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PubMed2002

Analysis of the relationship between mannose-binding lectin (MBL) genotype, MBL levels and function in an Australian blood donor population.

The mannose-binding lectin (MBL) pathway of complement activation is an important component of innate host defence. Numerous studies have described associations between the MBL genotype, MBL levels and disease susceptibility. However, genotyping and quantitative assays used in these studies have frequently been limited, and comprehensive data examining the interaction between structural and coding MBL genetic variants, MBL antigenic levels and MBL functional activity are lacking. Such data may be important for accurate planning and interpretation of studies of MBL and disease. This study has examined MBL in a cohort of 236 Australian blood donors. Five MBL promoter and coding single nucleotide polymorphisms were genotyped using polymerase chain reaction-sequence-specific priming (PCR-SSP). Plasma levels of MBL antigen were quantified using a double-antibody enzyme-linked immunosorbent assay (ELISA), and functional MBL levels were quantified using a mannan-binding assay. Activation of the complement pathway by MBL was measured in a C4-deposition assay. Significant associations were found between both coding and promoter polymorphisms and MBL antigenic and functional levels. There was significant correlation between the results of MBL double-antibody, mannan-binding and C4-deposition assays. Comprehensive MBL genotyping and functional MBL quantitation using mannan-binding and C4-deposition assays have the potential to be highly informative in MBL disease association studies.

Australia↗
PubMed1978

C3 component of complement secreted by established cell lines.

The hamster cell line NIL8 secretes the C3 component of complement as well as collagenous molecules and fibronectin (LETS protein). The C3 is found in the culture medium as a disulfidebonded complex of two polypeptides of 130,000 daltons (alpha) and 65,000 daltons (beta). The secreted C3 can be quantitatively cleaved to C3b and further cleaved by C3 inactivators. The activation of C3 to C3b is promoted by zymosan or by antibody-coated erythrocytes, demonstrating participation in both the classical and alternative complement pathways. The availability of this culture system has enabled us to show that C3 is synthesized as a 185,000 dalton precursor (proC3) which is biologically inactive and becomes cleaved to active C3. Some other established cell lines (NIL1 and BALB/c 3T3) also secrete C3, but some others do not.

Animals↗
PubMed1993

Localization and quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils.

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell-free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.

Blotting, Western↗
PubMed1985

Identification of immunostimulatory dendritic cells in the synovial effusions of patients with rheumatoid arthritis.

Dendritic cells in the circulation are leukocytes that are rich in Ia antigens and that actively stimulate T cell replication. We have identified dendritic cells in the joint effusions of patients with rheumatoid arthritis. By phase-contrast and immunofluorescence microscopy, synovial mononuclear cells contained 1-5% dendritic profiles that were rich in HLA-DR and DQ, had small amounts of C3bi receptor, and lacked a battery of monocyte and lymphocyte markers. These dendritic cells could be enriched to 60-80% purity by cytolytic depletion of monocytes and lymphocytes with a group of monoclonal antibodies (MAb) and complement. By transmission electron microscopy, the dendritic cell processes were bulbous in shape and lacked organelles. The cytoplasm had few lysosomes or endocytic vacuoles but contained a well-developed smooth reticulum that was comparable to that previously described in the Ia-rich interdigitating cells of lymphoid tissues. The growth of sodium periodate-modified T lymphocytes was used as a rapid quantitative assay of accessory cell function. Synovial mononuclear cells were some ten times more active than normal blood cells. Treatment with alpha-Ia MAb and complement ablated stimulatory function. In contrast, removal of monocytes (MAb, 3C10) or monocytes and B (MAb, BA-1) plus T (MAb, OKT3, or T101) lymphocytes did not significantly alter total activity, and the function per viable cell increased four- to eightfold. We conclude that rheumatoid arthritis synovial fluids contain cells that are comparable in function, phenotype, and structure to blood dendritic cells, although the frequency (1-5%) is 10 times greater in joints. The reason for their accumulation in the articular cavity is not known, but dendritic cells may be important in perpetuating the joint inflammation characteristic of this disease.

Animals↗
PubMed1986

Enzyme-linked immunolysis assay for tumour-specific surface antigens.

Expression of the activity of the cytosolic enzyme lactate dehydrogenase released on cell lysis by tumour specific antibodies in the presence of complement was investigated by direct assay of the expression of the occluded enzyme in tumour cell suspensions. This method, called Enzyme Linked Immunolysis Assay (ELILA) was shown to be more sensitive than conventional cytotoxic assays. The data fit well in the linear regression model, so that the technique can be used for quantitation of the antibody titres.

Animals↗
PubMed1993

Quantitation of vitronectin and clusterin. Pitfalls and solutions in enzyme immunoassays for adhesive proteins.

