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PubMed2003

Simultaneous onset of acute inflammatory response, sepsis-like symptoms and intestinal mucosal injury after cancer chemotherapy.

Chemotherapy is 1 method for the treatment of cancer, but serious side effects can sometimes limit the dosage given. Mild fever and diarrhea are common side effects of cancer chemotherapy. Gastrointestinal injury induced by chemotherapeutic agents may result in bacterial/endotoxin translocation from the gut into the systemic circulation. An experimental study was therefore conducted to clarify the effect of systemic chemotherapeutic agents on gastrointestinal barrier function. Male Wistar rats were divided into a 5-fluorouracil (5-FU) group (100 mg/kg/day for 4 days; n = 27) and a control group (n = 5). All rats were fasted and central venous catheterization was performed for total parenteral nutrition and blood sampling. Intestinal tissue was also sampled for pathological examination. Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFalpha) were determined by ELISA, bacterial translocation was quantified by lymph node culture and plasma endotoxin content of portal blood was measured by the Limulus-amebocyte-lysate test. In the 5-FU group on day 4, a proportion of rats exhibited severe watery diarrhea (73.9%) and occasional vomiting (86.2%). The levels of plasma TNFalpha and IL-6 were seen to increase, peaking at day 6 (IL-6, 350.0 +/- 67.8 pg/ml; TNFalpha, 26.1 +/- 3.2 pg/ml). The pathological findings also changed on day 4. On day 6, 90% of the rats in the 5-FU group showed dramatic sepsis-like manifestations, whereas the control group did not. Within the 5-FU group, only at day 6 was bacterial translocation in the rat mesenteric lymph nodes or significantly elevated levels of endotoxin evident. These results suggest that bacterial/endotoxin translocation might cause sepsis-like manifestations after systemic chemotherapy.

Animals↗
PubMedDate not supplied

Chemical and biological properties of a phenol-water extract from Leptospira interrogans. Evidence for the absence of lipopolysaccharide.

Leptospira interrogans, serovar copenhageni was extracted by the phenol-water method and the resulting preparation examined for chemical composition and endotoxic activity. Chemical analysis revealed that a number of sugars were present, however, the amount of lipid content was very low. Further, the preparation was devoid of characteristic endotoxic properties, like lethal toxicity, pyrogenicity and the property to induce the local Shwartzman reaction. The extract, however, was active in the limulus lysate gelation test and in the induction of monocyte activation. It is concluded that the leptospira preparation is devoid of endotoxin properties, both from the chemical and from the biological point of view.

Animals↗
PubMed2003

Endotoxinemia in the portal and the systemic circulation in obstructive jaundice.

Endotoxinemia in patients with obstructive jaundice is linked to acute renal failure and sepsis and remains a major cause of complications during postoperative treatment. The current study examines the mechanisms of endotoxinemia in the portal and the systemic circulation in obstructive jaundice. As an experimental model of the disease we used rabbits subjected to sham operation. Serum total bilirubin aminotransferases and endotoxin concentrations were determined at 2, 5, 8, and 13 days after operation. Endotoxin concentrations were estimated by the limulus lysate endotoxin test. A high frequency of portal or systemic endotoxinemia is observed in obstructive jaundice, but no difference between endotoxinemia levels in the portal and systemic circulation was observed.

Animals↗
PubMed2001

The detergent octylglucoside neutralizes lipopolysaccharide in a specific manner.

The stimulatory effect of lipopolysaccharide (LPS) on human macrophages was found to be neutralized by the detergent octylglucoside (OG). Both macrophage stimulation and reactivity in a limulus amebocyte lysate test were suppressed by suspension of LPS in OG at concentrations between 0.25 and 2.5 mM, whereas other stimulatory lipopeptides and lipid containing stimulants were unaffected by OG. LPS at concentrations causing maximal stimulation of macrophages could be completely neutralized by non-toxic concentrations of OG. In addition, it was found that the neutralization in complex mixtures of macromolecules, such as bacterial cell lysate, was specific for LPS and that the stimulatory activity of the other substances in the mixture was not affected by the OG.

