Endotoxin-induced Septic Shock Research Articles

Renin‐Angiotensin‐Aldosterone System During Extracorporeal Membrane Oxygenation (ECMO) in a Pig Model of Septic Shock

ECMO is used to provide cardiopulmonary support to critically ill patients, but the ability of the renin‐angiotensin‐aldosterone system to function appropriately during ECMO remains unclear. In this study, plasma renin activity (PRA) and aldosterone (ALDO) were compared at baseline and before and after stabilization on veno‐arterial ECMO for 2 hours in anesthetized piglets either during control (n=10) or endotoxin‐induced septic shock (n=9) states. In control piglets, ECMO was able to maintain mean arterial pressure (MAP) at baseline levels and PRA and ALDO remained unchanged. In contrast, endotoxin induced a 46% decrease (p<0.01) in MAP, and while ECMO could maintain flow at this decreased MAP, it did not return MAP to baseline. PRA appropriately increased in response to shock, but ultimately dropped by 54% (p<0.01) in early ECMO. ALDO increased in response to shock, and remained constant during ECMO. There was a strong correlation between PRA and ALDO at baseline (r=0.90, p<0.01) and on ECMO in control piglets (r= 0.70, p<0.01), but septic shock disrupted that relationship. Plasma sodium concentrations increased (p<0.01) from 137 ± 3.3 to 145 ± 0.7 mEq/L in shock, and with sustained ALDO, remained at this elevated level throughout ECMO. Results suggest that ALDO sodium regulating ability remains intact during ECMO but PRA is attenuated during ECMO in the endotoxic shock state.

Vasopressin Regulation During Extracorporeal Membrane Oxygenation (ECMO) in a Pig Model of Septic Shock

ECMO is used to provide cardiopulmonary support to critically ill neonates. Vasopressin (VP) plays a vital role in regulating mean arterial pressure (MAP) and regional blood flow in hypotensive disorders, but whether endogenous VP levels can be sustained during ECMO is unclear. In this study, 11 anesthetized, cardiac catheterized piglets were put on veno‐arterial ECMO either during control or endotoxin‐induced septic shock states. ECMO blood flow was delivered to maintain MAP at baseline levels. Plasma VP (pVP) was compared before and after stabilization on ECMO for 2 hours. ECMO caused pVP to drop by 30% (p<0.01) in both control and septic shock conditions. This drop may be due to the ECMO circuit adding to total circulating volume without additional release of VP. To test whether right atrial pressure (RAP) influence on VP release may be disrupted during ECMO due to partial cardiac bypass of blood flow, the relationship between pVP and hemodynamics during control was examined with stepwise regression. Pre‐ECMO, pVP negatively correlated with RAP and heart rate (r=0.83, p<0.05), but not MAP. In contrast, during ECMO, this pVP relationship with RAP was lost, but a strong positive correlation (r=0.76, p<0.05) between pVP and MAP became evident. Results indicate that during ECMO, low pressure atrial baroreceptors no longer regulate VP release, but VP control of MAP is maintained. Supported by US Army CRADA 0709‐T‐C454

Selective Regulation of Heme Oxygenase-1 Expression and Function by Insulin through IRS1/Phosphoinositide 3-Kinase/Akt-2 Pathway

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H(2)O(2)-induced NF-kappaB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Effect of lipid-bound apoA-I cysteine mutants on lipopolysaccharide-induced endotoxemia in mice

HDL has been shown to be able to neutralize the toxicity of lipopolysaccharide (LPS). Our previous study (J. Lipid Res. 2005. 46: 1303-1311) characterized the properties of secondary structure and in vitro functions of different cysteine mutants of apolipoprotein A-I. Here, we reconstituted recombinant HDLs (named rHDLwt, rHDL52, rHDL74, rHDL107, rHDL129, rHDL173, rHDL195, and rHDL228) by mixing wild type or those mutants with dipalmitoyl phosphatidylcholine and examined their in vivo effects on LPS-induced endotoxemia in mice. Our results showed that 24 h after injection, mice receiving rHDL74 or rHDL52 had a significant decrease of plasma tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), compared with control mice receiving either saline or rHDLwt (P < 0.05). Administration of rHDL74 to mice injected with LPS also led to a decrease of plasma IL-6, protection of lung against acute injury, and attenuation of endotoxin-induced clinical symptoms in mice, compared with controls injected with LPS only. However, injection of rHDL228 significantly increased plasma concentration of TNF-alpha and exacerbated LPS-induced lung injury. In summary, compared with rHDLwt, rHDL74 and rHDL52 exhibit higher anti-inflammation capabilities, whereas rHDL228 shows hyper-proinflammation by exacerbating LPS-induced endotoxemia in mice.

