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PubMed1973

Thermal sensitivity of lateral inhibition in Limulus eye.

The effectiveness of lateral inhibition, measured as spike response decrement in a test ommatidium, produced by activity in a group of neighboring ommatidia, decreases as temperature decreases (Q(10) of 2.6). The corresponding sensory transducer-spike encoding processes have a weaker temperature dependence (Q(10) of 1.6). Relative synaptic delay, the time difference between the latency of inhibition onset and the latency of test receptor excitation, has a strong temperature dependence (Q(10) of 5), while receptor potential onset latency (Q(10) of 1.4) and optic nerve spike conduction velocity (Q(10) of 1.7), two factors inherent in relative synaptic delay, are less temperature sensitive. Oscillations of optic nerve spike response ("bursting") may be produced by thermal adjustment of temperature-sensitive parameters of the lateral inhibitory network in the retina. Burst interval has a strong temperature dependence (Q(10) of 2.4) and broad interspike interval distribution compared to the thermal sensitivity (Q(10) of 1.4) and narrow spike interval spectrum of the response of a single unit within the bursting group.

Action Potentials↗
PubMed1986

Uridine-induced hyperthermia in the rabbit.

Uridine injection in 0.6% saline elevated rabbit temperatures (mean = 0.9 degree C) in the USP XX pyrogen test. Hyperthermia was delayed in onset and peaking 3-4 h post injection, but the injection was negative in the limulus amoebocyte lysate (LAL) assay. Uridine from five lots of different sources exceeded USP XX limits in the rabbit pyrogen test and proved negative in the LAL assay. Because the dose of uridine was high, several procedures were used to determine if an impurity was the cause of temperature elevation. Uridine remained pyrogenic in spite of ultrafiltration (10 000 nominal mol. wt), recrystallization and preparative scale HPLC. Sterile filtration and autoclaving also did not affect the response. Hyperthermia, therefore, appears to be an inherent property of uridine. Uridine was also found to release endogenous pyrogen in-vitro from human mononuclear cells. Uridine has been reported to induce fever in man, thus the USP rabbit pyrogen test predicted for the clinical response.

Animals↗
PubMed1982

Growth of nosocomial pathogens in multiple-dose parenteral medication vials.

The extent to which microbial contamination of medications dispensed in multiple-dose vials might serve as a source of infection to patients has not been fully investigated. To characterize the effects of microbial contamination, we studied the growth-supporting properties of eight medications dispensed in multiple-dose vials. Two medications, procainamide and methohexital, demonstrated no survival of any microbes 24 h after inoculation. Succinylcholine chloride, regular insulin, potassium chloride, heparin, and thiopental slowly killed or allowed limited survival of several of the microorganisms used as contaminants. Lidocaine allowed survival or proliferation of several microbial strains suspended in 0.25% peptone water in saline, but slowly killed all strains except Pseudomonas cepacia suspended in 0.9% saline. Endotoxin, measured by the Limulus amebocyte lysate assay, was found in the two medications tested, lidocaine contaminated with Pseudomonas cepacia and insulin contaminated with enterococci. Inadvertent microbial contamination of at least some parenteral medications in multiple-dose vials may result in the exposure of patients to viable organisms. The potential, however, for medications such as lidocaine to support growth of organisms under selected circumstances should be noted by those responsible for preparing and administering these drugs. The potential hazard to patients from endotoxin in contaminated medications under these circumstances has not been assessed. Additional studies of this type should be pursued to provide more complete information about the risk of microbial contamination of products for parenteral use.

Bacteria↗
PubMed1972

Activation of the adenosine triphosphatase of Limulus polyphemus actomyosin by tropomyosin.

Purified actin does not stimulate the adenosine triphosphatase (ATPase) activity of Limulus myosin greatly. The ATPase activity of such reconstituted preparations is only about one-fourth the ATPase of myofibrils or of natural actomyosin. Actin preparations containing tropomyosin, however, activate Limulus myosin fully. Both the tropomyosin and the actin preparations appear to be pure when tested by different techniques. Tropomyosin combines with actin but not with myosin and full activation is reached at a tropomyosin-to-actin ratio likely to be present in muscle. Tropomyosin and actin of several different animals stimulate the ATPase of Limulus myosin. Tropomyosin, however, is not required for the ATPases of scallop and rabbit myosin which are fully activated by pure actin alone. Evidence is presented that Limulus myosin, in the presence of ATP at low ionic strength, has a higher affinity for actin modified by tropomyosin than for pure actin.

