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PubMed2004

The short-term effects of treatment of chronic periodontitis on circulating levels of endotoxin, C-reactive protein, tumor necrosis factor-alpha, and interleukin-6.

BACKGROUND: The acute-phase response involves molecules including tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and C-reactive protein (CRP). This study aimed to determine whether subgingival scaling resulted in rapid changes in plasma concentrations of these molecules. METHODS: Twenty-three non-smoking adults with chronic periodontitis received subgingival scaling for 60 minutes. Venous blood samples were taken at 0, 15, 30, 60, and 120 minutes. TNF-alpha and IL-6 were assayed from all samples and CRP from the baseline and final samples. Lipopolysaccharide (LPS) was assayed at 0, 15, and 30 minutes using limulus lysate assay (LAL) and EndoCAb Ig assays. RESULTS: LPS assays were suggestive of a transient low-grade bacteremia, but changes in LPS approaching significance (P=0.061) were seen with LAL only. There was a significant increase in circulating TNF-alpha (P=0.0387) and IL-6 (P<0.0001), and the degree of change in TNF-alpha was correlated with the severity of periodontal breakdown (P=0.001). There was also a significant correlation between levels of IL-6 and TNF-alpha (P<0.001). CONCLUSIONS: Chronic periodontitis patients undergoing an episode of subgingival scaling show a significant elevation in circulating TNF-alpha and IL-6. This may account for anecdotal reports of pyrexia following treatment and may be significant in terms of the relationship between periodontal disease, bacteremia, and cardiovascular disease.

Adult↗
PubMed2004

Detection of bacterial endotoxin in human tissues.

Detection of bacterial lipopolysaccharide (LPS) in the absence of overt infection is a challenging problem in tissue homogenates and other complex samples. We found that conventional Limulus amebocyte lysate (LAL) assays are not suitable for this purpose due to interference from beta-glucan-like molecules. In contrast, a modified LAL assay that is unaffected by beta-glucan-like molecules was able to detect LPS in infected tissue and in a subset of clinically aseptic tissues. A two-step LAL assay was used to exclude the possibility of false positives due to nonspecific amidases. False positives due to sample color were also excluded, as were false negatives due to assay inhibition. This is the first report to successfully detect LPS in tissue in the absence of overt infection. This approach may be extremely useful in assessing recent hypotheses that subclinical levels of bacteria contribute to a wide range of chronic diseases.

Amidohydrolases↗
PubMed1994

Effect of feeding regimen on concentration of free endotoxin in ruminal fluid of cattle.

The influence of concentrate diets on endotoxin concentration in sterile filtrate of ruminal fluid was assessed in ruminally fistulated Jersey cows. Three cows underwent a change in diet from hay to a diet containing 3.0 kg and 14 d later 6.0 kg of a 12% CP concentrate. The cows had free access to water and a mineral-stone. A modified Limulus Amoebocyte Lysate technique was used for the endotoxin analyses. The endotoxin concentration in the ruminal fluid of cows fed on hay were 148 +/- 84 and 118 +/- 50 endotoxin units (EU)/mL (mean +/- SD) on two separate days and increased from the 2nd d of supplementation with 3.0 kg of concentrate to 408 +/- 198 EU/mL on d 5. When the cows were fed 6.0 kg of concentrate, the endotoxin concentrations increased to 1,599 +/- 944 EU/mL. To assess the influence on the ruminal endotoxin concentration of an adaptation of the ruminal environment to concentrate before hyperalimentation, four ruminally fistulated Jersey cows previously fed either hay or a high-concentrate diet for 1 mo were hyperalimented with 60 to 70 g of barley per kilogram BW. A relative increase in ruminal endotoxin concentration was determined only in the two cows previously fed concentrates. The results show that high-concentrate diets do not consistently relate to increases of ruminal endotoxin concentrations.

Adaptation, Physiological↗
PubMed1991

Increased spontaneous release of tumor necrosis factor-alpha/cachectin in headache patients. A possible correlation with plasma endotoxin and hypothalamic-pituitary-adrenal axis.

Tumor Necrosis Factor-alpha/cachectin (TNF-alpha/cachectin), Lipopolysaccharide (LPS), ACTH, beta-Endorphin (beta-EPH), and Cortisol (F) levels were determined in 33 Headache patients: 22 patients were affected with Migraine (M) and 11 patients with Chronic Type Tension Headache (CTTH). TNF-alpha/cachectin serum level was detected in 15 out of 22 migraneous patients and in no CTTH patients. Plasma LPS was observed in 11 out of 15 TNF-alpha/cachectin positive migraneous patients (73%) and in 3 out of 11 CTTH patients (27%). A negative correlation was observed between TNF-alpha/cachectin values and either ACTH or beta-EPH. In the group of migraneous patients the presence of serum TNF-alpha/cachectin showed a sensibility of .6 and a specificity of 1. The endocrine and immunological implications concerning these data are discussed.