Adhesins, Bacterial↗
PubMed1992

Potentiation of HIV envelope glycoprotein and other immunogens by endotoxin (ET) and its molecular fragments.

The structural requirements for the immunopotentiating (adjuvant) effect of endotoxin (ET) were investigated. Mild hydrolysis (0.2 N acetic acid at 95 degrees C) was applied to various ET preparations and the lipid rich (Lipid A) and polysaccharide-rich (PS) preparations obtained were tested as adjuvants on three immunogens: sheep red blood cells (SRBC), L-glutamine: L-lysine: L-alanine containing random synthetic polypeptide (GLA-40), and recombinant HIV viral envelope polypeptide (CBre3). It was found that not only the Lipid A precipitates, but under certain hydrolytic conditions the non-toxic PS preparations were also potent adjuvants. The exact conditions of hydrolysis which led to the isolation of immune adjuvant bacterial products were established. These materials were also tested for endotoxicity (Limulus lysate clotting, chick embryo lethality and local Shwartzman skin reactivity), as well as for TNF generating activities. It was found that TNF generation runs parallel with toxicity of the samples, but it does not follow the adjuvant activity of the isolates. Chemical analysis of the preparations indicated that they did not contain residual ET or Lipid A, however, they did not exclude that deacylated and dephosphorylated skeletal remains of ET are among those components in these preparations which have immunomodulatory activity.

Adjuvants, Immunologic↗
PubMed1990

Quantitative determination of endotoxins on surgical gloves.

The amount of endotoxin on surgical gloves was investigated by the limulus chromogenic substrate method. Heavy endotoxin contamination was observed on the outside of certain surgical gloves sold in Sweden. Given an endotoxin absorption of 10% to the patients, one of the types of gloves studied would cause fever, chills, headache and muscle pain. The amount of endotoxin on the gloves could be minimized by rinsing in endotoxin-free water. We conclude that the manufacturing process of latex gloves should be checked microbiologically for Gram-negative bacteria before sterilization. Alternatively, the amount of endotoxin should be checked on the product by the limulus ameobocyte lysate test before release.

Endotoxins↗
PubMed2004

In vivo studies of endotoxin removal by lysine-cellulose adsorbents.

A new type adsorbent for removal of bacterial endotoxins was prepared by immobilizing lysine covalently onto cellulose beads. Endotoxins (Escherichia coli O55: B5) were injected into 13 healthy New Zealand white rabbits to induce infectious symptoms. Hemoperfusion using the adsorbent column removed endotoxins in the blood of eight rabbits during 2h while other five rabbits were used as control. The mean blood endotoxin concentration was reduced significantly from 5.56 +/- 0.54 EU/ml (1 EU = 100 pg) before treatment to 0.41 +/- 0.26 EU/ml after perfusion as measured by the limulus amebocyte lysate test (Chromogenix). Liver function and renal function tests showed significant improvement of septic symptoms in contrast to the control group. Other parameters such as superoxide dismutase and malondialdehyde were ameliorated markedly after the treatment. Moreover, the adsorbent showed good results in mechanical strength, blood compatibility and cytotoxicity, which suggested that lysine-cellulose adsorbent was of high ET-binding efficacy without significant side effect. It has a high potential of clinical application for treatment of patients with severe sepsis.

Adsorption↗
PubMed2001

Endotoxemia and acute-phase proteins in major abdominal surgery.

