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Author(s): Ryoichi Kawaguchi [sup.1] , Naoyuki Hirata [sup.1] , Yasuyuki Tokinaga [sup.1] , Shunsuke Hayashi [sup.1] , Shigeaki Inoue [sup.2] , Nobuo Watanabe [sup.2] , Michiaki Yamakage [sup.1] Author Affiliations: [...], Purpose A specific therapeutic strategy in sepsis-induced myocardial dysfunction remains to be determined. Nitrite may have cardioprotective effects against sepsis-induced myocardial dysfunction. This study investigated the cardioprotective effects of nitrite on myocardial function, mitochondrial bioenergetics, and its underlying molecular mechanisms in severe septic rats. Methods Sepsis was induced in male Wistar rats by cecal ligation and puncture (CLP). After CLP, we administered normal saline (NS group) or nitrite (nitrite group) subcutaneously. We administered nitrite at different doses (0.1-10 mg/kg) to ascertain the most effective dose and examined cardiac function in an isolated heart experiment 8 h after CLP. We investigated mitochondrial bioenergetics and molecular mechanisms underlying the administration of nitrite in vitro. Results In isolated heart experiments, the left ventricular developed pressure (96 ± 5 mmHg) at a moderate nitrite dose (1.0 mg/kg) was significantly higher than that in the NS group (75 ± 4 mmHg, P < 0.05). Mitochondrial oxidative phosphorylation in the nitrite group was significantly higher than that in the NS group (P < 0.01). Immunoblotting revealed that nitrite significantly increased the phosphorylation of Akt (P < 0.05) and reduced the nuclear translocation of NF-[kappa]B (P < 0.05) compared with the NS group. Nitrite was also shown to improve the rate of survival in severe septic rats (P < 0.001). Conclusions Our results showed that a moderate nitrite dose improved septic myocardial dysfunction at organ, cellular, and molecular levels via modulation of stress signal responses, which resulted in an improvement in survival.

Introduction Sepsis is defined as systemic inflammatory response syndrome (SIRS) caused by infection (Russell 2006). Sepsis and severe sepsis (sepsis accompanied by acute organ dysfunction) are the leading causes of [...], Transforming growth factor p induced protein (TGFBIp) is an extracellular matrix protein expressed in several cell types in response to TGF-p. TGFBIp is released by human umbilical vein endothelial cells (HUVECs) and functions as a mediator of experimental sepsis. Pelargonidin (PEL) is a well-known red pigment found in plants, and has been reported as having important biological activities that are potentially beneficial for human health. This study was undertaken to investigate whether PEL can modulate TGFBIp-mediated inflammatory responses in HUVECs and in mice. The anti-inflammatory activities of PEL were determined by measuring permeability, leukocyte adhesion and migration, and activation of proinflammatory proteins in TGFBIp-activated HUVECs and mice. In addition, the beneficial effects of PEL on survival rate in a mouse sepsis model were tested. We found that PEL inhibited TGFBIp-induced barrier disruption, expression of cell adhesion molecules and adhesion/transendothelial migration of neutrophils to human endothelial cells. PEL also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that PEL possesses anti-inflammatory properties that result in inhibition of hyperpermeability, expression of cell adhesion molecules, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases. Key words: anthocyanin, anthocyanidin, pelargonidin, TGFBIp, sepsis. La proteine TGFBIp (acronyme de "transforming growth factor p induced protein") est une proteine de la matrice extracellulaire exprimee par plusieurs types cellulaires en reponse au TGF-p. La TGFBIp est liberee par les cellules endotheliales humaines de cordon ombilical (HUVEC) et elle agit comme mediateur de la sepsie experimentale. La pelargonidine (PEL) est un pigment rouge bien connu d&apos;origine vegetale et on rapporte qu&apos;il exerce d&apos;importantes activites biologiques potentiellement benefiques pour la sante humaine. Cette etude a ete entreprise afin d&apos;examiner si la PEL peut moduler les reponses inflammatoires mediees par la TGFBIp chez les HUVEC et la souris. Les activites anti-inflammatoires de la PEL ont ete determinees en mesurant la permeabilite, l&apos;adhesion et la migration des leucocytes et l&apos;activation de proteines pro-inflammatoires chez les HUVEC et les souris traitees a la TGFBIp. De plus, les effets benefiques de la PEL sur le taux de survie dans un modele de sepsie chez la souris ont ete testes. Les auteurs ont trouve que la PEL inhibait la rupture de la barriere, l&apos;expression de molecules d&apos;adhesion cellulaire et l&apos;adhesion/migration transendotheliale des neutrophiles aux cellules endotheliales humaines, phenomenes induits par la TGFBIp. La PEL supprimait aussi l&apos;hyperpermeabilite et la migration des leucocytes induites par la TGFBIp in vivo. Ces resultats suggerent que la PEL possede des proprietes anti-inflammatoires qui resultent en l&apos;inhibition de l&apos;hyperpermeabilite, l&apos;expression de molecules d&apos;adhesion cellulaire et l&apos;adhesion et la migration des leucocytes, soutenant ainsi son utilite comme therapie des maladies inflammatoires vasculaires. [Traduit par la Redaction] Mots-cles : anthocyanine, anthocyanidine, pelargonidine, TGFBIp, sepsie.

Although lipopolysaccharide (LPS)-induced proinflammatory molecules are indispensable for counteracting the growth and dissemination of Gram-negative bacteria, overproduction can lead to sepsis syndrome, also known as endotoxin shock. However, a prior [...], Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

Muscle wasting during sepsis is in part regulated by glucocorticoids. In recent studies, treatment of cultured muscle cells in vitro with dexamethasone upregulated expression arid activity of p300, a histone acetyl transferase (HAT), and reduced expression and activity of the histone deacetylases-3 (HDAC3) and -6, changes that favor hyperacetylation. Here, we tested the hypothesis that sepsis and glucocorticoids regulate p300 and HDAC3 and -6 in skeletal muscle in vivo. Because sepsis-induced metabolic changes are particularly pronounced in white, fast-twitch skeletal muscle, most experiments were performed in extensor digitorum longus muscles. Sepsis in rats upregulated p300 mRNA and protein levels, stimulated HAT activity, and reduced HDAC6 expression and HDAC activity. The sepsis-induced changes in p300 and HDAC expression were prevented by the glucocorticoid receptor antagonist RU38486. Treatment of rats with dexamethasone increased expression of p300 and HAT activity, reduced expression of HDAC3 and -6, and inhibited HDAC activity. Finally, treatment with the HDAC inhibitor trichostatin A resulted in increased muscle proteolysis and expression of the ubiquitin ligase atrogin-1. Taken together, our results suggest for the first time that sepsis-induced muscle wasting may be regulated by glucocorticoid-dependent hyperacetylation caused by increased p300 and reduced HDAC expression and activity. The recent development of pharmacological HDAC activators may provide a novel avenue to prevent and treat muscle wasting in sepsis and other catabolic conditions. acetylation; muscle wasting doi: 10.1152/ajpregu.00858.2009.

