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PubMed2002

Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

Animals↗
PubMed2006

Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone.

Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p<0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p<0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair.

Algorithms↗
PubMed1980

Microcomplement fixation and particle size of chicken lipoproteins.

Conformational changes of the lipoproteins of chicken serum and egg yolk were studied with the use of microcomplement fixation. Egg yolk very low density lipoproteins and serum total low density lipoproteins were separated by 2% agarose gel chromatography into four and five fractions, respectively, with different average particle diameters. A correlation between percent complement fixed at equivalence and particle diameter was noted. Thus, smaller particles fixed less complement. The shortening of the radius of curvature could alter the steric fit of apolipoprotein with antibody and, as a consequence, reduce complement fixation. The patterns obtained after delipidation of very low density lipoproteins indicate a general distortion of apolipoprotein conformation supporting the view that lipids are essential for maintaining the conformation of these apolipoproteins. On the other hand, the general conformation of the high density apolipoproteins does not appear to depend on lipid content. It has been reported that lipids restrain the free movement of these apolipoproteins. After delipidation, the greater degree of freedom allowed the apolipoprotein may increase the strength of the antigen-antibody union and therefore an increase in complement fixation is observed. These studies demonstrate the applicability of quantitative microcomplement fixation to the eludication of lipoprotein structure and function.

Animals↗
PubMed1987

Determination of C4b.C4-bp complex formed by the activation of classical complement pathway using an enzyme-linked immunosorbent assay.

We developed a quantitative enzyme-linked immunosorbent assay (ELISA) for the detection of C4b.C4-bp complex by incubating the sample on anti-C4-bp-coated plate and then developing with HRP-labeled anti-C4. The amount of C4b.C4-bp complex, generated in vivo by the interaction of purified C4b with C4-bp or normal human serum with aggregated human IgG, was measured by the ELISA. The complex, however, rapidly decreased in serum by the action of factor I. Six out of the 100 plasma samples from patients with various diseases were found positive in the ELISA. One plasma sample from a patient with SLE showed high level of C4b.C4-bp complex with decreased levels of factor I, C4, C4-bp and CH50. These results suggest that the detection of C4b.C4-bp complex is useful for monitoring the diseases in which the classical pathway activation is expected.

Arthritis, Rheumatoid↗
PubMed1982

Activation of human complement by immunoglobulin G antigranulocyte antibody.

The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia.

Arthritis, Rheumatoid↗
PubMed1987

An analysis of the levels of complement components in the synovial fluid in rheumatic diseases.

A linear relationship between the synovial fluid to serum concentration ratios and log molecular weight was found for six plasma proteins, which are largely synthesized by the liver. Production or utilization of a given protein in the joint can, therefore, be determined by its deviation from the calculated diffusion line. Based on this diffusion model the role of the complement system was investigated in the joint effusions of 48 patients with rheumatoid arthritis (RA), 6 patients with osteoarthritis (OA) and 7 patients with meniscus lesions (ML). Among these three groups quantitative differences were found in the metabolism or utilization of several complement components, based on the fact that the ratios were lower than expected for diffusion of proteins of similar molecular weight. The ratios for the RA group were the lowest. In the three patient groups, results showed increased consumption mainly of C3 and C4 locally in the joint. The existence of a real complement activation in the joints of the three different patient groups was further proved by the elevated levels of C3 breakdown products (C3d). Overall this kind of calculation provides us with a method for studying the role of other proteins which may be important in the inflammatory process of the joint.

Arthritis, Rheumatoid↗
PubMedDate not supplied

Electroblotting of proteins on agarose gel containing specific antibodies.

A method is described for electroblotting of proteins, separated by gradient polyacrylamide gel electrophoresis, onto an agarose gel matrix containing specific antibodies. Three proteins of different molecular weight, including human albumin, isolated and in plasma, human plasma transferrin and C3 complement were tested. Immunoblotting on agarose, compared with nitrocellulose, was quantitative and highly sensitive, with small amounts of protein (i.e., 100 pg) being detected. Moreover, albumin aggregates (i.e., dimer, trimer and tetramer) were blotted quantitatively in addition to the monomer, and their percentages were calculated. This method is sensitive, quantitative, reproducible, and includes fewer manipulations; furthermore, it is less expensive and does not require the use of toxic or carcinogenic agents.

Collodion↗
PubMed2003

[Present status and problems with rheumatoid factor as a laboratory test].