Pretreatment of Curcumin Protects Vascular Dysfunction in Endotoxin-induced Septic Shock in Mice

Pretreatment of Curcumin Protects Vascular Dysfunction in Endotoxin-induced Septic Shock in Mice

Evaluation of cardiac dysfunction in an animal model of endotoxin-induced septic shock

Aims: Besides serious inflammatory reactions and multiple organ failure, septic shock leads to cardiac dysfunction and acute heart failure. We aimed to establish a model to examine cardiac function in endotoxic shock in vivo and ex vivo.

Melatonin reverses lipopolysaccharide‐induced gastro‐intestinal motility disturbances through the inhibition of oxidative stress

A growing number of reports demonstrate that a pro-inflammatory and oxidative condition is related to the pathogenesis and the progression of endotoxin-induced septic shock and that antioxidants may have therapeutic potential in lipopolysaccharide (LPS)-induced sepsis. Melatonin has been shown to possess potent antioxidant properties in several models of inflammation in mice and rats. In the present study we focused on the possible protective mechanism of melatonin in preventing gastrointestinal (GI) disturbances induced by LPS in mice. In fact, mice treated with LPS showed a reduced gastric emptying of solid beads. Also the geometric center, representing the relative distribution of the solid beads throughout the entire GI tract, was significantly reduced in LPS-treated mice confirming that sepsis leads to a disturbed GI motility in mice. Melatonin completely reversed the LPS-induced motility disturbance. This beneficial effect of melatonin is associated with a reduction in lipid peroxidation, MAPK activation, NF-kappaB activation, iNOS transcription and expression and nitrite production in intestinal tissue from septic mice. These results demonstrate that melatonin prevents the LPS-induced GI disturbances in mice switching off the pro-oxidant pathways induced by the endotoxin. Therefore, it is reasonable to propose melatonin as a molecule with therapeutic potential for the treatment of systemic inflammation by interfering at the earliest steps of activation of the oxidative and pro-inflammatory cascade.

Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock

Adrenomedullin (AM) is a highly conserved peptide that can act as a potent vasodilator, anti-microbial factor and anti-inflammatory factor. Several studies have implicated diverse roles for AM in regulating the inflammatory and hemodynamic responses to septic shock. Moreover, during sepsis the receptors that mediate AM signaling [calcitonin receptor-like receptor ( calcrl) and receptor activity modifying proteins ( RAMP) 2 and 3] undergo dynamic and robust changes in their expression. Although numerous studies have used animal models to study the role of administered or increased AM in septic animals, genetic studies to determine the consequences of reduced AM during septic shock have not yet been performed. Here, we used a murine model of lipopolysaccharide (LPS)-induced septic shock to assess the inflammatory response in mice heterozygous for the AM gene. Following LPS challenge, AM +/− mice had higher expression of TNF-α and IL-1β than LPS-treated wild-type (WT) controls. Consequently, serum TNF-α was also significantly elevated in LPS-treated AM +/− mice compared to WT LPS-treated mice. We also observed higher serum levels of liver enzymes, suggesting more advanced end-organ damage in mice with genetically reduced AM. Finally, we found that RAMP2 and calcrl expression levels were markedly reduced in LPS-treated mice, whereas RAMP3 expression was significantly elevated. Importantly, these changes in receptor gene expression were conserved in AM +/− mice, demonstrating that AM peptide itself does not impact directly on the expression of the genes encoding its receptors. We, therefore, conclude that during septic shock the dynamic modulation of AM and its receptors primarily functions to dampen the inflammatory response.

Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock

Mice heterozygous for adrenomedullin exhibit a more extreme inflammatory response to endotoxin-induced septic shock

Etanercept Reduces Late Endotoxin-Induced Pulmonary Hypertension in the Pig

To evaluate whether etanercept, a tumor necrosis factor (TNF)-blocking agent, may counteract hemodynamic deterioration in endotoxemic shock, we designed a prospective, randomized placebo-controlled trial with parallel groups, consisting of 13 pigs aged 10-14 weeks receiving general anesthesia. Five pigs were given 25 mg of etanercept, 1 h before the start of a 4-h continuous infusion of endotoxin. Another 5 pigs were given the corresponding volume of saline, 1 h before the start of a 4-h continuous infusion of endotoxin. Three pigs were given 25 mg of etanercept, 1 hr before the start of a 4-h continuous infusion of saline. At 1 h of endotoxemia, mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance index (PVRI) increased identically in both groups of pigs receiving endotoxin. Thereafter, two distinct different patterns in hemodynamics were observed. TNF-blocked pigs showed significantly lower MPAP and PVRI compared to controls. In the etanercept-treated endotoxemic pigs, Doppler analysis of the diastolic mitral inflow demonstrated a significantly increased E/A-ratio (early mitral wave inflow was divided by the atrial wave) at 2 h. The TNFblocking agent etanercept normalized two hemodynamic features of endotoxin-induced septic shock in pigs: (1) the sustained pulmonary hypertension and (2) diastolic dysfunction.

Effects of verapamil and nifedipine on different parameters in lipopolysaccharide-induced septic shock

Septic shock has a high mortality rate due to the hypotension and circulatory disorder that occurs during its pathogenesis. Recently, humoral factors such as cytokines and nitric oxide became important in the complex pathophysiology of septic shock because there is a close relationship between the determined levels of these humoral factors and the responses to the therapy and survival periods. Verapamil and nifedipine are calcium channel blockers commonly used in the pharmacotherapy of cardiovascular disorders. In the present study these drugs were investigated in the rat septic shock model. In vivo hemodynamic parameters were recorded using a data acquisition system in endotoxin-induced septic shock in rats. The animals were followed for 5 h and blood pressure, rectal temperature, and ECG were recorded. Blood samples were collected at 1 h and 5 h time points after the injection of endotoxin, and serological samples were stored at -25 degrees C. Subsequently, tumor necrosis factor-alpha, interleukin-10 (enzyme-linked immunosorbent assay), and nitrite (Griess reagent) were determined in these serological samples. Significant correlations were observed between these humoral factors and the disordered hemodynamic factors. A reversal of changes was observed in the levels of serum cytokines, nitrite levels, and hemodynamic parameters with verapamil and nifedipine preadministration (P<0.05). Additionally, superoxide dismutase (SOD), catalase, and malondialdehyde (MDA) were determined in livers obtained from these animals at the end of the experiments, and these results were compared to hemodynamic parameters and cytokines. Nifedipine and verapamil increased the levels of MDA and SOD but did not change catalase activity.

Neutralization of endotoxin in vitro and in vivo by Bac7(1-35), a proline-rich antibacterial peptide.

Lipopolysaccharides (LPS), or endotoxins, are structural components of gram-negative bacteria implicated in the pathogenesis of septic shock. In this study the antiendotoxin activity of Bac7(1-35), a synthetic peptide based on the sequence of a proline-rich antibacterial peptide from bovine neutrophils, was investigated in vitro and in an experimental rat model of gram-negative septic shock. The ability of Bac7(1-35) to bind LPS from Escherichia coli O111:B4 was determined using a sensitive Limulus chromogenic assay. In the in vivo study, adult male Wistar rats were given an intraperitoneal injection of 1 x 10(9) colony-forming units of E. coli ATCC 25922. All animals were randomized to receive intraperitoneally 1 mg/kg Bac7(1-35), or isotonic sodium chloride solution (control group C1), 60 mg/kg of piperacillin and 1 mg/kg polymyxin B, 1 mg/kg of polymyxin B plus 60 mg/kg of piperacillin, and 1 mg/kg of Bac7(1-35) plus 60 mg/kg of piperacillin. Each group included 15 animals. Bac7(1-35) was found to completely inhibit the LPS procoagulant activity at approximately 10 microM peptide concentration, as determined by in vitro LAL chromogenic assay. Treatment with Bac7(1-35) resulted in significant decrease in plasma endotoxin levels and lethality rates compared with saline injected control animals. No statistically significant differences were noted between Bac7(1-35) and polymyxin B in reducing all variables measured. These results provide evidence for the ability of Bac7(1-35) to effectively bind LPS and protect animals from lethal effects of this molecule, and point to its potential use for the treatment of endotoxin-induced septic shock.

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20 mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-α levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10 mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase–Akt/protein kinase B cytoprotective pathway.

