Tumor Necrosis Factor-alpha Release Research Articles

Omega-3 polyunsaturated fatty acids reduce vascular tone and inflammation in human saphenous vein

Dietary intake of omega-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has been reported to have beneficial cardiovascular effects. However, little is known about the effect of EPA and DHA on human vascular tone. Therefore, the aim of this study is to evaluate the effect of EPA and DHA on vascular tone of the human saphenous vein (SV) obtained from patients undergoing coronary bypass operation under normal and inflammatory conditions. Moreover, we aimed to investigate the effect of EPA and DHA on the release of inflammatory mediators from SV. Pretreatment of SV with EPA and DHA (100μM, 18h) decreased the contractile response of SV to norepinephrine (NE) under normal and inflammatory conditions. Moreover, EPA and DHA pretreatment diminished increased Monocyte Chemoattractant Protein-1 (MCP-1) and Tumor Necrosis Factor-alpha (TNF-α) release from SV under inflammatory conditions. In conclusion, our results suggest that EPA and DHA pretreatment may be beneficial to counteract graft vasospasm and vascular inflammation in SV which are important factors in graft failure development. Therefore, dietary intake of EPA and DHA may have potential clinical applications in improving coronary bypass graft patency.

Importance of Autophagy in Mediating Human Immunodeficiency Virus (HIV) and Morphine-Induced Metabolic Dysfunction and Inflammation in Human Astrocytes.

Under physiological conditions, the function of astrocytes in providing brain metabolic support is compromised under pathophysiological conditions caused by human immunodeficiency virus (HIV) and opioids. Herein, we examined the role of autophagy, a lysosomal degradation pathway important for cellular homeostasis and survival, as a potential regulatory mechanism during pathophysiological conditions in primary human astrocytes. Blocking autophagy with small interfering RNA (siRNA) targeting BECN1, but not the Autophagy-related 5 (ATG5) gene, caused a significant decrease in HIV and morphine-induced intracellular calcium release. On the contrary, inducing autophagy pharmacologically with rapamycin further enhanced calcium release and significantly reverted HIV and morphine-decreased glutamate uptake. Furthermore, siBeclin1 caused an increase in HIV-induced nitric oxide (NO) release, while viral-induced NO in astrocytes exposed to rapamycin was decreased. HIV replication was significantly attenuated in astrocytes transfected with siRNA while significantly induced in astrocytes exposed to rapamycin. Silencing with siBeclin1, but not siATG5, caused a significant decrease in HIV and morphine-induced interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) release, while secretion of IL-8 was significantly induced with rapamycin. Mechanistically, the effects of siBeclin1 in decreasing HIV-induced calcium release, viral replication, and viral-induced cytokine secretion were associated with a decrease in activation of the nuclear factor kappa B (NF-κB) pathway.

Discovery of N-{4-[5-(4-Fluorophenyl)-3-methyl-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-acetamide (CBS-3595), a Dual p38α MAPK/PDE-4 Inhibitor with Activity against TNFα-Related Diseases.

The anti-inflammatory potential of p38 mitogen-activated protein kinase (MAPK) inhibitors was coincidentally expanded to a dual inhibition of p38α MAPK and phosphodiesterase 4 (PDE4), and the potential benefits arising from the blockage of both inflammation-related enzymes were thoroughly investigated. The most promising compound, CBS-3595 (1), was successively evaluated in in vitro experiments as well as in ex vivo and in vivo preclinical studies after administration of 1 to rodents, dogs, and monkeys. The resulting data clearly indicated a potent suppression of tumor necrosis factor alpha release. For reconfirming the findings of the animal studies when administering 1 to healthy human volunteers, a phase I clinical trial was conducted. Apart from further information regarding the pharmacokinetic and pharmacodynamic characteristics of 1, it was demonstrated that dual inhibition of p38α MAPK and PDE4 is able to synergistically attenuate the excessive anti-inflammatory response.

