High Lipopolysaccharide Levels Research Articles

Relationship between Microbial Translocation and Endothelial Function in HIV Infected Patients

BackgroundCirculating levels of microbial products are increased in HIV infection, and provoke endothelial dysfunction in other disease settings.Methodology/Principal FindingsWe examined data from a cross-sectional single site study at Indiana University (Indiana, N = 85) and a 24- week multicenter prospective study of antiretroviral therapy (ART) initiation (ACTG 5152s, N = 75). Brachial artery flow-mediated dilation (FMD) was measured by ultrasound. Plasma lipopolysaccharide (LPS) and soluble CD14 (sCD14) levels were measured from stored specimens and correlated with FMD values using Pearson correlations. The Indiana subjects were 63% male with a mean age of 39 years and a median CD4 count of 406 cells/mm3 (388 not on ART, 464 on ART). The 5152s subjects were 92% were male with a mean age of 35 years and a median CD4 count of 251 cells/mm3 at entry which increased to 396 cells/mm3 on ART. When analyzing the two cohorts individually or in combination neither sCD14 nor LPS correlated significantly with FMD. In a pre-specified subgroup analysis of the Indiana subjects receiving ART (N = 46, mean ART duration 40 months) LPS was inversely correlated with FMD (r = −0.33, p = 0.02), but not sCD14 (r = −0.01, p = 0.9). Multivariate analysis confirmed LPS as an independent predictor of FMD in this subgroup (p = 0.02).Conclusions/Significance In HIV- infected individuals on prolonged ART, higher LPS levels are associated with worse endothelial function but not in untreated subjects or at 24 weeks after ART initiation. Persistent microbial translocation may contribute to arterial dysfunction and the increased cardiovascular disease risk observed in individuals on long-term ART.

Microbial Translocation and Infectious Diseases: What Is the Link?

Microbial Translocation and Infectious Diseases: What Is the Link?

Epstein-Barr Virus load and immune activation in Human Immunodeficiency Virus type 1-infected patients

BackgroundPatients infected with HIV-1 are at high risk of developing Epstein-Barr Virus (EBV)-related diseases. Chronic immune activation is a hallmark of HIV-1 pathogenesis and may play a role in B-cell stimulation and expansion of EBV-infected cells. ObjectivesThe aim of the study was to define the relationship between parameters of immune activation and EBV load in HIV-1-infected subjects. Study designA total of 156 HIV-1-infected patients were studied. EBV types 1 and 2 were quantified on peripheral blood mononuclear cells by multiplex real-time PCR. Plasma levels of cytokines and lipopolysaccharide (LPS) were determined by immunoenzymatic assays. B-cell activation was analyzed by flow cytometry. ResultsEBV-DNA was detected in 114 patients, and in all but 3 was EBV type 1. The median [interquartile] EBV-DNA load was 43[1–151] copies/105 PBMC. EBV-DNA load was higher in patients with detectable HIV-1 plasma viremia, despite good immunological status (CD4>500 cells/μl), than in patients with undetectable HIV-1 plasma viremia regardless of immunological status (46[5–136] copies/105 cells vs 17[1–56] copies/105 cells, p=0.008). Patients with high EBV-DNA load (>median value) had higher levels of LPS and proinflammatory cytokines (IL-6, IL-10 and TNF-α) than patients with low EBV load. Furthermore, percentages of activated B-cells correlated with EBV-DNA load (rs=0.754; p<0.001). ConclusionsOverall, these findings indicate a strong association between HIV-1 viremia, markers of immune activation and EBV load and suggest that persistence of HIV-1 viremia and immune activation, regardless of peripheral CD4 cell depletion/repopulation, may favor expansion of EBV-infected cells and onset of EBV-related malignancies.

Effects of recombinant human elafin gene transfection on lipopolysaccharide-induced airway mucous hypersecretion

