Negative regulation of type I interferon signaling by integrin-linked kinase permits dengue virus replication.

Dengue virus (DENV) infection can induce life-threatening dengue hemorrhagic fever/dengue shock syndrome in infected patients. DENV is a threat to global health due to its growing numbers and incidence of infection in the last 50 years. During infection, DENV expresses ten structural and nonstructural proteins modulating cell responses to benefit viral replication. However, the lack of knowledge regarding the cellular proteins and their functions in enhancing DENV pathogenesis impedes the development of antiviral drugs and therapies against fatal DENV infection. Here, we identified that integrin-linked kinase (ILK) is a novel enhancing factor for DENV infection by suppressing type I interferon (IFN) responses. Mechanistically, ILK binds DENV NS1 and NS3, activates Akt and Erk, and induces NF-κB-driven suppressor of cytokine signaling 3 (SOCS3) expression. Elevated SOCS3 in DENV-infected cells inhibits phosphorylation of STAT1/2 and expression of interferon-stimulated genes (ISGs). Inhibiting ILK, Akt, or Erk activation abrogates SOCS3 expression. In DENV-infected mice, the treatment of an ILK inhibitor significantly reduces viral loads in the brains, disease severity, and mortality rate. Collectively, our results show that ILK is a potential therapeutic target against DENV infection.

Measurement of lipid droplets in peripheral immune cells shows an immunomodulatory effect on monocyte polarization in experimental dyslipidaemia.

Intracellular lipid droplet (LD) generation is the primary site of energy storage, which is necessary for physiological homeostasis but is related to pathological metabolic disorders. Lipid metabolism is critical for maintaining innate and adaptive immunity; however, it is mainly undefined in peripheral immune cells. Flow cytometry-based immune profiling in healthy peripheral blood cells showed significant original generation of LDs in dendritic cells (DCs, CD3-CD19-CD56-CD11+), monocytes (CD3-CD19-CD56-CD14+), natural killer cells (NK, CD3-CD19-CD56+), and B cells (CD3-CD19+). CD36, a common scavenger receptor of lipids, was also highly expressed in LD-accumulated DCs and monocytes. Following short-term treatment with oxidized LDL (oxLDL) in an experimental ex vivo model, CD14+ monocytes showed an effective increase in LD generation, but there were no alterations in the immune cell populations. Furthermore, oxLDL-treated CD14+ monocytes displayed CD36 expression. However, oxLDL-primed CD14+ monocytes showed a blockade in the uptake of extra oxLDL, even while expressing increased CD36, indicating a defect in lipid clearance. Exogenous treatment with oxLDL caused monocyte type 1 polarization accompanied by increased LD accumulation and CD36 expression. This study describes a method to monitor LD generation and CD36 expression in peripheral immune cells and identified an immunomodulatory effect of oxLDL on monocytes by tilting them towards type 1 polarization.

SHP2: The protein tyrosine phosphatase involved in chronic pulmonary inflammation and fibrosis.

Chronic respiratory diseases (CRDs), including pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), lung cancer, and asthma, are significant global health problems due to their prevalence and rising incidence. The roles of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in controlling tyrosine phosphorylation of targeting proteins modulate multiple physiological cellular responses and contribute to the pathogenesis of CRDs. Src homology-2 domain-containing PTP2 (SHP2) plays a pivotal role in modulating downstream growth factor receptor signaling and cytoplasmic PTKs, including MAPK/ERK, PI3K/AKT, and JAK/STAT pathways, to regulate cell survival and proliferation. In addition, SHP2 mutation and activation are commonly implicated in tumorigenesis. However, little is known about SHP2 in chronic pulmonary inflammation and fibrosis. This review discusses the potential involvement of SHP2 deregulation in chronic pulmonary inflammation and fibrosis, as well as the therapeutic effects of targeting SHP2 in CRDs.

Monocyte Distribution Width in Children With Systemic Inflammatory Response: Retrospective Cohort Examining Association With Early Sepsis.

