Pyrogen reactions associated with the infusion of normal serum albumin (human)

In November, 1974, eight patients in three hospitals had pyrogen reactions associated with the infusion of 25% Normal Serum Albumin from the same lot. The reactions were recognized because the same physician or nurse observed several patients having recurrent reactions or because a single patient receiving several vials had consecutive reactions. The remaining albumin in three vials associated with reactions had apparent endotoxin concentrations of 4, 16, and 32 ng/ml and that 22 vials from recalled supplies had a median concentration of 4 ng/ml (range: 2 to 64) as determined by the Limulus amebocyte lysate test, but the lot again passed the rabbit pyrogen test. In a prospective study to determine the efficacy of the Limulus test in quality control, patients had their temperatures taken hourly during albumin infusions and the remaining fluid was tested by the Limulus assay. The albumin in 443 of the 662 vials infused (65%) gave a positive test and 311 of these vials (45%) had apparent endotoxin concentrations of 4 to 64 ng/ml, but no patient had a reaction. Because of the limitations of both the rabbit pyrogen and Limulus test, the detection of some pyrogenic lots continues to depend on hospital surveillance and reporting os suspect reactions.     

A modified Limulus amebocyte lysate test with increased sensitivity for detection of bacterial endotoxin

We have developed a simple modification of the chromogenic Limulus amebocyte lysate test that increases the sensitivity for the detection of bacterial endotoxins. In this assay, free paranitroaniline, cleaved from synthetic chromogenic substrates by proteases that were generated by Limulus lysate after incubation with endotoxin, was then derived. Derivation was with p-dimethylaminocinnamaldehyde in the presence of strong acid, forming a stable Schiff base end product with much greater molar absorbancy than the parent chromogen. Conditions (times and temperatures of incubations, concentrations of reagents) for the augmented chromogenic procedure were optimized. A ten-fold or greater increase in sensitivity for bacterial endotoxin was obtained with the modified assay as compared with the standard chromogenic Limulus test, with unequivocal detection of endotoxin concentrations of less than 100 pg/ml. The greater sensitivity of this modified Limulus test increases its usefulness for a wide range of research applications and clinical investigations.     

Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 1

Recombinant human tumor necrosis factor (rTNF alpha) injected intravenously into rabbits produces a rapid-onset, monophasic fever indistinguishable from the fever produced by rIL-1. On a weight basis (1 microgram/kg) rTNF alpha and rIL-1 produce the same amount of fever and induce comparable levels of PGE2 in rabbit hypothalamic cells in vitro; like IL-1, TNF fever is blocked by drugs that inhibit cyclooxygenase. At higher doses (10 micrograms/kg) rTNF alpha produces biphasic fevers. The first fever reaches peak elevation 45-55 min after bolus injection and likely represents a direct action on the thermoregulatory center. During the second fever peak (3 h later), a circulating endogenous pyrogen can be shown present using passive transfer of plasma into fresh rabbits. This likely represents the in vivo induction of IL-1. In vitro, rTNF alpha induces the release of IL-1 activity from human mononuclear cells with maximal production observed at 50-100 ng/ml of rTNF alpha. In addition, rTNF alpha and rIFN-gamma have a synergistic effect on IL-1 production. The biological activity of rTNF alpha could be distinguished from IL-1 in three ways: the monophasic pyrogenic activity of rIL-1 was destroyed at 70 degrees C, whereas rTNF alpha remained active; anti-IL-1 neutralized IL-1 but did recognize rTNF alpha or natural cachectin nor neutralize its cytotoxic effect; and unlike IL-1, rTNF alpha was not active in the mitogen-stimulated T cell proliferation assay. The possibility that endotoxin was responsible for rTNF alpha fever and/or the induction of IL-1 was ruled-out in several studies: rTNF alpha produced fever in the endotoxin-resistant C3H/HeJ mice; the IL-1-inducing property of rTNF alpha was destroyed either by heat (70 degrees C) or trypsinization, and was unaffected by polymyxin B; pyrogenic tolerance to daily injections of rTNF alpha did not occur; levels of endotoxin, as determined in the Limulus amebocyte lysate, were below the minimum rabbit pyrogen dose; and these levels of endotoxin were confirmed by gas chromatography/mass spectrometry analysis for the presence of beta-hydroxymyristic acid. Although rTNF alpha is not active in T cell proliferation assays, it may mimic IL-1 in a T cell assay, since high concentrations of rTNF alpha induced IL-1 from epithelial or macrophagic cells in the thymocyte preparations. These studies show that TNF (cachectin) is another endogenous pyrogen which, like IL-1 and IFN-alpha, directly stimulate hypothalamic PGE2 synthesis. In addition, rTNF alpha is an endogenous inducer of IL-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Optimization of detection of bacterial endotoxin in plasma with the Limulus test

