Reactive oxygen species drive herpes simplex virus (HSV)-1-induced proinflammatory cytokine production by murine microglia

Abstract Background Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia. Methods Oxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H2DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production. Results Levels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK. Conclusions Data presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.</p

Herpes simplex virus induces neural oxidative damage via microglial cell Toll-like receptor-2

<p>Abstract</p> <p>Background</p> <p>Using a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis.</p> <p>Methods</p> <p>Purified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2^-/-mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from β-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia.</p> <p>Results</p> <p>Stimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2^-/-microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2^-/-microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2^-/-microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2^-/-mice.</p> <p>Conclusions</p> <p>These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.</p

Dentin Regeneration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66570/2/10.1177_08959374950090030501.pd

Production of mannitol by streptococcus mutans

Mannitol was produced as an end product when resting cell suspensions of strains of Streptococcus mutans were given high levels of sucrose or glucose. The presence of mannitol in the supernatant from glucose incubated cells and the occasional detection of mannitol-1-phosphate suggest that mannitol was formed by a reduction of fructose-6-phosphate. This pathway would permit the regeneration of NAD under anaerobic conditions, thereby permitting maximal catabolism of hexoses via the glycolytic pathway.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21913/1/0000320.pd

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice

<div><p>Congenital cytomegalovirus (CMV) infection is the most common non-hereditary cause of sensorineural hearing loss (SNHL) yet the mechanisms of hearing loss remain obscure. Natural Killer (NK) cells play a critical role in regulating murine CMV infection via NK cell recognition of the Ly49H cell surface receptor of the viral-encoded m157 ligand expressed at the infected cell surface. This Ly49H NK receptor/m157 ligand interaction has been found to mediate host resistance to CMV in the spleen, and lung, but is much less effective in the liver, so it is not known if this interaction is important in the context of SNHL. Using a murine model for CMV-induced labyrinthitis, we have demonstrated that the Ly49H/m157 interaction mediates host resistance in the temporal bone. BALB/c mice, which lack functional Ly49H, inoculated with mCMV at post-natal day 3 developed profound hearing loss and significant outer hair cell loss by 28 days of life. In contrast, C57BL/6 mice, competent for the Ly49H/m157 interaction, had minimal hearing loss and attenuated outer hair cell loss with the same mCMV dose. Administration of Ly49H blocking antibody or inoculation with a mCMV viral strain deleted for the m157 gene rendered the previously resistant C57BL/6 mouse strain susceptible to hearing loss to a similar extent as the BALB/c mouse strain indicating a direct role of the Ly49H/m157 interaction in mCMV-dependent hearing loss. Additionally, NK cell recruitment to sites of infection was evident in the temporal bone of inoculated susceptible mouse strains. These results demonstrate participation of NK cells in protection from CMV-induced labyrinthitis and SNHL in mice.</p></div

Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection

Методы повышения эффективности работы газотурбинных установок

Объект исследования: газотурбинная установка. Цель работы: аналитический обзор современных направлений повышения эффективности работы ГТУ, сравнительный расчет простой и регенеративной ГТУ при заданных равных условиях. Для достижения поставленной цели рассмотрены следующие задачи: проведение литературного обзора современных направлений повышения эффективности работы газотурбинных установок; сравнительный тепловой расчет простой и регенеративной ГТУ при заданных равных условиях; Определение сметной стоимости работ по монтажу регенератора; анализ вредныхThe object of the study: gas turbine plant. The purpose of the work: analytical review of modern directions of increasing the efficiency of GTP operation, comparative calculation of simple and regenerative GTP under given equal conditions. To achieve this goal, the following tasks are considered: conducting a literature review of modern technologies to increase the efficiency of gas turbine plants; comparative heat calculation of simple and regenerative gas turbine plant (efficiency, specific fuel consumption, heat rate) for given equal conditions; determination of the estimated cost of

Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a Km of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans

HSV Infection Induces Production of ROS, which Potentiate Signaling from Pattern Recognition Receptors: Role for S-glutathionylation of TRAF3 and 6

The innate immune response constitutes the first line of defense against infections. Pattern recognition receptors recognize pathogen structures and trigger intracellular signaling pathways leading to cytokine and chemokine expression. Reactive oxygen species (ROS) are emerging as an important regulator of some of these pathways. ROS directly interact with signaling components or induce other post-translational modifications such as S-glutathionylation, thereby altering target function. Applying live microscopy, we have demonstrated that herpes simplex virus (HSV) infection induces early production of ROS that are required for the activation of NF-κB and IRF-3 pathways and the production of type I IFNs and ISGs. All the known receptors involved in the recognition of HSV were shown to be dependent on the cellular redox levels for successful signaling. In addition, we provide biochemical evidence suggesting S-glutathionylation of TRAF family proteins to be important. In particular, by performing mutational studies we show that S-glutathionylation of a conserved cysteine residue of TRAF3 and TRAF6 is important for ROS-dependent activation of innate immune pathways. In conclusion, these findings demonstrate that ROS are essential for effective activation of signaling pathways leading to a successful innate immune response against HSV infection