BACKGROUNDLipopolysaccharide (LPS), the major glycolipid component of Gram-negative bacterial outer membranes, is a potent endotoxin responsible for many of the directly or indirectly induced ...symptoms of infection. Lipoproteins (in particular, high-density lipoproteins) sequester LPS, thereby acting as a humoral detoxification mechanism.
PATIENTSDifferences in the lipoprotein composition in human plasma and lymph of a control patient group (n = 5) without systemic inflammatory response syndrome (non-SIRS/MOF) and patients with SIRS and multiple organ failure (MOF, n = 9) were studied. The LPS binding capacity of the lipoproteins in SIRS/MOF and non-SIRS/MOF patients was investigated by rechallenge of the plasma and lymph with fluorescently labeled LPS ex vivo. The lipoprotein composition was analyzed using immunochemical techniques and high-performance gel permeation chromatography.
RESULTSIn the non-SIRS/MOF patient group, plasma and lymph levels of apolipoprotein A-I (600 and 450 mg/L, respectively), apolipoprotein B (440 and 280 mg/L, respectively), total cholesterol (2.88 and 1.05 mM, respectively), and total triglycerides (0.67 and 0.97 mM, respectively) were observed. In the SIRS/MOF group, a decrease of apolipoprotein A-I (−55% in plasma and lymph), a decrease of apolipoprotein B (−43% in plasma and −38% in lymph), and a decrease of total cholesterol levels (−54% in plasma and −37% in lymph) were demonstrated. However, the triglyceride levels in the SIRS/MOF group showed a 30% increase in plasma and a 47% decrease in lymph compared with the non-SIRS/MOF patients. In SIRS/MOF patients, a 2.8-fold increase in plasma and a 1.8-fold increase in lymph of the LPS low-density lipoprotein/high-density lipoprotein ratio was observed, indicating that the relative LPS binding capacity of the lipoproteins in the SIRS/MOF patient group showed a trend to be shifted mainly toward low-density lipoproteins. Furthermore, in plasma and lymph of four SIRS/MOF patients, a novel cholesterol-containing high-density lipoprotein–like particle was found that barely had LPS binding capacity (<5%).
CONCLUSIONSIn the SIRS/MOF patients, the changes in lipoprotein composition in lymph are a reflection of those in plasma, except for the triglyceride levels. In comparison with the non-SIRS/MOF patients, the SIRS/MOF patients show a shifted LPS binding capacity of high-density lipoproteins toward low-density lipoproteins in plasma and in lymph. Moreover, in plasma and lymph, novel cholesterol-containing particles, resembling high-density lipoprotein, were identified in the SIRS/MOF patient group.
We have synthesized a novel analog of the general anesthetic etomidate in which the ethoxy group has been replaced by an azide group, and which can be used as a photolabel to identify etomidate ...binding sites. This acyl azide analog is a potent general anesthetic in both rats and tadpoles and, as with etomidate, is stereoselective in its actions, with the R(+) enantiomer being significantly more potent than the S(-) enantiomer. Its effects on α1β2γ2s GABAA receptors expressed in HEK-293 cells are virtually indistinguishable from the parent compound etomidate, showing stereoselective potentiation of GABA-induced currents, as well as direct mimetic effects at higher concentrations. In addition, a point mutation (β2 N265M), which is known to attenuate the potentiating actions of etomidate, also blocks the effects of the acyl azide analog. We have investigated the utility of the analog to identify etomidate binding sites by using it to photolabel human serum albumin, a protein that binds ∼75% of etomidate in human plasma and which is thought to play a major role in its pharmacokinetics. Using HPLC/mass spectrometry we have identified two anesthetic binding sites on HSA. One site is the well-characterized drug binding site I, located in HSA subdomain IIA, and the second site is also an established drug binding site located in subdomain IIIB, which also binds propofol. The acyl azide etomidate may prove to be a useful new photolabel to identify anesthetic binding sites on the GABAA receptor or other putative targets.
The effects of steroids on the outcome of sepsis are dose dependent. Low doses appear to be beneficial, but high doses do not improve outcome for reasons that are insufficiently understood. The ...effects of steroids on systemic inflammation as a function of dose have not previously been studied in humans. To determine the effects of increasing doses of prednisolone on inflammation and coagulation in humans exposed to LPS, 32 healthy males received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before i.v. injection of Escherichia coli LPS (4 ng/kg). Prednisolone dose-dependently inhibited the LPS-induced release of cytokines (TNF-alpha and IL-6) and chemokines (IL-8 and MCP-1), while enhancing the release of the anti-inflammatory cytokine IL-10. Prednisolone attenuated neutrophil activation (plasma elastase levels) and endothelial cell activation (von Willebrand factor). Most remarkably, prednisolone did not inhibit LPS-induced coagulation activation, measured by plasma concentrations of thrombin-antithrombin complexes, prothrombin fragment F1+2, and soluble tissue factor. In addition, activation of the fibrinolytic pathway (tissue-type plasminogen activator and plasmin-alpha(2)-antiplasmin complexes) was dose-dependently enhanced by prednisolone. These data indicate that prednisolone dose-dependently and differentially influences the systemic activation of different host response pathways during human endotoxemia.
To determine whether translocation of bacteria or endotoxin occurred into the thoracic duct in patients with multiple organ failure (MOF).
