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Endotoxin and adjunctive therapies in gram-negative sepsis
Endotoxin and Adjunctive Therapies in Gram-negative Sepsis Michael J. Romano, MD Gram-negative infections remain a major cause of morbidity and mortality. The death rate from septicemia is increasing and now surpasses acquired immunodeficiency syndrome as a cause of death. Initial attempts at adjunctive therapy using monoclonal antibodies directed against the lipid A core of endotoxin (HA-1A, E5) have not provided consistent benefit in large-scale trials. The only other agent targeting endotoxin is bactericidal permeability increasing protein, for which a phase III trial in children with meningococcemia has been completed. Other strategies being developed include modifying the effect of mediators of endotoxin toxicity, including CD14, soluble CD14, and lipopolysaccharide binding protein; detoxifying endotoxin by binding with polymyxin B; and implementing antagonists of endotoxin or compounds that inhibit the biosynthesis of endotoxin. Copyright © 2001 by W.B. Saunders Company
eptic shock is a common and serious condition in adult and pediatric intensive care unit patients worldwide. The death rate from septicemia is increasing, and septicemia now ranks above human immunodeficiency virus infection as a cause of death.1 The relative proportion of gram-positive and fungal organisms is increasing, but gram-negative organisms account for most of the clinical isolates.2 A slight improvement in survival rates for septic shock may have occurred in recent years, but most studies continue to report mortality rates of 41 to 80 percent.2 Most adjunctive therapies for gram-negative sepsis focus on the central role of endotoxin (lipopolysaccharide or LPS) in what is clinically recognized as sepsis, systemic inflammatory response syndrome (SIRS), or septic shock. Endotoxemia, however, is not a consistent finding in gram-negative sepsis, and it occurs frequently in gram-positive, fungal, or culture-negative sepsis.3 Furthermore, the hemodynamic response to gram-positive sepsis is similar to that of gramnegative sepsis,4 complicating the early clinical recognition of patients who might be candidates for therapies directed against endotoxin.
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Endotoxin and LipopolysaccharideBinding Protein Endotoxin consists of three distinct components: (1) repeating oligosaccharide terminal side chains that provide O
From the Division of Pediatric Critical Care Medicine, Texas Tech University Health Sciences Center, Lubbock, TX. Address correspondence to Michael J. Romano, MD, Associate Professor of Clinical Pediatrics, Texas Tech University Health Sciences Center, 3601 4th St, Lubbock, TX 79430. Copyright © 2001 by W.B. Saunders Company 1045-1870/01/1201-0005$35.00/0 doi:10.1053/spid.2001.19231
antigen specificity and differ from strain to strain, (2) an oligosaccharide core, and (3) lipid A. Lipid A is highly conserved across gram-negative organisms and is responsible for the toxicity of LPS. Infusions of LPS produce a stereotypical pattern of cytokine activation characterized by rapid clearance of endotoxin from circulation and the sequential appearance of tumor necrosis factor (TNF) and interleukin (IL)-6.5 The biology of LPS receptors has been reviewed recently6-8 and is discussed here briefly as a basis for therapeutic interventions. LPS binds to numerous cell surface and serum molecules, including CD11/18, CD14, lipopolysaccharide-binding protein (LBP), and soluble CD14 (sCD14). The initial and key cellular event after the occurrence of endotoxemia appears to be the interaction of LPS with CD14. Knockout mice deficient in CD14 are resistant to endotoxin shock,9,10 and CD14-specific monoclonal antibodies inhibit monocyte production of TNF and IL-10 by LPS.11 Transfection of CD14 into cell lines that normally do not express it transforms those cells into LPS-responsive cells.12,13 Soluble CD14 facilitates the endotoxin-induced activation of cells that do not express surface-bound CD14.14,15 LBP is a glycoprotein that is present at low baseline levels in plasma16 and increases 2 log orders during inflammation.17 LBP is synthesized primarily in hepatocytes,18,19 with lesser amounts in other tissues, including the pulmonary artery.20,21 LBP catalyzes the transfer of LPS monomers from circulating aggregates to sCD14.22 In the presence of LBP, sensitivity to LPS is increased dramatically. Blood from LBP knockout mice that lack LBP activity is profoundly hyporesponsive to LPS stimulation.23 Interestingly, the in vivo TNF response of LBP knockout mice is essentially identical to that of LBP hemizygous mice, suggesting an LBP-independent, but CD14-dependent, mechanism of TNF production.
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LBP also appears to play a key role in LPS detoxification through its interaction with high-density lipoproteins (HDLs). LBP catalyzes the binding of LPS to HDL24 and facilitates the transfer of LPS from LPS-sCD14 complexes to HDL.25 This apparent paradox may represent a dosedependent effect of LBP on TNF production. Nanogram levels of LBP clearly enhance LPS sensitivity. Higher levels of LBP, in the microgram range, appear to decrease TNF production in vitro. Recombinant LBP injected into mice intraperitoneally (but not intravenously) attenuates the TNF-␣ and IL-6 responses to LPS administration and decreases the mortality from administration of LPS or live Escherichia coli.26 Analogous observations have been made in the case of sCD14. High doses of sCD14 inhibited the LBP-dependent activation of neutrophils, whereas lower doses stimulated neutrophil activation.27
Antibodies Directed Against Endotoxin Immunization with normal or “smooth” bacteria generates immunoglobulin that is highly protective in animals challenged with the homologous bacteria. Antibodies directed against O-antigen side chains do not neutralize LPS in vitro, so the presumed mechanism is via improved clearance of LPS or bacteria. The large number of gram-negative organisms causing clinical disease precludes this approach as practical. Immunization with mutants that lack the O-side chains, so called “rough” mutants based on their appearance in culture, generates antibodies directed against the core polysaccharide and highly conserved lipid A,28,29 yielding antibodies that may afford protection against a broad range of gram-negative pathogens.
