Co-degradation of interferon signaling factor DDX3 by PB1-F2 as a basis for high virulence of 1918 pandemic influenza.

The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.

Improvement of STING-mediated cancer immunotherapy using immune checkpoint inhibitors as a game-changer.

Various cancer therapies, such as surgery, radiotherapy, chemotherapy, and immunotherapy, have been used to treat cancer. Among cancer immunotherapies, stimulators of interferon genes (STING) activate various immune cells and induce them to attack cancer cells. However, the secretion of type I interferon (IFN α and ß) increases after stimulation of the immune cell as a side effect of STING agonist, thereby increasing the expression of programmed death-ligand 1 (PD-L1) in the tumor microenvironment (TME). Therefore, it is necessary to reduce the side effects of STING agonists and maximize cancer treatment by administering combination therapy. Tumor-bearing mice were treated with cisplatin, tumor-specific peptide, neoantigen, DMXAA (STING agonist), and immune checkpoint inhibitor (ICI). The combination vaccine group showed a reduction in tumor mass, an increased survival rate, and IFN-γ+ (interferon gamma) CD8+ (cluster of differentiation 8) T cells in the spleen and TME. The distribution of immune cells in the spleen and TME was confirmed, and the number of active immune cells increased, whereas that of immunosuppressive cells decreased. When measuring cytokine levels in the tumor and serum, the levels of pro-inflammatory cytokines increased and anti-inflammatory cytokines decreased. This study demonstrated that when various cancer therapies are combined to treat cancer, it can lead to an anticancer immune synergistic effect by increasing the immune response and reducing side effects.

Myostatin inhibitor YK11 as a preventative health supplement for bacterial sepsis.

Muscle wasting caused by catabolic reactions in skeletal muscle is commonly observed in patients with sepsis. Myostatin, a negative regulator of muscle mass, has been reported to be upregulated in diseases associated with muscle atrophy. However, the behavior of myostatin during sepsis is not well understood. Herein, we sought to investigate the expression and regulation of myostatin in skeletal muscle in mice inoculated with gram-negative bacteria. Interestingly, the protein level of myostatin was found to increase in the muscle of septic mice simultaneously with an increase in the levels of follistatin, NF-κΒ, myogenin, MyoD, p- FOXO3a, and p-Smad2. Furthermore, the inhibition of myostatin by YK11 repressed the levels of pro-inflammatory cytokines and organ damage markers in the bloodstream and in the major organs of mice, which originally increased in sepsis; thus, myostatin inhibition by YK11 decreased the mortality rate due to sepsis. The results of this study suggest that YK11 may help revert muscle wasting during sepsis and subdue the inflammatory environment, thereby highlighting its potential as a preventive agent for sepsis-related muscle wasting.

Improvement of DC-based vaccines using adjuvant TLR4-binding 60S acidic ribosomal protein P2 and immune checkpoint inhibitors.

Cancer immunotherapy has fewer side effects and higher efficiency than conventional methods. Dendritic cell (DC)-based vaccine, a cancer immunotherapeutic, is prepared by processing mature DCs and pulsing with tumor antigen peptide ex vivo, to induce the activation of tumor-specific T lymphocytes followed by tumor clearance in vivo. Unfortunately, clinical trials of this method mostly failed due to low patient response, possibly due to the absence of novel adjuvants that induce DC maturation through Toll-like receptor (TLR) signals. Interestingly, immune checkpoint inhibitor (ICI) therapy has shown remarkable anti-tumor efficacy when combined with cancer vaccines. In this study, we identified 60S acidic ribosomal protein P2 (RPLP2) through pull-down assay using human cancer cells derived proteins that binds to Toll-like receptor 4 (TLR4). Recombinant RPLP2 induced maturation and activation of DCs in vitro. This DC-based vaccine, followed by pulsing with tumor-specific antigen, has shown to significantly increase tumor-specific CD8+IFN-γ+ T cells, and improved both tumor prevention and tumor treatment effects in vivo. The adjuvant effects of RPLP2 were shown to be dependent on TLR4 using TLR4 knockout mice. Moreover, ICIs that suppress the tumor evasion mechanism showed synergistic effects on tumor treatment when combined with these vaccines.

Platelet-activating Factor Mediates Endotoxin Tolerance by Regulating Indoleamine 2,3-Dioxygenase-dependent Expression of the Suppressor of Cytokine Signaling 3.

