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PubMedDate not supplied

Dry heat inactivation of endotoxin on the surface of glass.

The thermal inactivation of three endotoxin preparations on the inner surface of glass capillary tubes was studied. The samples were exposed to precisely controlled dry heat conditions at study temperatures ranging from 170 degrees to 350 degrees C, and were assayed using the gel-clot method of the Limulus Amebocyte Lysate test. Plots of the log of the amount of pyrogenic material remaining versus heating time revealed apparently biphasic destruction curves. The initial slopes were linear to a minimum 3-log unit reduction, and were followed by slower destruction rates for the terminal slopes. D values were calculated from the initial slopes of the destruction data, and Z values were estimated from the D values. The D and Z values were found to vary with the initial charged amounts of endotoxin. A second-order equation was found to be an inappropriate model for the inactivation process at temperatures between 170 degrees and 250 degrees C, but was found to be suitable for temperatures between 250 degrees and 325 degrees C. The data were successfully fit to a biexponential equation for all the temperatures studied. The overall inactivation rate of the endotoxin material formulated with fillers was apparently faster than that for the pure endotoxin preparations.

Endotoxins↗
PubMedDate not supplied

[High-efficiency membranes and the dialysis solution].

During this work through the total bacterial count and of the pseudomonas and the determination of the positive substances to Limulus Amebocyte Lysate test, bacterial pollution of the liquid bicarbonate concentrate has been determined together with the influence that preparation conditions and the entity of interval between the said preparation and utilization of concentrates have on it. Through the evaluation of the cardiac rate and the systemic arterial pressure, we have studied the influence of the bacterial pollution on the clinical of the patient. Analysing the obtained data we can point out that the liquid bicarbonate concentrate present an elevated degree of contamination which feels the effects of the production and storage procedures. The remark of inverse correlation between systemic arterial pressure and endotoxin level of the dialysate seems to reconfirm the importance of the bacterial contamination on the clinical of the patient.

Bacteria↗
PubMed1988

Antigenic analysis of Pasteurella haemolytica serovars 1 through 15 by crossed immunoelectrophoresis.

Pasteurella haemolytica serovars 1 through 12, grown in broth and on agar plates, and 2 field isolates (types A1 and T10) were used to develop polyvalent crossed immunoelectrophoresis (XIE) reference systems. The maximal number of antigens was revealed by XIE when sonicates of agar plate-grown organisms were used as the immunogen (to produce antibodies) and as the soluble antigen for XIE. Antigens produced from agar plate-grown organisms were less contaminated (by antigenic components of the medium) than were those produced from organisms grown in broth. Seventy-two antigens were detected in sonicated preparations of agar plate-grown P haemolytica. The common antigen of gram-negative bacteria was identified in the P haemolytica XIE reference system; precipitation was observed with rabbit antiserum to the common antigen of gram-negative bacteria isolated from Escherichia coli, as well as with rabbit immunoglobulins (obtained from unvaccinated rabbits). Most preimmune sera from our vaccinated rabbits also precipitated the common antigen. Serovar-specific antigens in the P haemolytica XIE reference system were defined and presumptively identified as part of the bacterial lipopolysaccharide complex by use of the limulus amebocyte lysate test. Partial cross-reactions were found between serovar-specific antigens within each biovar (A and T). Pasteurella haemolytica biovar A-specific and biovar T-specific antigens were defined by crossed-line immunoelectrophoresis. When serovars A13, A14, and T15 were tested in the P haemolytica XIE reference system, they gave high matching coefficient values of 0.98, 0.98, and 0.87, respectively. The proposal to separate P haemolytica biovars A and T into 2 different species was supported by immunotaxonomic data obtained from crossed immunoelectrophoresis, but more extensive studies will be necessary to establish the appropriate taxonomic position of these 2 groups of organisms.

Animals↗
PubMed1987

C5a induction of human interleukin 1. Synergistic effect with endotoxin or interferon-gamma.

