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PubMed2003

Physicochemical characterization and biological activity of a glycoglycerolipid from Mycoplasma fermentans.

We report a comprehensive physicochemical characterization of a glycoglycerolipid from Mycoplasma fermentans, MfGl-II, in relation to its bioactivity and compared this with the respective behaviors of phosphatidylcholine (PC) and a bacterial glycolipid, lipopolysaccharide (LPS) from deep rough mutant Salmonella minnesota strain R595. The beta left arrow over right arrow alpha gel-to-liquid crystalline phase transition behavior of the hydrocarbon chains with Tc = 30 degrees C for MfGl-II as well as for LPS exhibits high similarity between the two glycolipids. A lipopolysaccharide-binding protein (LBP)-mediated incorporation into negatively charged liposomes is observed for both glycolipids. The determination of the supramolecular aggregate structure confirms the existence of a mixed unilamellar/cubic structure for MfGl-II, similar to that observed for the lipid A moiety of LPS. The biological data clearly show that MfGl-II is able to induce cytokines such as tumor necrosis factor-alpha (TNF-alpha) in human mononuclear cells, although to a significantly lower degree than LPS. In contrast, in the Limulus amebocyte lysate test, MfGl-II is completely inactive, and in the CHO reporter cell line it does not indicate any reactivity with the Toll-like receptors TLR-2 and -4, in contrast to control lipopeptides and LPS. These data confirm the applicability of our conformational concept of endotoxicity to nonlipid A structures: an amphiphilic molecule with a nonlamellar cubic aggregate structure corresponding to a conical conformation of the single molecules and a sufficiently high negative charge density in the backbone.

Acute-Phase Proteins↗
PubMed1978

Endotoxins in commercial vaccines.

Twenty samples of commercial vaccines intended for administration to humans were assayed for the presence of bacterial endotoxins by using the Limulus amebocyte lysate test. Sixteen of the vaccines contained more than 0.1 ng of endotoxin per ml (which corresponds to 103 bacterial cell wall equivalents per ml in the undiluted vaccines). These results suggest that at some stage of preparation, the vaccines have contained varying amounts of gram-negative bacteria and may indicate the presence of other bacterial products as well. It might be useful to list the level of endotoxins, phage, and other contaminants on each vaccine lot to facilitate studies on any side effects of these contaminants. Selection of vaccine lots with the least endotoxin might reduce some of the adverse effects of vaccinations.

Bacterial Toxins↗
PubMed1999

[The application of the endotoxin test for globulin and other blood products].

The application of the endotoxin test for globulin and other blood products were investigated by two different limulus amebocyte lysate (LAL) test methods, colorimetric and kinetic turbidimetric methods, using two endotoxin-specific reagents. By the dilution of the blood products in 40 times or more, spiked endotoxin in the products was recovered accurately showing neither inhibition nor enhancement. The definite difference was not shown between the results obtained by the two LAL test methods. According to the method of the endotoxin test described under General Tests of The Japanese Pharmacopeia (thirteenth edition), JPXIII, the maximum valid dilution (MVD) for these products will be calculated to be 40 or more, so it is capable to measure the endotoxin limit for each product. This study indicates that the endotoxin test is applicable to measure the endotoxin content in globulin and other blood products as an alternative method for the rabbit pyrogen test.

Colorimetry↗
PubMedDate not supplied

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)

Cells, Cultured↗
PubMed1985

Statistical test of linearity of photoreceptor transduction process: Limulus passes, others fail.

We present the results of a theoretical analysis of a completely general linear chain model for transduction in photoreceptors from which we have derived a statistical test for the intrinsic linearity of the single-photon transduction process. By linearity we mean comprising first-order chemical reactions only. We show results of our own measurements in Limulus ventral photoreceptors that pass this linearity test, suggesting that the single-photon transduction in Limulus may be a simple chain of first-order biochemical reactions (plus possible diffusional processes). However, we also demonstrate that published data show the existence of strong nonlinearities in the single-photon responses of toad and perhaps also of locust. Such nonlinearities are not difficult to construct from existing biochemical notions (feedback, cooperativity), but all but one [Kramer, L. (1975) Biophys. Struct. Mech. 1,239-257] of the published analytical models of the single-photon process have been linear. The test we have used is the distribution of "areas" (time integrals of conductance changes) of single-photon responses or "bumps." Reasonable molecular linear chain models do not allow distributions very sharply peaked at non-zero values. Such peaked distributions are seen in toad and locust but not in Limulus.

