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PubMed2001

Detection of anti-lipopolysaccharide antibodies to Vibrio cholerae O1 and O139 using a novel microtiter limulus amebocyte lysate (LAL) assay.

This paper describes a new assay for measuring antibodies to lipopolysaccharide (LPS) of Vibrio cholerae using blocking of the limulus amebocyte lysate (LAL) reaction in a microtiter plate. When V. cholerae LPS was coated onto a microtiter plate, and then LAL reagent was added, a typical gel reaction occurred. However, when the LPS-coated plates were first incubated with serum containing anti-cholera antibodies, the gel reaction did not occur. Blocking of the gel reaction was serotype specific, in that anti-O1 serum blocked the O1 LAL reaction but not the O139 LAL reaction, and anti-O139 serum neutralized the O139 reaction but not the O1 reaction. Preliminary data suggested that the LAL titers were comparable to the vibriocidal titers but that titers using the LAL assay may have been slightly higher. This study showed that antibodies to V. cholerae blocked the LAL reaction and suggested that the LAL blocking assay could be applicable for determining antibodies to other endotoxin-containing bacteria and microorganisms.

Animals↗
PubMed1986

Endotoxin levels in immunocompromised children with fever.

Febrile episodes for which no cause can be found are common in immunocompromised children. We postulated that circulating endotoxin, a known pyrogen, might be responsible for some of these episodes in the absence of documented infection. Plasma endotoxin levels were assayed using a recently developed Limulus amebocyte lysate assay enhanced in sensitivity and objectivity by the addition of a chromogenic substrate. Eighty-seven plasma endotoxin determinations were made in 36 immunocompromised children with fever. Convalescent endotoxin levels and levels in normal children were also obtained. It was concluded that a plasma endotoxin level of 35 pg (0.10 EU)/ml constitutes the upper limit of normal in children. Five children (14%) had elevated endotoxin levels in the course of the febrile episodes, in the absence of bacteremia or clinically diagnosed infection. In each case, the levels returned to normal during convalescence. It is concluded that endotoxemia is a possible cause or contributing cause of unexplained fever in immunocompromised children.

Adolescent↗
PubMed1983

The effect of ischemia of the dog's colon on transmural migration of bacteria and endotoxin.

The purpose of this investigation was to evaluate the effect of temporary ischemia created during the surgical preparation of the colon for resection on transmural migration of bacteria and passage of endotoxin through the ischemic wall of the canine colon. Eighteen dogs were used: in fourteen the colon was devascularized by ligating all marginal vessels. Aerobic and anaerobic cultures and washings for endotoxin assay were obtained from the surface of the bowel at intervals up to 6 hr after creating the ischemia. Peripheral and portal blood samples were also obtained at equal intervals for bacteriologic cultures and endotoxin assay. Bacterial transmural migration was examined in 10 dogs. In 6 of the dogs biologically marked bacteria were introduced into the colon via a rectal catheter before producing the ischemia. In 4 dogs radioactively labeled endotoxin was introduced into the colon in a similar fashion. In all the dogs, surface cultures, both routine and specific for the marked bacteria, were negative for the whole period of up to 6 hr after creation of ischemia. All portal and peripheral vein cultures were also negative. Transmural migration of endotoxin was investigated in 8 dogs, 4 of which served as controls and underwent a sham operation. In the 4 dogs in which the colon was devascularized endotoxin was discovered in peritoneal washings, and in portal and systemic blood samples, as early as 30 min after the preparation of the bowel was completed. In the control dogs endotoxin assays were negative throughout the experiment. In this model up to 6 hr of ischemia did not result in migration of bacteria through the ischemic wall. Endotoxin, however, entered the peritoneum and the blood very soon after producing the ischemia.

Animals↗
PubMed1999

Does steroid pretreatment increase endotoxin release during clinical cardiopulmonary bypass?

OBJECTIVE: The mechanism involved in the endotoxemia frequently recognized during cardiopulmonary bypass remains unclear. It has also been suggested that endotoxin levels were higher in steroid-pretreated patients undergoing cardiopulmonary bypass. METHODS: Twenty patients undergoing cardiopulmonary bypass were randomly pretreated with steroids (methylprednisolone, 30 mg/kg) or placebo. Blood samples for endotoxin measurement were drawn simultaneously from the superior and inferior venae cavae before heparin administration, 5 and 50 minutes after the onset of bypass, 5 minutes after aortic declamping, at the end of bypass, and 1, 2, and 20 hours after the end of cardiopulmonary bypass. RESULTS: The perioperative variables in the two groups were similar. Blood endotoxin levels were higher in the inferior vena cava than in the superior vena cava immediately after the onset of bypass. Endotoxin levels in inferior vena cava blood were significantly lower in steroid-pretreated patients than those in patients not receiving steroids. CONCLUSIONS: Endotoxin is released during cardiopulmonary bypass from the region drained by the inferior vena cava. Steroid pretreatment may actually reduce endotoxin release during bypass.