Interleukin-1 (IL-1) is a potent cytokine which possesses the ability to mediate systemic acute phase responses as well as local tissue inflammation. In these studies, we have examined the ability of C5a and C5a des Arg to induce IL-1 production in vitro. Human C5a and C5a des Arg were purified to homogeneity and were found to stimulate IL-1 release from freshly obtained human mononuclear cells into the extracellular medium. Only 2 hr of exposure to the purified complement components were necessary in order to stimulate IL-1 production. The minimal concentration of C5a required was 25 ng/ml, whereas 125 ng/ml of C5a des Arg induced comparable amounts of IL-1. This dose relationship was maintained at higher concentrations (150 ng/ml vs 750 ng/ml, respectively). That the effect was due to the anaphylatoxins themselves, and not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and employing preincubation of C5a/C5a des Arg with polymyxin B. The latter blocked a wide dose range of endotoxin-stimulated IL-1 production. However, when endotoxin was added to C5a or C5a des Arg, significant synergism in the stimulation of IL-1 production was observed, occurring at various concentrations of either agent. A similar synergism with C5a/C5a des Arg was seen with interferon-gamma. In these studies, IL-1 production was measured by bioassay employing cloned D . 10 . G4 . 1 murine T cells and by radioimmunoassay for human IL-1 beta; using C5a/C5a des Arg as stimulants, there was a high degree of correlation (r = 0.82) between the two assays. Since traumatic, infectious, and inflammatory diseases may result in the simultaneous appearance of these stimuli, the synergism described herein is likely to be clinically relevant.

Biological Assay↗
PubMed1986

Studies on Escherichia coli vaccines: an in vitro approach to establishing safe limits of endotoxins in vaccines.

A total of fifty production batch samples from six different vaccines for pigs, sheep and cattle were monitored for their endotoxin content. All vaccines recommended for parenteral administration into pregnant animals were aqueous and cellular with the exception of one comprising enterotoxin only. The Limulus amoebocyte lysate test was used for endotoxin determination. Total endotoxin of the vaccine was estimated and a method has been devised to estimate the enterotoxin bound either to the cells or the adjuvant. The highest endotoxin levels obtained for pigs, sheep and cattle vaccines were 47.9 micrograms ml-1, 4.7 micrograms ml-1 and 2.4 micrograms ml-1 respectively. All these vaccines have been found to be safe in pregnant animals. The non-cellular vaccine containing enterotoxin only had an endotoxin level of 0.47 microgram ml-1.

Bacterial Vaccines↗
PubMedDate not supplied

[Bacteriocins as cytotoxic agents in experimental neoplasms: considerations on the role of possible contaminants].

In most recent papers some Authors have demonstrated that several bacteriocins are able to markedly inhibit the division of various neoplastic mammalian cell lines; also in vivo the inhibition of tumor growth or even regression relative to controls are statistically significant. Since such preparations are "crude" and it's possible that contaminating agents may represent a possible source of variability in the results, mostly in vivo; we have examined a preparation of Pyocin employed in this study for the possible endotoxin contamination using the Limulus assay. Such test was positive in triplicate and semiquantitative determination has shown and endotoxin amount of 5 to 10 microgram/ml. This level is, in vitro, not anough to give cytotoxic effect, but in vivo is able to influence experimental results. In addition, since some bacteriocins and enterotoxins are produced under exactly identical conditions, there is this a possibility that the enterotoxin are coproduced in these preparations and are causing some of the cytotoxic effects on experimental tumors. A more complete separation and purification of preparations will be required before definitive comments.

Animals↗
PubMed1984

Immunological abnormalities in patients with chronic bronchial suppuration: a possible relationship with endotoxaemia.

Chronic bronchial suppuration has been associated with elevations in immunoglobulin levels, circulating autoantibodies and immune complexes but the cause is obscure. In order to investigate one possible explanation we studied eight male subjects (mean age 54.9 years, range 39-68) and 16 female subjects (mean age 45.2 years, range 24-68) with bronchial suppuration attributed to bronchiectasis. Gram-negative organisms were found in the sputum of 95% of subjects. Forty per cent (10 subjects) had elevations in immunoglobulin levels and 60% (15 subjects) had circulating autoantibodies. Eighty-eight per cent (21 subjects) had mild-moderate increases in the level of C-reactive protein. Endotoxin was detected using the limulus amoebocyte lysate test in 50% (12 subjects). Endotoxin was found only in patients with an immunological abnormality and was more likely to be detected in those with both elevated immunoglobulins and circulatory autoantibodies. It is suggested that the endotoxin may be of aetiological significance in the development of autoantibodies and elevations of immunoglobulin levels.

