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PubMed1988

Repeatability of interview-derived socio-demographic and medical information.

In this report, the repeatability of socio-demographic and medical information elicited by means of an interviewer-administered questionnaire is examined. One hundred and seven women who were controls in case-control studies of breast disease were re-interviewed approximately one year after initial interview. Both interviews addressed the same time period, namely that which preceded the first interview. For most of the quantitative variables studied, at least 70% of individuals provided two estimates (original and repeat) which were within one unit of each other. Also, comparison of the categorization of individuals on repeated measurements of qualitative variables (reports of events, operations and habits) revealed that most individuals were categorized consistently. This strong agreement between the original and repeat estimates was also reflected in the relatively high values of the summary measures of agreement (the intraclass correlation coefficient for quantitative variables and the Kappa statistic for qualitative variables) for most of the variables studied. The results of the present study on repeatability complement the results of previous research into the criterion validity of questionnaire-derived information and, taken together, suggest that information of relatively high quality may be obtained from such sources.

Breast Diseases↗
PubMed1982

Measurement of circulating immune complexes in human sera by enzyme immunoassay.

Two methods, based on enzyme immunoassay, are described for the detection and quantitation of circulating immune complexes (CIC) in human sera. Aggregated human gamma globulin (AHG) or immune complexes in human sera are bound to complement receptors on Raji cells or to Clq adsorbed on to the plastic surface of microtitre plates. The bound complexes are subsequently detected using peroxidase-conjugated anti-human immunoglobulin antisera. The assays offer a benefit over previously described assays in that they use cheap, commercially available antisera and enable the detection of immune complexes composed of varying immunoglobulin classes.

Animals↗
PubMed1998

PRIM: proximity imaging of green fluorescent protein-tagged polypeptides.

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

Amino Acid Sequence↗
PubMed1988

The initiator tRNA acceptance assay as a short-term test for carcinogens. 1. A standardized procedure.

A short-term test for carcinogens has been developed based on the interaction of chemical carcinogens with tRNA(FMet) in vitro. Transfer RNA from rat or rabbit liver is pre-treated with compounds to be tested in the presence of microsomal enzymes and NADPH. Re-isolated tRNA is then charged with L-methionine by aminoacyl-tRNA synthetases from E. coli B. Carcinogens induce a stimulation of tRNA charging whereas chemically similar non-carcinogenic compounds do not show this effect. Experiments with model substances N-methyl-N'-nitro-N-nitrosoguanidine (strong carcinogen) and aflatoxin G1 (weak carcinogen) revealed some differences in dose effect relationships. It is advisable to test unknown compounds at three different concentrations (10(-5), 10(-7) and 10(-9) mg/ml) with at least two different quantities of microsomal enzymes. Tests on greater than 150 different compounds performed so far indicate that the evaluation of results as % of stimulation (when compared with the control value obtained with the charging of tRNA treated with the solvent only) may allow a quantitative discrimination between weak and intermediate, and strong carcinogens. The procedure is rapid, well reproducible and relatively inexpensive and may be used to complement the other short-term tests for carcinogenicity.

Animals↗
PubMedDate not supplied

Distribution of zeranol in bovine tissues determined by selected ion monitoring capillary gas chromatography/mass spectrometry.

Bovine tissues, including liver, muscle, kidney, bile, serum, and urine, have been quantified by selected ion monitoring capillary gas chromatography/mass spectrometry to establish the distribution of the anabolic drug, zeranol, and its metabolites, taleranol and zearalanone, after administration of zeranol to 9 bovine animals. The method used to isolate, confirm, and quantify zeranol is undergoing validation by the United States Department of Agriculture, Food Safety Inspection Service (FSIS). Application of this method demonstrates utility in determining residue levels of zeranol in a variety of tissues with levels ranging over 4 orders of magnitude (i.e., 100 parts per trillion (ppt) to 1 part per million (ppm]. The analyte levels determined in this study complement previously reported pharmacokinetic data on the distribution of zeranol in addition to providing more specific information for taleranol and zearalanone. In this quantitative study it is shown that the liver is the main organ of deposition for zeranol, taleranol, and zearalanone, that taleranol is the main metabolite in the bovine, and that zeranol is efficiently eliminated.