Rheumatoid factor(RF) has generally been defined as antibodies against mammalian IgG Fc. In a majority of patients with rheumatoid arthritis(RA), RF can be regarded as an autoantibody because it binds to their own IgG. RF in RA, however, can bind to various other IgG antigens such as IgG allotypic antigens(Gm), isoallotypic antigens, species-specific IgG antigens, cross-reactive antigens shared by other mammalian IgG and neoantigens created within IgG by immune-complex formation. This multi-specific characterization of RF has caused interlaboratory variation and it has been difficult to conduct external quality assessment(EQA) using various qualitative and/or quantitative RF kits. In the future, RF measurement should be assessed by each kit's EQA. It is significant to make a distinction between the pathological and non-pathological roles of RF owing to improvement of vagueness in clinical evaluation of RF measurement. Complement activation by RF, measurement of functional affinity(avidity) and quantitative determination of IgG and/or IgA RF have been investigated by us and other investigators for evaluation of RF's pathological role. Quantitative determination of RF's physiological, innate and non-pathological role is so important in health and disease that we have developed IgM-class antiglobulin against neoantigens appearing not in heat-aggregated human IgG but in human IgG bound to antigen.

Humans↗
PubMed1983

Impact of automation on the quantitation of low molecular weight markers of hemostatic defects.

Through in depth studies, the biochemical pathways of hemostasis-related systems have been elucidated in terms of well-defined molecular mechanisms. The interrelationships of coagulation, fibrinolytic, kallikrein-kinin, platelets, prostaglandins, blood vessel, and complement systems are now well understood. Methods are currently developed to quantitate the molecular markers of each of these systems and define the involvement of each in disease and drug-related aberrations. Molecular markers allow for very early detection of disease states well before clinical manifestations are seen or current coagulation methods are affected. Therefore prophylactic or therapeutic treatment can begin before a disease state causes damage. Platelet factor 4 and beta-thromboglobulin are low molecular weight proteins released from the light (alpha) granules of platelets and provide a reliable index of endogenous activation and consumption of platelets. Serotonin and ADP are released during activation from the beta-granules and can be measured by high-performance liquid chromatography. Fibrinopeptide A is a molecular marker of the activation of the coagulation process and provides a useful index of the action of thrombin on fibrinogen. Elevated levels of this peptide are found in patients with hypercoagulable states or a thrombotic tendency. B beta 15-42 peptides are released at the early stages of fibrinolysis and are a useful collective parameter for the measurement of the activation of fibrinolysis. In both the primary and secondary fibrinolytic disorders this peptide is elevated. Circulating kinins provide information on the activation of the kallikrein system and are useful in monitoring coagulation and shock related disorders. Arachidonic acid metabolites, such as thromboxanes and prostacyclins, are products of platelet and vascular endothelium interactions. Their measurement in peripheral blood provides a useful tool to measure the vascular and platelet-related thrombotic defects. Furthermore, antiplatelet therapy can be monitored using these parameters. Numerous other metabolites of arachidonic acid such as the leukotrienes and PAFs also are generated in various immunopathologic disorders associated with hemostatic activation. Unlike the other coagulant tests, the measurement of molecular markers in native blood or plasma samples provides a true picture of the endogenous physiology. Since no activator or additive is added to influence the test, these markers provide the most relevant information on the pathophysiologic condition. Since most of these markers are proteins or low molecular weight products, isotopic and nonisotopic immunoassays, high performance liquid chromatography and fluorometric methods can be used to analyze their levels. Furthermore, multiple panels can be developed to profile various pathologic states.(ABSTRACT TRUNCATED AT 400 WORDS)

Arachidonic Acid↗
PubMed2005

Survival analytical techniques were used to assess agreement of a quantitative variable.

BACKGROUND AND OBJECTIVE: Survival-agreement plots have been suggested as a new graphical approach to assess agreement in quantitative variables. We propose that survival analytical techniques can complement this method, providing a new analytical insight for agreement. METHODS: Two survival-agreement plots are used to detect the bias between to measurements of the same variable. The presence of bias is tested with log-rank test, and its magnitude with Cox regression. RESULTS: An example on C-reactive protein determinations shows how survival analytical methods would be interpreted in the context of assessing agreement. CONCLUSION: Log-rank test, Cox regression, or other analytical methods could be used to assess agreement in quantitative variables; correct interpretations require good clinical sense.

Bias↗
PubMed1984

[Detection of circulating immune complexes by polyethyleneglycol precipitations complement consumption test in urological malignant diseases].

