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PubMed1989

Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants.

The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. We have constructed Nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, AL1, AL2, and AL3, from the left side of TGMV A. In the transgenic plants, the AL open reading frames are under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In TGMV infectivity assays, seven of 10 transgenic lines complemented TGMV A variants with mutations in AL1, AL2, or AL3 when co-inoculated with the B component. The 35S-AL construct was transcribed as a single RNA species in the transgenic plants, indicating that AL1, AL2, and AL3 were expressed from a polycistronic mRNA. This differs from the complex transcription pattern in TGMV-infected plants, which contains five AL transcripts. There was no quantitative correlation between the efficiency of complementation in the infectivity assay and the level of expression of transgenic AL RNA in the leaves of a transgenic line. One line that failed to complement defects in AL1, AL2, and AL3 in infectivity assays contained high levels of transgenic AL RNA and functional AL1 protein. These results provide evidence that chromosomal position can affect the cell- and tissue-specific transcription of the 35S promoter in transgenic plants. Comparison of the complementing plants and wild-type infected plants may provide insight into the TGMV infection process and the use of the CaMV 35S promoter for gene expression in transgenic plants.

Base Sequence↗
PubMed1995

A novel ELISA for the assessment of classical pathway of complement activation in vivo by measurement of C4-C3 complexes.

Measurements of complement split products by enzyme-linked immunosorbent assays (ELISA) are well established for the assessment of in vivo complement activation. We have combined two monoclonal antibodies (mAb) with specificities for C3b/iC3b/C3dg (mAb I3/15) and C4/C4b/C4d (mAb M4d2), respectively, in a sandwich ELISA to quantitate C4-C3 complexes as an indicator of complement activation. Serum incubated with heat aggregated IgG (HAG) was used as a standard and the C4-C3 levels expressed as microgram equivalent HAG/ml (microgram HAG-equ/ml). Normal values of C4-C3 complexes in plasma (EDTA) of healthy probands (n = 11) were 6.3 micrograms HAG-equ/ml +/- 1.5 (mean +/- 1 standard deviation (SD), with a range from 3.6 to 9.1). In patients with systemic lupus erythematosus (SLE, n = 23) C4-C3 values were clearly elevated (48.8 micrograms HAG-equ/ml +/- 52.9, range 7.5-184.7) as compared to samples from patients with idiopathic hypertension (IDH, n = 10) (6.5 micrograms HAG-equ/ml +/- 1.7, range 4.1-9.4). For SLE patients C4-C3 levels significantly correlated with values for C3b/iC3b/C3d (r = 0.69, p < 0.001) and C3 containing immune complexes (r = 0.68, p < 0.001), but not with the C4d fragment (r = 0.26). C4-C3 levels of 96% of the studied SLE patients were increased more than 2 SD above the normal mean as compared to 74% of C4d and activated C3 values, respectively. Serum treated with zymosan as an activator of the alternative pathway of complement did not exhibit higher C4-C3 values. These results demonstrate that the quantitation of in vivo generated C4-C3 complexes by ELISA provide a novel, sensitive parameter for classical pathway of complement activation.

Antibodies, Monoclonal↗
PubMedDate not supplied

Effects of nonionic contrast media (CM) on the components of coagulation and complement systems.

Several nonionic experimental contrast media (CM) were evaluated for their effects on coagulation cascade, platelet aggregation, and the activation of complement pathways. In an in vitro system, most of the contrast media tested showed a mild anticoagulant property by prolonging the clotting times, such as partial thromboplastin time and the thrombin time of pooled normal human plasma. However, aggregation of normal human platelets by adenosine-diphosphate (ADP), collagen, epinephrine, ristocetin, and thrombin was not blocked when the platelet-rich plasma was incubated with these agents. No quantitative or qualitative changes in the complement components C3 or C4 were detected when a mixture of CM and pooled normal human serum was analyzed by radial immunodiffusion, immuno- or crossed-immunoelectrophoresis techniques. These results indicate that nonionic contrast media produce certain transient hematologic changes and should be further tested for their immunologic properties in order to establish their absolute safety in diagnostic procedures.

Blood Coagulation↗
PubMed1991

The hematopoietic and mature blood cells of the rat: their morphology and the kinetics of circulating leukocytes in control rats.

Although the morphology of the hematopoietic and circulating blood cells of the rat differs slightly from that of human blood cells, the basophil is the only human blood cell for which the rat does not have a readily recognizable counterpart. The morphological classification of the rat's marrow and peripheral blood cells as based on our experience is described with reference to the differing observations of previous investigators and with comparison to the human hematopoietic system. Examination of the bone marrow and peripheral blood demonstrated that control rats injected i.v. with 1% normal rat serum in sterile saline exhibit a moderate transient lymphopenia as the only significant hematologic fluctuation induced by the stresses of injection, general anesthesia, and tail bleeding. Morphologic quantitation of marrow and peripheral blood cells complements the insights obtained by the culture of marrow cells. An appreciation of the morphology of the rat's blood cells and of the reproducible and quantitative nature of marrow and peripheral blood smear examinations may lead to more in vivo investigations of hematopoiesis and leukocyte trafficking.

