Accumulation Of Endotoxin Research Articles

Prognostic value of molecules of average mass in patients with chronic obstructive pulmonary disease

Background. Chronic obstructive pulmonary disease is a socially significant disease affecting patient’s quality of life. Assessment of endogenous intoxication in patients with chronic obstructive pulmonary disease will allow to understand pathogenetic features of different phenotypes of this disease, which can be taken into account when predicting its course.The aim of the study. To determine the prognostic value of levels of mediumand low-molecular-weight substances and oligopeptides in patients with chronic obstructive pulmonary disease.Materials and methods. One hundred and four patients with chronic obstructive pulmonary disease (COPD) and 110 somatically healthy individuals were examined. Molecular weight medium and low molecular weight substances (LMWSM) and oligopeptides (OP) were determined in blood plasma, erythrocytes and urine. Based on these indicators mathematically calculated indices of endogenous intoxication and coefficient of elimination were defined. Statistical processing of the data was performed using the SPSS 26.0 software package (IBM Corp., USA).Results. In all biological fluids, the levels of average molecules and calculated indices in the COPD patients’ group were statistically significantly different from those in the control group. The indices characterizing endotoxin accumulation were statistically significantly higher, while those characterizing toxin elimination were lower. The level of endotoxemia was correlated with the frequency of exacerbations, clinical manifestations severity, quality of life, COPD group and phenotype.Conclusions. Frequent exacerbations, groups C and D, bronchitic and mixed COPD phenotypes are characterized by more severe endotoxicosis manifested by high levels of LMWSM, OP and calculated indices.

Maternal diet disrupts the placenta-brain axis in a sex-specific manner.

High maternal weight is associated with detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression and communicative disorders. Despite widespread acknowledgement of sex biases in the development of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we show that a maternal high-fat diet causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation contributes to sex-specific behavioural outcomes in offspring. In male offspring exposed to a maternal high-fat diet, increased macrophage Toll-like receptor 4 signalling results in excess microglial phagocytosis of serotonin (5-HT) neurons in the developing dorsal raphe nucleus, decreasing 5-HT bioavailability in the fetal and adult brains. Bulk sequencing from a large cohort of matched first-trimester human samples reveals sex-specific transcriptome-wide changes in placental and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, fetal brain 5-HT levels decrease as placental triglycerides increase in male mice and male human samples. These findings uncover a microglia-dependent mechanism through which maternal diet can impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.

Macrophage malfunction in Triptolide-induced indirect hepatotoxicity.

Background and Objective: Indirect hepatotoxicity is a new type of drug-induced hepatotoxicity in which the character of a drug that may induce its occurrence and the underlying mechanism remains elusive. Previously, we proved that Triptolide (TP) induced indirect hepatotoxicity upon LPS stimulation resulting from the deficiency of cytoprotective protein of hepatocyte. However, whether immune cells participated in TP-induced indirect hepatotoxicity and the way immune cells change the liver hypersensitivity to LPS still need to be deeply investigated. In this study, we tried to explore whether and how macrophages are involved in TP-induced indirect hepatotoxicity. Method: Firstly, TP (500 μg/kg) and LPS (0.1 mg/kg) were administrated into female C57BL/6 mice as previously reported. Serum biochemical indicators, morphological changes, hepatic macrophage markers, as well as macrophage M1/M2 markers were detected. Secondly, macrophage scavenger clodronate liposomes were injected to prove whether macrophages participated in TP-induced indirect hepatotoxicity. Also, the ability of macrophages to secrete inflammatory factors and macrophage phagocytosis were detected. Lastly, reverse docking was used to find the target of TP on macrophage and the possible target was verified in vivo and in RAW264.7 cells. Results: TP pretreatment increased the liver hypersensitization to LPS accompanied by the recruitment of macrophages to the liver and promoted the transformation of macrophages to M1 type. Depletion of hepatic macrophages almost completely alleviated the liver injury induced by TP/LPS. TP pretreatment increased the secretion of pro-inflammatory factors and weakened the phagocytic function of macrophages upon LPS exposure. Reverse docking results revealed that MerTK might be the real target of TP. Conclusion: TP disrupts inflammatory cytokines profile and phagocytic function of hepatic macrophages, resulting in the production of massive inflammatory factors and the accumulation of endotoxin in the liver, ultimately leading to the indirect hepatotoxicity of TP. MerTK might be the target of TP on the macrophage, while the binding of TP to MerTK should be investigated in vivo and in vitro.