Relationship between TLR4/NF-κB signaling pathway and propofol-induced inhibition of endotoxin-induced release of TNF-α from alveolar macrophages of rats

Objective To evaluate the relationship between Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)signaling pathway and propofol-induced inhibition of endotoxin-induced release of tumor necrosis factor-alpha(TNF-α)from alveolar macrophages(AMs)of rats. Methods AMs extracted from adult male Sprague-Dawley rats were cultured and inoculated in 6-well plates(1×106 cells/well)and in 96-well plates(1×104 cells/well). The cells were divided into 5 groups(n=18 each)using a random number table: control group(group C), dimethyl sulfoxide group(group D), lipopolysaccharide(LPS)group(group L), propofol group(group P)and LPS plus propofol group(group L+ P). The cells were continuously cultured with phosphate buffer solution in group C. Dimethyl sulfoxide was added at the final concentration of 5 mg/ml in group D. LPS was added at the final concentration of 1 μg/ml in group L. Propofol was added at the final concentration of 25 μmol/L(4.46 μg/ml)in group P. LPS and propofol were added at the final concentration of 1 μg/ml and 25 μmol/L(4.46 μg/ml), respectively, in group L+ P.At 24 h of culture or incubation, the cell viability was detected by CCK-8 assay, the morphological changes of cells were observed using Wright′s staining, the concentration of TNF-α in the supernatant was determined by enzyme-linked immunosorbent assay, and TLR4 expression and NF-κB activities were measured by Western blot. Results Compared with group C, the cell viability and concentration of TNF-α in the supernatant were significantly increased, the expression of TLR4 was up-regulated, and the activity of NF-κB was enhanced in L and L+ P groups(P 0.05). Compared with group L, the cell viability and concentration of TNF-α in the supernatant were significantly decreased, the expression of TLR4 was down-regulated, and the activity of NF-κB was weakened(P<0.05), the morphological changes of cells were significantly attenuated, and the number of pseudopodia was reduced in group L+ P. Conclusion The mechanism by which propofol inhibits endotoxin-induced release of TNF-α from AMs is related to inhibited activation of TLR4/NF-κB signaling pathway in rats. Key words: Toll-like receptor 4; NF-kappa B; Propofol; Endotoxins; Macrophages, alveolar; Tumor necrosis factor-alpha

Effect of calcium carbonate particle shape on phagocytosis and pro-inflammatory response in differentiated THP-1 macrophages

Phagocytosis is a physiological process used by immune cells such as macrophages to actively ingest and destroy foreign pathogens and particles. It is the cellular process that leads to the failure of drug delivery carriers because the drug carriers are cleared by immune cells before reaching their target. Therefore, clarifying the mechanism of particle phagocytosis would have a significant implication for both fundamental understanding and biomedical engineering. As far as we know, the effect of particle shape on biological response has not been fully investigated. In the present study, we investigated the particle shape-dependent cellular uptake and biological response of differentiated THP-1 macrophages by using calcium carbonate (CaCO3)-based particles as a model. Transmission electron microscopy analysis revealed that the high uptake of needle-shaped CaCO3 particles by THP-1 macrophages because of their high phagocytic activity. In addition, the THP-1 macrophages exposed to needle-shaped CaCO3 accumulated a large amount of calcium in the intracellular matrix. The enhanced release of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) by the THP-1 macrophages suggested that the needle-shaped CaCO3 particles trigger a pro-inflammatory response. In contrast, no pro-inflammatory response was induced in undifferentiated THP-1 monocytes exposed to either needle- or cuboidal-shaped CaCO3 particles, probably because of their low phagocytic activity. We also found that phosphate-coated particles efficiently repressed cellular uptake and the resulting pro-inflammatory response in both THP-1 macrophages and primary peritoneal macrophages. Our results indicate that the pro-inflammatory response of macrophages upon exposure to CaCO3 particles is shape- and surface property-dependent, and is mediated by the intracellular accumulation of calcium ions released from phagocytosed CaCO3 particles.

