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Results for “quantitative complementation”

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PubMed1991

Inheritance of human plasma dopamine-beta-hydroxylase thermal stability.

Although the structural gene for human dopamine-beta-hydroxylase (DBH) has been cloned, the mechanism by which DBH physical properties and activity are regulated is not well understood. Previous reports have suggested that three-allele or two-locus models may account for the genetic regulation of these traits in human blood. It is an interesting challenge to determine the extent to which quantitative analyses will complement or guide molecular genetic studies. In this study we analyzed data on the physical property of DBH thermal stability and DBH activity in 230 individuals in 53 families in an attempt to clarify genetic mechanisms for the inheritance of these traits. Commingling and segregation analyses of the thermal stability data provided the first clear evidence of a major gene polymorphism for DBH thermal stability analyzed as a quantitative trait. Major gene transmission was supported within a mixed model (chi 2[3] = 13.39, P less than .004). In keeping with earlier findings, similar analyses of DBH activity provided strong evidence of genetic transmission. However, in our data support for a major gene polymorphism was equivocal (chi 2(2) = 2.99, P = .22).

Adolescent↗

PubMed1976

The Raji cell radioimmune assay for detecting immune complexes in human sera.

A sensitivie and simple procedure for the detection and quantitation of soluble complement (C)- fixing immune complexes in sera of patients with various disease states has been developed by utilizing C receptors on Raji cells. These cells lack membrane-bound immunoglobulin but have receptors for IgG Fc, C3b, C3d, and possibly with other C proteins. Uptake experiments showed that both aggregated human gamma globulin (AHG) and 7S IgG bound to receptors for IgG Fc; however, AHG reacted with C bound to cells only via receptors for C and this binding was much more efficient than via IgG Fc receptors. AHG was used as an in vitro model of human immune complexes and its uptake by Raji cells was quantitated by 125I-radiolabeled antihuman IgG. The limit of sensitivity of this test was 6 mug AHG/ml serum. The ability of Raji cells to detect AHG in serum depended on the amount of radioactive antibody used and the size of aggregates. The presence of an excess of C somewhat inhibited binding of AHG containing C to Raji cells. The efficient binding of AHG by receptors for C on Raji cells was used for the detection and quantitation of immune complexes in human sera. Raji cells were incubated with sera to be tested and then reacted with excess radiolabeled antihuman IgG; the amount of radioactivity bound to the washed cells was determined and referred to a standard curve of radioactive antibody uptake by cells previously incubated with increasing amounts of AHG in serum. Thereby immune complexes were detected and quantitated in serum hepatitis, systemic lupus erythematosus, vasculitis, subacute sclerosing panencephalitis, dengue hemorrhagic fever, and malignancies.

Antigen-Antibody Complex↗

PubMed1978

Demonstration and quantitation of activation of the first component of complement in human serum.

Activation of the first component of human complement (C1) in human sera can be readily detected in double immunodiffusion studies with anti-C1q, anti- C1r, and anti-C1s as it produces a characteristic pattern quite different from that of precursor C1. Native macromolecular C1 gives a continuous line of precipitation with antisera to C1q, C1r, and C1s in double diffusion studies. After activation of C1 by incubation of serum with complement activators, three major changes occurred in the Ouchterlony pattern. First, spurring of the C1s precipitin line over that of macromolecular C1, indicating release of C1s from C1, was observed with low doses of activator. Release of C1s was quantitated by single radial diffusion and shown to be complete with the highest activator dose examined. Second, C1q was released with larger activator doses as shown also by spurring of the precipitin line due to this component over the remaining macromolecular C1. Third, and most surprising, C1r antigenicity was progressively lost as the activator dose was increased and no C1r line remained with the highest dose of activator tested. This was not true with C1s as there was no change in the total C1s concentration in serum incubated with various activator doses. These observations provide two approaches to the quantitation of C1 activation in human serum. First, C1r and C1s can be quantitated by single radial diffusion. A decrease in the C1r:C1s ratio correlates with activation. Second, C1s released by the activation can be quantitated by single radial diffusion if the agarose contains high concentrations of anti-C1q to confine C1, also containing C1s, to the area near the application well, and lesser concentrations of anti-C1s to permit free C1s to produce a measurable ring. The extent of release of C1s also correlates with activation. These immunochemical techniques to quantitate C1 activation directly inserum do not require specialized reagents. It is hoped that they will be useful in screening pathological sera and in monitoring the status of the complement system in patients.

