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PubMed1999

[Further development of a cell culture model for the detection of bacterial pyrogens]

In many cases the limulus amoebocyte lysate assay (LAL) is now accepted to detect pyrogenic contaminations in parenteralia instead of the rabbit pyrogenicity test. As the LAL test is able to detect only compounds of gramnegative bacteria (endotoxins) and as it is difficult to test samples with high amounts of protein, we have now further developed our previously established in vitro assay for the detection of bacterial pyrogens in order to overcome the shortcomings of the LAL assay. Our test system is based on the stimulation of IFN-gamma-primed human (THP-1) and murine (J774A.1 and RAW 264.7) monocytic cell lines by bacterial pyrogens to form neopterin or nitrite, respectively. THP-1 in serum-containing media can be used as a high sensitivity assay, while RAW 264.7 in serum-free media is a very good screening system. We showed that the cell-free supernatants of various S. aureus cultures could not be assessed by the LAL assay while the here presented cell culture model detected them very sensitively. The stimulation of immune cells seems to be caused by substances of various molecular masses. In conclusion, we suggest to reduce or even replace the rabbit pyrogenicity test by using our cell culture model in addition to the LAL assay.

Journal Article↗
PubMed1994

Measurement of interleukin-6 production by monocytes for in vitro safety testing of hemoglobin solutions.

Induction of interleukin-6 (IL-6) production by isolated human mononuclear blood cells was taken as in vitro model for the induction of inflammatory reactions. The model was very sensitive to bacterial endotoxin (detection limit < 10pg/ml). Hemoglobin (Hb) solutions, prepared under non-sterile conditions also induced IL-6 production, which correlated with a positive reaction in the Limulus assay. Purification of the Hb solutions with a detergent prevented IL-6 production, showing that pure Hb itself does not activate the monocytes. We conclude that this assay is a useful and sensitive test of contamination with components that can induce inflammatory reactions, especially microbial products.

Acute-Phase Reaction↗
PubMed1984

Lectin grouping of Bacillus thuringiensis serovars.

Twelve lectins, 11 of plant and 1 of animal origin, were tested against the 28 serovars of Bacillus thuringiensis to study their agglutinating specificities. Except for the sialic specific lectin from Limulus polyphemus, tube agglutination assays were performed using lectin concentrations of 10, 50, and 100 micrograms against 0.2 mL of 10(9) cells/mL in a final volume of 1.0 mL. The agglutination studies with the Limulus lectin were performed using 10 and 50 micrograms of lectin. Tubes were incubated overnight at room temperature (25 degrees C) and observed for agglutination patterns. Ten of the 28 serovars were individualized according to their ability to bind with various lectins. The study shows that the various serovars have different carbohydrate residues which indicates that the O-somatic antigens differ.

Agglutination↗
PubMed1992

Reactivity of monoclonal antibody E5 with endotoxin. II. Binding to short- and long-chain smooth lipopolysaccharides.

The murine monoclonal IgM antibody E5 has been shown to significantly reduce the mortality and morbidity of patients with Gram-negative sepsis in a multicenter randomized placebo-controlled clinical trial. The in vitro binding characteristics of monoclonal antibody (mAb) E5 were studied using highly purified smooth lipopolysaccharide (LPS) isolated from a variety of clinically relevant, wild-type Gram-negative bacteria. Using a sensitive antibody-capture assay which involves immobilized mAb E5 and a chromogenic Limulus amebocyte lysate (LAL) LPS-detection system, mAb E5 was shown to bind to all 15 smooth LPS preparations tested, including LPS isolated from Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia and Yersinia species. When LPS was fractionated according to size by size-exclusion chromatography, mAb E5 was shown to bind to smooth LPS molecules that have long as well as short O-polysaccharide chains. These results confirm and extend those reported previously and demonstrate that the anti-lipid A mAb E5 binds specifically to a diverse spectrum of smooth LPS isolated from wild-type Gram-negative bacteria.

Animals↗
PubMed2002

Evaluation of endotoxin release and cytokine production induced by antibiotics in patients with Gram-negative nosocomial pneumonia.

