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PubMed1972

The relationship of the chemotactic behavior of the complement-derived factors, C3a, C5a, and C567, and a bacterial chemotactic factor to their ability to activate the proesterase 1 of rabbit polymorphonuclear leukocytes.

The inhibition profiles obtained when a series of p-nitrophenyl ethyl alkylphosphonates and of p-nitrophenyl ethyl chloroalkylphosphonates were used to interfere with the chemotactic activity of polymorphonuclear leukocytes stimulated by C3a, C5a, and bacterial factor were the same as found previously when C567 was the chemotactic agent. This indicates that as in the chemotactic activity induced by C567, an obligatory step in the chemotaxis caused by C3a, C5a, and bacterial factor is the activation of proesterase 1 of the rabbit polymorphonuclear leukocyte. C5a and C3a activate proesterase 1 of peripheral blood polymophonuclear leukocytes as measured by the increase of acetyl DL-phenylalanine beta-naphthyl esterase activity. Attempts to detect in a like manner the proesterase 1 of the same leukocytes using bacterial factor under varying circumstances have consistently failed. It is concluded that bacterial factor, for unknown reasons, is unable to activate proesterase 1 to the same extent as the complement-derived chemotactic factors. The hypothesis of there being a quantitative difference in the ability of bacterial factor to activate proesterase 1 compared with the complement-derived factors explains the previous observations that bacterial factor can not deactivate to itself or to the complement-derived factors, although these latter factors can deactivate to themselves, to each other, and to the bacterial factor. The quantitative difference in the ability of bacterial factor to activate proesterase 1 compared to the complement-derived factors is also associated with and explains the finding that the maximal chemotactic activity attainable when bacterial factor is the chemotactic agent is distinctly less than that obtained using either C3a, C5a, or C567. These results indicate that the activation of proesterase 1 is a general requirement for the chemotactic activity of rabbit polymorphonuclear leukocytes with known macromolecular chemotactic agents and suggest that under several different circumstances the level of chemotactic activity attained is related to the degree of such activation.

Animals↗
PubMed1982

Functional evidence for complement-activating immune complexes in the skin of patients with bullous pemphigoid.

Previous immunofluorescent studies showing deposits of immunoglobulin and complement at the cutaneous basement membrane zone have provided evidence supporting a role for immune complexes in the pathogenesis of bullous pemphigoid. In this study the functional activity of the deposits has been examined using leukocyte attachment, a method for detecting and quantitating the biological activity of complement-activating immune complexes in tissues. When peripheral blood leukocytes suspended in serum complement were incubated with cryostat sections of lesional and adjacent normal-appearing skin from 9 patients with pemphigoid, skin from 11 normal controls and lesional skin from 14 nonpemphigoid disease controls there was significantly greater attachment of leukocytes to the basement membrane zone of lesional bullous pemphigoid skin compared to normal-appearing pemphigoid skin and skin of both control groups. A significant reduction in attachment in the absence of serum complement suggested the reaction was dependent on activation of complement by tissue-deposited complexes. Although leukocyte attachment was greater in lesional than normal-appearing pemphigoid skin, a comparison of the incidence and intensity of cutaneous IgG and complement immunofluorescence between the 2 groups showed no significant differences. Furthermore, no correlation between leukocyte attachment and serum titers of immunoglobulin G or complement-binding anti-basement membrane zone antibodies was observed. These results suggest that immune reactants in lesional pemphigoid skin are functional complement-activating immune complexes, that differences exist between the activity of complexes in lesional and normal-appearing pemphigoid skin and may explain why lesions develop at some sites and not others.

Antigen-Antibody Complex↗
PubMed1981

Humoral immune killing of nucleated cells: mechanisms of complement-mediated attack and target cell defense.

The killing or lysis of nucleated cells, erythrocytes, bacteria, and other targets (e.g., liposomes) by antibody and complement is the result of complex series of actions and interactions between antibody, components of the complement system, and the cell. The complement attack mechanism has strict qualitative and quantitative requirements. For efficient activity, sufficient amounts of antibody must bind to the cell, the antibody must be of the complement-fixing type, and sufficient amounts of complement components must be activated and fixed to the surface of the target cells. Nucleated cells of different types differ in their sensitivity to the cytotoxic action of complement. This difference in sensitivity may be attributed to differences in metabolic properties and/or the chemical and physical composition of the cells. Since complement action occurs primarily on or in the cell membrane, the properties of the cell which may affect the outcome of complement-mediated attack should be linked to cell membrane function and integrity. The relationship between the susceptibility of nucleated cells to complement-mediated killing and the chemical and metabolic properties of the cells will be discussed in this review.