Animals↗
PubMed1995

Improving out-patient clinic waiting times: methodological and substantive issues.

In the Patients' Charter, a standard is laid down that all patients who attend out-patient clinics should be seen within 30 minutes of their appointment time. Discusses how a statistical monitoring system using a "low technology" approach was implemented at Leicester General Hospital during 1992-93. This was instrumental in raising the proportion of patients seen within 30 minutes from under 50 per cent to over 80 per cent in 15 months. Points to the fact that statistical monitoring alone is not sufficient to deliver quality improvements. Suggests that only enlightened management action which brings both management and clinicians into a quality improvement programme is likely to be effective. Discusses some of the measurement problems involved. Warns against the fact that over-reliance on purely quantitative measures (as indicated in "Charter" standards) to the neglect of more qualitative measures may be counterproductive. Suggests that qualitative approaches need to complement the normal statistical monitoring.

Appointments and Schedules↗
PubMed2004

Disruption of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen leads to abortive episome persistence.

Latent nuclear antigen (LNA) is implicated in Kaposi's sarcoma-associated herpesvirus (KSHV) episome persistence. LNA colocalizes with KSHV episomes on chromosomes in metaphase, and it maintains the stability and replication of KSHV terminal repeat-containing plasmids. In this study, we examined the function of LNA in episome persistence in the context of full-length KSHV genome by mutagenesis analysis. We generated a KSHV mutant, BAC36-DeltaLNA, with LNA disrupted by transposon-based mutagenesis with a KSHV BAC clone, BAC36, as a template. Immunofluorescence antibody staining revealed that the insertion of a transposon cassette into LNA disrupted its expression but had no effect on the expression of two adjacent genes, the vCyclin and vFLIP genes. Using a green fluorescent protein (GFP) cassette as a tracking marker for the KSHV episome, we found 8.7-fold-fewer GFP-positive cells in BAC36-DeltaLNA cultures than in wild-type BAC36 cultures at the early stage following episome delivery into 293 cells by transfection, which could be partially rescued by cotransfection with a LNA expression plasmid but not a control plasmid. Cells harboring BAC36-DeltaLNA with or without transient complementation rapidly lost episomes and became virus-free after 2 weeks of culture based on GFP expression and Gardella gel analysis and quantitative PCR assays for detecting KSHV genomes. In contrast, BAC36 episomes were stably maintained during the same period. Stable cultures with close to 100% of cells harboring KSHV episomes were readily established by hygromycin selection for BAC36 but not for BAC36-DeltaLNA. These results conclusively indicate that LNA is essential for the establishment and persistence of KSHV episomes in mammalian cells.

Antigens, Viral↗
PubMed2002

Intracranial mass lesions: dynamic contrast-enhanced susceptibility-weighted echo-planar perfusion MR imaging.

Dynamic contrast agent-enhanced perfusion magnetic resonance (MR) imaging provides physiologic information that complements the anatomic information available with conventional MR imaging. Analysis of dynamic data from perfusion MR imaging, based on tracer kinetic theory, yields quantitative estimates of cerebral blood volume that reflect the underlying microvasculature and angiogenesis. Perfusion MR imaging is a fast and robust imaging technique that is increasingly used as a research tool to help evaluate and understand intracranial disease processes and as a clinical tool to help diagnose, manage, and understand intracranial mass lesions. With the increasing number of applications of perfusion MR imaging, it is important to understand the principles underlying the technique. In this review, the essential underlying physics and methods of dynamic contrast-enhanced susceptibility-weighted echo-planar perfusion MR imaging are described. The clinical applications of cerebral blood volume maps obtained with perfusion MR imaging in the differential diagnosis of intracranial mass lesions, as well as the pitfalls and limitations of the technique, are discussed. Emphasis is on the clinical role of perfusion MR imaging in providing insight into the underlying pathophysiology of cerebral microcirculation.