The Intervention of Prebiotics on Depression via the Gut-Brain Axis.

The imbalance of intestinal microbiota can cause the accumulation of endotoxin in the main circulation system of the human body, which has a great impact on human health. Increased work and life pressure have led to a rise in the number of people falling into depression, which has also reduced their quality of life. The gut–brain axis (GBA) is closely related to the pathological basis of depression, and intestinal microbiota can improve depressive symptoms through GBA. Previous studies have proven that prebiotics can modulate intestinal microbiota and thus participate in human health regulation. We reviewed the regulatory mechanism of intestinal microbiota on depression through GBA, and discussed the effects of prebiotics, including plant polysaccharides and polyphenols on the regulation of intestinal microbiota, providing new clues for the prevention and treatment of depression.

Extracorporeal hemoperfusion therapy for sepsis: Multi-lamellar microspheres towards cascade endotoxin removal and broad-spectrum radical eliminating

Sepsis is a life-threatening condition originated from the accumulation of endotoxin in blood in response to infection (e.g., bacterial infection, COVID-19), and then aggravating by the systemic inflammatory responses, microcirculation disorders and oxidative stress, ultimately resulting in the dysfunction of multiple organs. Herein, advanced multi-lamellar microspheres (CPG-Ln-MSs) with cascade endotoxin adsorption and oxidative stress relief functions are constructed as an alternative adsorbent for hemoperfusion therapy towards sepsis. The CPG-Ln-MSs achieve effective endotoxin absorption (nearly 455.3 EU/g) and show excellent broad-spectrum radical scavenging activity for treating oxidative stress triggered by endotoxin accumulation. Specifically, the structural integrity of the multi-layered structure plays a vital role in promoting the efficiency of endotoxin removal and the subsequently scavenging of reactive oxygen species. Hemoperfusion simulation experiments demonstrate that the CPG-Ln-MSs could effectively remove endotoxin with a ratio of 92%, and the following oxidative stress state could be well alleviated, as confirmed by the reduced H2O2 and MDA levels in septic blood. Furthermore, endogenous antioxidants could be restored (recover SOD and CAT activity to 96.9 and 10.1 U/mL, respectively) and the red blood cells could be protected from oxidative damage. This study provides a promising therapeutic strategy and guides the design of future for septic blood purification.

Inhibition of indoleamine 2,3-dioxygenase attenuates endotoxin-mediated tolerance in granulosa cells through kynurenine pathway.

Ovarian granulosa cells (GCs) have been shown to have innate immune capabilities, which modulate their native endocrine functions through toll-like receptors (TLRs). We have recently shown that GCs exposed to lipopolysaccharide (LPS; 1.0 µg/mL) transiently regulate proinflammatory cytokine expression (interleukin 1β [IL-1β], IL-6, and tumor necrosis factor α) through chromatin remodeling. In the present study, we have demonstrated that GCs become tolerant to LPS on repeated exposure of LPS. To understand the mechanism of this endotoxin tolerance (ET) phenomenon in buffalo GCs, we have further studied the genome-wide transcriptomic analyses in buffalo GCs (unpublished data) and identified indoleamine 2,3-dioxygenase 1 (IDO1) gene, known to be involved in tryptophan catabolism, was found to be highly upregulated in endotoxin-tolerant GCs. Real-time gene expression analyses also showed similar results. Further analyses of tryptophan and tryptophan metabolite, kynurenine, showed that tryptophan was found to be depleted with the accumulation of kynurenine in the endotoxin-tolerant GCs. The effect of IDO1 induced ET was reversed when cells were pretreated with IDO1 inhibitor (1-methyl tryptophan, 1 mM). To the best of our knowledge, this is the first report describing the role of IDO1 gene in ET in GCs mimicked by repeated endotoxin exposure in vitro. In summary, the present study convincingly demonstrated that the tryptophan catabolism, through the kynurenine pathway, plays a crucial role as an immunomodulatory mechanism of ET in GCs. The finding could be exploited in developing potential therapeutics to treat impaired GCs function due to the ET underlying prolonged uterine or systemic infection leads to accumulation of endotoxin in follicular fluid.

