The most studied exogenous mediator is endotoxin. This macromolecule is found in gram-negative bacteria as one of the integral components of the outer bacterial lipopolysaccharide cell wall. The molecule can be functionally divided into 3 parts: a highly variable O-polysaccharide side-chain that provides the heat-stable serologic specificity of gram-negative bacteria and is the basis for the O (somatic) antigen type scheme; an oligosaccharide or R-core region composed of approximately 10 monosaccharides; and a lipid backbone referred to as lipid A. The lipid A portion of endotoxin is responsible for the majority of the molecule's toxicity; this portion is also highly conserved and is essentially invariable and ubiquitous.
Experimental studies of the administration of endotoxin have produced in physiologic changes paralleling those of septic shock. Administration of endotoxin in dogs results in severe hypotension. Administration of endotoxin in humans produces an increase in heart rate and cardiac index, and a decrease in peripheral vascular resistance. Later hemodynamic changes include a depressed cardiac state with a dilated left ventricle. Endotoxin administration in rabbits produces DIC and bilateral renal cortical necrosis. Endotoxin administration in both animals and humans results in fever.
Endotoxin (otherwise known as lipopolysaccharide) alone can induce septic shock. Recently, scientists have discovered a major cellular basis for the pathophysiologic response to LPS. Endotoxin (LPS), lipopolysaccharide-binding protein (LBP), and CD14 (a protein found on the surface of monocytes and macrophages) form a complex that is critical for macrophage and neutrophil activation by LPS. LBP is present in the circulation and concentrations are increased in the response to inflammatory stimuli. LBP binds LPS and this LPS-LBP complex then interacts with CD14. CD14 is thought to act as a carrier molecule presenting LPS to a signaling receptor. It is the LPS-sCD14 complex that is thought to be the major module that binds to endothelial cells and triggers the cascade of events leading to septic shock.
Many strategies have been developed to manipulate this complex, including enhanced LPS clearance (anti-LPS antibodies, direct removal of LPS via filtration and use of hemoglobin); direct neutralization of circulating LPS (anti-LPS antibodies, LPS neutralizing proteins); inhibition of LPS-LBP; and removal of both endotoxin and cytokines by hemofiltration, hemadsorption, or hemodialysis. Limited information from using continuous veno-venous hemofiltration (CVVH) on patients with sepsis and oliguric renal failure has suggested that CVVH may increase the predominance of circulating anti-inflammatory cytokines over pro-inflammatory cytokines. This change is thought to be beneficial. However, there are no randomized trial data to support the routine use of plasma filtration or hemodialysis in sepsis. Other interventions include the use of LPS-sCD14 complex modifiers (lipid A analogues, anti-LBP antibodies, anti-CD14 antibodies); blocking cellular LPS receptors (lipid A analogues, anti-CD14 antibodies); and inhibition of cell signal transduction via tryosine kinase or protein kinase C inhibitors.
J5 (POLYCLONAL ANTI-ENDOTOXIN ANTIBODY) Despite initial promise, five subsequent clinical trials using either J5 antiserum, immune plasma, or intravenous immunoglobulin have not improved survival in sepsis or septic shock.
E5 (MONOCLONAL ANTI-ENDOTOXIN ANTIBODY) Monoclonal antibodies were developed to produce a more specific anti-endotoxin therapy with less risk for transmission of infection. E5 is an IgM antiendotoxin monoclonal antibody produced by immunizing mice with the core lipopolysaccharide antigen of the E5 mutant of E. coli. E5 is manufactured by an antibody-producing hybridoma created by the fusion of mouse spleen cells with a murine myeloma cell line. Preliminary animal studies suggested potential efficacy in septic shock. Double-blind, placebo-controlled investigations of patients in sepsis and septic shock have produced mixed results. Overall mortality has not been consistently reduced in either sepsis or septic shock. The only consistent benefit appears to be a more rapid resolution of organ failure in patients with gram-negative sepsis without hypotension or shock. Allergic reactions to the murine antibody were minimal, and adverse effects were minimal. Overall, the benefits of E5 are too limited to justify the expense of routine use.
HA-1A (HUMAN-HYBRID MONOCLONAL ANTI-ENDOTOXIN ANTIBODY) While the hybridoma that produces HA-1A is about 80% murine, the antibody itself is predominately human. Studies of HA-1A in animals have met with varied results; some models showing benefit and others showing harm. The first human study with HA-1A originally reported a decrease in mortality and a more rapid resolution of organ system failure in patients with gram-negative bacteremia. HA-1A treatment did not improve survival in patients who had non-bacteremic gram-negative infections and in patients without gram-negative infections. Despite these favorable results, significant concerns were raised, and when the data were analyzed using the original analytic plan, HA-1A did not show a significant effect on survival. In the second placebo-controlled study with HA-1A, mortality in patients with gram-negative bacteremia was unaffected and mortality in patients without gram-negative bacteremia was actually worsened. Further development of HA-1A for the treatment of septic shock was halted.
T88 (MONOCLONAL ANTIBODY) This is an antibody to a common enterobacterial antigen that has shown promise in animal studies. A phase III trial of patients with sepsis has recently been completed and preliminary data suggest that no overall benefit resulted from the administration of this medication.
LPS-NEUTRALIZING PROTEIN A number of lipopolysaccharide-neutralizing proteins has been described of which BPI has been most extensively studied and is currently in clinical trials for gram-negative sepsis. BPI is a 55 to 60 kDa neutrophil primary granule protein with 45 percent sequence homology to LBP. BPI has a higher affinity for LPS than LBP and therefore displaces LPS from the LPS-LBP complex. In addition, BPI is cytotoxic for many gram-negative bacteria. Phase II and phase III clinical studies of BPI23 are in progress including a multicenter trial of patients with meningococcemia.
LIPID A ANALOGUES Inhibition of the LPS-LBP interaction is an attractive and potential method of intervention. Analogues based upon the structure of lipid A display reduced or absent cellular toxicity. The most attractive of these compounds is E5331. This compound has been shown to block endotoxin binding to cells, to inhibit LPS-induced TNFa release, and to protect mice from E. coli challenge. E5331 inhibits endotoxin-induced cytokine release in human volunteers in response to low-dose endotoxin infusion and phase I/II studies are in progress.
A number of conclusions can be made. Antiendotoxin core-directed antibodies have not shown a reproducible survival benefit for patients with sepsis in 10 clinical trials. However, both E5 and HA1A may have beneficial roles in a subgroup of patients presenting with sepsis. Further investigations are needed to determine if monoclonal antibody to endotoxin is an appropriate therapeutic modality for treatment of septic shock.
The setting where antiendotoxin antibody therapy is therapeutic and cost-effective remains to be defined. Neither E5 nor HA-1A benefited the approximately 30 percent of individuals who did not have a gram-negative infection. Separating patients with gram-positive sepsis from those with gram-negative sepsis based upon clinical symptoms and presenting signs alone is difficult. Early laboratory diagnosis of gram-negative bacteremia or endotoxemia is not currently available. The rapid identification of the presence of gram-negative organisms from potential sites of infection (UTI; pneumonia; abdominal infection; CNS infections; soft tissue infections; decubitus or cellulitis in the setting of diabetes or peripheral vascular disease) is critical for interventions and antiendotoxin antibody therapy. Gram's stain and other rapid assays remain the most valuable tests for rapid identification of gram-negative organisms in clinical specimens.
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