Endotoxin Sensitivity Research Articles

Characterization, protein modeling, and molecular docking of factor C from Indonesian horseshoe crab (Tachypleus gigas)

BackgroundHorseshoe crab (Tachypleus gigas) amebocytes are useful biomedical components for endotoxin detection, and their growing needs for biomedical purposes cause the horseshoe crab population to decline. Factor C synthesis via genetic engineering offers a solution to replace natural horseshoe crab’s factor C and prevent its excessive harvest from nature. In response to these concerns, this study aimed to characterize the amebocyte lysates and factor C protein modeling of T. gigas originated from Banyuasin South Sumatra Estuary. Methods and resultsSampling of T. gigas was carried out in Banyuasin South Sumatra Estuary, Indonesia. The endotoxin test or TAL (Tachypleus amebocyte lysates) assay was performed using gel coagulation method. Protein characterization of protease enzyme was conducted by protease activity, SDS-PAGE, and zymogram analysis. The cDNA of mitochondrial COI gene was amplified for molecular identification followed by cDNA cloning of factor C. Protein modeling was investigated by molecular docking and molecular dynamic (MD) simulation. Endotoxin test results showed that TAL-35 had endotoxin sensitivity in a range of 0.0156–1 EU/ml, while TAL 36 had a sensitivity between 00,625 and 1 EU/ml. T. gigas amebocytes have protease activity in molecular mass sizes less than 60 kDa, with 367 U/ml for TAL 35 and 430 U/ml for TAL 36. The molecular identification revealed 98.68% identity similarity to T. gigas. The docking results suggested three ligands; i.e., diphosphoryl lipid A, core lipid A, and Kdo2 lipid A can be activators of the factor C protein by binding to the region of the receptor to form a ligand-receptor complex. ConclusionsEndotoxins can be detected using horseshoe crab amebocytes. The presence of proteases is considered responsible for this ability, as evidenced by casein zymogram results. According to docking and MD analysis, we found that lipopolysaccharides (LPS) participate to the binding site of factor C.

RAPID COMMUNICATION: TLR4 expressed but with reduced functionality on equine B lymphocytes.

Varying susceptibility exists among mammalian species to the development of potentially fatal endotoxemia due to gram-negative bacteria molecular component, lipopolysaccharide (LPS). Toll-like receptor 4 (TLR4) is responsible for LPS-associated immune response and is expressed on numerous immune cells including B lymphocytes. TLR4 is expressed in a functional form on mouse B lymphocytes, a species much less susceptible to endotoxemia compared with humans who are highly sensitive to endotoxin. Humans possess B lymphocytes that are not responsive to LPS. Likewise, horses are highly susceptible to endotoxemia but the expression and function of TLR4 on horse B lymphocytes is not known. Colic, the major cause of mortality in horses, is often complicated by resultant endotoxemia. The objective of this study was to determine the expression and function of TLR4 on equine B lymphocytes. Lymphocytes were isolated from peripheral blood mononuclear cells that were collected from six horses, and the expression and function of TLR4 was analyzed for each horse. Flow cytometry results indicate TLR4 is expressed on horse B lymphocytes but stimulation with LPS did not alter this expression (P = 0.99) compared with unstimulated B lymphocytes after 24 h. After 72 h of in vitro LPS stimulation, analysis of cell proliferation dye by flow cytometry demonstrated that equine B lymphocytes did not proliferate, while mouse B lymphocytes predictably did. Furthermore, the total number of LPS stimulated equine B lymphocytes did not significantly differ from unstimulated cells after 72 h of culture (P = 0.92). Horse lymphocytes exhibited no significant differences in the measured TLR4 signaling pathway genes (TLR4, IL-10, IL-6, IFNβ, and TNFα) when expression was compared with LPS stimulated vs. unstimulated cells. In conclusion, while TLR4 is expressed on horse B lymphocytes, it appears minimally responsive to LPS in vitro, similar to results seen in human B lymphocytes. While further studies are still needed, our work reveals a potential link between B lymphocyte TLR4 expression and endotoxin sensitivity.

Systemic Treatment with a miR-146a Mimic Suppresses Endotoxin Sensitivity and Partially Protects Mice from the Progression of Acute Graft-versus-Host Disease.