OBJECTIVES: To investigate the association between increased monocyte distribution width (MDW) and pediatric sepsis in the emergency department (ED). DESIGN: Retrospective cohort study. SETTING: A single academic hospital study. PATIENTS: Patients from birth to the age of 18 years who presented at the ED of an academic hospital with systemic inflammatory response syndrome (SIRS) were consecutively enrolled. Sepsis was diagnosed using the International Pediatric Surviving Sepsis Campaign criteria. INTERVENTIONS: Antibiotic treatment was administrated once infection was suspected. MEASUREMENTS AND MAIN RESULTS: Routine complete blood cell count, neutrophil-to-lymphocyte ratio (NLR), and MDW, a new inflammatory biomarker, were evaluated in the ED. Logistic regression models were used to explore associations with early pediatric sepsis. We included 201 patients with sepsis and 1,050 without sepsis. In the multivariable model, MDW greater than 23 U (odds ratio [OR], 4.97; 95% CI, 3.42-7.22; p < 0.0001), NLR greater than 6 (OR, 2.06; 95% CI, 1.43-2.94; p = 0.0001), WBC greater than 11,000 cells/µL (OR, 6.52; 95% CI, 4.45-9.53; p < 0.0001), and the SIRS score (OR, 3.42; 95% CI, 2.57-4.55; p < 0.0001) were associated with pediatric sepsis. In subgroup analysis, MDW greater than 23 U remained significantly associated with sepsis for children 6-12 years old (OR, 6.76; 95% CI, 2.60-17.57; p = 0.0001) and 13-18 years (OR, 17.49; 95% CI, 7.69-39.76; p = 0.0001) with an area under the receiver operating curve of 0.8-0.9. CONCLUSIONS: MDW greater than 23 U at presentation is associated with the early diagnosis of sepsis in children greater than or equal to 6 years old. This parameter should be considered as a stratification variable in studies of pediatric sepsis.

Role of Glycogen Synthase Kinase-3 in Interferon-γ-Mediated Immune Hepatitis.

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase, is a vital glycogen synthase regulator controlling glycogen synthesis, glucose metabolism, and insulin signaling. GSK-3 is widely expressed in different types of cells, and its abundant roles in cellular bioregulation have been speculated. Abnormal GSK-3 activation and inactivation may affect its original bioactivity. Moreover, active and inactive GSK-3 can regulate several cytosolic factors and modulate their diverse cellular functional roles. Studies in experimental liver disease models have illustrated the possible pathological role of GSK-3 in facilitating acute hepatic injury. Pharmacologically targeting GSK-3 is therefore suggested as a therapeutic strategy for liver protection. Furthermore, while the signaling transduction of GSK-3 facilitates proinflammatory interferon (IFN)-γ in vitro and in vivo, the blockade of GSK-3 can be protective, as shown by an IFN-γ-induced immune hepatitis model. In this study, we explored the possible regulation of GSK-3 and the potential relevance of GSK-3 blockade in IFN-γ-mediated immune hepatitis.

Fractionated ionizing radiation facilitates interferon-γ signaling and anticancer activity in lung adenocarcinoma cells.

Fractionated ionizing radiation (FIR) is a radiotherapy regimen that is regularly performed as part of lung cancer treatment. In contrast to the growth inhibition caused by DNA damage, immunomodulation in post-irradiated cancer cells is not well documented. Interferon (IFN)-γ confers anticancer activity by triggering both growth inhibition and cytotoxicity. This study investigated the priming effects of FIR with immunomodulation on the anticancer IFN-γ. Cell morphology, cell growth, and cytotoxicity were observed in FIR-treated A549 lung adenocarcinoma. Induction of p53 and epithelial-mesenchymal transition (EMT) were monitored. Following FIR, activation of IFN-γ signaling pathways were detected. FIR caused changes in cell morphology, inhibited cell growth, and induced cytotoxicity. While p53 was induced by FIR, no epithelial-mesenchymal transition could be found. Following IFN-γ stimulation, FIR-induced p53-associated cell cytotoxicity was significantly enhanced. Additionally, FIR increased the downstream response to IFN-γ by facilitating IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) signaling without affecting the receptor expression. FIR-facilitated STAT1 activation through the mechanism involving mitogen-activated protein kinase activation and Src-homology 2 domain-containing tyrosine phosphatase 2 inactivation. These results demonstrate the FIR-facilitated IFN-γ signaling and its anticancer activity.

Therapeutic Effects of Monoclonal Antibody against Dengue Virus NS1 in a STAT1 Knockout Mouse Model of Dengue Infection.