Involvement of p38-βTrCP-Tristetraprolin-TNFα axis in radiation pneumonitis.

Early release of tumor necrosis factor-alpha (TNF-α) during radiotherapy of thoracic cancers plays an important role in radiation pneumonitis, whose inhibition may provide lung radioprotection. We previously reported radiation inactivates Tristetraprolin (TTP), a negative regulator of TNF-α synthesis, which correlated with increased TNF-α release. However, the molecular events involved in radiation-induced TTP inactivation remain unclear. To determine if eliminating Ttp in mice resulted in a phenotypic response to radiation, Ttp-null mice lungs were exposed to a single dose of 15 Gy, and TNF-α release and lung inflammation were analyzed at different time points post-irradiation. Ttp−/− mice with elevated (9.5±0.6 fold) basal TNF-α showed further increase (12.2±0.9 fold, p<0.02) in TNF-α release and acute lung inflammation within a week post-irradiation. Further studies using mouse lung macrophage (MH-S), human lung fibroblast (MRC-5), and exogenous human TTP overexpressing U2OS and HEK293 cells upon irradiation (a single dose of 4 Gy) promoted p38-mediated TTP phosphorylation at the serine 186 position, which primed it to be recognized by an ubiquitin ligase (E3), beta transducing repeat containing protein (β-TrCP), to promote polyubiquitination-mediated proteasomal degradation. Consequently, a serine 186 to alanine (SA) mutant of TTP was resistant to radiation-induced degradation. Similarly, either a p38 kinase inhibitor (SB203580), or siRNA-mediated β-TrCP knockdown, or overexpression of dominant negative Cullin1 mutants protected TTP from radiation-induced degradation. Consequently, SB203580 pretreatment blocked radiation-induced TNF-α release and radioprotected macrophages. Together, these data establish the involvement of the p38-βTrCP-TTP-TNFα signaling axis in radiation-induced lung inflammation and identified p38 inhibition as a possible lung radioprotection strategy.

Involvement of the Bradykinin B1 Receptor in Microglial Activation: In Vitro and In Vivo Studies.

The importance of brain inflammation to Alzheimer’s disease (AD) pathogenesis has been accepted of late, with it currently being held that brain inflammation aggravates AD pathology. One important aspect of brain inflammation is the recruitment and activation of microglia, a process termed microgliosis. Kinins and bradykinin (BK), in particular, are major pro-inflammatory mediators in the periphery, although all of the factors comprising the kinin system have also been described in the brain. Moreover, it was shown that the amyloid β (Aβ) peptide (a component of AD plaques) enhances kinin secretion and activates BK receptors that can, in turn, stimulate Aβ production. Still, the role of bradykinin in modulating brain inflammation and AD is not completely understood. In this study, we aimed to investigate the roles of the bradykinin B1 receptor (B1R) and bradykinin B2 receptor (B2R) in regulating microglial secretion of pro-inflammatory factors in vitro. Furthermore, the effects of intranasal administration of specific B1R and B2R antagonists on Aβ burden and microglial accumulation in the brains of transgenic AD mice were studied. The data obtained show that neither R-715 (a B1R antagonist) nor HOE 140 (a B2R antagonist) altered microglial cell viability. However, R-715, but not HOE 140, markedly increased lipopolysaccharide-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) release, as well as inducible nitric oxide synthase expression in BV2 microglial cells. Neither antagonist altered NO nor TNF-α production in non-stimulated cells. We also showed that intranasal administration of R-715 but not HOE 140 to 8-week-old 5X familial AD mice enhanced amyloid burden and microglia/macrophage accumulation in the cortex. To conclude, we provide evidence supporting a role of B1R in brain inflammation and in the regulation of amyloid deposition in AD mice, possibly with microglial/macrophage involvement. Further studies are required to test whether modulation of this receptor can serve as a novel therapeutic strategy for AD.