Role of Bacterial Contamination in Endometriosis

The human lower genital tract is constantly exposed to various microorganisms, which can infect the upper genital tract through ascending migration. We report on tests of the hypothesis that as normal microbial flora in the vagina, some gram-negative bacteria infect the uterine wall after ascending migration from the vagina, to contaminate menstrual blood, consequently resulting in accumulation of endotoxin in the menstrual/peritoneal fluid and initiation of pelvic inflammation. The aim of the present review is to assess possible intrauterine microbial colonization and to examine the stimulatory effect of E. coli-derived lipopolysaccharide (LPS) on the secretion of various macromolecules by macrophages (Mϕ) known to be involved in endometriosis. We also assess the growth-promoting effect of LPS on endometrial cells and the role of Toll-like receptor 4 (TLR4), a receptor recognizing LPS, in the secretion of macromolecules by Mϕ and growth of endometrial cells. We found higher intrauterine microbial colonization in women with endometriosis than in controls. The ligand from gram-negative bacteria is involved in TLR4-mediated proinflammation in the pelvis as well as growth of endometriosis. Treatment with gonadotropin-releasing hormone agonist (GnRHa) further promotes intrauterine microbial colonization compared with GnRHa-untreated women. Conclusions We propose the bacterial contamination hypothesis as a new concept in endometriosis. Based on our current findings, an estrogen-suppressing agent and an antimicrobial agent may have new therapeutic potential in women with endometriosis.

Mechanisms by which TLR4 and endotoxin promote hepatocellular carcinoma require further investigation

We thank Dr. Schwabe and colleagues for interest in our article.1, 2 We have carefully reviewed our published results and also the original data. Our experiments showed that carcinogen-induced apoptosis in mice can be protected by lipopolysaccharide (LPS), an endotoxin (Fig. 1), or other factors induced by diethylnitrosamine (DEN). This figure was originally included in the manuscript, but deleted in revision due to page limitations. The main question concerns why reduction of LPS by antibiotics (figure 7B)1 led to lower liver injury but LPS receptor Toll-like receptor 4 (TLR4) deficiency potentiated liver injury (figure 3A).1 Possible explanations are as follows: (1) The alanine aminotransferase level usually indicates liver damage and does not exclusively represent the degree of cell apoptosis. LPS protection as previously described takes effect by a certain time period (e.g., at 48 hours after injection; figure 6C-F),1 although it even enhances liver injury at 24 hours after DEN injection. We did not claim that no or low LPS levels and antibiotic suppression of LPS will increase liver injury. (2) Because treatment with antibiotics prevented the increase of plasma LPS (figure 7A),1 inhibition of LPS-induced inflammation upon DEN exposure might be responsible for the reduction of liver injury. We found that antibiotic-induced reduction of LPS and lack of TLR4 reduced the number and volume of tumors with the decrease of tumor necrosis factor α and interleukin-6. LPS pretreatment prevents death receptor ligands–induced apoptosis in vivo and in vitro. (A,C) Survival curves of LPS-preconditioned or control wild-type mice in response to treatment with (A) tumor necrosis factor/galactosamine (TNF/GaIN) and (C) Jo-2. (B,D) The typical gross and histological appearance of the above treated mice: (B) tumor necrosis factor/galactosamine (TNF/GalN); (D) Jo-2. Scale bars = 100 μm. (E,F) The cleavage of poly(adenosine diphosphate ribose) polymerase (PARP) protein was detected by western blot in LPS-pretreated (1μg/mL for 12 hours) or untreated primary wild-type or TLR4−/− hepatocytes (E) or Hepa1-6 cells (F) upon TNF + cycloheximide (CHX) treatment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. We had the same concerns about this discrepancy, because it appears to contradict the hypothesis that TLR4−/− mice should have less liver injury. However, in terms of the acute liver damage induced by DEN, the TLR4 knockout mice are different from wild-type mice in response to the short-term antibiotics treatment. In addition to LPS, TLR4 interacts with other endogenous ligands, such as high mobility group box 1 (HMGB1) and heat shock proteins (HSPs). TLR4 deficiency therefore ablated the protective effects of both LPS and endogenous ligands on hepatocytes, thus leading to more severe liver damage. Treatment with antibiotics mainly reduced the acute inflammatory responses to LPS in nonparenchymal cells, which is thought to be the major contributing factor to acute hepatocyte injury. It is conceivable that reduced production of proinflammatory cytokines such as tumor necrosis factor α was responsible for alleviation of DEN-induced acute liver damage. On the other hand, during the multistage process of hepatocarcinogenesis, accumulation of endotoxin promotes chronic tumorigenic inflammation and alleviates apoptosis in precancerous and cancerous cells. So, loss of TLR4 is not equivalent to LPS ablation in terms of the responsive cell types and inflammation status. As cited by Dr. Schwabe and colleagues, Karin's group at University of California San Diego reported an increase in DEN-induced hepatocellular carcinoma development when inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) was deleted in hepatocytes. However, the same group also found that hepatocellular carcinoma was suppressed in mice with IKKβ ablated in both hepatocytes and Kupffer cells.3 Furthermore, Pikarsky et al. found that inhibiting nuclear factor κB signaling suppressed hepatocarcinogenesis.4 Resolving these differences will lead to better understanding of hepatocarcinogenesis. Lexing Yu Ph.D.*, Hexin Yan Ph.D.*, Wen Yang Ph.D.*, Hongyang Wang M.D.* , * International Cooperation Laboratory on Signal Transduction, Liver Centre of SMMU, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China, National Laboratory for Oncogene and Related Genes, Cancer Institute of Shanghai Jiao Tong University, Shanghai, China.