Translocation of bacteria or endotoxin has been proposed as ...a causative factor for MOF in patients without an infectious focus, although it has rarely been demonstrated in patients at risk for MOF. Most studies have investigated the hematogenic route of translocation, but it has been argued that lymphatic translocation of bacteria or endotoxin by the thoracic duct is the major route of translocation.
The thoracic duct was drained for 5 days in patients with MOF caused either by generalized fecal peritonitis (n = 4) or by an event without clinical and microbiologic evidence of infection (n = 4). Patients without MOF who were undergoing a transthoracic esophageal resection served as controls. In lymph and blood, concentrations of endotoxin, proinflammatory cytokines, and antiinflammatory cytokines were measured.
Endotoxin concentrations in lymph and blood of patients with MOF ranged from 39 to 63 units per liter and were not significantly different from concentrations in patients without MOF. The quantity of endotoxin transported by the thoracic duct in the study group was small. In patients with MOF, low levels of proinflammatory cytokines and high levels of antagonists of these cytokines were found.
This study provides evidence that translocation (especially of endotoxin) occurs into the thoracic duct. However, these data do not support the concept that the thoracic duct is a major route of bacterial translocation in patients with MOF.
In patients with systemic inflammatory response syndrome (SIRS), tolerance of peripheral blood mononuclear cells to a second challenge with lipopolysaccharide (LPS) has been described. Thoracic duct ...lymph transports LPS and represents the extravascular, interstitial fluid compartment of the body. The aim of this study was to determine the capacity of lymph to influence LPS-induced cytokine production in vitro. Thoracic duct lymph was obtained from patients with SIRS and without SIRS (controls). The effect of lymph and simultaneously collected plasma on LPS-induced cytokine production by normal peripheral blood mononuclear cells was assessed. Both lymph and plasma of patients with SIRS reduced LPS-induced tumor necrosis factor-α and interleukin-6 production (P < .01); lymph of controls also inhibited cytokine production (P < .01), although to a lesser extent. This study suggests that LPS tolerance may occur both in the intra- and extravascular compartments.
Lipopolysaccharide (LPS) tolerance is characterized by an impaired proinflammatory cytokine production upon restimulation of mononuclear cells with LPS. LPS is considered the primary activator for ...this phenomenon. In response to major injury and extensive abdominal surgery, an immune reaction comparable to LPS tolerance has been described. Therefore, it was investigated whether primary stimuli other than LPS could induce cytokine downregulation. In eight patients who underwent a laparoscopic cholecystectomy, blood was obtained before and after induction of anaesthesia and 2, 6, and 24 hr postoperatively. Ex vivo stimulation of whole blood resulted in a transient reduction (nadir 2 hr postoperatively) of tumor necrosis factor-alpha, interleukin (IL)-1 beta, and interferon-gamma release, while IL-1 receptor antagonist production increased. Stress hormones, LPS-binding protein, and bactericidal/permeability-increasing protein do not seem to be involved. This study shows that minimally invasive surgery, in the absence of endotoxemia, can induce LPS desensitization. These data suggest that prior endotoxemia is not essential for the development of LPS tolerance.
Corticosteroids are widely used for the suppression of cell-mediated cytoxicity. This process is mediated by natural killer cells and cytotoxic T lymphocytes, and their activation can be monitored by ...levels of the chemokines CXCL9 and CXCL10, the degranulation product granzymes A and B, and by levels of secretory phospholipase A2. The current study aimed to determine the effects of increasing doses of prednisolone on the release of these mediators in healthy humans exposed to LPS. Therefore, 32 healthy men received prednisolone orally at doses of 0, 3, 10, or 30 mg (n = 8 per group) at 2 h before intravenous injection of Escherichia coil LPS (4 ng/kg). Prednisolone dose-dependently attenuated the LPS-induced rises in the plasma concentrations of the chemokines CXCL9 and CXCL10, as well as of granzymes A and B levels. CXCL10 and granzyme B release were most sensitive to prednisolone, with a significant inhibition already achieved at the lowest prednisolone dose (3 mg). The levels of secretory phospholipase A2 were increased after LPS administration but were not significantly affected by prednisolone. This study demonstrates that prednisolone differentially inhibits the systemic release of mediators involved in cell-mediated cytotoxicity in humans in vivo.
Bacterial translocation is postulated as a risk factor in the development of a systemic inflammatory response syndrome (SIRS). Research on this topic has focused on the detection of bacteria and ...endotoxin in blood or mesenteric lymph nodes (MLNs). We investigated whether bacterial translocation occurs beyond the MLNs into the thoracic duct in a setting of ischemia, partial resection and reperfusion of the porcine liver.
A porcine model of severe, extra-intestinal tissue injury, consisting of prolonged hepatic ischemia and reperfusion, in combination with hemihepatectomy, was used (experimental group, n = 5 pigs). To prevent venous congestion of the gut during ischemia, a temporary portal-caval shunt was created. In 5 animals (sham group) a sham portal-caval shunt was constructed while liver ischemia, partial resection and reperfusion were not induced. Thoracic duct lymph, portal blood and systemic blood were collected, and analyzed for the presence of bacteria and endotoxin.
In the experimental group, the incidence of bacterial translocation to the thoracic duct was significantly higher during early reperfusion compared to the sham group (5/5 animals versus 1/5 animals, p < 0.05).
This study demonstrates bacterial translocation into the thoracic duct. Translocation at this level leads to direct discharge of bacteria and endotoxin into the systemic circulation and therefore, may potentially enhance the development of SIRS.
PDF
