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PubMed1984

Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.

DNA, Recombinant↗
PubMed2000

Determination of endotoxins in caries: association with pulpal pain.

AIM: The aims of this study were: (i) to determine the presence or absence of endotoxins in the superficial and deep layers of carious lesions of symptomatic and asymptomatic teeth with vital pulps; (ii) to quantify the amount of endotoxin present; and (iii) to associate the presence of endotoxins with the acute pulpal pain derived from the irreversible pulpitis. METHODOLOGY: Two specimens of carious dentine were taken under aseptic conditions from symptomatic teeth with irreversible pulpitis (n = 9) and asymptomatic teeth with reversible pulpitis (n = 11). The first specimen was taken from a layer of superficial caries and the second from a deeper one. Sound dentine was also collected from intact teeth without restoration and used as a noncarious control group (n = 4). During the patient sampling procedure an effort was made to collect an equal quantity of caries and sound dentine in all cases (approximately 6 mg). The extraction of endotoxins was performed using the Phenol-water method. The assay and quantitative determination of endotoxins was performed by the Quantitative Chromogenic test using Limulus Lysate. Data were analysed statistically using either independent or paired t-tests. RESULTS: The results indicated that endotoxins were present in the superficial and deep layers of caries of all symptomatic teeth with irreversible pulpitis (0.15078 and 0.12111 ng mL-1, respectively), with significantly greater amount (P < 0.01) in the superficial compared to the deep layer. Endotoxins were found in superficial and deep layer of caries of all asymptomatic teeth with reversible pulpitis (0.12091 and 0.07163 ng mL-1, respectively), with significantly greater amounts (P < 0.001) in the superficial compared to the deep layer. The results also demonstrated that significantly greater concentrations (P < 0.005) of endotoxins were present in the superficial carious layer of symptomatic compared with asymptomatic teeth (0.15078 and 0.12091 ng mL-1, respectively). Likewise, significantly greater amounts (P < 0.001) of endotoxins were present in the deep carious layer of symptomatic compared with asymptomatic teeth (0.12111 and 0.07163 ng mL-1, respectively). In sound dentine no endotoxins were detected. CONCLUSIONS: This study demonstrates that endotoxins are present in carious lesions of symptomatic and asymptomatic teeth. The amount of endotoxin was significantly greater in the superficial compared to the deep layer of carious dentine. More endotoxins are present in caries of painful teeth compared with those without symptoms.

Adolescent↗
PubMedDate not supplied

A novel method for the continuous measurement of endotoxin concentration.

Conventional limulus amebocyte lysate tests involved procedures to prevent contamination by atmospheric endotoxins. To address this problem, the authors have proposed a technique in which the sampling, reagent mixing, and reaction steps are carried out consecutively in a single tube. Since reagents do not come in contact with the atmosphere, the new technique promises stable determination of the concentration of endotoxins in dialysate fluid. An aqueous solution of endotoxin simulating dialysate fluid was sampled in a silicone rubber tube from a sterile infusion bag, then mixed with an indicator in the same tube. After reaction at 310 K, measurements were made of light absorbance at 405 nm and its linearity with endotoxin concentration was determined. Results showed a high degree of linearity (correlation coefficient of not less than 0.99) at endotoxin concentrations of 0-15 pg/ml. The time for the reaction was shortened to 12 min, in which case the response time was 15 min. It is suggested that this new test for determining endotoxin concentration using limulus amebocyte lysate reagent, in which all three steps--sampling, mixing, and reaction--proceed continuously in a single tube, offers higher reliability, greater ease of operation, and shorter response time than conventional tests.

Calibration↗
PubMed1983

An improved chromogenic substrate endotoxin assay for clinical use.