Animals↗
PubMed2003

[Changes of ECR1 genomic density polymorphism, quantitative expression and the activity of ECR1 natural adhesion in patients with chronic hepatitis].

OBJECTIVE: To study the changes of genomic density polymorphism, quantitative expression and the adhesion activity of complement receptor type 1 (ECR1) on erythrocytes in patients with chronic hepatitis. METHODS: Polymerase chain reaction (PCR) and Hind restriction enzyme digestion, the quantitative assay of ECR1 and the activity of erythrocytes immune adhesion test were applied. RESULTS: The spot mutation rate (25.0%-30.3%) of ECR1 density gene in patients with chronic hepatitis was not significantly different from that of healthy individuals (28.0%). The amount of ECR1 in patients with chronic hepatitis, except for the diseases with normal liver function, was significantly lower than that of healthy individuals (t=9.87,P<0.000 1). The quantitative expression of ECR1 in decompensated cirrhosis was obviously lower than that of compensated cirrhosis (t=2.21,P<0.05). CONCLUSIONS: Defective expression of ECR1 in chronic hepatitis B may be acquired through central and/or peripheral mechanisms. It is very important to study the quantitative expression in the patients with chronic hepatitis.

Erythrocytes↗
PubMed2006

Quantitative analysis of protein co-localization on B cells opsonized with rituximab and complement using the ImageStream multispectral imaging flow cytometer.

Binding of the chimeric, humanized anti-CD20 mAb Rituximab (RTX) to B lymphocytes activates complement and promotes covalent deposition of C3 fragments (C3b/iC3b) on cells. Previous fluorescence microscopy studies, based on examination of B cell lines and of blood samples from RTX-treated CLL patients, suggest that C3b/iC3b is closely associated with cell-bound RTX. We examined Raji cells opsonized with serum and RTX with the ImageStream imaging flow cytometer. Cells were stained with fluorescently-labeled RTX and mAbs specific for C3b/iC3b fragments or for human IgG, and then imaged using the ImageStream cytometer and analyzed with an algorithm (Similarity Bright Detail Score, SBDS) which tests for co-localization of fluorescent probes. SBDS, calculated on 10,000 cells, verified that the majority of deposited C3b/iC3b is co-localized with bound RTX. In contrast, when cells were first opsonized in serum alone, washed and then reacted with RTX, SBDS confirmed that RTX and C3b/iC3b are poorly co-localized, thus demonstrating that cell-bound RTX directs deposition of C3b. In addition, a sulfhydryl-specific probe, maleimide conjugated to AF488, exhibited substantial co-localization with an anti-C3b/iC3b mAb on Raji cells opsonized with RTX and serum, thus validating maleimide labeling as an alternative for detecting cell-bound C3b/iC3b. The digital imaging method described should have wide applicability for quantitative analysis of co-localization.

Algorithms↗
PubMed2006

Inhibition of complement component C3 reduces vein graft atherosclerosis in apolipoprotein E3-Leiden transgenic mice.

BACKGROUND: Venous bypass grafts may fail because of development of intimal hyperplasia and accelerated atherosclerosis. Inflammation plays a major role in these processes. Complement is an important part of the immune system and participates in the regulation of inflammation. The exact role of complement in the process of accelerated atherosclerosis of vein grafts has not yet been explored, however. METHODS AND RESULTS: To assess the role of complement in the development of vein graft atherosclerosis, a mouse model, in which a venous interposition was placed in the common carotid artery, was used. In this model, vein graft thickening appeared within 4 weeks. The expression of complement components was studied with the use of immunohistochemistry on sections of the thickened vein graft. C1q, C3, C9, and the regulatory proteins CD59 and complement receptor-related gene y could be detected in the lesions 4 weeks after surgery. Quantitative mRNA analysis for C1q, C3, CD59, and complement receptor-related gene y revealed expression of these molecules in the thickened vein graft, whereas C9 did not show local mRNA expression. Furthermore, interference with C3 activation with complement receptor-related gene y-Ig was associated with reduced vein graft thickening, reduced C3 and C9 deposition, and reduced inflammation as assessed by analysis of influx of inflammatory cells, such as leukocytes, T cells, and monocytes. In addition, changes in apoptosis and proliferation were observed. When C3 was inhibited by cobra venom factor, a similar reduction in vein graft thickening was observed. CONCLUSIONS: The complement cascade is involved in vein graft thickening and may be a target for therapy in vein graft failure disease.