BACKGROUND: Translocation of endotoxin is a controversial issue. The ability of plasma to inactivate endotoxin is an indirect measure of endotoxemia. Endotoxin is a potent stimulator of the inflammatory response and affects the innate immune system. OBJECTIVE: To elucidate the kinetics of endotoxemia and the ability of plasma to inactivate endotoxin in patients with major abdominal operations. To demonstrate the early time course of the acute-phase proteins C-reactive protein (CRP), serum amyloid A (SAA), alpha(1)-antitrypsin, alpha(2)-macroglobulin, transferrin, and interleukin 6 (IL-6), and to correlate them with the amount of endotoxemia. METHODS: Twenty patients with elective major abdominal operation and 10 healthy controls were investigated. Blood was collected preoperatively, during the operation and regularly up to 12 days after surgery. Endotoxin was measured by Limulus amebocyte lysate test (LAL), the ability of plasma to inactivate endotoxin by modified LAL, the acute-phase proteins nephelometrically, and IL-6 by enzyme-linked immunosorbent assay (ELISA). RESULTS: Preoperative endotoxin plasma level (0.026 +/- 0.004 EU/mL) did not differ from healthy volunteers but increased during operation (0.09 +/- 0.02 EU/mL, P = 0.02). Endotoxemia peaked 1 hour after the surgical procedure (0.16 +/- 0.03 EU/mL; P <0.0001 versus preoperative) and decreased to almost normal values after 48 hours. The capability of plasma to inactivate endotoxin was significantly reduced during (recovery, 0.16 +/- 0.03 EU/mL), 1 hour (0.25 +/- 0.04 EU/mL) and 24 hours (0.16 +/- 0.02 EU/mL) after the operation compared with preoperative (0.068 +/- 0.01 EU/mL) values. Plasma IL-6 was significantly increased for 48 hours with a peak 1 hour after surgery (470 +/- 108 pg/mL). CRP peaked at 210 +/- 19 mg/L (P <0.0001 versus preoperative) 48 hours after operation and was significantly elevated for the rest of the observation period. SAA was significantly increased 24 hours after surgery (249 +/- 45 mg/L) and peaked additional 48 hours later (456 +/- 86 mg/L). alpha(1)-Antitrypsin, although a positive acute-phase protein, decreased initially to 1.38 +/- 0.1 g/L (preoperative, 2.33 +/- 0.18 g/L; P <0.0001) and increased thereafter until day 12 (3.05 +/- 0.35 g/L, P = 0.11 versus preoperative). The same was true for alpha(2)-macroglobulin (preoperative, 2.2 +/- 0.16 g/L; intraoperative, 1.36 +/- 0.13 g/L; day 5, 2.8 +/- 0.4 g/L). Transferrin decreased already during surgery (1.6 +/- 0.1 g/L versus preoperative 2.8 +/- 0.17 g/L, P <0.0001) and remained on this level for 5 days. Correlation analysis revealed a relationship between endotoxemia and the ability of plasma to inactivate endotoxin (r = 0.67, P <0.0001) and also a relation between intraoperative endotoxemia on one hand and alpha(2)-macroglobulin (-0.53 > r > -0.6, P <0.05) as well as alpha(1)-antitrypsin (0.64 > r >0.55, P <0.05) on the other. CONCLUSION: Major abdominal surgery is associated with transient endotoxemia and a transient reduced endotoxin inactivation capacity of the plasma. Endotoxemia correlates with the endotoxin inactivation capacity. The surgical procedure causes substantial changes in plasma concentrations of acute-phase proteins. alpha(2)-Macroglobulin and alpha(1)-antitrypsin correlate moderately with endotoxemia.

Abdomen↗
PubMed1996

Setting standards for the levels of endotoxin in the embryo culture media of human in vitro fertilization and embryo transfer.

OBJECTIVE: To clarify the optimum set standards for the levels of endotoxin in a culture medium for human IVF-ET programs. DESIGN: Retrospective and randomized study. SETTING: One hundred patients underwent 163 cycles of IVF-ET at Fukuoka University Hospital. INTERVENTIONS: Measurements for endotoxin were performed using the Limulus Amoebocyte Lysate test. The negative group was administered media with < 1 pg/mL of endotoxin and the positive group was given media with > or = 1 pg/mL of endotoxin. MAIN OUTCOME MEASURES: Human embryo development, clinical outcomes, and pregnancy outcomes were evaluated in each group and also the levels of endotoxin. RESULTS: The rates of clinical pregnancy (26.1%) and the live birth rates (20.7%) in the negative media were significantly higher than those in the positive media (9.9% and 5.6%, respectively). Rates of embryo development were decreased and a gestational sac and fetal heart beat were not detected in media with > 2 pg/mL of endotoxin. CONCLUSION: The set standard for the level of endotoxin in a medium should be < 1 pg/mL to obtain the best outcome. The allowable levels of endotoxin in a medium should be < or = 2 pg/mL in human IVF.