Actins↗
PubMed2001

Some inflammation-related parameters in patients following normo- and hypothermic cardio-pulmonary bypass.

AIMS: One of the complications of Cardio-Pulmonary Bypass is the Systemic Inflammatory Response Syndrome. Cardio-Pulmonary Bypass can be performed under either normothermic or hypothermic conditions. The aim of this study was to compare some inflammation-related parameters of patients following normothermic and hypothermic bypass. Moreover, attempts were undertaken to detect endotoxin, an inflammatory agent that has been implicated in the Systemic Inflammatory Response Syndrome, in the serum of patients. Levels of serum anti-endotoxin antibodies were estimated since they have been reported to negate the effect of endotoxin in the inflammatory syndrome. METHODS AND RESULTS: Seventeen normothermic and 20 hypothermic cases were studied. Blood specimens were collected pre-, off- and post-bypass. Pertinent clinical and surgical data were collected. Hematological parameters (leukocyte, neutrophil and platelet counts) and liver function tests were determined by standard procedures. Endotoxin was determined by the Limulus Lysate Assay and anti-endotoxin antibodies by an enzyme immunoassay. Complement (C3 and C4) levels were determined by radial immunodiffusion. There were increases in leukocyte and neutrophil, and a decline in platelet numbers in both groups of patients. There was a decline in C3 and C4 levels in both groups of patients. Endotoxin was not detected in sera, and anti-endotoxin antibody levels were similar, in both groups of patients. CONCLUSION: There were no significant differences in most of the altered inflammation-related parameters between the two groups of patients. Some of the findings might be partly due to hemo-dilution. The hydrophobic nature of endotoxin among other factors, might have hindered its detection in serum.

Adult↗
PubMedDate not supplied

Endotoxin concentration in treated water of all hemodialysis units in Greece and inquisition of influencing factors.

Mains and purified water samples from all hemodialysis centers in Greece were surveyed to assess hemodialysis water quality and the potential consequences on the dialysis population of any microbial and endotoxin contamination. Total heterotrophic bacteria counts were estimated employing the pour-plate method, the membrane filter technique, for total coliforms, fecal coliforms, enterococci and Pseudomonas spp., and the most probable number method for sulfite-reducing clostridia. Overall 170 paired samples were tested. Endotoxin concentrations were assayed in treated water samples, by the Limulus Amebocyte Lysate method, employing a chromogenic substrate. Endotoxin concentration varied from 0-30 EU/ml and exceeded 5 EU/ml in 30.6% of the centers. Endotoxin contaminated samples showed higher counts of total heterotrophic bacteria, fecal coliforms, enterococci and Pseudomonas spp. than the rest of the samples, though the difference was significant only for total heterotrophic bacteria and enterococci. Additionally, samples with endotoxin contamination derived from the mains water had significantly higher counts of fecal coliforms, enterococci and Pseudomonas spp. Endotoxin concentration was correlated with the presence of a deionization system, counts of fecal coliforms in supply water and counts of total coliforms, fecal coliforms and enterococci in treated water. These results demonstrate that hemodialysis centers in Greece need to monitor and preventively maintain the entire hemodialysis system, in order to ensure renal replacement therapy of good quality.

Endotoxins↗
PubMed2003

Removal of bacterial endotoxin from root surface with Er:YAG laser.

PURPOSE: To determine the potential of 2.94 microm Er:YAG laser radiation to remove bacterial endotoxin from root surfaces. MATERIALS AND METHODS: 40 extracted teeth were divided into four groups of 10 samples each. A 16 mm2 area of the root surface on each sample was inoculated with an aliquot of 7 microl of a lipopolysaccharide suspension at a concentration of 50 IU/ml. LPS was derived from a non-oral Escherichia coli strain (E. coli 055:B5). Source of laser radiation was an Er:YAG laser emitting pulsed infrared radiation at a wavelength of 2.94 microm, with a pulse duration of 250 micros, and a pulse repetition rate of 15 pps. Three specimen groups were irradiated with 105 laser pulses at a radiation energy of 60 mJ, 100 mJ and 140 mJ. One specimen group was untreated (control). The LPS concentration with each sample was determined using a chromogenic, quantitative Limulus-amoebocyte-lysate assay. Statistical analysis was ANOVA and Scheffé-test. RESULTS: Mean LPS yield from the untreated control samples was 50.1 (+/- 35.9) IU/ml. Following laser irradiation the average LPS on the root surfaces was 19.86 (+/- 14.4) IU/ml at 60 mJ, 12.86 (+/- 8.1) IU/ml at 100 mJ and 8.58 (+/- 4.9) IU/ml at 140 mJ.