Thymosin β 4 reduces lethality and down-regulates inflammatory mediators in endotoxin-induced septic shock

Thymosin β 4 (Tβ 4), a highly conserved peptide with immunomodulatory properties, is the major actin-sequestering peptide in mammalian cells. Recent studies have established that Tβ 4 can accelerate wound healing in full thickness skin wounds and following burn injuries to the cornea. In the eye studies, the accelerated healing due to Tβ 4 was accompanied by a significant reduction in polymorphonuclear leukocyte (PMN) infiltration and a several-fold decrease in interleukin-1β ( p≤0.015) and 6-keto-prostaglandin F 1α (6-keto-PGF1α, p≤0.05). Given the recognized role of proinflammatory cytokines in septic shock and of extracellular F- and G-actin in the pathophysiology of multiple organ dysfunction, we have investigated the role of Tβ 4 in sepsis. We report that an LD 50 dose of LPS (24 mg/kg) in rats resulted in a significant reduction of Tβ 4 levels in the blood. Furthermore, administration of 100 μg of Tβ 4 immediately following and at 2 and 4 h after an LD 50 dose of LPS (60 mg/kg) in mice significantly reduced mortality rates ( p≤0.024) and lowered blood levels of a number of inflammatory cytokines, eicosanoids, and other molecules that are highly elevated following endotoxin administration. In studies in human subjects given low doses of endotoxin (4 ng/kg LPS) and in patients with septic shock, we have also observed significant decreases in blood levels of Tβ 4. The rapid disappearance of Tβ 4 in the blood following LPS administration or during septic shock suggests that Tβ 4 may be involved in early events leading to activation of the inflammatory cascade and ultimately the clinical sequelae of sepsis. The results of this study indicate that Tβ 4 may have utility in the clinic in the treatment of septic shock and in syndromes associated with actin toxicities.

Review: Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application

Lactoferrin is an iron-binding glycoprotein present in various secretions ( e.g. milk, tears, saliva, pancreatic juice, etc.). It is also stored in specific granules of polymorphonuclear granulocytes from which it is released following activation. Lactoferrin exerts a bactericidal activity by damagingthe outer membrane of Gram-negative bacteria, as well as immunoregulatory functions by decreasing the release of interleukin-1 (IL-1), IL-2 and tumor necrosis factor- (TNF-) and enhancing monocyte and natural killer cell cytotoxicity. Lactoferrin binds with high affinity to lipid A, the toxic moiety of the lipopolysaccharide, or endotoxin from Gram-negative bacteria. Lipopolysaccharide interaction with monocytes/macrophages results in the production and release of TNF- , that plays an important role in inducing septic shock. In this respect, it has recently been demonstrated that lactoferrin inhibits the lipopolysaccharide interaction with CD14 on monocytes/macrophages by competition with the lipopolysaccharide binding protein. Therefore, besides its bactericidal activity, lactoferrin may also act by neutralizing the toxic effects of lipopolysaccharide and this protective role against endotoxin lethal shock has been demonstrated in animal models. Moreover, in vitro and in vivo neutralization of endotoxin by a human lactoferrin-derived peptide was also reported and lactoferrin or lactoferrin-derived peptides could represent useful tools for the treatment of endotoxin-induced septic shock. The recent production and characterization of monoclonal antibodies against different epitopes of human lactoferrin, including monoclonal antibodies selectively neutralizinglactoferrin binding to lipid A, may allow a better elucidation of the consequence of lactoferrin-lipopolysaccharideinteraction.

Pivotal role of G-protein-coupled adenosine A2a receptors in protection of the liver against inflammation and ischemia-reperfusion injury

Inappropriate or prolonged inflammation is the main cause of many diseases; for this reason it is important to understand the physiological mechanisms that terminate inflammation in vivo. Agonists for several G s -protein-coupled receptors, including cell-surface adenosine purinergic receptors, can increase levels of immunosuppressive cyclic AMP in immune cells; however, it was unknown whether any of these receptors regulates inflammation in vivo. Here we show that A2a adenosine receptors have a non-redundant role in the attenuation of inflammation and tissue damage in vivo. Sub-threshold doses of an inflammatory stimulus that caused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death of male animals deficient in the A2a adenosine receptor. Similar observations were made in studies of three different models of inflammation and liver damage as well as during bacterial endotoxin-induced septic shock. We suggest that A2a adenosine receptors are a critical part of the physiological negative feedback mechanism for limitation and termination of both tissue-specific and systemic inflammatory responses.