To explore the effects of recombinant human Elafin gene transfection on lipopolysaccharide (LPS)-induced airway mucous hypersecretion. An eukaryotic expression vector pEGFP-C1-Elafin was first constructed and then transfected into NCI-H292 cell. After co-incubation with polymorphonuclear granulocyte (PMN) plus LPS stimulation for 24 hour, the vital force unit of neutrophil elastase (NE) and the content of mucin (MUC) protein in culture media and the level of MUC 5AC mRNA in culture cells were detected with substrate detection technique, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) respectively. All parameters in control group showed a low level (0.13 ± 0.03, 0.28 ± 0.06, 0.29 ± 0.04 respectively). And transfecting NCI-H292 cell with pEGFP-C1 at different levels of LPS, all parameters were obviously different (all P < 0.01) and they all showed a dose-dependent relationship. After LPS stimulation (0.5, 5 mg/L), as compared with the transfection of NCI-H292 cell with pEGFP-C1-Elafin, the vital force unit of NE and the content of MUC5AC protein in culture media and the level of MUC 5AC mRNA in transfecting pEGFP-C1 NCI-H292 cell decreased obviously (all P < 0.05). But after LPS stimulation (25 mg/L), as compared with the transfection of NCI-H292 cell with pEGFP-C1-Elafin, no parameter of transfecting pEGFP-C1 NCI-H292 cell had any obvious alteration (0.67 ± 0.06, 0.64 ± 0.08, 0.67 ± 0.07 respectively, all P > 0.05). The transfection of recombinant human elafin gene into NCI-H292 cell may inhibit the force of inflammatory telo-effective factor NE induced by a certain level of LPS and airway mucous hypersecretion. But it can not inhibit the effects induced by a high level of LPS.

Effect of globular adiponectin on interleukin-6 and interleukin-8 expression in periodontal ligament and gingival fibroblasts

PurposeGlobular adiponectin (gAd) is a type of adipocytokine, which is mainly produced by adipose tissue. It has been reported that gAd acts as a pro- as well as an anti-inflammatory factor. Interleukin (IL)-6 and IL-8 are pro-inflammatory cytokines. To investigate the role of gAd on periodontal tissues, the expression of adiponectin receptor 1 (AdipoR1) and the effect of gAd on the expression of IL-6 and IL-8 were investigated in periodontal ligament (PDL) and gingival fibroblasts.MethodsPDL and gingival fibroblasts were cultured from human periodontal tissues. gAd derived from Escherichia coli and murine myeloma cells were used. The expression of AdipoR1 was estimated by reverse transcription-polymerase chain reaction and western blot. The expression of cytokines was measured by enzyme-linked immunosorbent assay.ResultsPDL and gingival fibroblasts expressed both mRNA and protein of AdipoR1. gAd derived from E. coli increased the production of IL-6 and IL-8, but polymyxin B, an inhibitor of lipopolysaccharide (LPS), inhibited IL-6 and IL-8 production induced by gAd in both types of cells. gAd derived from murine myeloma cells did not induce IL-6 and IL-8 production in those cells. gAd derived from E. coli contained higher levels of LPS than gAd derived from murine myeloma cells. LPS increased production of IL-6 and IL-8 in PDL and gingival fibroblasts, but pretreatment of cells with gAd derived from murine myeloma cells did not inhibit LPS-induced IL-6 and IL-8 expression.ConclusionsOur results suggest that PDL and gingival fibroblasts express AdipoR1 and that gAd does not act as a modulator of IL-6 and IL-8 expression in PDL and gingival fibroblasts.

Systemic toll-like receptor ligands modify B-cell responses in human inflammatory bowel disease

Bacteria have a central, although poorly understood, role in inflammatory bowel disease (IBD). Host-bacteria interactions primarily take place in the gastrointestinal tract, but cells may also encounter translocated bacteria in the bloodstream. IBD is associated with activated, circulating Toll-like receptor (TLR)2 and TLR4-expressing B cells suggesting that blood-borne microbial TLR ligands modulate B cell responses. Serum levels of lipopolysaccharide (LPS)/endotoxin and high mobility group box 1 (HMGB1), an endogenous TLR ligand, were quantified in Crohn's disease (CD) and ulcerative colitis (UC). Responses of purified B cells to LPS and HMGB1 were correlated with levels of systemic TLR ligands and clinical parameters of disease. While IBD patients have increased levels of blood LPS, the net effect of endotoxemia has unexpected characteristics illustrating that LPS has both pro- and antiinflammatory roles through TLR4+ B cells. Experimental treatment of B cells demonstrates that the antiinflammatory effect of LPS is due to its hypo-acylation of lipid A suggesting an increased prevalence of systemic, hypo-acylated LPS in CD. In contrast, high levels of LPS are associated with disease activity in UC. HMGB1 activates B cells through TLR2 and CD36. Serum levels of HMGB1 correlate with spontaneous IL-8 production by B cells suggesting that blood-borne TLR2 ligands increase B-cell activation in vivo. Systemic TLR ligands modulate B cells towards either proinflammatory or antiinflammatory activity depending on the predominant ligand(s). Further, the circulating B cell may represent an important proxy for quantifying the LPS lipid A acylation burden in patients with IBD.