Decontamination↗
PubMed1982

Gram-negative pathogens in septicaemic infections.

For the last two decades, gram-negative rod bacteremia has been the major infectious disease problem developing in patients hospitalized in the United States and many European countries. Mortality rates are high and have exceeded 30% in patients with serious underlying diseases. The most commonly isolated organisms are Escherichia coli, Klebsiella-Enterobacter-Serratia, and Pseudomonas aeruginosa but the latter organism has consistently been associated with the highest case fatality ratios. Stool sampling surveys in normal individuals reveal low carriage rates of both K. Pneumoniae and Pseudomonas, but prospective studies in neutropenic patients using the surveillance culture technique has indicated a higher prevalence of gastrointestinal colonization. In neutropenic patients asymptomatic colonization may occur weeks prior to onset of gram-negative rod bacteremia. The patients most likely to develop systemic infection are neutropenic subjects who have P. aeruginosa colonizing the gastrointestinal tract. Microbial pathogenetic factors associated with gram-negative bacillary infections are poorly understood. Antigens such as the O and K antigens appear to confer the property of resistance to phagocytosis. Both antibody deficiency and restricted activation of the alternative complement pathway are responsible for impaired phagocytosis of some bacteremic strains in both fresh normal human serum as well as in the serum or plasma of bacteremic patients. A number of studies have shown a direct correlation between type-specific and cross-reacting serum antibody levels with survival. Additionally, Exotoxin A of P. aeruginosa is a likely virulence factors of this species and levels of humoral antitoxic antibodies measured at onset of bacteremia correlate with improved prognosis. Antibodies to capsular antigens like K-1 antigen of E. coli enhance phagocytosis of strains with different O types. Despite a substantial amount of circumstantial evidence, a role for circulating toxins or other candidate virulence factors has not bee convincingly established in human sepsis. Bioassays like those involving the limulus lysate gelation have identified some patients with endotoxemia, but this test may be negative if circulating immune complexes are present. Additionally, other factors in serum may inactivate endotoxins of complex with endotoxins. Several human and experimental studies suggest that prophylaxis or treatment of gram-negative infections may be effective with antiserum against endotoxins antigens. The high mortality from gram-negative bacteremia in some patients strongly justifies further research in this area.

Cross Infection↗
PubMed1982

Induction of macrophage-mediated tumor lysis by an animal lectin, Sarcophaga peregrina agglutinin.

Several animal lectins, such as Sarcophaga peregrina agglutinin, Limulus polyhemus agglutinin, Helix pomatia agglutinin and Helix aspersa agglutinin, were tested for induction of tumor lysis mediated by macrophages. Among them, only S. peregrina agglutinin purified from the hemolymph of S. peregrina larvae lysed tumor cells in co-operation with macrophages from the peritoneal cavity of mice. S. peregrina agglutinin alone did not kill target tumor cells. Macrophages in the presence of this lectin could kill other syngeneic tumor cells. This lectin-dependent cytolysis by macrophages was inhibited by galactose, a sugar which is specifically recognized by S. peregrina agglutinin. These findings suggest that the animal lectin S. peregrina agglutinin is a ligand in macrophage-mediated cytolysis, inducing binding of effector macrophages to target cells which in turn triggers off lysis of the target cells.

Agglutinins↗
PubMed1988

Circadian rhythms in adaptation to light of Limulus photoreception.

1. The lateral eye of Limulus responds differently to light-adaptation depending upon whether the eye is tested during the day, or at night when a central circadian clock is known to activate efferent fibers that terminate in the retina. 2. At night, the decrement in retinal sensitivity due to light-adaptation is less pronounced immediately following a light-flash but is greater at longer times after the flash when compared with responses recorded during the day. 3. In our quantitative description of dark adaptation we find that at least two time constants are needed to describe the time course, and that the two decaying exponentials are differentially altered by circadian clock action. 4. We relate these day-to-night alterations in light-adaptive properties to circadian changes in physiology and morphology studied previously in Limulus photoreceptors.