J5 Mutant of E coli Ziegler et al30 tested this approach using the J5 mutant of E coli 0111:B4. J5 is a “rough” mutant lacking an epimerase enzyme responsible for attaching the O-side chain to the core polysaccharide.31 Healthy individuals donated one unit of serum before and after immunization with killed J5 vaccine. Patients (n ⫽ 304) were infused in a blinded fashion with either preimmunization or postimmunization serum. A single infusion of postimmunization J5 antiserum reduced mortality in the group with bacteremia by 37 percent. J5 antiserum has been evaluated in 3 other human trials. McCutchan et al32 gave a single unit as prophylaxis to patients at high risk of gram-negative infections. No effect on the incidence of bacteremia or mortality was seen. Baumgartner et al33 administered a unit of J5 antiserum every 5 days while patients were at high risk for gramnegative sepsis. J5 antiserum had no effect on the incidence of bacteremia, but the incidence of shock was reduced by 55 percent and death by 71 percent in the J5-treated group. J5 antiserum has provided no mortality benefit in children with infectious purpura.34 Questions have been raised regarding what component of the serum was protective. Calandra et al35 used an immunization method similar to that used by Ziegler but
separated the IgG subfraction of the serum. In this randomized, blinded trial, J5 IgG provided no protection against death or time to death.
HA-1A and E5 Advances in the production of monoclonal antibodies led to the development of 2 IgM class antibodies using J5 as the antigen; one of human origin36 (HA-1A; Centocor, Malvern, PA), the other murine37 (E5; XOMA, Berkeley, CA). Both antibodies have been evaluated in large-scale human trials. Ziegler et al38 studied 543 adults with suspected gramnegative infection and sepsis syndrome who received a single 100-mg dose of HA-1A or placebo. Overall, mortality was unchanged. In the subset of 200 patients with gramnegative bacteremia, 28-day mortality was decreased 39 percent in the treatment group. In 101 of these 200 patients in shock at enrollment, mortality was decreased 42 percent in the treatment group. No benefit was seen in patients with nonbacteremic gram-negative, gram-positive, or fungal infections. A second trial of HA-1A did not confirm decreased mortality in patients with gram-negative bacteremia, and it was stopped after the first planned interim analysis because a higher mortality rate occurred in patients who were receiving HA-1A and did not have gram-negative bacteremia.39 A French registry open-label study40 of 600 patients who received HA-1A found that mortality rates tended to be higher than predicted by severity of illness, especially among patients without gram-negative bacteremia. E5 has been evaluated in at least three phase III trials. The first trial41 enrolled 486 patients with suspected gramnegative infection and a systemic septic response. No difference was seen in the primary end point of 30-day mortality in patients with gram-negative sepsis or gramnegative sepsis with one or more organ failures at study entry. Subgroup analysis suggested a survival benefit in patients who were not in refractory shock at the time of study enrollment. A second trial, excluding patients in refractory shock, failed to show improved survival in patients with gram-negative sepsis or gram-negative sepsis with organ failure.42 A third trial was halted after interim analysis of 1000 patients.43
Bactericidal Permeability Increasing Protein The fourth agent with antiendotoxin activity that has entered large-scale human trials is an analogue of bactericidal permeability increasing protein (BPI). BPI originally was found in the azurophilic granules of human neutrophils.44 Subsequently, it has been identified in eosinophils45 and expressed on the surface of monocytes.46 BPI shares significant sequence homology with LBP, although the biologic effects are profoundly different. BPI has antibacterial activity against gram-negative organisms47 and certain cell wall– deficient gram-positive organisms.48 BPI inhibits TNF release from whole blood incubated with gram-negative organisms. A recombinant N-terminal fragment, BPI23, ap-
Adjunctive Therapies in Gram-negative Sepsis pears more potent than does the native protein.47 BPI or its analogues also have antiendotoxin effects. In human volunteers administered endotoxin, BPI23 inhibited activation of the coagulation and fibrinolytic systems49; significantly reduced serum levels of TNF, soluble TNF receptors p55 and p75, IL-6, IL-8, and IL-10; and attenuated neutrophil activation.50 A recombinant N-terminal 21-kD fragment of BPI (rBPI21, Neuprax; XOMA) has been evaluated in several diseases, including severe meningococcal sepsis in children. Meningococcemia is a unique gram-negative infection. It is predominantly a disease of the young; half of the endemic cases occur in children younger than 2 years, and in epidemics, most of the affected individuals are adolescents or young adults.51-53 Endotoxin levels may be profoundly elevated and correlate with the severity of the illness.54 In large series, mortality rates in this otherwise healthy population have been significant, ranging from 10 to 18.6 percent,55-57 and technologic advances in intensive care during the last 30 years have not appeared to appreciably improve the mortality rate.56 rBPI21 has been evaluated in 2 clinical trials of meningococcal sepsis in children. The first trial was an open-label, phase I/II dose-escalation trial in patients aged 1 to 18 years.58 Twenty-six patients received a loading dose followed by a 24-hour continuous infusion. No safety-related issues were identified. One patient died (3.8%), compared with expected mortality rates of at least 15 to 30 percent. A phase III, double-blind, placebo-controlled trial involving almost 400 pediatric patients with meningococcemia had completed enrollment, and the data were analyzed.59 The results of this study have been published.59a Three hundred ninety-three patients with severe meningococcal sepsis received rBPI21 (n ⫽ 190) or placebo (n ⫽ 203). Overall, there was no difference in the primary endpoint of mortality (rBPI21 ⫽ 7.4%, placebo ⫽ 9.9%; P ⫽ .48) or number of patients requiring amputations. There was a modest benefit noted in pediatric overall performance category (POPC) scales in the rBPI21 group. rBPI21 also has been evaluated in the setting of significant hemorrhage caused by trauma.60 Adults (n ⫽ 401) with acute hemorrhage secondary to trauma requiring at least 2 units of blood and within 12 hours of injury received rBPI21 or placebo in a double-blinded fashion. Among the 401 enrolled patients, no differences were found between drug and placebo groups in any of the primary or secondary end points, including mortality. Post hoc analysis suggested a decreased incidence of pneumonia or acute respiratory distress syndrome (ARDS) in patients receiving active drug (placebo group, 32%; rBPI21, 22%; hazard ratio ⫽ 0.66; post hoc P ⫽ .03). A second trial utilizing incidence of pneumonia or ARDS as the primary end point was halted after a planned interim analysis.61 Preliminary studies with rBPI21 are underway or completed in patients with cystic fibrosis who are having pulmonary exacerbations, patients undergoing partial hepatectomy, and patients with severe intraabdominal infections.