van der Hoven B, van Pelt H, Swart EL, Bonthuis F, Tilanus HW, Bakker J, Gommers D. Noninvasive functional liver blood flow measurement: comparison between bolus dose and steady-state clearance of sorbitol in a small-rodent model. Am J Physiol Gastrointest Liver Physiol 298: G177-G181, 2010. First published November 25, 2009; doi: 10.1152/ajpgi.90688.2008.--Plasma clearance of D-sorbitol, a nontoxic polyol, occurs predominantly in the liver and has been used to measure functional liver blood flow after bolus and steady- state intravenous administration. However, it is not known which of these two administration methods is superior. Therefore, plasma D-sorbitol clearance was studied in an animal model both after a bolus dose and under steady-state (SS) conditions and compared directly with liver blood flow, under normal conditions, and after the induction of endotoxin (LPS) sepsis. Adult male Wistar rats (526 [+ or -] 38 g body wt; n = 27) were anesthetized and mechanically ventilated. Hemodynamics, hepatic arterial flow, and portal venous flow were measured. Two groups were studied, namely healthy animals that served as controls and a sepsis group that received 5 mg/kg LPS intravenously (Escherichia coli O127:B8). Each animal received either a SS infusion (0.1 rag/100 g body wt per rain) or a bolus (3 rag/100 g body wt) of a 5% D-sorbitol solution intravenously in a randomized order. After the initial measurements and a 60-min pause time in between ([T.sub.1/2,sorbitol] = 9 min), a crossover was done. The hepatic clearance of D-sorbitol in the control group showed a good correlation between bolus and SS (Spearman's r = 0.7681, P = 0.0004), and both techniques correlated well with total liver blood flow (TLBF) (r = 0.7239, P = 0.0023 and r = 0.7226, P = 0.0023, respectively). Also in the sepsis group there was a good correlation between bolus and SS sorbitol clearance (r = 0.6655, P = 0.0182). In the sepsis group, only the SS clearance correlated with TLBF (r = 0.6434, P = 0.024). In conclusion, in normal and under septic conditions, hepatic clearance of D-sorbitol either by bolus or a SS infusion is comparable. In healthy animals, this also correlated well with TLBF but not in septic conditions. However, this is expected because of the changes in the liver microcirculation, shunting, and decreased hepatocyte function in sepsis. microcirculation; rats; sepsis doi: 10.1152/ajpgi.90688.2008.

Am J Physiol Renal Physiol 298: F196-F204, 2010. First published October 14, 2009; doi: 10.1152/ajprenal.90607.2008.--Acute renal failure (ARF) is frequently associated with polyuria and urine concentration defects and it is a severe complication of sepsis because it increases the mortality rate. Inhibition of NF-[kappa]B activation has been suggested to provide a useful strategy for the treatment of septic shock. However, the impact on sepsis-induced ARF is still unclear. Therefore, we examined the effect of pyrrolidine dithiocarbamate (PDTC) and of small interfering RNA (siRNA) silencing NF-[kappa]B p50/p105 on sepsis-induced downregulation of vasopressin [V.sub.2] receptors and aquaporin (AQP)-2 channels using a cecal ligation and puncture (CLP) mouse model. CLP caused a time-dependent downregulation of renal vasopressin [V.sub.2] receptor and of AQP2 expression without alterations in plasma vasopressin levels. Renal activation of NF-[kappa]B in response to CLP was attenuated by PDTC pretreatment, which also attenuated the downregulation of V2 receptor and AQP2 expression. Furthermore, a strong nuclear staining for the NF-[kappa]B p50 subunit throughout the whole kidney in response to CLP was observed, siRNA against NF-[kappa]B p50 attenuated the CLP-induced nuclear translocation of the p50 subunit and the CLP-induced downregulation of [V.sub.2] receptor and AQP2 expression. Additionally, PDTC and siRNA pretreatment inhibited the CLP-induced increase in renal TNF-[alpha] and IL-1[beta] concentration and NOS-2 mRNA abundance. Moreover, PDTC and siRNA pretreatment ameliorated CLP-induced hypotension and ARF. Our findings suggest that NF-[kappa]B activation is of importance for the downregulation of AQP2 channel and vasopressin [V.sub.2] receptor expression during sepsis. In addition, our data indicate that NF-[kappa]B inhibition ameliorates sepsis-induced ARF. cecal ligation and puncture; kidney; inflammation; vasopressin; cytokines

Byline: Martina Crivellari (1), Patrizia Della Valle (2), Giovanni Landoni (1), Federico Pappalardo (1), Chiara Gerli (1), Elena Bignami (1), Giovanni Marino (1), Alberto Zangrillo (1), Armando D'Angelo (2) Keywords: Bleeding; Cardiac surgery; Critically ill; Organ failure; Protein C concentrate; Septic shock Abstract: Purpose To describe outcome and changes in clotting and inflammatory parameters in an uncontrolled case series of consecutive patients with severe sepsis who received protein C concentrate soon after cardiac surgery. Methods From January 2007 to January 2008 nine consecutive adult patients with severe sepsis or septic shock and two or more organ failure after cardiac surgery received protein C concentrate, 50 IU/kg as a bolus followed by continuous infusion of 3 IU/kg per hour for 72 h. Results The increase in protein C levels was accompanied by an early drop in interleukins and near-normalization of prothrombin time, activated partial thromboplastin time, antithrombin and thrombin--antithrombin complex levels (p a$? 0.03). No patient experienced drug-related side effects. Thirty-day mortality was 11% (1 patient) compared to the expected mortality of 68%. Conclusions In this pilot, uncontrolled study of nine patients with sepsis-induced double organ failure following cardiac surgery, treatment with protein C concentrate was associated with significant improvement in clinical, inflammatory and clotting parameters, no bleeding and low 30-day mortality. Author Affiliation: (1) Department of Cardiac Anesthesia and Intensive Care, Universita Vita-Salute San Raffaele, Via Olgettina 60 Milano, Milan, 20132, Italy (2) Coagulation Service and Thrombosis Research Unit, Universita Vita-Salute San Raffaele, Milan, Italy Article History: Registration Date: 14/07/2009 Received Date: 26/01/2009 Accepted Date: 09/07/2009 Online Date: 01/08/2009 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00134-009-1584-3) contains supplementary material, which is available to authorized users.

