Quantitative in vitro assay of cytotoxic cellular immunity.
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Animals↗
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Results for “quantitative complementation”
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Quantitation of proenzyme C1r-C1s complexes in serum by electroimmunoassay.
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Adult↗
[Quantitative determination of circulating antivascular antibodies in atherosclerosis by the autocomplement consumption method].
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Aorta↗
The interest of multimodality evoked potentials in the evaluation of chronic coma.
The clinical usefulness of three-modality evoked potentials (TMEPs) has been widely demonstrated in acute coma, but is still a matter of debate in the persistent vegetative state. We observed that TMEPs in vegetative patients could be situated along a continuum, one extremity of which corresponds to absent cortical, but preserved brain-stem activities, and the other extremity to major abnormalities of somatosensory conductions at the midbrain level, but variable involvement of auditory pontine and visual cortical pathways. The former pattern is most often associated with ischemic-anoxic encephalopathy, while the latter is most often consecutive to head trauma. However, all intermediate states may be observed. TMEPs can usefully complement the clinical evaluation in answering two questions. The first one concerns the chances of subsequent improvement that are lower in the absence of quantitative TMEP evolution, but are likely to be higher in the presence of non-fixed mesencephalic dysfunction associated with a relative preservation of cortical function. The second question concerns the evaluation of remaining cognitive abilities that are more likely to be present if only mild abnormalities of cortical activities are found and if some cognitive EPs are still obtained.
Adult↗
A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b-9 complex enables selective detection and quantification of non-activated C9.
Recently, monoclonal antibodies recognizing epitopes exposed in activation products of complement but hidden in the native components have been characterized and used for selective quantification of the activated protein. We now demonstrate that an epitope in the native component C9 is hidden in the terminal SC5b-9 complex. A monoclonal antibody against this epitope enabled selective detection of C9 without influence of the amount of SC5b-9 present. This antibody recognizing native soluble C9 was used to construct a quantitative double-antibody ELISA with unique sensitivity and specificity. Combination of this assay with an assay previously described for selective quantification of the SC5b-9 complex provides an important tool for evaluating terminal pathway activation of complement.
Animals↗
Candidate quantitative trait loci and naturally occurring phenotypic variation for bristle number in Drosophila melanogaster: the Delta-Hairless gene region.
Delta (Dl) and Hairless (H) are two chromosome 3 candidate neurogenic loci that might contribute to naturally occurring quantitative variation for sensory bristle number. To evaluate this hypothesis, we assessed quantitative genetic variation in abdominal and sternopleural bristle numbers among homozygous isogenic third chromosomes sampled from nature and substituted into the Samarkand (Sam) inbred chromosome 1 and 2 background; among homozygous lines in which the wild-derived Dl-H gene region was introgressed into the Sam chromosome 3 background; and among Dl-H region introgression lines as heterozygotes against the Sam wild-type strain and derivatives of Sam into which mutant Dl and H alleles had been introgressed. Variation among the Dl-H region introgression lines accounted for 36% (8.3%) of the total chromosome 3 among line variance in abdominal (sternopleural) bristle number and for 53% of the chromosome 3 sex x line variance in abdominal bristle number. Naturally occurring alleles in the Dl-H region failed to complement a Dl mutant allele for female abdominal bristle number and sternopleural bristle number in both sexes, and an H mutant allele for both bristle traits in males and females. These results are consistent with the hypothesis that naturally occurring alleles at Dl and H contribute to quantitative genetic variation in sensory bristle number.
Analysis of Variance↗
C5 convertase of the alternative pathway of complement. Kinetic analysis of the free and surface-bound forms of the enzyme.