Endotoxin Accumulation Prevents Carcinogen-Induced Apoptosis and Promotes Liver Tumorigenesis in Rodents

Increasing evidence suggests that the presence of endotoxemia is of substantial clinical relevance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS amplifies the tumorigenic response of the liver. We found that the circulating levels of LPS were elevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPS using antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4 (TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigation revealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-κB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxicity, but lessens inflammation-mediated compensatory proliferation. Reconstitution of TLR4-expressing myeloid cells in TLR4-deficient mice restored diethylnitrosamine (DEN)-induced hepatic inflammation and proliferation, indicating a paracrine mechanism of LPS in tumor promotion. Meanwhile, deletion of gut-derived endotoxin suppressed DEN-induced cytokine production and compensatory proliferation, whereas in vivo LPS pre-challenge promotes hepatocyte proliferation. Our data indicate that sustained LPS accumulation represents a pathological mediator of inflammation-associated hepatocellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterial translocation and endotoxin absorption may favorably influence liver function in patients with cirrhosis who are at risk of developing HCC.

Endotoxin-Directed Innate Immunity in Tracheal Aspirates of Mechanically Ventilated Human Neonates

Mechanical ventilation of preterm infants is associated with pulmonary inflammation. Intubated infants often develop bacterial tracheal colonization, but little is known about endotoxin in tracheal aspirates (TAs) or the mobilization of innate immunity toward endotoxin, a potent stimulus that contributes to inflammatory disease. We characterized mobilization of endotoxin-directed innate immunity in TAs from an observational cohort of mechanically ventilated neonates. TA supernatants (n = 42; GA = 23-40 wk, postnatal age = 1-71 d) were assayed for endotoxin (Limulus amoebocyte lysate assay) and endotoxin-modulating proteins: bactericidal/ permeability-increasing protein (BPI), LPS-binding protein (LBP), and soluble cell differentiation antigen 14 (sCD14). TA cellular BPI was measured by ELISA, Western blot, flow cytometry, and bactericidal assay. TA mRNAs encoding endotoxin-modulating proteins were measured by quantitative real-time PCR (qRT-PCR). Endotoxin in TA supernatants was proportional to both postnatal age and polymorphonuclear leukocytes (PMN). Neonatal TAs were rich in PMN containing BPI and expressed mRNAs encoding Toll-like receptor (TLR) 4, CD14, and myeloid differentiation protein 2 (MD-2). Extracellular BPI was consistently detectable and correlated with TA PMN and GA. Both extracellular- and cellular-BPI increased during the first postnatal week. TA extracellular BPI, LBP, and sCD14 were positively correlated. TAs from intubated neonates demonstrate endotoxin accumulation and mobilization of endotoxin-directed innate immunity, potentially contributing to pulmonary inflammation.