Systemic inflammation induces a multiple organ dysfunction syndrome that contributes to morbidity and mortality in septic patients. Since increasing plasma apolipoprotein A-I (apoA-I) and HDL may reduce the complications of sepsis, we tested the hypothesis that the apoA-I mimetic peptide 4F confers similar protective effects in rats undergoing cecal ligation and puncture (CLP) injury. Male Sprague-Dawley rats were randomized to undergo CLP or sham surgery. IL-6 levels were significantly elevated in plasma by 6 h after CLP surgery compared with shams. In subsequent studies, CLP rats were further subdivided to receive vehicle or 4F (10 mg/kg) by intraperitoneal injection, 6 h after sepsis induction. Sham-operated rats received saline. Echocardiographic studies showed a reduction in left ventricular end-diastolic volume, stroke volume, and cardiac output (CO) 24 h after CLP surgery. These changes were associated with reduced blood volume and left ventricular filling pressure. 4F treatment improved blood volume status, increased CO, and reduced plasma IL-6 in CLP rats. Total cholesterol (TC) and HDL were 79 [+ or -] 5 and 61 [+ or -] 4 mg/dl, respectively, in sham rats. TC was significantly reduced in CLP rats (54 [+ or -] 3 mg/dl) due to a reduction in HDL (26 [+ or -] 3 mg/dl). 4F administration to CLP rats attenuated the reduction in TC (69 [+ or -] 4 mg/dl) and HDL (41 [+ or -] 3 mg/dl) and prevented sepsis-induced changes in HDL protein composition. Increased plasma HDL in 4F-treated CLP rats was associated with an improvement in CO and reduced mortality. It is proposed that protective effects of 4F are related to its ability to prevent the sepsis-induced reduction in plasma HDL. sepsis; apolipoprotein A-I mimetic peptide; high-density lipoprotein; cardiac function; inflammation

Byline: Patrick Scheiermann (1), Devan Ahluwalia (2), Sandra Hoegl (1), Andrea Dolfen (2), Marc Revermann (2), Bernhard Zwissler (1), Heiko Muhl (3), Kim A. Boost (1), Christian Hofstetter (2) Keywords: Levosimendan; Cecal ligation and incision; Severe sepsis; Cytokines; Apoptosis; Rat Abstract: Purpose We aimed at comparing the effects of intravenous (i.v.) and inhaled (inh.) levosimendan (LEVO) on survival, inflammatory cytokines and the apoptotic mediator caspase-3 in a rat model of severe sepsis induced by cecal ligation and incision (CLI). Methods Twenty-eight anesthetized/ventilated male Sprague--Dawley rats (body weight 528 +- 20 g) underwent laparotomy. Cecal mobilisation served as control (SHAM, n = 7). In all other groups, severe sepsis was induced by CLI. No further intervention occurred in the CLI-group (n = 7). 180 min after CLI, 24 ug/kg i.v. LEVO was administered in the CLI + LEVO-IV-group (n = 7), and 24 ug/kg inh. LEVO was administered via jet nebulizer in the CLI + LEVO-INH-group (n = 7). Results CLI induced arterial hypotension, with i.v. and inh. LEVO attenuating blood pressure decrease over 390 min [CLI 34(31/50), CLI + LEVO-IV 82(69/131)*, CLI + LEVO-INH 78(62/85)* mmHg median(25/75% quartile), *P < 0.05]. CLI induced metabolic acidosis. I.v. and inh. LEVO avoided arterial pH [CLI 7.18(7.16/7.2), CLI + LEVO-IV 7.27(7.24/7.31)*, CLI + LEVO-INH 7.26(7.24/7.28)*] and base excess deterioration [CLI -19(-21.8/-17.9), CLI + LEVO-IV -13(-14.8/-12)*, CLI + LEVO-INH -12.7(-14/-12.2)* mmol/l]. Overall mortality in the CLI-group was 57% compared to 0%* in both LEVO-treated groups after 390 min. LEVO administration significantly attenuated the increase in proinflammatory interleukin (IL)-1[beta] [CLI 896(739/911), CLI + LEVO-IV 302(230/385)*, CLI + LEVO-INH 346(271/548) pg/ml] and IL-6 [CLI 35651(31413/35816), CLI + LEVO-IV 21156(18397/28026), CLI + LEVO-INH 13674(10105/24843) pg/ml] in the plasma and reduced cleaved caspase-3 expression in the spleen. Conclusions In a rat model of severe sepsis induced by CLI, i.v. and inh. LEVO equally attenuated arterial hypotension, metabolic acidosis and prolonged survival. Moreover, i.v. and inh. LEVO inhibited proinflammatory mediator release and reduced splenic caspase-3 expression. Author Affiliation: (1) Department of Anaesthesiology, Hospital of the Ludwig-Maximilians-University Munich, Marchioninistr. 15, 81377, Munich, Germany (2) Department of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany (3) Pharmazentrum/ZAFES, Hospital of the Johann Wolfgang Goethe-University, Frankfurt/Main, Germany Article History: Registration Date: 30/03/2009 Received Date: 18/08/2008 Accepted Date: 13/02/2009 Online Date: 15/04/2009 Article note: Parts of this study have been presented at the 2008 meeting of the German Society of Anaesthesiology and Intensive Care Medicine (DGAI) in Wurzburg, Germany.

Byline: Alex Gnaegi (1), Francois Feihl (1), Olivier Boulat (2), Bernard Waeber (1), Lucas Liaudet (1,3) Keywords: ARDS; Sepsis; Hypercapnia; Mechanical ventilation; Energy metabolism Abstract: Purpose Low tidal volume ventilation and permissive hypercapnia are required in patients with sepsis complicated by ARDS. The effects of hypercapnia on tissue oxidative metabolism in this setting are unknown. We therefore determined the effects of moderate hypercapnia on markers of systemic and splanchnic oxidative metabolism in an animal model of endotoxemia. Methods Anesthetized rats maintained at a PaCO.sub.2 of 30, 40 or 60 mmHg were challenged with endotoxin. A control group (PaCO.sub.2 40 mmHg) received isotonic saline. Hemodynamic variables, arterial lactate, pyruvate, and ketone bodies were measured at baseline and after 4 h. Tissue adenosine triphosphate (ATP) and lactate were measured in the small intestine and the liver after 4 h. Results Endotoxin resulted in low cardiac output, increased lactate/pyruvate ratio and decreased ketone body ratio. These changes were not influenced by hypercapnia, but were more severe with hypocapnia. In the liver, ATP decreased and lactate increased independently from PaCO.sub.2 after endotoxin. In contrast, the drop of ATP and the rise in lactate triggered by endotoxin in the intestine were prevented by hypercapnia. Conclusions During endotoxemia in rats, moderate hypercapnia prevents the deterioration of tissue energetics in the intestine. Author Affiliation: (1) Division of Clinical Pathophysiology and Medical Teaching, Faculty of Biology and Medicine, University Hospital Center, CHUV-BH 08-621, 1011, Lausanne, Switzerland (2) Central Laboratory of Clinical Chemistry, Faculty of Biology and Medicine, University Hospital Center, CHUV-BH 08-621, 1011, Lausanne, Switzerland (3) Department of Intensive Care Medicine, Faculty of Biology and Medicine, University Hospital Center, 1011, Lausanne, Switzerland Article History: Registration Date: 30/03/2009 Received Date: 28/10/2008 Accepted Date: 22/03/2009 Online Date: 17/04/2009