Lipopolysaccharide‐induced carotid body inflammation in cats: functional manifestations, histopathology and involvement of tumour necrosis factor‐α

In the absence of information on functional manifestations of carotid body (CB) inflammation, we studied an experimental model in which lipopolysaccharide (LPS) administration to pentobarbitone-anaesthetized cats was performed by topical application upon the CB surface or by intravenous infusion (endotoxaemia). The latter caused: (i) disorganization of CB glomoids, increased connective tissue, and rapid recruitment of polymorphonuclear cells into the vascular bed and parenchyma within 4 h; (ii) increased respiratory frequency and diminished ventilatory chemoreflex responses to brief hypoxia (breathing 100% N(2) for 10 s) and diminished ventilatory chemosensory drive (assessed by 100% O(2) tests) during normoxia and hypoxia; (iii) tachycardia, increased haematocrit and systemic hypotension in response to LPS i.v.; and (iv) increased basal frequency of carotid chemosensory discharges during normoxia, but no change in maximal chemoreceptor responses to brief hypoxic exposures. Lipopolysaccharide-induced tachypnoea was prevented by prior bilateral carotid neurotomy. Apoptosis was not observed in CBs from cats subjected to endotoxaemia. Searching for pro-inflammatory mediators, tumour necrosis factor-alpha (TNF-alpha) was localized by immunohistochemistry in glomus and endothelial cells; reverse transcriptase-polymerase chain reaction revealed that the CB expresses the mRNAs for both type-1 (TNF-R1) and type-2 TNF-alpha receptors (TNF-R2); Western blot confirmed a band of the size expected for TNF-R1; and histochemistry showed the presence of TNF-R1 in glomus cells and of TNF-R2 in endothelial cells. Experiments in vitro showed that the frequency of carotid nerve discharges recorded from CBs perfused and superfused under normoxic conditions was not significantly modified by TNF-alpha, but that the enhanced frequency of chemosensory discharges recorded along responses to hypoxic stimulation was transiently diminished in a dose-dependent manner by TNF-alpha injections. The results suggest that the CB may operate as a sensor for immune signals, that the CB exhibits histological features of acute inflammation induced by LPS, that TNF-alpha may participate in LPS-induced changes in chemosensory activity and that some pathophysiological reactions to high levels of LPS in the bloodstream may originate from changes in CB function.

Microbial Translocation Is Associated with Increased Monocyte Activation and Dementia in AIDS Patients

Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4+ T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

Human Immunodeficiency Virus-Related Microbial Translocation and Progression of Hepatitis C

Human immunodeficiency virus (HIV)-1 infection has been associated with enhanced microbial translocation, and microbial translocation is a mechanism through which alcohol and some enteric conditions cause liver disease. We hypothesized that HIV promotes liver disease by enhancing microbial translocation. We studied human cohorts in which hepatitis C virus (HCV) and HIV outcomes were carefully characterized. HIV-related CD4(+) lymphocyte depletion was strongly associated with microbial translocation as indicated by elevated levels of circulating lipopolysaccharide (LPS), LPS-binding protein, soluble CD14, and fucose-binding lectin (AAL) reactive to immunoglobulin G specific for the alpha-galactose epitope and suppressed levels of endotoxin core antibodies (EndoCAb IgM) in HIV-infected subjects compared with the same persons before they had HIV infection and compared with HIV-uninfected subjects. The same measures of microbial translocation were strongly associated with HCV-related liver disease progression (cirrhosis), eg, LPS, odds ratio, 19.0 (P = .002); AAL, odds ratio, 27.8 (P < .0001); in addition, levels of LPS were elevated prior to recognition of cirrhosis. Microbial translocation may be a fundamental mechanism through which HIV accelerates progression of chronic liver disease.