Brain Diseases↗
PubMed1999

[Risk increase and economic consequences of the introduction of contagious animal diseases in the Netherlands].

The paper describes the development of the Monte Carlo simulation model VIRiS (Virus Introduction Risk Simulation) and the results obtained with this model. VIRiS simulates the introduction of Classical Swine Fever (CSF) and Foot and Mouth Disease (FMD) into the Netherlands. The model is based on objective information (research, databases), complemented by expert knowledge. Various questionnaire techniques (Conjoint Analysis, ELI) were used in order to elicit the experts' estimates in an objective and quantitative way. VIRiS provides information on the number of primary outbreaks in a certain period, their geographic location, causative risk factor, and causative country (or region). The information provided by VIRiS in combination with the outcome of models describing the spread and economic consequences of epidemics provides a tool that can be used to evaluate prevention strategies for their ability to reduce annual losses due to outbreaks.

Animals↗
PubMedDate not supplied

Quantitative evaluation of 99mTc-hexamethylpropylenamineoxime brain SPECT in childhood-onset epilepsy.

In 10 patients with childhood-onset epilepsy, quantitative evaluation (QE) of interictal regional cerebral blood flow (rCBF) using technetium-99m-hexamethylpropylenamine oxime (99mTc HMPAO) single-photon emission computed tomography (SPECT) was correlated with seizure foci localized by electroencephalography (EEG). Eighty regions of interest (5 x 5 pixels) were placed at five different neocortical areas in both cerebral hemispheres, in coronal and transaxial planes. Mean counts/pixel were calculated for each region and multiple right-left comparisons were done. Quantitative evaluation of 99mTc-HMPAO increased detection of rCBF abnormalities by 26%. Abnormal EEG patterns correlated with abnormal rCBF in 75% of cases (Phi = 0.788, p < 0.001), and with visually detected abnormalities in 50% (Phi = 0.493, p < 0.001). Discordance between EEG and SPECT was most frequent in patients with non-localizing EEG (n = 3). In childhood-onset epilepsy, quantitative evaluation of brain perfusion scans provides: (a) objective estimates of interictal rCBF; (b) more precise detection of epileptogenic foci, when used to complement EEG.

Adolescent↗
PubMed1985

Electrical charge of the antigen determines its localization in the mouse knee joint. Deep penetration of cationic BSA in hyaline articular cartilage.

Intraarticular injection of cationic bovine serum albumin (BSA) induces a chronic arthritis in immunized mice, whereas the negatively charged native BSA fails to cause a protracted joint inflammation. In this study the authors examined the role of antigenic charge as a determinant of antigen retention and exact localization within the knee joint. Immune and nonimmune mice received an intraarticular injection of either radiolabeled native BSA (125I-BSA) or charge-modified BSA rendered cationic by amidation (aBSA), and autoradiographs were prepared of whole joint sections at various days after injection. As has been shown in the rabbit, the retention of the negatively charged native BSA is largely dependent upon the presence of antibodies. In nonimmune mice the radiolabeled antigen was hardly detectable after Day 1. In immune mice antibody-mediated retention of BSA was found in the ligaments and fibrous cartilage structures of the joint but appeared to be absent at the hyaline cartilage. In contrast, large amounts of the cationic aBSA were retained at all collagenous structures of the joint, the most striking observation being the deep penetration in the dense hyaline cartilage. This was found both in immune and nonimmune mice, which indicates that the deep penetration was not due to cartilage damage occurring under inflammatory conditions. With different dosages of aBSA it was found that the presence of antibodies may modulate the retention pattern in immune mice. Deep diffuse penetration into the dense hyaline cartilage, together with some surface labeling, was observed after injection of a high dose (60 micrograms), whereas mere surface labeling was found with the low dose (6 micrograms). Distinct superficial labeling was not seen in nonimmune mice, which suggests that this pattern represents immune complex formation at the cartilage surface. Immunofluorescence studies on undecalcified whole joint sections confirmed the deep penetration of the cationic antigen and supported the presence of immune complexes at the cartilage surface, because intense complement and Ig staining was detectable at this site. Our data indicate that antigenic charge determines the antigen retention in the joint both quantitatively and qualitatively. Negatively charged native BSA has no affinity for cartilage, high amounts of antibodies are needed for its retention in the joint, retention by this immune complex formation is largely restricted to the loose collagenous tissues, and the capacity to retain anionic antigen in the joint is therefore low.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Specific anti-erythrocyte focus formation as a measure of autoantibody secreting cells in NZB mice.