Byline: Erica L. T. Akker (1,2,4), Jan W. Koper (2), Koen Joosten (3), Frank H. Jong (2), Jan A. Hazelzet (3), Steven W. J. Lamberts (2), Anita C. S. Hokken-Koelega (1) Keywords: Children; Corticosteroids; Cortisol sensitivity; Sepsis; Septic shock Abstract: Objective Corticosteroids are used in sepsis treatment to benefit outcome. However, discussion remains on which patients will benefit from treatment. Inter-individual variations in cortisol sensitivity, mediated through the glucocorticoid receptor, might play a role in the observed differences. Our aim was to study changes in mRNA levels of three glucocorticoid receptor splice variants in neutrophils of children with sepsis. Patients and design Twenty-three children admitted to the pediatric intensive care unit with sepsis or septic shock were included. Neutrophils were isolated at days 0, 3 and 7, and after recovery (>3 months). mRNA levels of the glucocorticoid receptor splice variants GR-[alpha] (determining most of the cortisol effect), GR-P (increasing GR-[alpha] effect) and GR-[beta] (inhibitor of GR-[alpha]) were measured quantitatively. Main results Neutrophils from sepsis patients showed decreased levels of glucocorticoid receptor mRNA of the GR-[alpha] and GR-P splice variants on day 0 compared to after recovery. GR-[alpha] and GR-P mRNA levels showed a gradual recovery on days 3 and 7 and normalized after recovery. GR-[beta] mRNA levels did not change significantly during sepsis. GR expression was negatively correlated to interleukin-6 (a measure of disease severity, r = -0.60, P = 0.009). Conclusions Children with sepsis or septic shock showed a transient depression of glucocorticoid receptor mRNA in their neutrophils. This feature may represent a tissue-specific adaptation during sepsis leading to increased cortisol resistance of neutrophils. Our study adds to understanding the mechanism of cortisol sensitivity in immune cells. Future treatment strategies, aiming at timing and tissue specific regulation of glucocorticoids, might benefit patients with sepsis or septic shock. Author Affiliation: (1) Division of Pediatric Endocrinology, Department of Pediatrics, Erasmus MC, Rotterdam, The Netherlands (2) Department of Internal Medicine, Erasmus MC, Rotterdam, The Netherlands (3) Division of Pediatric Intensive Care, Department of Pediatrics, Erasmus MC, Rotterdam, The Netherlands (4) Division of Pediatric Endocrinology, Department of Pediatrics, Erasmus MC, Room sp 1536, PO Box 2060, 3000 CB, Rotterdam, The Netherlands Article History: Registration Date: 23/03/2009 Received Date: 19/05/2008 Accepted Date: 22/02/2009 Online Date: 17/04/2009

Byline: Michael R. Marchick (1), Jeffrey A. Kline (1), Alan E. Jones (1) Keywords: Hypotension; Sepsis; Shock; Mortality; Emergency medicine Abstract: Objective Few studies have documented the incidence and significance of non-sustained hypotension in emergency department (ED) patients with sepsis. We hypothesized that ED non-sustained hypotension increases risk of in-hospital mortality in patients with sepsis. Methods Secondary analysis of a prospective cohort study. ED patients aged >17 years admitted to the hospital with explicitly defined sepsis were prospectively identified. Inclusion criteria Evidence of systemic inflammation (>1 criteria) and suspicion for infection. Patients with overt shock were excluded. The primary outcome was in-hospital mortality. Results Seven hundred patients with sepsis were enrolled, including 150 (21%) with non-sustained hypotension. The primary outcome of in-hospital mortality was present in 10% (15/150) of patients with non-sustained hypotension compared with 3.6% (20/550) of patients with no hypotension. The presence of non-sustained hypotension resulted in three times the risk of mortality than no hypotension (risk ratio = 2.8, 95% CI 1.5--5.2). Patients with a lowest systolic blood pressure Conclusion Non-sustained hypotension in the ED confers a significantly increased risk of death during hospitalization in patients admitted with sepsis. These data should impart reluctance to dismiss non-sustained hypotension, including a single measurement, as not clinically significant or meaningful. Author Affiliation: (1) Emergency Medicine Research, Department of Emergency Medicine, Carolinas Medical Center, PO Box 32861, Charlotte, NC, 28232-2861, USA Article History: Registration Date: 10/02/2009 Received Date: 03/01/2009 Accepted Date: 03/02/2009 Online Date: 24/02/2009 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00134-009-1448-x) contains supplementary material, which is available to authorized users.

Byline: Martin W. Dunser (1), Jukka Takala (1), Hanno Ulmer (2), Viktoria D. Mayr (3), Gunter Luckner (3), Stefan Jochberger (3), Fritz Daudel (1), Philipp Lepper (1), Walter R. Hasibeder (4), Stephan M. Jakob (1) Keywords: Hypotension; Mean arterial blood pressure; Mean perfusion pressure; Sepsis Abstract: Objective To evaluate the association between arterial blood pressure (ABP) during the first 24 h and mortality in sepsis. Design Retrospective cohort study. Setting Multidisciplinary intensive care unit (ICU). Patients and participants A total of 274 septic patients. Interventions None. Measurements and results Hemodynamic, and laboratory parameters were extracted from a PDMS database. The hourly time integral of ABP drops below clinically relevant systolic arterial pressure (SAP), mean arterial pressure (MAP), and mean perfusion pressure (MPP = MAP - central venous pressure) levels was calculated for the first 24 h after ICU admission and compared with 28-day-mortality. Binary and linear regression models (adjusted for SAPS II as a measure of disease severity), and a receiver operating characteristic (ROC) analysis were applied. The areas under the ROC curve were largest for the hourly time integrals of ABP drops below MAP 60 mmHg (0.779 vs. 0.764 for ABP drops below MAP 55 mmHg P a$? 0.01) and MPP 45 mmHg. No association between the hourly time integrals of ABP drops below certain SAP levels and mortality was detected. One or more episodes of MAP < 60 mmHg increased the risk of death by 2.96 (CI 95%, 1.06--10.36, P = 0.04). The area under the ROC curve to predict the need for renal replacement therapy was highest for the hourly time integral of ABP drops below MAP 75 mmHg. Conclusions A MAP level aY= 60 mmHg may be as safe as higher MAP levels during the first 24 h of ICU therapy in septic patients. A higher MAP may be required to maintain kidney function. Author Affiliation: (1) Department of Intensive Care Medicine, Inselspital, 3010, Bern, Switzerland (2) Institute of Medical Biostatistics, Innsbruck Medical University, Innsbruck, Austria (3) Department of Anesthesiology and Critical Care Medicine, Innsbruck Medical University, Innsbruck, Austria (4) Department of Anesthesiology and Critical Care Medicine, Krankenhaus Ried im Innkreis, Ried im Innkreis, Austria Article History: Registration Date: 22/01/2009 Received Date: 29/05/2008 Accepted Date: 19/01/2009 Online Date: 03/02/2009 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00134-009-1427-2) contains supplementary material, which is available to authorized users.

