31 research outputs found

    Bench-to-bedside review: Developmental influences on the mechanisms, treatment and outcomes of cardiovascular dysfunction in neonatal versus adult sepsis

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    Sepsis is a significant cause of morbidity and mortality in neonates and adults, and the mortality rate doubles in patients who develop cardiovascular dysfunction and septic shock. Sepsis is especially devastating in the neonatal population, as it is one of the leading causes of death for hospitalized infants. In the neonate, there are multiple developmental alterations in both the response to pathogens and the response to treatment that distinguish this age group from adults. Differences in innate immunity and cytokine response may predispose neonates to the harmful effects of pro-inflammatory cytokines and oxidative stress, leading to severe organ dysfunction and sequelae during infection and inflammation. Underlying differences in cardiovascular anatomy, function and response to treatment may further alter the neonate's response to pathogen exposure. Unlike adults, little is known about the cardiovascular response to sepsis in the neonate. In addition, recent research has demonstrated that the mechanisms, inflammatory response, response to treatment and outcome of neonatal sepsis vary not only from that of adults, but vary among neonates based on gestational age. The goal of the present article is to review key pathophysiologic aspects of sepsis-related cardiovascular dysfunction, with an emphasis on defining known differences between adult and neonatal populations. Investigations of these relationships may ultimately lead to 'neonate-specific' therapeutic strategies for this devastating and costly medical problem

    The ABC130 barrel module prototyping programme for the ATLAS strip tracker

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    For the Phase-II Upgrade of the ATLAS Detector, its Inner Detector, consisting of silicon pixel, silicon strip and transition radiation sub-detectors, will be replaced with an all new 100 % silicon tracker, composed of a pixel tracker at inner radii and a strip tracker at outer radii. The future ATLAS strip tracker will include 11,000 silicon sensor modules in the central region (barrel) and 7,000 modules in the forward region (end-caps), which are foreseen to be constructed over a period of 3.5 years. The construction of each module consists of a series of assembly and quality control steps, which were engineered to be identical for all production sites. In order to develop the tooling and procedures for assembly and testing of these modules, two series of major prototyping programs were conducted: an early program using readout chips designed using a 250 nm fabrication process (ABCN-25) and a subsequent program using a follow-up chip set made using 130 nm processing (ABC130 and HCC130 chips). This second generation of readout chips was used for an extensive prototyping program that produced around 100 barrel-type modules and contributed significantly to the development of the final module layout. This paper gives an overview of the components used in ABC130 barrel modules, their assembly procedure and findings resulting from their tests.Comment: 82 pages, 66 figure

    MAPK signaling drives inflammation in LPS-stimulated cardiomyocytes: the route of crosstalk to G-protein-coupled receptors.

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    Profound cardiovascular dysfunction is an important cause of mortality from septic shock. The molecular underpinnings of cardiac dysfunction during the inflammatory surge of early sepsis are not fully understood. MAPKs are important signal transducers mediating inflammation whereas G-protein signaling pathways modulate the cardiac response to stress. Using H9c2 cardiomyocytes, we investigated the interaction of MAPK and G-protein signaling in a sepsis model to test the hypothesis that the cardiomyocyte inflammatory response is controlled by MAPKs via G-protein-mediated events. We found that LPS stimulated proinflammatory cytokine production was markedly exacerbated by siRNA knockdown of the MAPK negative regulator Mkp-1. Cytokine production was blunted when cells were treated with p38 inhibitor. Two important cellular signaling molecules typically regulated by G-protein-coupled receptors, cAMP and PKC activity, were also stimulated by LPS and inflammatory cytokines TNF-α and IL-6, through a process regulated by Mkp-1 and p38. Interestingly, neutralizing antibodies against Gα(s) and Gα(q) blocked the increase in cellular cAMP and PKC activation, respectively, in response to inflammatory stimuli, indicating a critical role of G-protein coupled receptors in this process. LPS stimulation increased COX-2 in H9c2 cells, which also express prostaglandin receptors. Blockade of G-protein-coupled EP4 prostaglandin receptor by AH 23848 prevented LPS-induced cAMP increase. These data implicate MAPKs and G-proteins in the cardiomyocyte inflammatory response to LPS as well as crosstalk via COX-2-generated PGE(2). These data add to our understanding of the pathogenesis of septic shock and have the potential to guide the selection of future therapeutics

    LPS transiently activates MAP kinases in H9c2 cardiomyocytes.

