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PubMedDate not supplied

Differentiation between endotoxin and non-endotoxin pyrogens in human albumin solutions using an ex vivo whole blood culture assay.

Purified E.coli endotoxin, Gram negative bacteria and Gram positive bacteria induce IL-6 secretion by whole blood cultures (WBC's). Polymyxin B at concentrations greater than 2 U/ml completely inhibits IL-6 secretion caused by 10 EU/ml of endotoxin. Polymyxin B has no effect on IL-6 secretion by WBC's in the absence of endotoxin. The inhibition of endotoxin induced IL-6 secretion is Polymyxin B concentration dependent at concentrations less than 1 U/ml. IL-6 induction caused by E.coli is only partially inactivated by 8 U/ml Polymyxin B. Polymyxin B has no effect on IL-6 secretion caused by B.subtilis. Two pyrogenic batches of human serum albumin (HSA), as tested by the rabbit assay for pyrogens, were also investigated. Polymyxin B at 4 U/ml inhibits less than 40 % of IL-6 secretion caused by these pyrogenic HSA batches. All the endotoxin activity in HSA samples spiked with purified endotoxin is inhibited by Polymyxin B indicating that HSA does not protect endotoxin against Polymyxin B inhibition. These results indicate that the pyrogenicity of these HSA batches are caused by Polymyxin B inhibitable and non-inhibitable fractions. This study shows that pyrogenic substances other than endotoxin can contaminate batches of pharmaceutical products and that results obtained using the Limulus Amoebocyte Lysate (LAL) assay does not necessarily indicate the pyrogenic status of pharmaceutical products. The WBC assay for pyrogens, having a broader sensitivity range than the LAL assay, is a better indicator of the pyrogenic status of pharmaceutical products.

Animals↗
PubMed1988

Reaction of Bacillus subtilis products with amebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentatus.

Autoclaved aqueous extracts of Candida albicans cells (and the glucans isolated from them) give a positive reaction with a chromogenic substrate combined with amebocyte lysates of the Japanese horseshoe crab, Tachypleus tridentatus (CS-TAL). The extracts and glucans activate the lysate enzyme compound G, which in turn activates clotting enzyme. Activated clotting enzyme causes a positive CS-TAL reaction. C. albicans extracts and glucans react positively with a commercially available, unaltered CS-TAL preparation (Toxicolor), but they give a negative reaction with a CS-TAL from which compound G has been excluded (Endospecy). An autoclaved, sterile preparation of Sabouraud glucose broth used as a control in one experiment gave (like Candida extracts) a positive reaction with Toxicolor and a negative reaction with Endospecy. We found that the peptone powder used to make the Sabouraud glucose broth was contaminated with a strain of Bacillus subtilis. Autoclaved aqueous extracts of culture-grown B. subtilis cells were positive with Toxicolor and negative with Endospecy. This was also the case with two other strains of B. subtilis. Polysaccharides obtained from these extracts gave the same result. Endotoxin activates clotting enzyme through activation of the lysate enzyme compound C, which is present in both Toxicolor and Endospecy. Endotoxin, therefore, reacts with both CS-TAL preparations. Simultaneous assay with Toxicolor and Endospecy distinguishes endotoxin from fungal products, but since products of fungi and B. subtilis both give a positive Toxicolor and a negative Endospecy test, a simultaneous assay cannot differentiate them. However, this does not decrease the clinical value of the simultaneous Toxicolor-Endospecy assay for distinguishing fungal infection from endotoxemia because B. subtilis so rarely causes disease that it can be excluded from clinical consideration.

Animals↗
PubMed1984

[Importance of postoperative endotoxin determination. A pilot study].

In a prospective pilot study a daily test for the presence of endotoxin was performed on the blood of 145 patients that were admitted to the intensive care unit for at least 3 days. Endotoxin positive patients showed a significantly higher mortality rate and the period of admittance to the intensive care unit and the period of ventilation were prolonged.

Adolescent↗
PubMed1984

Comparative investigation of a quantitative chromogenic endotoxin assay and blood cultures.

In a prospective study, the clinical relevance of a quantitative chromogenic endotoxin assay in plasma (detection limit 10 ng/L, assay time 2.5 hours) versus blood cultures was evaluated in 51 critically ill patients with increased susceptibility for infectious complications. Of the 400 samples tested, the endotoxin assay and bacterial culture both were negative in 342 samples. In 21 samples from 15 patients, gram-negative aerobic microorganisms were cultured. Corresponding endotoxin assays were positive in 14 samples (mean 100 ng/L). Twenty-three samples grew gram-positive bacteria. The associated endotoxin assays all were negative. Twelve samples were found to be endotoxin-positive without a corresponding gram-negative bacterial culture. In 7 of these 12 positive endotoxin assays, a laboratory or clinical explanation for these positive tests could be provided. In view of the high sensitivity, specificity, and predictive values obtained, the authors conclude that the endotoxin assay used is a useful clinical adjunct for both the detection and exclusion of gram-negative septicemia.