OBJECTIVE: To determine the plasma concentrations of lipopolysaccharide, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 in a homogeneous group of septic patients and to evaluate the effect of antibiotic treatment, imipenem or ceftazidime, on the release of lipopolysaccharide and cytokines. DESIGN: Prospective, randomized study. SETTING: Sixteen-bed multidisciplinary intensive care unit. PATIENTS: Twenty-four septic patients with documented Gram-negative nosocomial pneumonia. Controls were 20 patients admitted without sepsis and 20 healthy volunteers. INTERVENTIONS: Septic patients were randomized between imipenem and ceftazidime. Blood samples were collected before (0 hrs) and after (4 and 12 hrs) antibiotic treatment. Concentrations of lipopolysaccharide were measured by using the limulus assay, and cytokine concentrations were measured by enzyme-linked immunosorbent assay. Statistical analyses were performed by Kruskal-Wallis test, Mann-Whitney U test, and Student's t-test. MEASUREMENTS AND MAIN RESULTS: The mean age was 48.5 +/- 19.5. The mean Acute Physiology and Chronic Health Evaluation II score was 18.4 +/- 4.5. Overall mortality rate was 45.4%. All septic patients showed significant higher concentrations of lipopolysaccharide (p <.001), tumor necrosis factor-alpha (p <.04), and interleukin-6 (p <.001) than the controls, but interleukin-1 beta was never detected. We did not find statistically significant changes in lipopolysaccharide or cytokine plasma concentrations over time within any of the two arms of the study (ceftazidime vs. imipenem). There were no statistically significant differences in lipopolysaccharide and interleukin-6 plasma concentrations between the two antibiotic treatments. Although tumor necrosis factor-alpha plasma concentrations were significantly higher in the group treated with ceftazidime compared with the group treated with imipenem at the baseline and 4 hrs later, these differences were not statistically significant after 12 hrs of initiation of both treatments. CONCLUSIONS: Patients with Gram-negative nosocomial pneumonia have high plasma concentrations of lipopolysaccharide, interleukin-6, and tumor necrosis factor-alpha, but the antibiotic therapy evaluated did not significantly modify these concentrations.

Anti-Bacterial Agents↗
PubMed1982

The specificity of Centruroides sculpturatus Ewing (Arizona lethal scorpion) hemolymph agglutinins.

C. sculpturatus sera agglutinate human erythrocytes independently of the ABO blood group, enzyme treatment, incubation temperature or sex of the scorpions. Tested with human lymphocytes and reptile and bird erythrocytes, C. sculpturatus serum reacts like an anti-sialic acid agglutinin. With leukemic lymphocytes, titers are higher than with normal lymphocytes. Mammalian erythrocytes show characteristic agglutination patterns for C. sculpturatus for Limulus polyphemus (horseshoe crab) that suggest different receptors for agglutinins of both species. Cross absorption and elution experiments indicate the presence of at least two specific agglutinins in C. sculpturatus serum. Agglutination is inhibited by N-acetylneuraminic acid and N-glycolyneuraminic acid, for all erythrocytes tested. Calcium is required for optimal activity of C. sculpturatus agglutinins. C. sculpturatus agglutinating activity is destroyed at 65% degrees C for 20 minutes. Titers are decreased by 2-mercaptoethanol, and more so after alkylation with iodoacetic acid suggesting that disulfide bonds are present in C. sculpturatus agglutinin molecules.

ABO Blood-Group System↗
PubMed1999

Effect of time post mortem on the concentration of endotoxin in rat organs: implications for sudden infant death syndrome (SIDS).

