Systemic endotoxin levels in chronic indolent periodontal infections

Endotoxin classically describes the biologic activity of the lipopolysac-charide structure comprising the outer membrane of gram-negative bacteria (1). Lipopolysaccharide, through engagement of lipopolysaccharide-binding protein (2,3) in serum and CD14 antigen on various cell types, particularly monocytes/macrophages, activates host cells at very low concentrations (3–5). In addition, Toll-like receptor-2 and -4, as cell-surface pattern-recognition receptors, bind lipo-polysaccharide as a ligand to initiate a cascade of multiple intracellular signaling pathways (4,6–8). These interactions between lipopolysaccharide and host cells result in up-regulation of the transcription of numerous genes, leading to the elevated production of an array of cytokines, chemokines and lipid mediators that contribute to both acute and chronic inflammatory responses to control the infection (3,9). Systemic bacteremia with gram-negative bacteria, resulting in a septic infection, delivers lipopolysaccharide to the circulation. Circulating endotoxin in the vasculature triggers the release of cytokines and acute-phase reactants, contributing to disseminated intravascular coagulation pathways leading to multiple organ system failure (10–12). In contrast to overt septic infections, various human diseases are expressed as chronic infections. These are usually localized to specific tissues or organs and, through induction of a chronic inflammatory response, can lead to loss of function of the tissues/organs. Moreover, the chronic local inflammation can release factors that may elicit systemic inflammatory responses (13,14). Periodontitis is a disease with localized tissue destruction, reflecting a chronic immunoinflammatory lesion (15–17) initiated by oral infection with a complex microbial ecology in sub-gingival biofilms (18–20). The tissue destruction undermines the integrity of the epithelial barrier in the periodontium and allows bacteria from the biofilms to translocate into the circulation (21–23). This can occur as a result of simple toothbrushing, but clearly the severity and extent of periodontitis accentuates the ability for bacteria to enter the bloodstream (24– 26). However, in contrast to overt septic infections, the bacteremia being seeded from the oral cavity is generally at low levels and relatively transient for individual episodes (25–28). Nevertheless, the chronicity of the disease and infection creates an indolent challenge to the systemic circulation that has been proposed to be a contributing facet of systemic health changes that accompany periodontitis (24,29–34). The primordial nature of the necessity for both vertebrates and invertebrates to protect against bacterial infections leading to endotoxicity was recognized by the identification of phagocytic cells in these species that detect and react to lipopolysaccharide/endotoxin (35–37). The ability of endotoxin to activate these phagocytic cells led to the development of a sensitive assay for this toxic contaminant using amoebocytes from the horseshoe crab, Limulus polyphemus (38). The original assay documented coagulation of the amoebocytes and has been used to determine endotoxin contamination of medical and biological reagents, as well as to estimate the endotoxin levels in biological fluids during septic infections (38,39). These early methods have been adapted to more quantitative colorimetric methods and kinetic assessments to enhance sensitivity of the assays (39,40). However, these techniques have not been effectively used to estimate the endotoxin levels in the sera of patients with indolent infections and low levels of serum endotoxin, such as those with periodontitis. This report describes a modification of a commercial assay for endotoxin that substantially increased the sensitivity of the detection of this toxin in serum and demonstrates its utility in documenting endotoxin levels in the sera of patients with periodontitis.