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Other Approaches to Adjunctive Therapy Active immunization with LPS derivatives is being investigated. A detoxified vaccine utilizing LPS from E coli J5 and an outer membrane protein from group B meningococcus is highly immunogenic.62 Polyclonal antibodies produced in response to the vaccine bound a variety of gram-negative organisms and were protective in a neutropenic rat model of Pseudomonas aeruginosa bacteremia.63 This approach has been reviewed recently.64 Antibodies to CD14 have been shown to be protective in 2 animal models of endotoxemia. Leturcq et al65 pretreated monkeys with 1 of 2 murine monoclonal antibodies against CD14 (28C5 or 18E12), followed by an 8-hour infusion of endotoxin. Both antibodies had a significant impact on the severe, sustained hypotension seen in control animals: 28C5 completely blocked this response, whereas animals treated with 18E12 had an initial hypotensive response similar to that of controls but which resolved in 5 hours. The TNF-␣ response was delayed and reduced in both treatment groups compared with controls. Significant decreases in IL-1 and IL-6 responses also were seen. Schimke et al66 investigated a different antibody in a rabbit model involving sequential exposures to endotoxin during a 24-hour period. This model was considered to more closely represent the development of human septic shock. Animals received three injections of endotoxin at 0, 5, and 24 hours and an anti-CD14 antibody on a variety of timetables, including a prophylactic treatment group and delayed treatment extending to 23 hours. At 48 hours, mortality was 32 percent in the control group, compared with zero in animals receiving anti-CD14. Treatment with antibody also eliminated renal cortical necrosis (84% in controls) and pulmonary injury (61% in controls) and prevented the gradual decrease in blood pressure seen in controls. Most importantly, the treatment effect was not dependent on time of administration; delayed treatment was as effective as was prophylactic therapy. Soluble CD14 occupies a paradoxic niche in the cytokine cascade. It is a key factor in facilitating the cytokine response to endotoxin and in detoxifying endotoxin. Soluble CD14 inhibits LPS-induced production of TNF in vitro.67 Finally, infusions of sCD14 decreased production of TNF and prevented mortality in a mouse model of endotoxemia.68 Numerous approaches to detoxifying LPS are being investigated. HDL binds LPS with high affinity and stability and is cleared via hepatic mechanisms. HDL can attenuate the biologic effects of endotoxin. Transgenic mice that express elevated levels of human apolipoprotein (the principal protein component of HDL) are resistant to endotoxin challenge,69 and rats made hypolipidemic have increased mortality from endotoxin challenge.70 Infusions of recombinant human HDL (rHDL) have been evaluated in a phase I trial in humans.71,72 rHDL reduced endotoxin-induced release of TNF, IL-6, and IL-8, activation of coagulation, and fibrinolysis. Monocytes from rHDL-treated whole blood showed reduced expression of CD14 and attenuation of TNF production with endotoxin challenge.