Acute GVHD (aGVHD) is driven by interactions between the allogenic T cell response, inflammation, tissue injury and microbial products that enter the circulation when protective barriers such as the intestinal epithelium become compromised. Mice with aGVHD become hypersensitive to LPS, secreting large quantities of inflammatory mediators that exacerbate tissue injury. We hypothesized that microRNA (miR) modulators could be used in vivo to mitigate LPS hypersensitivity, altering the course of aGVHD. Using the C57BL/6 → (C57BL/6 × DBA/2)F1 -hybrid model of aGVHD, we measured intestinal permeability over time and used a qPCR array to detect concomitant changes in the expression levels of certain microRNAs (miRs) in the intestine. Large increases in permeability were seen on day 15, when endotoxemia becomes detectable and GVHD-associated histopathological lesions develop. Amongst the miRs with altered expression levels were some that regulate sensitivity to endotoxin. We chose to focus on miR-146a and treated recipient mice systemically with a miR-146a mimic early in the GVH reaction. This led to a reduction in the burst of IFNγ that likely plays a priming role in the mechanism underlying heightened sensitivity to endotoxin. LPS-induced TNFα release and GVHD-associated weight loss were also diminished and survival was prolonged. In summary, systemic treatment with a miR-146a mimic dampens the heightened sensitivity to LPS that occurs concomitantly with increased intestinal permeability and provides partial protection from the progression of acute GVHD.

Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

A critical role for human caspase-4 in endotoxin sensitivity.

Response to endotoxins is an important part of the organismal reaction to Gram-negative bacteria and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared with experimental animals such as mice. Inflammatory caspases mediate endotoxin-induced IL-1β secretion and lethality in mice, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following LPS challenge. Using bone marrow-derived macrophages, we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1β and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent a unique mechanism in humans, as compared with laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represents targets for systemic inflammatory response syndrome, sepsis, septic shock, and related disorders.

Diminished IL-10 production is Associated with Impaired Versatility of Monocytes in Familiar Mediterranean Fever

Purpose: The nature of the heightened endotoxin sensitivity state observed in Familial Mediterranean Fever (FMF) at present remains unknown. To assess the possibility that IL-10 plays a role in setting the inflammatory threshold, we studied IL-10 production by monocytes and dendritic cells as well as endotoxin tolerance induction in FMF patients.Methods: 46 attack-free FMF patients were included in this study. The production of IL-10 by NLR- or TLRagonist- stimulated monocytes and dendritic cells were assayed either by conventional ELISA or flow cytometry. The versatility of monocytes was studied by measuring the production of IL-10 and IL-1β after stimulation by pro- and anti-inflammatory agents, and after stimulation arrest or a further counter stimulation. Monocyte endotoxin tolerance and cross-tolerance induction were assayed by measuring the production of IL-1β, IL-10, TNF-α and IFN-γ after prestimulation by NLR- or TLR-ligands and after re-stimulation with LPS.Results: In FMF patients, we observed down-regulation of circulating CD36+ peripheral blood lymphoid cells but not monocytes, constitutively producing IL-10. The production of IL-10 by TLR- and NLR-agonist-stimulated monocytes and dendritic cells declines in FMF patients. Monocytes isolated from FMF patients failed to switch from a pro-inflammatory activated state to anti-inflammatory phenotype and still produce IL-1β but not IL-10, which cause impaired endotoxin tolerance and cross-tolerance induction. The IL-10 production and endotoxin tolerance induction by monocytes and dendritic cells were restored by NOD2- ligand MDP and colchicine treatment.Conclusion: The reduced IL-10 production was associated with the impaired setting of feedback inhibition of inflammatory response and caused impaired resolution of inflammation and endotoxin tolerance induction.