Detection and quantification of bacterial endotoxin in plasma by the Limulus amebocyte lysate test (or other assays for endotoxins) is hindered by the presence of inhibitors. Treatment of plasma to overcome inhibitory activities is required before plasma can be successfully assayed for endotoxin. We have conducted an investigation comparing the three most commonly used procedures (dilution-heating, trifluoroacetic acid oxidation, and chloroform extraction) for treatment of plasma before its assay for endotoxin with the chromogenic Limulus test. Initially, conditions were optimized for treatment of plasma by each of these methods. Subsequently, a direct comparison of the three plasma treatment procedures was performed with plasma spiked with known concentrations of endotoxin. The optimized dilution-heating procedure resulted in the most sensitive detection of endotoxin, with sensitivity approximately 10 times greater than the optimized trifluoroacetic acid oxidation procedure and approximately 100 times greater than treatment of plasma by chloroform extraction. Maximal detection of low concentrations of endotoxin by the chromogenic Limulus test was obtained by dilution of plasma fourfold with 0.15 mol/L NaCl followed by heating at 60 degrees C for 30 minutes. This procedure was simple, rapid, and did not involve addition of any reagents to plasma that could potentially add contaminating endotoxin.     

Endotoxin, thrombin, and the Limulus amebocyte lysate test.

The Limulus amebocyte lysate, a proteinaceous composite isolated from the hemolymph cells of the horseshoe crab (Limulus polyphemus) is sensitive to picogram quantities of Gram-negative bacterial lipopolysaccharides. However, a controversy currently exists as to whether the Limulus amebocyte lysate is specifically sensitive to Gram-negative bacterial endotoxins as a result of a recent report that the blood coagulation protease, thrombin, can mimic endotoxins in the Limulus amebocyte lysate test. Experiments including those employing two highly purified fractions isolated from the Limulus lystae have provided us with evidence that thrombin per se is unable to mimic endotoxin.     

IN ACTIVATION OF B. SUBTILIS SPORES AND E. COLI ENDOTOXIN BY ETHYLENE OXIDE

Exposure of Bacillus subtilis spores to ethylene oxide (EO) showed correlation between the killing rate and the EO concentration, when the temperature was kept at 55 degrees C and the relative humidity at 100%. The co-efficient of dilution was calculated to be 0.9. The effect of EO on Escherichia coli endotoxin was investigated by the chromogenic Limulus Amebocyte Lysate (LAL) test. A solution of endotoxin was dried on glass tubes and exposed to 450 or 900 mg EO/l during 1-46 h under the same conditions as the spore inactivation. The LAL activity of the endotoxin was reduced to about 30%. The EO-treated endotoxin was tested in the rabbit pyrogen test. The summed temperature increase for three rabbits was 0.9 degrees C, while the same assay using untreated test pieces showed an increment of 3.7 degrees C. Administration of the same quantity of EO-treated and untreated endotoxin to the rabbits, as adjusted by the LAL-test, produced the same temperature increment. The addition of polymyxin B (PB) to an endotoxin solution reduced the LAL activity by 75%. Had the endotoxin been exposed to EO, thereby reducing the LAL activity by 70%, addition of PB further reduced the activity by 99%. The reaction of EO on the endotoxin reduced the LAL activity as well as the pyrogenic response and increased the affinity to PB.     

Production of modified crosslinked cell-free hemoglobin for human use: the role of quantitative determination of endotoxin contamination

In vivo toxicity remains a major barrier to the successful use of cell- free hemoglobin (Hb) as an oxygen carrier in humans. Bacterial endotoxin (lipopolysaccharide, LPS) is known to contribute to the in vivo toxicity of Hb preparations, and the prevention of LPS contamination is a critical aspect of the effort to create an efficacious Hb blood substitute. Limulus amebocyte lysate assays for endotoxin were performed on multiple Hb samples from 26 independent production runs for the preparation of human crosslinked cell-free hemoglobin (alpha alpha Hb). High levels of LPS contamination (1- > 100 ng/mL) of alpha alpha Hb solutions were detected in multiple samples during many of the initial production runs. It was observed that LPS contamination of alpha alpha Hb solutions could occur at any step during the production sequence. Substantial enhancement by alpha alpha Hb of the biologic effects of LPS was demonstrated by two independent assays for endotoxin (the Limulus amebocyte lysate test and a mononuclear cell procoagulant assay), whereas LPS biologic activity was only slightly increased by human serum albumin and substantially diminished by IgG. These results suggest that the prevention of LPS- related toxicities in vivo may be more important to the clinical use of Hb solutions than to the use of other intravenous protein products. Therefore, it was encouraging to note that, with the careful monitoring for LPS in the production facility and in multiple samples during cell- free Hb production, sources of LPS contamination were recognized and the appropriate sites were made endotoxin-free.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pilot study on the Limulus test and apyrogenic injectable preparations]

After some notices on the Limulus amebocyte lysate test the AA. expose the results of an investigation for in vitro pyrogen detection in parenteral preparations.     