Complement C1↗

PubMed1996

Polygenic mutation in Drosophila melanogaster: genetic interactions between selection lines and candidate quantitative trait loci.

We have investigated genetic interactions between spontaneous mutations affecting abdominal and sternopleural bristle number that have accumulated in 12 long-term selection lines derived from an inbred strain, and mutations at 14 candidate bristle number quantitative trait loci. The quantitative test for complementation was to cross the selection lines to an inbred wild-type strain (the control cross) and to a derivative of the control strain into which the mutant allele at the candidate locus to be tested was substituted (the tester strain). Genetic interactions between spontaneous mutations affecting bristle number and the candidate locus mutations were common, and in several cases the interaction effects were different in males and females. Analyses of variance of the (tester- control) differences among and within groups of replicate lines selected in the same direction for the same trait showed significant group effects for several candidate loci. Genetically, the interactions could be caused by allelism of, and/ or epistasis between, spontaneous mutations in the selection lines and the candidate locus mutations. It is possible that much of the response to selection was from new mutations at candidate bristle number quantitative trait loci, and that for some of these loci, mutation rates were high.

Analysis of Variance↗

PubMed1987

Complement activation by hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Certain biomaterials, including nylon oxygenator and cellulosic dialysis membranes, are potent activators of human complement. In this study, the effect of polymers containing 2-hydroxyethyl methacrylate (HEMA) on the human complement system was investigated. Copolymers of HEMA with ethylmethacrylate (EMA) varying from 100 to 40% HEMA in the monomer made by radiation initiation were used to coat glass discs. These were equilibrated with human plasma in vitro and the degree of complement activation was quantitated by C3a radio immunoassay. Significant activation was caused by copolymers made from monomers containing 60% or greater HEMA. A direct relationship between the amount of activation and the percentage of HEMA was found. The degree of activation by poly HEMA, when corrected for surface area, was quite similar to that observed for dialysis and oxygenator membranes. Similar observations were made when solid casts of crosslinked HEMA/N-vinylpyrrolidone (NVP) copolymer gels were tested, but the magnitude of activation was much greater. The results are significant because complement activation may play an important role in the response to foreign surfaces, in both extravascular and intravascular settings. A new concept of molecular biocompatibility is proposed in which surfaces eliciting molecular transformations in any of the biological defense systems are deemed nonbiocompatible. By this criterion, the hydrogel poly-HEMA, which has so frequently been thought of as biocompatible, is considered to be a molecularly non-biocompatible material.

Biocompatible Materials↗

PubMed1997

Spatial distributions of sulfur-rich proteins in cornifying epithelia.

We have used energy-filtered electron microscopy and electron energy loss spectroscopy (EELS) to characterize the distributions of sulfur-rich proteins in granular layer cells of squamous stratifying epithelia and their redistribution in the cornified layer, with particular attention to assembly of the cornified cell envelope (CE). Our measurements provide quantitative information that complements highly specific but qualitative data from immunocytochemistry. Spatial distributions of sulfur, phosphorus, and nitrogen were mapped in unstained thin sections of mouse epidermis and forestomach, using a postcolumn energy filter. Nitrogen images were indicative of total protein while phosphorus images provided a control to validate the algorithms used to calculate the elemental maps. Sulfur was found at high levels in round L-granules in the granulocyte cytoplasm and in the cornified CE, correlating with the presence of the protein, loricrin ( approximately 7% Cys/Met residues). EELS confirmed these observations quantitatively: either L-granules consist exclusively of loricrin or any additional components must have an equally high net sulfur content. These data also confirm the large fraction (approximately 75%) of loricrin in the CE, as inferred from modeling of its amino acid composition. We also observed extracellular deposits between cornified squames in fetal mouse epidermis that we call peripheral granules. Their sulfur content is at least as high as that of L-granules but they do not label with anti-loricrin antibodies, suggesting the presence of another sulfur-rich protein.