Adrenocorticotropic Hormone↗
PubMed1986

Oral administration of antibiotics and intestinal flora associated endotoxin in mice.

The contribution of aerobic and anaerobic gram-negative intestinal bacteria to the release of endotoxin in the intestinal tract was investigated during oral administration of various nonabsorbable antimicrobial drugs to C3H/Law mice. The intestinal endotoxin release was studied by determination of the endotoxin concentration in faecal supernatants with the Limulus amebocyte lysate assay. Selective elimination of aerobic gram-negative bacteria by oral treatment with polymyxin, aztreonam or temocillin resulted in a reduction of the endotoxin concentration of faecal supernatants to 10% of the untreated control. Further decrease of the endotoxin concentration to 1% was achieved by total decontamination of the intestinal tract by oral cephalothin/neomycin treatment. Endotoxin determination with the Limulus amebocyte lysate assay appeared to be unaffected by the antibiotics present in the faecal supernatants after oral treatment. On basis of these experiments, it is concluded that in mice 90% of the faeces derived endotoxin can be ascribed to release of endotoxin by intestinal aerobic gram-negative bacteria.

Administration, Oral↗
PubMed1987

Endotoxin liberation from Neisseria meningitidis isolated from carriers and clinical cases.

Endotoxin liberation was studied in a blinded material of 121 Neisseria meningitidis isolates; from nasopharynx of 58 carriers and from cerebrospinal fluid or blood of 63 cases with meningococcal disease. Endotoxin activity in culture filtrates was determined by a Limulus lysate test. Meningococci isolated from clinical cases were significantly more frequently endotoxin-liberating (E+) (84.1%) than in carriers (25.9%); p less than 0.001. Serogroupable carrier isolates had a significantly higher frequency of E+ meningococci (61.9%) than non-groupable ones (5.4%); p less than 0.002. Serogroup B case isolates, which generally had a larger amount of capsular polysaccharide than B meningococci from carriers, had a significantly higher proportion of E+ meningococci than group B from carriers; p = 0.007. All 7 serogroup C isolates were E+ (5 cases and 2 carriers). No correlation was found between endotoxin liberation and the serotype: subtype 15:P1.16, tested by a selection of monoclonal antibodies, or between endotoxin liberation and sulfonamide resistance, when carrier and case isolates were studied separately. Meningococci isolated from cases had the following mean endotoxin titres: 320.5 in the meningitis group, 408.2 in the septicaemic group, 462.1 in the septicaemic and meningitis group, and 123.7 in the group with other systemic disease. E+ meningococci were isolated from 5/6 fatal cases. Thus, endotoxin liberation from meningococci is strongly, but not completely associated with establishment of meningococcal disease and with the presence of capsular polysaccharide.

Carrier State↗
PubMed1992

Delayed antibiotic-induced lysis of Escherichia coli in vitro is correlated with enhancement of LPS release.

A kinetic turbidimetric Limulus amebocyte lysate (LAL) assay was used to study the effects of gentamicin, amoxycillin and ciprofloxacin (16 x MIC) upon release of lipopolysaccharide at different stages of a growing Escherichia coli 055:B5:H culture in vitro. In this model a linear correlation was present between the logarithms of colony counts and free LAL activities. Untreated E. coli grew from log values of 4.9 +/- 0.15 (low inoculum) and 6.8 +/- 0.08 cfu/ml (high inoculum) at t = 0 to 8.9 +/- 0.05 and 9.1 +/- 0.13 cfu/ml at t = 6 h, respectively. The log values of basal free LAL activities at low and high inoculum sizes were 1.9 +/- 0.07 and 3.3 +/- 0.14 endotoxin units/ml, increasing 2100- and 69-fold, respectively during a 6-h growth. Amoxycillin-induced lysis was not significantly associated with an increase in free LAL activity. Efficacy of bacterial killing by gentamicin was high, but free LAL activity increased only 3.2- and 7.7-fold at the low and high inoculum experiments, respectively. Ciprofloxacin induced cell filamentation during the experiments. At low and high inoculum conditions exposure to ciprofloxacin induced a 43- and 68-fold increase in free LAL activities, respectively. Our data indicate that (a) LPS is released as long as E. coli remain structurally intact; (b) LPS release is enhanced when bacterial biomass increases; and (c) are taken as evidence against the concept of lysis-correlated LPS release.

