Endotoxemia: methods of detection and clinical correlates.

As an assay for endotoxin, the Limulus amebocyte lysate assay has several desirable properties: sensitivity, specificity, and potential for adaptation to a quantitative format. Several modifications have been developed to enhance its potential for clinical application. The modifications that allow quantitative measurement of endotoxin and also improve its application to blood samples are described in this review. In fluids other than blood, the detection of endotoxin with the Limulus amebocyte lysate assay can be used as an aid to identify the presence of gram-negative bacteria, and the assay has established utility. With blood, however, there are a range of factors that interfere with the detection of endotoxemia and there are disparate views with respect to the diagnostic and prognostic significance of the test results. In general, the clinical significance of the finding of endotoxemia broadly parallels the frequency and importance of gram-negative sepsis in the patient groups studied and a decline in endotoxin levels accompanies clinical improvement. However, with therapies designed to reduce levels of endotoxin, or to antagonize its effects, it is unclear whether clinical improvement occurs as a consequence of changes in the levels of endotoxemia.     

Comparable endotoxic properties of lipopolysaccharides are manifest in diverse clinical isolates of gram-negative bacteria

In general there is a poor correlation between serum lipopolysaccharide (LPS; the biologically active constituent of endotoxin) levels and mortality in septic patients. The objective of this study was to determine if chemical, structural, or biological differences among LPS from different clinical isolates of gram-negative bacteria might explain this discrepancy. LPS preparations were made using the hot phenol-water extraction method from eight clinical isolates of gram-negative bacteria. As a percentage of the total weight of the LPS, the phosphate content ranged from 3.0 to 13.8% (average, 6.7 +/- 3.6%), and the 2-keto-3-deoxyoctonate content ranged from 1.9 to 27.4% (average, 8.9 +/- 8.5%). These values were not dissimilar to those obtained for a reference endotoxin. In a standard measure of LPS activity, the Limulus amoebocyte lysate assay, there was approximately a twofold difference between the least and most active preparations. The two preparations with the greatest difference in their ability to elicit the secretion of tumor necrosis factor alpha from a mouse peritoneal macrophage cell line were similar in lethality when administered to mice sensitized to the effects of LPS by D(+)-galactosamine. These relatively minor differences in LPS activity seem unlikely to explain the generally observed discrepancy between serum endotoxin levels and mortality in patients with gram-negative sepsis.     

The host response to endotoxin, antilipopolysaccharide strategies, and the management of severe sepsis

Endotoxin, more accurately referred to as lipopolysaccharide (LPS), is recognized as the most potent microbial mediator implicated in the pathogenesis of sepsis and septic shock. Despite its discovery over one century ago, the fundamental role of endotoxin in most patients with septic shock remains enigmatic and its value as a target for therapeutic intervention continues to be a contentious clinical issue. LPS is viewed by the host as an alarm molecule indicating microbial invasion by gram-negative bacterial pathogens. The release of large quantities of LPS into the bloodstream is clearly deleterious to the host, and this event can precipitate the induction of a potentially lethal array of inflammatory mediators and procoagulant factors. The host response to highly purified LPS can create diffuse endothelial injury, tissue hypo-perfusion, disseminated intravascular coagulation, and refractory shock. Numerous attempts to block endotoxin activity in clinical trials with septic patients have met with inconsistent and largely negative results. The tremendous knowledge gained within the past decade into the precise molecular basis for LPS-mediated cellular activation and tissue injury has generated a new generation of therapies that specifically disrupt LPS signaling. This information should provide the necessary insights to specifically target anti-LPS interventions in the ongoing effort to improve the management of sepsis.     

SPLENECTOMY AND SEPSIS: THE ROLE OF THE SPLEEN IN THE IMMUNE-MEDIATED BACTERIAL CLEARANCE

Over the past few years, many observations of overwhelming post splenectomy bacterial infections have been reported. Streptococcus pneumoniae is the aetiologic agent in about 80% of cases, but also gram-negative bacteria are involved in the development of fatal infections in splenectomized patients. Functionally, the spleen plays a fundamental role in bacterial clearance either by antibody response or macrophage bactericidal capacity. At the same time, there is evidence that the spleen also contributes to bacterial endotoxin detoxification. Finally, the mechanisms responsible for gram-positive and gram-negative sepsis in the splenectomized host and possible therapeutical approaches able to neutralize bacterial products endowed with noxious effects are discussed.     

