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PubMed1976

Comparative trial of influenza vaccines. II. Adverse reactions in children and adults.

Commercially prepared zonally and chromatographically purified bivalent (A/England-B/Mass) and monovalent (B/Hong Kong) inactivated influenza vaccines were given to 438 individuals 6-33 years old. The vaccines had been examined for antigen content by chick cell agglutination (CCA) tests and electron microscopic particle count determinations. Endotoxin and pyrogen content were determined by limulus amebocyte lysate (LAL) and rabbit pyrogenicity assays; and egg-associated protein contamination was estimated by total protein and single radial immunodiffusion assays. Although great differences (10-200-fold) were found in the amount of endotoxin or pyrogen in the vaccines, no significant differences were found in the febrile responses they induced. Both bivalent and monovalent vaccines induced fever of greater than or equal to 38 C at a rate of approximately 3 1/2-4% above background. The febrile responses were most frequent at 24 hours after inoculation and a higher rate was observed in children than adults. Local reactions consisting of tenderness, erythema or induration were seen in from 20-57% of the recipients and also were unrelated to the pyrogenic or host-derived materials in the vaccines. Adults had higher local reaction rates than children and some vaccines containing larger amounts of viral antigen induced significantly higher rates of reactivity than did vaccines containing smaller amounts of antigen. Although 37-51% of all recipients experienced either a local and/or febrile reaction to influenza immunization, the reactions were in general mild and would not consitute a significant disadvantage in the immunization of children over 6 years and adults to prevent influenza infection and its sequelae.

Adolescent↗
PubMed2001

Influence of in-vivo endotoxin liberation on anti-anaerobic antimicrobial efficacy.

The ability of cefoxitin, clindamycin, imipenem, meropenem, metronidazole and piperacillin-tazobactam to cause gram-negative anaerobic bacteria to release endotoxin and the influence of such liberated endotoxin on antibiotic efficacy were investigated in in-vivo experiments in animal models. Experimental infections in various animal models (mice, hamster and infant rats) with cultures of wild and reference strains of Bacteroides fragilis group and Fusobacterium spp. were carried out by injecting these animals with different inocula (10(6), 10(7) and 10(8) cfu/ml) of the bacterial suspension, Appropriate doses of the test antibiotics were then injected and the plasma lipopolysaccharide (endotoxin) release measured by the Limulus Amoebocyte Lysate (LAL) Assay. Evidence of worsening of the outcome of the infections post-therapy was assessed, including histopathological changes in the internal organs. Infection with generalized septicemia was established with F. nucleatum in the mice and hamster models while with the B. fragilis group, infections only led to intra-abdominal abscess formation. Plasma endotoxin release was higher in animals infected with F. nucleatum than B. fragilis and was unrelated to the bacterial inoculum. Imipenem, meropenem and cefoxitin, in that order, induced the highest levels of endotoxin activities in the animal model, particularly following F. nucleatum infection. Histological examination of the internal organs of various animals showed variation in the pattern of histopathological changes; grades 3-4 inflammatory changes in the liver were observed in the Fusobacterium-infected animals that were treated with the carbapenems and cefoxitin. Therapy with the other antibiotics did not exacerbate anaerobic sepsis. In this study, bacteremia did not lead to massive endotoxin release and antibiotic therapy appeared not to have negatively influenced the outcome of most of the gram-negative anaerobic infections, except for infections caused by Fusobacterium spp. However, it is conceivable that if the gastrointestinal tract is the source of the endotoxin in patients with systemic inflammatory response syndrome, then the obligate anaerobes like Bacteroides and Fusobacterium species, which are members of the gut flora, may play a major role in the unfavorable outcome of antibiotic therapy in some of these infections.

Animals↗
PubMed2003

Evaluation of endotoxin content of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines that interfere with the bacterial endotoxin test.

Applicability of the endotoxin test to diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines was examined. We found some DTaP vaccines that strongly interfered with Limulus amoebocyte lysate (LAL) activity of endotoxin without affecting lethal activity of endotoxin in D-galactosamine-treated mice. LAL activity that was interfered in such vaccines increased apparently after the treatment with phosphate buffer at 4 degrees C for a week. The DTaP vaccines that interfered with the endotoxin test showed no significant effect on endotoxin activity in inducing tumor necrosis factor-alpha (TNF-alpha) in rabbit peripheral blood. The in vitro TNF-alpha induction assay was, therefore, suggested to be an appropriate assay method for the quantitative detection of the endotoxin activity in DTaP vaccines.

Animals↗
PubMed1979

Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis.

Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU-e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.

Adsorption↗
PubMed1980

Biological and chemical characterization of endotoxin from Capnocytophaga sputigena.