Adult↗
PubMed1980

[Endotoxinaemia in liver cirrhosis (author's transl)].

23 patients with liver cirrhosis of diverse etiology and of different activity levels and grades of compensation of the portal circulation were examined with regard to the systemic endotoxinaemia. The endotoxin was determined using the Limulus-Amoebocyte-Lysat-test. Endotoxin was traceable in the venous blood of 7 out of 23 patients (= 30,4%). No relation was found between endotoxaemia and the activity or grade of severity of liver cirrhosis; nor were there any accumulation of endotoxinaemia in patients with collateral circulation or portal decompensation. Consequently, as a result of our findings, the prognostic value of the symptom endotoxinaemia remains debatable, then endotoxinaemia does not always mean endotoxicosis. Particularly high immunoglobulin concentration and low albumin values in serum of endotoxin-positive cirrhotics indicate a "spillovet" of antigen substances and also of endotoxines in the body circulation resulting from insufficiency of the liver-RHS. Parallel analysis of microbian small bowel flora in 10 patients indicate that coli-dysbiosis appears to play a role in the development of systemic endoxinaemia, although the latter was also traceable in borderline cases of eubiosis/dysbiosis. Our findings strengthen the view that it is not possible to simply translate the findings in animals on to human liver diseases. Quite a number of the mechanisms being discussed are still with uncleared details and they give only a blurred picture of the pathophysiology of endotoxinaemia.

Adult↗
PubMed1995

[Biochemistry, molecular mechanism of action and biological effects of endotoxin].

This review is a brief attempt at providing an overview of a subject of enormous complexity-endotoxins and the mediators associated with its biological effects. More specifically it deals with biochemistry and biology of endotoxin, detection of endotoxin with the Limulus amebocyte lysate test, the molecular mechanisms and biological effects, and in the last part with future aspects of therapeutical strategies. It seems certain that the subject will become even more complex and possibly controversial as scientific knowledge further involves. However, because of the high mortality rate of patients suffering from gram-negative sepsis all efforts have to be made to find effective therapeutical strategies.

Animals↗
PubMed1975

Consequences of microbial contamination during extended intravenous therapy using inline filters.

A study was conducted to determine: (1) the effectiveness of 0.22-mum and 0.45-mum pore size membrane filters in retaining microorganisms over a period of 72 hours of intravenous fluid administration; (2) the growth and multiplication characteristics of microorganisms retained on the filter; and (3) the possibility of pyrogens being released by microorganisms trapped on the filter. Lactated Ringer's injection was used as the i.v. solution. The 0.45-mum filter was found to be effective in retaining all microorganisms studied for at least 6 hours. However, Esherichia coli and Pseudomomas aeruginosa began passing through the 0.45-mum filter between 6 and 8 hours after seeding. All other microorganisms (Candida albicans, Citrobacter freundii, Klebsiella pneumoniae, Staphyloccus aureus) were retained for at least 72 hours. None of the microorganisms passed through the 0.22-mum filter during the 72-hour study period. With viable microorganisms on the filter for 72 hours, no detectable endotoxins or pyrogens were found in the filtrates as examined by the limulus and rabbit tests.

Animals↗
PubMed1994

Co-ligation of CD31 and Fc gamma RII induces cytokine production in human monocytes.

The CD31 (PECAM-1) cell surface glycoprotein is considered to be involved in intercellular recognition and adhesion. Cytokines play a major role in cellular interactions, and therefore it was of interest to study whether engagement of CD31 affects synthesis and release of proadhesive cytokines. Here we demonstrate that immobilized CD31 mAb 1B5 induces the release of TNF-alpha, IL-1 beta, and IL-8 from human PBMCs. CD11b mAb VIM12 and HLA-D mAb VID1, both of which are of the same Ig subclass as mAb 1B5 (IgG1), as well as nonbinding isotype control mAb VIAP, were ineffective. That the effect was caused by the mAb, but not endotoxin contamination, was shown by negative Limulus amebocyte lysate tests and coculture with polymyxin B, which did not abolish TNF-alpha release. Cytokine production through intact mAb 1B5 was completely blocked by soluble F(ab) fragments of anti-IgG Fc gamma RII mAb IV.3, suggesting a significant contribution of that FcR. Cross-linking of neither CD31 nor Fc gamma RII molecules with the respective F(ab) fragments induced TNF-alpha release, but nonbinding control IgG1 Ab was able to restore the response of PBMC to 1B5 F(ab) fragments, when both Ab preparations were coated concomitantly. Therefore, only coligation of CD31 and Fc gamma RII appears to transduce activation signals leading to cytokine production. Our findings thus indicate a novel functional aspect of CD31 molecules that might play an important role in the propagation of an ongoing immune response as well as in the regulation of cell-cell interactions during inflammatory reactions.