Animals↗
PubMed1985

Guideline for validation of the LAL test as an end-product endotoxin test for human and biological drug products.

The draft guideline sets forth acceptable conditions for use of the Limulus Amebocyte Lysate (LAL) test. It also describes procedures for using this methodology as an end-product endotoxin test for human (including biological products) and animal injectable drugs, and medical devices. Manufacturers are allowed to switch from the rabbit pyrogen test to the LAL test, without preclearance from FDA, if they follow the guideline.

Chromogenic Compounds↗
PubMed1997

Lipopolysaccharides and beta-glucuronidase activity in choledochal bile in relation to choledocholithiasis.

Common duct gallstones are mainly of the brown pigment type, which are usually attributed to bacterial factors. Bacterial beta-glucuronidase most probably plays a role in the pathogenesis in many but not all patients. The role of other bacterial factors is more undecided. The aims of this study were to investigate a possible association between lipopolysaccharides (LPS) and choledocholithiasis, and to examine the interrelationship to beta-glucuronidase. Common duct bile obtained at endoscopic retrograde cholangiography in 86 patients was assayed for LPS by a limulus amebocyte lysate test, and beta-glucuronidase activity at pH 7.0 was measured. We found that both elevated concentration of LPS and the presence of juxtapapillary duodenal diverticula were associated with common duct stones (p < 0.01, both). Patients who had their common duct stones removed recently had a lower LPS concentration and a lower activity of beta-glucuronidase than those who had a stone in situ (p < 0.01, both), but still higher LPS concentration than those without choledocholithiasis at all (p < 0.01). In multiple logistic regression analysis, elevated LPS was the significant predictor of common duct stones (p < 0.01), and not confounding with neither beta-glucuronidase nor juxtapapillary diverticula. We conclude that gram-negative bacteria convey bacterial factors associated with choledocholithiasis, by mechanisms independent of, and additional to beta-glucuronidase.

Aged↗
PubMed1988

Lipopolysaccharides in a traditional pertussis vaccine.

Analysis of the lipopolysaccharide (LPS, endotoxin) in cell sonicates of four Danish vaccine strains of Bordetella pertussis (3803, 3825, 3843 and 3860) and of purified strain 3803 LPS in sodium dodecyl sulphate-polyacrylamide gel electrophoresis by silver staining, showed identical profiles. The LPS profile revealed a dominant, brownish LPS II band and a minor, faster-migrating, black-stained LPS I band. However, the ratio of LPS I to LPS II in the preparation of purified LPS differed slightly from the cell sonicates. Using marker LPS, the molecular weights of LPS I and LPS II were estimated at 5.4 and 6.0 kD, respectively. Seven different lots of whole cell pertussis vaccine were assayed for LPS in the Limulus Amoebocyte Lysate test and were found to contain 0.9-2.8 micrograms LPS/ml. No significant difference in the content of LPS in similar dilutions of the individual strains was observed. In addition, the distribution of free and cell-bound LPS in four pertussis vaccines was investigated. Most of the LPS was found to exist as free LPS. During several months, the course of both LPS and pertussis toxin (Pt) release in freshly killed B. pertussis preparations was followed. In the first few weeks, 35-50% of the LPS was released and after 5-6 months of storage 60-80% had been released. In contrast, less than 10% of the biologically active pertussis toxin was released during the experimental period. The possibility of producing a safer whole cell pertussis vaccine by reducing the amount of free LPS without reducing the protective value correspondingly is discussed.

Animals↗
PubMed2004

In vivo effects of bifidobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in Balb/c mice.

The aim of the present study was to examine the effects of oral supplementation of newborn Balb/c mice with bifidobacteria (B. infantis, B. bifidum) and iron-free apo-lactoferrin (bovine, human) on gut endotoxin concentration and mucosal immunity. Endotoxin concentration was measured in ileocecal filtrates at 7, 14, 21, and 28 days postdelivery by a quantitative limulus amebocyte lysate test. While endotoxin levels in bifidobacteria-fed mice showed a steady rise over time, they were consistently lower than that observed in control animals. Results of lactoferrin supplementation varied depending on the specific time point, but overall by day 28, all treatment groups showed lower intestinal endotoxin concentrations compared to saline fed animals. Neither bifidobacteria nor lactoferrin stimulated an increase in B or T cells, or in cytokine production (IL-6, TNF-alpha, INF-gamma), in Peyer's patches as measured by flow cytometry. Bifidobacteria and lactoferrin were well tolerated as dietary supplements and showed promising potential to reduce gut endotoxin levels.