Bacterial Infections↗
PubMed1982

Isolation immunosuppressive serum components following thermal injury.

There has been increasing recognition in recent literature that immunoregulatory factors can often be detected in the serum of patients with thermal and traumatic injuries. We, too, have shown that a significant number of patients with severe thermal injuries are profoundly immunosuppressed. This immunosuppression was mediated by substances which circulate in the serum which could be easily detected using in vitro lymphocyte assays. The suppressive material was not present in normal serum, and exerted its effects through the activity of a specific (suppressor) subpopulation of lymphocytes. In this study, we have analyzed serum samples obtained from burn patients by plasmapheresis for suppressive activity, then fractionated each using Sephadex G-200. Individual fractions were tested for suppressive activity in mixed lymphocyte cultures, and approximate molecular weights established for suppressive peaks by means of chromatography calibration standards. Evidence linking suppressive activity of the sera to the presence of endotoxin, prostaglandin E, interferon, and "cutaneous burn toxin' is discussed.

Adolescent↗
PubMed1982

The polyethylene glycol precipitation technique and the particle-counting immunoassay for detection of circulating immune complex-like material in liver cirrhosis and septicemia.

Both the PEG-N technique and the PACIA were applied to determine ICLM in sera from patients with liver cirrhosis and gram-negative septicemia, that have been shown to yield positive reactions for endotoxemia with the LAL test. The precision of the PEG precipitation technique, with a stabilized fraction of human HAG of known molecular size used as a calibrator, was satisfactory. In liver cirrhosis, the detectable ICLM was mainly composed of IgG and C3, whereas in gram-negative septicemia IgM, IgG, and C3 were found. PACIA also measured significantly elevated levels of ICLM with rheumatoid factor-binding activity in both patient groups, although the two assay systems did not correlate. PEG precipitates from both patient groups analyzed by gel-chromatography and passive hemolysis test contained large immune aggregates and various amounts of immunoglobulins with specificity directed toward lipid A, the least variable portion of endotoxin. These compositional differences of detected substances may imply the presence of different types of ICLM in patients with liver cirrhosis and gram-negative septicemia.

Antigen-Antibody Complex↗
PubMed1982

Formulae for maximum valid dilution.

The preparation of test specimens using the MVD calculation represents a reasonable approach to equating the sensitivity of the in vitro and in vivo tests for endotoxin. Labeling of LAL reagents and control standard endotoxins are a point of confusion for the parenterals industry. Government and reagent suppliers must work together expeditiously to provide test materials that are explicitly labeled as to reactivity in EU/ml and ng/ml. The relatively greater reactivity of LAL reagent to environmental endotoxin than the rabbit bioassay provides a greater margin of safety for the in vitro approach than previously anticipated, and suggests that rabbit retest of LAL failures may be appropriate for specific products.

Animals↗
PubMed1992

[Plasma endotoxin measured by the combination of new perchloric acid pretreatment method and endotoxin specific assay in liver cirrhosis].

Using with the newly modified perchloric acid pretreatment method and endotoxin specific assay (Endospecy), the plasma endotoxin in the patients with liver cirrhosis was investigated. The mean value of plasma endotoxin in control (n = 20) was below 9.8 pg/ml. The plasma endotoxin level in liver cirrhosis was 5.7 +/- 5.3 pg/ml (n = 70, mean +/- SD), and 20% of the patients (15 cases) showed above 9.8 pg/ml. Endotoxin level significantly correlated with the severity of liver function based on the Child-Turcotte classification (p less than 0.01). Plasma endotoxin positively correlated with total bilirubin (r = 0.417) and ICG clearance test (r = 0.298) and negatively correlated with prothrombin time (%) (r = 0.497) and HDL-cholesterol (r = 0.578) in the patients with liver cirrhosis. There was no correlation between esophageal varices and plasma endotoxin level. Plasma endotoxin was slightly detected in the patients with decompensated cirrhosis, and these data suggest that plasma endotoxin level in cirrhosis is not so elevated.

Bilirubin↗
PubMed1994

Combination of DTP and Haemophilus influenzae type b conjugate vaccines can affect laboratory evaluation of potency and immunogenicity.