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Polymyxin B is a cationic polypeptide antibiotic that neutralizes most biologic activities of lipid A.73,74 Numerous approaches have been attempted to overcome its significant renal and central nervous system toxicity; the most advanced of these attempts is binding polymyxin B to the fibers of a hemoperfusion cartridge.75 Aoki et al76 described 16 patients who underwent 2 hours of veno-venous hemoperfusion through such a cartridge. Cardiopulmonary parameters showing improvement after therapy included an increase in mean systolic blood pressure in patients who were hypotensive when beginning therapy and a decrease in cardiac index in patients who were hyperdynamic before therapy. Serum endotoxin levels decreased from 76 pg/mL to 21 pg/mL after treatment (P ⬍ .05), an effect that has been confirmed in subsequent trials.77-79 Survival has been improved compared with that of untreated controls.79 Each of these trials is limited by small sample size and the lack of a sham treatment control group. Numerous antagonist analogues of lipid A have been developed. Lipid X is a precursor of lipid A and has weak antagonistic activity with some beneficial effects in mice80 and sheep.81 Modifications of the lipid X molecule have yielded molecules with much greater antagonistic activity.82 DT-5461 is a synthetic analogue of E coli lipid A, with mixed agonist and antagonist activity. It has been studied as an antitumor agent by virtue of its ability to stimulate TNF production,83,84 although at levels 5 to 6 log orders lower than those produced by native LPS.85 In mice, DT-5461 protects against LPS-induced mortality.85 E5531 is a potent, pure LPS antagonist that is protective in a mouse model of endotoxemia and gram-negative bacteremia.86,87 E5531 is in phase II trials in the United States. A final approach has been to develop molecules that selectively inhibit enzymatic steps in the synthesis of lipid A. The second step in the synthesis of lipid A is deacetylation of UDP-3-O-[R-3-hydroxymyristoyl)-GlcNAc.88 Mutant E coli that lack this enzyme are profoundly sensitive to antibiotics such as erythromycin, rifampin, and clindamycin, which normally are not active against gram-negative organisms.89 Numerous molecules that inhibit this deacetylase have been developed. The most potent of these (L161,240) has been shown to cure mice in a lethal model of E coli peritonitis.90 Unfortunately, the agent has little activity against other gram-negative organisms such a Serratia marcescens or P aeruginosa.
Conclusions The clinical application of antiendotoxin strategies in particular and immunologic modification of the inflammatory process in general must be ranked by clinicians as one of the major disappointments of the last 10 years. None of the myriad number of agents that have been evaluated has shown consistent benefit in clinical trials, and none has survived the Food and Drug Administration review process to enter clinical practice. What have we learned from almost 20 years of clinical trials? The evolution of antiendotoxin strategies, from basic science through highly publicized trials and subsequent
failures, has been critiqued exhaustively.91,92 Basic assumptions about this approach have been questioned: J5 antiserum does not appear to bind LPS93; HA-1A and E5 will bind to lipid A or LPS under some circumstances,36,94,95 but neither appears able to neutralize the biologic activity of LPS.95 What is the appropriate animal model? Infusions of endotoxin commonly are used to model sepsis and septic shock, despite the number of physiologic differences between this model and others utilizing infusions of live or killed gram-negative organisms96 or a cecal ligation and puncture model simulating peritonitis.97,98 What are appropriate end points in clinical trials? Is all-cause mortality valid, or should one evaluate resolution of organ system failure or resolution of hemodynamic perturbations? How are patients identified for inclusion? Evidence of SIRS frequently is included in the criteria, but SIRS is by no means specific for gram-negative bacteremia.99 Should a cost-benefit analysis be part of the approval process, and, if so, where is the line drawn (and by whom)?
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inhibits LPS-induced tumor necrosis factor-␣ production by cells in whole blood. J Immunol 152:5868-5876, 1994 Haziot A, Rong GW, Lin XY, et al: Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide). J Immunol 154:6529-6532, 1995 Levine DM, Parker TS, Donnelly TM, et al: In vivo protection against endotoxin by plasma high density lipoprotein. Proc Natl Acad Sci USA 90:12040-12044, 1993 Feingold KR, Funk JL, Moser AH, et al: Role for circulating lipoproteins in protection from endotoxin toxicity. Infect Immun 63:2041-2046, 1995 Pajkrt D, Doran JE, Koster F, et al: Antiinflammatory effects of reconstituted high-density lipoprotein during human endotoxemia. J Exp Med 184:1601-1608, 1996 Pajkrt D, Lerch PG, van der Poll T, et al: Differential effects of reconstituted high-density lipoprotein on coagulation, fibrinolysis and platelet activation during human endotoxemia. Thromb Haemost 77:303-307, 1997 Corrigan JJ Jr, Bell BM: Endotoxin-induced intravascular coagulation: Prevention with polymyxin B sulphate. J Lab Clin Med 77:802-810, 1971 Morrison DC, Jacobs DM: Binding of polymyxin B to the lipid A portion of bacterial lipopolysaccharides. Immunochemistry 13:813-818, 1976 Shoji H, Tani T, Hanasawa K, et al: Extracorporeal endotoxin removal by polymyxin B immobilized fiber cartridge: Designing and antiendotoxin efficacy in the