Animals↗
PubMed1980

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots.

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.

Journal Article↗
PubMed1983

Quantitation of in vitro opsonic activity of human antibody induced by a vaccine consisting of the type III-specific polysaccharide of group B streptococcus.

Human antibody, induced by a vaccine consisting of undegraded and highly purified extracellular type III-specific polysaccharide of group B streptococcus, was shown to increase the rate of phagocyte-mediated killing of bacteria of the homologous type. The bactericidal effect was mediated by type III-specific antibody and was complement dependent. An assay which permitted quantitation of "opsonic activity" was developed. In this assay, loss of CFUs occurred at a constant rate, and the rate constant was used as a measure of opsonic activity of antisera. A linear relationship between type III-specific antibody concentration (40 to 500 ng/ml) and the rate constant of killing was observed. When sets of immune sera were tested, some sera reacted anomalously, mediating significantly higher or lower rates than expected on the basis of their antibody content. Since type III-specific antibody in immune sera was found almost exclusively in the immunoglobulin G class, we hypothesize that differences in immunoglobulin G subclass distribution of specific antibody may have been the source of this variation.

Antibodies, Bacterial↗
PubMed1966

Quantitative assay of Coxiella burnetii in mice.

Experimental data are presented which demonstrate that the complement-fixing antibody response in individual mice can be used for quantitative assay of Coxiella burnetii. The method allows the replacement of a single guinea pig with a single mouse, thus resulting in considerable savings in caging requirements and animal costs.

Animals↗
PubMed1983

Platelet associated immunoglobulins and complement in idiopathic thrombocytopenic purpura.

A double antibody sandwich enzyme linked immunosorbent assay (ELISA) was applied to quantitate platelet associated (PA) immunoglobulins G, A and M and complement factors C3c and C4. Fifteen patients with acute and 29 patients with chronic idiopathic thrombocytopenic purpura (ITP) were studied as well as 35 normal controls. Forty-three out of 44 (98%) patients had elevated platelet associated immunoglobulins. PAIgG was elevated in 95%, PAIgA in 82% and PAIgM in 74% of the patients. PAC3 was increased in 86% and PAC4 in 57% of the patients. There was strong correlation between PAC3 and PAIgG but not between PAC4 and PAIgG in acute ITP. In chronic ITP, however, PAC4 correlated strongly and even better than PAC3 with PAIgG. This strengthens the conjecture that the pathogenesis of ITP in many acute cases differs from that of chronic ITP.

Adult↗
PubMed2006

Simulations of optimized anguilliform swimming.

The hydrodynamics of anguilliform swimming motions was investigated using three-dimensional simulations of the fluid flow past a self-propelled body. The motion of the body is not specified a priori, but is instead obtained through an evolutionary algorithm used to optimize the swimming efficiency and the burst swimming speed. The results of the present simulations support the hypothesis that anguilliform swimmers modify their kinematics according to different objectives and provide a quantitative analysis of the swimming motion and the forces experienced by the body. The kinematics of burst swimming is characterized by the large amplitude of the tail undulations while the anterior part of the body remains straight. In contrast, during efficient swimming behavior significant lateral undulation occurs along the entire length of the body. In turn, during burst swimming, the majority of the thrust is generated at the tail, whereas in the efficient swimming mode, in addition to the tail, the middle of the body contributes significantly to the thrust. The burst swimming velocity is 42% higher and the propulsive efficiency is 15% lower than the respective values during efficient swimming. The wake, for both swimming modes, consists largely of a double row of vortex rings with an axis aligned with the swimming direction. The vortex rings are responsible for producing lateral jets of fluid, which has been documented in prior experimental studies. We note that the primary wake vortices are qualitatively similar in both swimming modes except that the wake vortex rings are stronger and relatively more elongated in the fast swimming mode. The present results provide quantitative information of three-dimensional fluid-body interactions that may complement related experimental studies. In addition they enable a detailed quantitative analysis, which may be difficult to obtain experimentally, of the different swimming modes linking the kinematics of the motion with the forces acting on the self-propelled body. Finally, the optimization procedure helps to identify, in a systematic fashion, links between swimming motion and biological function.

Animals↗
PubMed1982

Monoclonal antibodies to human prostate and bladder tumor-associated antigens.

