediator of apoptosis and the use of JNK inhibitors or jnk3 gene deletion each protect against brain injury in adults. However, little is known about the role of JNK3 or its mechanism of action in neonatal brain injury. The aim of the present study was to compare the vulnerability of neonatal JNK3 knockout (JNK3 KO) mice and wild-type (WT) mice to cerebral hypoxic-ischaemic injury (HII) using unilateral-carotid occlusion combined with transient hypoxia. The degree of neural tissue loss in JNK3 KO mice was substantially reduced compared with WT mice (JNK3 KO 27.8%+/-2.8% versus WT 48.3%+/-2.0%, P

Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial decidualization.

The integrity of the feto-maternal interface is critical for survival of the conceptus. This interface, consisting of the maternal decidua and the invading placental trophoblast, is exposed to profound changes in oxygen tension during pregnancy. We demonstrate that human endometrial stromal cells become extraordinarily resistant to oxidative stress-induced apoptosis upon decidualization in response to cAMP and progesterone signaling. This differentiation process is associated with the induction of the forkhead transcription factor FOXO1, which in turn increases the expression of the mitochondrial antioxidant manganese superoxide dismutase. However, silencing of FOXO1 did not increase the susceptibility of decidualized cells to oxidative cell death. Comparative analysis demonstrated that hydrogen peroxide, a source of free radicals, strongly induces FOXO3a mRNA and protein expression in undifferentiated human endometrial stromal cells but not in decidualized cells. Expression of a constitutively active FOXO3a mutant elicited apoptosis in decidualized cells. Furthermore, silencing of endogenous FOXO3a in undifferentiated cells abrogated apoptosis induced by hydrogen peroxide. These results suggest that the induction of FOXO1 may enhance the ability of decidualized cells to prevent oxidative damage while the simultaneous repression of FOXO3a expression disables the signaling pathway responsible for oxidative cell death. The differential regulation of FOXO expression provides the decidua with a robust system capable of coping with prolonged episodes of oxidative stress during pregnancy.