The balance between endotoxin accumulation and clearance during particle‐induced osteolysis in murine calvaria

Bacterial endotoxin may contribute to aseptic loosening of orthopedic implants even in the absence of clinical or microbiological evidence of infection. One potential source of endotoxin during aseptic loosening is systemically circulating endotoxin, derived from intestinal flora, minor infections, or dental procedures, that may bind to wear particles. The current study demonstrates that systemically derived endotoxin accumulates when 'endotoxin-free' titanium and polyethylene particles are implanted on murine calvaria. Time-course experiments and experiments using germ-free mice rule out the possibility that the observed endotoxin accumulation may be due to bacterial contamination. In contrast, endotoxin is cleared from titanium particles that originally carry high amounts of adherent endotoxin. The mechanism of endotoxin clearance is not dependent on induction of a respiratory burst. Taken together, these results indicate that a balance between endotoxin accumulation and endotoxin clearance controls the steady-state level of endotoxin surrounding orthopedic wear particles implanted on murine calvaria. This balance may regulate the rate of osteolysis in the murine calvaria model as well as in patients with aseptic loosening.

Relation of Endotoxin, Endotoxin Binding Proteins and Macrophages to Severe Alcoholic Liver Injury and Multiple Organ Failure

Our retrospective analysis of 105 patients with alcoholic liver injury confirmed that patients with severe alcoholic hepatitis (SAH) showed severe hyperbilirubinemia, reduced hepatic biosynthetic capacity, and marked acute inflammatory reactions, and developed multiple organ failure (MOF). Multivariate analysis using the Cox proportional hazards model showed serum C-reactive protein and DIC as significant independent prognostic factors among SAH, LC+AH, and AH groups. Improved assay showed an increase of plasma endotoxin with the progression of alcoholic liver injury. In most survivors, plasma Et levels decreased in the recovery phase. Serum interleukin (IL)-6 and IL-8 levels in the acute phase were high in patients with AH and LC+AH, especially in non-survivors and in patients with SAH. In the recovery phase, these cytokine levels in survivors tended to decrease, but in non-survivors, IL-6 remained high, and IL-8 further increased. Serum levels of HDL and albumin, which are protective against endotoxicity by inhibiting endotoxin uptake and TNF production by macrophages, were decreased with the progression of alcoholic liver injury. Animal experiments supported that the increase in endotoxin-binding capacity of HDL and albumin may serve as a protective mechanism against endotoxin in chronic ethanol-loaded rats and that an addition of high-dose ethanol to these rats may lead to impaired binding and inactivation of endotoxin. Lipopolysaccharide-binding protein (LBP) which enhances endotoxin uptake and TNF production by macrophages, was generally increased in patients with alcoholic liver injury. This imbalance among endotoxin binding proteins in the blood may induce overproduction of cytokines by macrophages in patients with severe alcoholic liver injury. Our animal experiments further revealed that an additional administration of a high-dose ethanol to chronic alcohol-fed rats led to decrease of endotoxin clearance, increased extrahepatic accumulation of endotoxin and elevation of plasma TNF. The splenic macrophages and pulmonary alveolar macrophages are demonstrated to be important for endotoxin uptake, and excessive production of TNF in rats given large amounts of alcohol. An in vitro culture experiment in the presence of rat LBP suggested a role of these macrophages in excessive production of TNF-alpha. When the functions of various macrophages were compared in rats given alcohol, maximum TNF-alpha secretion was noted in alveolar macrophages, In conclusion, endotoxemia and its effects on extrahepatic macrophages may play key roles in the progression of severe alcoholic liver injury and MOF.