Stimulation of autophagy prevents apoptosis of neonatal rat cardiomyocytes exposed to lipopolysaccharide

Unlike adult cardiomyocytes, neonatal rat ventricular cardiomyocytes exhibit a protective phenotype towards high levels of lipopolysaccharide (LPS). The physiological basis of this protection is not known. We have previously shown that neonatal cells exposed to LPS exhibit profound changes to their functional mitochondria, these changes include a temporal loss of mitochondria membrane potential and an increased expression of uncoupling protein 3 (UCP 3). Over time these measurements return to pretreatment levels and are not associated with apoptosis induction. We demonstrate that mitochondrial biogenesis is stimulated by LPS treatment. Concomitant with an increase in mitochondrial biogenesis we also observed an increase in the level of autophagy within LPS treated cells. Furthermore, inhibition of autophagy induces apoptosis in the presence of LPS. LPS treatment is associated with an increase in the expression of inducible nitric oxide synthase (iNOS) and increased nitric oxide levels. Our results demonstrate that production of nitric oxide is necessary for the stimulation of autophagy. We propose that the stimulation of autophagy in neonatal cells is a protective phenotype removing mitochondria damaged by LPS, thereby possibly decreasing the production of reactive oxygen species and limiting cell damage. Supported by the Department of Defense # DAMD W81XWH-04-02-0035

Lipopolysaccharide-enhanced, Toll-like Receptor 4–dependent T Helper Cell Type 2 Responses to Inhaled Antigen

Allergic asthma is an inflammatory lung disease initiated and directed by T helper cells type 2 (Th2). The mechanism involved in generation of Th2 responses to inert inhaled antigens, however, is unknown. Epidemiological evidence suggests that exposure to lipopolysaccharide (LPS) or other microbial products can influence the development and severity of asthma. However, the mechanism by which LPS influences asthma pathogenesis remains undefined. Although it is known that signaling through Toll-like receptors (TLR) is required for adaptive T helper cell type 1 (Th1) responses, it is unclear if TLRs are needed for Th2 priming. Here, we report that low level inhaled LPS signaling through TLR4 is necessary to induce Th2 responses to inhaled antigens in a mouse model of allergic sensitization. The mechanism by which LPS signaling results in Th2 sensitization involves the activation of antigen-containing dendritic cells. In contrast to low levels, inhalation of high levels of LPS with antigen results in Th1 responses. These studies suggest that the level of LPS exposure can determine the type of inflammatory response generated and provide a potential mechanistic explanation of epidemiological data on endotoxin exposure and asthma prevalence.

Oral administration of lipopolysaccharide to Atlantic salmon ( Salmo salar L.) fry. Uptake, distribution, influence on growth and immune stimulation

Atlantic salmon fry were fed Aeromonas salmonicida lipopolysaccharide (LPS)-coated feed for 62 days and then challenged with virulent A. salmonicida bacteria. The fry were fed LPS-coated feed also after challenge. Fry that were fed LPS feed (0.1% LPS) showed a higher mortality throughout the challenge period (accumulated mortality of 57% after 42 days) compared with fry fed control feed (accumulated mortality of 36.5% after 42 days). Fry receiving LPS-coated feed showed an increase in mean weight of 10.7% at day 62 compared with fry receiving control feed. The increase was, however, not statistically significant. Sixty days of feeding with LPS-coated feed did not result in measurable amounts of specific antibodies against A. salmonicida LPS in the homogenised fry. The total amount of immunoglobulins (Igs) was, however, slightly increased. Studies of immunohistochemical localisation and radioactive LPS distribution revealed high levels of LPS in the intestinal epithelial cells. Head kidney, liver and heart showed low levels of radioactivity and no immunohistochemical staining. In another set of experiments, Atlantic salmon fry were fed LPS-coated feed (0.03% and 0.01%, respectively) for 64 days and then challenged with A. salmonicida and Vibrio anguillarum. The accumulated mortality in fry challenged with A. salmonicida was 40.9%, 34.3% and 30.8% after feeding 0.03% and 0.01% LPS and control feed, respectively. The accumulated mortality in fry challenged with V. anguillarum was 47.0%, 55.0% and 55.1% after feeding 0.03% and 0.01% LPS and control feed, respectively. Fry receiving 0.03% and 0.01% LPS-coated feed showed a statistically significant increase in mean weight at day 64 compared with fry receiving control feed.

