Endotoxic Shock Model Research Articles

Activated protein C improves lipopolysaccharide-induced cardiovascular dysfunction by decreasing tissular inflammation and oxidative stress*

Recombinant human activated Protein C (APC) is used as an adjunctive therapeutic treatment in septic shock. APC seemingly acts on coagulation-inflammation interaction but also by decreasing proinflammatory gene activity, thus inhibiting subsequent production of proinflammatory cytokines, NO and NO-induced mediators, reactive oxygen species production and leukocyte-endothelium interaction. The hemodynamic effects of APC on arterial pressure and cardiac function are now well established in animal models. However, the specific effects of APC on heart and vessels have never been studied. To investigate the potential protective properties of therapeutic ranges of APC on a rat endotoxic shock model in terms of anti-inflammatory and cytoprotective pathways. Laboratory investigation. University medical center research laboratory. Rats were exposed to lipopolysaccharide (LPS) (10 mg/Kg intravenous). Endotoxic shock was treated with infusion of saline with or without APC (33 microg/kg/h) during 4 hrs. Hemodynamic parameters were continuously assessed and measurements of muscle oxygen partial pressures, NO and superoxide anion (O2(-)) by spin trapping, of NF-kappaB, metalloproteinase-9 (MMP-9) and inducible NO synthase (iNOS) by Western blotting, as well as leukocyte infiltration and MMP-9 activity were performed at both the heart and aorta level (tissue). APC partially prevented the reduction of blood pressure induced by LPS and improved both vascular hyporeactivity and myocardial performance. This was associated with a decreased up-regulation of NF-kappaB, iNOS and MMP-9. LPS-induced tissue increases in NO and O2(-) production were decreased by APC. Furthermore, APC decreased tissue leukocyte infiltration/activation as assessed by a decrease in myeloperoxidase and matrix metalloproteinase 9 activity. These data suggest that APC improves cardiovascular function: 1) by modulating the endotoxin induced-proinflammatory/prooxidant state, 2) by decreasing endothelial/leukocyte interaction and 3) by favoring stabilization of the extracellular matrix.

Dexamethasone inhibits lipopolysaccharide‐induced hydrogen sulphide biosynthesis in intact cells and in an animal model of endotoxic shock

Dexamethasone (1 mg/kg, i.p.) administered either 1 hr before or 1 hr after E. coli lipopolysaccharide (LPS, 4 mg/kg, i.p.) in conscious rats inhibited the subsequent (4 hrs) rise in plasma cytokine (interleukin [IL]-1beta, tumour necrosis factor [TNF]-alpha), nitrate/nitrite (NO(x)), soluble intercellular adhesion molecule-1 (sICAM-1) concentration and lung/liver myeloperoxidase activity indicative of an anti-inflammatory effect. Dexamethasone also reduced the LPS-evoked rise in plasma hydrogen sulphide (H(2)S) concentration, liver H(2)S synthesizing activity and expression of cystathionine gamma lyase (CSE) and inducible nitric oxide synthase (iNOS). Mifepristone (RU-486) inhibited these effects. Dexamethasone (1-10 microM) reduced the LPS-evoked release of IL-1beta, TNF-alpha and L-selectin, decreased expression of CSE and iNOS and diminished nuclear factor kappaB (NF-kappaB)-DNA binding in isolated rat neutrophils. In contrast, NaHS (100 microM) increased L-selectin release from rat neutrophils. Dexamethasone also reduced LPS-induced up-regulation of CSE in foetal liver cells. 6-amino-4-(4-phenoxyphenylethylamino) quinazoline (QNZ, 10 nM), a selective inhibitor of transcription via the NF-kappaB pathway, abolished LPS-induced up-regulation of CSE expression. We propose that inhibition of CSE expression and reduction in formation of the pro-inflammatory component of H(2)S activity contributes to the anti-inflammatory effect of dexamethasone in endotoxic shock. Whether H(2)S plays a part in the anti-inflammatory effect of this steroid in other forms of inflammation such as arthritis or asthma warrants further study.