An endotoxin was isolated from Capnocytophaga sputigena strain 4 by a modification of the hot phenol-water method. The extraction procedure yielded a lipopolysaccharide which accounted for approximately 1.5% of the dry weight of the cells. The material was composed of 18.6% lipid (as C(15) fatty acid), 46.5% neutral sugar including 9.6% hexose, 18.3% 6-deoxy sugar, 1.0% 2-keto-3-deoxy sugar, and 4.8% heptose. Hexosamine, protein, and phosphorus were found in quantities amounting to 9.0, 2.9, and 2.0% of the dry weight, respectively. No pentose or nucleic acid was detected. Acid hydrolysis resulted in the release of the constituent sugars and the formation of an insoluble precipitate. The lipopolysaccharide was tested for numerous biological activities characteristic of endotoxins. The pyrogenicity was relatively low; the fever index 40 was 17 mug, and 10 mug was required to give the characteristic biphasic fever response. The toxicity of the extract was very low, with a 50% chicken embryo lethal dose of 15.6 mug and a 50% mouse embryo lethal dose of greater than 8 mg. Similarly, the C. sputigena endotoxin had modest effects on leukocytes when compared with endotoxin standards from other organisms. The extract exhibited little or no mitogenicity when tested on mouse spleen lymphocytes. It was not toxic to human peripheral polymorphonuclear leukocytes and caused the release of only a small (13%) portion of lysosomal enzymes. Although the C. sputigena lipopolysaccharide caused significant activation of mouse peritoneal macrophages, the dose required was twice that of an Escherichia coli endotoxic standard. However, the Limulus amoebocyte lysate clotting activity of the lipopolysaccharide was comparable to that of an Serratia marcescens lipopolysaccharide standard, and passive hemagglutination tests revealed that 1 mug of the lipopolysaccharide was capable of sensitizing 1 ml of a 2% sheep erythrocyte suspension for agglutination with an antiserum prepared against C. sputigena whole cells.

Animals↗
PubMed1984

Differentiation between Trypanosoma cruzi and Trypanosoma rangeli on the basis of their sialic acid content.

Four day-old culture forms of Trypanosoma rangeli strain San Augustin were tested with the following lectins: Canavalia ensiformis, Pisum sativum, Ricinus communis-120, Sojahispida, Aaptos papillata II, Triticum vulgaris, Limulus polyphemus, lberis amara and Arachis hypogaea. These flagellates were only agglutinated by the lectins from Canavalia ensiformis and Pisum sativum. Furthermore four day-old culture forms of the T. cruzi strains Morcego 1354, P-60, Téhuantépéc and WA 301/130 as well as the T. rangli strains San Augustin, Venezuela and DA-3412 were treated with neuraminidase and the supernatant examined by the Aminoff TBA assay. While the supernatant of the T. cruzi strains showed a red colour complex the supernatant of the T. rangeli strains was weak yellow-green. In contrast to the T. cruzi strains no N-acetylneuraminic acid was detectable on the cell surface of T. rangeli. Therefore the Aminoff test can be used for the differentiation between T. cruzi and T. rangeli.

Agglutination↗
PubMed1999

Functionalized nanoparticles for endotoxin binding in aqueous solutions.

Nanoparticles consisting of a polystyrene core and a polyglycidyl methacrylate shell were prepared by a two-step emulsion polymerization. The size and surface properties of the particles were characterized by scanning electron microscopy, dynamic light scattering and polyelectrolyte titration techniques. Particles were found to be monodisperse with a mean diameter of about 85 nm. Parent particles were modified with a number of different ligands including diamines of increasing chain length, amino acids and corresponding amines and higher molecular weight ligands like polymyxin B. The modified particles were tested for their endotoxin (ET) binding capacity in water and physiological sodium chloride solution with the Limulus amebocyte lysate (LAL) assay. It was found that the ET binding properties of the different ligands depend both on the ability of the ligand to form Coulomb- and van der Waals-interactions with the ET molecule influenced by the nature of the suspension medium. Therefore, the choice of ligands for particle modification has to consider minutely the conditions under which ET has to be removed, e.g. removal from pure water, dialysis fluids, plasma or blood.

Biocompatible Materials↗
PubMed1959

The additivity of contrast in the human eye.

Using the method of binocular brightness matching, simultaneous brightness contrast effects were measured on two observers. The effects of a given pattern were invariably smaller than the summation of the effects of the pattern's components. This failure of additivity was valid both for patterns with isolated components as well as for those with components exactly contiguous with one another. This failure was more pronounced the farther the inducing patterns were from the test patch. These findings are interpreted as indicating that in the human (just as in the Limulus) eye, the amount of inhibition exerted by a given region on its neighbors depends upon the inhibition exerted against it as well as its excitation state.