乳糖酸阿奇霉素注射液细菌内毒素检测方法的研究

目的对乳糖酸阿奇霉素注射液进行凝胶法干扰试验,检测乳糖酸阿奇霉素注射液细菌内毒素。方法按照中国药典细菌内毒素检查方法（2010,~版二部）,对2批样品进行干扰试验和细菌内毒素检查。结果浓度为0．83mg／mL的样品稀释液对标示灵敏度为0．25EU／mL的鲎试剂无干扰作用。结论使用细菌内毒素检查法检查乳糖酸阿奇霉素注射液中的细菌内毒素是可行的,可用细菌内毒素检查法代替家兔热原检查法。     

The Limulus amoebocyte lysate assay for prediction of chlamydial infection in males with non-gonococcal urethritis: an evaluation.

Chlamydia trachomatis produces small amounts of an endotoxin-like material. The Limulus amoebocyte lysate assay was used to evaluate chlamydial cell cultures and also the exudates from adult male patients with non-gonococcal urethritis, as a possible method to subdivide this condition into chlamydial and nonchlamydial urethritis. In vitro endotoxin assays were conducted in McCoy cell media using the Limulus assay, and endotoxin levels were consistently 10-fold less at 24 h than at 0, 48, 72, and 96 h, which may be accounted for by the unique growth cycle of chlamydia. In 75 males with non-gonococcal urethritis, urethral exudates were collected, serially diluted and assayed for endotoxin content. Of these, 27 (36%) had positive chlamydial cultures and 48 were negative. There was no statistically significant correlation between the level of endotoxin present and a positive or a negative culture for C. trachomatis (P greater than 0.05). Sensitivity and specificity of the assay were only 59% and 56%, respectively, at a 1 in 8 dilution; it was not useful in predicting chlamydial culture results in male patients with non-gonococcal urethritis.     

A new sensitive microplate assay of plasma endotoxin.

We have developed a microplate method for determining endotoxin in platelet-rich plasma-using Endospecy, an endotoxin-specific chromogenic Limulus test reagent. Nonspecific activators and inhibitors of the test were eliminated by exposing samples (5 microliters) to the alkali reagent consisting of KOH, CaCl2, Triton X-100, ethyleniminepolymer and N,N-bis(2-hydroxyethyl)glycine. The recoveries of various endotoxins were almost complete and not enhanced by dilution. The dose-response curve was linear over endotoxin concentrations of 2-400 pg/ml with good precision (C.V. less than 5.0%). Normal human plasmas (n = 30) contained less than 5.0 pg/ml of endotoxin in reference to that of Escherichia coli 0111: B4. All plasma samples with high concentration of endotoxin by a conventional method showed high values by the microplate assay as well. Since it does not require centrifugation, the new treatment allows the whole reactions to proceed on the same microplate. This permits us to apply the Limulus test to an automated assay system, making plasma endotoxin determination simpler and more rapid than a conventional test tube method.     

Detection of endotoxin in antibiotic solutions with Limulus amoebocyte lysate

Twenty-eight antibiotics were tested with the Limulus amoebocyte lysate assay to determine their non-inhibitory concentrations (NICs). The Limulus amoebocyte lysate assay was found to be a valid test for most of the antibiotics tested; the NICs were found to be greater than the minimum valid test concentrations. Borderline results were obtained with cefamandole nafate and neomycin sulfate. Polymyxin B and colistimethate contained too much endotoxin to permit determination of NICs. The NIC of tetracycline hydrochloride was dependent on the initial concentration of antibiotic. This dependence was most likely caused by the amount of base required to adjust the pH before testing.     