Infections produce severe respiratory muscle dysfunction. It is known that the proteasome proteolytic system is activated in skeletal muscle in sepsis, and it has been postulated that this degradative pathway is responsible for inducing skeletal muscle weakness and wasting. The objective of this study was to determine if administration of proteasomal inhibitors (MGI32, epoxomicin, bortezomib) can prevent sepsis-induced diaphragm weakness. Rats were given either 1) saline (0.5 ml ip), 2) endotoxin (12 mg/kg ip), 3) endotoxin plus MG132 (2.5 mg/kg), 4) endotoxin plus epoxomicin (1 [micro]mo1/kg), or 5) endotoxin plus bortezomib (0.05 mg/kg). Animals were killed either 48 or 96 h after injections, and assessments were made of diaphragm proteolysis, force-frequency relationships, mass, protein content, and caspase activation. Endotoxin increased proteolysis (P caspase; sepsis; skeletal muscle

Byline: Jaume Mesquida (1), Jordi Masip (1), Gisela Gili (1), Antoni Artigas (1), Francisco Baigorri (1) Keywords: Near-infrared spectroscopy; Sepsis; Microcirculation; Tissue oxygenation Abstract: Objective To validate thenar oxygen saturation (StO.sub.2) measured by near-infrared spectroscopy (NIRS) as a noninvasive estimation of central venous saturation (ScvO.sub.2) in septic patients. Design Prospective observational study. Setting A 26-bed medical--surgical intensive care unit at a university-affiliated hospital. Patients Patients consecutively admitted to the ICU in the early phase of severe sepsis and septic shock, after normalization of blood pressure with fluids and/or vasoactive drugs. Measurements We recorded demographic data, severity score, hemodynamic data, and blood lactate, as well as ScvO.sub.2, and StO.sub.2 measured simultaneously on inclusion. Patients were divided into two groups according to ScvO.sub.2 values: group A, with ScvO.sub.2 < 70%, and group B, with ScvO.sub.2 aY= 70%. Results Forty patients were studied. StO.sub.2 was significantly lower in group A than in group B (74.7 +- 13.0 vs. 83.3 +- 6.2, P 0.018). No differences in age, severity score, hemodynamics, vasoactive drugs, or lactate were found between groups. Simultaneously measured ScvO.sub.2 and StO.sub.2 showed a significant Pearson correlation (r = 0.39, P 0.017). For a StO.sub.2 value of 75%, sensitivity was 0.44, specificity 0.93, positive predictive value 0.92, and negative predictive value 0.52 for detecting ScvO.sub.2 values lower than 70%. Conclusions StO.sub.2 correlates with ScvO.sub.2 in normotensive patients with severe sepsis or septic shock. We propose a StO.sub.2 cut-off value of 75% as a specific, rapid, noninvasive first step for detecting patients with low ScvO.sub.2 values. Further studies are necessary to analyze the role of noninvasive StO.sub.2 measurement in future resuscitation algorithms. Author Affiliation: (1) Critical Care Center, Hospital de Sabadell, Corporacio Parc Tauli, Institut Universitari Parc Tauli, CIBER Enfermedades Respiratorias, Universitat Autonoma de Barcelona, Parc Tauli s/n, 08208, Sabadell, Spain Article History: Registration Date: 16/01/2009 Received Date: 18/04/2008 Accepted Date: 03/12/2008 Online Date: 29/01/2009 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00134-009-1410-y) contains supplementary material, which is available to authorized users.

The roles of intracellular calcium concentration ([[[Ca.sup.2+].sub.i]) and [Ca.sup.2+] sensitization in lipopolysaccharide (LPS)-induced vascular smooth muscle (VSM) hyporesponsiveness are incompletely understood. To investigate these roles, contraction responses to endothelin-1 (ET-1) and 80 mM KC1; relaxation responses to nifedipine; the expression levels of mRNAs of ET-1 and its receptors ([ET.sub.A] or [ET.sub.B]); the expression levels of protein kinase C (PKC) and phosphorylation of Rho kinase (ROK[alpha], CPI-17, and myosin phosphatase target subunit-1 (MYPT1); and changes in aortic VSM cell [[[Ca.sup.2+].sub.i] were measured in LPS-treated aortic rings from male Wistar rats (250-300 g). LPS (10 [micro]g/ml, 20 h) decreased contraction induced by ET-1 (0.3-100 nM) or 80 mM KC1. LPS-induced hypocontractility was not observed in the absence of external [Ca.sup.2+], but LPS-treated aorta remained hypocontractile on subsequent stepwise restoration of extracellular [Ca.sup.2+] (0.01-10 mM). Vascular relaxation to nifedipine; mRNA expression levels of ET-I, [ET.sub.A], or [ET.sub.B]; protein expression levels of PKC; and phosphorylation levels of ROK[alpha], CPI-17, and MYPT1 were not affected by LPS. In isolated aortic VSM cells, ET-1 caused a transient initial increase in [[[Ca.sup.2+].sub.i], followed by a maintained tonic increase in [[[Ca.sup.2+].sub.i], which was decreased by LPS pretreatment and was dependent on external [Ca.sup.2+]. Subsequent restoration of extracellular [Ca.sup.2+] increased [[[Ca.sup.2+].sub.i], but this increase was lower in the LPS-treated group. This difference in response to extracellular [Ca.sup.2+] addition was not affected by diltiazem, but was abolished by SKF-96365. Therefore, LPS induces hyporeactivity to ET-1 in rat aorta that depends on external [Ca.sup.2+] influx through non-voltage-operated [Ca.sup.2+] channels, but not on ET-1 receptor expression or [Ca.sup.2+] sensitization. calcium sensitization; sepsis; vascular smooth muscle

To investigate the potential role of the local expression of alternative complement factor B (hBf) in human sepsis, we examined the induction of Bf gene expression in human peripheral blood monocytes (PBMCs) from patients with septic shock and the mechanisms of hBf gene regulation by tumor necrosis factor (TNF)-[alpha], interferon (IFN)-[gamma], and lipopolysaccharide (LPS) in human monocytes. PBMCs from septic shock patients showed increased hBf mRNA expression when compared with control patients. Costimulation with TNF-[alpha] and IFN-[gamma] or stimulation with LPS demonstrated a time-and dose-dependent induction of hBf mRNA expression in human PBMCs. A region of the hBf promoter between -735 and + 128 bp was found to mediate IFN-[gamma], TNF-[alpha], and LPS responsiveness as well as the synergistic effect of IFN-[gamma]/TNF-[alpha] on hBf promoter activity. Site-directed mutagenesis of a IFN-[gamma]-activation site (GAS) cis element (-90 to -82 bp) abrogated IFN-[gamma] responsiveness. Mutagenesis of a nuclear factor (NF)-[kappa]B cis element at -466 to -456 bp abrogated TNF-[alpha] and LPS responsiveness of the Bf promoter. Thus hBf gene expression is induced in PBMCs from septic shock patients, and the induction of hBf by IFN-[gamma] TNF-[alpha], and LPS is through GAS and NF-[kappa]B cis-binding sites on the hBf promoter. Furthermore, activated protein C (APC) inhibited LPS-stimulated hBf promoter activity and protein expression in human monocytes suggesting that the beneficial effect of APC therapy in sepsis may in part be due to inhibition of complement induction and/or activation via the alternative pathway. septic shock; monocytes/macrophages; complement factor B; cytokines

Malaria-induced sepsis is associated with an intense proinflammatory cytokinemia for which the underlying mechanisms are poorly understood. It has been demonstrated that experimental infection of humans with Plasmodium falciparum primes Toll-like receptor (TLR)-mediated proinflammatory responses. Nevertheless, the relevance of this phenomenon during natural infection and, more importantly, the mechanisms by which malaria mediates TLR hyperresponsiveness are unclear. Here we show that TLR responses are boosted in febrile patients during natural infection with P. falciparum. Microarray analyses demonstrated that an extraordinary percentage of the up-regulated genes, including genes involving TLR signaling, had sites for IFN-inducible transcription factors. To further define the mechanism involved in malaria-mediated 'priming,' we infected mice with Plasmodium chabaudi. The human data were remarkably predictive of what we observed in the rodent malaria model. Malaria-induced priming of TLR responses correlated with increased expression of TLR mRNA in a [TLR9.sup.-/-], MyD88-, and IFN[gamma]-dependent manner. Acutely infected WT mice were highly susceptible to LPS-induced lethality while [TLR9.sup.-/-], [IL12.sup.-/-] and to a greater extent, [IFN[gamma].sup.-/-] mice were protected, Our data provide unprecedented evidence that TLR9 and MyD88 are essential to initiate IL12 and IFN[gamma], responses and favor host hyperresponsiveness to TLR agonists resulting in overproduction of proinflammatory cytokines and the sepsis-like symptoms of acute malaria.

Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from Gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of Gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. To validate the concept of TLR4-targeted treatment strategies in Gram-negative sepsis, we first showed that [TLR4.sup.-/-] and myeloid differentiation primary response gene 88 [(MyD88).sup.-/-] mice were fully resistant to Escherichia coli-induced septic shock, whereas [TLR2.sup.-/-] and wild-type mice rapidly died of fulminant sepsis. Neutralizing anti-TLR4 antibodies were then generated using a soluble chimeric fusion protein composed of the N-terminal domain of mouse TLR4 (amino acids 1-334) and the Fc portion of human IgG1. Anti-TLR4 antibodies inhibited intracellular signaling, markedly reduced cytokine production, and protected mice from lethal endotoxic shock and E. coli sepsis when administered in a prophylactic and therapeutic manner up to 13 h after the onset of bacterial sepsis. These experimental data provide strong support for the concept of TLR4-targeted therapy for Gram-negative sepsis. endotoxic shock | Gram-negative bacteria | lipopolysaccharide | TLR4

The CD5 lymphocyte surface receptor is a group B member of the ancient and highly conserved scavenger receptor cysteine-rich superfamily. CD5 is expressed on mature T and Bla cells, where it is known to modulate lymphocyte activation and/or differentiation processes. Recently, the interaction of a few group B SRCR members (CD6, Sp[alpha], and DMBT1) with conserved microbial structures has been reported. Protein binding assays presented herein indicate that the CD5 ectodomain binds to and aggregates fungal cells (Schizosaccharomyces pombe, Candida albicans, and Cryptococcus neoformans) but not to Gram-negative (Escherichia coli) or Gram-positive (Staphylococcus aureus) bacteria. Accordingly, the CD5 ectodomain binds to zymosan but not to purified bacterial cell wall constituents (LPS, lipotheicoic acid, or peptidoglycan), and such binding is specifically competed by [beta]-glucan but not by mannan. The [K.sub.d] of the rshCD5/(1[right arrow]3)-[beta]-D-glucan phosphate interaction is 3.7 [+ or -] 0.2 nM as calculated from tryptophan fluorescence data analysis of free and bound rshCD5. Moreover, zymosan binds to membrane-bound CD5, and this induces both MAPK activation and cytokine release. In vivo validation of the fungal binding properties of the CD5 ectodomain is deduced from its protective effect in a mouse model of zymosan-induced septic shock-like syndrome. In conclusion, the present results indicate that the CD5 lymphocyte receptor may sense the presence of conserved fungal components [namely, (1[right arrow]3)-[beta]-D-glucans] and support the therapeutic potential of soluble CD5 forms in fungal sepsis. [beta]-glucans | fungal sepsis | lymphocytes | scavenger receptor

Sepsis causes over 200,000 deaths yearly in the US; better treatments are urgently needed. Administering bone marrow stromal cells (BMSCs--also known as mesenchymal stem cells) to mice before or shortly after inducing sepsis by cecal ligation and puncture reduced mortality and improved organ function. The beneficial effect of BMSCs was eliminated by macrophage depletion or pretreatment with antibodies specific for interleukin-10 (IL-10) or IL-10 receptor. Monocytes and/or macrophages from septic lungs made more IL-10 when prepared from mice treated with BMSCs versus untreated mice. Lipopolysaccharide (LPS)-stimulated macrophages produced more IL-10 when cultured with BMSCs, but this effect was eliminated if the BMSCs lacked the genes encoding Toll-like receptor 4, myeloid differentiation primary response gene-88, tumor necrosis factor (TNF) receptor-1a or cyclooxygenase-2. Our results suggest that BMSCs (activated by LPS or TNF-[alpha]) reprogram macrophages by releasing prostaglandin E2 that acts on the macrophages through the prostaglandin EP2 and EP4 receptors. Because BMSCs have been successfully given to humans and can easily be cultured and might be used without human leukocyte antigen matching, we suggest that cultured, banked human BMSCs may be effective in treating sepsis in high-risk patient groups., Sepsis, a serious medical condition that affects 18 million people per year worldwide, is characterized by a generalized inflammatory state caused by infection. Widespread activation of inflammation and coagulation pathways [...]

Byline: Joel M. Dulhunty (1), Jeffrey Lipman (1,2), Simon Finfer (2,3) Keywords: Sepsis; Severe sepsis; SIRS; Epidemiology Abstract: Objective To compare the time course of organ dysfunction/failure, mortality and cause of death in patients with severe sepsis (SS) and patients with severe non-infectious systemic inflammatory response syndrome (SNISIRS). Design Secondary analysis of a multi-centre inception cohort study. Setting Twenty-three multidisciplinary intensive care units (ICUs) in Australia and New Zealand. Patients and participants 3,543 ICU admissions aY=48 h or Interventions None. Measurements and results ICU prevalence of SS and SNISIRS was 20% (707/3,543) and 28% (980/3,543), respectively. ICU mortality was similar in patients with SNISIRS and with SS (25 vs. 27%, P = 0.40). Central nervous system (CNS) failure occurred more frequently in patients with SNISIRS (33 vs. 22%, P < 0.001) and resulted in death more commonly than in SS (relative risk = 1.6, 95% confidence interval 1.4--1.7, P < 0.001). The time to peak organ dysfunction (0.67 vs. 0.91 days, P = 0.004), overall episode length (3.6 vs. 5.6 days, P < 0.001) and ICU stay (geometric mean: 4.1 vs. 5.8 days, P < 0.001) were significantly shorter in patients with SNISIRS. Conclusions Whilst SNISIRS and SS have similarities, including their crude mortality rate, important differences exist. SNISIRS is more common on admission to the ICU, and is more commonly coupled with CNS dysfunction and death from neurological failure. Descriptors SIRS/sepsis: clinical studies. Author Affiliation: (1) Department of Intensive Care Medicine, Royal Brisbane and Women's Hospital, and Burns, Trauma and Critical Care Research Centre, University of Queensland, Herston, QLD, 4029, Australia (2) ANZICS Clinical Trials Group, 10 Ievers Terrace, Carlton, VIC, 3053, Australia (3) Royal North Shore Hospital and The George Institute for International Health, University of Sydney, Sydney, Australia Article History: Registration Date: 13/05/2008 Received Date: 18/02/2008 Accepted Date: 11/05/2008 Online Date: 27/05/2008

