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PubMed1991

A microassay for the pore-forming activity of complement, perforin, and other cytolytic proteins based on confocal laser scanning microscopy.

A fluorescence microscopic assay for the activity of complement, perforin, and other cytolytic proteins which form transmembrane pores in cellular membranes is described. The assay was worked out and tested with red blood cell membranes (ghosts) and was then applied to intact hemoglobin-free cells. Resealed human erythrocyte ghosts were incubated with complement or perforin. A small polar fluorescent probe (fluorescein-labeled 1-kDa dextran, FD1) which permeates through complement and perforin pores but not through normal cell membranes was added to the samples. The capability of the confocal laser scanning microscope (CLSM) to generate thin optical sections was exploited to visualize and quantitate fluorescence inside single ghosts and thus determine the fraction of ghosts which had become permeable for FD1. The activity of complement or perforin was quantitated by plotting the fraction of permeable cells versus the concentration of the pore-forming protein. The results were in good agreement with those of a conventional hemolytic assay. The CLSM-based assay was then applied to intact hemoglobin-free cells for which only few alternative assays are available. Compared to conventional hemolytic assays for the activity of pore-forming proteins the assay described here can be applied to a large variety of natural and artificial membrane systems. The assay can be performed under nonlysing conditions. Furthermore, the assay is simple, relatively fast, and requires only extremely small amounts of cells and pore-forming proteins.

Animals↗
PubMed1984

"Functional polarity" of the spinal cord caused by its longitudinal electric dipole moment.

Specific reactions of the vertebrates that occur through lengthwise application of direct-current (DC) electric fields in the spinal cord and are dependent on the direction of the current (e.g., electric narcosis and electric excitation) can be explained by the existence of a longitudinal dipole moment in the spinal cord. The relationship between "functional polarity of the spinal cord" and its longitudinal dipole moment was established in two different experimental series. 1) Reactions of functional polarity were quantitatively tested on Amphioxus (lancelet) and on numerous Teleostei. On this point the findings of other researchers were confirmed and complemented. 2) Fresh, almost-live spinal cord preparations (cow) were quantitatively tested using pyroelectric (PE) methods. A longitudinal electric dipole moment was demonstrated along the entire length of the white matter. Moreover fresh spinal cord preparations showed a polar orientation on sudden temperature change in a high-voltage DC field. The PE behavior showed a pronounced electric field dependence. Because the white matter is largely composed of longitudinally arranged nerve fibers, individual axons may also have a longitudinal electric dipole moment. Consequently one might expect the following: polar orientation of the growing neurite by an applied DC field, an electric dipole field inside and outside the axon, and a transport of charge carriers along the lines of flux of this dipole field. Moreover PE and piezoelectric (PZE) properties of the neuron should be provable.

Animals↗
PubMed2000

Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma.

The prevalence and severity of allergic asthma continue to rise, lending urgency to the search for environmental triggers and genetic substrates. Using microarray analysis of pulmonary gene expression and single nucleotide polymorphism-based genotyping, combined with quantitative trait locus analysis, we identified the gene encoding complement factor 5 (C5) as a susceptibility locus for allergen-induced airway hyperresponsiveness in a murine model of asthma. A deletion in the coding sequence of C5 leads to C5-deficiency and susceptibility. Interleukin 12 (IL-12) is able to prevent or reverse experimental allergic asthma. Blockade of the C5a receptor rendered human monocytes unable to produce IL-12, mimicking blunted IL-12 production by macrophages from C5-deficient mice and providing a mechanism for the regulation of susceptibility to asthma by C5. The role of complement in modulating susceptibility to asthma highlights the importance of immunoregulatory events at the interface of innate and adaptive immunity in disease pathogenesis.

Animals↗
PubMed1990

Expression of three plant glutamine synthetase cDNA in Escherichia coli. Formation of catalytically active isoenzymes, and complementation of a glnA mutant.