Antibodies, Monoclonal↗
PubMed1977

Remarks on some properties and effects of sodium nucleinate.

The pyrogen standard of the Pharmacopoeia of the German Democratic Republic--which is recommended in the Compendium Medicamentorum and the Pharmacopoeia of the CMEA countries, consists of a selected batch of sodium nucleinate, the pyrogenicity of which had been demonstrated in very many rabbits. One ampoule of this standard contains 10 mg freeze-dried sodium nucleinate. After inoculation of 0.4 mg per kg of body weight into rabbits an increase in temperature of 0.8 degrees C or more will occur. Westphal assumes that the reason for the pyrogenicity of sodium nucleinate is possibly due to its obligatory contamination with bacterial lipopolysaccharides. Some investigations to find out if the properties and effects of this standard are different from those of isolated bacterial lipopolysaccharides have been performed by the author: adsorption by plastic granulates, inactivation by heating and by incubation with horse serum, temperature changes produced by inoculation into rabbits and results of investigations using the limulus amebocyte lysate test. The results of the investigations did not show significant differences between sodium nucleinate and isolated bacterial lipopolysaccharides. This is in agreement with Westphal's hypothesis mentioned above.

Adsorption↗
PubMed1995

Comparison of the blood clearance of triple- and single-helical schizophyllan in mice.

(1-->3)-beta-D-Glucans exhibit a variety of biological and immunopharmacological activities, and the degree of these activities depends on the nature of the individual glucans e.q. molecular weight, degree of branching and conformation. Based on the generally accepted evidence that the conformation of Sonifilan (SPG) used clinically is a triple helix, we prepared alkali-denatured SPG (SPG-OH) as a single helix conformer. In this report, we measured the concentration of beta-glucan administered to mice by using a beta-glucan-specific reagent prepared from limulus amebocyte lysate (Gluspecy [G test], Seikagaku Corporation, Tokyo) and discuss the blood clearance of SPG and SPG-OH following intraperitoneal (i.p.) or intravenous (i.v.) administration. Comparing the clearance of SPG-OH from the blood with that of SPG, SPG-OH was removed faster than SPG following both i.p. and i.v. administration. This strongly suggests that the clearance of beta-glucans is dependent on their conformation.

Animals↗
PubMedDate not supplied

[Lipopolysaccharides of Rickettsiaceae and the Limulus endotoxin assay].

The A.A. have examined by Limulus endotoxin assay some LPS preparations from R. typhi, R. slovaka, C. burnetti phase I and II, as a demonstration of endotoxicity. All the preparations tested were able to gel the Limulus amoebocyte lysates, even if to a different degree of reactivity. The results add credibility to the hypothesis that LPS from Rickettsiaceae can represent at least in part the pathogenetic mediators of some manifestations described in Rickettsiosis; however, without endotoxin assay in the blood of patients, one cannot assume to this be true.

Coxiella↗
PubMed1991

Plasma concentrations of endotoxin following jugular or portal injections of endotoxin and following gastrointestinal ischemia due to hemorrhage.