Animals↗
PubMed1995

Acquired deficiency in C1-inhibitor associated with signet ring cell gastric adenocarcinoma: a probable connection of antitumor-associated antibodies, hemolytic anemia, and complement turnover.

BACKGROUND: Acquired deficiency in C1-inhibitor (C1-INH) associated with malignancy is often asymptomatic because clinical manifestations are not dependent on a critical complement threshold (in contrast to hereditary C1-INH deficiency). Increased complement consumption involving different kinds of antibodies is the postulated mechanism for this disease, but other factors must play an important role. CASE REPORT: A 76-year-old woman with unremarkable medical history experienced three episodes of angioedema over 6 months. Investigations revealed a complement profile characteristic of acquired deficiency in C1-INH, a hemolytic anemia, and a signet ring cell adenocarcinoma (linitis plastica). A gastrectomy and a splenectomy were performed. The postoperative course was characterized by a complete disappearance of the symptoms of angioedema and hemolytic anemia. A local recurrence of the tumor 5 months later could not be resected. The patient died 17 months after the initial surgery was performed. RESULTS: Quantitative and functional analyses of the complement factors showed persistent excessive complement consumption. Markers of hemolytic anemia disappeared after tumor removal but recurred in the second part of the disease evolution. Immunohistochemical findings in tumor tissue showed loss of normal blood group antigens but expression of Lea antigen, as well as C1q deposition. CONCLUSION: To explain the whole clinical and laboratory picture, we hypothesize a connection between tumor immunohistochemical profile, complement consumption, and hemolytic anemia. Tumor cell surface antigens might lead to a permanent but asymptomatic complement consumption that is worsened and becomes clinically manifest by superimposed hemolytic anemia caused by cross-reactive antibodies to newly expressed blood group antigens on tumor cells. This hypothesis should be confirmed by other observations.

Aged↗
PubMed1991

Complement-opsonized IgG antibody/dsDNA immune complexes bind to CR1 clusters on isolated human erythrocytes.

We used fluorescence microscopy, quantitative FACS analyses, and radioimmunoassays to examine the distribution of complement (C3b)-opsonized antibody/dsDNA immune complexes (IC) bound to normal human erythrocytes (RBCs) via immune adherence (IA). IC were detected with fluorescent anti-human IgG, and the RBC IA-receptor (CR1) was revealed with monoclonal antibodies (Mabs) to CR1 and fluorescent anti-mouse IgG. Under saturating conditions RBCs exhibit a large heterogeneity in binding; a significant fraction binds no IC. The positions of bound IC coincide with CR1 clusters on RBCs, as predicted by several investigators. FACS experiments indicate an excellent correlation between CR1 number and IC binding within an RBC population. The number of CR1 clusters able to bind IC is proportional to the number of CR1 per RBC. However, IC (fluorescent spots) detected per RBC (on average less than 10) are less than the average number of IC bound (20-30). This suggests that each fluorescent spot represents a small number of aggregated IC bound to a CR1 cluster. These "patches" of aggregated complexes may facilitate transfer of RBC-bound IC to cells of the mononuclear phagocytic system. Finally, not all the CR1 clusters bind IC, suggesting that proper geometric alignment of multiple C3b per IC with several CR1 in the cluster is required for IA.

Antigen-Antibody Complex↗
PubMed1987

Quantitative comparison of direct and cold-target competition assay in monocyte- and complement-mediated lysis of sensitized erythrocytes.

Differences in the number of the antigenic determinants of A1 and B erythrocytes were exploited to study the influence of the sensitization on antibody-dependent monocyte- and complement-mediated cytotoxicity. Lysis was compared in direct and cold target competition assays. The findings were: (1) Monocyte mediated lysis of both targets was similar; A1 erythrocytes, however, showed stronger competitive inhibition than B cells; (2) the competitive inhibition was influenced by the antiserum concentration in a range where the direct lysis was not; (3) in the presence of optimal concentrations of hyperimmune antisera and complement, A1 erythrocytes were lysed, while B erythrocytes were not; and (4) A1 cells exposed to suboptimal concentrations of complement consumed complement, while B cells did not. It was concluded that the cold target competition assay is more sensitive than the direct cytotoxicity test to detect differences in the sensitization of erythrocytes.