Byline: Guillermo Gutierrez (1), Pablo Comignani (2), Luis Huespe (3), F. Javier Hurtado (4), Arnaldo Dubin (5), Vinayak Jha (1), Yanina Arzani (2), Silvio Lazzeri (3), Lombardo Sosa (3), Juan Riva (6), Wolf Kohn (6), Daniela Suarez (4), Gonzalo Lacuesta (4), Daniela Olmos (5), Carlos Mizdraji (3), Alejandra Ojeda (3) Keywords: Mixed venous; Central venous; Sepsis; Myocardial metabolism; Tissue oxygenation Abstract: Objective Blood O.sub.2 saturation and lactate concentration gradients from superior vena cava (SVC) to pulmonary artery (PA) occur in critically ill patients. These gradients (ISO.sub.2 and I[Lac]) may be positive or negative. We tested the hypothesis that positive ISO.sub.2 and I[Lac] are associated with improved survival in critically ill patients. Design and setting Multinational, prospective observational study conducted in six medical and surgical ICUs. Patients Consecutive sample of 106 adults requiring insertion of a pulmonary artery catheter (PAC). Average age was 59.5 +- 15.5 years, APACHE II score was 15.5 +- 6.7 (mean +- SD). Main outcome measure was 28-day mortality. Interventions None. Measurements and results We drew blood samples from the proximal and distal ports of PACs every 6 h from the time of PAC insertion (Initial measurement) until its removal (Final measurement). Samples were analyzed for SO.sub.2, [Lac], glucose concentration and blood gases. Hemodynamic measurements were obtained after blood samples. We monitored patients for 30.9 +- 11.0 h. Overall mortality rate was 25.5%. More survivors had mean and final ISO.sub.2 aY= 0 and I[Lac] aY= 0 than decedents (p < 0.01 p < 0.05 respectively). On the average, ISO.sub.2 and I[Lac] were positive in survivors and negative in decedents. Survival odds ratios for final measurements of ISO.sub.2 aY= 0 and I[Lac] aY= 0 were 19.22 and 7.70, respectively (p < 0.05). Conclusions A strong association exists between positive ISO.sub.2 and I[Lac] and survival in critically ill patients. Whether therapy aimed at increasing ISO.sub.2 and I[Lac] results in improved ICU survival remains to be determined. Author Affiliation: (1) Pulmonary and Critical Care Medicine Division, The George Washington University Medical Center, 2150 Pennsylvania Ave., N.W, Washington, DC, 20037, USA (2) Fundacion Favaloro, Buenos Aires, Argentina (3) Hospital Escuela Corrientes, Corrientes, Argentina (4) Centro de Tratamiento Intensivo, Hospital de Clinicas, Montevideo, Uruguay (5) Servicio de Terapia Intensiva, Sanatorio Otamendi y Miroli, Buenos Aires, Argentina (6) Centro Cardiologico del Sanatorio Americano, Montevideo, Uruguay Article History: Registration Date: 11/04/2008 Received Date: 21/12/2007 Accepted Date: 01/04/2008 Online Date: 30/04/2008 Article note: Electronic supplementary material The online version of this article (doi: 10.1007/s00134-008-1128-2) contains supplementary material, which is available to authorized users.

Byline: Lisiane Tuon (1), Clarissa M. Comim (1), Fabricia Petronilho (2), Tatiana Barichello (2), Ivan Izquierdo (4), Joao Quevedo (1), Felipe Dal-Pizzol (2) Keywords: Sepsis; Learning; Memory; Depression; Anxiety; Central nervous system Abstract: Objective To evaluate the cognitive performance in rats that survived sepsis induced by cecal ligation and puncture (CLP) after 10, 30 and 60 days. Design Prospective, controlled experiment. Setting Animal basic science laboratory. Subjects Male Wistar rats, weighing 300--350 g. Interventions The rats were sham-operated or submitted to CLP (sepsis group) with 'basic support' (saline, s.c. at 50 mL/kg immediately and 12 h after CLP plus ceftriaxone, s.c. at 30 mg/kg and clindamycin, s.c. at 25 mg/kg 6, 12 and 18 h after CLP). Measurements and main results The animals underwent six behavioral tasks 10, 30 and 60 days after surgery: (a) habituation to the open field (b) inhibitory avoidance task (c) continuous multiple trials step-down inhibitory avoidance task (d) object recognition (e) elevated plus-maze and (f) forced swimming test. We demonstrated that survivors 10 days after CLP presented deficits on the habituation to the open field, step-down inhibitory avoidance, continuous multiple-trials step-down inhibitory avoidance, object recognition and forced swimming. After 30 days of sepsis induction, survivors maintained deficits on the step-down inhibitory avoidance, continuous multiple-trials step-down inhibitory avoidance and forced swimming. However, after 60 days all behavior deficits were reversed. Conclusions These results indicate that the impairment of memory and learning, demonstrated 10 days after the induction of sepsis, persist 30 days after the CLP. The cognitive impairments did not persist after 60 days suggesting that this model can help in the understanding of the biological mechanisms associated with sepsis-induced sickness behavior. Author Affiliation: (1) Laboratorio de Neurociencias, Programa de Pos-Graduacao Ciencias da Saude, Unidade Academica de Ciencias da Saude, Universidade do Extremo Sul Catarinense, 88806-000, Criciuma, SC, Brazil (2) Laboratorio de Fisiopatologia Experimental, PPGCS, UNASAU, Universidade do Extremo Sul Catarinense, 88806-000, Criciuma, SC, Brazil (4) Centro de Memoria, Instituto de Pesquisas Biomedicas, Pontificia Universidade Catolica do Rio Grande do Sul, 90610-000, Porto Alegre, RS, Brazil Article History: Registration Date: 11/04/2008 Received Date: 28/11/2007 Accepted Date: 08/04/2008 Online Date: 30/04/2008 Article note: Supported, in part, by UNESC (Brazil), FAPESC (Brazil) and CNPq (Brazil).

Endothelial hyperpermeability leading to vascular leak is an important consequence of sepsis and sepsis-induced lung injury. We previously reported that heat shock protein (hsp) 90 inhibitor pretreatment improved pulmonary barrier dysfunction in a murine model of sepsis-induced lung injury. We now examine the effects of hsp90 inhibitors on LPS-mediated endothelial hyperpermeability, as reflected in changes in transendothelial electrical resistance (TER) of bovine pulmonary arterial endothelial cells (BPAEC). Vehicle-pretreated cells exposed to endotoxin exhibited a concentration-dependent decrease in TER, activation of [pp60.sup.src], phosphorylation of the focal adhesion protein paxillin, and reduced expression of the adherens junction proteins, vascular endothelial (VE)-cadherin and [beta]-catenin. Pretreatment with the hsp90 inhibitor, radicicol, prevented the decrease in TER, maintained VEcadherin and [beta]-catenin expression, and inhibited activation of [pp60.sup.Src] and phosphorylation of paxillin. Similarly, when BPAEC hyperpermeability was induced by endotoxin-activated neutrophils, pretreatment of neutrophils and/or endothelial cells with radicicol protected against the activated neutrophil-induced decrease in TER. Increased paxillin phosphorylation and decreased expression of [beta]-catenin and VE-cadherin were also observed in mouse lungs 12 h after intraperitoneal endotoxin and attenuated in mice pretreated with radicicol. These results suggest that hsp90 plays an important role in sepsisassociated endothelial barrier dysfunction. endothelial permeability; radicicol; endotoxin; acute respiratory distress syndrome