Animals↗

PubMed1987

Quantitative analysis of the interaction between immune complex and C1q complement subcomponent. The role of interdomain interactions in rabbit IgG in binding of C1q to immune precipitates.

A novel method was developed for the analysis of the interaction of large multivalent ligands with surfaces (matrices) to analyse the binding of complement subcomponent C1q to immune precipitates. Our new evaluation method provides quantitative data characteristic of the C1q-immune-complex interaction and of the structure of the immune complex as well. To reveal the functional role of domain-domain interactions in the Fc part of IgG the binding of C1q to different anti-ovalbumin IgG-ovalbumin immune complexes was studied. Immune-complex precipitates composed of rabbit IgG in which the non-covalent or covalent bonds between the heavy chains had been eliminated were used. Non-covalent bonds were abolished by splitting off the CH3 domains, i.e. by using Facb fragments, and the covalent contact was broken by reduction and alkylation of the single inter-heavy-chain disulphide bond. The quantitative analysis of the binding curves provides a dissociation constant (K) of 200 nM for the interaction between C1q and immune precipitate formed from native IgG. Surprisingly, for immune precipitates composed of Facb fragments or IgG in which the inter-heavy-chain disulphide bond had been selectively reduced and alkylated, stronger binding (K = 30 nM) was observed. In this case, however, changes in the structure of the immune-complex matrix were also detected. These structural changes may account for the strengthening of the C1q-immune-complex interaction, which can be strongly influenced by the flexibility and the binding-site pattern of the immune-complex precipitates. These results suggest that domain-domain interactions in the Fc part of IgG affect the segmental mobility of IgG molecules and the spatial arrangement of the immune-complex matrix rather than the affinity of individual C1q-binding sites on IgG.

Animals↗

PubMed1977

C1 subcomponents in acute pneumococcal otitis media in children.

Twenty children with acute pneumococcal otitis media were studied. In 6 children the infection ran a normal course and healed after the first episode and in 14 it relapsed. The serum levels of the immunoglobulins IgG, IgA and IgM were normal in all 20 children. Specific antibodies to pneumococcal polysaccharide were found in all cases, with no differences in the titers between the relapsed cases and those that healed. The complement components were quantitated with electroimmuno assay. G1q proved depressed in 60 per cent of the relapsed cases and in 16 per cent of the healed cases. C1r and C1s were disproportionally high compared with the C1q levels. Furthermore, crossed immunoelectrophoresis revealed abnormal complexes composed of C1r and C1s, and complexes composed of C1r, C1s and C1 IA. These complexes were more pronounced in sera from the children with relapsing otitis media.

Acute Disease↗

PubMed1990

Immunohistochemical detection of terminal complement complex and S protein in normal and pre-eclamptic placentae.

Terminal complement complex and S protein were searched for in term placentae obtained from 13 women with normal pregnancy and 15 patients with moderate or severe form of pre-eclampsia. Terminal complement complex was found to localize in the fibrinoid material of the decidua of the basal plate, in the stroma of the chorionic villi and in the vessel walls, as subendothelial deposits. S protein had a quite different distribution, being detected in the syncytiotrophoblast located both in the chorionic villi and in the decidua of the basal plate (DBP) and also on the endothelial cells of fetal stem vessels. Mild deposits of C3 were found in the decidua of the basal plate and also in the stroma and on the basal membranes of the villi. Reactivity for C9 neoantigen was also observed in the cytoplasm of some cells, which were recognized to be macrophages by the presence in their cytoplasm of acid phosphatase and by their reaction with a monoclonal antibody specific for macrophages. Differences in complement deposition in normal and pre-eclamptic placentae were essentially quantitative. Possible mechanisms of complement activation are discussed.

