The Limulus Coagulation Test for Endotoxin. A Comparison with Other Assay Methods

Rojas–Corona, R. R.; Skarnes, Robert C.; Tamakuma, Shoetsu; Fine, Jacob

doi:10.3181/00379727-132-34268

Cited by 90 publications

(41 citation statements)

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“…Where indicated, LPS concentration in medium or serum was determined using the kinetic colorimetric Limulus amebocyte assay (Charles River Laboratories) in pyrogen-free tubes, according to the manufacturer's instructions, and expressed as endotoxin units per ml (EU/ml) of fluid (39). For the purpose of the in vitro experiments, a concentration of 50 g/ml LPS was used, given that this corresponds to ϳ15-20 EU/ml as determined by Limulus assay, which is within the range of serum LPS that we measure in experimental NEC.…”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

Leaphart

Cavallo

Gribar

et al. 2007

The Journal of Immunology

417

507

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Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.

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Section: Cell Culture and Reagentsmentioning

confidence: 99%

A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

Leaphart

Cavallo

Gribar

et al. 2007

The Journal of Immunology

417

507

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“…During the last year we embarked on a new study involving bacterial for the detection of endotoxins (Levin and Bang, 1964;Rojas-Corona et al, 1969;Reinhold and Fine, 1971;Levin et al, 1970 Since the test is specific for bacterial lipopolysaccharide4 the bacterial biomass can easily be distinguished from the biomass of other organisms .…”

Section: Endotoxin Studiesmentioning

confidence: 99%

MARINE MICROBIOLOGY. Five-Year Progress Report, 1967--1972.

Watson¹

1972

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“…injection of LPS both by check of pyrogenicity and by the Limulus test, an in vitro specific assay for LPS (21,22 (25). Sodium, potassium and calcium were deter mined by an atomic absorption spectro photometer (Perkin Elmer, model 290) (26).…”

mentioning

confidence: 99%

“…Consequently in this report, the Limu/us test, an in vitro specific and highly sensitive assay for LPS (21,22), was utilized in addition to the pyrogen test. The results of the test was positive only in the CSF with drawn from the rabbits injected with a higher dose of LPS (500 /cg/kg, i.v.)…”

mentioning

confidence: 99%

Involvement of central action of lipopolysaccharide in pyrogen fever.

Ogawa

Kanoh

1984

Jpn.J.Pharmacol

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Abstract-To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoe bocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. co/i LPS at graded doses of 0, 1, 10 and 50 ,gig/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 ,ug/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 jig/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyper thermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

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The Limulus Coagulation Test for Endotoxin. A Comparison with Other Assay Methods

Cited by 90 publications

References 0 publications

A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

MARINE MICROBIOLOGY. Five-Year Progress Report, 1967--1972.

Involvement of central action of lipopolysaccharide in pyrogen fever.

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