Gelation Of Limulus Amebocyte Lysate Research Articles

A method to prevail false positive responses due to excess cations and viscous nature of Radiopharmaceuticals in Limulus Amebocyte Lysate Gel Clot test

Objective(s):Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized.Methods:GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD.Results:It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes.Conclusions:Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.

Nanoparticle interactions on a cellular scale

Nanoobjects are distinguished from other materials by their nanoscale size. The special physico-chemical properties of nanoobjects attributed to size-effects are the basis for their high application potential. Some of these properties are considered to be the basis for biologically relevant effects of nanoobjects. These are promoted by a) specific entry pathways and distribution within the body and tissues, b) uptake into single cells and specific intracellular distribution, and c) a high chemical reactivity, influencing dissolution, adsorption patterns and kinetics, or agglomeration. To take this even further, the composition and morphology of nanoobjects, their surface properties as well as the composition of the surrounding media influence the interactions of nanoobjects with molecules and living entities. Focussing on the intracellular location of nanoparticles, we are using well-defined and fluorescently labelled nanoparticles. In order to detect cell-associated 25 – 130nm silica particles, confocal and the recently developed STED (STimulated Emission Depletion) microscopy are applied. An introduction into the latter technique, allowing for sub-diffraction resolution, is given and results of microscopic studies are presented. Furthermore, the location of 3 – 15nm gold nanoparticles after uptake into A549 cells as model for type II alveolar cells was studied using light and correlative light and electron microscopy. The distribution pattern of 3nm gold nanoparticles seemed to be different compared with larger nanoparticles. At relevant concentrations, particles induced changes of the cellular morphology, involving the cytoskeleton. Biochemical assays are generally used to analyse nanoparticle effects. Nanotoxicity studies using conventional assays are prone to interference of the particles with the test systems. As an example for direct effects of nanoparticles on such assays, the interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay was analysed. In the presence of particles, not only inhibition, but also enhancement of the test system was observed. The influence of nanoparticle properties on biological assays is discussed.

Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay

Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.

Characterization of the cell surface glycolipid from Spirochaeta aurantia

Spirochaeta aurantia is a free-living saprophytic spirochete that grows easily in simple laboratory media, and thus can be used as a model for the investigation of surface carbohydrate structures in spirochetae, which are normally not available in sufficient amounts. Freeze-substitution electron microscopy indicated the presence of a capsule-like material projecting from the surface of S. aurantia. Extraction of cells gave two major glycolipids, the one with a higher molecular mass glycolipid was designated large glycolipid A (LGLA). LGLA contained small amount of branched and unsaturated O-linked fatty acids, L: -rhamnose, L: -fucose, D: -xylose, D: -mannose, D: -glucosamine, D: -glycero-D: -gluco-heptose (DDglcHep), D: -glycero-D: -manno-heptose (DDHep), and a novel branched tetradeoxydecose monosaccharide, which we proposed to call aurantose (Aur). The carbohydrate structure of LGLA was extremely complex and consisted of the repeating units built of 11 monosaccharides, arrangement of nine of them was determined as: - [- 3 - beta - DDglcHep - 3 - beta - D - GlcNAc - 2 - beta - D - Man - ] - which wasdeduced from the NMR and chemical data on the LGLA and its fragments, obtained by various degradations. Tentative position of two remaining sugars is proposed. LGLA was negative for gelation of Limulus amebocyte lysate, did not contain lipid A, and was unable to activate any known Toll-like receptors.

Prevotella bivia as a source of lipopolysaccharide in the vagina

Objectives To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). Methods Vaginal washes obtained from patients with ( n = 43) and without ( n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. Results The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log 10 CFU/ml, respectively, P < 0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS). The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV. Conclusion P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.

Analysis of Endotoxin Residues on Cleaned Implant Materials

Abstract In this study we have investigated the effect of bacteria-containing metal working fluids on the biological residues, which may be found on orthopedic implants. Thus, test coupons and unfinished implants made from commercial pure titanium and titanium aluminum niob were immersed in metal-working fluids containing Pseudomonas pseudoalcaligenes bacteria. Subsequently, these samples were cleaned by means of a multi-tank industrial cleaning scheme, which is commonly used in the medical device industry. The samples were then analyzed with a rapid screening test for bacteria, a Ps. pseudoalcaligenes, specifically, quantitative polymerase chain reaction (PCR) protocol for DNA detection and the Limulus Amebocyte Lysate gel clot test for endotoxins. None of the test pieces showed any biological residues after the cleaning process. There is no increased risk regarding biological residues for metallic implants that were in contact with bacteria containing metal-working fluids if the devices are properly cleaned. A higher risk for biological contamination would more likely arise from any processes after the final cleaning, i.e., from handling, environmental factors, or packaging of the materials.

Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins

The gelation of Limulus amebocyte lysate (LAL) evoked by bacterial endotoxins can be detected earlier than with usual methods by using laser scattering photometry to recognize the formation of small particles of clotted enzyme produced when the reaction mixture is agitated. The appearance of these small particles means that the influence of endotoxins has stimulated activation of the clotting enzyme across the LAL cascade, and the timing of their appearance is related to endotoxin concentration. This new method can be used for quick and sensitive endotoxin assay. The average endotoxin level of healthy volunteers was assayed to be 0.0738 pg/ml [0.0312–0.3445 pg/ml] ( n = 11) within 70 min from the start of the assay.

Airborne Endotoxin and Its Relationship to Pulmonary Function among Workers in an Indian Jute Mill

Air samples from various processing areas of an Indian jute mill were examined for endotoxin. The authors assessed work-related respiratory symptoms and pulmonary function changes in the dust-exposed workers from the different processing areas using a standard questionnaire and spirometry. Endotoxin was estimated in water extract of jute dust from 3 milling areas, and in outside air, by the Limulus amebocyte lysate gel clot technique. The batching, spinning, and weaving areas of the jute mill showed endotoxin levels of 0.22-4.42 microg/m3, 0.04-1.47 microg/m3, and 0.01-0.07 microg/m3, respectively, values similar to those found in Indian cotton mills. Respiratory morbidities among the workers included typical byssinotic symptoms, along with acute changes in postshift forced expiratory volume in 1 s (FEV1.0) (31.8%). Results of this study demonstrated that increased exposure to bacterial endotoxin in airborne dust is related to byssinotic symptoms among Indian jute mill workers. Findings were similar to those reported previously for workers in the cotton, flax, and hemp industries.

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products.

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.

Biological characterization of Campylobacter fetus lipopolysaccharides

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Biological characterization of Campylobacter fetus lipopolysaccharides.

Lipopolysaccharides (LPS) of three strains of Campylobacter fetus (subspp. fetus and venerealis, and serotypes A and B), a bacterium of veterinary importance but also a cause of various infections in humans, were assessed for their ability to induce mitogenicity, gelation of Limulus amebocyte lysate, lethal toxicity in mice, and pyrogenicity in rabbits. All C. fetus LPS exhibited activities lower than those of Salmonella typhimurium LPS. LPS of C. fetus subsp. fetus serotype A had the lowest activity in all assays. Since the majority of C. fetus subsp. fetus isolates from humans are serotype A, the lower biological activities of this LPS may aid the pathogenesis of such strains. The lower activities of C. fetus LPS compared with those of S. typhimurium LPS may reflect the presence of longer fatty acid chains in the lipid A of C. fetus LPS, whereas interstrain differences in C. fetus LPS bioactivities may be related to some property influenced by composition of the saccharide moiety.

Lipopolysaccharides from periodontal pathogens prime neutrophils for enhanced respiratory burst: differential effect of a synthetic lipid a Precursor IVA(LA‐14‐PP)

When neutrophils are incubated with bacterial lipopolysaccharide (LPS), they become primed for enhanced release of superoxide anion (O2-) in response to stimulation by FMLP. We investigated the human neutrophil-priming activity of LPS from the periodontal pathogens, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Actinobacillus actinomycetemcomitans (Aa) in comparison with that of LPS from Escherichia coli (E. coli). The optimum conditions for LPS to prime neutrophils were assessed for every LPS and found to be as follows: Neutrophils were incubated with LPS in the presence of 10% heat-inactivated plasma and 1 mM EDTA at 37 degrees C for 30 min and then stimulated with 1 microM FMLP at 37 degrees C for 7 min. Under these conditions, half-maximum priming was observed at 6.2 ng/ml Pg-LPS, 45 ng/ml Pi-LPS, 1.5 ng/ml Aa-LPS and 1.5 ng/ml E. coli-LPS. The priming activity of each LPS was neutralized by polymyxin B. Anti-CD14 monoclonal antibody inhibited priming by all LPS. The priming by Aa-LPS and E. coli-LPS was inhibited by LA-14-PP, a synthetic lipid A precursor IVA, but that by Pg-LPS and Pi-LPS was not. Priming by tumor necrosis factor alpha was not affected by polymyxin B, anti-CD14 antibody or LA-14-PP. Gelation of Limulus amebocyte lysate occurred at 10 pg/ml Pg-LPS, 30 pg/ml Pi-LPS, 3 pg/ml Aa-LPS and 3 pg/ml E. coli-LPS. Thus LPS from different periodontal pathogens primed neutrophils with different efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a recombinant endotoxin-neutralizing protein on endotoxin shock in rabbits.