SPLENECTOMY AND SEPSIS: THE ROLE OF THE SPLEEN IN THE IMMUNE-MEDIATED BACTERIAL CLEARANCE

Over the past few years, many observations of overwhelming post splenectomy bacterial infections have been reported. Streptococcus pneumoniae is the aetiologic agent in about 80% of cases, but also gram-negative bacteria are involved in the development of fatal infections in splenectomized patients. Functionally, the spleen plays a fundamental role in bacterial clearance either by antibody response or macrophage bactericidal capacity. At the same time, there is evidence that the spleen also contributes to bacterial endotoxin detoxification. Finally, the mechanisms responsible for gram-positive and gram-negative sepsis in the splenectomized host and possible therapeutical approaches able to neutralize bacterial products endowed with noxious effects are discussed.     

Effect of methylprednisolone on bacterial clearance and endotoxin liberation during experimental sepsis induced by gram-negative bacteria.

To determine the effect of methylprednisolone administration on the clearance of bacteremia and the release and clearance of endotoxin during antibiotic therapy of gram-negative bacterial sepsis, Escherichia coli K1 sepsis was induced in paired rabbits. Moxalactam and either methylprednisolone or placebo were administered to infected rabbits 1.5 h after intraperitoneal administration of live bacteria. Serial blood samples were obtained for quantitation of bacteremia and endotoxemia, arterial blood gases, and complete blood count. Arterial blood pressure, heart rate, and core body temperature were also monitored. There were no significant differences between the methylprednisolone-treated and placebo-treated groups in either the levels of bacteremia or endotoxemia or in the physiologic, metabolic, or hematologic parameters that were measured. We conclude that methylprednisolone administration has no acute effect on bacterial clearance or on the kinetics of endotoxin release and clearance during antibiotic therapy of gram-negative bacterial sepsis in this experimental model.     

Hemoglobin increases mortality from bacterial endotoxin.

Cell-free hemoglobin (Hb) is being developed as an erythrocyte substitute. We have previously demonstrated that cell-free Hb is an endotoxin-binding protein which disaggregates endotoxin and subsequently increases the biological activity of endotoxin in several in vitro assays. Because much of the morbidity and mortality associated with gram-negative bacterial infection is the result of pathophysiologic responses to bacterial lipopolysaccharide (LPS; endotoxin), we studied the effect of Hb on LPS-mediated mortality. Hb infused intravenously into mice before, coincident with, or after intraperitoneal LPS injection substantially increased LPS-related mortality from <5% to 50 to 70% 24 h after administration of LPS and from 50% to 60 to 90% at 48 h. Enhanced mortality was observed over a range of doses of injected LPS. At a given LPS dose, enhancement of mortality was shown to be dependent on the dose of Hb administered. Unmodified native human Hb, alpha-alpha-cross-linked human Hb, and beta-beta-cross-linked human or bovine Hb all were shown to enhance LPS-mediated mortality. Depressed reticuloendothelial cell function may have contributed to the enhanced mortality from LPS in the presence of Hb. Therefore, Hb-based blood substitutes, which are currently undergoing clinical trials, may intensify the potentially fatal effects of the sepsis syndrome in patients with trauma, infection, or hypotension who receive Hb for erythrocyte replacement.     

Treatment of gram-negative bacteremia and septic shock with HA-1A human monoclonal antibody against endotoxin. A randomized, double-blind, placebo-controlled trial. The HA-1A Sepsis Study Group