Autoregulative function in the brain in an endotoxic rat shock model

Autoregulative function in the brain gets relevant in hypodynamic conditions of a sepsis syndrome. We investigated the temporal pattern and dose dependent effects of LPS-induced shock on autoregulative function in rats. Chloralose-anesthetized and mechanically ventilated male CD-rats (n = 30). Animals were subjected to vehicle, 1 or 5 mg/kg b.w. lipopolysaccharide (LPS) from E. coli given intravenously. Autoregulative function was tested repeatedly with a carotid compression technique assessing the transient hyperemic response ratio (THRR) in the cortex with laser Doppler flowmetry up to 270 min. THRR data from exsanguination experiments served as controls. Despite lower blood pressure levels in the high dose group (control: 114 +/- 7 mmHg; 1 mg/kg LPS group: 82 +/- 16 mmHg; 5 mg/kg LPS group: 62 +/- 16 mmHg; p < 0.05) progressive cerebral hyperemia occurred similarly in both groups. Compared to exsanguinations experiments autoregulative compensation for lower blood pressure levels was lacking in the high LPS dose group at the end of experiments. Cerebral autoregulation was affected by LPS-induced shock supporting the notion of vasoregulative failure in endotoxic shock.

Myocardial Dysfunction in Early State of Endotoxemia Role of Heme-Oxygenase-1

The triggers and cellular mechanisms of cardiac dysfunction have not been clearly established during the early period following challenge with lipopolysaccharides (LPS) (<1 h post-LPS). The aim of the study was to evaluate the myocardial depression during early stage of endotoxemia, the relationship between oxidative stress production and cardiac dysfunction in a rat model of endotoxic shock, and its inhibition by heme-oxygenase-1 (HO-1) overexpression. LPS-induced myocardial deformation was assessed by tissue Doppler imaging and invasive hemodynamic measurements in rats 2 h after LPS challenge. Myocardial samples were processed for the measurements of tumor necrosis factor alpha (TNFalpha), nitric oxidase synthase II (NOSII), HO-1 gene expression, reactive oxygen species (ROS) production, and reduced glutathione/oxidized glutathione (GSH/GSSH) ratio. Myocardial systolic and diastolic deformation was evident as determined by tissue Doppler imaging but left ventricular conventional echocardiographic parameters did not show significant alterations. Myocardial deformation was significantly associated with reactive oxygen species and TNFalpha overproduction. Pretreatment with hemin to induce HO-1 resulted in decreased oxidative stress and TNFalpha production, and prevented LPS-induced alterations in myocardium. These preliminary results suggest myocardial alteration at a very early stage after LPS challenge associated with oxidative stress response. Manipulation of the HO-1 pathway may represent a future therapeutic strategy to counteract oxidative stress of endotoxemia and perhaps may limit future myocardial deformation.

LPS-induced endotoxic shock does not cause early brain edema formation – An MRI study in rats

Early microcirculatory failure is assumed as a key factor in the development of a septic encephalopathy. However, brain edema is also a common finding in sepsis syndromes possibly interfering with the vasoregulative mechanisms of the brain. We assessed the occurrence of brain edema in a rat model of endotoxic shock. Eleven mechanically ventilated male CD-rats. Intravenous application of 5 mg/kg LPS (n = 8) or vehicle (n = 3). Apparent diffusion coefficient (ADC) and T2-relaxation time (T2RT) were quantified on cerebral MRI at baseline and repeatedly for up to 3.5 h after LPS-injection. Change in blood pressure was compensated with norepinephrine. Brain water content was quantified using the wet/dry method. All LPS-treated rats developed endotoxic shock. No significant difference in T2RT or ADC was detectable before and after LPS-injection (T2RT: baseline 60.33 +/- 1.21; after 3.5 h 60.15 +/- 0.59; ADC: baseline 6.86 +/- 0.51; after 3.5 h 6.75 +/- 0.33). Post-mortem analysis did not indicate a difference in brain water content between septic and non-septic animals. Reports of early microcirculatory failure seem not to be related to the occurrence of early (< or =3.5 h) brain edema.

The role of hydrogen sulfide in acute lung injury during endotoxic shock and its relationship with nitric oxide and carbon monoxide