clinical application. Ther Apher 2:3-12, 1998 Aoki H, Kodama M, Tani T, et al: Treatment of sepsis by extracorporeal elimination of endotoxin using polymyxin Bimmobilized fiber. Am J Surg 167:412-417, 1994 Nakamura T, Ebihara I, Shimada N, et al: Effects of hemoperfusion with polymyxin B-immobilized fibre on serum neopterin and soluble interleukin-2 receptor concentrations in patients with septic shock. J Infect 37:241-247, 1998 Nakamura T, Suzuki Y, Shimada N, et al: Hemoperfusion with polymyxin B-immobilized fiber attenuates the increased plasma levels of thrombomodulin and von Willebrand factor from patients with septic shock. Blood Purif 16:179-186, 1998 Nakamura T, Ebihara I, Shoji H, et al: Treatment with polymyxin B-immobilized fiber reduces platelet activation in septic shock patients: Decrease in plasma levels of soluble P-selectin, platelet factor 4 and beta-thromboglobulin. Inflamm Res 48: 171-175, 1999 Golenbock DT, Leggett JE, Rasmussen P, et al: Lipid X protects mice against fatal Escherichia coli infection. Infect Immun 56:779-784, 1988 Golenbock DT, Will JA, Raetz CR, et al: Lipid X ameliorates pulmonary hypertension and protects sheep from death due to endotoxin. Infect Immun 55:2471-2476, 1987 Augy-Dorey S, Billington DC, Camara JA, et al: Synthesis and biological evaluation of carbocyclic analogues of lipid X: New nonpolar antagonists of LPS induced TNF production. Drug Des Discov 16:41-48, 1999 Jimbo T, Akimoto T, Tohgo A: Systemic administration of a synthetic lipid A derivative, DT-5461a, reduces tumor blood flow through endogenous TNF production in hepatic cancer model of VX2 carcinoma in rabbits. Anticancer Res 16:359364, 1996 Kumazawa E, Jimbo T, Akimoto T, et al: Antitumor effect of DT-5461, a lipid A derivative, against human tumor xenografts is mediated by intratumoral production of tumor necrosis fac-
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tor and affected by host immunosuppressive factors in nude mice. Cancer Invest 15:522-530, 1997 Sato K, Yoo YC, Fukushima A, et al: A novel synthetic lipid A analog with low endotoxicity, DT-4561, prevents lethal endotoxemia. Infect Immun 63:2859-2866, 1995 Christ WJ, Asano O, Robidoux AL, et al: E5531, a pure endotoxin antagonist of high potency. Science 268:80-83, 1995 Kawata T, Bristol JR, Rossignol DP, et al: E5531, a synthetic non-toxic lipid A derivative blocks the immunobiological activities of lipopolysaccharide. Br J Pharmacol 27:853-862, 1999 Sorensen PG, Lutkenhaus J, Young K, et al: Regulation of UDP-3-O-[R-3-hydroxymyristoyl]-N-acetylglucosamine deacetylase in Escherichia coli. The second enzymatic step of lipid A biosynthesis. J Biol Chem 271:25898-25905, 1996 Vuorio R, Vaara M: The lipid A biosynthesis mutation 1pxA2 of Escherichia coli results in drastic antibiotic supersusceptibility. Antimicrob Agents Chemother 4:826-829, 1992 Onishi HR, Pelak BA, Gerckens LS, et al: Antibacterial agents that inhibit lipid A biosynthesis. Science 274:980-982, 1996 Warren HS, Danner RL, Munford RS: Anti-endotoxin monoclonal antibodies. N Engl J Med 326:1153-1157, 1992 Stephenson J: Reflecting and regrouping after failed trials, sepsis researchers forge on. JAMA 275:823-824, 1996 Heumann D, Baumgartner JD, Jacot-Guillarmod H, et al: Antibodies to core lipopolysaccharide determinants: Absence of
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cross-reactivity with heterologous lipopolysaccharides. J Infect Dis 163:762-768, 1991 Gazzano-Santoro H, Parent JB, Wood EL, et al: Reactivity of E5 monoclonal antibody to smooth lipopolysaccharides. Program and Abstract of the 31st Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, IL, Am Soc Microbiol, 1991 (asbtr 788) Warren HS, Amato SF, Fitting C, et al: Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide. J Exp Med 177:8997, 1993 Brooke M, Lingnau W, Hinder F, et al: Endotoxin versus bacteremia: A comparison focusing on clinical relevance. Prog Clin Biol Res 392:393-403, 1995 Hadjiminas DJ, McMasters KM, Peyton JC, et al: Tissue tumor necrosis factor mRNA expression following cecal ligation and puncture or intraperitoneal injection of endotoxin. J Surg Res 56:549-555, 1994 Astiz ME, Saha DC, Brooks K, et al: Comparison of the induction of endotoxin tolerance in endotoxemia and peritonitis by monophosphoryl lipid A and lipopolysaccharide. Circ Shock 39:194-198, 1993 Bone RC: Toward an epidemiology and natural history of SIRS (systemic inflammatory response syndrome). JAMA 268:34523455, 1992
eptic shock is a common and serious condition in adult and pediatric intensive care unit patients worldwide. The death rate from septicemia is increasing, and septicemia now ranks above human immunodeficiency virus infection as a cause of death.1 The relative proportion of gram-positive and fungal organisms is increasing, but gram-negative organisms account for most of the clinical isolates.2 A slight improvement in survival rates for septic shock may have occurred in recent years, but most studies continue to report mortality rates of 41 to 80 percent.2 Most adjunctive therapies for gram-negative sepsis focus on the central role of endotoxin (lipopolysaccharide or LPS) in what is clinically recognized as sepsis, systemic inflammatory response syndrome (SIRS), or septic shock. Endotoxemia, however, is not a consistent finding in gram-negative sepsis, and it occurs frequently in gram-positive, fungal, or culture-negative sepsis.3 Furthermore, the hemodynamic response to gram-positive sepsis is similar to that of gramnegative sepsis,4 complicating the early clinical recognition of patients who might be candidates for therapies directed against endotoxin.