Comparative Study of Several Lymphocyte Functions in Two Strains of Mice with Different Models of Endotoxic Shock.

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFa production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFa released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFa release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.

Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage.

Inappropriate or prolonged inflammation is the main cause of many diseases; for this reason it is important to understand the physiological mechanisms that terminate inflammation in vivo. Agonists for several Gs-protein-coupled receptors, including cell-surface adenosine purinergic receptors, can increase levels of immunosuppressive cyclic AMP in immune cells; however, it was unknown whether any of these receptors regulates inflammation in vivo. Here we show that A2a adenosine receptors have a non-redundant role in the attenuation of inflammation and tissue damage in vivo. Sub-threshold doses of an inflammatory stimulus that caused minimal tissue damage in wild-type mice were sufficient to induce extensive tissue damage, more prolonged and higher levels of pro-inflammatory cytokines, and death of male animals deficient in the A2a adenosine receptor. Similar observations were made in studies of three different models of inflammation and liver damage as well as during bacterial endotoxin-induced septic shock. We suggest that A2a adenosine receptors are a critical part of the physiological negative feedback mechanism for limitation and termination of both tissue-specific and systemic inflammatory responses.

Glutamine reduces cytokine release, organ damage, and mortality in a rat model of endotoxemia.

Clinical trials have demonstrated that glutamine (GLN) supplementation can decrease infectious morbidity and improve survival in a number of settings of critical illness. The mechanism of this protection remains unclear. The objective of this study was to evaluate the effect of GLN on cytokine release, organ injury, and survival from endotoxin-induced septic shock. Endotoxemia was induced in Male Sprague-Dawley rats by intravenous administration of 5 mg/kg Escherichia coli lipopolysaccharide (LPS). Concomitantly, animals were fluid resuscitated with a lactated ringers (LR) solution and given GLN (0.75 g/kg i.v.) or LR alone. Blood samples were obtained at multiple time points post-LPS injury for cytokine analysis. Survival rates were monitored for 72 h. Organ injury was evaluated in a separate set of animals via pathologic exam of tissues harvested 6 h post-LPS injury. A single dose of GLN significantly attenuated the release of TNF-alpha at 2 h (P < 0.005) and IL-1 beta at 4 h (P < 0.0001). This attenuation of cytokine release was associated with a significant decrease in mortality (P < 0.003). Pathologic exam demonstrated significant protection of both lung and small bowel tissue by GLN. Blood gas values 6-h post-LPS injury showed increased PaO2 and bicarbonate concentration in GLN treated animals. These data indicate that GLN can significantly attenuate pro-inflammatory cytokine release, protect against end-organ damage, and decrease mortality from endotoxemia. GLN confers protection even when administered at the onset of endotoxemia, rather then as pre-treatment. Thus, one explanation for the clinical benefits observed from GLN-supplementation may be related to the attenuation of pro-inflammatory cytokines.

High‐affinity LPS binding domain(s) in recombinant factor C of a horseshoe crab neutralizes LPS‐induced lethality

SSCrFCES is a biologically active, recombinant fragment of factor C, which is the endotoxin-sensitive serine protease of the LAL coagulation cascade. The approximately 38 kDa protein represents the LPS binding domain of factor C. A novel secretory signal directs the secretion of SSCrFCES into the culture supernatant of Drosophila cells, and hence it is readily purified. By differential ultrafiltration followed by preparative isoelectric membrane electrophoresis, SSCrFCES was purified as an isoelectrically homogeneous and stable monomeric protein. The ability of SSCrFCES to bind lipid A was analyzed using an ELISA-based assay as well as surface plasmon resonance. SSCrFCES exhibits high positive cooperativity of binding to two or three lipid A molecules, with a Hill's coefficient of 2.2. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is approximately 0.069 microM, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as little as 1 microM of SSCrFCES inhibits the LPS-induced secretion of hTNF-alpha and hIL-8 by THP-1 and human peripheral blood mononuclear cells with greater potency than polymyxin B. SSCrFCES is noncytotoxic, with a clearance rate of 4.7 ml/min. The L.D.(90) of SSCrFCES for LPS lethality is achieved at 2 microM. These results demonstrate the endotoxin-neutralizing capability of SSCrFCES in vitro and in vivo and its potential use for the treatment of endotoxin-induced septic shock.