To explore the role of hydrogen sulfide (H2S) in acute lung injury (ALI) during endotoxic shock (ES) and its relationship with nitric oxide (NO) and carbon monoxide (CO). Sixty-four adult male SD rats were randomly divided into 4 equal groups: control group injected with normal saline via the caudal vein, lipopolysaccharide (LPS)-treated group injected with LPS to establish ES model, LPS + NaHS group injected with LPS and sodium hydrosulfide (NaHS, an exogenous H2S donor], and LPS + PPG group injected with LPS and polypropylene glycol (PPG, a H2S synthase inhibitor). The mean artery pressure (MAP) was measured via a polyethylene catheter in the right common carotid artery for 6 h. Then the rats were sacrificed with their lungs taken out to determine the lung water content, lung tissue malonyldialdehyde (MDA), NO, and CO contents, as well as lung tissue cystathionine-gamma-lyase (CSE), myeloperoxidase (MPO), nitric oxide synthase (NOS), and heme oxygenase (HO) activities. The H2S content in blood plasma was detected also. Morphological changes of the lung tissues were observed under light microscope and the index of quantitative assessment (IQA) of lung injury was calculated. Immunohistochemistry and Western blotting were used to detect the lung tissue inducible NOS (iNOS) and HO-1 protein expression. Compared with the control group, the MAP of the LPS group was significantly lower, the pathological changes in lung tissue was more obvious, and the IQA, lung water content, lung MDA content, lung MPO and CSE activities as well as plasma H2S content were all significantly higher (P < 0.05 or P < 0.01). Compared to the LPS group, the plasma H2S and lung CSE activity of the LPS + NaHS group were higher, the lung injury was more severe, and the MAP was lower. And compared to the LPS group, the MAP of the LPS + PPG group was higher, and the lung injury was milder (both P < 0.05). The eNOS activity in the lung tissue of the LPS group was (5.26 +/- 0.25) Uxmg(-1)xprot(-1), significantly lower than that of the control group [(6.45 +/- 0.42) Uxmg(-1)xprot(-1)]; and the iNOS activity and NO content of the LPS group were (12.6 +/- 0.6) Uxmg(-1)xprot(-1) and (144 +/- 25) micromol/L respectively, both higher than those of the control group [(10.5 +/- 0.7) Uxmg(-1)xprot(-1) and (68 +/- 5) micromol/L respectively] (P < 0.05 or P < 0.01). Compared with the LPS group, the lung tissue eNOS activity of the LPS + PPG group was significantly higher, and the iNOS activity [(10.2 +/- 0.4) Uxmg(-1)xprot(-1)], iNOS protein expression, and NO content [(74 +/- 5) micromol/L]were all significantly lower (P < 0.05 or P < 0.01). Compared with the LPS group, the lung tissue eNOS activity of the LPS + NaHS group [(4.81 +/- 0.23) Uxmg(-1)xprot(-1)] was significantly lower, and the iNOS activity [(14.6 +/- 0.4) Uxmg(-1)xprot(-1)], iNOS protein expression, and NO content [(217 +/- 18) micromol/L] were significantly higher (P < 0.05 or P < 0.01). The lung tissue HO activity [(173 +/- 31) pkat/g], HO protein expression, and CO content [(3.63 +/- 0.24)%] of the LPS group were all significantly higher than those of the control group [(125 +/- 22) pkat/g, (2.48 +/- 0.33)%, both P < 0.05], and the LPS + PPG group [(88 +/- 17) pkat/g, (2.98 +/- 0.23)%, both P < 0.05]. Compared to the LPS group, the lung tissue HO activity [(263 +/- 37) pkat/g], HO protein expression, and CO content [(4.35 +/- 0.32)%] of the LPS + NaHS group were all significantly higher (all P < 0.05). The increase of H(2)S generation participates in the lung tissue injury during ES and this event is related to eNOS activity decrease, iNOS activity increase that causes the production of large amount of NO. H2S up-regulates the HO-1/CO system in the lung tissues during ES, which may be the endogenous compensatory response against the injury.

Activation of the PGI 2 /IP System Contributes to the Development of Circulatory Failure in a Rat Model of Endotoxic Shock

Prostacyclin levels are increased in septic patients and several animal models of septic shock, and selective inhibition of cyclooxygenase-2 improved cardiovascular dysfunction in rats treated with lipopolysaccharide (LPS). Here, we examine the specific role of prostacyclin and of the receptor for prostacyclin (IP) in the development of LPS-induced circulatory failure. Intravenous injection of LPS (10 mg/kg) into male Sprague-Dawley rats caused a strong increase in plasma prostacyclin levels, which was paralleled by a decrease in blood pressure and an increase in heart rate. Moreover, LPS injection increased the mRNA expression of the IP receptor in the heart, aorta, lung, liver, adrenal glands, and kidneys. Cotreatment with the IP antagonist CAY-10441 (1, 10, 30, and 100 mg/kg) dose-dependently moderated the LPS-induced changes in mean arterial blood pressure, heart rate, cardiac output, and systemic vascular resistance. The development of cardiovascular failure was ameliorated by CAY-10441 in spite of the typical LPS-induced increases in plasma levels of cytokines and NO. In vitro, cytokines dose- and time-dependently induced IP expression in rat vascular smooth muscle cells. Incubation of cells with the stable IP agonist iloprost in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-mehylxanthine resulted in higher cAMP levels in cytokine-treated cells compared with untreated cells. Taken together, our data demonstrate a prominent role of the prostacyclin/IP system in the development of LPS-induced cardiovascular failure.