Lyn-Dependent Signaling Regulates the Innate Immune Response by Controlling Dendritic Cell Activation of NK Cells

The innate immune response is a first line of defense against invading pathogens; however, the magnitude of this response must be tightly regulated, as hyper- or suboptimal responses can be detrimental to the host. Systemic inflammation resulting from bacterial infection can lead to sepsis, which remains a serious problem with high mortality rates. Lyn tyrosine kinase plays a key role in adaptive immunity, although its role in innate immunity remains unclear. In this study, we show that Lyn gain-of-function (Lyn(up/up)) mice display enhanced sensitivity to endotoxin and succumb to upregulated proinflammatory cytokine production at a dose well tolerated by control animals. Endotoxin sensitivity in Lyn(up/up) mice depends on dendritic cells (DCs) and NK cells and occurs though a mechanism involving increased maturation and activation of the DC compartment, leading to elevated production of IFN-γ by NK cells. We further show that modulation of endotoxin-induced signal transduction in DCs by Lyn involves the phosphatases Src homology 2 domain-containing phosphatase-1 and SHIP-1. Collectively, we demonstrate that Lyn regulates DC physiology such that alterations in Lyn-dependent signaling have profound effects on the nature and magnitude of inflammatory responses. Our studies highlight how perturbations in signaling pathways controlling DC/NK cell-regulated responses to microbial products can profoundly affect the magnitude of innate immune responses.

Effects of Hepatocyte CD14 Upregulation during Cholestasis on Endotoxin Sensitivity

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats.

Parenteral iron formulations differentially affect MCP-1, HO-1, and NGAL gene expression and renal responses to injury

Despite their prooxidant effects, ferric iron compounds are routinely administered to patients with renal disease to correct Fe deficiency. This study assessed relative degrees to which three clinically employed Fe formulations [Fe sucrose (FeS); Fe gluconate (FeG); ferumoxytol (FMX)] impact renal redox- sensitive signaling, cytotoxicity, and responses to superimposed stress [endotoxin; glycerol-induced acute renal failure (ARF)]. Cultured human proximal tubule (HK-2) cells, isolated proximal tubule segments (PTS), or mice were exposed to variable, but equal, amounts of FeS, FeG, or FMX. Oxidant-stimulated signaling was assessed by heme oxygenase-1 (HO-1) or monocyte chemoattractant protein (MCP)-1 mRNA induction. Cell injury was gauged by MTT assay (HK-2 cells), %LDH release (PTS), or renal cortical neutrophil gelatinase-associated lipoprotein (NGAL) protein/mRNA levels. Endotoxin sensitivity and ARF severity were assessed by TNF-alpha and blood urea nitrogen concentrations, respectively. FeS and FeG induced lethal cell injury (in HK-2 cells, PTS), increased HO-1 and MCP-1 mRNAs (HK-2 cells; in vivo), and markedly raised plasma ( approximately 10 times), and renal cortical ( approximately 3 times) NGAL protein levels. Both renal and extrarenal (e.g., hepatic) NGAL production likely contributed to these results, based on assessments of tissue and HK-2 cell NGAL mRNA. FeS pretreatment exacerbated endotoxemia. However, it conferred marked protection against the glycerol model of ARF (halving azotemia). FMX appeared to be "bioneutral," as it exerted none of the above noted FeS/FeG effects. We conclude that 1) parenteral iron formulations that stimulate redox signaling can evoke cyto/nephrotoxicity; 2) secondary adaptive responses to this injury (e.g., HO-1/NGAL induction) can initiate a renal tubular cytoresistant state; this suggests a potential new clinical application for intravenous Fe therapy; and 3) FMX is bioneutral regarding these responses. The clinical implication(s) of the latter, vis a vis the treatment of Fe deficiency in renal disease patients, remains to be defined.

The Kinase Akt1 Controls Macrophage Response to Lipopolysaccharide by Regulating MicroRNAs

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Functional impact of endotoxin receptor CD14 polymorphisms on transcriptional activity

The polymorphism rs2569190 within the CD14 endotoxin (lipopolysaccharide, LPS) receptor gene is associated with various disease conditions that are assumed to rely on endotoxin sensitivity. In vitro experiments suggest that the T allele sensitizes the host for exogenous or endogenous LPS via an enhanced CD14 expression. To prove the impact of this single nucleotide polymorphism in its natural genomic context in vivo, two parameters of gene transcription were analyzed in peripheral blood mononuclear cells (PBMC) from single healthy individuals: (a) recruitment of RNA polymerase II by haplotype-specific chromatin immunoprecipitation and (b) the relative amount of transcripts by allele-specific transcript quantification (ASTQ). RNA polymerase II was found to be twice as much bound to the most prevalent haplotype, C-T-C-G, the only one carrying a T at the position rs2569190 of interest. ASTQ employing two independent read-out assays revealed, however, similar transcript numbers originating from C-T-C-G and non-C-T-C-G haplotypes. Total CD14 mRNA levels from freshly isolated PBMC, moreover, were neither related to donors’ geno- nor haplogenotypes. Our data argue for a functional impact of the rs2569190 polymorphism in terms of a stronger transcription initiation on T allele gene variants even if preferential allele-specific binding does not result in an increase in transcript numbers. Endotoxin sensitivity associated with this genetic variation appears not to rely solely on a cis-acting regulatory impact of rs2569190 on CD14 gene transcription in PBMC.