Indoleamine 2,3-dioxygenase (IDO) mediates immune tolerance, and suppressor of cytokine signaling 3 (SOCS3) negatively regulates the JAK/STAT signal transduction pathway. We determined previously that platelet-activating factor (PAF) protects mice against LPS-induced endotoxic shock, but its detailed mechanism of action was unknown. We performed survival experiments in IDO+/+ and IDO-/- mice using an LPS-induced endotoxemia model and rated organ injury (neutrophil infiltration and liver function). Using ELISA and Western blotting, we also investigated the mechanism of PAF-mediated endotoxin tolerance during endotoxemia. PAF-mediated endotoxin tolerance was dependent on IDO in vivo and in vitro and was not observed in IDO-/- mice. JAK/STAT signaling, crucial for SOCS3 expression, was also impaired in the absence of IDO. In an IDO- and STAT-dependent manner, PAF mediated a decrease in IL-12 and a dramatic increase in IL-10 and reduced mouse mortality. In addition, PAF attenuated LPS-mediated neutrophil infiltration into the lungs and interactions between neutrophil-like (THP-1) and endothelial cells (human umbilical vein endothelial cells). These results indicate that PAF-mediated endotoxin tolerance is initiated via IDO- and JAK/STAT-dependent expression of SOCS3. Our study has revealed a novel tolerogenic mechanism of IDO action and an important association between IDO and SOCS3 with respect to endotoxin tolerance.

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation.

The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

Tamarixetin Exhibits Anti-inflammatory Activity and Prevents Bacterial Sepsis by Increasing IL-10 Production.

Sepsis is a systemic inflammatory response to pathogenic infection that currently has no specific pharmaceutical interventions. Instead, antibiotics administration is considered the best available option, despite increasing drug resistance. Alternative strategies are therefore urgently required to prevent sepsis and strengthen the host immune system. One such option is tamarixetin (4'- O-methylquercetin), a naturally occurring flavonoid derivative of quercetin that protects against inflammation. The purpose of this study was to determine whether the anti-inflammatory effects of tamarixetin protect against the specific inflammatory conditions induced in lipopolysaccharide (LPS) or Escherichia coli K1 models of sepsis. Our study showed that tamarixetin reduced the secretion of various inflammatory cytokines by dendritic cells after activation with LPS. It also promoted the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and specifically increased the population of IL-10-secreting immune cells in LPS-activated splenocytes. Tamarixetin showed general anti-inflammatory effects in mouse models of bacterial sepsis and decreased bacteria abundance and endotoxin levels. We therefore conclude that tamarixetin has superior anti-inflammatory properties than quercetin during bacterial sepsis. This effect is associated with an increased population of IL-10-secreting immune cells and suggests that tamarixetin could serve as a specific pharmaceutical option to prevent bacterial sepsis.

Neoagarooligosaccharides prevent septic shock by modulating A20-and cyclooxygenase-2-mediated interleukin-10 secretion in a septic-shock mouse model.

Analysis of the signaling mechanism triggered by endotoxin-mediated toll-like receptor-4 activation using immune cell systems or rodent models may help identify potential agents for the prevention of Gram-negative bacteria infection. ß-agarase cleaves the ß-1,4-linkages of agar to produce neoagarooligosaccharides (NAOs), which have various physiological functions. The aim of this study was to investigate the efficacy of NAOs in preventing experimental sepsis caused by the administration of endotoxin or Gram-negative bacteria. Organ damage and neutrophil infiltration in an endotoxemia and septic-shock mouse model were suppressed by NAOs. Pro-inflammatory cytokine level was decreased, but IL-10 level was increased by NAO-treatment. Further induction by NAOs in the presence of endotoxin was associated with a significant induction of A20 and cyclooxygenase (COX)-2 expressions. Our data suggest that NAOs have a beneficial preventive effect in septic shock correlated with the enhancement of IL-10 via the induction of A20 and COX-2.

A Novel Therapeutic Approach Using Mesenchymal Stem Cells to Protect Against Mycobacterium abscessus.