Using a modification of the antibody forming cell (AFC) focus assay, it is possible to quantitate the spontaneous anti-erythrocyte autoantibody response of New Zealand Black (NZB) mice at the level of the autoantibody secreting cell. This complement independent assay is specific for an antigen(s) present on unmodified mouse erythrocytes. A comparison with the direct Coombs' test showed that the focus assay detected autoantibody responses earlier and demonstrated a wide range of AFC levels in responding mice. The focus assay provides a method for evaluating autoimmunity at the cellular level and for investigating the activities of cells responding to autoantigens.

Anemia, Hemolytic, Autoimmune↗
PubMed1997

Issues in the comparison of risk estimates for the population in the Techa River region and atomic bomb survivors.

Plutonium production in the former Soviet Union began in 1949 at the Mayak Production Association located between the cities of Chelyabinsk and Ekaterinbourg in the southern Ural mountains about 1200 km east to Moscow. During the first few years of Mayak's operation, almost 30,000 people living on the banks of the Techa River received significant internal and external exposures as a consequence of the release of large quantities of radioactive materials from Mayak. Studies of levels of radioactive contamination and health effects in this population began in the early 1950s. A systematic follow-up of a fixed cohort that includes all people who were living in Techa River villages in 1949 was begun about 30 years ago. In this paper we describe the Techa River cohort, outline the nature of the exposures and discuss the status of follow-up for the period from 1950 through 1989. While noting the limitations of the current epidemiological follow-up data, we also compare the demographic and mortality structure of the Techa River cohort with the Life Span Study cohort of Japanese atomic bomb survivors. It is seen that, despite a number of limitations, the current data suggest that the risks of mortality from leukemia and other cancers increase with increasing radiation dose in the Techa River cohort. This finding suggests that, with continued improvements in the quality of the follow-up and dosimetry, the Techa River cohort has the potential to provide quantitative estimates of the risks of chronic low-dose-rate radiation exposures for an unselected general population that will be an important complement to the estimates based on the Life Span Study that are used as the primary basis for numerical assessments of radiation risk.

Environmental Exposure↗
PubMed1984

Expression of Ia antigens on macrophages is reduced after stimulation with homologous C3b.

The complement cleavage product C3b was examined for its ability to influence the expression of Ia antigens on macrophages. Monoclonal anti-Ia antibodies were applied for quantitation of Ia antigens by using FACS analysis. Albumin-elicited guinea pig peritoneal macrophages were cultured for 48 hr in the presence of C3b. C3b at final concentrations varying from 10 to 80 micrograms/ml reduced the proportion of Ia-positive macrophages from 87 to 25%. The C3b-stimulated loss of Ia antigens from the macrophage surface was mediated by metabolites of the cyclooxygenase pathway as shown by the inhibitory effect of indomethacin. Exposure of macrophages to C3b for 5 min was sufficient to induce Ia reduction, seen after subsequent 48-hr incubation. C3b generated in vivo in the course of inflammatory reactions might therefore influence the interaction of immunocompetent cells by modulating macrophage Ia expression.

Animals↗
PubMed2006

Measurement by ELISA of complement factor 4 (C4) in the rat brain: necessity for removal of cerebrovascular proteins.