Maternal High‐Fat Diet Causes Cardiac Dysfunction and Increased Endotoxin Sensitivity in Offspring in Mice

"The fetal origins hypothesis" has been recognized for decades. The relationship between nutritional uptake and fetal cardiovascular development has not been defined. We hypothesized that maternal high‐fat diet (HFD) affects innate immunity, and thereby predisposes the offspring to cardiovascular disease in adulthood. Female C57BL/6 mice were fed with either control diet (CD) or HFD while breeding and 3 weeks after delivery. Cardiac profiles were assessed in offspring at 5‐week with echocardiogram. Offspring were then given a single dose of bacterial endotoxin (LPS, 1mg/kg, ip). Plasma serum amyloid A (SAA) levels were measured with ELISA at 0, 2h, and 24h after treatment. There were no differences on total‐, LDL‐cholesterol, and triglycerides between control and prenatal HFD offspring. While basal heart rate, cardiac output, and LV fractional shortening (FS) showed no differences, offspring exposed to prenatal HFD had significant less isoproteronol (2.5 mg/kg, ip) induced increases of FS (p<0.05), indicating a reduced cardiac reserve capacity. An elevation of plasma SAA level was detected at 24h after LPS treatment (p<0.001), which was more significant in mice with prenatal HFD. We concluded that maternal HFD has permanent effect on cardiac function in offspring especially under stress. It also causes an increase in LPS sensitivity, which could be explained by altered innate immunity.

Impaired Endotoxin Tolerance Induction in Patients with Familial Mediterranean Fever

Objective: To investigate periodic disturbances in proinflammatory activation of neutrophils and monocytes in patients with familial Mediterranean fever (FMF) both during an attack and in remission. Methods: 20 FMF patients, who were naive to colchicine treatment and did not have amyloidosis, and 10 patients with Behçet’s disease (BD) were enrolled in this study. Phagocytosis, respiratory burst, CD11a/CD18 expression and intracellular cytokine synthesis were determined by flow cytometry. Endotoxin tolerance induction was defined by a reduced capacity of monocytes to respond to lipopolysaccharide (LPS) activation following a first exposure to LPS. Results:In FMF patients, we observed upregulation of neutrophil and monocyte phagocytic activity and oxidative burst during remission and downregulation of phagocytic activity and stimulus-dependent oxidative burst during an attack. A comparative analysis of oxidative burst has revealed that while the neutrophil population shows a certain periodicity in the increase (during remission) and decrease (during attacks) in the spontaneous and inducible respiratory burst, periodicity in the monocyte population is very poor. In addition, LPS-induced oxidative burst and CD11a/CD18 integrin surface expression is higher in patients during an attack compared to patients in remission. The induction of homologous tolerance of monocytes to the repeated action of LPS is observed in FMF patients during an attack, normal donors and patients with BD, whereas monocytes from patients in remission failed to induce LPS homologous tolerance and exhibited heightened sensitivity to bacterial endotoxin. We found that colchicine is able to restore impaired LPS homologous tolerance induction in FMF patients in remission upon increased synthesis of IL-4 in FMF patient monocytes. Conclusion: Chronic inflammation during FMF is characterized by periodic changes in monocyte and neutrophil activation and heightened sensitivity to endotoxin, which is associated with the episodic nature of FMF. Increased endotoxin sensitivity in the period of remission could result from a shift in the monocyte activation program from ‘alternatively’ into ‘classically’ activated monocytes, which may have important implications for the treatment of FMF.