Adult↗
PubMed1983

The preparation of pyrogen-free foetal calf serum for use in cell and tissue cultured.

A method is described for the preparation of foetal calf serum from blood drawn by heart puncture from calf foetuses obtained from a local municipal abattoir. This method differs from that previously described in that the Seitz Supra EK filter pads used in preliminary purification steps have been abandoned. These pads contain significant amounts of pyrogenic bacterial lipopolysaccharides and attempts to depyrogenate them with sodium hypochlorite, hydrogen peroxide and 1% foetal calf serum were unsuccessful. The modified method uses pyrogen-free glass fibre and cellulose fibre filters and provides an entirely satisfactory serum as proved by negative Limulus amoebocyte lysate tests and the excellent growth of cells.

Animals↗
PubMed1988

Determination of environmental levels of peptidoglycan and lipopolysaccharide using gas chromatography with negative-ion chemical-ionization mass spectrometry utilizing bacterial amino acids and hydroxy fatty acids as biomarkers.

D-Alanine and diaminopimelic acid originating from bacterial peptidoglycans and hydroxy fatty acids from lipopolysaccharides (endotoxins) were analysed by gas chromatography using a chiral column (Chirasil-Val as stationary phase) and selected-ion monitoring detection with negative-ion chemical-ionization mass spectrometry. The amino acids were analysed as N-heptafluorobutyryl isobutyl esters after rapid hydrolysis of peptidoglycan followed by isolation of the amino acids with disposable ion-exchange columns. Racemization of amino acid enantiomers was controlled by using deuterium chloride in the hydrolysis. The hydroxy acids were analysed as O-pentafluorobenzoyl methyl esters. Most of the bacteria present in airborne dust from a poultry confinement building were found to be Gram-positive according to the analytical chemical method whereas the Limulus amoebocyte lysate test suggested the presence of appreciable amounts of lipopolysaccharides of Gram-negative bacteria. Further studies are required to compare the utility of these two methods for determining endotoxins in complex environments.

Amino Acids↗
PubMed2000

Monoclonal anti-double-stranded DNA autoantibody stimulates the expression and release of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha from normal human mononuclear cells involving in the lupus pathogenesis.

In our previous reports, we found polyclonal anti-double-stranded DNA antibodies (anti-dsDNA) purified from patients with active systemic lupus erythematosus (SLE) exerted inhibitory effect on [3H]thymidine incorporation of human mononuclear cells (MNC). However, the other immunological effects of anti-dsDNA on the functions of MNC have not yet been reported. In this study, two monoclonal antibodies, 12B3 and 9D7, with different anti-dsDNA activity were evaluated for their effects on the expression and release of different cytokines from human MNC. We confirmed absence of endotoxin in the two monoclonal antibody preparations and the used medium as detected by Limulus amoebocyte lysate test. The mRNA expression and release of different cytokines including interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were measured. We found the two monoclonal anti-dsDNA not only dose-responsively suppressed the phytohaemagglutinin (PHA)-induced thymidine uptake of human MNC but stimulated the mRNA expression of IL-1beta, IL-6 and IL-8 in normal human MNC detected by reverse transcription-polymerase chain reaction (RT-PCR). Enzyme-linked immunosorbent assay (ELISA) measurement of cytokines in MNC culture supernatants revealed that anti-dsDNA enhanced IL-1beta, IL-8, TNF-alpha and IL-10 release from resting MNC. These effects of anti-dsDNA antibodies were not affected by polymyxin B, a potent binder and neutralizer of lipopolysaccharide (LPS). These in vitro studies suggest that anti-dsDNA possess a dual effect on normal human MNC: (a) to enhance the release of proinflammatory cytokines (IL-1beta, IL-8 and TNF-alpha) from MNC to augment inflammatory reaction; and (b) to polarize the immune reaction towards the T helper 2 (Th2) (increased IL-10 production) pathway. This unique effect of anti-dsDNA may play a role in lupus pathogenesis by augmenting inflammatory reactions and autoantibody production which are commonly found in patients with active SLE.