Administration, Oral↗
PubMed1986

Endotoxin and dengue haemorrhagic fever.

Limulus amoebocyte lysate test (LALT) was used to detect endotoxin-like substances in the plasma of 57 patients with dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS), four patients with dengue fever and 20 control patients with other diseases. The LALT positivity rates in DHF/DSS and dengue fever patients were 43.9 and 25 per cent respectively, whereas all control patients were negative (p less than 0.0025). LALT positivity was highest on 5th and 6th days of admission with positive rates of 46 and 50 per cent respectively whereas the positive rates in those admitted on fourth and seventh days of admission were 29 and 33 per cent respectively. A follow-up in LALT positive patients showed a decline in the positive rates after admission. LALT positivity was observed in 48.8 per cent of DHF/DSS patients with shock and in 26.6 per cent of patients without shock.

Adolescent↗
PubMed1986

Microbial filtrates activate granulocytes without complement or prostaglandins.

UNLABELLED: Cardiorespiratory dysfunction in sepsis may be mediated by circulating complement, activated leukocytes, prostaglandins, or by a direct effect of endotoxin. The purposes of this study were to determine if pathogenic microbes produce these substances and to evaluate the direct effects of substances released by micro-organisms on granulocyte aggregation (GA). Escherichia coli, (E. coli), Aeromonas hydrophila (Aeromonas h.), Staphylococcus aureus (S. aureus), and Candida albicans, (Candida a.) were incubated in broth to a concentration of 10(9)/ml. Broth was filtered and analyzed by radioimmunoassay for complement components C3a and C5a, thromboxane B2 (TxB), and prostaglandin 6-keto-F1 alpha (PGI) and by the limulus amebocyte lysate test (LAL) for endotoxin. GA, % of maximum zymosan activated aggregation (% max. T), was performed with broth, microbial filtrates, and endotoxin or normal purified human leukocytes in HBSS. Organisms were incubated in broth (B), broth + 0.0135 mg/ml arachidonic acid (BA), and broth + arachidonic acid + indomethacin (BAI). Broth alone was the control (C). RESULTS: C3a, C5a, TxB, and PGI were not detectable in C broth or in any microbian filtrate. LAL was positive in all filtrates, but negative in C broth. GA responses were significantly greater in E. coli (56 +/- 5% max T) and Aeromonas h. (57% +/- 8% max T) compared to S. aureus (10 +/- 5% max T), Candida a. (14 +/- 8% max T) and C broth (1 +/- 1% max T). GA with purified E. coli endotoxin at concentrations measured in the filtrates was not related to the GA responses the original filtrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria↗
PubMed1999

Identification and characterisation of a cytotoxic porin-lipopolysaccharide complex from Campylobacter jejuni.

A clinical isolate of Campylobacter jejuni, previously found to produce a toxin active in cell culture assays, was used for identification and characterisation of a cytotoxic porin-lipopolysaccharide (LPS) complex. This cytotoxic complex was isolated by high-performance liquid chromatography of crude concentrated culture supernate and DEAE-anion exchange chromatography. The complex had a toxic activity of 20.1 tissue culture dose50 (TCD50)/microg of protein for HEp-2 cells, 7.49 TCD50/microg of protein for HeLa cells and 1.87 TCD50/microg of protein for Chinese hamster ovary cells. Analysis by SDS-PAGE revealed a single protein band of 45 kDa and a high mol. wt carbohydrate moiety. The complex gave a positive result in the Limulus amoebocyte lysate test, indicating that the co-purifying carbohydrate was LPS, and had specificity for the lectins Galanthus nivalis agglutinin, Maackia amurensis agglutinin and Datura stramonium agglutinin. The cytotoxic activity associated with the complex was heat-labile at 70 degrees C, resistant to inactivation with trypsin and retained activity after treatment with sodium metaperiodate and the glycosidases neuraminidase and N-glycosidase F. Sequencing of the N-terminus of the protein component of the complex revealed 97% homology with the major outer-membrane porin protein from C. jejuni. The cytotoxic activity of the complex was neutralised by a polyclonal, homologous antiserum, which reacted on Western blot with the 45-kDa protein, but not by polyclonal antisera raised against a number of other bacterial toxins.