The TLR4 +896 polymorphism is not associated with lipopolysaccharide hypo-responsiveness in leukocytes

Toll-like receptor 4 (TLR-4) is required for detection of Gram negative bacterial infections by binding lipopolysaccharide (LPS) and for the initiation of inflammatory signaling. Recent studies have demonstrated that a nonsynonymous single-nucleotide polymorphism (Asp299Gly, A+896G) is associated with decreased endotoxin responsiveness and poor outcomes from sepsis. We show that human carriers of this polymorphism show no deficit in LPS induced peripheral blood mononuclear cell (PBMC) mitogen-activated protein kinase (MAPK) activity, no reduction in sensitivity to endotoxin, and variable differences in whole-blood inflammatory cytokine production. These results indicate that this mutation is not a primary determinant of human endotoxin sensitivity.

Adipocyte differentiation induces dynamic changes in NF-kappaB expression and activity.

The adipocyte exerts an important role in energy homeostasis, both as depot for energy-rich triglycerides and as a source for metabolic hormones. Adipocytes also contribute to inflammation and the innate immune response. Although it can be physiologically beneficial to combine these two functions in a single cell type under some circumstances, the proinflammatory signals emanating from adipocytes in the obese state can have local and systemic effects that promote atherosclerosis and insulin resistance. The transcriptional machinery in the adipocyte that mediates these pro-inflammatory responses has remained poorly characterized to date. In particular, no information is currently available on the NF-kappaB family of transcription factors. Here, we show that adipogenesis is associated with changes in amount and subunit composition of the NF-kappaB complexes. NF-kappaB subunits p65 (RelA), p68 (RelB), and IkappaB are upregulated during fat cell differentiation. Correspondingly, basal NF-kappaB nuclear gel shift and luciferase reporter assays are induced in parallel during differentiation. Surprisingly, endotoxin sensitivity of the classical NF-kappaB pathway is substantially delayed and attenuated despite increased overall inflammatory response in the mature adipocyte, as judged by induction of IL-6 and TNF-alpha. As a reflection of the constitutively elevated NF-kappaB activity in the mature adipocyte, adipocytes (but not preadipocytes) exert a strong inflammatory stimulus on macrophages in vitro, suggesting a cross talk between adipocytes and interstitial macrophages in adipose tissue in vivo. These effects are mediated by a secretory product of adipocytes that is unlikely to be IL-6 or TNF-alpha.

Endotoxin sensitivity and immune competence in chronic heart failure

Background: Raised concentrations of endotoxin (lipopolysaccharide, LPS) are found in patients with chronic heart failure (CHF). Tolerance of monocytes to LPS can be induced by LPS itself resulting in a downregulation of cytokine response to LPS challenge. This phenomenon of LPS desensitization has also been suggested for CHF. Methods: We investigated whether CHF patients really show a desensitization to LPS stimuli at rest or after physical exercise, which was used as a model of limited inflammatory reaction. Thirty-five patients with CHF (59±12 years, 8 women) and 30 healthy control subjects were prospectively studied with cardiopulmonary exercise testing. At rest and directly after exercise blood samples were taken for the quantitative determination of HLA-DR expression of monocytes as a measure for immune competence and for the measurement of TNFα generation after ex vivo stimulation by LPS. Results: HLA-DR expression was comparable in CHF patients and controls at rest as well as after exercise. TNFα production by LPS-stimulated monocytes ex vivo was higher in CHF patients compared to controls at rest and after exercise. Conclusions: Thus, our data are the first to show that patients with stable CHF show a cellular hypersensitivity to LPS with a higher TNFα generation capacity at rest and after exercise compared to controls. CHF patients seem to have a marked susceptibility to low inflammatory stimuli and no desensitization to LPS.

Diverse regulation of NF-kappaB and peroxisome proliferator-activated receptors in murine nonalcoholic fatty liver.