Adaptation, Physiological↗
PubMed1988

Laboratory aids in the diagnosis of bacterial meningitis.

Bacterial meningitis is a uncommon but serious infectious disease. Critical care personnel have a great deal of difficulty in differentiating aseptic meningitis from partially treated meningitis from bacterial meningitis. Such a differentiation has obvious implications for therapy and prognosis. This article provides guidelines in using clinical criteria as well as cerebrospinal fluid parameters to diagnosis the commonly encountered infectious causes of meningitis. The current interpretation of laboratory tests such as countercurrent immunoelectrophoresis, latex agglutination, enzyme-linked immunosorbent assays, limulus assays, and lactic acid levels are discussed.

Bacterial Infections↗
PubMed2004

Cytoskeletal organization of limulus amebocytes pre- and post-activation: comparative aspects.

One of the major functions of circulating Limulus amebocytes is to effect blood coagulation upon receipt of appropriate signals. However, the hypothesis that Limulus amebocytes are fundamentally similar to vertebrate thrombocytes and platelets has not been tested sufficiently in previous studies of their cytoskeletal organization. Whereas the earlier data were derived from transmission electron microscopy (TEM) of thin sections of a limited number of cells, improved fluorescence labeling methods that retain cell morphology have now enabled us to survey F-actin and microtubule organization in intact individual amebocytes and in large amebocyte populations pre- and post-activation. Anti-tubulin immunofluorescence showed the marginal band (MB) of microtubules to be ellipsoidal in most unactivated cells, with essentially no other microtubules present. However, minor subpopulations of cells with discoidal or pointed shape, containing corresponding arrangements of microtubules suggestive of morphogenetic intermediates, were also observed. Texas-red phalloidin labeled an F-actin-rich cortex in unactivated amebocytes, accounting for MB and granule separation from the plasma membrane as visualized in TEM thin sections, and supporting earlier models for MB maintenance of flattened amebocyte morphology by pressure against a cortical layer. Shape transformation after activation by bacterial lipopolysaccharide was attributable principally to spiky and spreading F-actin in outer cell regions, with the MB changing to twisted, nuclei-associated forms and eventually becoming unrecognizable. These major pre- and post-activation cytoskeletal features resemble those of platelets and non-mammalian vertebrate thrombocytes, supporting recognition of the Limulus amebocyte as a representative evolutionary precursor of more specialized clotting cell types.

Actins↗
PubMedDate not supplied

Production, purification and analysis of an experimental DNA vaccine against rabies.

BACKGROUND: The basic and applied research efforts devoted to the development of DNA vaccines must be accompanied by manufacturing processes capable of being scaled up and delivering a clinical-grade product. This work describes a rapid process of this kind, based on hydrophobic interaction chromatography (HIC) for the production of milligram quantities of an experimental DNA rabies vaccine. Its properties and protective activity are tested in comparison with the same plasmid DNA purified with a commercial kit. METHODS: The experimental DNA vaccine encoding the rabies virus glycoprotein was amplified in vivo in Escherichia coli. The plasmid was isolated by alkaline lysis, pre-purified and concentrated by isopropanol and (NH4)2SO4 precipitation, and purified by HIC and dialysis. Product quality was controlled by using high-performance liquid chromatography (HPLC), Southern slot blotting, agarose gel electrophoresis, the kinetic-QCL Limulus amoebocyte lysate assay, and protein assays. The expression of the rabies virus glycoprotein was tested in vitro in neuroblastoma cells. The production of rabies-virus-neutralising antibodies and the protection against an intracerebral virus challenge were tested in mice. RESULTS: One hundred and forty-two milligrams of the plasmid, with an HPLC purity greater than 99% were obtained from 4.5 l medium. Control analysis showed that the vaccine conforms to specifications in terms of impurities (endotoxins, genomic DNA, RNA, proteins). Furthermore, the final experimental vaccine induces rabies-virus-neutralising antibodies and protects mice against a rabies virus challenge. CONCLUSIONS: This study demonstrates that the method developed for the purification of milligram amounts of plasmid delivers an endotoxin-free, experimental rabies DNA vaccine, with protective activity similar to that obtained with the vaccine purified using a commercial kit.