Dengue virus (DENV) is the causative agent of dengue fever, dengue hemorrhagic fever, and dengue shock syndrome and is endemic to tropical and subtropical regions of the world. Our previous studies showed the existence of epitopes in the C-terminal region of DENV nonstructural protein 1 (NS1) which are cross-reactive with host Ags and trigger anti-DENV NS1 Ab-mediated endothelial cell damage and platelet dysfunction. To circumvent these potentially harmful events, we replaced the C-terminal region of DENV NS1 with the corresponding region from Japanese encephalitis virus NS1 to create chimeric DJ NS1 protein. Passive immunization of DENV-infected mice with polyclonal anti-DJ NS1 Abs reduced viral Ag expression at skin inoculation sites and shortened DENV-induced prolonged bleeding time. We also investigated the therapeutic effects of anti-NS1 mAb. One mAb designated 2E8 does not recognize the C-terminal region of DENV NS1 in which host-cross-reactive epitopes reside. Moreover, mAb 2E8 recognizes NS1 of all four DENV serotypes. We also found that mAb 2E8 caused complement-mediated lysis in DENV-infected cells. In mouse model studies, treatment with mAb 2E8 shortened DENV-induced prolonged bleeding time and reduced viral Ag expression in the skin. Importantly, mAb 2E8 provided therapeutic effects against all four serotypes of DENV. We further found that mAb administration to mice as late as 1 d prior to severe bleeding still reduced prolonged bleeding time and hemorrhage. Therefore, administration with a single dose of mAb 2E8 can protect mice against DENV infection and pathological effects, suggesting that NS1-specific mAb may be a therapeutic option against dengue disease.

Escape from IFN-γ-dependent immunosurveillance in tumorigenesis.

Immune interferon (IFN), also known as IFN-γ, promotes not only immunomodulation but also antimicrobial and anticancer activity. After IFN-γ binds to the complex of IFN-γ receptor (IFNGR) 1-IFNGR2 and subsequently activates its downstream signaling pathways, IFN-γ immediately causes transcriptional stimulation of a variety of genes that are principally involved in its biological activities. Regarding IFN-γ-dependent immunosurveillance, IFN-γ can directly suppress tumorigenesis and infection and/or can modulate the immunological status in both cancer cells and infected cells. Regarding the anticancer effects of IFN-γ, cancer cells develop strategies to escape from IFN-γ-dependent cancer immunosurveillance. Immune evasion, including the recruitment of immunosuppressive cells, secretion of immunosuppressive factors, and suppression of cytotoxic T lymphocyte responses, is speculated to be elicited by the oncogenic microenvironment. All of these events effectively downregulate IFN-γ-expressing cells and IFN-γ production. In addition to these extrinsic pathways, cancer cells may develop cellular tolerance that manifests as hyporesponsiveness to IFN-γ stimulation. This review discusses the potential escape mechanisms from IFN-γ-dependent immunosurveillance in tumorigenesis.

Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of Gamma Interferon via Signaling Attenuation.

UNLABELLED: Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms. IMPORTANCE: Immunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.

Helicobacter pylori infection activates Src homology-2 domain-containing phosphatase 2 to suppress IFN-γ signaling.

Helicobacter pylori infection not only induces gastric inflammation but also increases the risk of gastric tumorigenesis. IFN-γ has antimicrobial effects; however, H. pylori infection elevates IFN-γ-mediated gastric inflammation and may suppress IFN-γ signaling as a strategy to avoid immune destruction through an as-yet-unknown mechanism. This study was aimed at investigating the mechanism of H. pylori-induced IFN-γ resistance. Postinfection of viable H. pylori decreased IFN-γ-activated signal transducers and activators of transcription 1 and IFN regulatory factor 1 not only in human gastric epithelial MKN45 and AZ-521 but also in human monocytic U937 cells. H. pylori caused an increase in the C-terminal tyrosine phosphorylation of Src homology-2 domain-containing phosphatase (SHP) 2. Pharmacologically and genetically inhibiting SHP2 reversed H. pylori-induced IFN-γ resistance. In contrast to a clinically isolated H. pylori strain HP238, the cytotoxin-associated gene A (CagA) isogenic mutant strain HP238(CagAm) failed to induce IFN-γ resistance, indicating that CagA regulates this effect. Notably, HP238 and HP238(CagAm) differently caused SHP2 phosphorylation; however, imaging and biochemical analyses demonstrated CagA-mediated membrane-associated binding with phosphorylated SHP2. CagA-independent generation of reactive oxygen species (ROS) contributed to H. pylori-induced SHP2 phosphorylation; however, ROS/SHP2 mediated IFN-γ resistance in a CagA-regulated manner. This finding not only provides an alternative mechanism for how CagA and ROS coregulate SHP2 activation but may also explain their roles in H. pylori-induced IFN-γ resistance.