Three cDNA clones encoding the closely related glutamine synthetase (GS) alpha, beta and gamma polypeptides of Phaseolus vulgaris (French bean) were recombinantly expressed in Escherichia coli. The GS expression plasmids correctly synthesised the recombinant alpha, beta and gamma polypeptides which then assembled into catalytically active homo-octameric isoenzymes. These isoenzymes behaved similarly to their native homologues on ion-exchange and gel-filtration chromatography. Furthermore, the alpha and gamma isoenzymes complemented a GS(glnA)-deficient mutant, thus demonstrating their physiological activity in E. coli. Differences were observed between the three recombinant GS plasmids in their quantitative expression of the GS polypeptides and their ability to complement the E. coli mutant. These differences were correlated to the degree of solubility of the polypeptide, which was observed to be dependent on the temperature of expression. The production of active GS isoenzymes in E. coli facilitates the isolation and characterisation of the individual P. vulgaris homo-octameric GS isoenzymes.

Base Sequence↗
PubMed1977

Virulence and immunogenicity of types 1 and 3 Neisseria gonorrhoeae in guinea pig subcutaneous chambers.

The relative virulence and immunogenicity of type 1 (T1) and type 3 (T3) cells of Neisseria gonorrhoeae were determined by tests with two different kinds of subcutaneous chambers in guinea pigs. In tests with a tissue nonencapsulated (NE) chamber, T1 gonococci were found to be greater than 1000 times more virulent as well as about 1000 times more immunogenic than T3 cells of the same gonococcal strain. However, T1 and T3 cells were found to be equally virulent for a tissue encapsulated (TE) chamber in guinea pigs. Analysis of fluids from the two types of chambers in a complement-dependent bactericidal assay revealed that the NE chamber fluid contained a substantially higher level of complement activity than fluid from TE chambers. The decline in complement level of chamber fluids due to tissue encapsulation was also confirmed by quantitation with rocket gel electrophoresis. A greater resistance of T1 cells to the bactericidal effects of complement appeared to provide a mechanism by which the T1 cells were most virulent than T3 cells for subcutaneous chambers in guinea pigs. Consequently, the NE chamber implant would appear to provide a more relevant environment for studying the virulence, as well as immunological characteristics of gonococcal strains and experimental immunogens.

Animals↗
PubMed1997

Bispecific monoclonal antibody complexes facilitate erythrocyte binding and liver clearance of a prototype particulate pathogen in a monkey model.

We used Anger camera imaging in a monkey model to investigate the organ localization of a prototype particulate pathogen, 131I-labeled bacteriophage phi X174, after it was bound to the primate erythrocyte complement receptor and then cleared from the circulation. This 131I-labeled phi X174 was infused into the circulation of an immunized monkey, and the nascently formed immune complexes showed rapid and quantitative binding to erythrocytes via the immune adherence reaction (complement-mediated binding). Alternatively, phi X174 was infused into the circulation of a naive animal, and then cross-linked bispecific mAb complexes (heteropolymers, anti-CR1 x anti-phi X174) were infused into the circulation. The infused heteropolymers also facilitated rapid and quantitative binding of phi X174 to erythrocytes. In both cases, after a short lag period, the erythrocyte-bound phi X174 was rapidly cleared from the circulation, and the vast majority of the radiolabel was cleared to the liver, with a small amount clearing to the spleen. Further liver imaging confirmed that within 24 h most of the bacteriophage previously cleared to the liver via the heteropolymer system was phagocytosed and destroyed. The findings in this model system provide additional evidence for the potential utility of heteropolymers to facilitate the safe and rapid clearance of blood-borne pathogens as a potential treatment for infectious diseases.

Animals↗
PubMed1986

Anaphylatoxins: possible roles in disease.

Anaphylatoxins, in particular C3a and C5a, have various biological activities which suggest a role as mediators of inflammatory reactions: they cause contraction of smooth muscle, histamine release, increase in capillary permeability, adhesion of leukocytes to vascular endothelium, leukocyte chemotaxis, and aggregation of platelets and leukocytes. Most of these effects are supported by the cooperation of other mediators, in particular arachidonic acid derivatives which may be produced by anaphylatoxin-stimulated cells, e.g. leukocytes or endothelium. In vivo effects of the complement peptides depend very much on the site of their generation: intravascular release in the general circulation leads to adverse symptoms such as adult respiratory distress syndrome and shock lung, mainly due to leukocyte activation, aggregation and their accumulation in lung vessels. Intravascular release may be induced by certain drugs, and by contact of blood with the surfaces of bypass or dialysis apparatus. Induction of local inflammatory and defense reactions requires release of anaphylatoxins in tissue spaces. Tissue fluid differs quantitatively from blood plasma in its concentration of complement components. This raises some problems of how efficient concentrations of C3a and C5a can be attained at the site of a lesion to generate a chemotactic gradient capable of attracting blood leukocytes.