S
Endotoxin and LipopolysaccharideBinding Protein Endotoxin consists of three distinct components: (1) repeating oligosaccharide terminal side chains that provide O
From the Division of Pediatric Critical Care Medicine, Texas Tech University Health Sciences Center, Lubbock, TX. Address correspondence to Michael J. Romano, MD, Associate Professor of Clinical Pediatrics, Texas Tech University Health Sciences Center, 3601 4th St, Lubbock, TX 79430. Copyright © 2001 by W.B. Saunders Company 1045-1870/01/1201-0005$35.00/0 doi:10.1053/spid.2001.19231
antigen specificity and differ from strain to strain, (2) an oligosaccharide core, and (3) lipid A. Lipid A is highly conserved across gram-negative organisms and is responsible for the toxicity of LPS. Infusions of LPS produce a stereotypical pattern of cytokine activation characterized by rapid clearance of endotoxin from circulation and the sequential appearance of tumor necrosis factor (TNF) and interleukin (IL)-6.5 The biology of LPS receptors has been reviewed recently6-8 and is discussed here briefly as a basis for therapeutic interventions. LPS binds to numerous cell surface and serum molecules, including CD11/18, CD14, lipopolysaccharide-binding protein (LBP), and soluble CD14 (sCD14). The initial and key cellular event after the occurrence of endotoxemia appears to be the interaction of LPS with CD14. Knockout mice deficient in CD14 are resistant to endotoxin shock,9,10 and CD14-specific monoclonal antibodies inhibit monocyte production of TNF and IL-10 by LPS.11 Transfection of CD14 into cell lines that normally do not express it transforms those cells into LPS-responsive cells.12,13 Soluble CD14 facilitates the endotoxin-induced activation of cells that do not express surface-bound CD14.14,15 LBP is a glycoprotein that is present at low baseline levels in plasma16 and increases 2 log orders during inflammation.17 LBP is synthesized primarily in hepatocytes,18,19 with lesser amounts in other tissues, including the pulmonary artery.20,21 LBP catalyzes the transfer of LPS monomers from circulating aggregates to sCD14.22 In the presence of LBP, sensitivity to LPS is increased dramatically. Blood from LBP knockout mice that lack LBP activity is profoundly hyporesponsive to LPS stimulation.23 Interestingly, the in vivo TNF response of LBP knockout mice is essentially identical to that of LBP hemizygous mice, suggesting an LBP-independent, but CD14-dependent, mechanism of TNF production.
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LBP also appears to play a key role in LPS detoxification through its interaction with high-density lipoproteins (HDLs). LBP catalyzes the binding of LPS to HDL24 and facilitates the transfer of LPS from LPS-sCD14 complexes to HDL.25 This apparent paradox may represent a dosedependent effect of LBP on TNF production. Nanogram levels of LBP clearly enhance LPS sensitivity. Higher levels of LBP, in the microgram range, appear to decrease TNF production in vitro. Recombinant LBP injected into mice intraperitoneally (but not intravenously) attenuates the TNF-␣ and IL-6 responses to LPS administration and decreases the mortality from administration of LPS or live Escherichia coli.26 Analogous observations have been made in the case of sCD14. High doses of sCD14 inhibited the LBP-dependent activation of neutrophils, whereas lower doses stimulated neutrophil activation.27
Antibodies Directed Against Endotoxin Immunization with normal or “smooth” bacteria generates immunoglobulin that is highly protective in animals challenged with the homologous bacteria. Antibodies directed against O-antigen side chains do not neutralize LPS in vitro, so the presumed mechanism is via improved clearance of LPS or bacteria. The large number of gram-negative organisms causing clinical disease precludes this approach as practical. Immunization with mutants that lack the O-side chains, so called “rough” mutants based on their appearance in culture, generates antibodies directed against the core polysaccharide and highly conserved lipid A,28,29 yielding antibodies that may afford protection against a broad range of gram-negative pathogens.
J5 Mutant of E coli Ziegler et al30 tested this approach using the J5 mutant of E coli 0111:B4. J5 is a “rough” mutant lacking an epimerase enzyme responsible for attaching the O-side chain to the core polysaccharide.31 Healthy individuals donated one unit of serum before and after immunization with killed J5 vaccine. Patients (n ⫽ 304) were infused in a blinded fashion with either preimmunization or postimmunization serum. A single infusion of postimmunization J5 antiserum reduced mortality in the group with bacteremia by 37 percent. J5 antiserum has been evaluated in 3 other human trials. McCutchan et al32 gave a single unit as prophylaxis to patients at high risk of gram-negative infections. No effect on the incidence of bacteremia or mortality was seen. Baumgartner et al33 administered a unit of J5 antiserum every 5 days while patients were at high risk for gramnegative sepsis. J5 antiserum had no effect on the incidence of bacteremia, but the incidence of shock was reduced by 55 percent and death by 71 percent in the J5-treated group. J5 antiserum has provided no mortality benefit in children with infectious purpura.34 Questions have been raised regarding what component of the serum was protective. Calandra et al35 used an immunization method similar to that used by Ziegler but
separated the IgG subfraction of the serum. In this randomized, blinded trial, J5 IgG provided no protection against death or time to death.