Chorionic Villi↗

PubMed2000

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Adjuvants, Immunologic↗

PubMedDate not supplied

Assessment of complement activation in vivo.

Hemolytic assays that measure the functional integrity of the complement system and the quantitation of individual components by immunochemical techniques have been widely used in the past for the assessment of in vivo complement activation. However, the complement system comprises a large number of interacting serum proteins which are subject to independent synthetic and catabolic processes. The fact that complement proteins are rapidly metabolized under in vivo conditions adds to the complexity of complement analysis. Assays that are based on monoclonal antibodies with specificities for activation-dependent neoepitopes now allow the direct determination of complement fragments in plasma. These methods are superior to the quantitation of native proteins. Several parameters that differentially affect the generation or the catabolism of individual complement activation products still have to be taken into account when elevated plasma levels of complement fragments suggest in vivo complement activation. These factors include the binding to complement fragment receptors, the degradation by serum proteases and renal or hepatic clearance. An accurate estimation of complement activation in vivo requires the simultaneous determination of both the native components and the activation products.

Animals↗

PubMed2002

Laboratory assays for predicting the severity of haemolytic disease of the fetus and newborn.

Haemolytic disease of the fetus and newborn (HDFN) is characterised by the presence of IgG antibodies in the maternal circulation which cause haemolysis in the fetus by crossing the placenta and sensitising red cells for destruction by macrophages in the fetal spleen. Serological, quantitative and cellular assays have all been developed to predict the severity of HDFN. These assays measure and/or characterise alloantibodies in the maternal circulation. Quantitative assays which accurately measure antibody levels correlate with disease severity better than serological assays which are inherently less precise. Nevertheless, high antibody levels are found in some cases of mild HFDN and relatively low antibody levels are found in some severe cases. This suggests that disease severity is influenced by factors in addition to antibody concentration. These factors remain to be fully elucidated but may include: the subclass and glycosylation of maternal antibodies; the structure, site density, maturational development and tissue distribution of blood group antigens; the efficiency of IgG transport to the fetus; the functional maturity of the fetal spleen; polymorphisms which affect Fc receptor function; and the presence of HLA-related inhibitory antibodies. Cellular assays which are sensitive to factors affecting antibody function have, therefore, been developed in an attempt to improve the prediction of disease severity. Although these assays are cumbersome, there are now sufficient data to suggest that some cellular assays provide clinically useful information to complement serological and quantitative assays.

ABO Blood-Group System↗

PubMed1998

A comparison of in vitro tests for predicting the severity of haemolytic disease of the fetus and newborn.

Haemolytic disease of the newborn (HDN) is characterized by the presence of IgG antibodies in the maternal circulation which cause haemolysis in the fetus by crossing the placenta and sensitizing red cells for destruction by macrophages in the fetal spleen. Numerous serological, quantitative and cellular assays have been developed to predict the severity of HDN. These assays all measure and/or characterize alloantibodies in the maternal circulation. Quantitative assays which accurately measure antibody levels correlate with disease severity better than serological assays which are inherently less precise. Nevertheless, high antibody levels are found in some cases of mild HDN and relatively low antibody levels are found in some severe cases. This suggests that disease severity is influenced by factors in addition to antibody concentration. These factors remain to be fully elucidated but may include the subclass and glycosylation of maternal antibodies, the structure, site density, maturational development and tissue distribution of blood group antigens, the efficiency of IgG transport to the fetus, the functional maturity of the fetal spleen, polymorphisms which affect Fc receptor function, and the presence of HLA-related inhibitory antibodies. Cellular assays which are sensitive to factors affecting antibody function have therefore been developed in an attempt to improve the prediction of disease severity. Although these assays are cumbersome, there are now sufficient data to suggest that some cellular assays, when used as part of a structured approach to diagnostic testing, may provide clinically-useful information to complement serological and quantitative assays.