Animals↗
PubMedDate not supplied

Evaluation of bacterial contamination in blood processing.

In the routine deglycerolization of frozen red blood cells, a bottle of solution intended as a final wash (0.8% NaCl, 0.2% dextrose) was found to be abnormally colored and slightly turbid. Close inspection revealed a small crack in the base of the bottle. A pure growth of Klebsiella pneumoniae was cultured. The rabbit pyrogen test on the solution was positive using 2.5 mul as was the limulus amebocyte lysate (LAL) test at a dilution of 1:10-5 for endotoxin. Studies of the growth of this organism are described and the clinical implications are discussed.

Animals↗
PubMed2000

Isothermic hemodialysis and ultrafiltration.

The increase in patient temperature during hemodialysis is explained by hemodynamic compensation during ultrafiltration and hypovolemia that leads to peripheral vasoconstriction and reduced heat losses. We analyzed 51 stable high-efficiency hemodialysis treatments in 27 patients during isothermic dialysis in which body temperature was maintained at a constant level (+/-0.1 degrees C) using the temperature-control option of the Blood Temperature Monitor (BTM; Fresenius Medical Care, Bad Homburg, Germany). Hemodialysis was delivered using ultrapure water (limulus amebocyte lysate test < 0. 06 endotoxin units/mL) at mean blood flows of 410 +/- 40 mL/min. During treatments lasting 178 +/- 23 minutes, 4.8% +/- 1.4% of postdialysis body weight (W%) and 9.5% +/- 2.5% of postdialysis body water were removed using mean ultrafiltration rates of 1.1 +/- 0.3 L/h. Dialysate temperatures significantly decreased from 35.9 degrees C +/- 0.3 degrees C to 35.6 degrees C +/- 0.6 degrees C during hemodialysis. During these treatments, 187 +/- 69 kJ of thermal energy were removed from the patients through the extracorporeal circulation using cool dialysate. Extracorporeal heat flow was 17 +/- 6 W. Energy expenditure (H) estimated from anthropometric data was 65 +/- 12 W. Thus, 28% +/- 10% of estimated energy expenditure (H%) was removed during isothermic dialysis. A highly significant correlation was observed between H% and W% (H% = -5.6 * W%; r(2) = 0.91; P < 0.0001). This result is in support of the volume hypothesis of intradialytic heat accumulation and provides a rule of thumb to estimate extracorporeal cooling requirements for isothermic dialysis. Approximately 6% of H must be removed through the extracorporeal circulation for each percent of ultrafiltration-induced body-weight change. The importance of body temperature control during hemodialysis increases with increased ultrafiltration requirements.

Body Temperature↗
PubMed1995

Endotoxemia and mediator release during colonoscopy.

BACKGROUND AND STUDY AIMS: Previous clinical and experimental studies have shown evidence of a leakage of whole bacteria and bacterial products after major trauma through the gut barrier. By determining plasma endotoxin levels, products of the arachidonic pathway, interleukin-6, and the endotoxin-neutralizing capacity (ENC) of plasma during colonoscopy, we studied the gut barrier function and the pathogenetic sequelae of mediator release during a minimally invasive procedure. PATIENTS AND METHODS: Thirty-two patients were enrolled in a controlled prospective study. Endotoxin and ENC were determined by a chromogenic modification of the limulus amebocyte lysate test. Prostanoids and interleukin-6 were measured using commercially available ELISA tests. C-reactive protein levels were checked by nephelometry. RESULTS: Twenty-one of the 32 patients had elevated endotoxin plasma levels during colonoscopy. In one patient, gut-derived bacteria were detected in plasma. ENC decreased after 5 min, and thromboxane B2 levels also started to increase at that time. No acute-phase response took place after 24 h. CONCLUSION: During colonoscopy, endotoxin can be detected in blood. ENC measurement was shown to be even more sensitive. The pathogenetic sequelae leading to gut barrier failure remain unclear, because mediator release and endotoxemia, as checked by ENC, took place simultaneously.