Recent studies have demonstrated the therapeutic potential of mesenchymal stem cells (MSCs) for the treatment of acute inflammatory injury and bacterial pneumonia, but their therapeutic applications in mycobacterial infections have not been investigated. In this study, we demonstrated the use of MSCs as a novel therapeutic strategy against Mycobacterium abscessus (M. abscessus), which is the most drug-resistant and difficult-to-treat mycobacterial pathogen. The systemic intravenous injection of MSCs not only improved mouse survival but also enhanced bacterial clearance in the lungs and spleen. Additionally, MSCs enhanced IFN-γ, TNF-α, IL-6, MCP-1, nitric oxide (NO) and PGE2 production and facilitated CD4(+) /CD8(+) T cell, CD11b(high) macrophage, and monocyte recruitment in the lungs of M. abscessus-infected mice. To precisely elucidate the functions of MSCs in M. abscessus infection, an in vitro macrophage infection system was used. MSCs caused markedly increased NO production via NF-κB activation in M. abscessus-infected macrophages cultured in the presence of IFN-γ. Inhibiting NO or NF-κB signaling using specific inhibitors reduced the antimycobacterial activity of MSCs. Furthermore, the cellular crosstalk between TNF-α released from IFN-γ-stimulated M. abscessus-infected macrophages and PGE2 produced by MSCs was necessary for the mycobacterial-killing activity of the macrophages. Finally, the importance of increased NO production in response to MSC administration was confirmed in the mouse M. abscessus infection model. Our results suggest that MSCs may offer a novel therapeutic strategy for treating this drug-resistant mycobacterial infection by enhancing the bacterial-killing power of macrophages. Stem Cells 2016;34:1957-1970.

Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER⁺ Breast Cancer Cells.

Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER⁺) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the PXN gene promoter. We also investigated the migration and invasive ability of nobiletin in ER⁺ cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER⁺ breast cancer cells.

Glycogen Synthase Kinase-3ß (GSK-3ß) Inhibition Enhances Dendritic Cell-based Cancer Vaccine Potency via Suppression of Interferon-γ-induced Indoleamine 2,3-Dioxygenase Expression.

Indoleamine 2,3-dioxygenase (IDO) functions as a crucial mediator of tumor-mediated immune tolerance by causing T-cell suppression via tryptophan starvation in a tumor environment. Glycogen synthase kinase-3ß (GSK-3ß) is also involved in immune and anti-tumor responses. However, the relativity of these proteins has not been as well defined. Here, we found that GSK-3ß-dependent IDO expression in the dendritic cell (DC) plays a role in anti-tumor activity via the regulation of CD8(+) T-cell polarization and cytotoxic T lymphocyte activity. By the inhibition of GSK-3ß, attenuated IDO expression and impaired JAK1/2-Stat signaling crucial for IDO expression were observed. Protein kinase Cδ (PKCδ) activity and the interaction between JAK1/2 and Stat3, which are important for IDO expression, were also reduced by GSK-3ß inhibition. CD8(+) T-cell proliferation mediated by OVA-pulsed DC was blocked by interferon (IFN)-γ-induced IDO expression via GSK-3ß activity. Specific cytotoxic T lymphocyte activity mediated by OVA-pulsed DC against OVA-expressing EG7 thymoma cells but not OVA-nonexpressing EL4 thymoma cells was also attenuated by the expressed IDO via IFN-γ-induced activation of GSK-3ß. Furthermore, tumor growth that was suppressed with OVA-pulsed DC vaccination was restored by IDO-expressing DC via IFN-γ-induced activation of GSK-3ß in an OVA-expressing murine EG7 thymoma model. Taken together, DC-based immune response mediated by interferon-γ-induced IDO expression via GSK-3ß activity not only regulates CD8(+) T-cell proliferation and cytotoxic T lymphocyte activity but also modulates OVA-pulsed DC vaccination against EG7 thymoma.

Annexin A5 increases survival in murine sepsis model by inhibiting HMGB1-mediated pro-inflammation and coagulation.

The identification of HMGB1 as a late-mediator in sepsis has highlighted HMGB1 as a promising therapeutic target for sepsis treatment. Recent studies have revealed that annexin A5, a 35 kDa Ca2+-dependent phospholipid binding protein, exerts anti-inflammatory effect by inhibiting LPS binding to TLR4/MD2 complex. Annexin A5 administration has been shown to protect against endotoxin lethality even when the treatment was given after the early cytokine response, which prompted our group to suspect that annexin A5 may inhibit the binding of HMGB1, as well as endotoxin to TLR4. Here we suggest annexin A5 as a new inhibitor of HMGB1-mediated pro-inflammatory cytokine production and coagulation in sepsis. We first confirmed the inhibitory role of annexin A5 in LPS-induced production of pro-inflammatory cytokines both in vitro and in vivo. We observed that annexin A5 protects against tissue damage and organ dysfunction during endotoxemia in vivo. We then assessed the inhibiting role of annexin A5 in HMGB1/TLR4 interaction, and showed that annexin A5 treatment reduces HMGB1-mediated cytokines IL6 and TNFα both in vitro and in vivo. Finally, we confirmed that anticoagulant property of annexin A5 persists in various septic conditions including elevated HMGB1. Overall, we suggest annexin A5 as an alternative therapeutic approach for controlling HMGB1-mediated pro-inflammation and coagulation in patients with sepsis.