Humans↗
PubMed1985

Endotoxinemia and thrombocytopenia during neonatal necrotizing enterocolitis.

Thrombocytopenia frequently complicates neonatal necrotizing enterocolitis (NEC) and has been postulated to result from absorption of bacterial endotoxins from the injured gut. The authors tested blood obtained during 47 episodes of NEC for endotoxin-like activity (ELA), using a Limulus amoebocyte lysate assay and found 23 patients (49%) had positive results. Concentrations of ELA in plasma ranged from 0.26 to 300 ng/mL of Escherichia coli equivalent activity, with a geometric mean of 1.1 ng/mL. Serial platelet measurements were available from 40 infants, 11 (28%) of whom had nadir counts below 100,000/mm3 following NEC onset. Nine of 19 infants (47%) with ELA in plasma and only 2 of 21 without (9.5%, P less than 0.05) developed thrombocytopenia, suggesting that endotoxinemia may indeed contribute to platelet depletion during NEC.

Ascitic Fluid↗
PubMed1992

Rapid detection of gram-negative endotoxin contamination of contact lens saline solutions.

The use of the limulus amoebocyte lysate for the early and rapid detection of gram-negative endotoxin contamination of contact lenses and their solutions could reduce the risk of a keratitis developing that is associated with these devices. Using multiple aliquots from 17 unopened brands of commercially available contact lens saline solutions (15 for soft, two for hard), plus multiple aliquots from these solutions mixed with bacterial endotoxin, we evaluated the ability of two limulus amoebocyte lysate (0.125 endotoxin units/mL) products to detect the presence or absence of gram-negative endotoxin contamination. Sensitivity ranged from 65% (11/17) to 82% (14/17) when the solutions were tested undiluted. When diluted 1:8, the sensitivity increased to 100%. Specificity was 100% for undiluted and diluted specimens. The solutions used for hard contact lenses had the highest false-negative results. The limulus amoebocyte lysate can be used to detect the presence of gram-negative endotoxin in contact lens solutions.

Antimicrobial Cationic Peptides↗
PubMed1982

The excitatory actions of a series of piperidine dicarboxylates on central neurons of the rat, snail, leech and horseshoe crab and on crab neuromuscular junction.

1. Intracellular recordings were made from Helix, Hirudo and Limulus central neurons and from Eupagurus leg muscle and extracellular recordings from rat lateral geniculate neurons. The actions of 2,3-, 2,4-, 2,5- and 2,6-piperidine dicarboxylates were tested on those preparations for both direct effects and interactions with the response to L-glutamate. 2. All four piperidine analogues were devoid of either reproducible direct or indirect effects on Helix and Limulus neurons save that the 2,5-analogue did mimic the action of L-glutamate on Limulus neurons which were inhibited by glutamate. 3. On Hirudo and rat neurons and on Eupagurus muscle, all four piperidines mimicked the action of L-glutamate, all were weak agonists on rat neurones while on Hirudo neurons the 2,5-analogue was the most potent but on Eupagurus muscle, the 2,3- and 2,6-analogues were the most potent. 4. All four piperidines potentiated the action of L-glutamate on rat and Hirudo neurons and on Eupagurus leg muscle and of muscle ejps. 5. The piperidine analogues indicate that there are differences between the glutamate receptors used in the present study and provide some supportive evidence that the preferred conformation for glutamate on crab leg muscle is partially folded while for Hirudo neurons it is more extended.

Animals↗
PubMed1973

Rapid detection of contaminated intravenous fluids using the Limulus in vitro endotoxin assay.

Intravenous fluids and administration sets may become contaminated with gram-negative bacteria during use and result in a life-threatening situation to the patient. The Limulus in vitro assay for endotoxin was used in two patients whose parenteral fluids had become contaminated with Pseudomonas aeruginosa. This test allowed rapid detection of the contaminated intravenous fluids and demonstrated a concomitant endotoxemia in both patients. The same strains of pseudomon were subsequently cultured from each patient's blood, intravenous catheter tip, and parenteral fluid and administration set. A different serotype of pseudomonas was unique to each patient, indicating two separate and unrelated cases of accidental contamination of the administration sets. Endotoxin-like activity was also demonstrated from several brands of commercial human serum albumin, which may contribute low-level activity detectable by the Limulus assay.

Bacteriological Techniques↗
PubMed1977

Enhancement and phototransduction in the ventral eye of limulus.