Although proteolytic activation of the complement protein C5 initiates important defensive and occasionally pathological inflammatory reactions, the enzymatic properties of the enzymes responsible for this cleavage have never been examined. We have studied the kinetic parameters of the C5 convertase of the alternative pathway of complement, either bound to a zymosan surface or in its monomeric soluble form. C5 convertase enzymatic activity was measured as a function of C5 concentration by quantitating production of C5b,6 under physiological conditions of temperature, pH, and ionic strength. The C5 convertases appeared to follow Michaelis-Menten kinetics and exhibited similar catalytic rate constants (kcat). However, the surface-bound enzyme, ZymC3b,Bb had a Km (1.4 microM) that was 17 times lower than that of the soluble monomeric form of the enzyme, C3b,Bb (Km = 24 microM). The kcat for the cell-bound enzyme, ZymC3b,Bb was 0.0048 s-1 and that for soluble C3b,Bb was 0.0110 s-1. Both forms of the enzyme had a low turnover number at Vmax (0.23 to 0.68 C5/min/enzyme). Substituting Mg2+ for Ni2+ did not alter the kinetic parameters but lowered the half-life of the enzyme by 5-7-fold. The kinetic data presented demonstrate that the fluid phase C5 convertase, C3b,Bb, can cleave C5 without the aid of a second C3b molecule. The results also show that the greater enzymatic activity previously observed for the surface-bound C5 convertases is not due to higher catalytic efficiency but is solely due to higher affinity for the substrate C5. In blood, C5 concentrations are 3-4-fold below the Km determined for the surface-bound C5 convertase suggesting a direct correlation between the local C5 concentration and production of the anaphylatoxin C5a and the cytolytic C5b-9 complex.
Cell Membrane↗
Detection by radioimmunoassay of antibodies in human smallpox patients and vaccinees.
A radioimmunoassay procedure was developed for determining smallpox and vaccinia antibodies in human sera. The test detected and measured both primary and secondary immune responses in persons infected with variola virus or vaccinia virus. The antibody titers obtained by complement fixation, hemagglutination inhibition, plaque reduction neutralization, and radioimmunoassay methods were compared. In sequential serum specimens, the radioimmunoassay test indicated fourfold or greater increases in all of the smallpox patients and in six of eight vaccinated persons. Both the complement fixation and the hemagglutination inhibition tests were less effective. In persons who had been vaccinated, radioimmunoassay and plaque reduction neutralization tests appeared to measure the same immune response. However, in smallpox patients the immune response was readily detected by radioimmunoassay, whereas an immune response was not detected by the plaque reduction neutralization test when vaccinia virus was the antigen in the test system. Radioimmunoassay is an operationally simple procedure which provides objective and quantitative end-point titers in serological determinations.
Antibodies, Viral↗
Surface antigenic specificities of human thymus-derived lymphocytes.
Surface antigenic specificities of human thymus-derived (T) lymphocytes were studied by cytotoxicity tests using a heterologous rabbit anti-human thymus serum. This serum showed higher cytotoxic titres on thymocytes by comparison with peripheral lymphocytes. After proper absorption the antiserum was non-toxic for chronic lymphatic leukaemia cells, but lysed the majority of thymocytes. It also lysed some of peripheral lymphocytes, corresponding to those lymphocytes which bound sheep erythrocytes (E) but not erythrocyte-antibody-complement complexes (EAC). Pretreatment of lymphocytes with the absorbed antiserum and complement completely abrogated rosette formation with E but spared EAC-binding lymphocytes. It also eliminated their reactivity to phytohaemagglutinin and concanavalin A. These findings indicate that the absorbed serum causes selective lysis of T cells. The results obtained from quantitative absorption studies suggest that a certain loss of T-cell antigens is brought about during the differentiation of thymocytes into peripheral T cells.
Animals↗
A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis.
We have developed a new two-color fluorescent method for the quantitative measurement of adherence and ingestion of Streptococcus pneumoniae by human monocytes. The method employs a fluorescent naphthalimide, Lucifer Yellow VS, that has been covalently linked to the bacterial cell wall. Bacteria were opsonized and allowed to adhere to monocytes. Lucifer Yellow did not alter the bacterial interaction with complement in serum or with the phagocytic cell. The ability of monocytes to ingest the adherent bacteria was tested under a variety of conditions. Rabbit antibody to Lucifer Yellow derivatized with Texas Red was used to detect monocyte-bound, but uningested bacteria. Dual laser flow cytometry simultaneously quantitated the total number of monocyte-associated S. pneumoniae and the number that remained surface adherent. This method allows separate analysis of the opsonins and receptors involved in bacterial adherence to phagocytes and in the ingestion process.
Adhesiveness↗
Large-scale expression study of human mesial temporal lobe epilepsy: evidence for dysregulation of the neurotransmission and complement systems in the entorhinal cortex.