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.

Evaluation of cardiac dysfunction in an animal model of LPS-induced endotoxic shock

Evaluation of cardiac dysfunction in an animal model of LPS-induced endotoxic shock

Evaluation of cardiac function in an animal model of LPS-induced endotoxic shock

Evaluation of cardiac function in an animal model of LPS-induced endotoxic shock

Role of levosimendan in sepsis and septic shock

To present the pharmacologic and biologic profile of levosimendan and discuss potential indications in the treatment of sepsis and septic shock, with a special focus on myocardial and pulmonary dysfunction. In animal models of endotoxic shock, levosimendan improved both left ventricular systolic and diastolic dysfunction, as well as ventriculovascular coupling. In addition, positive effects have been reported on right ventricular performance and pulmonary circulation. Two randomized, controlled trials in patients with septic shock revealed levosimendan provided consistent beneficial effects on cardiopulmonary performance, global oxygen transport, splanchnic perfusion and renal function. These effects have been reported as superior to placebo and the classic inotropic agent dobutamine. Due to its vasodilatory effects, combination with vasoconstrictor agents may be crucial in the presence of arterial hypotension. There is increasing evidence that levosimendan exerts beneficial effects in the treatment of sepsis-induced myocardial and pulmonary dysfunction. Future large-scale multicenter clinical trials are now needed to clarify whether levosimendan improves the overall outcome of patients with sepsis and septic shock.

Noninvasive rodent cardiac output: comparison of a compact clinical monitor with echocardiography and proposal of a simple correction factor

Introduction Rodent models are often studied in critical care research. Noninvasive cardiac output (CO) measurement is desirable but often impractical. The present study aimed to compare a commercially available human CO monitor, USCOM® (USCOM Ltd, Sydney, Australia), with specialised rodent echocardiography for noninvasive measurement of rat CO. Methods With institutional ethics committee approval (UQ AEC protocol 675/05), 21 anaesthetised, mechanically ventilated male Sprague–Dawley rats (573 ± 96 g) were studied during refinement and study of an endotoxic shock model. Pulsed–wave Doppler echocardiography (15 MHz rodent probe) was used to measure the left ventricular outflow velocity and to calculate the stroke volume and CO. USCOM (v1.7, human neonatal algorithm; 2.2 MHz) measurements followed each echocardiographic examination. USCOM CO was measured by combining continuous wave Doppler with the predicted outflow tract diameter (OTD-U). Results Twenty-one paired measurements were analysed. The mean echocardiographic CO was 113 ml/min (range 46–236). The mean USCOM CO was 245 ml/min (range 75–553). Paired echocardiographic and USCOM measurements demonstrated significant correlations for heart rate (r = 0.92, P ≤ 0.0001) and CO (r = 0.68, P = 0.001). Bland–Altman analysis of CO demonstrated a mean bias of -131 ml/min and precision of 52 ml/min. Linear regression analysis yielded a simple correction factor for USCOM OTD estimation. Following application of this correction factor (0.68 × OTD-U), the mean bias improved to -0.1 ml/min with precision of 38 ml/min. Conclusion Measurement of rat CO using the USCOM human neonatal algorithm (v1.7) is not interchangeable with specialised pulsed-wave Doppler echocardiography. We propose a simple correction factor that should improve performance of this device in the rodent laboratory. Incorporation into a rat-specific algorithm should be evaluated prospectively across a range of potential applications.