Limulus ventral photoreceptors were voltage clamped to the resting (dark) potential and stimulated by a 20-ms test flash and a 1-s conditioning flash. At a constant level of adaptation, we measured the response to the test flash given in the dark (control) and the incremental response produced when the test flash occurred within the duration of the conditioning flash. The incremental response is defined as the response to the conditioning and test flashes minus the response to the conditioning flash given alone. When the test flash was presented within 100 ms after the onset of the conditioning flash we observed that: (a) for dim conditioning flashes the incremental response equaled the control response; (b) for intermediate intensity conditioning flashes the incremental response was greater than the control response (we refer to this as enhancement); (c) for high intensity conditioning flashes the incremental response nearly equaled the control response. Using 10-mum diam spots of illumnination, we stimulated two spatially separate regions of one photoreceptor. When the test flash and the conditioning flash were presented to the same region, enhancement was present; but when the flashes were applied to separate regions, enhancement was nearly absent. This result indicates that enhancement is localized to the region of illumination. We discuss mechanisms that may account for enhancement.

Action Potentials↗
PubMed2001

Interfering effect of diphtheria-tetanus-acellular pertussis combined (DTaP) vaccines on the bacterial endotoxin test.

We evaluated applicability of the endotoxin test to DTaP vaccines. Although no DTaP vaccine batches showed immediate interference with the activating activity of spiked endotoxin on the clotting of Limulus amaebocyte lysate (LAL), a gradually progressing interference depending on the time after spiking was seen for some of the batches. The interfering DTaP vaccine batches, however, showed no significant effect onin vitro TNF-alpha induction in RAW264.7 cells and pyrogenicity in rabbits of spiked endotoxin. Aluminium hydroxide gel in the vaccines was suggested to be one of the causes of the interference. Accordingly, a careful evaluation of the interfering effect was assumed crucial for the effective application of the endotoxin test to check residual biological activity of endotoxin in DTaP vaccines.

Animals↗
PubMed2001

Detrimental effects of nitric oxide inhibition on hepatic encephalopathy in rats with thioacetamide-induced fulminant hepatic failure.

Hepatic encephalopathy is a complex neuropsychiatric syndrome seen secondary to acute liver failure, chronic parenchymal liver disease, or portal-systemic anastomosis. Vasodilatation induced by nitric oxide (NO) may be involved in the development of hepatic coma. However, there are no comprehensive data concerning the effects of NO inhibition on the severity of hepatic encephalopathy. Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg kg-1 day-1) for 3 days. Rats were divided into two groups to receive either NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 day-1 via intragastric gavage) or normal saline (N/S) from 2 days prior to TAA administration for 5 days. Severity of encephalopathy was assessed by counts of motor activity and neurobehaviour test scores. Plasma levels of endotoxin, tumour necrosis factor-alpha and nitrate/nitrite were determined by the chromogenic Limulus assay, enzyme-linked immunosorbent assay and colorimetric assay, respectively. Compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving L-NAME (59% vs. 18%, P < 0.01). Inhibition of NO had detrimental effects on the counts of motor activities (P < 0.05) and neurobehaviour score (P < 0.01). Rats treated with L-NAME had significantly higher plasma levels of endotoxin (26.7 +/- 3.8 pg mL-1) and tumour necrosis factor-alpha (29.4 +/- 6.5 pg mL-1) compared with rats treated with N/S (13.2 +/- 2.7 pg mL-1 and 11.2 +/- 2.6 pg mL-1, respectively, P < 0.01). Plasma levels of endotoxin and tumour necrosis factor-alpha, but not of nitrate/nitrite, were significantly correlated with the severity of hepatic encephalopathy (P < 0.05). Chronic L-NAME administration had detrimental effects on the severity of encephalopathy in TAA-treated rats, suggesting a protective role of NO in the development of fulminant hepatic failure.

Animals↗
PubMed1958

Spatial summation of inhibitory influences in the eye of Limulus, and the mutual interaction of receptor units.

The inhibitory influences exerted mutually among the receptor units (ommatidia) of the lateral eye of Limulus are additive. If two groups of receptors are illuminated together the total inhibition they exert on a "test receptor" near them (decrease in the frequency of its nerve impulse discharge in response to light) depends on the combined inhibitory influences exerted by the two groups. If the two groups are widely separated in the eye, their total inhibitory effect on the test receptor equals the sum of the inhibitory effects they each produce separately. If they are close enough together to interact, their effect when acting together is usually less than the sum of their separate effects, since each group inhibits the activity of the other and hence reduces its inhibitory influence. However, the test receptor, or a small group illuminated with it, may interact with the two groups and affect the net inhibitory action. A variety of quantitative effects have been observed for different configurations of three such groups of receptors. The activity of a population of n interacting elements is described by a set of n simultaneous equations, linear in the frequencies of the receptor elements involved. Applied to three interacting receptors or receptor groups equations are derived that account quantitatively for the variety of effects observed in the various experimental configurations of retinal illumination used.

Action Potentials↗