Amino Acid Sequence↗
PubMed1991

Demonstration of an interaction between transferrin and lipopolysaccharide--an in vitro study.

Transferrin is reported to be a major lipopolysaccharide binding protein of human plasma, at least in vitro. By use of the limulus-amebocyte-lysate test the influence of transferrin on endotoxicity was studied. In the absence of any other protein human iron-free transferrin was able to strongly enhance endotoxicity in a concentration-dependent manner. Similar results were obtained when transferrin was added to primarily heat-inactivated plasma. Even in this assay the endotoxin recovery increased when transferrin was exogenously added. On the other hand, transferrin inhibited endotoxicity when inactivation of the plasma samples was performed after the addition of endotoxin and transferrin. These results lead to the conclusion that transferrin in fact interacts with lipopolysaccharide in a biologically important manner. In order to achieve neutralization of endotoxin, however, other plasma constituents are needed. The hypothetical function of transferrin is possibly a disaggregation of lipopolysaccharide micelles, following the interaction between the two molecules. The present data should justify further studies in order to clarify a possible benefit of the substitution of transferrin during gram-negative sepsis.

Blood Physiological Phenomena↗
PubMed2004

Impact of detection of bacterial endotoxin in menstrual effluent on the pregnancy rate in in vitro fertilization and embryo transfer.

OBJECTIVE: To examine whether bacterial endotoxin is detectable in menstrual effluent and to analyze a possible association between endotoxin levels and a pregnancy rate after IVF-ET. DESIGN: Prospective observational study. SETTING: University hospital. PATIENT(S): Thirty-eight infertile women undergoing endotoxin assay and IVF-ET. INTERVENTION(S): Endotoxin was assayed by the limulus amoebocyte lysate test. MAIN OUTCOME MEASURE(S): Levels of bacterial endotoxin and a pregnancy rate. RESULT(S): In 38 samples of menstrual effluent taken from 38 women, bacterial endotoxin was detected with a range of 7.1 to >1,000 pg/mL in 37 samples and was not detected in 1 sample. After IVF-ET, pregnancy occurred in 9 of the 38 women. The mean (+/- SD) endotoxin level in these 9 pregnant women was 71.3 +/- 52.5 pg/mL and was significantly lower compared with >236.2 +/- 333.6 pg/mL in the 29 nonpregnant women. All pregnancies occurred in 28 women with an endotoxin level of </=200 pg/mL, whereas no pregnancies occurred in 10 women with an endotoxin level of >200 pg/mL, producing the significantly higher pregnancy rate in the former group than in the latter. CONCLUSION(S): Bacterial endotoxin was detectable in menstrual effluent from infertile women. The pregnancy rate after IVF-ET was significantly higher in women with an endotoxin level of </=200 pg/mL than in women with an endotoxin level of >200.0 pg/mL.

Adult↗
PubMedDate not supplied

Endotoxin levels in steam and reservoirs of table-top steam sterilizers.

PURPOSE: To document endotoxin levels in "Statim" cassette sterilizer reservoirs and in steam delivered to the cassette in the unwrapped instrument cycle. To document endotoxin levels in sterilizer reservoir water using different management protocols. METHODS: Endotoxin levels were determined using the Limulus Amebocyte Lysate test. Endotoxin preparations were from Escherichia coli and Ralstonia pickettii. All samples were collected in depyrogenated glassware and stored at -20 degrees C until assayed. RESULTS: The majority of water samples contained < 1.0 Endotoxin Unit (EU)/ml. The highest level found in sterilizers in clinical use was 5.3 EU/ml. Endotoxin was not detected in steam condensate within the limits of the assay. When the endotoxin level in the reservoir water was experimentally enhanced to 200 EU/ml, cassette steam condensate endotoxin levels were from 0.5% to 5% of the reservoir level. Daily and weekly emptying of the cassette reservoir consistently yielded low endotoxin levels as did monthly emptying, but with the latter there was a trend toward higher levels that favors weekly emptying as a precautionary measure. CONCLUSIONS: Endotoxin levels in the reservoirs of 23 sterilizers involving 240 samplings were never high enough to yield detectable endotoxin levels in steam in the sterilizer cassette. Regular weekly emptying of sterilizer reservoirs would eliminate the risk of endotoxin transfer during steam sterilization.

Ambulatory Care↗
PubMed1985

Factors contributing to the development of cerebral malaria. II. Endotoxin.