An improved quantitative assay for endotoxin in plasma was developed after evaluating three different chromogenic substrates and seven methods for removal of plasma inhibitors. Optimal storage conditions for plasma samples prior to assay were also determined. Using chromogenic substrate S2423 with plasma diluted 1/10 in water and heated to 75 degrees C for 5 min to remove inhibitors, a within-batch coefficient of variation of 4% was obtained at levels of endotoxin likely to be encountered clinically. The limit of assay sensitivity was less than 10 pg/ml. This assay provides a sensitive quantitative test for single episodes of endotoxaemia in individual patients but variable activation of the Limulus proenzyme by endotoxin from different bacterial strains limits quantitative comparisons between patients.

Blood Preservation↗
PubMed1984

Bacterial endotoxins in umbilical cord blood of neonates.

We tested umbilical cord blood from 255 infants for evidence of bacterial endotoxins. Using a Limulus lysate gelation technique, endotoxin-like activity (ELA) was detected in 13 (9.1%) of 142 term infants and in 23 (20.3%) of 113 preterm infants (p less than 0.05), in concentrations ranging from 30 to 3,000 pg Escherichia coli equivalent activity per milliliter of plasma. No factors predisposing to endotoxemia could be detected in term pregnancies but among ELA+ premature infants, placental cultures more frequently revealed gram-negative species (p less than 0.05). Cord blood endotoxemia in the concentrations observed was without obvious consequence in mothers and term infants. Our data confirm the existence of natal endotoxemia, a potential mechanism of perinatal injury.

Endotoxins↗
PubMedDate not supplied

[Effects of endotoxin of gram-negative intestinal microflora on adaptation to physical exercise].

The study of plasm concentration of endotoxin (ET), activity of antiendotoxinic immunity (AEI) and early adaptation to exercise in 61 athletes and 13 students who were not athletes has revealed correlation between the former two and the latter parameters. ET was elevated in all the examinees prior to exercise. In exercise maladaptation both athletes and non-athletes demonstrated high ET levels in blood plasm and low AEI. It is suggested that ET may have a negative effect on exercise adaptation. Elevation of lipopolysaccharide concentration in systemic blood flow and depressed AEI may provoke acute maladaptation after exercise.

Adaptation, Physiological↗
PubMed1993

The bacterial endotoxin test in the PET facility.

A method by which the gel-clot Limulus amebocyte lysate test may be performed in 20 rather than 60 min with sufficient sensitivity to satisfy the needs of the nuclear medicine or positron emission tomography laboratories has been developed and validated for use as a substitute for the Bacterial Endotoxin Test described in the United States Pharmacopeia, 22nd revision. Using this method, results may be obtained from the test prior to the human administration of radiopharmaceuticals without extensive loss of activity and with increased safety when compared to tests performed after administration. Additionally, studies on the shelf-lives of the reagents used in the test were conducted. When refrigerated between use, control standard endotoxin dilutions of 5 EU/ml or greater may be used for at least 1 mo after preparation and reconstituted lysate retains its labeled sensitivity for at least 10 days, considerably longer than the manufacturer's stated shelf-lives.

Ammonia↗
PubMedDate not supplied

Resolving liposomal inhibition of quantitative LAL methods.

Bacterial endotoxin is composed of Lipid A as well as variable polysaccharide regions, and is capable of eliciting a pyrogenic response in vivo (1). NeXstar has developed a quantitative LAL assay at maximum sensitivity to provide assurance that its products are well below the endotoxin pyrogenic threshold. This test uses rigorous treatment of 1.0 mL liposome samples with sodium dodecyl sulfate, an anionic surfactant, or CHAPS, a zwitterionic surfactant to disrupt the lipids present in the liposome sample including endotoxin. This liposome disruption allows potentially undetectable endotoxin to become available to the LAL cascade for quantification and overcomes inhibition of the LAL test by liposomes.

Endotoxins↗
PubMed1992

Mandatory biological testing of Spictra Cardiotomy Reservoir.

