TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS.

Lipopolysaccharide (LPS), a cell wall component of Gram negative anaerobic bacteria, has been implicated in the pathogenesis of periapical disease resulting from infected root canals. Calcium hydroxide [Ca(OH)2] has been shown to be an effective medicament in such infections, reducing the microbial titre within the canal. It has been proposed that the therapeutic effect of Ca(OH)2 may also be the result of direct inactivation of LPS. The aim of this study was to investigate whether the toxic potential of an Escherichia coli LPS could be reduced or eliminated by Ca(OH)2. Four concentrations of E. coli LPS ranging from 1-1000 ng/ml sterile water were incubated in duplicate either with 25 mg Ca(OH)2 or sterile water alone. Controls consisted of Ca(OH)2 without LPS or sterile water only. Monocytes were collected from peripheral blood by centrifuging through a gradient and plated to a specific density. Adherent monocytes were incubated for 4 days at 37 degrees C with 5% CO2 in M199 medium with 10% autologous serum. The different LPS solutions were added to the wells on day 5. After 4 h the supernatants were collected and quantitatively assayed for TNF-alpha using a commercial ELISA kit. Statistical analysis was performed with ANOVA. Results indicated that Ca(OH)2 is able to eliminate the ability of an E. coli LPS to stimulate TNF-alpha production in peripheral blood monocytes (P < 0.0001).

Specific inhibition of Physarum polycephalum DNA-polymerase-alpha-primase by poly(L-malate) and related polyanions.

Poly(L-malate) is an unusual polyanion found in nuclei of plasmodia of Physarum polycephalum. We have investigated, by enzymatic and fluorimetric methods, whether poly(L-malate) and structurally related polyanions can interact with DNA-polymerase-alpha-primase complex and with histones of P. polycephalum. Poly(L-malate) is found to inhibit the activities of the DNA-polymerase-alpha-primase complex and to bind to histones. The mode of inhibition is competitive with regard to DNA in elongation and noncompetitive in the priming of DNA synthesis. Spermidine, spermine, and histones from P. polycephalum and from calf thymus bind to poly(L-malate) and antagonize the inhibition. The polyanions poly(vinyl sulfate), poly(acrylate), poly(L-malate), poly(D,L-malate), poly(L-aspartate), poly(L-glutamate) have been examined for their potency to inhibit the DNA polymerase. The degree of inhibition is found to depend on the distance between neighboring charges, given by the number of atoms (N) interspaced between them. Poly(L-malate) (N = 5) and poly(D,L-malate) (N = 5) are the most efficient inhibitors, followed by poly(L-aspartate) (N = 6), poly(acrylate) (N = 3), poly(L-glutamate) (N = 8), poly(vinyl sulfate) (N = 3). It is proposed that poly(L-malate) interacts with DNA-polymerase-alpha-primase of P. polycephalum. According to its physical and biochemical properties, poly(L-malate) may alternatively function as a molecular chaperone in nucleosome assembly in the S phase and as both an inhibitor and a stock-piling agent of DNA-polymerase-alpha-primase in the G2 phase and M phase of the plasmodial cell cycle.

Possible effect of secretor locus on plasma concentration of factor VIII and von Willebrand factor.

A significant fraction (30%) of the genetically determined variance in plasma concentration of the von Willebrand factor antigen (vWf:Ag) has been shown to be related to ABH determinants. Individuals with blood group O, who have the highest amounts of blood group H substance, have the lowest concentration of vWf:Ag. The Lewis substances, Le(a) and Le(b), are biochemically closely related to the ABH substances as both can be produced from the same precursor substance. We studied the effect of the presence of the Lewis antigens on the plasma concentration of vWf:Ag and factor VIII antigen (VIII:Ag) in 323 individuals of different ABO groups from a series of twins and in 58 blood donors of blood group O. Among persons belonging to blood group O, those with the Le(a) antigen had a higher concentration of both vWf:Ag and VIII:Ag than individuals lacking Le(a). Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. Thus, the lowest concentration of vWf:Ag and VIII:Ag was found in group O secretors. The effect is most likely due to an effect of the secretor locus. This finding may be of importance for the detection of carriers of hemophilia A and for the diagnosis of type I von Willebrand disease.

Use of monoclonal antibodies in an enzyme immunoassay for factor VIII-related antigen.