Abstract 978: Novel Endothelial-protective Effect of High Density Lipoprotein: Down-regulation of Toll-like Receptor 4

Introduction: Endothelium-derived Toll-like receptor (TLR) 4 is known to be the key molecule in lipopolysaccharide (LPS)-induced neutrophil sequestration into lungs. The aim of our study was to investigate whether the endothelial-protective effects of HDL also include regulation of Toll-like receptor (TLR) 4. Therefore, we analyzed the effect of HDL supplementation on TLR4 expression in human aortic endothelial cells (HAEC) and the effect of increased HDL following adenoviral apo AI ( Ad.AI) gene transfer (GT) on lung TLR4 expression in an experimental model of LPS-induced endotoxic shock. Methods: HDL (50 μg/ml) was supplemented to HAEC in the presence/absence of LPS (100 ng/ml). Surface-TLR4 expression on HAEC was analyzed by FACS. C57BL/6 mice were i.v. injected with 5 x 10 10 total particles of Ad.AI or with Ad.Null . Fourteen days hereafter, mice were i.p. injected with LPS (80 mg/kg) or with saline and 20 hours afterwards sacrificed. For survival studies, endotoxic shock was induced in 25 mice per group. Lung myeloperoxidase activity (MPO), as a marker of neutrophil infiltration was analysed. Lung TLR4-mRNA expression was determined by real-time PCR, plasma interferon-gamma (INFγ) levels by ELISA. Results: HDL supplementation significantly reduced TLR4 expression on HAEC. Ad.AI resulted in human apo AI expression levels of 80 ± 10 mg/dl at day 14. By 24 hours after LPS injection, 81% of LPS-treated Ad.Null and saline mice died, in contrast to 50% of Ad.AI LPS-treated mice. This was associated with a 1.6- and 1.7-fold (p&lt;0.05) lower lung/body weight ratio in Ad.AI compared to Ad.Null and saline LPS-injected mice. LPS-induced lung TLR4 mRNA expression was 2.0-fold (p&lt;0.05) reduced after human apo AI-GT compared to LPS controls. Moreover, human apo AI-GT significantly reduced neutrophil infiltration in LPS-treated mice, as indicated by 1.8-fold (p&lt;0.05) and 1.9-fold (p&lt;0.05) reduced MPO activity compared to controls, respectively. Plasma INFγ-levels were 2.8-fold and 2.2-fold (p&lt;0.05) lower versus LPS-treated Ad.Null and saline mice, respectively. Conclusion: The HDL-mediated reduction in lung TLR4 expression contributes for the reduced neutrophil infiltration and subsequently may account for improved mortality rate.

Intestinal preconditioning prevents inflammatory response by modulating heme oxygenase-1 expression in endotoxic shock model

Gut mucosal injury observed during ischemia-reperfusion is believed to trigger a systemic inflammatory response leading to multiple organ failure. It should be interesting to demonstrate this relationship between gut and multiple organ failure in a sepsis model. Intestinal preconditioning (PC) can be used as a tool to assess the effect of intestinal ischemia in inflammatory response after LPS challenge. The aim of this study was to investigate the protective effect of PC against LPS-induced systemic inflammatory and intestinal heme oxygenase-1 (HO-1) expression. ES was performed with LPS (10 mg/kg iv) with or without PC, which was done before LPS. Rats were first subjected to sham surgery or PC with four cycles of 1 min ischemia and 4 min of reperfusion 24 h before LPS challenge or saline administration. PC significantly reduced fluid requirements, lung edema, intestinal lactate production, and intestinal injury. Inflammatory mRNA expressions for intestine and lung ICAM and TNF were significantly reduced after PC, and these effects were significantly abolished by zinc-protoporphyrin (a specific HO-1 activity inhibitor) and mimicked by bilirubin administration. Intestinal PC selectively increased HO-1 mRNA expression in intestine, but we have observed no expression in lungs. These findings demonstrate that intestinal injury is a important event for inflammatory response and multiple organ injury after LPS challenge. Intestinal HO-1 expression attenuates LPS-induced multiple organ failure by modulating intestine injury and its consequences on inflammatory response. Identification of the exact mechanisms responsible for intestine HO-1 induction may lead to the development of new pharmacological interventions.