Monoclonal antibodies to human prostate adenocarcinoma membrane antigens were produced by fusion of P3X63/Ag8 mouse myeloma cells with spleen cells from BALB/c mice immunized against the prostate cancer cell line DU145. The hybrids were screened for antibody production using glutaraldehyde-fixed cells in a solid-phase radioimmunoassay. Antibody-binding specificity was also checked by quantitative adsorption, membrane immunofluorescence, and complement-dependent cytotoxicity assays. A hybridoma clone (83.21) was isolated that secreted antibodies which preferentially bound to several prostate and bladder cancer cell lines but did not bind to a variety of other normal and malignant human cell lines. This antibody also reacted with a cytomegalovirus-transformed human embryonic lung cell line but not to normal human embryonic lung cells. Quantitative adsorption studies demonstrated that the 83.21 monoclonal antibody was strongly reactive to membrane preparations from human prostate adenocarcinoma tissue and a liver metastasis of prostate carcinoma. Little or no binding activity was observed against two other prostate carcinomas, bening prostatic hyperplasia, normal prostate, or normal liver. Binding studies indicate that the 83.21 monoclonal antibody does not bind to alpha-fetoprotein, carcinoembryonic antigen, prostatic acid phosphatase, human leukocyte antigen, beta 2-microglobulin, HLA-Dr antigens, fibronectin, or prostate antigen. The data indicate that we have isolated a monoclonal antibody that binds to an antigen(s) expressed by several urogenital carcinoma cell lines as well as human prostate tumor tissue and that the antibody is not directed against well-known human tumor cell markers.

Animals↗
PubMed1982

Rapid complementation method for classifying excision repair-defective xeroderma pigmentosum cell strains.

A rapid method has been developed that permits demonstration of complementation between different cell strains from ultraviolet-sensitive xeroderma pigmentosum patients. Combining polyethylene glycol-mediated cell fusion with low doses of ultraviolet light to eliminate unfused sensitive cells, the method permits assignment of cell strains to complementation groups by visual inspection, avoiding use of laborious methods involving autoradiography. This method can be augmented by measuring DNA repair synthesis, which shows large quantitative differences between fusions that result in complementation and those that do not.

Cell Line↗
PubMed1976

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.

Animals↗
PubMed2004

Quantitative trait loci for apolipoprotein B, cholesterol, and triglycerides in familial combined hyperlipidemia pedigrees.

OBJECTIVE: Familial combined hyperlipidemia (FCHL) is a genetically complex lipid disorder that is diagnosed in families by combinations of increased cholesterol, triglycerides, and/or apolipoprotein B (apoB) levels in patients and their first-degree relatives. Identifying the predisposing genes promises to reveal the primary risk factors and susceptibility pathways and suggest methods of prevention and treatment. As with most genetically complex disorders, a clinical definition of disease may not be the most useful phenotype for finding the complement of predisposing genes, and the quantitative traits used to define the disorder can provide important information. This is a report of a quantitative trait loci (QTL) analysis of FCHL. METHODS AND RESULTS: A full genome scan of 377 multi-allelic markers genotyped at approximately 10 centimorgan (cM) intervals was conducted in 150 sibling pairs from 22 nuclear families in FCHL pedigrees. These data were analyzed by 2 multipoint QTL linkage methods using the nonparametric and Haseman-Elston procedures of the Genehunter software. Using a criterion of P<0.001 by the nonparametric analysis, we found evidence of 2 apoB QTL at 1p21-31 (P<0.000009) and 17p11-q21 (P<0.000009), a total serum cholesterol QTL at 12p13 (P<0.0001), and a serum triglycerides QTL at 4p15-16 (P<0.0002). Using the criterion of P<0.03 for at least 2 traits at the same locus, additional evidence for cholesterol (P<0.01) and a triglycerides P<0.02) was observed at 17p11-21, as well as suggestive evidence for apoB (P<0.02) and triglycerides (P<0.01) at 4q34-35, and cholesterol (P<0.01) and triglycerides (P<0.02) and a binary FCHL trait (lod=1.5) at 16p12-13. CONCLUSIONS: QTL analyses of the traits that define FCHL are effective for localizing disease-predisposing genes.

Apolipoproteins B↗
PubMed1985

[Mechanisms of the mediated neutrophil reactivity to Escherichia coli peptidoglycan].