In Vitro and In Vivo Stimulation of Toll-Like Receptor 9 by CpG Oligodeoxynucleotides Incorporated Into Polypod-Like DNA Nanostructures

Cytosine-phosphate-guanine (CpG) DNA is known to increase the potency of vaccines. Here, in vitro and in vivo stimulation of toll-like receptor 9 by CpG DNA incorporated into polypod-like DNA nanostructures was evaluated by measuring the levels of tumor necrosis factor alpha released from macrophage-like RAW 264.7 cells and plasma interleukin (IL)-12p40 in vivo following intravenous injection into mice. Phosphodiester CpG1668 was selected as the CpG DNA, and tripodna and hexapodna, which were CpG1668-containing tripod and hexapod-like DNA nanostructures, respectively, were designed. CpG-tripodna and CpG-hexapodna induced tumor necrosis factor alpha release from RAW 264.7 cells about 10- and ∼30-fold higher than single-stranded CpG1668 (CpG-SS). Moreover, in all cases examined, plasma IL-12p40 concentrations increased after intravenous injection into mice, with peak levels depending on the samples and the doses. The area under the plasma concentration-time curves indicated that the CpG-hexapodna was approximately 20-fold more efficient in inducing IL-12p40 production than CpG-SS. The efficiency of CpG-tripodna and CpG-hexapodna to increase the potency of CpG-SS in vivo was comparable to that observed in cultured RAW 264.7 cells. These results provide experimental evidence that in vitro studies can be used to estimate the in vivo immunostimulatory activity of CpG DNA incorporated into DNA nanostructures.

Hypertonic saline attenuates expression of Notch signaling and proinflammatory mediators in activated microglia in experimentally induced cerebral ischemia and hypoxic BV-2 microglia

BackgroundIschemic stroke is a major disease that threatens human health in ageing population. Increasing evidence has shown that neuroinflammatory mediators play crucial roles in the pathophysiology of cerebral ischemia injury. Notch signaling is recognized as the cell fate signaling but recent evidence indicates that it may be involved in the inflammatory response in activated microglia in cerebral ischemia. Previous report in our group demonstrated hypertonic saline (HS) could reduce the release of interleukin-1 beta and tumor necrosis factor-alpha in activated microglia, but the underlying molecular and cellular mechanisms have remained uncertain. This study was aimed to explore whether HS would partake in regulating production of proinflammatory mediators through Notch signaling.ResultsHS markedly attenuated the expression of Notch-1, NICD, RBP-JK and Hes-1 in activated microglia both in vivo and in vitro. Remarkably, HS also reduced the expression of iNOS in vivo, while the in vitro levels of inflammatory mediators Phos-NF-κB, iNOS and ROS were reduced by HS as well.ConclusionOur results suggest that HS may suppress of inflammatory mediators following ischemia/hypoxic through the Notch signaling which operates synergistically with NF-κB pathway in activated microglia. Our study has provided the morphological and biochemical evidence that HS can attenuate inflammation reaction and can be neuroprotective in cerebral ischemia, thus supporting the use of hypertonic saline by clinicians in patients with an ischemia stroke.

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™−hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10μM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.

Potential anti-inflammatory, antioxidant and antimicrobial activities of Sambucus australis