A commonly used Diphtheria-Tetanus-Pertussis (whole-cell) vaccine was combined with each of three different Haemophilus influenzae type b (Hib) capsular polysaccharide vaccines. Each Hib vaccine incorporated one of three different protein conjugates: tetanus toxoid, diphtheria CRM197 toxoid or group B Neisseria meningitidis outer membrane vesicles. The effects of these combinations on the subsequent laboratory control testing were examined. The addition of the Hib vaccines had no significant effect on the reactogenicity or the potency of the whole-cell pertussis component. The potency of, and antibody responses to, the diphtheria component were also unaffected in all three combinations. However, combination with the Hib vaccine comprising polysaccharide conjugated to tetanus toxoid had dramatic effects on tetanus potency and immunogenicity when assayed in mice. This combination resulted in a five-fold potentiation of the tetanus potency and a similarly large increase in the antibody responses to tetanus toxin and toxoid. The level of the antibody response to the Hib polysaccharide in this vaccine was also elevated, more than 20-fold, as a result of the combination. Such phenomena were not evident with combinations involving the other two Hib vaccines. These results have implications for the control testing of combined vaccines containing a whole-cell pertussis component and Hib polysaccharide-tetanus protein conjugate vaccine.

Animals↗
PubMed1987

Usefulness of LAL assay for rapid detection of gram-negative bacteriuria.

LAL assay is a rapid and reliable and easy to perform and interpret urine screen for clinically significant gram-negative bacteriuria. Chromogenic assays are rapid (5 min) and obviate problems associated with gel endpoints, such as inadvertant dissolution of the gel, either during incubation or reading. LAL urine screening is characterized by both high positive and negative predictive values. LAL urine assay identifies gram-negative urinary tract infections by measuring urine endotoxin content as an index for significant numbers of urinary tract pathogens, thus eliminating large numbers of false-positive tests characteristic of other urine screening methods. Screening urine for greater than or equal to 10(5) CFU/ml appears to be an acceptable criterion for defining a UTI in patient populations with low prevalence rates (2-10%). Thus, LAL urine assay may have potential usefulness for screening of asymptomatic populations, such as pregnant women and the elderly.

Bacteriuria↗
PubMed1992

Early diagnosis of gram-negative peritonitis in continuous ambulatory peritoneal dialysis patients with the Lymulus amebocyte lysate assay.

The treatment of peritonitis in continuous ambulatory peritoneal dialysis patients is empiric until the bacteriological results are available. The Lymulus amebocyte lysate assay (LAL) is a very sensitive method for the detection of endotoxin, a structural component of gram-negative bacteria. We performed the LAL assay in a prospective study in 36 consecutive episodes of peritonitis. The LAL assay was positive in all 10 episodes of gram-negative peritonitis (100% specificity). Treatment directed specifically against gram-negative or -positive infection was started based on the LAL assay result. In 26 episodes with LAL-negative test, a gram-positive bacterium was cultured in 23 episodes, in 1 there was fungal infection and 2 were sterile. In summary: the LAL assay is a rapid (1 h) and sensitive method for the differentiation of gram-positive or -negative peritonitis and enables starting an immediate and more appropriate antibiotic therapy.

Endotoxins↗
PubMed2004

Locally produced bovine bone sponge as a haemostatic agent.

The aim of this study was to evaluate the morphological and biological properties of a locally produced "Bovine Bone Sponge" for use in dentistry. Bovine bone sponge was prepared from local calf bone. Endotoxin level and surface properties were investigated. The pore size and water uptake ability were measured and results were compared with the commercial haemostatic agent. The material was tested for its haemostatic property and its inhibition of alveolar bone resorption in a sheep model following dental extraction. Results revealed a significant difference in haemostatic effect, and a shorter bleeding time and a lower rate of alveolar bone resorption in bovine bone sponge compare to a commercial haemostatic agent.

Alveolar Bone Loss↗
PubMed1991

[Polysaccharides from Nerium oleander: structure and biological activity].

A water extraction of crushed leaves of Nerium oleander yielded 2.3% of a crude polysaccharide. The main fraction (67%) represents a pectic polysaccharide mainly composed of galacturonic acid besides rhamnose, arabinose and galactose. The polysaccharide structure was characterized by NMR, mild acid- and pectinase treatment combined with GC-MS analyses. In vivo tests for a possible antitumor activity did not result in a significant action. Investigation of immunomodulating activity brought some indications for mitogenic activity and a weak macrophage-mediated cytotoxicity. Increase of phagocytosis could not be doubtlessly assigned to the polysaccharide due to the high amount of endotoxin in the homogenous fraction OLE 2.

Animals↗
PubMed1985

Parallel line assays of endotoxins with the LAL chromogenic substrate method.

All endotoxins from the gram negative microorganisms studied sofar contain lipid A, which is considered to be responsible for the biological activity. Endotoxins may therefore react in a similar way in quantitative assays. The varying non-lipid A part of different endotoxins may potentiate or reduce the activity of the identical lipid A part without changing the slope of the log-dose vs. response curve of these endotoxins. To test whether 13 endotoxins from different microorganisms react similarly with amoebocyte lysate (LAL) log-dose response curves were made using a chromogenic substrate method. A balanced incomplete block design with 13 blocks and four replications of each endotoxin was applied. A group of eight endotoxins with a common slope was defined with the Student-Newman-Keuls procedure. Two independent potency estimates of these endotoxins were in good agreement.