Adult↗
PubMed1983

Endotoxaemia following colonoscopy.

In 100 consecutive patients the occurrence of bacteraemia and endotoxaemia after colonoscopy was studied. The Limulus Amoebocyte Lysate Test was used to detect endotoxaemia. No bacteraemia was detected after colonoscopy. Growth of Staph, epidermidis was considered as contamination. In 9% of the patients endotoxaemia was discovered after colonoscopy. The occurrence of endotoxaemia was found to be unrelated to the duration of the procedure, the number of the biopsies taken, or the cleansing of the bowel prior to colonoscopy. In patients with liver cirrhosis endotoxaemia occurred more often than in patients without liver disease. No clinical consequences of endotoxaemia were found in patients who developed endotoxaemia after colonoscopy. We conclude that colonoscopy is followed by endotoxaemia quite frequently. The clinical implication of these conditions, especially in patients with liver cirrhosis merits further consideration.

Adolescent↗
PubMed1986

[Postoperative determination of endotoxin levels. Prognostic and diagnostic value in intensive care units].

197 patients monitored postoperatively for more than two days in an intensive-care unit were followed up prospectively for nine months. Endotoxin plasma levels were determined daily by means of the limulus amoebocyte lysate test. In endotoxin-positive patients there was a statistically significantly higher incidence of peritonitis, sutural insufficiency, pneumonia, and sepsis. The diagnostic value of the test was found to be higher if the patients were considered "septic" only when endotoxin was positive for more than two days. In that case the specificity of the test was 80.6%; this was superior to that of a corresponding rise in leukocyte count or in body temperature. The positive predictive value for septic complications was 67.6%. Determination of the course of the endotoxin titre is more relevant than recording an isolated rise. This allows differentiation between septic and non-septic diseases.

Endotoxins↗
PubMed2001

Exposure to endotoxin decreases the risk of atopic eczema in infancy: a cohort study.

BACKGROUND: Previous studies have shown a protective effect of early exposure to cats and dogs on the development of atopic eczema, asthma, allergic rhinitis, and atopic sensitization in later life. In particular, a higher microbial exposure to endotoxin in early childhood might contribute to this effect. OBJECTIVE: We examined the associations between bacterial endotoxin in house dust and atopic eczema, infections, and wheezing during the first year of life in an ongoing birth cohort study (LISA). METHODS: Data of 1884 term and normal-weight neonates with complete information on exposure to biocontaminants and confounding variables were analyzed. House dust from the mothers' and the children's mattresses was sampled 3 months after birth. Endotoxin content was quantified by using a chromogenic kinetic limulus amoebocyte lysate test. RESULTS: During the first 6 months of life, the risk of atopic eczema was significantly decreased by endotoxin exposure in dust from mothers' mattresses in the fifth quintile (odds ratio [OR], 0.50; 95% CI, 0.28-0.88), whereas the risk was increased for respiratory infections (OR, 1.69; 95% CI, 1.25-2.28) and cough with respiratory infection, bronchitis, or both (OR, 1.73; 95% CI, 1.28-2.33). The risk of wheezing was also significantly increased during the first 6 months of life (OR, 2.37; 95% CI, 1.40-4.03). For the entire first year of life, these associations attenuated, except for the risk of wheezing, which remained significant (OR, 1.60; 95% CI, 1.10-2.30). CONCLUSION: Our findings support the hygiene hypothesis that exposure to high concentrations of endotoxin very early in life might protect against the development of atopic eczema within the first 6 months of life, along with an increased prevalence of nonspecific respiratory diseases.

Air Pollution, Indoor↗