Macrophage migration inhibitory factor triggers chemotaxis of CD74+CXCR2+ NKT cells in chemically induced IFN-γ-mediated skin inflammation.

IFN-γ mediates chemically induced skin inflammation; however, the mechanism by which IFN-γ-producing cells are recruited to the sites of inflammation remains undefined. Secretion of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, from damaged cells may promote immune cell recruitment. We hypothesized that MIF triggers an initial step in the chemotaxis of IFN-γ-producing cells in chemically induced skin inflammation. Using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears, MIF expression was examined, and its role in this process was investigated pharmacologically. The cell populations targeted by MIF, their receptor expression patterns, and the effects of MIF on cell migration were examined. TPA directly caused cytotoxicity accompanied by MIF release in mouse ear epidermal keratinocytes, as well as in human keratinocytic HaCaT cells. Treatment with the MIF antagonist (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester considerably attenuated TPA-induced ear swelling, leukocyte infiltration, epidermal cell proliferation, and dermal angiogenesis. Inhibition of MIF greatly diminished the dermal infiltration of IFN-γ(+) NKT cells, whereas the addition of exogenous TPA and MIF to NKT cells promoted their IFN-γ production and migration, respectively. MIF specifically triggered the chemotaxis of NKT cells via CD74 and CXCR2, and the resulting depletion of NKT cells abolished TPA-induced skin inflammation. In TPA-induced skin inflammation, MIF is released from damaged keratinocytes and then triggers the chemotaxis of CD74(+)CXCR2(+) NKT cells for IFN-γ production.

Effectiveness and Mechanism of Preoperative Lugol Solution for Reducing Thyroid Blood Flow in Patients with Euthyroid Graves' Disease.

BACKGROUND: To reduce intraoperative and postoperative complications, using Lugol solution to preoperatively prepare patients with Graves' disease has (1) rapidly reduced the severity of thyrotoxicosis and (2) reduced the vascularity of the thyroid gland. The vascularity reduction normally accompanies reducing the severity of thyrotoxicosis. However, the effects and mechanism of Lugol solution for reducing blood flow have not been well investigated in the patients with euthyroid (normally functioning thyroid) Graves' disease. METHODS: Twenty-five patients with euthyroid Graves' disease being preoperatively treated with Lugol solution for 10 days were measured, at baseline and on the operative day, for (1) superior thyroid artery blood flow; (2) systemic angiogenic factor (VEGF); and (3) systemic inflammatory factor [interleukin (IL)-16]. RESULTS: All three parameters were significantly (p < 0.0001) lower after 10 days of Lugol solution treatment. The average reductions were blood flow: 60% (0.294 vs. 0.117 L/min), serum VEGF: 55% (169.8 vs. 76.7 pg/mL), and serum IL-16: 50% (427.2 vs. 214.2; pg/mL). CONCLUSION: Lugol solution significantly reduced thyroid arterial blood flow, VEGF, and IL-16, even in patients with euthyroid Graves' disease. We recommend routine preoperative Lugol solution treatment for all patients with Graves' disease.

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ.

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cells also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ.

Anti-dengue virus nonstructural protein 1 antibodies cause NO-mediated endothelial cell apoptosis via ceramide-regulated glycogen synthase kinase-3ß and NF-κB activation.