Circulating immune complexes (CIC) were detected and quantitated in 49 patients with urological malignant diseases (9 cases of renal cell cancer, 3 cases of renal pelvic and ureter cancer, 21 cases of bladder cancer and 16 cases of prostatic cancer), 9 patients with urological benign diseases and in normal subjects by the polyethylene-glycol precipitation complement consumption test (PEG-CC test). The average CIC level was 2.7 +/- 3.0% in 18 normal subjects and the normal range was less than 10% of the CIC level. CIC level of patients with renal cell cancer was 14.3 +/- 20.1%, being elevated in 3 of the 9 patients, that of patients with renal pelvic and ureter cancer was 4.7 +/- 4.6%, being within the normal range in 3 cases, that of patients with bladder cancer was 4.7 +/- 4.4%, being elevated in 1 of 21 patients, and that of patients with prostatic cancer was 8.9 +/- 15.4%, being elevated in 3 of 16 patients. In urological malignant diseases such as renal cell cancer and prostatic cancer the CIC values were relatively high.

Antigen-Antibody Complex↗
PubMed1989

Ba and Bb fragments of factor B activation: fragment production, biological activities, neoepitope expression and quantitation in clinical samples.

Factor B is a centrally important component of the alternative complement pathway. Alternative pathway activation results in factor B cleavage and production of the amino-terminal Ba and the carboxyl-terminal Bb fragments which have molecular weights of approximately 30,000 and 63,000 daltons, respectively. Both Ba and Bb fragments have been reported to express a variety of biological activities in vitro. Thus, binding of Ba and Bb fragments to specific B lymphocyte surface receptors modulates proliferation of prestimulated B cells. In addition, the enzymatically active Bb fragment induces activation and spreading of human and murine macrophages and monocytes as well as regulates C5a des Arg chemotactic activity. The fractional catabolic rate and metabolism of factor B in vivo is similar to that of C3, C4 and C5 complement proteins, which are among the most metabolically active plasma proteins in the circulatory system. Factor B hyperconsumption and increased catabolism, concomitant with factor B fragment production, occurs in a wide variety of diseases, including gram-negative sepsis, autoimmune diseases and burns. Measurement of alternative pathway activation in vivo has been attempted utilized a number of different techniques to quantitate factor B fragments in biological fluids. However, the recent development of enzyme immunoassays (EIA) employing monoclonal antibodies (MoAbs) reactive with factor B fragment neoepitopes provides the best approach currently available for the quantitation of factor B activation fragments. Results obtained using these new MoAb-based EIAs have indicated that factor B fragment concentrations were elevated, as compared with normal donor levels, in EDTA plasma samples obtained from patients with rheumatoid arthritis and systemic lupus erythematosus (SLE). Plasma concentrations of factor B fragments, especially Ba fragment levels, in these patients showed a positive correlation with disease activity scores. One of the highest disease activity correlations was obtained with Ba fragment measurements in SLE plasma samples. In fact, the results strongly suggested that quantitation of Ba fragment levels in SLE plasma samples more accurately reflected disease activity and was a more sensitive predictor of impending flare in these patients than any other test(s) currently available.

Antibodies, Monoclonal↗
PubMed2004

Quantitative trait loci affecting starvation resistance in Drosophila melanogaster.

The ability to withstand periods of scarce food resources is an important fitness trait. Starvation resistance is a quantitative trait controlled by multiple interacting genes and exhibits considerable genetic variation in natural populations. This genetic variation could be maintained in the face of strong selection due to a trade-off in resource allocation between reproductive activity and individual survival. Knowledge of the genes affecting starvation tolerance and the subset of genes that affect variation in starvation resistance in natural populations would enable us to evaluate this hypothesis from a quantitative genetic perspective. We screened 933 co-isogenic P-element insertion lines to identify candidate genes affecting starvation tolerance. A total of 383 P-element insertions induced highly significant and often sex-specific mutational variance in starvation resistance. We also used deficiency complementation mapping followed by complementation to mutations to identify 12 genes contributing to variation in starvation resistance between two wild-type strains. The genes we identified are involved in oogenesis, metabolism, and feeding behaviors, indicating a possible link to reproduction and survival. However, we also found genes with cell fate specification and cell proliferation phenotypes, which implies that resource allocation during development and at the cellular level may also influence the phenotypic response to starvation.

Analysis of Variance↗
PubMed2005

A mouse model for study of systemic HIV-1 infection, antiviral immune responses, and neuroinvasiveness.

We created a model of HIV-1 infection of conventional mice for investigation of viral replication, control, and pathogenesis. To target HIV-1 to mice, the coding region of gp120 in HIV-1/NL4-3 was replaced with that of gp80 from ecotropic murine leukemia virus, a retrovirus that infects only rodents. The resulting chimeric virus construct, EcoHIV, productively infected murine lymphocytes, but not human lymphocytes, in culture. Adult, immunocompetent mice were readily susceptible to infection by a single inoculation of EcoHIV as shown by detection of virus in splenic lymphocytes, peritoneal macrophages, and the brain. The virus produced in animals was infectious, as shown by passage in culture, and immunogenic, as shown by induction of antibodies to HIV-1 Gag and Tat. A second chimeric virus based on clade D HIV-1/NDK was also highly infectious in mice; it was detected in both spleen and brain 3 wk after tail vein inoculation, and it induced expression of infection response genes, MCP-1, STAT1, IL-1beta, and complement component C3, in brain tissue as determined by quantitative real-time PCR. EcoHIV infection of mice forms a useful model of HIV-1 infection of human beings for convenient and safe investigation of HIV-1 therapy, vaccines, and potentially pathogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed1982

Monoclonal antibodies reactive with human myeloid leukaemia cells.