Context: Sambucus australis Cham. & Schltdl. (Adoxaceae) is used in Brazilian folk medicine to treat inflammatory disorders.Objective: To evaluate the in vitro anti-inflammatory, antioxidant and antimicrobial properties of S. australis.Materials and methods: The anti-inflammatory activity of ethanol extracts of the leaf and bark of S. australis (1–100 μg/mL) were studied in lipopolysaccharide/interferon γ stimulated murine macrophages RAW 264.7 cells (24 h incubation) by investigating the release of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) and in the TNF-α-induced nuclear factor kappa (NF-κB) assay. Minimum inhibitory concentration (MIC) was determined by the microdilution test (24 h incubation). Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and the NO scavenging assays. Chemical composition was assessed by LC-MS/MS.Results: Antioxidant activities in the DPPH (IC50 43.5 and 66.2 μg/mL), FRAP (IC50 312.6 and 568.3 μg/mL) and NO radical scavenging assays (IC50 285.0 and 972.6 μg/mL) were observed in the leaf and bark ethanol extracts, respectively. Solely the leaf extract showed significant inhibition of NO and TNF-α production in RAW264.7 cells at concentrations of 2 and 100 μg/mL, respectively, and suppression of TNF-α inhibition of NF-κB by 12.8 and 20.4% at concentrations of 50 and 100 μg/mL, respectively. The extract also exhibited antibacterial activity against Salmonella typhimurium (MIC 250 μg/mL) and Klebsiella pneumoniae (MIC 250 μg/mL). LC-MS/MS revealed the presence of chlorogenic acid and rutin as major compounds.Discussion and conclusion: The results indicate that the ethanol leaf extract of S. australis exhibit prominent anti-inflammatory effects.

Carboxymethyllysine, an Advanced Glycation End-Product, Promotes the Invasion and Migration of Lung Cancer A549 Cells

Effects of carboxymethyllysine (CML), an advanced glycation end-product (AGE), at 1, 2, 4, 8 or 16 &mu;mol/l upon invasion and migration of A549 cells, non-small cell lung cancer (NSCLC) cells, were investigated. Results showed that CML at used test doses did not affect A549 cell growth. However, CML at 4-16 &mu;mol/l enhanced both invasion and migration, and stimulated the release of reactive oxygen species, interleukin-6 and tumor necrosis factor-alpha in A549 cells. CML at 2-16 &mu;mol/l increased protein expression of AGE receptor, p47^phox, intercellular adhesion molecule-1, fibronectin, kappa-B (NF- &kappa;B) p65 and p-p38 in A549 cells. CML only at 4-16 μmol/l increased matrix metalloproteinase-2 expression in A549 cells. These findings indicated that CML might benefit NSCLC metastasis through promoting invasion and migration.

Ionizing radiation promotes CCL27 secretion from keratinocytes through the cross talk between TNF-α and ROS.

The skin-associated chemokine CCL27 and its receptor CCR10 mediate the immune response of skin-homing T cells. The CCL27 secreted from keratinocytes was reportedly involved in inflammatory skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis. However, whether ionizing radiation increases the levels of CCL27 secretion still remains unclear. In HaCaT cells, a human keratinocyte cell line, CCL27 secretion was markedly increased after X-ray irradiation. We further found that irradiation boosted the generation of reactive oxygen species (ROS), which was concomitant with the release of tumor necrosis factor-alpha (TNF-α). Moreover, alteration of ROS in irradiated HaCaT cells correlated with TNF-α secretion, indicating a positive loop of TNF-α secretion and ROS generation. This positive loop regulated the secretion of CCL27 from irradiated cells. We therefore concluded that the cross talk between TNF-α and ROS after keratinocytes was exposed to radiation, triggered CCL27 secretion for subsequent inflammation response.

Malondialdehyde-Acetaldehyde-Adducted Surfactant Protein Alters Macrophage Functions Through Scavenger Receptor A.