The aim of the study was to test the following hypotheses: (i) that endotoxin injected 40 min prior to death can be detected in rat organs post mortem and (ii) that endotoxin levels do not change with increasing time post mortem. Rats were injected with or without endotoxin in buffered saline, 40 min prior to being killed. Endotoxin levels in rat organs were assessed using a Limulus amoebocyte assay. The effect of storage time post mortem was assessed by following various storage regimes at 25 degrees C and 8 degrees C. Significant differences (P = < 0.001) in endotoxin levels of all samples tested were found between rats injected with and without endotoxin. A significant increase in detectable endotoxin was observed between 0 h and 6 h post mortem in rats injected with or without endotoxin. No difference in detectable endotoxin levels in the kidney, liver and spleen was observed from 30 h to 102 h post mortem in rats injected with or without endotoxin. In rats injected with endotoxin, detectable endotoxin levels in the heart were raised between 0 h and 6 h, 6 h and 54 h, and 30 h and 78 h. Endotoxin injected into rats 40 min prior to death can be detected post mortem. For rats injected with saline or endotoxin prior to death levels in the kidney, liver and spleen were not affected by storage at 8 degrees C for 30-102 h, after initial storage at room temperature for 6 h. Levels of endotoxin detected in the hearts of rats injected with saline were not affected by storage up to 102 h. In rats injected with endotoxin prior to death, detectable levels in the heart were significantly affected by increasing time in storage.

Animals↗
PubMed1988

Biophysical evidence that light adaptation in Limulus photoreceptors is due to a negative feedback.

The steady-state stimulus-response curve of the Limulus ventral photoreceptor comprises a linear portion followed by a less-than-unity power law dependence, which is maintained over at least 4 decades of intensity. This progressive desensitization corresponds to light adaptation. For flash stimulation of dark-adapted cells, the stimulus-response curve again has an initial linear portion, but this is followed by a region of supralinearity before the curve saturates. In a previous article, we showed that the distribution of time integrals of the single-photon responses is consistent with a model of a single chain of first-order reactions. Starting with such a model, we have looked at relevant elementary nonlinear biochemical mechanisms to determine which of them can modulate the enzymatic amplifications of the chain in such a way as to lead to these behaviors. We assume that each of the two phenomena, adaptation and supralinearity, derives from a single mechanism that acts on a single enzymatic stage. We then conclude that the adaptation must be a cooperative negative feedback, in which an accessory material activated by a late stage of the transduction chain acts cooperatively to inhibit an earlier enzymatic amplification. In Limulus, the number of molecules that cooperate is between 3 and 5. We were not able to discard any of the mechanisms tested for the supralinearity, except to say that they must act at a stage of the chain later than that on which the adaptive material acts. If we assume the conclusions of a previous work which shows that the supralinearity mechanism is active during the steady state, we can also conclude that the supralinearity stage must precede the stage that is the source of the adaptive material.

Adaptation, Physiological↗
PubMed1985

Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

Animals↗
PubMed1975

The early diagnosis of gram negative septicemia in the pediatric surgical patient.

Ninety-three postoperative patients 1 day to 13 years of age had blood cultures, limulus lysate assay, determination of fibrin degradation products, white blood cell and platelet counts. Seven groups were studied. The limulus lysate assay was often positive (64%) in the presence of gram negative septicemia but there were false positives and negatives. The tests for fibrin degradation products were inconsistent. The white blood cell count was low in babies with gram negative septicemia. One hundred per cent of the infants with gram negative septicemia had a platelet count below 150,000; 71% below 100,000 (average 67,000 septic babies, 257,000 non-septic babies). The drop in platelet count with gram negative septicemia was abrupt---as much as 222,000 in 24 hours. Platelets increased when therapy was effective. Two children with gram negative septicemia had platelet counts of 50,000 and 20,000. The platelet count for patients with gram positive septicemia was 299,000, and above 150,000 in all children with ruptured and non-ruptured appendicitis and major surgery without gram negative septicemia. It was concluded that serial measurements of platelet count in the postoperative infant and child was a rapid and reliable method for early detection of gram negative septicemia and changes in platelet count in response to treatment was an indicator of the effectiveness of therapy.

Abdominal Muscles↗
PubMed1989

Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins.