Hybridomas producing monoclonal antibodies that bind to determinants on myelogenous leukaemia blast cells were developed using fresh leukaemia blasts as immunogens. These monoclonal antibodies were used to quantitate the amount of a variety of antigens on both leukaemia and normal blood cells in a radioimmunoassay. The ability of these antibodies to mediate complement-dependent lysis of leukaemia cells and normal blood cells was evaluated. Significant quantitative differences in the expression of a variety of cell surface antigens were observed among different myeloid leukaemia cell samples, normal cells, and other forms of leukaemia. However, none of the monoclonal antibodies studied were found to be specific for myeloblasts. Despite the fact that these antibodies bound to both leukaemia and normal cells, several of them mediated complement-dependent cytotoxicity which was restricted to leukaemic myeloblasts (AML-1-211, AML-2-30, CML-18, CML-75, CML-115, and CML-150). None of these clones, alone or in any combination, were capable of lysing normal lymphocytes or monocytes. Others were specifically cytotoxic to leukaemia cell lines (AML-1-99), B cells and some leukaemia samples (AML-2-9), and myelomonocytic leukaemia cell samples and normal monocytes (AML-2-23). Several of the monoclonal antibodies from this panel appear to be promising for future clinical applications.

Antibodies, Monoclonal↗
PubMed1953

Quantitative studies of the virus-host relationship in chimpanzees after inapparent infection with Coxsackie viruses. II. The development of complement-fixing antibodies.

Eight chimpanzees were fed and/or inoculated with a single Coxsackie (C) virus or combinations of different ones. The responses in terms of complement-fixing (c-f) antibodies to 6 to 10 such viruses were measured throughout periods of 1 to 2 years. Although the animals usually responded with rises in homologous antibodies after the feeding or the inoculation of viruses of special immunological types, a variable number of heterologous c-f antibodies were observed to increase significantly at the same time. When immune animals were challenged with homologous virus, they failed to become virus carriers again and no rises in neutralizing antibodies were detected. However, the challenge usually resulted in a boost in the titer of the homologous c-f antibody and often of heterotypic c-f antibodies. This was particularly striking in those chimpanzees in which the c-f antibody had fallen below a detectable level. In contrast to this, the infection of chimpanzees with agents outside the Coxsackie family (poliomyelitis viruses and Egyptian strains of West Nile virus) failed to influence the level of Coxsackie antibodies even during the periods when c-f antibodies to these non-Coxsackie viruses were rapidly rising.

Animals↗
PubMed1983

Stage-related correlations between immunoglobulins and complement components in preoperative sera from patients with gastric carcinoma.

Immunoglobulins A, G and M and complement components C3, C4 and C1-INH were quantitated in sera taken preoperatively from 168 patients with gastric carcinoma. The values were grouped according to stages (pTNM). The concentrations of C4 and C1-INH increased with advancing stage of disease and were above normal mean values in all stages. The concentration of IgG was below the normal mean value among all the patients and with the lowest concentration in stage III. Concentrations of IgA and C3 were above normal means but without significant relation to stages. There was a positive correlation between the concentrations of IgG and C1-INH in sera from patients with stage IV carcinoma, while the same correlation was negative in stages I-III. Although the results varied among the patients within each stage, the profiles of immunoglobulin and complement concentrations are of value in the preoperative staging of the disease.

Adult↗
PubMed1975

[The reactivity of lupus erythematosus serum with native and denatured DNA of various origins].

The serum of a female suffering from Lupus erythematosus visceralis was investigated by complement fixation for the reaction with native and denatured DNA's of various base composition. The reaction with native DNA is independent on the (G+C)-content of the DNA. It is apparent that the responsible antibodies react with determinants of the helical conformation, which are identical in the various DNA-molecules. Quantitative differences are found with denatured DNA's. The strongest complement fixation is observed with (G+C)-rich denatured DNA. The reaction with denatured DNA is only partially inhibited by DNA digest. These antibodies obviously react with sequential determinants containing bases. Therefore, they are induced by a different mechanism of sensitization.

Adult↗