HA-1A and E5 Advances in the production of monoclonal antibodies led to the development of 2 IgM class antibodies using J5 as the antigen; one of human origin36 (HA-1A; Centocor, Malvern, PA), the other murine37 (E5; XOMA, Berkeley, CA). Both antibodies have been evaluated in large-scale human trials. Ziegler et al38 studied 543 adults with suspected gramnegative infection and sepsis syndrome who received a single 100-mg dose of HA-1A or placebo. Overall, mortality was unchanged. In the subset of 200 patients with gramnegative bacteremia, 28-day mortality was decreased 39 percent in the treatment group. In 101 of these 200 patients in shock at enrollment, mortality was decreased 42 percent in the treatment group. No benefit was seen in patients with nonbacteremic gram-negative, gram-positive, or fungal infections. A second trial of HA-1A did not confirm decreased mortality in patients with gram-negative bacteremia, and it was stopped after the first planned interim analysis because a higher mortality rate occurred in patients who were receiving HA-1A and did not have gram-negative bacteremia.39 A French registry open-label study40 of 600 patients who received HA-1A found that mortality rates tended to be higher than predicted by severity of illness, especially among patients without gram-negative bacteremia. E5 has been evaluated in at least three phase III trials. The first trial41 enrolled 486 patients with suspected gramnegative infection and a systemic septic response. No difference was seen in the primary end point of 30-day mortality in patients with gram-negative sepsis or gramnegative sepsis with one or more organ failures at study entry. Subgroup analysis suggested a survival benefit in patients who were not in refractory shock at the time of study enrollment. A second trial, excluding patients in refractory shock, failed to show improved survival in patients with gram-negative sepsis or gram-negative sepsis with organ failure.42 A third trial was halted after interim analysis of 1000 patients.43
Bactericidal Permeability Increasing Protein The fourth agent with antiendotoxin activity that has entered large-scale human trials is an analogue of bactericidal permeability increasing protein (BPI). BPI originally was found in the azurophilic granules of human neutrophils.44 Subsequently, it has been identified in eosinophils45 and expressed on the surface of monocytes.46 BPI shares significant sequence homology with LBP, although the biologic effects are profoundly different. BPI has antibacterial activity against gram-negative organisms47 and certain cell wall– deficient gram-positive organisms.48 BPI inhibits TNF release from whole blood incubated with gram-negative organisms. A recombinant N-terminal fragment, BPI23, ap-
Adjunctive Therapies in Gram-negative Sepsis pears more potent than does the native protein.47 BPI or its analogues also have antiendotoxin effects. In human volunteers administered endotoxin, BPI23 inhibited activation of the coagulation and fibrinolytic systems49; significantly reduced serum levels of TNF, soluble TNF receptors p55 and p75, IL-6, IL-8, and IL-10; and attenuated neutrophil activation.50 A recombinant N-terminal 21-kD fragment of BPI (rBPI21, Neuprax; XOMA) has been evaluated in several diseases, including severe meningococcal sepsis in children. Meningococcemia is a unique gram-negative infection. It is predominantly a disease of the young; half of the endemic cases occur in children younger than 2 years, and in epidemics, most of the affected individuals are adolescents or young adults.51-53 Endotoxin levels may be profoundly elevated and correlate with the severity of the illness.54 In large series, mortality rates in this otherwise healthy population have been significant, ranging from 10 to 18.6 percent,55-57 and technologic advances in intensive care during the last 30 years have not appeared to appreciably improve the mortality rate.56 rBPI21 has been evaluated in 2 clinical trials of meningococcal sepsis in children. The first trial was an open-label, phase I/II dose-escalation trial in patients aged 1 to 18 years.58 Twenty-six patients received a loading dose followed by a 24-hour continuous infusion. No safety-related issues were identified. One patient died (3.8%), compared with expected mortality rates of at least 15 to 30 percent. A phase III, double-blind, placebo-controlled trial involving almost 400 pediatric patients with meningococcemia had completed enrollment, and the data were analyzed.59 The results of this study have been published.59a Three hundred ninety-three patients with severe meningococcal sepsis received rBPI21 (n ⫽ 190) or placebo (n ⫽ 203). Overall, there was no difference in the primary endpoint of mortality (rBPI21 ⫽ 7.4%, placebo ⫽ 9.9%; P ⫽ .48) or number of patients requiring amputations. There was a modest benefit noted in pediatric overall performance category (POPC) scales in the rBPI21 group. rBPI21 also has been evaluated in the setting of significant hemorrhage caused by trauma.60 Adults (n ⫽ 401) with acute hemorrhage secondary to trauma requiring at least 2 units of blood and within 12 hours of injury received rBPI21 or placebo in a double-blinded fashion. Among the 401 enrolled patients, no differences were found between drug and placebo groups in any of the primary or secondary end points, including mortality. Post hoc analysis suggested a decreased incidence of pneumonia or acute respiratory distress syndrome (ARDS) in patients receiving active drug (placebo group, 32%; rBPI21, 22%; hazard ratio ⫽ 0.66; post hoc P ⫽ .03). A second trial utilizing incidence of pneumonia or ARDS as the primary end point was halted after a planned interim analysis.61 Preliminary studies with rBPI21 are underway or completed in patients with cystic fibrosis who are having pulmonary exacerbations, patients undergoing partial hepatectomy, and patients with severe intraabdominal infections.