Reactive aldehydes such as acetaldehyde and malondialdehyde generated as a result of alcohol metabolism and cigarette smoke exposure lead to the formation of malondialdehyde-acetaldehyde-adducted proteins (MAA adducts). These aldehydes can adduct to different proteins such as bovine serum albumin and surfactant protein A or surfactant protein D (SPD). Macrophages play an important role in innate immunity, but the effect of MAA adducts on macrophage function has not yet been examined. Because macrophage scavenger receptor A (SRA; CD204) mediates the uptake of modified proteins, we hypothesized that the effects of MAA-modified proteins on macrophage function are primarily mediated through SRA. We tested this hypothesis by exposing SPD-MAA to macrophages and measuring functions. SPD-MAA treatment significantly stimulated pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) release in the macrophage cell line, RAW 264.7. A significant reduction in phagocytosis of zymosan particles was also observed. SPD-MAA stimulated a significant dose-dependent increase in TNF-α and interleukin (IL)-6 release from peritoneal macrophages (PMs) of wild-type (WT) mice. But significantly less TNF-α and IL-6 were released from PMs of SRA-/- mice. We observed a significant reduction in phagocytosis of zymosan particles in PMs from WT mice treated with SPD-MAA. No further SPD-MAA-induced reduction was seen in PMs from SRA-/- mice. SPD-MAA treatment significantly increased SRA mRNA expression, but had no effect on surface receptor protein expression. Protein kinase C alpha inhibitor and NF-κB inhibitor significantly reduced pro-inflammatory cytokine release in response to SPD-MAA. In conclusion, our data demonstrate that SRA is important for MAA-adducted protein-mediated effect on macrophage functions.

Systemic over-release of interleukin-17 in acute kidney injury after septic shock: Clinical and experimental evidence

In order to investigate the role of T-helper 17 (Th17) cell activation in acute kidney injury (AKI) after septic shock, a two-stage approach was used. Firstly, peripheral blood mononuclear cells (PBMCs) and CD4-lymphocytes were isolated the first 24h after septic shock from 26 patients with AKI and 18 patients with chronic renal disease (CRD) without AKI and stimulated for the release of tumour necrosis factor-alpha (TNFα), interleukin (IL)-10, IL-17, IL-22 and interferon-gamma (IFNγ). Results were compared with 15 healthy volunteers and 13 patients with uncomplicated sepsis. Secondly, a murine model of multiple organ dysfunction (MODS) complicated with AKI and bacterial gut translocation was studied, and IL-10, IL-17, IL-22 and IFNγ were measured in kidney homogenates. IL-17 was the only cytokine produced at greater quantities from PBMCs and CD4-lymphocytes of patients with septic shock and AKI than comparators. When PBMCs of patients with septic shock and AKI were ex-vivo stimulated, intracellular staining for IL-17 was greater in CD3(+)/CD4(+)/CD196(+) cells compared to patients with septic shock and CRD. IL-17 was released at greater amounts from PBMCs of non-survivors by septic shock and AKI but not of septic shock and CRD. In the murine model of MODS, a gradual decrease of IL-17, but not of IL-10, IL-22 and IFNγ, of kidney homogenates was found indicating over-consumption. These results suggest that AKI after septic shock is driven through IL-17 release by Th17 cells; this is gradually consumed in the kidney.

Dynorphin 1-17 and Its N-Terminal Biotransformation Fragments Modulate Lipopolysaccharide-Stimulated Nuclear Factor-kappa B Nuclear Translocation, Interleukin-1beta and Tumor Necrosis Factor-alpha in Differentiated THP-1 Cells.

Dynorphin 1–17, (DYN 1–17) opioid peptide produces antinociception following binding to the kappa-opioid peptide (KOP) receptor. Upon synthesis and release in inflamed tissues by immune cells, DYN 1–17 undergoes rapid biotransformation and yields a unique set of opioid and non-opioid fragments. Some of these major fragments possess a role in immunomodulation, suggesting that opioid-targeted therapeutics may be effective in diminishing the severity of inflammatory disorders. This study aimed to examine the immunomodulatory effects of DYN 1–17 and major N-terminal fragments found in the inflammatory environment on nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) from lipopolysaccharide (LPS)-stimulated, differentiated THP-1 cells. The results demonstrate that NF-κB/p65 nuclear translocation was significantly attenuated following treatment with DYN 1–17 and a specific range of fragments, with the greatest reduction observed with DYN 1–7 at a low concentration (10 nM). Antagonism with a selective KOP receptor antagonist, ML-190, significantly reversed the inhibitory effects of DYN 1–17, DYN 1–6, DYN 1–7 and DYN 1–9, but not other DYN 1–17 N-terminal fragments (DYN 1–10 and 1–11) on NF-κB/p65 nuclear translocation. DYN 1–17 and selected fragments demonstrated differential modulation on the release of IL-1β and TNF-α with significant inhibition observed with DYN 1–7 at low concentrations (1 nM and 10 pM). These effects were blocked by ML-190, suggesting a KOP receptor-mediated pathway. The results demonstrate that DYN 1–17 and certain N-terminal fragments, produced in an inflamed environment, play an anti-inflammatory role by inhibiting NF-κB/p65 translocation and the subsequent cytokine release through KOP receptor-dependent and independent pathways.