Antibodies↗
PubMed1994

Complement C3, eosinophil cationic protein and symptom evaluation in interstitial cystitis.

Interstitial cystitis is a painful, irritative voiding dysfunction of unknown etiology. In this study 19 women undergoing treatment for interstitial cystitis and 6 healthy women of similar age provided 2 urine and serum specimens with at least a 3-month interval between collections. Complement C3 and eosinophil cationic protein were determined by immunoassay methods, and symptom severity was quantitated with a visual analog scale questionnaire. Concentrations of complement C3 and eosinophil cationic protein from either serum or urine were not significantly different between interstitial cystitis patients and controls at either determination, although substantial differences were noted even between individual initial and followup determinations. Normalization of urine osmolality did not alter these results. Symptom severity scores were significantly greater in interstitial cystitis patients compared to controls but failed to correlate with the concentrations of complement C3 or eosinophil cationic protein. Therefore, the hypothesis that complement C3 and eosinophil cationic protein may be etiological factors of interstitial cystitis is not supported.

Adult↗
PubMed1992

Quantitative ultrasonic imaging: tissue characterization and instantaneous quantification of cardiac function.

Quantitative myocardial tissue characterization is being developed to complement and expand conventional echocardiography by delineating the physical state of myocardium under diverse pathophysiologic conditions. Real-time quantitative integrated backscatter imaging has already been applied to patients with ischemic heart disease, hypertrophic cardiomyopathy, and cardiac allograft rejection in clinical investigations performed in the United States, Europe, and Japan. A recently introduced modification of imaging processing algorithms employed for characterization of tissue facilitates automatic detection of endocardial-blood interfaces and on-line quantification of ventricular size and function. Further progress and anticipated developments in quantitative ultrasonic imaging will undoubtedly augment the clinical applications of tissue characterizations based on myocardial integrated backscatter for improved diagnosis, elucidation of pathophysiology, and assessment of cardiac function.

Animals↗
PubMed2006

Studying patient safety in health care organizations: accentuate the qualitative.

BACKGROUND: The study of patient safety can benefit from greater methodological diversity to improve scientific knowledge and to increase the effectiveness and tailoring of strategies aimed at improving it. METHODOLOGICAL DIVERSITY TO BETTER CAPTURE CAUSAL MECHANISMS AND PROCESSES: Additional methods for studying patient safety and errors to reflect the complexity of what goes on within health care organizations should be made routine. Interviews, focus groups, and observation--the predominant methods used in qualitative research--are infrequently used in health services research, generally and specifically in the study of errors and patient safety. However, they offer several advantages over quantitative designs. They often are less expensive and quicker to implement; they may not need a lot of advance work; and they can be used to study retrospectively a particular failure event, outcome, or situation. ACTION STEPS: Organizations can use an action agenda to better implement and promote the use of qualitative methods. Implementing these action steps can help achieve the attributes-trust, honesty, communication, participation, and efficiency-necessary to facilitate the qualitative approach in health care work settings. SUMMARY AND CONCLUSION: Qualitative approaches should be used in studying patient safety as a complement to (not a substitute for) quantitative approaches. They can be implemented more easily in organizations through structural and cultural adjustments that provide a more supportive foundation for this work.

Health Knowledge, Attitudes, Practice↗
PubMedDate not supplied

[Demonstration and differentiation of circulating immune complexes in chronic inflammatory rheumatic diseases using a semiautomated PEG-precipitation laser nephelometer test].

Circulating immune complexes were determined in rheumatoid arthritis and systemic lupus erythematosus using a semiautomated PEG-precipitation laser-nephelometer technique. Immunoglobulins IgA, IgG and IgM as well as complement proteins C3c and C4 were quantitatively determined in 2.75% PEG-precipitates. The PEG-precipitates obtained from the sera of healthy controls, SLE patients, and RA subjects displayed different protein patterns. In RA sera a significant elevation of all investigated proteins was found. In SLE sera, a significant increase of IgG, IgA, IgM, and C3 was found, whereas C4 was significantly reduced as compared to the controls. Typical values for a calculated IgG/C4 ratio in circulating immune complexes were obtained for RA and SLE. In RA this ratio remained unchanged in different activity stages and was found in seropositive as well as in seronegative sera. Thus circulating immune complexes from SLE and RA sera differ with respect to the degree of complement-dependent solubilization.

Adult↗
PubMed1961

Humoral aspects of the immune response to homografts. II. Relationship between the hemagglutinating and cytotoxic activities of certain isoimmune sera.