BACKGROUND. HA-1A is a human monoclonal IgM antibody that binds specifically to the lipid A domain of endotoxin and prevents death in laboratory animals with gram-negative bacteremia and endotoxemia. METHODS. To evaluate the efficacy and safety of HA-1A, we conducted a randomized, double-blind trial in patients with sepsis and a presumed diagnosis of gram-negative infection. The patients received either a single 100-mg intravenous dose of HA-1A (in 3.5 g of albumin) or placebo (3.5 g of albumin). Other interventions, including the administration of antibiotics and fluids, were not affected by the study protocol. RESULTS. Of 543 patients with sepsis who were treated, 200 (37 percent) had gram-negative bacteremia as proved by blood culture. For the patients with gram-negative bacteremia followed to death or day 28, there were 45 deaths among the 92 recipients of placebo (49 percent) and 32 deaths among the 105 recipients of HA-1A (30 percent; P = 0.014). For the patients with gram-negative bacteremia and shock at entry, there were 27 deaths among the 47 recipients of placebo (57 percent) and 18 deaths among the 54 recipients of HA-1A (33 percent; P = 0.017). Analyses that stratified according to the severity of illness at entry showed improved survival with HA-1A treatment in both severely ill and less severely ill patients. Of the 196 patients with gram-negative bacteremia who were followed to hospital discharge or death, 45 of the 93 given placebo (48 percent) were discharged alive, as compared with 65 of the 103 treated with HA-1A (63 percent; P = 0.038). No benefit of treatment with HA-1A was demonstrated in the 343 patients with sepsis who did not prove to have gram-negative bacteremia. For all 543 patients with sepsis who were treated, the mortality rate was 43 percent among the recipients of placebo and 39 percent among those given HA-1A (P = 0.24). All patients tolerated HA-1A well, and no anti-HA-1A antibodies were detected. CONCLUSIONS. HA-1A is safe and effective for the treatment of patients with sepsis and gram-negative bacteremia.     

Intracellular and extracellular enzymatic deacylation of bacterial endotoxin during localized inflammation induced by Escherichia coli.

Acyloxyacyl hydrolase (AOAH), an enzyme that removes the secondary acyl chains of gram-negative bacterial lipid A (endotoxin), has been identified previously in human neutrophils and mouse macrophages. We report here that bovine leukocytes also contain AOAH activity. Although bovine AOAH deacylates bacterial lipopolysaccharide in a manner similar to human AOAH, it is active in vitro over a broader pH range, from 4.0 to 7.0. By using Escherichia coli infection of the bovine mammary gland as a model of localized gram-negative bacterial disease and associated tissue inflammation, AOAH activity per leukocyte increased. In addition, AOAH activity increased in the cell-free portion of infected mammary secretions. These data indicate that AOAH activity increases in leukocytes associated with inflammation induced by gram-negative bacteria and provide additional evidence of its potential involvement in the defense against the effects of bacterial endotoxin.     

A model of infected burn wounds using Escherichia coli O18:K1:H7 for the study of gram-negative bacteremia and sepsis

A difficulty that has emerged in the development and preclinical evaluation of adjuvant therapies for gram-negative sepsis is the lack of easily studied animal models that closely mimic human infection. An objective of this study was to adapt a previously described model of infection in burned mice to rats with a defined bacterial strain of Escherichia coli. Challenge with two colonies of live E. coli O18:K1:H7 bacteria into an 8% full-thickness burn of the dorsal skin surface of rats produced predictable bacteremia at 24 to 48 h and 80 to 100% mortality at 3 to 4 days. E. coli O18:K1:H7 was approximately 10-million-fold more virulent than several other gram-negative bacterial strains. The model should be a useful tool in studying the pathogenicity of burn wound infections and in evaluating the efficacy of novel adjuvant therapies for gram-negative sepsis.     