Limulus anti-lipopolysaccharide factor, an 11.8-kilodalton peptide isolated from amebocytes of Limulus polyphemus inhibits the biologic activities of endotoxin in vitro, including gelation of Limulus amebocyte lysate. A recombinant version of Limulus anti-lipopolysaccharide factor, termed endotoxin neutralizing protein, has now been expressed in yeast. Endotoxin-neutralizing protein was evaluated for its potential prophylactic and therapeutic effects in rabbits challenged with Escherichia coli endotoxin. Controlled animal trial. Animal research laboratory. A total of 112 New Zealand white rabbits were studied. Rabbits were challenged with an LD80 dose of E. coli endotoxin (100 micrograms/kg); control animals (n = 52) were treated with saline solution at the time of endotoxin challenge; experimental animals received endotoxin-neutralizing protein 2.5 mg/kg prechallenge (n = 20), 5.0 mg/kg prechallenge (n = 20), or 5.0 mg/kg 30 mins postchallenge (n = 20).

C‐Reactive Proteins, Limunectin, Lipopolysaccharide‐Binding Protein, and Coagulin

In 1964, Levin and Bang discovered that gram-negative bacterial endotoxin could rapidly induce gelation of Limulus amebocyte lysate. This observation has led to the development of the most sensitive and specific method for the detection of bacterial endotoxin in pharmaceuticals and drugs intended for human use. Over 10 years ago, Bang injected endotoxin into young horseshoe crabs and observed a time and dose-dependent coagulation of the whole hemolymph. Limunectin, LEBP-PI, and Limulus CRP are found together with coagulin as part of the hemolymph clot at the time of endotoxin-induced exocytosis of amebocytes. In this manner, these molecules with agglutinin/lectin activities could work in concert to assist in the recognition and eventual removal of invading microorganisms from the circulating system. Although the mechanism of endotoxin-induced clot formation is to a large extent understood, the mechanism of clot dissolution and removal in the Limulus hemolymph remains to be clarified.

Limulus antilipopolysaccharide factor protects rabbits from meningococcal endotoxin shock.

Limulus antilipopolysaccharide factor (LALF), an 11.8-kDa peptide isolated from amebocytes of Limulus polyphemus, neutralizes meningococcal lipooligosaccharide (LOS)-induced gelation of limulus amebocyte lysate. Rabbits challenged with an LD90 of LOS (10 micrograms/kg) premixed with LALF in vitro (n = 10) had significantly higher mean arterial pressure, arterial pH, serum bicarbonate concentrations, and survival (90% vs. 8%, P = .005) than did rabbits challenged with LOS alone. Relative to untreated controls, rabbits pretreated with LALF intravenously (iv) at 1.2 mg/kg (n = 21) also had significant improvements in physiologic measurements and higher survival (52% vs. 8%, P = .003). Even when LALF (1.2 mg/kg iv) was given 1/2 h after LOS challenge, animals showed significant improvements in physiologic measurements and survival (33% vs. 8% in untreated controls P = .028). LALF-treated animals also had significantly lower circulating endotoxin activity and tumor necrosis factor concentrations. Thus, LALF attenuates the toxic effects of meningococcal LOS in rabbits even when administered after LOS challenge and deserves further evaluation as a potential therapeutic agent for treating gram-negative septic shock.

Ability of gonococcal and meningococcal lipooligosaccharides to clot Limulus amebocyte lysate.

We investigated whether the striking difference in severity of coagulopathy observed between bacterial sepsis involving Neisseria meningitidis and Neisseria gonorrhoeae species is related to species-dependent abilities to directly activate coagulation. Using lipooligosaccharide (LOS)-activated gelation of Limulus amebocyte lysate, we compared the relative abilities of outer membrane LOS of 10 N. meningitidis and 10 N. gonorrhoeae strains to initiate coagulation. A wide range of procoagulant potencies was observed for each species, and there was significant overlap of potencies between species. Relative biological activities did not correlate with the oligosaccharide components as defined by LOS molecular weight or specific antigenic epitopes. Purified lipid A of two LOS strains of different potency demonstrated relative procoagulant biological activities similar to those of their parent LOSs. When these lipid A preparations were further separated by thin-layer chromatography, the most polar component of each lipid A possessed the majority of the procoagulant activity. We concluded that the ability of neisserial LOS to initiate coagulation of Limulus lysate is a property of the lipid A portion of the molecule and is most likely determined by fine structural differences in the lipid A which are independent of species.