Aged↗
PubMed2000

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation.

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.

Animals↗
PubMed1995

Induction of acute pancreatitis in germ-free rats: evidence of a primary role for tumor necrosis factor-alpha.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of the systemic manifestations associated with acute pancreatitis. The purpose of this study was to show that TNF-alpha expression in pancreatitis is a primary response and is not the result of endotoxemia. METHODS: Severe acute pancreatitis was induced in germ-free rats, which have no source of endogenous endotoxin, by ductal infusion of artificial bile. Control animals underwent sham operation and ductal infusion of saline solution. TNF-alpha levels were measured by the WEHI bioassay. Endotoxin was measured by the Limulus assay. RESULTS: TNF-alpha levels remained low in the sham group (mean, 24.6 +/- 8.0 pg/ml) but were significantly elevated in normal rats with pancreatitis (181 +/- 26.8 pg/ml; p < 0.001 versus sham group) and in germ-free rats with pancreatitis (213 +/- 90 pg/ml; p < 0.002 versus sham group). No endotoxin was detected in any of the experimental rats. CONCLUSIONS: Our results indicate that TNF-alpha levels are elevated in acute pancreatitis despite the absence of endotoxin, indicating a primary role of TNF-alpha in this disease.

Acute Disease↗
PubMed1989

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis.

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.

Animals↗
PubMed1991

Modeling bacterial damage to pulpal cells in vitro.

There is increasing evidence that access to patent dentinal tubules by bacteria and their products rather than trauma from restorative materials is responsible for subsequent pulpitides. The purpose of this study was to compare the relative cytotoxicity of centrifugal fractions of two bacteria, Fusobacterium nucleatum and Treponema denticola, on L929 cells in monolayer cultures and in the "in vitro pulp chamber." Neutrophilic chemotaxis assays and Limmulus assays were performed to verify biological activity of the various fractions of these bacteria. It was found that T. denticola inhibits new protein synthesis in cultured cells to a much greater extent than F. nucleatum, but that only F. nucleatum fractions are chemoattractive for human neutrophils in the absence of serum. While the chemical nature and molecular weights of the "toxic" materials were not determined, it appeared that eukaryotic protein synthesis inhibition caused by the T. denticola pellet fraction in the in vitro pulp chamber was at least 1000 times less than that caused by the same concentrations in monolayer cultures.

Cells, Cultured↗
PubMed1989

Cytotoxicity against various cell lines of lipopolysaccharides purified from Bacteroides, Fusobacterium, and Veillonella isolated from infected root canals.

The cytotoxicity against two mesenchymal cell lines, L-929 and WI-38, and two epithelial cell lines, KB and HeLa S-3, of lipopolysaccharide (LPS) purified from strains of Bacteroides gingivalis, Fusobacterium nucleatum, and Veillonella parvula isolated from infected root canals was investigated. The inhibition of cell growth by these LPS's was considerable for mesenchymal cell lines, but mild for epithelial cell lines. The cytotoxic effect of F. nucleatum LPS was the greatest and that of B. gingivalis LPS was the least.

Bacteroides↗
PubMed1975

Pyrogenic reactions during haemodialysis caused by extramural endotoxin.

Between July 24 and Aug. 19, 1974, an outbreak of pyrogenic reactions occurred in patients at a private haemodialysis centre in a suburb of Washington, D.C. 49 reactions characterised by chills, fever, and hypotension occurred in twenty-three of the seventy patients dialysed during this period. No infections could be documented in any of the affected individuals. Despite the fact that only low levels of gram-negative bacterial contamination of the haemodialysis system were found, high levels of endotoxin contamination of dialysis fluid and endotoxaemia in patients experiencing overt reactions were recorded using the Limulus lysate test. The cause of these reactions was traced to an increase in endotoxin contamination of the tap water used to prepare dialysate, possibly caused by an increase in the algae levels in the local water source. The installation of a reverse osmosis system for water treatment may be a solution to the problem of endotoxin contamination of water used to prepare dialysis fluid.

Adult↗