Two monoclonal antibodies (MAb 53, MAb D7) were produced, each having specificity for Factor VIII-related antigen (FVIIIR:Ag), but exhibiting no inhibitory effect on either procoagulant activity or the ability of von Willebrand factor to agglutinate platelets in the presence of the antibiotic ristocetin. For quantification of FVIIIR:Ag, we used the antibodies in a competitive enzyme-linked immunosorbent assay (ELISA). Binding of either of the MAb's to solid-phase antigen was inhibited by free FVIIIR:Ag in the test sample. Dose-response curves for the reference standards were consistently linear (r2 greater than 0.990) and reproducible. The normal range of FVIIIR:Ag detected in plasma (normal defined as 1000 units/L) was similar to that reported for polyclonal heterologous antibodies in similar ELISA or immunoradiometric (IRMA) systems, and the assay was sensitive to 10 units of FVIIIR:Ag per liter. Inter- and intra-assay precision was good, coefficients of variation being less than 11%. Studies on patients showed good correlations between values measured by MAb ELISAS and IRMA (polyclonal rabbit antibody) over FVIIIR:Ag concentrations ranging from less than 10 to 2700 units/L (r = 0.971, p less than 0.001 for MAb 53; r = 0.938, p less than 0.001 for MAb D7). Both ELISAS could be used to quantify FVIIIR:Ag in other mammalian species. The assay is inexpensive and simple, and all reagents required for it are commercially available.

Characterization of the catalytic subunit of factor XIII by radioimmunoassay.

Plasma factor XIII is composed of two subunits, a and b, whereas platelet and other intracellular zymogens have only a-subunits. The catalytic subunit, a, is the same in all forms. In order to characterize the interactions of 1- with b-chains in the plasma zymogen and a-chains with other molecules and to correlate factor XIII activity with a-protein, a specific, sensitive radioimmunoassay was developed. With the polyclonal antisera used, the assay recognizes all molecular forms of a (zymogens, activation intermediates, enzyme) equally well. The assay can be used to determine a-chain concentration in plasma and serum and in purified test systems. Fibrinogen in high concentrations affects the assay, probably by interfering with the interactions of 125I-a with antibody. However, at the plasma dilutions used in the assay, there is no significant fibrinogen effect. With this assay, the a-chain concentration in normal plasma is approximately 15 micrograms/ml. This compares with 14 micrograms/ml b-chain in plasma and indicates that all of the a- and b-chains in plasma probably circulate in the form of an equimolar zymogen complex. The serum concentration of a-protein is about 6% of the plasma concentration. There is a high correlation between a-protein and factor XIII activity.

125I radioimmunoassay of delta-9-tetrahydrocannabinol in blood and plasma with a solid-phase second-antibody separation method.

In this sensitive and specific radioimmunoassay for delta-9-tetrahydrocannabinol, an iodinated tracer with high specific activity and a solid-phase separation are used. Within-run coefficients of variation for 5.0 and 30.0 microgram/L concentrations were 7.8 and 4.2% for plasma and 14 and 10.6% for hemolyzed blood specimens, respectively. Day-to-day coefficients of variations ranged from 7.3 to 13.6% (for 7.6 to 33.0 microgram/L concentrations) for plasma and 13.4 to 18.1% (3.0 to 52.1 microgram/L) for hemolyzed blood specimens. Data for time after start of smoking of a standard THC-containing cigarette vs the concentration of delta-9-tetrahydrocannabinol in the plasma were similar to those obtained by others. Positive plasma specimens from the smoking study were analyzed by gas chromatography-mass spectrometry and by our radioimmunoassay. Nonparametric statistical comparison and linear regression (r2 = 0.972) showed that results by the two methods of analysis correlate well. The sensitivity of the assay was at least 0.3 microgram/L for plasma, 1.1 microgram/L for hemolyzed blood.

[Scope and limitations of cranial computerized tomography (author's transl)]. / Möglichkeiten und Grenzen der kranialen Computertomographie

Cranial computerized tomography (CT) has revolutionized the scope of diagnostic procedures in intracranial diseases. The method is based on the quantitative registration of x-ray absorption and enables many different pathological features within the skull to be directly visualized for the first time on the tomogram. Cerebral haemorrhage and encephalomalacia can be immediatley differentiated on the basis of the totally different appearance on CT. In the case of tumours, the additional administration of the contrast medium intravenously provides enhancement of the diagnostic evidence. Atrophic processes can be diagnosed by means of CT without recourse to additional neuroradiological methods and this facilitation is of particular value in neuropaediatrics. Orbital processes can be easily spotted due to the high definiation matrix of image. The limitations of CT are based on physical conditions.

[The place of echoencephalography in neurolgy]. / Die Ultaschallechographie in der Neurologie

A short survey is given of the use of ultrasound examinations in neurology. Special attention is paid to the original immersion technique of Dussik, the A-scan echoencephalographic technique and the non-directional and directional Doppler technique, which are described with particular reference to their applications in the field of neurological diagnosis.