Assessment of complement 4 (C4) levels in experimental animals is used as a marker for activation of the classical complement pathway. The objective of this study was to develop a method for measuring C4 concentrations in the rat brain. An ELISA (sensitivity = 0.5 ng C4/ml) was used to measure C4 in regional brain homogenates from Fischer rats cardiac-perfused with phosphate buffered saline to remove cerebrovascular contents, and from sham-perfused rats. Ventral midbrain C4 levels were increased (p < 0.001) versus frontal cortex and striatum in sham-perfused rats, whereas after perfusion there were no differences between brain regions. Removal of cerebrovascular contents decreased C4 by 43% in striatum, 52% in frontal cortex, and 69% in ventral midbrain (all p < 0.01 versus sham-perfused means). These results indicate that C4 in the rat brain can be measured quantitatively by ELISA provided that cerebrovascular proteins are removed by perfusion.

Animals↗
PubMed1999

Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm.

The thiol/disulfide oxidoreductase DsbA is the strongest oxidant of the thioredoxin superfamily and is required for efficient disulfide bond formation in the periplasm of Escherichia coli. To determine the importance of the redox potential of the final oxidant in periplasmic protein folding, we have investigated the ability of the most reducing thiol/disulfide oxidoreductase, E.coli thioredoxin, of complementing DsbA deficiency when secreted to the periplasm. In addition, we secreted thioredoxin variants with increased redox potentials as well as the catalytic a-domain of human protein disulfide isomerase (PDI) to the periplasm. While secreted wild-type thioredoxin and the most reducing thioredoxin variant could not replace DsbA, all more oxidizing thioredoxin variants as well as the PDI a-domain could complement DsbA deficiency in a DsbB-dependent manner. There is an excellent agreement between the activity of the secreted thioredoxin variants in vivo and their ability to oxidize polypeptides fast and quantitatively in vitro. We conclude that the redox potential of the direct oxidant of folding proteins and in particular its reactivity towards reduced polypeptides are crucial for efficient oxidative protein folding in the bacterial periplasm.

Bacterial Proteins↗
PubMed1989

Analysis of complement factor B activation by PAGE-immunoblotting.

Initiation of complement activation via the alternative pathway results in cleavage of Factor B into two fragments Ba and Bb. Employing PAGE and immunoblotting analysis we have demonstrated Ba and Bb activation fragments following in vitro and in vivo activation in a range of biological fluids including cerebrospinal fluid and synovial fluid. Quantitative linearity was obtained over the range 5-20 micrograms/ml of Factor B and its fragments in body fluids. Using Davis-PAGE a previously unrecognised triple component heterogeneity of the Ba fragment was observed. This study demonstrates that immunoblotting may be used as a sensitive and quantitative tool for the detection and characterisation of the individual cleavage products of Factor B in complex biological fluids without prior purification.

Cardiopulmonary Bypass↗
PubMed1984

Inhibition of C9 polymerization within the SC5b-9 complex of complement by S-protein.

The effect of S-protein on the polymerization of C9 during assembly of the C5b-9 complex was examined. Utilizing SDS polyacrylamide gradient slab gel electrophoresis, tubular poly C9 was quantitated as SDS resistant protein of 1.1 to 1.3 X 10(6) molecular weight. Poly C9 formation occurred upon incubation of purified C5b-6, C7, C8 and C9 at molar ratios 1:1:1:12. Addition of purified S-protein to the protein mixture or to preassembled C5b-7 or C5b-8 blocked formation of poly C9 in a dose dependent fashion and gave rise to SC5b-9. SC5b-9 assembled from purified proteins or in zymosan-activated serum was visualized in the electron microscope as a wedge-shaped structure of 350 to 400 A length and 30 to 250 A width which lacked tubular poly C9 seen in images of the membrane attack complex (MAC). Using biotinyl-S-protein and colloidal gold particles coated with avidin, S-protein was located at the wide end of the wedge-like SC5b-9 complex. It is concluded that S-protein has a dual function in SC5b-9 assembly. It blocks the membrane site of C5b-7 and it inhibits C9 polymerization by SC5b-8. Accordingly, the main structural difference between SC5b-9 and the MAC is the lack of tubular poly C9.

Binding Sites↗