Distinct Roles of Myosins in Aspergillus fumigatus Hyphal Growth and Pathogenesis.

Myosins are a family of actin-based motor proteins found in many organisms and are categorized into classes based on their structures. Class II and V myosins are known to be important for critical cellular processes, including cytokinesis, endocytosis, exocytosis, and organelle trafficking, in the model fungi Saccharomyces cerevisiae and Aspergillus nidulans However, the roles of myosins in the growth and virulence of the pathogen Aspergillus fumigatus are unknown. We constructed single- and double-deletion strains of the class II and class V myosins in A. fumigatus and found that while the class II myosin (myoB) is dispensable for growth, the class V myosin (myoE) is required for proper hyphal extension; deletion of myoE resulted in hyperbranching and loss of hyphal polarity. Both myoB and myoE are necessary for proper septation, conidiation, and conidial germination, but only myoB is required for conidial viability. Infection with the ΔmyoE strain in the invertebrate Galleria mellonella model and also in a persistently immunosuppressed murine model of invasive aspergillosis resulted in hypovirulence, while analysis of bronchoalveolar lavage fluid revealed that tumor necrosis factor alpha (TNF-α) release and cellular infiltration were similar compared to those of the wild-type strain. The ΔmyoE strain showed fungal growth in the murine lung, while the ΔmyoB strain exhibited little fungal burden, most likely due to the reduced conidial viability. These results show, for the first time, the important role these cytoskeletal components play in the growth of and disease caused by a known pathogen, prompting future studies to understand their regulation and potential targeting for novel antifungal therapies.

Investigation of enhanced hemocompatibility and tissue compatibility associated with multi-functional coating based on hyaluronic acid and Type IV collagen.

The biocompatibility of cardiovascular devices has always been considered crucial for their clinical efficacy. Therefore, a biofunctional coating composed of Type IV collagen (CoIV) and hyaluronan (HA) was previously fabricated onto the titanium (Ti) substrate for the application of promoting vascular smooth muscle cell contractile phenotype and improving surface endothelialization. However, the anti-inflammation property, blood compatibility and in vivo tissue compatibility of the HA/CoIV coating, as paramount consideration of cardiovascular materials surface coating, have not been investigated. Thus, in this study, the three crucial properties of the HA/CoIV coating were tested. The platelet adhesion/activation test and the dynamic whole blood experiment implied that the HA/CoIV coating had better blood compatibility compared with Ti substrate and pure CoIV coating. The macrophage adhesion/activation and inflammatory cytokine release (tumor necrosis factor-alpha and interleukin-1) results indicated that the HA/CoIV coating could significantly improve the anti-inflammation property of the Ti substrate. The in vivo implantation of SD rats for 3 weeks’ results demonstrated that the HA/CoIV coating caused milder tissue response. All these results suggested that the multi-functional HA/CoIV coating possessed good biocompatibility. This research is anticipated to be potentially applied for the surface modification of cardiovascular stents.