The opsonic properties of normal serum with respect to E. coli peptidoglycan was studied under the actual conditions of the oxygen-dependent metabolism of neutrophils. In the course of the differentiated study of the influence of antibodies, the classical and the alternative cascades of complement the serum was heated, treated with ethylenediaminetetraacetate and ethylene glycol tetraacetate, exhausted in the cold. In serial experiments the opsonic activity of purified fibronectin was studied. The indirect reactions were shown to be the leading mechanisms of the neutrophil-stimulated activity of E. coli peptidoglycan. IgG was found to be in the center of the opsonic cooperation and thus to determine the quantitative manifestation of the total phenomenon. Complement proved to be of lesser importance; depending on the conditions of the experiment, the activation of complement occurred by the alternative way (after the removal of antibodies) or the classical way (whole serum). The actual contribution of IgG-independent and complement-independent opsonins was insignificant. Fibronectin in physiological concentrations showed no opsonic activity.

Antibodies, Bacterial↗
PubMed2005

Classification analysis of the transcriptosome of nonlesional cultured dermal fibroblasts from systemic sclerosis patients with early disease.

OBJECTIVE: To compare the transcriptosome of early-passage nonlesional dermal fibroblasts from systemic sclerosis (SSc) patients with diffuse disease and matched normal controls in order to gain further understanding of the gene activation patterns that occur in early disease. METHODS: Total RNA was isolated from early-passage fibroblasts obtained from nonlesional skin biopsy specimens from 21 patients with diffuse SSc (disease duration <5 years in all but 1) and 18 healthy controls who were matched to the cases by age (+/-5 years), sex, and race. Array experiments were performed on a 16,659-oligonucleotide microarray utilizing a reference experimental design. Supervised methods were used to select differentially expressed genes. Quantitative polymerase chain reaction (PCR) was used to independently validate the array results. RESULTS: Of the 8,324 genes that passed filtering criteria, classification analysis revealed that <5% were differentially expressed between SSc and normal fibroblasts. Individually, differentially expressed genes included COL7A1, COL18A1 (endostatin), DAF, COMP, and VEGFB. Using the panel of genes discovered through classification analysis, a set of model predictors that achieved reasonably high predictive accuracy was developed. Analysis of 1,297 gene ontology (GO) classes revealed 35 classes that were significantly dysregulated in SSc fibroblasts. These GO classes included anchoring collagen (30934), extracellular matrix structural constituent (5201), and complement activation (6958, 6956). Validation by quantitative PCR demonstrated that 7 of 7 genes selected were concordant with the array results. CONCLUSION: Fibroblasts cultured from nonlesional skin of patients with SSc already have detectable abnormalities in a variety of genes and cellular processes, including those involved in extracellular matrix formation, fibrillogenesis, complement activation, and angiogenesis.

Dermis↗
PubMed2005

The inferior colliculus of the rat: a quantitative analysis of monaural frequency response areas.

Frequency response areas (FRAs) were measured for 237 single units in the inferior colliculus (IC) of urethane-anesthetized pigmented rats using monaural pure-tone stimulation. Based on qualitative criteria [J Neurosci 21 (2001) 7303], FRAs were classified as V-shaped in 69% of neurons, non-V-shaped in 29%, and unclassifiable in the remaining 2%. Non-V-shaped FRAs were heterogeneous, comprising a number of subtypes including narrow, closed, low- and high-tilt, multipeaked, U-shaped, mosaic and inhibitory. To complement this subjective classification, we applied quantitative measures used by others (e.g. [J Neurophysiol 84 (2000) 1012]), including the inverse slope of the upper and lower FRA borders, Q-values, and other measures of bandwidth. The results suggest that FRAs in the rat IC are best described as forming a continuous distribution among subtypes, rather than clustering into discrete categories. Moreover, there is a broad range of frequency tuning characteristics and FRA types across the entire frequency spectrum. Within this general pattern, however, there are some frequency-specific differences in FRA type distribution. The relative proportion of V-shaped FRAs was greatest at the high and low ends of the auditory range, with the highest proportion of non-V-shaped FRAs in the mid-range from 6 to 12 kHz. For most neurons with multipeaked FRAs, the peak frequencies were not harmonically related. Frequency tuning in the pigmented rat IC is generally similar to that in other species. Comparison of Q values across auditory nuclei shows little evidence that FRAs are sharpened at levels above the auditory nerve. Rather, there is a broad range of frequency tuning properties at each level.

Acoustic Stimulation↗