Regulation of interactions of endotoxin with host cells

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [14C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein: endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates ( agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (Mr ≥ 20 × 106) to monomeric (M ~55 × 10 3) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 with rLOS agg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (Mr ~1—20 × 106) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

Regulation of interactions of endotoxin with host cells.

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [(14)C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein:endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates (agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (M(r) > or = 20 x 10(6)) to monomeric (M(r) approximately 55 x 10(3)) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 with LOSagg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (M(r) approximately 1-20 x 10(6)) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

Endotoxin in pooled pericardial blood contributes to the systemic inflammatory response during cardiac surgery.

Although endotoxin has been implicated as an important contributor to the systemic inflammatory response (SIR) during cardiopulmonary bypass (CPB), its source remains unclear. While gut translocation has traditionally been perceived as the primary source of endotoxemia, accumulation of endotoxin in pooled pericardial blood may represent an additional source of endotoxin that is continually reinfused into the CPB circuit. Eighteen patients undergoing primary coronary revascularization procedures were prospectively evaluated. Shed blood pooled in the pericardial space was returned to the CPB circuit through cardiotomy suction catheters at 45 min after placement of the aortic cross-clamp. Simultaneous samples of pooled pericardial and peripheral arterial blood were obtained and analyzed by a limulus amebocyte lysate assay for the determination of endotoxin concentration, and an enzyme-linked immunosorbent assay for tumor necrosis factor (TNF-alpha) levels. Significant elevations in endotoxin were demonstrated in pooled pericardial blood samples compared with arterial blood (3.5 +/- 0.5 vs 0.8 +/- 0.2 pg/ml; p < 0.05). TNF-alpha levels were below the limits of detection in both samples. These data implicate pooled pericardial blood as an important primary source of endotoxin that, when continually reinfused throughout CPB, may contribute to the overall SIR. Because endotoxemia has been identified as an important predictor of adverse outcomes following cardiac surgery, removal of endotoxin antigen in shed pericardial blood, prior to its reinfusion into the CPB circuit, may provide a directed means to improve perioperative outcome without compromising established blood conservation techniques.

Abnormal accumulation of endotoxin in biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis

Backgrounds/Aims: Previous studies have revealed the involvement of Kupffer cells and hepatocytes in the metabolism of endotoxin in the liver. The aim of this study was to investigate the in vivo localization of endotoxin in liver cells, including Kupffer cells, hepatocytes, and biliary epithelial cells, in primary biliary cirrhosis and primary sclerosing cholangitis. We also examined the effect of ursodeoxycholic acid on the intrahepatic distribution of endotoxin in primary biliary cirrhosis. Methods: The immunohistochemical localization of endotoxin was examined in liver specimens from 30 cases of primary biliary cirrhosis and seven of primary sclerosing cholangitis using a monoclonal antibody against lipid A. Controls were seven cases of obstructive jaundice, ten of hepatitis C virus-related liver cirrhosis, 14 of chronic hepatitis C, and five histologically normal liver cases. Semi-quantitative analysis of endotoxin accumulation was performed to measure the intensity of fluorescence for endotoxin. Nine of the 30 patients with primary biliary cirrhosis underwent a second liver biopsy for evaluation of the ursodeoxycholic acid treatment. Results: In primary biliary cirrhosis and primary sclerosing cholangitis, biliary epithelial cells showed strong immunostaining for endotoxin as well as hepatocytes and Kupffer cells. Biliary epithelial cells of primary biliary cirrhosis and primary sclerosing cholangitis showed more intense immunoreactivity than those of other controls. In primary biliary cirrhosis, ursodeoxycholic acid reduced the immunoreactivity to endotoxin in biliary epithelial cells, and increased, the immunoreactivity to endotoxin in Kupffer cells, but did not affect that in hepatocytes. Conclusions: Our results revealed that in primary biliary cirrhosis and primary sclerosing cholangitis, endotoxin accumulates abnormally in biliary epithelial cells. In addition, we found that ursodeoxychlic acid treatment in primary biliary cirrhosis may provide a beneficial effect on the intrahepatic metabolism of endotoxin.