BIOLOGICAL PROPERTIES OF PARENT ENDOTOXINS AND LIPOID FRACTIONS, WITH A KINETIC STUDY OF ACID-HYDROLYZED ENDOTOXIN

The biological potencies of a number of lipid fractions separated from endotoxins by acid hydrolysis, including the material known as lipid A, were determined in parallel with those of their parent endotoxins, employing bio-assays based on the following dose-related host responses: fever, resistance to infection, tumor damage, primary inflammation of skin, and lethality. Without exception, lipid fractions dispersed by detergents exerted less than 1 per cent of the biological activity of the potent endotoxins from which they were derived. A study was made of the rate at which biologic activities diminished in relation to the release of bound lipid during progressive hydrolysis of Salmonella enteritidis endotoxin with dilute acid. Each of the five assays for endotoxin revealed that biological activity had been reduced to negligible proportions prior to any significant liberation from the endotoxin of water-insoluble firmly bound lipid. The major pharmacological activity of endotoxins, therefore, is acid-labile and cannot be accounted for in isolated lipids. This conclusion is also supported by the finding that lipids with activity similar to that of lipid A could be obtained by non-hydrolytic methods without diminishing the potency of the parent endotoxins.     

Immunochemical study of a bacterial endotoxin: Shigella flexneri type Z

The endotoxins of the Gram-negative bacteria have similar biological and chemical properties. The toxic antigen of Shigella flexneri Type Z was selected as a representative endotoxin, and it was confirmed that the antigen consists of a polysaccharide conjugated with phospholipid and protein. By the technique of zone electrophoresis, the polysaccharide of the purified endotoxin was shown to be conjugated with each of three different proteins, and each conjugate proved toxic and antigenic for the rabbit. Two of the protein conjugates were digested by trypsin and the released polysaccharide appeared to conjugate with the remaining trypsin-resistant protein. Immunological analysis revealed that the purified toxic antigen is heterogeneous and that the polysaccharide and protein components possessed serological activity. The trypsin-treated toxin had a single electrophoretic zone and its precipitation in gel suggested immunological purity. Proteolytic treatment of the endotoxin did not destroy its toxicity or antigenicity for the rabbit.     

Biological Activity, Lipoprotein-binding Behavior, and In Vivo Disposition of Extracted and Native Forms of Salmonella typhimurium Lipopolysaccharides

Although phenol-extracted gram-negative bacterial lipopolysaccharides (LPS) have been used to study the properties of endotoxins for many years, nothing is known about the behavior of native (unextracted) LPS in vivo. Accordingly, we have compared extracted and native forms of LPS with regard to their biological activity, their ability to bind to plasma high density lipoproteins (HDL), and their fate after intravenous injection into rats. The LPS of Salmonella typhimurium G-30 were labeled with [³H]galactose, and whole bacteria, bacterial outer membranes, outer membrane fragments (harvested from the bacterial culture supernatant), and phenol extracts of the bacteria were prepared. After defining the LPS, phospholipid, and protein composition of these preparations, we compared the activity of the LPS in phenol extracts and membrane fragments in two assays. In both the limulus lysate assay and the rabbit pyrogen test, the LPS in phenol extracts were slightly more potent than the LPS in membrane fragments. We next studied the ability of the LPS in each preparation to bind to rat lipoproteins in vitro, and each preparation was then injected intravenously into rats for measurements of LPS-HDL binding and tissue uptake in vivo. Two patterns of lipoprotein binding were observed. Less than 25% of the LPS in both outer membranes and whole bacteria bound to HDL in vitro. When the outer membranes and whole bacteria were injected into rats, their LPS again bound poorly to HDL and they were rapidly removed from plasma into the liver and spleen. In contrast, >50% of the LPS in both culture supernatant membrane fragments and phenol-water extracts bound to HDL in vitro. When these preparations were injected into rats, ~50% of the LPS in the membrane fragments and phenol-water extracts bound to HDL and remained in the plasma over the 10-min study period. Moreover, the LPS in these preparations accumulated in the ovary and the adrenal gland, two tissues that use HDL-cholesterol for hormone synthesis. Binding to HDL thus greatly influenced the plasma half-life and tissue uptake of both extracted and native LPS.     

An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate.

A method for in vitro pyrogen testing using Limulus amoebocyte lysate (LAL) has been described. The method is based upon the measurement of endotoxin-precipitable protein and can be used to measure picogram quantities equivalent to E. coli endotoxin in unknown solutions. When increasing concentrations of E. coli endotoxin are added to a constant amount of LAL and the reaction is allowed to proceed to completion, there is a proportional increase in the protein precipitated by endotoxin. Therefore, by measuring the amount of protein precipitated from LAL, it is possible to determine the equivalent E. coli endotoxin concentration in unknown solutions, when samples of the unknowns are run simultaneously with E. coli endotoxin standards and negative controls. The endotoxin proportional precipitation of protein occurs in reaction mixture showing gelation as well as in reaction mixture where the levels of endotoxin are lower than required for gelation. Determination of precipitated protein provides greater sensitivity for endotoxin detection than the gelation methods currently in use.     