Viscosity monitoring with a piezoelectric quartz crystal and its application to determination of endotoxin by gelation of limulus amebocyte lysate

A piezo-electric quartz crystal is used to monitor viscosity changes in a liquid. A resistance, included in the electrical equivalent circuit of the crystal, and the resonance frequency change of the crystal, measured by an impedance analyzer, are used for the viscosity measurement. Endotoxin concentrations are determined with this system by the gelation of Limulus amebocyte lysate. The gelation time is computed by using an approximation to a polynomial equation from the resistance or the resonance frequency change. The gelation time measured was in good agreement with that obtained conventionally. The detection limit is 1 pg ml −1. The maximum rate of change of the resistance was confirmed as a good index of endotoxin concentration in order to shorten the measurement time to ⩽ 40 min. The changes in the resistance or resonance frequency were also considered as indices of endotoxin concentration.

Isolation, purification, and partial characterization of a lipopolysaccharide from Haemophilus pleuropneumoniae.

Lipopolysaccharide (LPS) from Haemophilus pleuropneumoniae 1536, serotype 2, was isolated and purified by a procedure designed to be equally satisfactory for both smooth- and rough-type LPS. The LPS yield was 53%. Analysis of the preparations revealed that protein, nucleic acid, and cellular phospholipid contamination was negligible (less than 0.1%). Analysis of the sugar content of the LPS by gas-liquid chromatography and colorimetric analysis revealed the presence of rhamnose, mannose, galactose, glucose, heptose, glucosamine, galactosamine, and 3-deoxy-D-manno-2-octulosonic acid. The heptose and glucose contents appeared to be unusually high. The fatty acids of the LPS consisted of a mixture of C14:0 and C16:0 in a ratio of about 4.5:1 (50% of the total) and 3-hydroxy C14:0. When used as a preparatory dose for the dermal Shwartzman reaction, as little as 10 micrograms of the LPS injected intradermally in rabbits produced reddening and swelling. After intravenous injection of a 100-micrograms LPS provoking dose, necrosis was observed at all intradermal injection sites. Limulus amebocyte lysate gelation was observed with an LPS concentration as low as 0.5 ng/ml. A typical biphasic fever response was noted in rabbits injected with as little as 0.25 ng of LPS per kg of body weight.

Biological activities of chemically synthesized partial structure analogues of lipid A.

Analogues of the nonreducing sugar part of lipid A were chemically synthesized and tested for biological activities such as Limulus amebocyte lysate gelation, interferon- and tumor necrosis factor-induction, lethal toxicity in galactosamine-sensitized mice, and pyrogenicity. A 4-O-monophosphorylglucosamine derivative possessing 2-N-3-tetradecanoyl-oxytetradecanoyl and 3-O-tetradecanoyl groups (GLA-27) exhibited all activities tested except for pyrogenicity. Alteration of the acyl substituents or dephosphorylation as well as acylation or phosphorylation of the 6-OH caused most activities of GLA-27 to diminish or disappear altogether. On the other hand, the biological activities expressed by GLA-27 were not significantly affected even when the glucosamine backbone was changed to 1-deoxy type, epimer type at C-3 (allose form), or 3-amino type. These results indicate that the acyl substituents and the phosphorylation positions rather than the backbone structures in these partial structure analogues of lipid A affect the expression of biological activities of endotoxin. The results also clearly indicate that some biological activities of endotoxin can be expressed separately from pyrogenicity.

Observations on the measurement and evaluation of endotoxemia by a quantitative limulus lysate microassay.

A quantitative microassay for bacterial endotoxin in whole blood, based on Limulus amebocyte lysate gelation and involving the use of a chromogenic substrate, was studied. The lower limit of detection was 10 pg/ml, and the mean level of recovery from plasma spiked with 1 ng of endotoxin/ml was 0.93 ng/ml. At clinically significant levels endotoxin was more easily recovered from plasma than from serum. Platelet binding had no adverse effect on the assay. Despite the use of conventional methods for the removal of nonspecific activators and inhibitors, simultaneous assay of six specimens spiked with the same amount of endotoxin resulted in variation in recovery by greater than 100%. These experiments may explain in part the conflicting results of studies of endotoxemia in humans.