Analysis of Variance↗
PubMed1999

Endotoxin release due to ciprofloxacin measured by three different methods.

Antibiotics are known to induce the release of bioactive endotoxin (LPS) from gram-negative bacterial cells. Because varying data have been published on the influence of quinolone antibiotics on LPS liberation, we studied the effect of ciprofloxacin on a culture of Escherichia coli by determining bacterial killing and free LPS concentrations in comparison with imipenem and ceftazidime. LPS levels were measured by three different methods, namely (1) the Limulus amebocyte lysate test, (2) an ELISA method based on capture of LPS by monoclonal antibodies, and (3) indirect determination by measuring the ability of antibiotic-induced LPS to trigger TNFalpha release from a monocytic cell line. With both the Limulus and ELISA tests, a low endotoxin-releasing activity of ciprofloxacin was confirmed. In contrast to previous studies, this LPS also had low bioactivity in terms of TNFalpha induction. Limulus LPS determinations correlated more precisely with LPS bioactivity than did ELISA values, an observation which underlines the crucial role of LPS determination methods in studies of antibiotic-induced LPS release.

Anti-Infective Agents↗
PubMed1985

Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant.

A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood polymorphonuclear leukocytes, and induction of a serum factor that is cytostatic and cytocidal to L-929 cells in Mycobacterium bovis BCG-primed mice. Relative potencies of test synthetic compounds depended on the assay systems and varied from one system to another. Dephospho-compound 503 lacked most of the biological activities that were definitely observed with phosphorylated compounds, probably because of its insolubility. This study demonstrates the successful chemical synthesis of an E. coli-type lipid A.

Adjuvants, Immunologic↗
PubMed1986

Some biological activities of Eikenella corrodens major outer membrane proteins.

Major outer membrane proteins of Eikenella corrodens, an organism frequently isolated from patients with periodontal disease, were tested for some biological activities. Mouse peritoneal macrophages, exposed at low concentrations of the above-mentioned proteins (between 0.05 and 5 micrograms/ml), showed evident and marked morphological modifications consisting of increases in the size and vacuolation of the cells. Higher concentrations showed a toxic effect. Low concentrations resulted in a selective release of lysosomal enzymes without any significant release of lactatedehydrogenase, and cytoplasmic marker; while concentrations of 25-50 micrograms/ml, which were toxic in trypan-blue exclusion test, increased LDH release. Eikenella corrodens major proteins increased the platelet aggregation of ADP and thrombin. The residual complement activity of serum samples incubated with various amounts of proteins at 37 degrees C for 30 minutes appeared strongly reduced with respect to controls, thus showing a consumption of the complement components. These results suggested that Eikenella corrodens major proteins may play a role in the development of periodontal lesions.

Animals↗
PubMed1986

Immunobiological activities of synthetic lipid A analogs with low endotoxicity.

Synthetic lipid A analogs, beta(1-6)glucosamine disaccharide 1,4'-bisphosphates, which possesses four tetradecanoyl groups at the 2- and 2'-amino, and 3- and 3'-hydroxyl groups (LA-17-PP), and each two of the (R)-3-hydroxytetradecanoyl and tetradecanoyl groups at the 2- and 2'-amino and 3- and 3'-hydroxyl groups, respectively (LA-18-PP), were far less endotoxic than synthetic (506, LA-15-PP) and bacterial Escherichia coli type lipid A's; neither compound showed any detectable lethal toxicity in chicken embryos or preparatory activity for the local Shwartzman reaction in rabbits. Also both compounds were only weakly pyrogenic and comparably less lethally toxic in galactosamine-loaded mice than the reference synthetic and bacterial lipid A's and a synthetic counterpart to biosynthetic lipid A precursor Ia (406, LA-14-PP). Nevertheless, LA-17-PP and LA-18-PP exhibited definite in vivo immunoadjuvant activity in mice, and the ability to induce a possible tumor necrosis factor and alpha/beta interferon in Mycobacterium bovis BCG and Propionibacterium acnes-primed mice, respectively, although these activities were weaker than those of the reference lipid A's. 4'-Monophosphate analogs of the above two test compounds exhibited neither endotoxic nor beneficial activities, but they showed remarkable in vitro bioactivities comparable to those of the corresponding bisphosphate compounds; the ability to activate the human complement system and the clotting enzyme cascade of horseshoe crab amoebocyte lysate, stimulatory effects on guinea pig and murine peritoneal macrophages, and murine splenocytes.

Adjuvants, Immunologic↗