Immunopathogenetic mechanisms of dengue virus (DENV) infection are involved in hemorrhagic syndrome resulting from thrombocytopenia, coagulopathy, and vasculopathy. We have proposed a mechanism of molecular mimicry in which Abs against DENV nonstructural protein 1 (NS1) cross-react with human endothelial cells and cause NF-κB-regulated immune activation and NO-mediated apoptosis. However, the signaling pathway leading to NF-κB activation after the binding of anti-DENV NS1 Abs to endothelial cells is unresolved. In this study, we found that anti-DENV NS1 Abs caused the formation of lipid raftlike structures, and that disrupting lipid raft formation by methyl-ß-cyclodextrin decreased NO production and apoptosis. Treatment with anti-DENV NS1 Abs elevated ceramide generation in lipid rafts. Pharmacological inhibition of acid sphingomyelinase (aSMase) decreased anti-DENV NS1 Ab-mediated ceramide and NO production, as well as apoptosis. Exogenous ceramide treatment induced biogenesis of inducible NO synthase (iNOS)/NO and apoptosis through an NF-κB-regulated manner. Furthermore, activation of glycogen synthase kinase-3ß (GSK-3ß) was required for ceramide-induced NF-κB activation and iNOS expression. Notably, anti-DENV NS1 Abs caused GSK-3ß-mediated NF-κB activation and iNOS expression, which were regulated by aSMase. Moreover, pharmacological inhibition of GSK-3ß reduced hepatic endothelial cell apoptosis in mice passively administered anti-DENV NS1 Abs. These results suggest that anti-DENV NS1 Abs bind to the endothelial cell membrane and cause NO production and apoptosis via a mechanism involving the aSMase/ceramide/GSK-3ß/NF-κB/iNOS/NO signaling pathway.

Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression.

Detection of reactive oxygen species during the cell cycle under normal culture conditions using a modified fixed-sample staining method.

We developed an alternative method of simultaneously monitoring the generation of reactive oxygen species (ROS) and cellular oxidative responses using the oxidation-sensitive fluorescent probe dichlorofluorescein (DCF) in fixed samples. In this study, we evaluated the ability of this method to detect ROS generation during the cell cycle under normal culture conditions using flow cytometric analyses. Among the fixatives tested, only acetone and paraformaldehyde did not alter the endogenous oxidation of the responsive dye 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), which is a chloromethyl derivative of H2DCFDA. Only acetone fixation followed by staining with propidium iodide was able to detect ROS generation during the cell cycle without altering DCF oxidation. Further thymidine treatment led to cell cycle arrest at the G1 phase followed by the downregulation of total intracellular ROS. Paraformaldehyde-based fixation enabled the evaluation of ROS generation by immunostaining at a different phase of the cell cycle, whereas MPM2 co-staining enabled identification of the specific mitotic phase. This study demonstrates a modified fixed-sample method that can be used to measure intracellular ROS production during the cell cycle using standard immunostaining techniques.

Anesthetic propofol overdose causes vascular hyperpermeability by reducing endothelial glycocalyx and ATP production.

Prolonged treatment with a large dose of propofol may cause diffuse cellular cytotoxicity; however, the detailed underlying mechanism remains unclear, particularly in vascular endothelial cells. Previous studies showed that a propofol overdose induces endothelial injury and vascular barrier dysfunction. Regarding the important role of endothelial glycocalyx on the maintenance of vascular barrier integrity, we therefore hypothesized that a propofol overdose-induced endothelial barrier dysfunction is caused by impaired endothelial glycocalyx. In vivo, we intraperitoneally injected ICR mice with overdosed propofol, and the results showed that a propofol overdose significantly induced systemic vascular hyperpermeability and reduced the expression of endothelial glycocalyx, syndecan-1, syndecan-4, perlecan mRNA and heparan sulfate (HS) in the vessels of multiple organs. In vitro, a propofol overdose reduced the expression of syndecan-1, syndecan-4, perlecan, glypican-1 mRNA and HS and induced significant decreases in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio and ATP concentrations in human microvascular endothelial cells (HMEC-1). Oligomycin treatment also induced significant decreases in the NAD+/NADH ratio, in ATP concentrations and in syndecan-4, perlecan and glypican-1 mRNA expression in HMEC-1 cells. These results demonstrate that a propofol overdose induces a partially ATP-dependent reduction of endothelial glycocalyx expression and consequently leads to vascular hyperpermeability due to the loss of endothelial barrier functions.

Panton-Valentine leukocidin facilitates the escape of Staphylococcus aureus from human keratinocyte endosomes and induces apoptosis.