Adult↗

PubMed1983

Complement components in 100 newborns and their mothers determined by electroimmunoassay.

Samples of blood were obtained from 100 healthy full-term women in labour and, after delivery, from the umbilical cord of their infants. By electroimmunoassay, complement components were quantitated in serum (C1q, C1r, C1s, C1 IA, C2, P, D. I, H, C6 and C7) or in EDTA-plasma (C4, C3, B, C5. and C). The concentrations of C7 in cord serum was twice that found by others using a functional assay. Concentrations of C1r, I and C6 in the cord sample were 50-60 per cent of those in healthy blood donors used as reference, and that of D was about 130 per cent. The cord serum and plasma concentrations of the remaining components agreed with previously reported values. The maternal levels of C2, C4, C3, B, H, C5 were 40-60 per cent higher than those of the reference.

Complement C4↗

PubMed2000

Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semi-quantitative indexes of fibrosis.

BACKGROUND/AIMS: Liver fibrosis is one of the most important and characteristic histologic alterations in progressive and chronic liver diseases. Thus, in both clinical and experimental practice, it is fundamental to have a reliable and objective method for its precise quantification. Several semi-quantitative scoring systems have been described. All are time-consuming and produce partially subjective fibrosis evaluations that are not very precise. This paper describes the design and validation of an original image analysis-based application, FibroQuant, for automatically and rapidly quantifying perisinusoidal, perivenular and portal-periportal and septal fibrosis and portal-periportal and septal morphology in liver histologic specimens. METHODS: The implemented image-processing algorithms automatically segment interstitial fibrosis areas, while extraction of portal-periportal and septal region is carried out with an automatic algorithm and a simple interactive step. For validation, all automatically extracted areas were also manually segmented and quantified. RESULTS: Statistical analysis showed significant intra- and interoperator variability in manual segmentation of all areas. Automatic quantifications did not significantly differ from mean manual evaluations of the same areas. Comparison of our image analysis quantifications with staging histologic evaluations of liver fibrosis showed significant correlations (Spearman's, 0.72<r<0.83; p<0.0001) and that the latter are based more on the distribution patterns than on the quantity of fibrosis. CONCLUSIONS: FibroQuant is a sensitive, precise, objective and reproducible method of fibrosis quantification, which complements semi-quantitative histologic evaluation systems. This novel tool could be of special value in clinical trials and for improving the prognosis and follow-up among patients with fibrosis-inducing hepatic diseases.

Algorithms↗

PubMed1996

New mutations and phenotypes associated with glutamate and aspartate transport in Chinese hamster ovary (CHO-K1) cells.

Two new Chinese hamster ovary cell (CHO-K1) mutants lacking amino acid transport System X-AG activity were isolated by [3H]aspartate suicide selection. These null mutants, Dd-B6 and Dd-B7, were analyzed by somatic cell hybridization, along with previously described partial-function mutants, Ed-A1 and Ed-B8. With respect to System X-AG activity, all four mutations fell into a single complementation group. By quantitative assay, the mutations in Ed-A1 and Ed-B8 behaved as simple recessives in fusions with wild type cells, while those in Dd-B6 and Dd-B7 were codominant. We have discovered that Ed-A1 and Ed-B8 are highly permeable to small neutral molecules. This high permeability phenotype was dominant to wild-type. Northern, Southern, and Western analyses indicated that System X-AG in CHO is not closely related to any of the three well characterized glutamate transporters represented by GLT-1, EAACI or GLAST.

Amino Acid Transport System X-AG↗

PubMed1997

Transcriptional analysis of type 1 capsule genes in Staphylococcus aureus.