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    <p>(<i>A</i>) The kinetics of p38 and ERK activation and Mkp-1 induction in response to LPS stimulation. H9c2 cardiomyocytes were stimulated with LPS (5 µg/ml) for indicated periods of time prior to harvest harvested. Levels of Mkp-1, phospho-38 and phospho-ERK were assessed by Western blot. The membrane was stripped and reblotted with an antibody against β-actin to verify comparable loading. (<i>B</i>) The effects of Mkp-1 knockdown on p38 and ERK activity in LPS-stimulated H9c2 cells. H9c2 cells were transiently transfected with either siRNA of <i>Mkp1</i> (si<i>Mkp1</i>) or scrambled RNA (SC). Twenty-four h after transfection, the cells were stimulated with LPS for an additional 18 h. Cell lysates were subjected to Western blot. (<i>C</i>) Verification of siRNA transfection by fluorescent microscopy. H9c2 cells were transiently transfected with fluorescent siRNA of Lamin A/C. After 48 h, cells were fixed, stained with DAPI, and subjected to fluorescent microscopy. Note that nuclei were stained blue and cytosolic compartments red due to the uptake of DY-547-labeled Lamin siRNA. Transfection efficiency was estimated to be >90%. Images presented are representative results (40× magnification).</p

    The production of IL-1β and IL-6 in response to LPS stimulation is positively regulated by p38 and negatively controlled by Mkp-1 in cardiomyocytes.

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    <p>H9c2 cells were transfected with <i>Mkp1</i> siRNA or a scrambled RNA control (SC). Twenty-four h after transfection, the cells were pretreated with either DMSO, as control (Con), or with the p38 inhibitor ED1428 for 30 min, and subsequently stimulated with 5 µg/mL LPS for 18 h. The concentrations of IL-6 (<i>A</i>) and IL-1β (<i>B</i>) in the culture medium were determined by ELISA. Data are presented as means ± SE from at least 3 independent experiments. *, <i>P</i><0.05.</p

    Mkp-1 negatively regulates cytokine expression in LPS-stimulated H9c2 cardiomyocytes.

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    <p>H9c2 cells were transfected with <i>Mkp1</i> siRNA or a scrambled RNA control (SC). After 48 h, cells were stimulated with 5 µg/mL LPS for either 1 or 4 h. Total RNA was harvested, and cDNA synthesized using reverse transcriptase. Expression levels of TNF-α (<i>A</i>) and IL-6 (<i>B</i>) were assessed by qPCR. The expression of TNF-α and IL-6 were normalized to the housekeeping gene RP30, and presented as fold change relative to scramble RNA-transfected, unstimulated cells. Data are means ± SE from at least 3 independent experiments. *, <i>P</i><0.05.</p

    PGE<sub>2</sub> receptor EP4 plays an important role in mediating increased intracellular cAMP during the cardiomyocyte response to LPS.

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    <p>(<i>A</i>) The effects of <i>Mkp1</i> knockdown on COX-2 mRNA induction by LPS. H9c2 cardiomyocytes were transfected with either scrambled RNA (SC) or <i>Mkp1</i> siRNA. After 48 h, cells were stimulated with LPS (5 µg/mL) for 4 h or left unstimulated. Total RNA was harvested and COX-2 expression was assessed by PCR. COX-2 expression is presented as fold change relative to unstimulated SC controls. (<i>B</i>) The effects of LPS on COX-2 protein expression. H9c2 cells were stimulated with LPS (5 µg/mL) for the indicated periods of time. COX-2 protein in lysates was assessed by Western blot. The membrane was stripped and reblotted with a β-actin antibody to verify comparable protein loading. (<i>C</i>) Detection of the mRNA transcripts of different prostaglandin receptors in H9c2 cardiomyocytes. Total RNA was extracted from unstimulated H9c2 cells. The transcripts of the receptors of PGE<sub>2</sub> (EP1–4) and PGD<sub>2</sub> (DP) were amplified by RT-PCR. PCR products were separated by electrophoresis on a 2% agarose gel. (<i>D</i>) The differential effects of prostaglandin receptor antagonists on LPS-induced increases in cAMP. H9c2 cells were first pretreated with DMSO (as control (Con)), 10 µM AH23848 (an inhibitor of EP4), or 10 µM AH 6809 (inhibitor of EP1, EP2, EP3, and DP) for 60 min, and then stimulated with LPS for an additional 18 h. Cells were harvested and intracellular cAMP levels were determined using an EIA kit. Data represent means ± SE of at least 3 experiments. *, <i>P</i><0.05. Images shown in panels <i>B</i> and <i>C</i> are representative results.</p
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