C1 inhibitor: biologic activities that are independent of protease inhibition

C1 inhibitor therapy improves outcome in several animal models of inflammatory disease. These include sepsis and Gram negative endotoxin shock, vascular leak syndromes, hyperacute transplant rejection, and ischemia-reperfusion injury. Furthermore, some data suggest a beneficial effect in human inflammatory disease. In many inflammatory conditions, complement system activation plays a role in pathogenesis. The contact system also very likely is involved in mediation of damage in inflammatory disease. Therefore, the beneficial effect of C1 inhibitor has been assumed to result from inhibition of one or both of these systems. Over the past several years, several other potential anti-inflammatory effects of C1 inhibitor have been described. These effects do not appear to require protease inhibition and depend on non-covalent interactions with other proteins, cell surfaces or lipids. In the first, C1 inhibitor binds to a variety of extracellular matrix components including type IV collagen, laminin, entactin and fibrinogen. The biologic role of these reactions is unclear, but they may serve to concentrate C1 inhibitor at extravascular inflammatory sites. The second is a non-covalent interaction with C3b that results in inhibition of formation of the alternative pathway C3 convertase, a function analogous to that of factor H. The third is an interaction with E and P selectins on endothelial cells that is mediated by the Lewis(x) tetrasaccharides that are expressed on C1 inhibitor. These interactions result in suppression of leukocyte rolling and transmigration. The fourth interaction is the binding of C1 inhibitor to Gram negative bacterial endotoxin that results in suppression of endotoxin shock by interference with the interaction of endotoxin with its receptor complex on macrophages. Lastly, C1 inhibitor binds directly to Gram negative bacteria, which leads to suppression of the development of sepsis, as demonstrated in the cecal ligation and puncture model. These observations suggest that C1 inhibitor is a multi-faceted anti-inflammatory protein that exerts its effects through a variety of mechanisms including both protease inhibition and several different non-covalent interactions that are unrelated to protease inhibition.     

Does gram-negative bacteraemia occur without endotoxaemia? A meta-analysis using hierarchical summary ROC curves

The limulus assay for endotoxin has been studied as a method for the rapid identification of gram-negative (GN) bacteraemia. The chromogenic (C-limulus) version is 100-fold more sensitive to an internal endotoxin standard than the earlier gelation version (G-limulus). The objective of this analysis is to compare the concordance between GN bacteraemia and endotoxaemia as determined in clinical studies using either version of the limulus assay. The summary results for the diagnostic odds ratio (DOR), sensitivity and specificity were derived using a hierarchical summary receiver operating characteristic (HSROC) method of meta-analysis. Fifty-eight studies (25 G-limulus and 33 C-limulus) were included. Surprisingly, the mean DOR (4.9; 3–7.9 versus 10.7; 5.2–21.8) was inferior for studies using the C-limulus versus the original G-limulus version of the assay. Moreover, among studies limited to sepsis syndrome patients, the mean DOR remains poor at 4.2 (1.8–9.5). The proportion of GN bacteraemic patients for whom endotoxaemia is not detectable with either version of the limulus assay is >20% among the 58 studies overall, but >30% after the exclusion of studies with <25 patients and >20% among studies of patients with sepsis syndrome. These findings help to reconcile seemingly disparate study results.     

The chronic consequences of severe sepsis

The early events of severe sepsis set in motion a cascade of events that significantly contributes to the morbidity and mortality observed during the first few days of this syndrome. Although sepsis is a deadly, acute disease, survivors also suffer long-term consequences. Clinical data underscore subsequent high mortality rates associated with patients who are long-term survivors of the acute septic episode. Within 1 year of surviving severe sepsis, there is a 26% predicted mortality rate, and many patients succumb to lung complications. In this review, we focus on the cellular and molecular mechanisms that dictate the longer-term sequela of sepsis and related lung injury. We have established a murine model of experimental sepsis [cecal ligation and puncture (CLP)], which results in an approximate 60% survival rate. Our studies have demonstrated that these survivors are susceptible to a fungal infection with 100% mortality when challenged 3 days or 15 days post-recovery from the initial CLP. This increased mortality correlates with changes in cytokines and Toll-like receptor expression and alterations in lung leukocyte populations. We hypothesize that the lung becomes predisposed to nosocomial infections for extended periods of time after severe sepsis via mechanisms that include alterations in inflammatory cytokines and an increase in immunomodulatory chemokines, such as monocyte chemoattractant protein-1 and C10. These mediators may alter the innate-immune response by affecting dendritic cells and macrophages, which could provide a mechanism for the immunosuppression observed following sepsis.     