Stoking Up BK Ca Channels in Hemorrhagic Shock

See related article, pages 493–502 Large conductance calcium-activated potassium channels (BKCa) are abundantly expressed in smooth muscle cells (SMCs) lining the blood vessel wall. They are composed of an α-subunit (Slo) and a modulatory β1-subunit, which serves to maintain the normal high voltage- and Ca2+-sensitivity of the pore-forming α-subunit (reviewed in1,2). In the vasculature, BKCa operate by limiting Ca2+ entry and arterial contraction by repolarizing SMCs and closing voltage-dependent Ca2+ channels previously opened by pressure or vasoconstrictor agents.1 BKCa can also mediate cellular hyperpolarization and vasorelaxation as a result of spontaneous transient outward currents (STOCs) activated by the localized release of micromolar concentrations of Ca2+ (Ca2+ sparks) from ryanodine receptors located in the sarcoplasmic reticulum (SR).1 Moreover, increased frequency of Ca2+ sparks may underlie activation of BKCa by endogenous vasodilators,1 though other mechanisms undoubtedly contribute.2,3 Genetic experiments also highlight BKCa as important regulators of vascular tone and blood pressure. Deletion of the α-subunit in mice results in membrane depolarization, a complete lack of STOCs, and attenuates cGMP relaxation in isolated blood vessels.4 On the other hand, deletion of β1 impairs the coupling of Ca2+ sparks to the activation of hyperpolarizing BKCa currents and enhances agonist-induced vasoconstriction without affecting nitric oxide (NO) mediated vasorelaxation.3 Knockout of both genes leads to systemic hypertension, though in BKCa β1–null mice this is more pronounced3,4 suggesting physical interactions of the β1-subunit with other proteins, possibly other ion-conducting pores.5 Moreover, depending on the hypertensive model, β1-subunit expression can either increase2,6 or decrease.7 The latter might argue that β1 acts as a compensatory mechanism to limit development of hypertension. Consistent with this, a gain-of-function mutation in the human β1-subunit gene …

Protection from endotoxic shock by EVK-203, a novel alkylpolyamine sequestrant of lipopolysaccharide

Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality in the critically ill patient. The only therapeutic option aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide is Toraymyxin™, an extracorporeal hemoperfusion device using solid phase-immobilized polymyxin B (PMB). While PMB is known to effectively sequester LPS, its severe systemic toxicity proscribes its parenteral use, and hemoperfusion may not be feasible in patients in shock. In our continuing efforts to develop small-molecule mimics which display the LPS-sequestering properties, but not the toxicity of PMB, a series of mono- and bis-substituted dialkylpolyamines were synthesized and evaluated. We show that EVK-203, an alkylpolyamine compound, specifically binds to and neutralizes the activity of LPS, and affords complete protection in a murine model of endotoxic shock. EVK-203 is without any apparent toxicity when administered to mice at multiples of therapeutic doses for several days. The specific endotoxin-sequestering property along with a very favorable therapeutic index renders this compound an ideal candidate for preclinical development.

Neutralizing and cross-reactive antibodies against enterobacterial lipopolysaccharide

Lipopolysaccharide (LPS, endotoxin) elicits an immune reaction which is responsible for many of the harmful effects seen in septic shock patients. The eradication of bacteria by antibiotics is insufficient to resolve the pathology due to the lack of LPS neutralization. LPS-neutralizing antibodies have been described; however, these were specific for the serotype of the infecting bacteria and thus not useful for the treatment of septic shock patients. Structural analyses revealed that the LPS structures of Escherichia coli and Salmonella are structurally conserved in the inner core region. Using whole LPS and a panel of neoglycoconjugates containing purified LPS oligosaccharides, which we have obtained from all E. coli core types (K-12, R1, R2, R3 and R4), Salmonella enterica, and the mutant strain E. coli J-5, we have identified an epitope which is bound with high affinity by the monoclonal antibody WN1 222-5, which has been shown previously shown to be cross-reactive against a large collection of blood, fecal, and urinary isolates of E. coli, S. enterica, some Citrobacter, independently of the serotype [Di Padova, F.E., Brade, H., Barclay, G.R., Poxton, I.R., Liehl, E., Schuetze, E., Kocher, H.P., Ramsay, G., Schreier, M.H., McClelland, D.B., Rietschel, E.T., 1993. A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides. Infect. Immun. 61, 3863–3872]. Importantly, WN1 222-5 was protective in various models of endotoxic shock. The minimal structural element necessary for high-affinity binding consists of R 1- α- d-Glc p-(1→3)-[ l- α- d-Hep p-(1→7)]- l- α- d-Hep p 4P- (1→3)-R 2 (R 1, R 2=additional sugars of LPS) in which the side-chain heptose and the 4-phosphate on the branched heptose are the main determinants of the epitope. Additional sugars of the outer core (R 1) enhance the affinity, whereas loss of an intact Kdo region and/or lipid A (R 2) prevent binding. The identification of the epitope provides the structural basis for the rational development of a potential vaccine against E. coli LPS.