Isoimmune sera, prepared in BALB/c mice by immunization with living or lyophilized tissue from C57Bl/6 mice, were found to possess both hemagglutinating and cytotoxic activity for donor cells. Comparative titrations indicated a good correspondence between these two activities. Absorbtion with living or dead donor tissue caused removal or proportionate reduction of both activities. The results are consistent with the existence in isoimmune sera of a single class of isoantibodies, capable of agglutinating donor cells or of sensitizing them for complement lysis. Living and lyophilized tissue differ quantitatively, but not qualitatively, in their capacity to induce formation of these antibodies.

Allografts↗
PubMedDate not supplied

Prediction of complement-inhibitory activity of benzamidines using topological and geometric parameters.

A hierarchical approach to quantitative structure-activity relationship (QSAR) modeling has been used to estimate the complement-inhibitory potency of 105 benzamidines. This hierarchical approach uses topostructural, topochemical, and geometric parameters in a stepwise fashion to build increasingly more complex models. The results show that topostructural indices alone, specifically I(D), predict inhibitory potency reasonably well. The addition of topochemical and geometrical parameters to the set of descriptors provides only marginal improvement in predictive power. However, when taken alone, the geometric parameter (3D)W provides a more stable model than the topostructural one.

Animals↗
PubMed1994

Quantitative analysis of steroid hormone receptors and their messenger ribonucleic acids.

Ligand binding will remain the basic technique for receptor determinations in many laboratories. It can easily be set up and it is inexpensive. It has, however, the disadvantage of requiring relatively large amounts of tissue or cultured cells, if the proper multipoint measurements for Scatchard plots are to be done. Furthermore, each of the bound/free separation techniques mentioned above has its own caveat that has to be respected if analytically correct results are to be obtained. The great advantage of receptor immunoassays is their technical simplicity and the possibility of measuring a single dose of a receptor sample (in contrast to the Scatchard plot approach in ligand-binding assays). The possibility of using a single dose (even if assayed in duplicate) is a very valuable feature in all instances when only small tissue samples are available for assay. It would be very attractive for many laboratories to set up their own receptor immunoassays. The availability of suitable antibodies may not be a major obstacle. However, the necessity of possessing a supply of a highly purified receptor standard preparation may pose a problem. This is why commercial kits seem to be used so frequently. The analysis of receptor mRNA is a complement of or alternative to receptor quantitation. It must be realized, however, that special skills, as well as a molecular biology laboratory environment and equipment, are required for successful analytic work in this area. Solution hybridization is to be preferred as an approach to obtain results of a quantitative character. However, the specificity of hybridization should be checked by Northern blots. The same is true for dot/slot hybridization, which is a suitable method for semiquantitative assessments of a series of samples. Last but not least, the in situ hybridization provides invaluable information on the tissue and cell distribution of the mRNA analyzed.

Animals↗
PubMed1983

Activation of the complement system and accumulation of hemoglobin-haptoglobin complexes in plasma during an adverse reaction to penicillin treatment.

A patient treated with penicillin intravenously developed a serum sickness-like reaction. Classical pathway complement (C) activation was indicated by quantitation of the split products C3c and C3d as well as demonstration of C4 conversion. Circulating immune complexes could, however, not be detected by the solid-phase Clq and PEG-precipitation methods. A concomitant accumulation of circulating hemoglobin-haptoglobin complexes and a marked fall in serum-fibronectin concentrations suggested saturation of the reticuloendothelial system. The plasma became a deep-red color, and a diffuse intravascular coagulation followed. The patient recovered completely upon discontinuation of penicillin administration.

Antigen-Antibody Complex↗
PubMed2001

Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.

Anemia, Hemolytic↗
PubMed1986

Activated terminal complement in cerebrospinal fluid in Guillain-Barré syndrome and multiple sclerosis.

A quantitative enzyme-linked immunosorbent assay was used to measure the concentration of fluid-phase complement C5b-9 complexes (SC5b-9) in the cerebrospinal fluid (CSF) of 14 patients with acute monophasic Guillain-Barré Syndrome (GBS), 21 patients with multiple sclerosis (MS), and 11 patients with noninflammatory central nervous system (CNS) diseases. SC5b-9 complexes were detected in the CSF of 13 of 14 patients with acute GBS (mean, 3.08 micrograms/ml; range, 0 to 7.1 micrograms/ml) and 16 of 21 patients with MS (mean, 1.83 micrograms/ml; range, 0 to 7.5 micrograms/ml). In the control group of patients with noninflammatory CNS diseases, SC5b-9 was not detected in eight of 11 and was present in low concentrations in the remaining three patients (mean, 0.28 micrograms/ml; range, 0 to 1.7 micrograms/ml). The finding of SC5b-9 complexes in the CSF of patients with GBS and MS suggests that terminal complement components may participate in the tissue-damaging processes in these diseases.

Complement Activation↗
PubMed2002

Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation.

Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4 degrees C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.

Animals↗