Anaphylatoxins↗
PubMed1999

Multicenter trial of the quantitative BTA TRAK assay in the detection of bladder cancer.

BACKGROUND: Human complement factor H-related protein (hCFHrp) is produced by several bladder cancer cell lines and may be useful as a cancer marker. The aim of this study was to compare urinary hCFHrp and cytology for the detection of bladder cancer found by cystoscopy in patients with suggestive signs, symptoms, or preliminary test results. METHODS: The BTA TRAK assay, a quantitative enzyme immunoassay for the bladder tumor-associated antigen in urine, was compared with exfoliative cytology in 220 patients (155 men, 65 women; mean age, 64.2 years) presenting with signs, symptoms, or preliminary diagnostic results suggestive of this disease. Cystoscopy was the standard of detection. RESULTS: In the 100 patients found to have bladder cancer, the overall sensitivities of the BTA TRAK assay (at a previously determined decision threshold of 14 kilounits/L) and cytology were 66% (66 of 100) and 33% (33 of 100), respectively (P <0.001). The BTA TRAK assay proved to be statistically more sensitive than cytology for tumor grades I and II and for stage Ta and T1 tumors. In contrast, the overall specificity of the BTA TRAK assay in the 120 patients without cystoscopically confirmed bladder cancer was 69% (83 of 120) and that of cytology was 99% (119 of 120; P <0.001). The specificity of the BTA TRAK assay was higher in patients without benign or malignant genitourinary disease other than bladder cancer (76%; n = 89) than in patients with these conditions. When the BTA TRAK assay and cytology were used together such that a positive result in either test was scored as positive and the results compared with those of the BTA TRAK assay alone, increases in overall sensitivity and equivalent specificity were observed. CONCLUSION: Because of its relatively high sensitivity, the BTA TRAK assay could complement cytology as an adjunct to cystoscopy in the diagnosis and follow-up of most patients with bladder cancer.

Adult↗
PubMed1972

Role of antibody and complement in the immune clearance and destruction of erythrocytes. I. In vivo effects of IgG and IgM complement-fixing sites.

A model which permits evaluation in molecular terms of the role of antibody and of complement in the immune destruction of erythrocytes was established in the guinea pig. IgM and IgG immunoglobulins were isolated from rabbit anti-guinea pig erythrocyte antisera and were used to sensitize (51)Cr-labeled guinea pig erythrocytes. The average number of complement-fixing sites per erythrocyte formed by antibody was determined for each of the various preparations by the Cla fixation and transfer test. The rate of clearance and of organ localization was determined for cells sensitized with either IgM or IgG antibodies, and dose-response curves were established in normal guinea pigs and guinea pigs with a genetically controlled, complete absence of the fourth component of complement (C4). At least 60 complement-fixing sites per cell were required for accelerated clearance of IgM-sensitized erythrocytes. The bulk of cells with IgM sites were cleared by the liver within 5 min after injection and were then slowly returned to the circulation where they survived normally. There was no accelerated clearance whatsoever of IgM-sensitized erythrocytes in C4-deficient guinea pigs. As few as 1.4 IgG complement-fixing sites per cell resulted in decreased erythrocyte survival. There was no evidence of immediate tissue sequestration and release. Progressive trapping and destruction of erythrocytes by the spleen was responsible for most of the clearance of IgG-sensitized cells. Clearance of IgG-sensitized cells was markedly impaired in guinea pigs with C4 deficiency; however, there was some decrease over normal survival. The data indicate that IgG and IgM antibodies interact with complement in vivo by mechanisms which are qualitatively or quantitatively different and produce different biologic effects.

Anemia, Hemolytic↗
PubMed2004

Quantitative methods for ecological network analysis.