Amoxicillin↗
PubMed1994

Toxicity of hemoglobin solutions: hemoglobin is a lipopolysaccharide (LPS) binding protein which enhances LPS biological activity.

Administration of alpha alpha-crosslinked stroma-free hemoglobin (SFH) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of SFH, we examined mixtures of SFH and E. coli LPS for evidence of LPS-SFH complex formation. LPS-SFH complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through 5% sucrose, which distinguished denser LPS alone from LPS-SFH complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of complexes compared to SFH alone. Interaction of LPS with SFH was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of cultured human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several LPS physical characteristics and enhancement of LPS biological activities.

Acute-Phase Proteins↗
PubMed1983

Sulfonamide resistance in Neisseria meningitidis strains liberating various amounts of free endotoxin.

The relationship between sulfonamide susceptibility and the ability to liberate endotoxin was studied in 50 strains of Neisseria meningitidis. The endotoxin activity was investigated by the Limulus lysate test in growing cultures of 25 sulfonamide-resistant and 25 sensitive strains. The sulfonamide-resistant strains tended to have a higher endotoxin activity in cultures grown for 6 h than sensitive ones, and had also a higher amount of free, filtrable endotoxin.

Anti-Bacterial Agents↗
PubMed2005

Determination of platelet-activating factor by reverse phase high-performance liquid chromatography and its application in viral hepatitis.

AIM: To detect the platelet-activating factor (PAF) and the plasma or serum levels of tumor necrosis factor-alpha (TNF-alpha) malondialdehyde (MDA), endotoxin (ET) and to discuss their significance in various types of viral hepatitis. METHODS: PAF, TNF-alpha, MDA, and ET levels in 60 controls, 16 cases of acute viral hepatitis, 71 cases of chronic viral hepatitis, 19 cases of severe viral hepatitis were detected by reverse phase high-performance liquid chromatography (rHPLC), bio-assay, ELISA, thiobarbituric acid (TBA), and limulus lysate test (LLT), respectively. RESULTS: The rHPLC was more sensitive and specific than bio-assay (r = 0.912, P<0.01). The plasma levels of PAF, TNF-alpha, MDA, and ET in patients with viral hepatitis were higher than those in controls (P<0.01). CONCLUSION: rHPLC is more reliable and accurate for the detection of PAF.

Adult↗
PubMed2000

Salmonella-type heptaacylated lipid A is inactive and acts as an antagonist of lipopolysaccharide action on human line cells.

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-alpha induction and NF-kappaB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulus gelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-alpha release and activated NF-kappaB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-alpha and NF-kappaB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-alpha induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-alpha release and NF-kappaB activation in THP-1 cells.

Acylation↗
PubMed1979

The effect of triethylenetetramine dihydrochloride on the in vivo susceptibility of Pseudomonas aeruginosa to gentamicin.

A chelating agent, triethylenetetramine dihydrochloride (TRIEN dihydrochloride) increased the efficacy of gentamicin in vivo against a clinical isolate of P. aeruginosa, designated Ps 15. Mice which were inoculated with 10 X LD50 of Ps 15 and treated with doses of 2 approximately 16 mg of gentamicin per kg per day all died. However, treatment with 8 mg of gentamicin per kg body weight per day plus 30 mg of Trien dihydrochloride per day markedly reduced the mortality. The combined therapy also reduced the number of viable organisms that accumulated in the kidney during a 24-hour period post inoculation. When a dosage level of 8 mg of gentamicin was exceeded in the combined treatment regimen, all of the infected mice died, and a high concentration of endotoxin could be detected in the mouse sera by the limulus assay.

Animals↗
PubMed1980

Pyrogenic reactions after inadvertent infusion of endotoxin during cardiac catheterizations.

In April 1976 the attack rate of chills and fever with or without falls in blood pressure increased in association with cardiac catheterizations. Fevers were associated with coronary angiography and right and left heart catheterizations. Blood cultures were negative, and reactions did not correlate with amounts of contrast materials infused or with procedures done by a single operator. Significant numbers of Acinetobacter calcoaceticus (var. anitratus) and a Pseudomonas species were cultured from hospital-reservoir distilled water when it was flushed through a catheter before gas sterilization. This same water after ethylene oxide sterilization contained 2 x 10(5) ng/mL of endotoxin by limulus lysate test and was positive by rabbit pyrogen test. When washed reusable cardiac catheters were sterilized daily or when disposable catheters were substituted, febrile reactions ended. Pyrogenic reactions in patients undergoing cardiac catheterizations correlated with emptying retained endotoxin with injected contrast material from reused washed-sterilized catheters.

Acinetobacter↗