Melioidosis: epidemiology, pathophysiology, and management

Melioidosis, caused by the gram-negative saprophyte Burkholderia pseudomallei, is a disease of public health importance in southeast Asia and northern Australia that is associated with high case-fatality rates in animals and humans. It has the potential for epidemic spread to areas where it is not endemic, and sporadic case reports elsewhere in the world suggest that as-yet-unrecognized foci of infection may exist. Environmental determinants of this infection, apart from a close association with rainfall, are yet to be elucidated. The sequencing of the genome of a strain of B. pseudomallei has recently been completed and will help in the further identification of virulence factors. The presence of specific risk factors for infection, such as diabetes, suggests that functional neutrophil defects are important in the pathogenesis of melioidosis; other studies have defined virulence factors (including a type III secretion system) that allow evasion of killing mechanisms by phagocytes. There is a possible role for cell-mediated immunity, but repeated environmental exposure does not elicit protective humoral or cellular immunity. A vaccine is under development, but economic constraints may make vaccination an unrealistic option for many regions of endemicity. Disease manifestations are protean, and no inexpensive, practical, and accurate rapid diagnostic tests are commercially available; diagnosis relies on culture of the organism. Despite the introduction of ceftazidime- and carbapenem-based intravenous treatments, melioidosis is still associated with a significant mortality attributable to severe sepsis and its complications. A long course of oral eradication therapy is required to prevent relapse. Studies exploring the role of preventative measures, earlier clinical identification, and better management of severe sepsis are required to reduce the burden of this disease.     

The endotoxin-binding bactericidal/permeability-increasing protein (BPI): a target antigen of autoantibodies

The bactericidal/permeability-increasing protein (BPI) is an endotoxin-binding neutrophil leukocyte-granule protein with antibacterial and anti-endotoxin properties. A recombinant form of BPI (rBPI21) has been developed and is being tested as a therapeutic agent to treat gram-negative bacterial infections and exposure to gram-negative bacterial endotoxin. BPI is also a target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA). BPI-ANCA are present in cystic fibrosis, inflammatory bowel disease, vasculitis, and primary sclerosing cholangitis; presence of BPI-ANCA appears associated with a higher inflammatory disease activity and greater organ damage. BPI-ANCA as well as ANCA directed at other neutrophil-granule proteins may exacerbate inflammation by nonspecific effects of extracellular and cell-associated immune complexes. BPI-ANCA may further worsen inflammation by reducing the ability of BPI to promote clearance of gram-negative bacteria and bacterial-associated endotoxin.     

Gelatinase B deficiency protects against endotoxin shock

Gelatinase B or matrix metalloproteinase-9 (MMP-9) is stored in the tertiary granules of polymorphonuclear leukocytes. These cells are key effectors in acute inflammatory diseases such as sepsis. Endotoxin leads to rapid release of gelatinase B from these granules in vitro and in vivo, but the role of this enzyme in bacterial sepsis and endotoxin shock remains unclear. We studied the clinical course of endotoxinemia and its relation with the expression of gelatinase B from the pool of circulating leukocytes in adult as well as in young mice in a model of endotoxin-induced shock and compared wild-type with gelatinase B-deficient mice. The gelatinase B-deficient mice were resistant to endotoxin shock, which implies that specific MMP-9 inhibition constitutes anapproach for the treatment of septic shock syndromes.     

Human neutrophils secrete gelatinase B in vitro and in vivo in response to endotoxin and proinflammatory mediators

Bacterial sepsis is characterized by a systemic inflammatory state, with activation of numerous cell types. Phagocytes participate in this phenomenon by secreting various proinflammatory cytokines and enzymes. Matrix metalloproteinases (MMPs) such as gelatinases are produced by phagocytes and are thought to play an important role in processes of cell transmigration and tissue remodeling. In this work, we show that endotoxin (lipopolysaccharide [LPS]) and other inflammatory mediators, such as tumor necrosis factor (TNF), interleukin-8, and granulocyte colony-stimulating factor, induce a rapid (within 20 min) release of gelatinase-B (MMP-9) zymogen in whole human blood, as determined by gelatin zymography. The polymorphonuclear neutrophil was identified as the cell responsible for this rapid secretion, as a result of the release of preformed enzymes stored in granules. Normal human subjects given LPS intravenously showed a similar pattern of proMMP-9 secretion, with maximum plasma levels reached 1.5 to 3 h after LPS administration (P = 0.0009). Prior administration of TNF receptor:Fc, a potent TNF antagonist, to subjects given LPS, only partially blunted the release of proMMP-9 (P = 0.033). Ibuprofen, a cyclooxygenase inhibitor, did not alter this pattern of release. Increased levels of proMMP-9 and proMMP-2, as well as activated forms of MMP-9, were found in plasma from two patients with gram-negative sepsis. The levels of MMPs paralleled the severity of clinical condition and a marker of the severity of sepsis, plasma procalcitonin. These data indicate that MMPs are released in whole blood in response to various inflammatory mediators and that they could serve as sensitive and early markers for cell activation during the course of bacterial sepsis.     