Differential clearance and induction of host responses by various administered or released lipopolysaccharides

The clearance and activity of different types of lipopolysaccharide (LPS) released during infection with Gram-negative bacteria were investigated. When highly purified preparations differing in their specific endotoxin activity were administered intravenously to mice, the clearance of rough (R)-form LPS preparations from Salmonella minnesota and Escherichia coli was much faster than that of a smooth (S)-form LPS preparation from Salmonella abortus equi, but slower than that of lipo-oligosaccharides (LOS) preparations from Bordetella pertussis and Helicobacter pylori. After intraperitoneal infection with 10(7) and 10(8) CFU E. coli O111:B4, relatively high levels of LPS were detected dose-dependently in the plasma of infected mice and persisted for a long time. In addition, plasma sCD14 levels in infected mice were higher than in LPS-administered mice. These results indicate that continuously higher levels of plasma LPS followed by stronger host responses occur during infection and suggest that these differences between LPS-administered and infected mice should be taken into consideration when analyzing host responses induced by LPS.

The preparation of the chronic hyper-endotoxemia experimental animal model by means of a drug delivery system

A drug delivery system (DDS) consisting of lipopolysaccharide (LPS) as a drug and 2-hydroxyethyl methacrylate (HEMA)-diethylene glycol dimethacrylate (2G) or –polyethylene glycol dimethacrylate (4G, 9G) copolymer was prepared, and used for the efficient preparation of an experimental animal model of chronic hyper-endotoxemia. The release profiles of LPS in the in-vitro test were greatly influenced by the composition of HEMA-2G, 4G, 9G in the copolymer. It was found that LPS release from the DDS continued gradually and constantly throughout 2 weeks. In the in-vivo experiment with rats, the DDS maintained a high blood concentration level of LPS for 3 days. These results strongly suggest the possibility of convenient and reproducible preparation of a chronic hyper-endotoxemia animal model.

Sensitivity of human melanoma cells to adherent leukocytes depends on the ratio between them, the activation status of adherent leukocytes and the metastatic potential of tumor cells.

This study examined the interaction of the poorly metastatic human melanoma cell line M4Be and the highly metastatic clone 4 derived from M4Be, with respect to fresh adherent leukocytes (AL) isolated from 17 different healthy blood donors. These AL contained 80% (73%-93%) monocytes, 15% (6%-20%) B lymphocytes and 5% (1%-8%) T lymphocytes. The survival of these tumor cells against the stress exerted by these AL was estimated with a clonogenic assay where isolated tumor cells were co-cultured for 14 days in contact with AL and lipopolysaccharide (LPS). For a given blood donor, AL either stimulates or inhibits the colony formation of the tumor cells (T) depending on the AL/T ratio, the AL activation status and the metastatic potential of tumor cells. At low AL/T ratios (< 10/1) in the presence of low (8 ng/ml) and trace (8 pg/ml) levels of LPS, hydrogen peroxide (H2O2) release is significantly reduced, and tumor cells significantly increase their colony formation; the feeder effect of AL is suggested to be due to low concentrations of soluble tumor necrosis factor-alpha (TNF-alpha). At high AL/T ratios (> 10/1), whatever the characteristics of the blood donor, clone 4 is significantly more sensitive than M4Be to AL activated with medium containing low (8 ng/ml) or high (1,000 ng/ml) levels of LPS; this killing effect is suggested to be due to TNF-alpha, both soluble and membrane-bound, but not to be due to release of H2O2. These data suggest that the regulatory role of AL, which remove the majority of human melanoma cells and stimulate the colony formation of a small fraction of them, is partly due to TNF-alpha.

Dental unit waterline contamination and its possible implications during periodontal surgery.

Dental unit waterline contamination has become a concern to clinical dentistry. This concern arises from the fact that bacteria sloughed from established biofilms in dental unit waterlines increase heterotrophic bacteria counts in water exiting these units. Scanning microscopy and bacterial viability staining were used to examine the sessile and planktonic biofilm present in dental unit waterlines and water samples, respectively. In addition, the limulus amebocyte assay was used to measure the lipopolysaccharide (LPS) levels in water samples. All dental unit waterlines were coated with a well-established biofilm made up of filamentous and bacillus-like microorganisms. Water samples collected from these dental units contained high numbers of individual bacteria and bacterial aggregates. A viability staining technique identified significantly more bacteria in water than could be cultured, and 64% of the total bacterial population stained as nonvital. Since the bacterial load (viable and nonviable) was high, we examined the LPS in dental unit water samples. The mean LPS levels in water collected from high-speed and air/water lines in use were 480 and 1,008 endotoxin units (EU)/ml. This was significantly higher than the mean level of 66 EU/ml found in water samples collected from adjacent clinic sinks. The LPS level at the start of the day (2,560 EU/ml) was reduced by 70% with 1 minute of flushing (800 EU/ml). Flushing times of 5 and 10 minutes were not able to reduce LPS levels to zero. The presence of high heterotrophic bacterial counts, sloughing biofilm, and high LPS levels are discussed in relation to patient risk and periodontal wound healing biology.

Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta

A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta. Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken. In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations. Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum. Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 × 10 4 Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively). However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows. IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL). High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli , black pigmented Gram-negative anaerobes and Clostridium spp. shortly after calving, and Arcanobacterium pyogenes and Gram-negative anaerobes in the uterus at 14 days postpartum. These results suggest that the presence of E. coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A. pyogenes and Gram-negative anaerobes later postpartum. LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins

Background: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. Results: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. Conclusions: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Influence of human anti-lipopolysaccharide immunoglobulins on tissue distribution and clearance of lipopolysaccharide in rats.

To examine the influence of passive immunization on the biological fate of injected lipopolysaccharide (LPS), we used a human IgG preparation (anti-LPS IgG) rich in antibodies to a large panel of smooth and rough purified LPS extracts as well as a normal IgG preparation (standard IgG). Our approach was to compare the uptake of 125I-labeled LPS by the tissues of saline or IgG-treated rats. After intravenous injection, one fraction of 125I-labeled Escherichia coli O55:B5 LPS is rapidly taken up by tissues, while another fraction remained in the blood. Uptake of 125I-labeled LPS was principally observed into the liver and spleen. In rats treated prophylactically with standard IgG, these tissues accumulated significantly larger amount of LPS than the tissues of rats treated with anti-LPS IgG. Nevertheless, both IgG preparations increased the specific binding of LPS by the liver and spleen. High levels of homologous unlabeled LPS decreased the uptake of LPS by the liver, presumably by occupying tissue receptors, whereas in the presence of E. coli O127:B8 LPS, an increase of the uptake of 125I-labeled LPS by the liver and lungs was observed. The pharmacokinetics and tissue distribution of LPS-IgG complexes pre-formed in vitro were compared. In the presence of standard IgG, a unexpected increase of the uptake of LPS by the tissues was recorded, whereas LPS-anti-LPS IgG complexes decreased the binding of 125I-labeled LPS to the tissues. On the other hand, the vascular effects induced by LPS did not appear to be modified in rats pretreated with either IgG preparation. In conclusion, although passive immunization against LPS slightly modified the uptake and clearance of LPS, neither in vitro nor in vivo formation of LPS-anti-LPS IgG complexes afforded a very significant protection against the toxic effects of LPS.

An antipyretic role for interleukin-10 in LPS fever in mice.

Interleukin (IL)-10 inhibits the synthesis of proinflammatory cytokines implicated in fever, including IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. We hypothesized that IL-10 functions as an antipyretic in the regulation of fevers to lipopolysaccharide (LPS) and turpentine. Body temperature was measured by biotelemetry. Swiss Webster (SW) mice treated with recombinant murine IL-10 were resistant to fever induced by a low dose of LPS (100 microgram/kg ip) and to the hypothermic and febrile effects of a high (septiclike) dose of LPS (2.5 mg/kg ip). IL-10 knockout mice developed an exacerbated and prolonged fever in response to a low dose of LPS (50 microgram/kg ip) compared with their wild-type counterparts. At 4 h after injection of the low dose of LPS, plasma levels of IL-6, but not TNF-alpha, were significantly elevated in the IL-10 knockout mice compared with their wild-type controls (ANOVA, P < 0.05). After injection of the same high dose of LPS injected into SW mice, wild-type mice developed a fever at 24 h whereas IL-10 knockout mice immediately developed a profound hypothermia that lasted through 41 h (ANOVA, P < 0.05). Body weight and food intake were more significantly depressed in response to the high dose of LPS in the knockout mice compared with their wild-type controls. Only 30% of the IL-10 knockout mice survived compared with 100% of the wild-type mice (Fisher's exact test, P < 0.05). Fever in response to the injection of turpentine (100 microliter/mouse sc) did not differ between wild-type and IL-10 knockout mice. These data support the hypotheses that 1) IL-10 functions as an endogenous antipyretic following exposure to LPS, 2) a putative mechanism of the early antipyretic action of IL-10 is through the inhibition of plasma levels of IL-6, 3) IL-10 has a protective role in the lethal effects of exposure to high levels of LPS, and 4) endogenous IL-10 does not have a role in fever induced by turpentine.