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Other Approaches to Adjunctive Therapy Active immunization with LPS derivatives is being investigated. A detoxified vaccine utilizing LPS from E coli J5 and an outer membrane protein from group B meningococcus is highly immunogenic.62 Polyclonal antibodies produced in response to the vaccine bound a variety of gram-negative organisms and were protective in a neutropenic rat model of Pseudomonas aeruginosa bacteremia.63 This approach has been reviewed recently.64 Antibodies to CD14 have been shown to be protective in 2 animal models of endotoxemia. Leturcq et al65 pretreated monkeys with 1 of 2 murine monoclonal antibodies against CD14 (28C5 or 18E12), followed by an 8-hour infusion of endotoxin. Both antibodies had a significant impact on the severe, sustained hypotension seen in control animals: 28C5 completely blocked this response, whereas animals treated with 18E12 had an initial hypotensive response similar to that of controls but which resolved in 5 hours. The TNF-␣ response was delayed and reduced in both treatment groups compared with controls. Significant decreases in IL-1 and IL-6 responses also were seen. Schimke et al66 investigated a different antibody in a rabbit model involving sequential exposures to endotoxin during a 24-hour period. This model was considered to more closely represent the development of human septic shock. Animals received three injections of endotoxin at 0, 5, and 24 hours and an anti-CD14 antibody on a variety of timetables, including a prophylactic treatment group and delayed treatment extending to 23 hours. At 48 hours, mortality was 32 percent in the control group, compared with zero in animals receiving anti-CD14. Treatment with antibody also eliminated renal cortical necrosis (84% in controls) and pulmonary injury (61% in controls) and prevented the gradual decrease in blood pressure seen in controls. Most importantly, the treatment effect was not dependent on time of administration; delayed treatment was as effective as was prophylactic therapy. Soluble CD14 occupies a paradoxic niche in the cytokine cascade. It is a key factor in facilitating the cytokine response to endotoxin and in detoxifying endotoxin. Soluble CD14 inhibits LPS-induced production of TNF in vitro.67 Finally, infusions of sCD14 decreased production of TNF and prevented mortality in a mouse model of endotoxemia.68 Numerous approaches to detoxifying LPS are being investigated. HDL binds LPS with high affinity and stability and is cleared via hepatic mechanisms. HDL can attenuate the biologic effects of endotoxin. Transgenic mice that express elevated levels of human apolipoprotein (the principal protein component of HDL) are resistant to endotoxin challenge,69 and rats made hypolipidemic have increased mortality from endotoxin challenge.70 Infusions of recombinant human HDL (rHDL) have been evaluated in a phase I trial in humans.71,72 rHDL reduced endotoxin-induced release of TNF, IL-6, and IL-8, activation of coagulation, and fibrinolysis. Monocytes from rHDL-treated whole blood showed reduced expression of CD14 and attenuation of TNF production with endotoxin challenge.
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Polymyxin B is a cationic polypeptide antibiotic that neutralizes most biologic activities of lipid A.73,74 Numerous approaches have been attempted to overcome its significant renal and central nervous system toxicity; the most advanced of these attempts is binding polymyxin B to the fibers of a hemoperfusion cartridge.75 Aoki et al76 described 16 patients who underwent 2 hours of veno-venous hemoperfusion through such a cartridge. Cardiopulmonary parameters showing improvement after therapy included an increase in mean systolic blood pressure in patients who were hypotensive when beginning therapy and a decrease in cardiac index in patients who were hyperdynamic before therapy. Serum endotoxin levels decreased from 76 pg/mL to 21 pg/mL after treatment (P ⬍ .05), an effect that has been confirmed in subsequent trials.77-79 Survival has been improved compared with that of untreated controls.79 Each of these trials is limited by small sample size and the lack of a sham treatment control group. Numerous antagonist analogues of lipid A have been developed. Lipid X is a precursor of lipid A and has weak antagonistic activity with some beneficial effects in mice80 and sheep.81 Modifications of the lipid X molecule have yielded molecules with much greater antagonistic activity.82 DT-5461 is a synthetic analogue of E coli lipid A, with mixed agonist and antagonist activity. It has been studied as an antitumor agent by virtue of its ability to stimulate TNF production,83,84 although at levels 5 to 6 log orders lower than those produced by native LPS.85 In mice, DT-5461 protects against LPS-induced mortality.85 E5531 is a potent, pure LPS antagonist that is protective in a mouse model of endotoxemia and gram-negative bacteremia.86,87 E5531 is in phase II trials in the United States. A final approach has been to develop molecules that selectively inhibit enzymatic steps in the synthesis of lipid A. The second step in the synthesis of lipid A is deacetylation of UDP-3-O-[R-3-hydroxymyristoyl)-GlcNAc.88 Mutant E coli that lack this enzyme are profoundly sensitive to antibiotics such as erythromycin, rifampin, and clindamycin, which normally are not active against gram-negative organisms.89 Numerous molecules that inhibit this deacetylase have been developed. The most potent of these (L161,240) has been shown to cure mice in a lethal model of E coli peritonitis.90 Unfortunately, the agent has little activity against other gram-negative organisms such a Serratia marcescens or P aeruginosa.
Conclusions The clinical application of antiendotoxin strategies in particular and immunologic modification of the inflammatory process in general must be ranked by clinicians as one of the major disappointments of the last 10 years. None of the myriad number of agents that have been evaluated has shown consistent benefit in clinical trials, and none has survived the Food and Drug Administration review process to enter clinical practice. What have we learned from almost 20 years of clinical trials? The evolution of antiendotoxin strategies, from basic science through highly publicized trials and subsequent
failures, has been critiqued exhaustively.91,92 Basic assumptions about this approach have been questioned: J5 antiserum does not appear to bind LPS93; HA-1A and E5 will bind to lipid A or LPS under some circumstances,36,94,95 but neither appears able to neutralize the biologic activity of LPS.95 What is the appropriate animal model? Infusions of endotoxin commonly are used to model sepsis and septic shock, despite the number of physiologic differences between this model and others utilizing infusions of live or killed gram-negative organisms96 or a cecal ligation and puncture model simulating peritonitis.97,98 What are appropriate end points in clinical trials? Is all-cause mortality valid, or should one evaluate resolution of organ system failure or resolution of hemodynamic perturbations? How are patients identified for inclusion? Evidence of SIRS frequently is included in the criteria, but SIRS is by no means specific for gram-negative bacteremia.99 Should a cost-benefit analysis be part of the approval process, and, if so, where is the line drawn (and by whom)?
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