The analysis of networks of ecological trophic transfers is a useful complement to simulation modeling in the quest for understanding whole-ecosystem dynamics. Trophic networks can be studied in quantitative and systematic fashion at several levels. Indirect relationships between any two individual taxa in an ecosystem, which often differ in either nature or magnitude from their direct influences, can be assayed using techniques from linear algebra. The same mathematics can also be employed to ascertain where along the trophic continuum any individual taxon is operating, or to map the web of connections into a virtual linear chain that summarizes trophodynamic performance by the system. Backtracking algorithms with pruning have been written which identify pathways for the recycle of materials and energy within the system. The pattern of such cycling often reveals modes of control or types of functions exhibited by various groups of taxa. The performance of the system as a whole at processing material and energy can be quantified using information theory. In particular, the complexity of process interactions can be parsed into separate terms that distinguish organized, efficient performance from the capacity for further development and recovery from disturbance. Finally, the sensitivities of the information-theoretic system indices appear to identify the dynamical bottlenecks in ecosystem functioning.

Animals↗
PubMed1992

Generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome.

OBJECTIVE: To determine the generation of anaphylatoxin C3a in plasma and bronchoalveolar lavage fluid in trauma patients at risk for the adult respiratory distress syndrome (ARDS). DESIGN: Prospective study. SETTING: ICU in a university hospital. PATIENTS: Severely traumatized patients at risk for the ARDS (n = 25). INTERVENTION: EDTA plasma samples and bronchoalveolar lavage fluid were obtained. MEASUREMENTS AND MAIN RESULTS: Complement proteins C3, C4, C5, and the inhibitors C1-inhibitor, Factor H, and Factor I were quantitated in EDTA-plasma samples obtained every 6 hrs during the first 48 hrs after ICU admission and every morning from days 4 to 14 after injury. In bronchoalveolar lavage fluid, the complement activation production of C3a-desArg was quantitated and the volume of epithelial lining fluid was calculated. All patients showed a decrease of the complement proteins C3, C4, C5 and of the inhibitors C1-inhibitor, Factor H, and Factor I during the first 24 hrs, indicating complement consumption. Patients developing ARDS (n = 11) showed significantly higher C3 concentrations and a higher C3a/C3 ratio in the first few hours after multitrauma. Follow-up bronchoalveolar lavages demonstrated highly increased amounts of C3a in epithelial lining fluid during the first 24 hrs, mainly in ARDS patients and, to a lesser degree, in non-ARDS patients. To determine the origin of C3a in bronchoalveolar lavages, the ratio of C3a in epithelial lining fluid and plasma was calculated. CONCLUSION: The C3a of epithelial lining fluid to plasma ratio was extremely high in patients developing ARDS, but even the non-ARDS group had a ratio greater than 1, indicating that a substantial local complement activation occurs in the lung.

Adult↗
PubMed1975

Detection and quantitation in plasma and synovial fluid of a fragment of human C4 with alpha mobility generated during the activation of the complement system.

A newly identified fragment of human C4 was detected, using a particular antiserum, in human serum after activation by heat-aggregated immunoglobulins, but not after activation of the complement alternative pathway. This fragment was shown to have a sedimentation velocity of approximately 2.5S, to be heat stable, and to exhibit alpha mobility in immunoelectrophoresis. This C4alpha mobility fragment was not generated in human C4 deficient serum but was generated in human C2 deficient serum after incubation with heat-aggregated immunoglobulin. After precipitation of native C4 and its higher molecular weight fragments from serum by polyethylene glycol, it was possible to quantitate the lower molecular weight C4 alpha mobility fragment by radial immunodiffusion. In kinetic experiments, it was shown that the C4alpha mobility fragment was generated after some delay when compared to the disappearance of C4 hemolytic activity. Quantitation of the C4alpha mobility fragment may be of further use in human diseases for the evaluation of the catabolism of C4: joint fluids of patients suffering from rheumatoid arthritis contained high levels of the C4alpha mobility fragment, and low concentrations were found in patients with degenerative joint disease.

Animals↗
PubMed2006

Computational methods in developing quantitative structure-activity relationships (QSAR): a review.

Virtual filtering and screening of combinatorial libraries have recently gained attention as methods complementing the high-throughput screening and combinatorial chemistry. These chemoinformatic techniques rely heavily on quantitative structure-activity relationship (QSAR) analysis, a field with established methodology and successful history. In this review, we discuss the computational methods for building QSAR models. We start with outlining their usefulness in high-throughput screening and identifying the general scheme of a QSAR model. Following, we focus on the methodologies in constructing three main components of QSAR model, namely the methods for describing the molecular structure of compounds, for selection of informative descriptors and for activity prediction. We present both the well-established methods as well as techniques recently introduced into the QSAR domain.