Antigens of Haemophilus influenzae.

Three antigenic preparations were obtained from a non-capsulated strain of Haemophilus influenzae by ultrasonic disintegration, hot phenol extraction and from a fluid culture. They were designated H. influenzae cytoplasmic antigen (H(1-5); H. influenzae cell wall antigen (HCW); and H. influenzae culture filtrate antigen (HCF). Studies showed that H(1-5) antigen contained heat stable and heat labile components. The heat stable fraction stained positively for polysaccharide, had a positive limulus lysate test and there was immunological cross-reactivity between this and heat stable fractions of HCW and HCF. Limulus lysate assay indicated the presence of endotoxin in HCW and HCF preparations. Heat stable as well as heat labile antigens of H. influenzae should be given consideration in future studies regarding the pathogenicity of this organism in the lower respiratory tree. The specificity of the heat stable antigen of H. influenzae needs to be determined.     

Combination of antimicrobial and endotoxin-neutralizing activities of novel oleoylamines

A combination of antimicrobial and endotoxin-neutralizing activities is desired in order to prevent progression from infection to sepsis due to the release of lipopolysaccharide from dying gram-negative bacteria. Lipopolyamines have emerged as a new type of endotoxin-neutralizing compound, but their antimicrobial activity has not been investigated. We synthesized a series of 10 oleoylamines differing in the polyamino head group, particularly in the number and separation between nitrogen atoms and the position of the oleoyl moiety. Compounds showed activity against both gram-negative and gram-positive bacteria in the micromolar range. Compounds were able to provide penetration of ethidium bromide into bacteria, indicating effects on the bacterial membrane. Oleoylamines neutralized endotoxin in Limulus amoebocyte lysate assays and by neutralization of tumor necrosis factor alpha release in human blood. Comparison of biological activities of compounds identified structural properties responsible for antimicrobial activity, and quantitative structure-property relationship analysis provided a quantitative model for prediction of activity of oleoylamines.     

Studies on the pathogenesis of fever. IX. Characteristics of endogenous serum pyrogen and mechanisms governing its release

The "endogenous serum pyrogen" that appears in the circulating blood after a single intravenous injection of endotoxin does not produce leukopenia in normal animals, fails to provoke the local Shwartzman reaction, and elicits no "tolerance" when injected daily. Suppression of the febrile response to endotoxin by prednisone does not prevent the appearance of pyrogen in the blood. Animals given large amounts of endotoxin daily continue to respond with high fevers despite failure of endogenous serum pyrogen to appear in detectable amounts after the first two or three injections. Analysis of the response to daily injections shows clearly that the fever during the first 2 hours after administration of endotoxin is unrelated to levels of endogenous serum pyrogen; in contrast, the magnitude of the fever after the 2nd hour correlates well with endogenous pyrogen in some instances. The leukopenic response to endotoxin could not be correlated with the appearance of endogenous serum pyrogen. The differences between endotoxin and endogenous pyrogen and the similarities between leukocyte extracts (sterile exudates) and endogenous pyrogen are summarized and discussed. Dissociation of the febrile response to bacterial endotoxin and levels of endogenous serum pyrogen are discussed and it is concluded that a mechanism involving both direct and indirect action of endotoxins offers the best explanation for the pyrogenic action of these bacterial products.     

An invertebrate coagulation system activated by endotoxin: evidence for enzymatic mediation

Lysates prepared from the amebocytes of Limulus polyphemus, the horseshoe crab, are gelled by endotoxin. Studies were carried out to characterize the components of amebocyte lysate and to examine the kinetics of their reaction with endotoxin. Analysis of amebocyte lysate using sucrose density gradients showed two peaks at 46% and 86% gradient volumes. G50 and G75 Sephadex column chromatography resulted in three protein peaks. One fraction contained a clottable protein, which had a molecular weight of approximately 27,000, and was heat stable. Another fraction contained a high molecular weight, heat labile material, which was activated by endotoxin and reacted with the clottable protein to form a gel. The rate of the reaction between endotoxin and amebocyte lysate was dependent upon the concentration of endotoxin and the concentration of the fraction containing the high molecular weight material. The activity of this fraction was inhibited by diisopropyl fluorophosphate, parachloromercuribenzoate, and para-chloromercuriphenyl sulfonate, suggesting that enzymatic activity depended upon serine hydroxyl and sulfhydryl groups. The reaction between endotoxin and the fractions of lysate was temperature and pH dependent. The data suggest that endotoxin activates an enzyme which then gels the clottable protein contained in amebocyte lysate.