Proinsulin c-peptide exerts beneficial effects in endotoxic shock in mice

Insulin connecting peptide (c-peptide) aids the folding of proinsulin and has been considered to have little biological activity. Recently, c-peptide has been shown to improve diabetic neuropathy and nephropathy as well as vascular inflammation. In vitro studies have reported that c-peptide may activate peroxisome proliferator-activated receptor-gamma, a nuclear transcription factor that plays a regulatory role in inflammation. This study was designed to investigate the biological effects of c-peptide during endotoxemia. Prospective, randomized laboratory investigation that used an established murine model of endotoxic shock. University hospital laboratory. Mice were subjected to endotoxic shock by intraperitoneal administration of Escherichia coli lipopolysaccharide. Mice received vehicle or c-peptide (70-140 nmol/kg) intraperitoneally at 3 hrs and 6 hrs after lipopolysaccharide. Mortality was monitored for 96 hrs. In a separate experiment, mice were killed at 4, 7, and 18 hrs after lipopolysaccharide administration. Lungs and plasma were collected for biochemical assays. In vehicle-treated mice, endotoxic shock resulted in lung injury and was associated with a 41% survival rate and elevation in plasma tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-1, and keratinocyte-derived chemokine levels. Lung nuclear levels of phosphorylated extracellular signal-regulated kinases 1 and 2 were significantly increased in vehicle-treated mice. On the other hand, lung nuclear expression and DNA binding of proliferator-activated receptor-gamma were decreased in comparison to control animals. Treatment with c-peptide (140 nmol/kg) improved survival rate (68%) and reduced plasma levels of tumor necrosis factor-alpha, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1, but it did not exert hypoglycemic effects. Treatment with c-peptide also up-regulated lung nuclear expression and DNA binding of proliferator-activated receptor-gamma and reduced phosphorylation of extracellular signal-regulated kinases 1 and 2 in comparison to vehicle-treated mice. Our data show that c-peptide has beneficial effects in endotoxic shock, and this therapeutic effect is associated with activation of proliferator-activated receptor-gamma.

NAFAMOSTAT MESILATE INHIBITS HIGH-MOBILITY GROUP BOX 1 BY LIPOPOLYSACCHARIDE STIMULATION IN MURINE MACROPHAGE RAW 264.7

In recent investigations, high-mobility group box 1 (HMGB1) has been recognized to be an important factor in the development of sepsis. On the other hand, a serine protease inhibitor, nafamostat mesilate (NM) inhibits the enzyme activities of various protease and coagulation factors. We examined whether NM could inhibit HMGB1 in a rat sepsis model and thus could potentially be useful for treating sepsis. We administered NM to rats before administering lipopolysaccharide and thereafter measured the HMGB1 levels of the serum and lung tissue. We used a mouse macrophage cell line and we performed lipopolysaccharide stimulation under NM administration and thereafter measured the quantity of HMGB1 and cytokines in the supernatant, and cell signal in the cells. We were thereby able to reduce the degree of injury to pulmonary tissue by administering NM. The HMGB1 levels of the serum and lung tissue were thus found to be inhibited. This action was confirmed at the cell level, and the release of HMGB1 and cytokines from the cell decreased. Regarding the cell signal in each cell, we observed the inhibition of the phosphorylation of IkappaB. We thus concluded that it is possible to prevent the occurrence of pulmonary disorders in an endotoxic shock model by administering NM, however, this also inhibits the cell signal in a cell, mainly by the phosphorylation of IkappaB, thereby inhibiting the HMGB1 levels. Our findings thus suggest that the administration of NM may therefore potentially improve the condition of patients who have septic shock.

Polycationic Sulfonamides for the Sequestration of Endotoxin

Lipopolysaccharides (LPS) play a key role in the pathogenesis of septic shock, a major cause of mortality in the critically ill patient. We had previously shown that monoacylated polyamine compounds specifically bind to and neutralize the activity of LPS with high in vitro potency and afford complete protection in a murine model of endotoxic shock. Fatty acid amides of polyamines may be rapidly cleared from systemic circulation due to their susceptibility to nonspecific serum amidases and, thus, would be predicted to have a short duration of action. In a systematic effort to increase the likelihood of better bioavailability properties together with structural modifications that may result in gains in activity, we now report structure-activity relationships pertaining to endotoxin-binding and -neutralizing activities of homologated polyamine sulfonamides.