Models, Molecular↗
PubMed1990

Surface phagocytosis and host defence in the peritoneal cavity during continuous ambulatory peritoneal dialysis.

Since patients on continuous ambulatory peritoneal dialysis are at high risk for peritonitis, the opsonins in peritoneal dialysis effluent responsible for phagocytosis and a neutrophil chemiluminescence response to surface-adherent Staphylococcus epidermidis were examined. In surface phagocytosis assays uninfected dialysate was as opsonic as 1% serum. The opsonic activity was heat stable and equal to that of purified IgG at the same concentration (0.1 mg/ml). In contrast, optimal chemiluminescence to surface-adherent Staphylococcus epidermidis was dependent on complement. C3 deposition on Staphylococcus epidermidis opsonized in dialysate was quantitated by an enzyme immunoassay (EIA) and represented 13% of control C3 deposited with opsonization in 10% serum. Unused dialysate was found to be inhibitory to neutrophil phagocytosis and complement deposition. A combination of the low pH and high dextrose concentration of dialysate was responsible, but restoration of the pH to 7.4 largely restored both indices. During peritonitis there was a parallel increase in IgG levels and C3 deposition (r = 0.8), and surface phagocytosis was also enhanced. On further analysis, subjects with a single episode of peritonitis had a significantly more opsonic peritoneal effluent than those who had two infections during the study. This latter group had a poor IgG response to infection. This study demonstrates the relative deficiencies of host defence in the peritoneal cavity and indicates that measures to improve opsonin delivery and reduce the inhibitory effects of dialysate would be beneficial.

Aged↗
PubMed1984

Anti-complement activity in a series of substituted 2-aryl-1,3-indandiones; absence of correlation with the anti-carrageenan oedema effects.

It was found that a number of substituted 2-phenyl-1,3-indandiones possesses a considerable inhibitory effect on the classical pathway of complement. Furthermore it appeared that in this series a reasonable correlation (r = 0.86) exists between this anti-complement effect and the inhibition of the prostaglandine biosynthesis. A quantitative structure-activity relation study revealed that both the electronic sigma parameter of the substituent involved and the lipophilicity of the corresponding compound are important for anti-complement activity. Mainly the C5 step in the complement process is inhibited by these compounds. The complement-inhibiting phenylindandiones also inhibit the alternative pathway. This effect may also be due to an inhibition of C5, as C5 is common to both pathways. Remarkably, the 2-(3,5-dichlorophenyl)-1,3-indandione, which is most active in the anti-complement test, showed no reduction of the carrageenan-induced oedema in the rat after both oral and local application. Possibly the absence of this anti-inflammatory effect is caused by the pharmacokinetic profile of the compound.

Animals↗
PubMed1990

Structures of the asparagine-linked oligosaccharides of guinea-pig factor B of the alternative complement pathway.

This paper describes the structures of the asparagine-linked oligosaccharides of two forms of guinea-pig Factor B of the alternative complement pathway with different Mr values. Oligosaccharides were quantitatively liberated from both glycoproteins by hydrazinolysis, fractionated by paper electrophoresis and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. Both glycoproteins were shown to have the same biantennary complex-type oligosaccharides but it is suggested that they contain different numbers of oligosaccharide chains.

Animals↗
PubMed1987

Activation of glomerular mesangial cells by the terminal membrane attack complex of complement.

Treatment of cultured renal glomerular mesangial cells (MC) with nonlytic concentrations of the purified components (C5b-9) of the terminal membrane attack complex (MAC) of complement induced significant functional alterations characteristic of cellular activation. C5b-9-treated MC released large quantities of primarily vasodilatory prostaglandins. In addition, the secretion of an MC-derived auto-growth factor (MC interleukin 1) was greatly enhanced. Examination of the action of C5b-9 on MC phospholipid metabolism indicated that complement induced the activation of phospholipases, leading to quantitative changes in the fatty acid profile of MC membrane phospholipids. These findings demonstrate that cultured MC are highly responsive to nonlytic concentrations of the C5b-9 complex, and suggest that the mesangial deposition of the MAC in many forms of glomerular disease, with resultant cellular activation, may play a major role in the hemodynamic and cellular proliferative events characteristic of these disorders.

Animals↗