Serotype 1 capsule of Staphylococcus aureus plays an important role in staphylococcal infections. A 14.6 kb chromosomal DNA fragment containing 13 cap1 genes responsible for the synthesis of type 1 capsule in S. aureus strain M has been previously cloned and sequenced in our laboratory. These genes are closely linked and are transcribed in one orientation. To study the transcription of these genes, we employed genetic complementation using a set of plasmids with various deletions in the 14.6 kb region to complement mutants mapped in each of the 13 genes. We found that there were six transcriptional units. By Northern hybridization using the entire gene cluster as a probe, we identified a large 14 kb band and several smaller bands ranging from 0.3 to 6 kb. The 14 kb band was also identified by various individual gene probes, suggesting that all 13 genes are transcribed into a large transcript. The 14 kb transcript and the smaller bands were not detected when a 533 bp fragment containing the potential promoter region upstream of the first gene (cap1A) was deleted from the chromosome, suggesting that the small transcripts are the degradation (processing) products of the 14 kb transcript. The activities of the promoters identified by genetic complementation tests were quantitatively measured by transcriptional fusions using xylE as the reporter gene. The result showed that the promoter preceding the first cap1 gene was 45-198-fold higher than the downstream promoters. The result from gene-fusion studies is in agreement with the Northern analysis in that only the transcript transcribed from the first promoter was detected. Taken together, these results suggest that the cap1 genes are organized as a large operon with at least five weak internal promoters, and the long polycistronic transcript is processed (degraded) into several smaller transcripts. The transcription initiation site of the primary transcript was mapped by S1 nuclease mapping and the major promoter was defined by deletion analysis of the promoter-xylE fusion. In addition, we showed that the internal promoters, despite their weak activities, were sufficiently effective at expressing their downstream genes required for producing capsule, compared to the wild-type strain.

Bacterial Capsules↗

PubMed1985

Complement activation in type 1 human diabetes.

Complement activation was quantitated in serum and plasma of diabetic and normal subjects by sensitive competitive equilibrium radioimmunoassays (RIA) for C3a, C4a, C5a, Factor B, and a newly described C5 neoantigen (termed C5 activation antigen, and abbreviated C5-AA) in a stable 54-kDa fragment of C5. Plasma C3a levels were significantly elevated in 8 of 16 patients with newly diagnosed Type 1 diabetes (P less than 0.0005) with the mean C3a concentration for these patients being more than 10-times greater than the mean value of normal controls. C4a levels were also elevated in 2 of these patients (P less than 0.02), but C5a levels, although higher than normal, were not significantly increased. In contrast, the levels of C5-AA in the serum of all patients (11/11) with chronic Type 1 diabetes were significantly higher than in control Type 2 patients (noninsulin-dependent diabetes) (P less than 0.0005) and 4 of 7 patients with new onset insulin-dependent diabetes mellitus also had significantly higher levels of C5-AA than the Type 2 patients (P less than 0.01). The levels of Factor B in the serum of 5 of 9 patients with new onset diabetes were significantly higher than normal (P less than 0.0025). Five recent onset Type 1 diabetes patients were evaluated longitudinally for C3a, C4a, and C5a: in 3 the levels of C3a were elevated during new onset disease decreasing into the normal range during remission; in 2 of these patients C4a was also significantly elevated and the levels decreased during remission; and in 3 patients the levels of C5a were not significantly elevated but they decreased during remission. Purified human complement proteins and complement hemolytic assays were used to measure complement activation in serum during incubation with rat pancreatic islet cells. With diluted normal human serum, less than 20% of C3 or Factor B were consumed during 30 min at 37 degrees C, while with new onset Type 1 diabetic patient sera up to 90% of C3 and Factor B were consumed in 5/6 sera and 4/6 sera, respectively. These findings suggest (a) that complement activation fragments C3a, C4a, and C5a are generated in vivo in new onset Type 1 diabetes; (b) that both the classical and the alternative complement pathways may be activated; and (c) that this may result in a measurable activation of C5 generating biologically and immunologically active C5a and other C5 activation fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