Endotoxin enhancement of lymphocyte adherence to cultured sheep lung microvascular endothelial cells.

The most common predisposing factor for development of the adult respiratory distress syndrome is gram-negative sepsis. Our previous studies have shown that a single infusion of Escherichia coli endotoxin into sheep causes early sequestration of lymphocytes in the lungs' microcirculation. In this report, we examined the effects of endotoxin on sheep lymphocyte adherence to sheep pulmonary microvascular endothelial cells in vitro. Endothelial cells were exposed to endotoxin, and subsequent adherence of 51Cr-labeled lymphocytes was measured in a monolayer adhesion assay. Endotoxin enhanced adherence of lymphocytes isolated from blood and caudal mediastinal node (CMN) lymph in a time- and dose-dependent manner. Adherence of CMN lymphocytes increased from a control value of 13.6 +/- 1.6% to 29.9 +/- 3.1% after 4 h of treatment with 1 microgram/ml endotoxin. Both B and T lymphocytes contributed to the increased adherence. Pretreatment of the endothelial cells with cycloheximide revealed that the endotoxin-enhanced adherence was partially dependent upon protein synthesis. Morphologic studies revealed that enhanced adherence was accompanied by a 5-fold increase in migration of lymphocytes between endothelial cells. In contrast to human umbilical vein endothelial cells, antibodies to the known lymphocyte adherence molecules, lymphocyte function-associated antigen (LFA-1), CD-44, and the lymphocyte homing receptor (LECAM-1), were ineffective in blocking adherence to the sheep pulmonary endothelial cells. We conclude that the acute sequestration of lymphocytes in the pulmonary microcirculation of sheep after endotoxin administration is due to increased adhesive properties of the endothelial cells. Our data suggest that this adherence is mediated by as yet undescribed mechanisms that may be unique to pulmonary microvascular endothelium.     

Proteolytic activity and fatal gram-negative sepsis in burned mice: effect of exogenous proteinase inhibition.

Circulating proteolytic activity (PA) increases following burn or surgical trauma. Challenging traumatized mice with the yeast Candida albicans further increases PA. Once a PA threshold has been passed, mortality increases as PA increases. The purposes of this study were to determine (i) if gram-negative bacterial challenge affects circulating PA and mortality as Candida challenge does and (ii) if proteinase inhibitor treatment with aprotinin, antithrombin III, and alpha 1-proteinase inhibitor decreases circulating PA and increases the survival of burned mice infected with a bacterium. For all bacteria tested (Proteus mirabilis, Pseudomonas aeruginosa, and Klebsiella pneumoniae), burn plus challenge significantly elevated PA and mortality above levels in mice that were only burned or only challenged. Quantitative culture counts indicated that the mice died of sepsis. Proteinase inhibitor treatment of mice burned and challenged with K. pneumoniae significantly decreased circulating PA, decreased the hepatic microbial load, and increased survival. Hence, in traumatized mice challenged with either C. albicans or gram-negative bacteria, a relationship exists between proteolytic load and subsequent septic death. Parallels between these animal studies and human studies are discussed.     

Recent Advances in Therapy of Sepsis

The inability to reduce the high mortality due to overwhelming bacterial infection and sepsis has prompted a search for new therapeutic agents. Among these may be a wide range of endogenous antibiotic polypeptides that are prominent components of effective antimicrobial host defences. One of these polypeptide antibiotics is the bactericidal/permeability-increasing protein (BPI), a protein of approximately 55kD which is present in human and other mammalian neutrophils. BPI is toxic for Gram-negative bacteria and binds to endotoxin, resulting in its clearance and neutralisation. A recombinant 21kD N-terminal BPI fragment is at least as active as holo-BPI and protects both animals and humans against the effects of Gram-negative infections and their complications. Phase II/III clinical trials in fulminant paediatric meningococcaemia, haemorrhagic trauma, hepatectomy and severe peritoneal infections are in progress.