H., Maier R. Shands and Chun [5], ? American Society for Biochemistry and Molecular Biology. (C) Diagram showing release of outer membrane vesicles (blebs) by growing culture supernatant showing blebs. Original bar (upper left), 100 nm. Originally published in Vesy et al. [43], ? 2000, American Society for Microbiology. (D) Maximal lipid A sensing by human MD-2CTLR4 requires phosphates at both 1 and 4 around the diglucosamine backbone, 4 glucosamine-linked acyl chains, and 2 secondary (acyloxyacyl) chains (reddish). Both lipid A and lipid IVa (also known as compound 406) are active in the LAL assay, which requires the 4 phosphoryl diglucosamine backbone and aggregated acyl chains. In contrast, aggregation is not necessary for lipid A with an attached and certain other Gram-negative aerobes, whereas the LPSs produced by and are even less stimulatory. Unlike enterobacterial LPS, the LPSs made by most sp. and many other Gram-negative anaerobes lack the 4-phosphate, have 5 acyl chains, and are very poor agonists; some may even inhibit the ability of LPS to stimulate human cells in vitro [8C10]. There can also be great variability in LPS structure, even within the same bacterial species: bacteria, for example, produce Polyphyllin VII LPSs that are TLR4 agonists, TLR4 antagonists, or nonstimulatory [11]. As will be discussed below, the lipid A structures that can be sensed by animal cells are not necessarily ones that are detected by assays for endotoxin in plasma. HOW DOES ENDOTOXIN GET INTO THE BLOODSTREAM? Plasma endotoxin may be derived from bacteria in an infected local tissue, the blood, the GI or respiratory tract, or food or other ingested matter. Movement from infected extravascular sites to blood In their landmark review of bacteremia, Bennett and Beeson [12] concluded that bacteria almost always make their way from a local site of contamination to the bloodstream via lymphatics. This topic has received little attention in recent years, but there is evidence that LPS also reaches the blood from tissues largely through lymphatic channels [13, 14]. Traffic via lymphatics may allow LPS to bind inhibitory proteins (observe below) before reaching the blood. Release from blood-borne bacteria Convincing evidence that bacteria can release LPS directly into the human bloodstream was reported by Brandtzaeg and colleagues [15], who obtained images of blebs in the plasma of children with meningococcemia and provided biochemical confirmation by demonstrating the presence of 3-OH-12:0, a component of lipid A, in the same samples. Meningococcemia is an outstanding case, Polyphyllin VII however. bacteria can colonize and invade vascular endothelial cells [16] and reach very high titers in blood (>108 DNA copies/ml [17]), whereas the Gram-negative bacteria that are most often isolated from blood cultures (such as LPS, injected directly into the bloodstream, can elicit fever and other responses in volunteers. Most of the natural routes to the blood are via lymphatic channels, not veins; they deliver smaller amounts of endotoxin into the blood gradually (and intermittently) over time, and they provide more opportunities for the bodys LPS-neutralizing mechanisms (observe below) to do their job. WHAT HAPPENS TO LPS THAT ENTERS THE BLOODSTREAM? Takeshita et al. [38] used circulation cytometry to detect binding of the E5 anti-LPS antibody (observe below) to blood monocytes. They reported that LPS was found on almost half of the CD14+ monocytes obtained from 5 children with Gram-negative bacteremia and on none of the monocytes from 4 children with Gram-positive bacteremia. The amount of cell-bound LPS could not be quantitated, but other evidence suggests that only a small fraction of bloodborne LPS is bound to cells. When Roth et al. [39] incubated LPS in citrated human blood for 15 min at room temperature, they found <5% of the LPS bound to cells (largely platelets), whereas >2/3 was bound to HDL. LPS may also be found on erythrocytes [40C42]. Plasma proteins can transfer LPSs from bacterial membranes to lipoproteins and other acceptors [43]. LBP and phospholipid transfer protein pass LPS to HDL and other plasma lipoproteins, many of which bind LPS in a way that prevents lipid A from interacting with MD-2CTLR4. These transfers can occur very quickly: when bacterial outer-membrane blebs were injected intravenously into rats, 50% of the LPS in the blebs experienced bound to lipoproteins within 1 min [44]. Rapid sequestration of lipid A also likely occurs when LPS translocates from your intestine via lymphatics. LPS that IL13RA1 antibody in the beginning binds Polyphyllin VII 1 lipoprotein class (usually HDL) can move to a different lipoprotein class,.