Enhancement of tumor-specific T cell-mediated immunity in dendritic cell-based vaccines by Mycobacterium tuberculosis heat shock protein X.

Despite the potential for stimulation of robust antitumor immunity by dendritic cells (DCs), clinical applications of DC-based immunotherapy are limited by the low potency in generating tumor Ag-specific T cell responses. Therefore, optimal conditions for generating potent immunostimulatory DCs that overcome tolerance and suppression are key factors in DC-based tumor immunotherapy. In this study, we demonstrate that use of the Mycobacterium tuberculosis heat shock protein X (HspX) as an immunoadjuvant in DC-based tumor immunotherapy has significant potential in therapeutics. In particular, the treatment aids the induction of tumor-reactive T cell responses, especially tumor-specific CTLs. The HspX protein induces DC maturation and proinflammatory cytokine production (TNF-α, IL-1ß, IL-6, and IFN-ß) through TLR4 binding partially mediated by both the MyD88 and the TRIF signaling pathways. We employed two models of tumor progression and metastasis to evaluate HspX-stimulated DCs in vivo. The administration of HspX-stimulated DCs increased the activation of naive T cells, effectively polarizing the CD4(+) and CD8(+) T cells to secrete IFN-γ, as well as enhanced the cytotoxicity of splenocytes against HPV-16 E7 (E7)-expressing TC-1 murine tumor cells in therapeutic experimental animals. Moreover, the metastatic capacity of B16-BL6 melanoma cancer cells toward the lungs was remarkably attenuated in mice that received HspX-stimulated DCs. In conclusion, the high therapeutic response rates with tumor-targeted Th1-type T cell immunity as a result of HspX-stimulated DCs in two models suggest that HspX harnesses the exquisite immunological power and specificity of DCs for the treatment of tumors.

Antituberculosis Activity of a Naturally Occurring Flavonoid, Isorhamnetin.

Isorhamnetin (1) is a naturally occurring flavonoid having anticancer and anti-inflammatory properties. The present study demonstrated that 1 had antimycobacterial effects on Mycobacterium tuberculosis H37Rv, multi-drug- and extensively drug-resistant clinical isolates with minimum inhibitory concentrations of 158 and 316 µM, respectively. Mycobacteria mainly affect the lungs, causing an intense local inflammatory response that is critical to the pathogenesis of tuberculosis. We investigated the effects of 1 on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells. Isorhamnetin suppressed the release of tumor necrosis factor (TNF)-α and interleukin (IL)-12. A nontoxic dose of 1 reduced mRNA expression of TNF-α, IL-1ß, IL-6, IL-12, and matrix metalloproteinase-1 in IFN-γ-stimulated cells. Isorhamnetin inhibited IFN-γ-mediated stimulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase and showed high-affinity binding to these kinases (binding constants: 4.46 × 10(6) M(-1) and 7.6 × 10(6) M(-1), respectively). The 4'-hydroxy group and the 3'-methoxy group of the B-ring and the 5-hydroxy group of the A-ring of 1 play key roles in these binding interactions. A mouse in vivo study of lipopolysaccharide-induced lung inflammation revealed that a nontoxic dose of 1 reduced the levels of IL-1ß, IL-6, IL-12, and INF-γ in lung tissue. These data provide the first evidence that 1 could be developed as a potent antituberculosis drug.

Characteristic Study of Boron Doped Carbon Nanowalls Films Deposited by Microwave Plasma Enhanced Chemical Vapor Deposition.

In this research, catalyst-free vertically aligned boron doped carbon nanowalls films were fabricated on silicon (100) substrates by MPECVD using feeding gases CH4, H2 and B2H6 (diluted with H2 to 5% vol) as precursors. The substrates were pre-seeded with nanodiamond colloid. The fabricated CNWs films were characterized by Scanning Electron Microscopy (SEM) and Raman Spectroscopy. The data obtained from SEM confirms that the CNWs films have different density and wall thickness. From Raman spectrum, a G peak around 1588 cm(-1) and a D band peak at 1362 cm(-1) were observed, which indicates a successful fabrication of CNWs films. The EDX spectrum of boron doped CNWs film shows the existence of boron and carbon. Furthermore, field emission properties of boron doped carbon nanowalls films were measured and field enhancement factor was calculated using Fowler-Nordheim plot. The result indicates that boron doped CNWs films could be potential electron emitting materials.