Animals↗
PubMedDate not supplied

[Endotoxemia and phagocytic function in premature subjects].

The AA. have evaluated some parameters of leukocyte function (phagocytosis, killing and nitroblu tetrazolium NBT test) from premature children, comparing such activities with endotoxemia, detected by means of limulus assay. The results obtained point out that there is not relationship between impaired leukocyte functions and endotoxemia.

Endotoxins↗
PubMed2007

In vitro pyrogen test--A new test method for solid medical devices.

Medical devices manufactured for implantation into humans must be free of any contamination with viable bacteria. However, remnants of dead bacteria and bacterial components alone may induce an inflammatory immune response. Pyrogen tests for such inflammatory contaminations are generally performed either by determining the content of lipopolysaccharide in rinsing solutions of batch samples by limulus amoebocyte lysate assay, by injecting the rinsing solutions into rabbits or by implanting batch samples into rabbits and measuring change of body temperature. In this study, we show that the in vitro pyrogen test (IPT), which measures the release of the inflammatory cytokine IL-1beta in fresh or cryopreserved human whole blood, can be used to assess the pyrogenic contamination of implantable medical devices. This test was used to check neurosurgical implants, namely aneurysm clips, as a proof of principle. Owing to the direct contact of the test material with the blood cells, this test does not require rinsing procedures, which have variable efficacy. The use of human blood ensures the detection of all substances that are pyrogenic for humans and reflects their relative potency. The safety of the products as delivered could be confirmed. The effects of sterilization and depyrogenization procedures on intentional pyrogenic contaminations of samples could be followed. This new application of the already internationally validated method promises to replace further rabbit pyrogen tests. It generates extremely sensitive results with an extended range of detectable pyrogenic contaminants.

Equipment and Supplies↗
PubMed1984

Two latent period processes in Limulus ventral photoreceptors.

The latent period of Limulus ventral photoreceptor potentials consists of two contiguous time segments, S1 and S2, which exhibit the following properties: (1) the duration of S1 is inversely dependent on the incident energy provided by the first stimulating light flash; (2) the duration of S1 is prolonged in the presence of GTP-gamma-S [guanosine-5'-0- (3-thiotriphosphate)] or papaverine; (3) the duration of S1 is abbreviated in the presence of sodium vanadate or chlorobutanol; (4) the duration of S2 is abbreviated by light adaptation; and (5) the duration of S2 is prolonged more than S1 in a low temperature environment. The existence of these two components of the Limulus ventral photoreceptor potential latent period is demonstrable both in dark adapted photoreceptors, using single constant intensity light pulses of varying duration, or in partially light adapted photoreceptors, using a stimulating conditioning pulse-test pulse sequence. On the basis of these results it is tentatively concluded that the two segments of the latent period of Limulus ventral photoreceptor potentials are occupied by different processes. The first process, occupying the S1 segment, generates a critical concentration of a photoproduct which, directly or indirectly, eventually alters the conductance of the active photoreceptor membrane. The second process, occupying the S2 segment, is concerned with the initiation of the SPFs which sum to produce the receptor potential. It is possible that the hydrolysis of GTP actively participates in the first process.

Aminophylline↗
PubMed1983

Polyphemin: a teichoic acid-binding lectin from the horseshoe crab, Limulus Polyphemus.

A Staphylococcus aureus-agglutinating lectin, capable of binding to N-acetyl-D-glucosamine, was isolated from the serum of Limulus polyphemus. The monosaccharide alone was incapable of inhibiting bacterial agglutination by this lectin. Quantitative precipitation studies with purified cell wall-derived teichoic acids, either devoid of or containing N-acetyl-D-glucosamine, confirmed the carbohydrate-binding specificity of the lectin and suggested that secondary, non-specific interactions contribute to binding biomolecules containing this sugar. The agglutination pattern with various S. aureus strains having N-acetyl-D-glucosamine-associated teichoic acid, teichoic acid without this sugar, and no teichoic acid indicated that this cell wall component is not the sole binding site for the lectin on intact S. aureus cells. Affinity gel chromatography, using N-acetyl-D-glucosamine-associated teichoic acid as the specific absorbent, has been used to isolate this lectin from Limulus serum.

Agglutination Tests↗