Infection of polymorphonuclear leukocytes (PMNL) with influenza virus causes depression of PMNL metabolic and bactericidal activities. The studies reported here were undertaken to determine whether the hemagglutinin (HA) glycoprotein of influenza virus mediates this depression. PMNL were incubated with purified HA and the oxidative responses to exogenous stimuli were measured. The results indicate that HA, in either liposomes or protein aggregates referred to as rosettes, depressed PMNL oxidative responses. Depression was observed within 2 min of initial interaction of HA with PMNL and lasted more than 2 h. The membrane fusion activity of HA requires proteolytic cleavage of the HA, whereas the receptor binding activity does not. There was no difference in the ability of virions with cleaved or uncleaved HA to depress PMNL responses suggesting that the fusion event is not required for PMNL dysfunction. Inasmuch as the HA glycoprotein binds to sialic acid-containing receptors on the surface of the PMNL, we tested whether other sialic acid-specific binding proteins can mediate the reduction of PMNL responses. Sialic acid-specific lectins from Limulus polyphemus or Limax flavus were incubated with PMNL before measuring their responses to secondary stimulus. Depression was observed upon incubation with the lectins similar to that seen upon incubation with the HA or influenza virus. These results suggest that attachment of influenza virus to sialic acid-containing receptors is responsible at least in part, for suppressing PMNL oxidative responses.

Animals↗
PubMed2003

[Biological activity of lipopolysaccharides from clinical Bacteroides fragilis strains isolated in Poland determined in reaction with limulus amoebocyte lysate].

The aim of this study was to determine a biological activity of lipopolysaccharides (LPS) from clinical Bacterioides fragilis strains isolated in Poland by means of quantitative, photometric BET (LAL) method with Limulus polyphemus amoebocyte lysate and chromogenic substrate S-2423. Lipopolysaccharides were extracted from nine clinical B. fragilis strains by the procedure of Westphal and Jann (1965). Crude LPS preparations were purified with ultracentrifugation. Biological activities of bacterial endotoxins were determined by quantitative BET method with chromogenic substrate S-2423 (ENDOCHROME kit). Tests were performed according to the recommendations of the producer (Charles River Endosafe Ltd., USA). E. coli O55:B5 LPS and LPS preparations from reference B. fragilis strains were applied to compare the results of examinations. Activities of endotoxins from clinical B. fragilis strains isolated in Poland determined in reaction with Limulus amoebocyte lysate were differentiated. Among endotoxins of clinical B. fragilis strains the most active was the preparation from strain cultured in the case of pancreatic ulcer (B. fragilis 80/81 LPS). Lipopolysaccharides of examined B. fragilis strains were less active in BET test than E. coli O55:B5 LPS.

Bacteroides fragilis↗
PubMed1985

Calcium ion, an intracellular messenger of light adaptation, also participates in excitation of Limulus photoreceptors.

Photoreceptor cells of Limulus ventral eyes were bathed in artificial sea water (ASW) that contained 10 mM-EGTA and no added Ca2+ (EGTA-ASW). Test flashes elicited responses that increased to a maximum size within 10 min in EGTA-ASW but did not change further when dark-adapted cells were bathed for an additional 35 min in this solution. Light responses progressively declined from this maximum size if the cells were repetitively illuminated in EGTA-ASW. In this state of reduced responsiveness, response amplitudes were further reduced by intracellular ionophoretic injection of EGTA; response amplitudes were increased by intracellular ionophoretic injection of Ca2+. Both of these findings are opposite to what is normally observed for cells bathed in ASW. Also, after repetitive illumination in EGTA-ASW, both the slope of the response versus intensity relationship became steeper and light responses often had a delayed increase in amplitude. The light responses and the response versus intensity relation returned to normal when the bathing medium was changed back to ASW containing 10 mM-Ca2+. The light-induced rise in luminescence recorded from photoreceptors injected with the photoprotein aequorin (the 'aequorin response') declined by at most 50% after dark-adapted photoreceptors were bathed in EGTA-ASW for 45 min. However, the aequorin response progressively declined by 98% if cells were repetitively illuminated while bathed in EGTA-ASW. The total intracellular Ca content of whole end-organs was measured by atomic absorption spectroscopy. Total intracellular Ca content did not change significantly while photoreceptors were bathed in EGTA-ASW even after repetitive illumination. We suggest that cytosolic Ca2+ is required by one or more steps in the mechanisms that link rhodopsin isomerization to both (i) an increase in the conductance of the cell membrane to Na+ and (ii) a release of Ca2+ from a light-labile store.