Interaction of Kupffer cells to splenic macrophages and hepatocytes in endotoxin clearance: effect of alcohol.

An additional administration of high dose ethanol to chronic alcohol-fed rats led to a decrease in endotoxin clearance and an increase in endotoxin accumulation in the spleen accompanied by an elevation of tumour necrosis factor (TNF) levels in the portal vein. Endotoxin uptake and TNF production by Kupffer cells (KC) and splenic macrophages in the chronic ethanol load rats were significantly greater than those in the control rats. When these cells were precultured in the medium containing 10 to 100 mmol/L ethanol, the endotoxin uptake and TNF production of KC were decreased. However, this did not affect the endotoxin uptake and TNF production of splenic macrophages. The hepatic production of endotoxin binding protein was increased when KC were preincubated in the medium containing ethanol and the resultant culture supernatant was added to the hepatocyte culture system. This endotoxin binding protein was proved to enhance the uptake of endotoxin and suppressed the production of TNF in the KC. When KC and hepatocytes were isolated from chronically alcohol-fed rats, further addition of ethanol to the culture medium of KC did not affect the hepatic production of endotoxin binding protein. The increase in hepatic production of endotoxin binding protein may serve as a defence mechanism against endotoxicity. There is a possibility that an impairment of this defence mechanism has a pivotal role in the development of endotoxaemia and endotoxicity in chronic alcoholics.

Increase of Endotoxin Concentration in Gingival Washings During Experimental Gingivitis in Man

man repeatedly has shown the major role of plaque in the etiology of gingivitis. It has been demonstrated also that as the flora of developing plaque becomes more Gram-negative, the signs of gingivitis become more evident.2 If bacteria, per se, probably cannot cross the gingival epithelial barrier (for review see Cimasoni, de Crousaz and Rebstein3), certain toxic components of the microorganisms, such as endotoxin, can do so, at least in experimental animals.4-6 In man, the accumulation of endotoxin in the gingival environment has been studied only in cross-sectional investigations. Simon, Goldman, Ruben and Baker79 have determined endotoxin in the crevicular fluid from 15 patients and have shown a direct relationship between oral endotoxin levels and the severity of gingival inflammation. Similarly, Shapiro, Lodato, Courant and Stallard10 determined the endotoxin content of crevicular fluid, dental plaque, gingival tissue and saliva in 37 patients with varying degrees of periodontal inflammation and observed a positive correlation between the lipopolysaccharide concentrations and gingival inflammation. An appliance described by Oppenheim11 has been used in our laboratory to study the enzyme contents of gingival sulcus;12'13 it allows the collection of gingival washings from the same individuals throughout long periods of time. It was used in the present longitudinal investigation, in order to follow the endotoxin concen-

Accumulation of activated lymphocytes in liver of endotoxin treated rabbits

Intravenous administration of endotoxin in doses, 1.0 mg/kg, 0.1 mg/kg or 0.01 mg/kg caused activation (in the sense of nucleolar RNA synthesis) of the lymphocytes in peripheral blood, kidney and liver in rabbits. The evaluation of the ratio of lymphocyte count to the number of organ tissue cells leads to the conclusion that the accumulation of endotoxin activated lymphocytes occurs in liver.

Impaired detoxification as a mechanism of lead acetate-induced hypersensitivity to endotoxin.

SummaryActinomycin D was employed to profoundly enhance the sensitivity of mice to endotoxin in order to bioassay submicrogram amounts of endotoxin. Homogenates of rat liver and spleen were able to detoxify endotoxin as bioassayed in actinomyein-treated mice. Lung, kidney, heart, and brain homogenate did not possess the ability to significantly detoxify endotoxin. Lead acetate, which profoundly sensitizes rats to endotoxin, enhanced the vascular clearance of colloidal carbon and also reduced detoxification of endotoxin by liver and spleen homogenates. Enhanced phagocytosis, resulting in accumulation of endotoxin in macrophages, coupled with the failure in endotoxin inactivation or detoxification, appears to be the mechanism of lead acetate-induced endotoxin hypersensitivity.