Limulus amoebocyte lysate test (LALT) was used to detect endotoxin-like substances in the plasma of 15 patients with cerebral malaria, 28 patients with uncomplicated falciparum malaria and 30 healthy controls. On admission, 67% of cerebral malaria patients were positive, whereas only 21.4% of uncomplicated malaria patients and none of controls were positive. Among uncomplicated malaria cases, four of eight patients with parasitaemia over 90,000/mm3 were LALT positive whereas only two of 20 patients with parasitaemia of less than 90,000/mm3 were positive. A follow-up study in cerebral malaria patients showed some variation in LALT positivity rate from day to day (85.7% on day 1, 53.3% on day 3 and all negative on discharge from hospital). LALT positivity bore no relationship to gram negative bacteraemia. Leucocytosis and elevated serum enzymes were more frequently found in LALT-positive patients. Our results suggest that endotoxin (LALT positivity) of the plasma of malaria patients is derived from either the parasites themselves or from the gut. It relates to parasitaemia, leucocytosis and elevated serum enzymes, but not to the clinical syndrome of cerebral malaria.

Bilirubin↗
PubMed1995

Influence of various dust sampling and extraction methods on the measurement of airborne endotoxin.

The influence of various filter types and extraction conditions on the quantitation of airborne endotoxin with the Limulus amebocyte lysate test was studied by using airborne dusts sampled in a potato processing plant. Samples were collected with an apparatus designed to provide parallel samples. Data from the parallel-sampling experiment were statistically evaluated by using analysis of variance. In addition, the influence of storage conditions on the detectable endotoxin concentration was investigated by using commercially available lipopolysaccharides (LPS) and endotoxin-containing house dust extracts. The endotoxin extraction efficiency of 0.05% Tween 20 in pyrogen-free water was seven times higher than that of pyrogen-free water only. Two-times-greater amounts of endotoxin were extracted from glass fiber, Teflon, and polycarbonate filters than from cellulose ester filters. The temperature and shaking intensity during extraction were not related to the extraction efficiency. Repeated freeze (-20 degrees C)-and-thaw cycles with commercial LPS reconstituted in pyrogen-free water had a dramatic effect on the detectable endotoxin level. A 25% loss in endotoxin activity per freeze-thaw cycle was observed. Storage of LPS samples for a period of 1 year at 7 degrees C had no effect on the endotoxin level. House dust extracts showed a decrease of about 20% in the endotoxin level after they had been frozen and thawed for a second time. The use of different container materials (borosilicate glass, "soft" glass, and polypropylene) did not result in different endotoxin levels. This study indicates that the assessment of endotoxin exposure may differ considerably between groups when different sampling, extraction, and storage procedures are employed.

Air Pollutants↗
PubMed1987

Role of interleukin-1 in augmenting serum neutralization of bacterial lipopolysaccharide.

We have previously described an assay to quantify the serum neutralization of bacterial lipopolysaccharide which is based on a spectrophotometric Limulus amoebocyte lysate test (T.J. Novitsky, P.F. Roslansky, G.R. Siber, and H.S. Warren, J. Clin. Microbiol. 21:211-216, 1985). Studies since have shown that serum samples drawn from patients with leukemia and fever, gram-negative or gram-positive bacterial infections, or shock caused by gram-negative bacteria neutralize approximately 10-fold more lipopolysaccharide than do samples from normal controls. These findings suggested that the increased neutralization might reflect an acute-phase response and raised the question of whether it might be under the control of interleukin-1. To answer this question, we studied the neutralization of lipopolysaccharide in serum samples drawn from rabbits before and after the administration of crude interleukin-1, prepared from activated macrophage supernatants, and recombinant human interleukin-1. Crude interleukin-1 induced a 5.7-fold increase in serum neutralization 24 h after intravenous injection, and cloned interleukin-1 induced a 3.0-fold increase (P less than or equal to 0.01 and 0.05, respectively). In individual rabbits given identical doses of crude interleukin-1 on a weight basis, the serum-neutralizing ability correlated significantly with three activities of interleukin-1: rise in temperature (r2 = 0.558; P less than or equal to 0.01), decrease in serum iron (r2 = 0.534; P less than or equal to 0.01), and increase in serum copper (r2 = 0.323; P less than or equal to 0.05). We conclude that the increase in neutralization of bacterial lipopolysaccharide by serum samples drawn from patients with inflammatory states is mediated, at least in part, by interleukin-1, presumably through the induction of acute-phase serum proteins.

Animals↗