Endotoxin (LPS) was quantitated in canine plasma using the Limulus amebocyte lysate (LAL) chromogenic testing procedure. The assay was validated for sensitivity (25 pg/ml), recovery (90-110%), intra-assay precision (CV = 5.5), interassay precision (CV = 10), and stability of diluted, heat-treated, frozen samples (greater than or equal to 60 days). Canine plasma samples were analyzed for endotoxin following sublethal IV injections (cephalic and portal, bolus and slow infusion) of LPS. Pharmacokinetic analysis using the two-compartment open model on plasma LPS levels was possible for portal bolus, cephalic bolus, and portal slow infusion dogs. The results revealed that LPS given via cephalic bolus route had a lower clearance rate than LPS given via portal bolus route. Slow infusion of LPS into the portal vein revealed an increased distribution phase t1/2 in plasma and a slower elimination kel and beta rate than observed following a portal bolus injection of LPS. During a clinical endo(to)xemia, LPS enters the circulation slowly, and is therefore probably cleared more slowly; the prolonged low level of LPS may be responsible for many pathophysiological changes observed. Low levels of endotoxin were detected in plasma following hemorrhage, indicating that intestinal ischemia results in low levels of LPS leaking into the circulation.

Animals↗
PubMed1983

Interactions of lipid a and liposome-associated lipid A with Limulus polyphemus amoebocytes.

Lipid A or lipid A fractions and liposomes containing lipid A were tested for the ability to gel Limulus amoebocyte lysates and for effects on intact Limulus amoebocytes. Liposomes having a relatively low concentration of lipid A did not produce coagulation of lysate and were designated as Limulus-negative, but liposomes having a high concentration of lipid A were Limulus-positive. Limulus-negative liposomes had no effect on intact amoebocytes. Limulus-positive liposomes caused a striking transformation in the appearance of amoebocytes in that the cells sent out long filamentous extensions that formed a tangled network of processes between cells. The filamentous projections were similar to those that have been previously observed in the presence of gram-negative bacteria. We conclude that amoebocytes have the ability to recognize Limulus-positive liposomes, but the lack of activation of Limulus lysate or the absence of amoebocyte recognition does not prove the absence of liposomal lipid A. We also found that individual lipid A fractions were heterogeneous in their ability to gel lysate. Of eight fractions tested, one (fraction 1) had no detectable activity above the background, and the seven others had activity that ranged from 10-fold to 10,000-fold above the background. The heterogeneity of lipid A fractions detected in assays with amoebocyte lysate was consistent with the finding of heterogeneity in other functional assays of lipid A fractions.

Animals↗
PubMed1997

Initial experience with reuse of coronary angioplasty catheters in the United States.

OBJECTIVES: We sought to evaluate the performance of angioplasty catheters, restored under a strict manufacturing process, in patients with coronary artery disease. BACKGROUND: Most countries outside the United States routinely reuse disposable medical equipment, resulting in significant cost savings. Because of quality and legal concerns, reuse in the United States has been limited. We investigated the reuse of percutaneous transluminal coronary angioplasty (PTCA) balloon catheters, restored by a process strictly controlled for bioburden and sterility, in patients undergoing PTCA. METHODS: Used PTCA balloon catheters were shipped to a central facility and were decontaminated, cleaned and tested for endotoxin using the limulus amebocyte lystate (LAL) gel clot method. Physical testing and quality assurance were performed. The products were packaged and sterilized with ethylene oxide. Catheter performance was assessed in a pilot study powered to detect a 5% difference in the angiographic failure rates of new and reused balloons (beta 0.8). RESULTS: The study enrolled 107 patients. The indication for PTCA was stable angina pectoris in 69 patients, unstable angina in 22 and acute myocardial infarction in 16. Of the 107 patients enrolled, 106 had a successful laboratory outcome, and 1 required coronary artery bypass graft surgery after failed rescue stenting. There were 122 lesions attempted (American College of Cardiology/American Heart Association classification A, n = 32; B1, n = 43; > or = B2, n = 35; C, n = 12). Of the 110 lesions initially approached with restored PTCA catheters, 108 were crossed and dilated. Sixty-four required no further procedures. Stenting was performed in 37 patients (29 planned, 8 rescue). Thus, the angiographic failure rate was 7% (10 of 108, 95% confidence interval 2% to 12%), comparable to the 10% rate seen with new balloons in other studies. CONCLUSIONS: Restoration of disposable coronary angioplasty catheters using a highly controlled process appears to be safe and effective, with success rates similar to those of new products and no detectable sacrifice in performance. Cost analysis suggests that implementation of reuse technology for expensive disposable equipment may offer cost savings for U.S. hospitals, without sacrifice of quality.

Angioplasty, Balloon, Coronary↗