Thermal Conductivity of Diamond Packed Electrospun PAN-Based Carbon Fibers Incorporated with Multi Wall Carbon Nanotubes.

Multi wall carbon nanotubes (MWCNTs) and diamond are renowned as superlative material due to their relatively high thermal conductivity and hardness while comparing with any bulk materials. In this research, polyacrylonitrile (PAN) solution incorporated with MWCNTs at an alteration of mass fractions (0 wt%, 0.6 wt%, 1 wt%, 2 wt%) were fabricated via electrospinning under optimized parameters. Dried composite nanofibers were stabilized and carbonized, after which water base polytrafluorethylene (PTFE) mixed with nano diamond powder solution was spin coated on them. Scanning electron microscopy, Raman spectroscopy, X-ray scattering and Laserflash thermal conductivity were used to characterize the composite nanofiber sheets. The result shows that the thermal conductivity increased to 4.825 W/m K from 2.061 W/mK. The improvement of thermal conductivities is suggesting the incorporation of MWCNTs.

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells.

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.

An indoxyl compound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice.

Mast cells, constituents of virtually all organs and tissues, are critical cells in IgE-mediated allergic responses. The aim of this study was to investigate the effect and mechanism of an indoxyl chromogenic compound, 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, on IgE-mediated mast cell activation and allergic responses in mice. CAC-0982 reversibly suppressed antigen-stimulated degranulation in murine mast cells (IC50, ~3.8µM) and human mast cells (IC50, ~3.0µM). CAC-0982 also inhibited the expression and secretion of IL-4 and TNF-α in mast cells. Furthermore, CAC-0982 suppressed the mast cell-mediated allergic responses in mice in a dose-dependent manner (ED50 27.9mg/kg). As for the mechanism, CAC-0982 largely suppressed the phosphorylation of Syk and its downstream signaling molecules, including LAT, Akt, Erk1/2, p38, and JNK. Notably, the tyrosine kinase assay of antigen-stimulated mast cells showed that CAC-0982 inhibited Fyn kinase, one of the upstream tyrosine kinases for Syk activation in mast cells. Taken together, these results suggest that CAC-0982 may be used as a new treatment for regulating IgE-mediated allergic diseases through the inhibition of the Fyn/Syk pathway in mast cells.

The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K(+) (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) value of 0.81µM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77±0.04µM(-1)s(-1) and 2.55±1.50s(-1), respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition.

Rhamnus davurica leaf extract inhibits Fyn activation by antigen in mast cells for anti-allergic activity.

BACKGROUND: Complementary and alternative herbal medicines are recently considered as a promising approach for treating various diseases. We screened approximately 100 plant extracts for anti-allergic activity. Rhamnus davurica leaf extract showed the most potent inhibitory effect on the activation of RBL-2H3 mast cells. Although Rhamnus davurica extract has been used to treat pruritus, dysuresia, and constipation as a traditional herbal medicine in some Asian countries, an anti-allergic effect of Rhamnus davurica has not yet been demonstrated. We aimed to investigate the effect and mechanism of the leaf extract of Rhamnus davurica (LERD) on mast cells in vitro and allergic responses in vivo. METHODS: The effects of LERD on the activation of mast cells and mast cell-mediated passive cutaneous anaphylaxis (PCA) were measured in mice and two types of mast cells, mouse bone marrow-derived mast cells (BMMCs) and RBL-2H3 cells in vitro. A mechanistic study of its inhibitory effect was performed by using degranulation assay, reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting analysis. RESULTS: LERD reversibly suppressed antigen-stimulated degranulation in BMMCs and RBL-2H3 cells, and also inhibited mRNA expression and secretion of TNF-α and IL-4 in a dose-dependent manner. In a PCA animal model, LERD significantly inhibited antigen-induced allergic response and degranulation of ear tissue mast cells. As for the mechanism of action, LERD inhibited the activation of Syk, which is the pivotal signaling protein for mast cell activation by antigen. Furthermore, LERD also impeded the activations of well-known downstream proteins such as LAT, Akt and three MAP kinases (Erk, p38 and JNK). In an in vitro kinase assay, LERD suppressed the activation of Fyn in antigen-stimulated mast cells. CONCLUSION: This study demonstrated for the first time that LERD has anti-allergic effects through inhibiting the Fyn/Syk pathway in mast cells. Therefore, this study provides scientific evidence for LERD to be used as an herbal medicine or health food for patients with allergic diseases.