Sepsis is characterized by a systemic response to severe infection. Although the inflammatory phase of sepsis helps eradicate the infection, it can have detrimental consequences if left unchecked. Therapy directed against inflammatory mediators of sepsis has shown little success and has the potential to impair innate antimicrobial defenses. Heme oxygenase-1 (HO-1) and the product of its enzymatic reaction, CO, have beneficial antiinflammatory properties, but little is known about their effects on microbial sepsis. Here, we have demonstrated that during microbial sepsis, HO-1-derived CO plays an important role in the antimicrobial process without inhibiting the inflammatory response. HO-1-deficient mice suffered exaggerated lethality from polymicrobial sepsis. Targeting HO-1 to SMCs and myofibroblasts of blood vessels and bowel ameliorated sepsis-induced death associated with Enterococcus faecalis, but not Escherichia coli, infection. The increase in HO-1 expression did not suppress circulating inflammatory cells or their accumulation at the site of injury but did enhance bacterial clearance by increasing phagocytosis and the endogenous antimicrobial response. Furthermore, injection of a CO-releasing molecule into WT mice increased phagocytosis and rescued HO-1-deficient mice from sepsis-induced lethality. These data advocate HO-1-derived CO as an important mediator of the host defense response to sepsis and suggest CO administration as a possible treatment for the disease., Introduction Sepsis represents a complex process of cellular events induced by a severe underlying infection (1), (2). Sepsis has been defined clinically as a syndrome characterized by both the presence [...]

The reduction of circulating neutrophil migration to infection sites is associated with a poor outcome of severe sepsis. [alpha]-1-Acid glycoprotein (AGP) was isolated from the sera of severely septic patients by HPLC and acrylamide gel electrophoresis and identified by mass spectrometry. Both the isolated protein and commercial AGP inhibited carrageenin-induced neutrophil migration into the rat peritoneal cavity when administered i.v. at a dose of 4.0 [micro]g per rat (95 pmol per rat). Analysis by intravital microscopy demonstrated that both proteins inhibited the rolling and adhesion of leukocytes in the mesenteric microcirculation. The inhibitory activity was blocked by 50 mg/kg aminoguanidine, s.c., and was not demonstrable in inducible nitric oxide synthase (iNOS) knockout mice. Incubation of AGP with neutrophils from healthy subjects induced the production of NO and inhibited the neutrophil chemotaxis by an iNOS/NO/cyclic guanosine 3,5-monophosphate-dependent pathway. In addition, AGP induced the L-selectin shedding by neutrophils. The administration of AGP to rats with mild cecal ligation puncture sepsis inhibited neutrophil migration and reduced 7-day survival from [approximately equal to] 80% to 20%. These data demonstrate that AGP, an acute-phase protein, inhibits neutrophil migration by an NO-dependent process and suggest that AGP also participates in human sepsis.

Our previous studies have shown that norepinephrine (NE) upregulates proinflammatory cytokines by activating [[alpha].sub.2]-adrenoceptor. Therefore, modulation of the sympathetic nervous system represents a novel treatment for sepsis. We have also shown that a novel stomach-derived peptide, ghrelin, is downregulated in sepsis and that its intravenous administration decreases proinflammatory cytokines and mitigates organ injury. However, it remains unknown whether ghrelin inhibits sympathetic activity through central ghrelin receptors [i.e., growth hormone secretagogue receptor la (GHSR-1a)] in sepsis. To study this, sepsis was induced in male rats by cecal ligation and puncture (CLP). Ghrelin was administered through intravenous or intracerebroventricular injection 30 min before CLP. Our results showed that intravenous administration of ghrelin significantly reduced the elevated NE and TNF-[alpha] levels at 2 h after CLP. NE administration partially blocked the inhibitory effect of ghrelin on TNF-[alpha] in sepsis. GHSR-1a inhibition by the administration of a GHSR-la antagonist, [D-[Arg.sup.1], D-[Phe.sup.5], D-[Trp.sup.7,9], [Leu.sup.11]]substance P, significantly increased both NE and TNF-[alpha] levels even in normal animals. Markedly elevated circulating levels of NE 2 h after CLP were also significantly decreased by intracerebroventricular administration of ghrelin. Ghrelin's inhibitory effect on NE release was completely blocked by intracerebroventricular injection of the GHSR-la antagonist or a neuropeptide Y (NPY)/[Y.sub.1] receptor antagonist. However, ghrelin's downregulatory effect on TNF-[alpha] release was only partially diminished by these agents. Thus ghrelin has sympathoinhibitory properties that are mediated by central ghrelin receptors involving a NPY/Y1 receptordependent pathway. Ghrelin's inhibitory effect on TNF-[alpha] production in sepsis is partially because of its modulation of the overstimulated sympathetic nerve activation. ghrelin; sepsis; sympathetic nervous system; norepinephrine; cytokines

Using an intrauterine bacterial inoculation method, our objective was to determine the effects of acute sepsis and level of dietary metabolizable Met (MM) on splanchnic metabolism of AA in ewes. Twenty-four nulliparous yearling Rambouillet-cross ewes (initial BW = 65.1 [+ or -] 0.6 kg), surgically fitted with chronic-indwelling catheters in hepatic and portal veins, a mesenteric vein and artery, and the uterine lumen, were assigned to a 2 x 2 factorial arrangement of treatments. Factors were intrauterine bacterial inoculation (no inoculated vs. inoculated) and level of MM [low (2.28 g/d) vs. high (3.91 g/d)]. Beginning 12 h before sampling, inoculated and noninoculated ewes received 10-mL intrauterine infusions of Escherichia coli (9.69 x [10.sup.11] cfu) +Arcanobacterium pyogenes (2.76 x [10.sup.12] cfu) and of sterile saline, respectively. Uterine infection was induced in ewes that received intrauterine bacterial inoculations, but not in ewes infused with sterile saline. Bacterial inoculation resulted in increased hepatic release and plasma concentrations of aromatic AA used for acute-phase protein synthesis, increased hepatic removal and decreased plasma concentrations of AA used for glutathione synthesis, and decreased plasma concentrations of some gluconeogenic and acetogenic AA used for glucose recycling and anaerobic energy production, respectively (P < 0.05). In ewes fed high-MM diets, compared with low-MM diets, a consistent net hepatic uptake of Phe occurred throughout the sampling period, more Asp was released from the portal-drained viscera, and hepatic vein glucose concentrations were greater (P < 0.05). We conclude that Met seemed to be limiting in low-MM ewes, and as such, would continue to be limiting during sepsis. However, additional MM, in excess of the dietary requirement, would not necessarily result in a benefit to ewes experiencing acute sepsis. Key words: methionine, amino acid, sepsis, sheep, uterine infection

The origins and applications of some successful replacement methods used in fundamental medical research and in regulatory testing are reviewed. Focus is laid on three areas of research namely respiratory illnesses, pain and sepsis.