Fatty liver is highly sensitive to inflammatory activation. Peroxisome proliferator-activated receptors (PPAR) have anti-inflammatory effects and regulate lipid metabolism in the fatty liver. We hypothesized that fatty liver leads to endotoxin sensitivity through an imbalance between pro- and anti-inflammatory signals. Leptin-deficient, ob/ob mice and their lean littermates were challenged with single or double insults and pro- and anti-inflammatory pathways were tested on cytokine production and activation of nuclear regulatory factors NF-kappaB and peroxisome proliferator receptor element (PPRE). Ob/ob mice produced significantly higher serum tumor necrosis factor alpha (TNF-alpha) and interleukin (IL) 6 and showed increased hepatic NF-kappaB activation compared to lean littermates after stimulation with a single dose of lipopolysaccharide (LPS) or alcohol. In ob/ob mice, double insults with alcohol and LPS augmented proinflammatory responses mediated by increased degradation of inhibitory kappaB (IkappaB)-alpha and IkappaB-beta and preferential induction of the p65/p50 NF-kappaB heterodimer. In lean mice, in contrast, acute alcohol attenuated LPS-induced TNF-alpha, IL-6 production, and NF-kappaB activation through reduced IkappaB-alpha degradation and induction of p50/p50 homodimers. PPRE binding was increased in fatty but not in lean livers after alcohol or LPS stimulation. However, cotreatment with alcohol and LPS reduced both PPRE binding and nuclear levels of PPAR-alpha in fatty livers but increased those in lean livers. In conclusion, our results show opposite PPRE and NF-kappaB activation in fatty and lean livers. PPAR activation may represent an anti-inflammatory mechanism that fails in the fatty liver on increased proinflammatory pressure. Thus, an imbalance between PPAR-mediated anti-inflammatory and NF-kappaB-mediated proinflammatory signals may contribute to increased inflammation in the fatty liver.

Metabolic disturbance and sensitivity to endotoxin in patients with advanced cancer: relationship to lymphocyte reactivity, tumour necrosis factor (TNF) production and survival.

Increased TNF production and impaired lymphocyte function have been individually linked with metabolic disturbance, endotoxaemia and mortality in humans. The inter-relationship between these observations was investigated in humans with cancer. In 13 patients with metastatic colorectal cancer and seven healthy volunteers, observations (n = 23) included peripheral blood mononuclear cell (PBMC) TNF production, IL-2 production and phytohaemagglutinin (PHA) response; the acute-phase protein response (APPR) (serum C-reactive protein (CRP), albumin, CRP/albumin ratio), and survival. APPR correlated with survival (CRP, r = -0.689, P = 0.006; CRP/albumin, r = -0.758, P = 0.002; albumin, r = 0.655, P = 0.011), but not with TNF production. TNF production in response to in vitro endotoxin correlated with impaired lymphocyte function in patients (r = 0.567, P = 0.043) and in the whole group (r = 0.65, P = 0.001). The ratio (basal PBMC TNF production)/(lymphocyte function) correlated with CRP (r = 0.569, P = 0.042), CRP/albumin (r = 0.617, P = 0.025), endotoxin sensitivity (r = 0.567, P = 0.043) and survival (r = -0.545, P = 0.038) in patients, and the whole group (P < 0.002). Impaired lymphocyte function may influence TNF production, endotoxin sensitivity and metabolic disturbance in humans with cancer. (r = Spearman correlation coefficient.)

P3244 Increased endotoxin sensitivity in patients with chronic heart failure

P3244 Increased endotoxin sensitivity in patients with chronic heart failure

Kupffer cell phagocytosis blockade decreases morbidity in endotoxemic rats with obstructive jaundice.

The consequences of Kupffer cell phagocytosis blockade were studied in endotoxemic rats with obstructive jaundice. 159 male Wistar rats. Obstructive jaundice was induced by bile duct ligation (BDL). Gadolinium chloride (1 mg/100 g iv) was given 6 days after BDL to inhibit Kupffer cell activity and the animals were challenged with 1 microg/g endotoxin 24 h later. Endotoxin sensitivity, tumor necrosis factor-alpha and interleukin-6 production were studied, liver and lung injury were assessed by neutrophil infiltration assay, tissue adenosine triphosphate, aspartate aminotransferase, alanine aminotransferase level determinations and histology, respectively. For statistics non-parametric methods were used. BDL sensitized the animals to endotoxin, increased endotoxin-induced tumor necrosis factor-alpha and interleukin-6 production and reduced ATP contents of the liver and the lung. Kupffer cell blockade significantly increased the resistance against endotoxin, diminished the inflammatory cytokine release and reduced endotoxin-induced tissue injury in BDL animals. Attenuation of Kupffer cell function decreases endotoxin-induced lethality and morbidity in obstructive jaundice.