Disposable rigid Spictra Cardiotomy Reservoir is one of the units used in the extracorporeal techniques during open-heart surgery. It was designed, developed, and tested by different groups of our Institute as an indigenous product to offset the prohibitive cost of the imported one. The finished device was subjected to mandatory biological tests, such as pyrogen (in vivo rabbits and in vitro Limulus Amebocyte Lysate test) and sterility tests. It can be concluded from the case study that the device deemed to have passed the test for apyrogenicity and sterility.

Animals↗
PubMed2002

Comparison and validation of novel pyrogen tests based on the human fever reaction.

The absence of pyrogens (fever-inducing contamination) in injectable drugs is a crucial safety control because pyrogens can pose a life-threatening risk to the patient. The field of applications for pyrogen-testing is expanding and becoming more diverse due to innovative high-tech products such as medical devices, cellular therapies and species-specific agents (e.g. recombinant proteins). The overall aim of the consortium is to develop, evaluate and validate a method based on the human fever reaction to replace the rabbit pyrogen test and the Limulus assay. The network brings together the six most prominent test systems developed recently in Europe for trans-national comparison and subsequent validation of the most promising models. This pre-competitive development will initiate further applications and exploitations for new fields of pyrogen testing such as cellular therapies, medical devices and pyrogen pollution control in the work place. A method for introduction into the European Pharmacopoeia will be developed as a replacement for the rabbit pyrogen test for end-product control.

Animal Use Alternatives↗
PubMed1989

Particles and molecules in endodontic leakage.

It is assumed that above a critical level, microleakage causes periapical disease and jeopardizes periapical tissue repair. The purpose of this investigation was first to find out to what degree obturation prevents leakage of bacteria-sized particles or large protein molecules, and second if leakage of the commonly used dye methylene blue is comparable with that of a small bacterial metabolic product of similar molecular size. Sections, 9 mm long, obtained from 46 extracted human incisor roots had their root canals prepared in a standardized manner and obturated in four experimental ways using gutta-percha. The root sections were mounted in the middle of tubes that were closed at both ends with rubber membrane stoppers, which permitted sampling from the apical reservoir. The coronal reservoir was filled with a solution of 1 mg/ml latex beads, 4 micrograms/ml endotoxin, 0.5 per cent butyric acid, 0.1 per cent valeric acid and 0.1 per cent methylene blue water. After 1 and 2 weeks, samples were taken from the apical reservoir and tested for the presence of latex beads by phase-contrast microscopy, for endotoxin with a limulus lysate test, for butyric acid with gas-chromatography and for methylene blue with spectrophotometric analysis. Leakage of bacteria-sized particles and large-sized protein molecules could be prevented only when both sealer and pressure were used in obturating root canals with gutta-percha. Leakage of butyric acid proved to be comparable with leakage of methylene blue; microleakage of these small molecules could not be prevented in this study whatever method of filling was used.

Dental Leakage↗
PubMed2002

Comparison and validation of novel pyrogen tests based on the human fever reaction.

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests, based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.

Animal Testing Alternatives↗
PubMed1977

Immunogenicity in weanling rabbits of a polyribophosphate complex from Haemophilus influenzae type b.

Polyribophosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, is more effectively immunogenic when it is associated with the bacterium than when it is in the purified form that is being tested as a vaccine for humans. In an effort to analyze this difference, we isolated from H. influenzae type b a high-molecular-weight, soluble complex, in which PRP appears to be combined with protein (about 7% protein). The pyrogenicity and limulus lysate gelation activity of the complex suggest that a small amount of lipopolysaccharide also is present. The protein was resolved into five polypeptides by electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate. In weanling rabbits, which do not respond to purified PRP, the complex induces high titers of antibody of PRP, in an anamnestic pattern. Bactericidal antibody to other bacterial components was also elicited. Equilibrium density gradient centrifugation of the complex indicated that most of the immunogenicity of PRP resides in the least dense fractions, which are high in protein, low in polysaccharide, and active in the limulus lysate test; denser fractions that react strongly with limulus lysate but are poor in protein were much less immunogenic.

Animals↗