Human mesial temporal lobe epilepsies (MTLE) are the most frequent form of partial epilepsies and display frequent pharmacoresistance. The molecular alterations underlying human MTLE remain poorly understood. A two-step transcriptional analysis consisting in cDNA microarray experiments followed by quantitative RT-PCR validations was performed. Because the entorhinal cortex (EC) plays an important role in the pathophysiology of the MTLE and usually discloses no detectable or little cell loss, resected EC and each corresponding lateral temporal neocortex (LTC) of MTLE patients were used as the source of disease-associated and control RNAs, respectively. Six genes encoding (i) a serotonin receptor (HTR2A) and a neuropeptide Y receptor type 1 (NPY1R), (ii) a protein (FHL2) associating with the KCNE1 (minK) potassium channel subunit and with presenilin-2 and (iii) three immune system-related proteins (C3, HLA-DR-gamma and CD99), were found consistently downregulated or upregulated in the EC of MTLE patients as compared with non-epileptic autopsy controls. Quantitative western blot analyses confirmed decreased expression of NPY1R in all eight MTLE patients tested. Immunohistochemistry experiments revealed the existence of a perivascular infiltration of C3 positive leucocytes and/or detected membrane attack complexes on a subset of neurons, within the EC of nine out of eleven MTLE patients. To summarize, a large-scale microarray expression study on the EC of MTLE patients led to the identification of six candidate genes for human MTLE pathophysiology. Altered expression of NPY1R and C3 was also demonstrated at the protein level. Overall, our data indicate that local dysregulation of the neurotransmission and complement systems in the EC is a frequent event in human MTLE.
Adult↗
Detection of multiple tumor suppressor genes for Syrian hamster fibrosarcomas by somatic cell hybridization.
Identification of tumor suppressor gene loci in rodent cell culture systems has relied upon the use of somatic cell hybridization studies. Although normal rodent fibroblasts are capable of suppressing the tumorigenicity of a variety of tumor cells, the lack of complementation in tumor cell x tumor cell hybrids has left the possibility that a single tumor suppressor gene may be responsible for tumor suppression in a particular rodent cell culture system. Using this same approach, we found no evidence for complementation resulting in suppression of the transformed phenotype when three viral oncogene-transformed Syrian hamster embryo (SHE) cell lines and one spontaneously transformed baby hamster kidney (BHK) cell line were fused to benzo[a]pyrene-transformed SHE cells (BP6T-M3). However, v-src oncogene-transformed cell line (srcT) x BP6T-M3 hybrids did demonstrate limited suppression of the transformed phenotype, suggesting at least two complementing tumor suppressor genes in this system. We were able to confirm and extend this finding using another experimental approach with preneoplastic hamster cell lines that are immortal in culture but nontumorigenic in nude mice. We propose that fusion of these preneoplastic cells to various tumor cells may reveal tumor suppressor genes not evident in the tumor cell x tumor cell complementation studies. Subclones of two nontumorigenic, immortal hamster cell lines, 10W and DES4, displayed differing abilities to suppress BP6T-M3 cells in somatic cell hybrids, as quantitated by the ability of the hybrid cells to form colonies in soft agar. With a panel of preneoplastic hamster cell x BP6T-M3 hybrids, a distinct pattern of suppression or expression of the transformed phenotype was observed. Marked differences in this pattern were seen when the same 10W and DES4 subclones were fused to other hamster fibrosarcoma cell lines, indicating different tumor suppressing activities of multiple tumor suppressor genes. Analysis of this data suggests that as few as three or as many as six different tumor suppressor genes may be active in the Syrian hamster embryo cell culture system. Thus, this system may provide a useful model for identifying and studying the effects and regulation of a number of different tumor suppressor genes for fibrosarcomas.
Animals↗
Fibrinogen binding inhibits the fixation of the third component of human complement on surface of groups A, B, C, and G streptococci.
Effects of fibrinogen binding to M protein-positive and -negative streptococci on fixation of the third component of human complement (C3) were determined. In all test cultures of serological groups A, B, C, and G fixation of C3 was observed in normal human serum as revealed by quantitative fluorescent immunoassay. Fibrinogen binding inhibited the fixation of C3 on streptococci. The degree of inhibition was proportional to the extent of fibrinogen binding. Thus, inhibition of C3 fixation was most pronounced in strongly fibrinogen-positive streptococci of groups A, C, and G and not demonstrable in fibrinogen-negative cultures of groups C and G. Trypsinization of the streptococci destroyed their capacity to bind fibrinogen and consequently the inhibitory effects on C3 fixation. The carboxymethylated alpha and beta chains of fibrinogen moderately inhibited C3 fixation whereas gamma chain had no influence. These studies may indicate that fibrinogen binding structures other than M protein could also be involved in streptococcal pathogenicity.
Binding Sites↗
Quantitative detection and typing of HBs Ag and anti-HBs by radioimmunoassay techniques.