Skin and soft-tissue infections (SSTIs) caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have emerged as major health problems throughout the world. Most SSTI CA-MRSA strains produce Panton-Valentine leukocidin (PVL), but its contribution to CA-MRSA pathogenesis is poorly defined. Here, we used an endemic PVL-positive SSTI-causing CA-MRSA strain from Taiwan, together with an isogenic PVL-knockout mutant (Δpvl) and complemented PVL-positive derivative, to evaluate the role of PVL in the pathogenesis of CA-MRSA in the RHEK-1 human keratinocyte cell line and a rabbit skin infection model. We found that both PVL-positive CA-MRSA and isogenic Δpvl strains attached and were engulfed into endosomes of RHEK-1 cells within 1 hour following infection. However, by 2 hours after infection PVL-positive CA-MRSA more effectively disrupted endosomes, escaped into the cytoplasm, and replicated intracellularly. By 6 hours after infection, the PVL-positive strain caused significantly more caspase-dependent keratinocyte apoptosis than the isogenic Δpvl mutant. In the rabbit infection model, 1 week following infection the wild-type strain produced significantly more widespread lesions and cell apoptosis than the isogenic Δpvl mutant. These findings indicate that PVL is an important virulence factor that enables CA-MRSA to produce necrotizing skin infections by allowing the bacteria to escape from endosomes, replicate intracellularly, and induce apoptosis.

An increase in integrin-linked kinase non-canonically confers NF-κB-mediated growth advantages to gastric cancer cells by activating ERK1/2.

BACKGROUND: Increased activity or expression of integrin-linked kinase (ILK), which regulates cell adhesion, migration, and proliferation, leads to oncogenesis. We identified the molecular basis for the regulation of ILK and its alternative role in conferring ERK1/2/NF-κB-mediated growth advantages to gastric cancer cells. RESULTS: Inhibiting ILK with short hairpin RNA or T315, a putative ILK inhibitor, abolished NF-κB-mediated the growth in the human gastric cancer cells AGS, SNU-1, MKN45, and GES-1. ILK stimulated Ras activity to activate the c-Raf/MEK1/2/ERK1/2/ribosomal S6 kinase/inhibitor of κBα/NF-κB signaling by facilitating the formation of the IQ motif-containing GTPase-activating protein 1 (IQGAP1)-Ras complex. Forced enzymatic ILK expression promoted cell growth by facilitating ERK1/2/NF-κB signaling. PI3K activation or decreased PTEN expression prolonged ERK1/2 activation by protecting ILK from proteasome-mediated degradation. C-terminus of heat shock cognate 70 interacting protein, an HSP90-associated E3 ubiquitin ligase, mediated ILK ubiquitination to control PI3K- and HSP90-regulated ILK stabilization and signaling. In addition to cell growth, the identified pathway promoted cell migration and reduced the sensitivity of gastric cancer cells to the anticancer agents 5-fluorouracil and cisplatin. Additionally, exogenous administration of EGF as well as overexpression of EGFR triggered ILK- and IQGAP1-regulated ERK1/2/NF-κB activation, cell growth, and migration. CONCLUSION: An increase in ILK non-canonically promotes ERK1/2/NF-κB activation and leads to the growth of gastric cancer cells.

Desmethylclomipramine triggers mitochondrial damage and death in TGF-ß-induced mesenchymal type of A549 cells.

Lung cancer is the leading cause of cancer deaths, where the metastasis often causes chemodrug resistance and leads to recurrence after treatment. Desmethylclomipramine (DCMI), a bioactive metabolite of clomipramine, shows the therapeutic efficacy with antidepressive agency as well as potential cytostatic effects on lung cancer cells. Here, we demonstrated that DCMI effectively caused transforming growth factor (TGF)-ß1-mediated mesenchymal type of A549 cells to undergo mitochondrial death via myeloid cell leukemia-1 (Mcl-1) suppression and activation of truncated Bid (tBid). TGF-ß1 induced epithelial mesenchymal transition in A549 cells with the increase of fibronectin and decrease of E-cadherin, the activation of Akt/glycogen synthase kinase-3ß (GSK-ß)/Mcl-1 axis, and the hypo-responsiveness to cisplatin. DCMI initiated a dose-dependent cytotoxicity on TGF-ß1-mediated mesenchymal type of A549 cells through inactivating Akt/GSK-ß/Mcl-1 axis, in which mitochondria instability and caspase-9/3 activation also occurred concurrently. Pharmacological inhibition of caspase-8 and cathepsin B partly reversed tBid expression and mitochondrial damage to further attenuate DCMI-mediated cytotoxicity. Additionally, DCMI presented partial therapeutic effects in treating mesenchymal type of A549 tumor bearing nude mice through an acceleration of cancer cell death. Taken together, DCMI exerts antitumor effects via initiating the mechanisms of Akt/GSK-ß/Mcl-1 inactivation and cathepsin B/caspase-8-regulated mitochondrial death, which suggests its potential role in mesenchymal type of cancer cell therapy.