Evaluation of the effects of active fractions of chinese medicine formulas on IL-1β, IL-6, and TNF-α release from ANA-1 murine macrophages

Ethnopharmacological relevanceYaotongning (YTN) is a traditional Chinese Medicine (TCM) that contains ten component medicinal materials (CMMs) and uses Chinese rice wine as a vehicle to enhance its efficacy. YTN has been used for rheumatoid arthritis (RA) treatment in China for decades and has been reported to have anti-inflammatory and analgesic effects, as well as to strengthen the immune system. Aim of the studyThe present work quantitatively evaluated the in vitro effects of active fractions from the ten CMMs that make up YTN and eight additional herbs commonly used in TCM formulas for RA treatment, as well as different combinations of these active fractions, on cellular immune response; the findings were used to determine which active fractions are responsible for promoting an immune response, and to assess whether YTN is superior to other similar formulas and whether YTN can be improved by simplifying its formula from the point of its cellular immunomodulatory activity. Materials and methodsUsing the YTN formulation principles and some concepts in combinatorial chemistry, 27 TCM samples were designed by combining some or all of the active fractions of YTN and other eight herbs used for RA treatment. Release of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) from ANA-1 murine macrophages was measured using an enzyme-linked immunosorbent assay (ELISA). The immunoregulatory effects of the TCM samples were evaluated by comparing their half-effective concentrations (EC50) for stimulating the release of these cytokines. ResultsAmong the investigated active fractions, the flavonoids from Carthamus tinctorius (Fct), Davallia mariesii (Fdm), and Cinnamomum cassia Twig volatile oils (Vca) from the eight selected herbs effectively promoted IL-1β and IL-6 release from ANA-1 cells. Saponins from the YTN CMM Glycyrrhiza uralensis (Sgu) were the most potent promoters of IL-1β and TNF-α release. The aqueous extract of YTN CMM Eupolyphaga sinensis (Ves) strongly enhanced the release of IL-1β, IL-6, and TNF-α from ANA-1 cells. The EC50 values for stimulating the secretion of IL-1β, IL-6, and TNF-α could be determined for only six samples. The full YTN formula and the sample containing 50% Glycyrrhiza uralensis saponins, 25% of the mixture of alkaloids, and 25% of the mixture of all flavonoids exhibited good comprehensive cellular immunomodulatory activity. The immunomodulatory activity of the complete YTN formula was better than that of the sample containing all active fractions of YTN without Chinese rice wine (the YTN vehicle). ConclusionsSgu and Ves are the primary active fractions of YTN involved in stimulating immune responses. The YTN prescription was reasonably effective at promoting cellular immune responses. Chinese rice wine, the YTN vehicle, strengthened the immunoregulatory activity of YTN. The results of this study demonstrate that the YTN recipe could be improved by reducing the number of CMMs and altering some active fractions without reducing its activity to promote cellular immune responses.

Chemical Hybridization of Vizantin and Lipid A to Generate a Novel LPS Antagonist.

Lipopolysaccharide (LPS) antagonists have attracted considerable interest as promising candidates for the treatment of severe sepsis triggered by Gram-negative bacteria. In this article, we describe the development of a novel LPS antagonist based on chemical hybridization of vizantin and the hydrophobic molecular unit of LPS (lipid A). Vizantin, 6,6'-bis-O-(3-nonyldodecanoyl)-α,α'-trehalose, was designed as an immunostimulator from a structure-activity relationship (SAR) study with trehalose 6,6'-dicorynomycolate (TDCM). Our recent study indicated that vizantin displays adjuvant activity by specifically binding to the Toll-like receptor 4 (TLR4)/MD2 protein complex. Because lipid A unit (or LPS) is also known to trigger an inflammatory response via the same TLR4/MD2 complex as vizantin, we designed a hybrid compound of vizantin and lipid A with the aim of developing a novel biofunctional glycolipid. Focusing on the antagonism to Escherichia coli LPS in an in vitro model with human macrophages (THP-1 cells), we identified a potent LPS antagonist among the synthesized hybrid compounds. The novel LPS antagonist effectively inhibited LPS-induced release of tumor necrosis factor-alpha (TNF-α) in a dose-dependent manner with an IC50 value of 3.8 nM, making it a candidate for the treatment drug of Gram-negative sepsis and/or septic shock.