Small polystyrene tubes coated with purified anti-HBs were used as solid-phase substrate for the detection of HBsAg and anti-HBs by RIA procedures. This substrate yields highly sensitive, reproducible and homogeneous results. The sensitivity and the kinetics of various solid-phase RIA procedures for anti-HBs detection were explored. The preliminary heating of sera at 56 degrees C for 30 min is needed, in order to remove a thermolabile aspecific inhibitor, which is not identifiable with complement. In particular, an RIA procedure is suggested, based upon preincubation of serum to be tested with a serum containing low amounts of HBsAg (count ratio to negative sera = 10:1) and suitable for the combined detection and quantitation of HBsAg and of anti-HBs. The results are expressed as HBs Units, which provide a simple and standardizable unit allowing estimation of HBsAg and anti-HBs amounts over a range of as much as one million values. A number of RIA procedures were also studied for typing HBsAg and anti-HBs, which are also suitable to evaluate 'in vitro' the degree of cross immunity among the various subtypes.
Animals↗
Selective adsorption of immunoglobulins and glucosylated proteins on phenylboronate-agarose.
Aminophenylboronate-substituted agarose in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid, pH 8.5, selectively adsorbs immunoglobulins and complement factors C3 and C4 from human serum. The selectivity of binding is strongly influenced by the presence of magnesium chloride in the sample buffer. Adsorbed immunoglobulins are quantitatively eluted by sorbitol, but only partially by ethylene glycol or methylcellosolve. Aniline-agarose of a similar degree of substitution shows only weak adsorption of serum proteins under similar experimental conditions, thus indicating the important contribution of the boronate moiety to this interaction. Immunoglobulin adsorption seems not to be due to the cis-diol complexation used extensively for the chromatographic determination of non-enzymatically glucosylated proteins. Hydrophobic and pi-pi interactions with the aromatic structure of the ligand seem also to contribute to protein binding. The behaviour of aminophenylboronate-liganded agarose is, in some respects, rather similar to that of the so-called "thiophilic adsorbents".
Adsorption↗
A new immunoassay for C3. Application of the cell-linked antigen radioactive antibody (CLARA) technique.
The cell-linked antigen radioactive antibody (CLARA) method of immunoassay was applied to the C3 component of the complement system. C3 from whole complement was fixed on erythrocytes sensitized with IgM antibody. The IgG1 fractions of monospecific sheep anti-C3 were labelled with 125I and measurement of their specific binding to C3-bearing erythrocytes was used to quantitate the relative amount of fixed C3. Competititive inhibition of this specific binding was used to assay fluid phase C3. 0.3-0.6mug/ml of murine C3 were detected in the supernatant fluid of in vitro cultures of mouse spleen cells.
Animals↗
Quantitation of cholinergic synaptosomes from guinea pig brain.
An antiserum raised to nerve terminal sacs derived from the electric organ and Torpedo marmorata was used to lyse guinea pig brain synaptosomes in the presence of complement. From the release of the cytoplasmic enzymes choline acetyltransferase, lactate dehydrogenase, tyrosine hydroxylase and glutamate decarboxylase it appears that the antiserum binds specifically to cholinergic terminals. The amount of lactate dehydrogenase released was used to estimate the proportion of cholinergic nerve terminals in different synaptosome preparations.
Acetylcholine↗
Quantitative synthesis in systematic reviews.
The final common pathway for most systematic reviews is a statistical summary of the data, or meta-analysis. The complex methods used in meta-analyses should always be complemented by clinical acumen and common sense in designing the protocol of a systematic review, deciding which data can be combined, and determining whether data should be combined. Both continuous and binary data can be pooled. Most meta-analyses summarize data from randomized trials, but other applications, such as the evaluation of diagnostic test performance and observational studies, have also been developed. The statistical methods of meta-analysis aim at evaluating the diversity (heterogeneity) among the results of different studies, exploring and explaining observed heterogeneity, and estimating a common pooled effect with increased precision. Fixed-effects models assume that an intervention has a single true effect, whereas random-effects models assume that an effect may vary across studies. Meta-regression analyses, by using each study rather than each patient as a unit of observation, can help to evaluate the effect of individual variables on the magnitude of an observed effect and thus may sometimes explain why study results differ. It is also important to assess the robustness of conclusions through sensitivity analyses and a formal evaluation of potential sources of bias, including publication bias and the effect of the quality of the studies on the observed effect.
Bias↗
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