Action Potentials↗
PubMed2005

Surface sampling for endotoxin assessment using electrostatic wiping cloths.

OBJECTIVES: Much of the cost of exposure assessment for studies of residential cohorts is in scheduling and travel time for field staff. One way to reduce costs is to simplify methods such that subjects can sample their own residence. Analysis of settled dust is being widely used for assessment of exposures to allergens, lead and pesticides and can also be used for endotoxins. While vacuum sampling is the most common surface sampling method, wipe sampling has the advantage that it can be readily performed by the resident when convenient and samples can then be mailed to researchers. Thus, we evaluated the feasibility of wipe sampling for endotoxin environmental assessment using electrostatic wipes with or without the use of disposable examination gloves. METHODS: Multiple lots of six types of commercial wipes and eight types of gloves were extracted and analyzed for endotoxin content using the kinetic chromogenic Limulus amebocyte lysate assay. Wipes were compared across brands, between lots, within lots, between pairs depending on proximity to cardboard packaging, and in wipe tests with or without gloves. Collected dust samples of known concentration were also tested in spiking assays for endotoxin recovery. RESULTS: The most striking finding was the high variability of endotoxin contamination of both wipes and gloves across brands and between various lots. The content of endotoxin in unused gloves ranged from <1.5 to 5810 endotoxin units (EU). The range for unused wipes was 3.6-87.8 EU. Surfaces of equal loading and area were sampled using three types of cloths that had low initial endotoxin contamination. The cloths were very good at collecting dust and endotoxin could be assayed from aqueous extracts of the wipes. Samples collected using cloths with bare washed hands yielded higher endotoxin loading per mass of collected dust versus samples collected wearing endotoxin-free gloves. This demonstrated additional endotoxin loading from the subject's hand. CONCLUSION: This study shows that wipe sampling while wearing medical gloves can be an effective method for collecting and assessing endotoxin on surfaces, so long as each lot of wipes and gloves have been tested and determined to be low in endotoxin.

Air Pollutants↗
PubMed1991

Endotoxin-neutralizing properties of the 25 kD N-terminal fragment and a newly isolated 30 kD C-terminal fragment of the 55-60 kD bactericidal/permeability-increasing protein of human neutrophils.

The bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes (PMN) is a potent cytotoxin, specific for Gram-negative bacteria, that also inhibits endotoxin activity by neutralizing isolated bacterial lipopolysaccharides (LPS). We have previously shown that an isolated 25 kD N-terminal fragment of human BPI carries all the antibacterial activities of the parent 55-60 kD molecule. In this study we have compared the LPS-neutralizing activities of human holo-BPI, the N-terminal fragment and a 30 kD C-terminal fragment that we have now isolated. We show that the N-terminal fragment also has LPS-neutralizing activity as detected by inhibition (up to 95%) of (a) activation by LPS of procoagulant proteases in Limulus amebocyte lysates, (b) LPS "priming" of PMN, and (c) LPS-mediated production of tumor necrosis factor in whole human blood. Holo-BPI and the 25 kD fragment have similar neutralizing potency (in nanomolar range) in all assays toward "smooth" LPS from Escherichia coli O111:B4 and O55:B5 (possessing long chain polysaccharide or O-antigen), and "deep rough" LPS from Salmonella minnesota Re595 mutant (possessing no O-antigen). The C-terminal fragment of BPI is devoid of antibacterial activity when tested against BPI-sensitive E. coli J5, but does have endotoxin-neutralizing activity. This activity is weak relative to holo-BPI and the 25 kD N-terminal fragment in the Limulus and PMN-priming assay, but is comparable for inhibition of TNF production in whole blood. We conclude that the principal determinants for LPS recognition and neutralization, like those for antibacterial action, reside in the N-terminal half of the BPI molecule, but that sites within the C-terminal half can also contribute to BPI-LPS interaction once LPS is detached from the bacterial envelope.

Antimicrobial Cationic Peptides↗
PubMedDate not supplied

Bacteriological activity in unfiltered calf sera collected for tissue culture use.

Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found were Bacillus species and streptococci and 63% of the sera coagulated Limulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain of Escherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.

Bacillus↗

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