Vitronectin (S protein) and clusterin (SP-40,40/cytolysis inhibitor) are non-homologous, multifunctional proteins which both inhibit complement lysis. Vitronectin is an adhesive protein which binds strongly to polystyrene by hydrophobic interactions. The current study demonstrated that clusterin adsorbed even more efficiently to polystyrene than did vitronectin. This adsorption increased in the presence of Tween 20 and was not abolished by blocking or by the use of other detergents. In double antibody enzyme immunoassays such non-specific binding might invalidate the results. However, the non-specific binding of both proteins was efficiently abolished by the following experimental format: Dynatech Immulon 2 microtiter plate, acidic sample buffer (pH 6.0) containing 0.2% Tween 20 and high sample dilution. Vitronectin was successfully quantitated using this approach, but the measurement of clusterin was not reliable because of high inter-well variation of binding. However, since few serum proteins adsorb to polystyrene in the presence of detergents, clusterin was successfully quantitated in a single antibody enzyme immunoassay in which samples were coated directly onto Nunc Maxisorp plates in the presence of 0.2% Tween 20. In normal blood donors the serum concentration (median and 2.5-97.5 percentile) of vitronectin was 0.34 g/l (0.24-0.53) and of clusterin 0.34 g/l (0.25-0.42).

Adsorption↗
PubMed1975

Assessment of reactivities of natural antibodies to endogenous RNA tumor virus envelope antigens and virus-induced cell surface antigens.

The autogenous humoral immune response of mice to their endogenous leukemia virus (MuLV) has been examined with respect to the reactivities of natural antibodies to MuLV envelope antigens and virus-induced cells surface antigens. The natural reactivity of MuLV envelope antigens was evaluated by means of a radioimmune precipitation assay of intact and disrupted virus, as well as by virus neutralization tests. The specificity of natural antibody for MuLV envelope antigens was determined by immunoelectron microscopy and radioimmune precipitation. Antibody reactivity to virus-induced cell-surface antigens was evaluated by immunoelectron microscopy and a complement-dependent cytotoxicity test. The strains of mice seleced for study were C57BL/6, C3H/Anf, and the C57BL/6 X C3H/Anf F1 hybrid. Although there were quantitative differences in the antibody levels among these various strains, the naturally recognized antigenic determinants of the virus were consistent, i.e., gp68, gp-43, and p15. High levels of neutralizing antibody against the xenotropic BALB:virus-2 were detected in these various normal sera with the focus reduction assay; however, only marginal levels of neutralizing activity against Moloney leukemia virus were detected with the XC virus assay. No anticellular antibody could be detected in these normal sera with the complement-dependent cytotoxicity assay.

Animals↗
PubMed1981

Cellular and thylakoid-membrane glycolipids of Chlamydomonas reinhardtii 137+.

The glycolipids of the green alga Chlamydomonas reinhardtii 137+ have been quantitated in the phototrophically-cultured cell and in its thylakoid membrane. Three lipids, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the sulfated glycolipid sulfoquinovosyldiacylglycerol (SL), constitute the total glycolipid complement and some 70-80% of the total polar lipid at both levels. About 70% of the alga's sulfolipid, but only about half of its galactolipid, is localized in the thylakoid. The three cellular and thylakoid glycolipids all contain prominent hexadecanoic and octadecanoic fatty acids. Quantitatively, each lipid has a distinctive acyl profile, making SL the most highly saturated and MGDG the least saturated glycolipid. Differences between the fatty acid profiles of each corresponding cellular and thylakoid glycolipid indicate that discrimination of the glycolipid species assembled into photosynthetic membrane takes place during thylakoid membrane biogenesis.

Cell Membrane↗
PubMed1992

Specificity of three anti-complement factor 3 monoclonal antibodies.

Monoclonal antibodies which recognize specific C3 fragments may be used to distinguish C3 cleavage products bound to organisms. We defined the specificity of three commercially available monoclonal antibodies by Western immunoblot analysis, enzyme-linked immunosorbent assay, and a quantitative flow cytometric technique. Two monoclonal antibodies with specificity for (i) an erythrocyte-bound C3d epitope or (ii) an erythrocyte-bound C3c epitope retained their specificity in all assays. However, the third monoclonal antibody with selectivity for erythrocyte-bound C3bi failed to retain specificity for C3bi bound to non-erythrocyte surfaces in each of our assays; binding instead to all C3 fragments containing the C3g domain. We postulate that erythrocyte C3b-binding surface proteins may alter the availability of certain C3b epitopes and influence observed anti-C3 monoclonal antibody specificity. We conclude that the specificity of monoclonal antibodies for C3 fragments should be confirmed with assays which do not employ erythrocytes or other surfaces bearing C3 receptors. Also, our quantitative flow cytometric technique is a potentially valuable tool for the enumeration of particle-bound C3 fragments.

Adult↗
PubMed1979

Soluble proteins in the human atherosclerotic plaque. With spectral reference to immunoglobulins, C3-complement component, alpha 1-antitrypsin and alpha 2-macroglobulin.

A number of soluble proteins contained in human aortic intimal tissue was extracted into buffered saline (pH 7.4) and identified and quantitated by immunoelectrophoresis and immunodiffusion. The proteins included IgA, IgG, IgM, B1C (C3), alpha 1-antitrypsin, alpha 2-macroglobulin, fibrinogen, albumin, LDL, HDL, alpha 1-acid glycoprotein, beta 2-glycoprotein, transferrin and ceruloplasmin. The concentration of soluble proteins was significantly higher in the atherosclerotic intima than in the normal intima. The diseased intima also contained a small amount of tissue-bound IgG, IgA and B1C which was extractable with citrate buffer at pH 3.2. The vascular band IgG, and B1C were shown by enzymatic and immunohistochemical studies to be closely associated with the collagenous tissue of the plaque. The Ig contained in the atherosclerotic plaque may be derived in part from the biosynthesis of Ig by the artery, since the incorporation of 14C-labeled leucine into IgG by the atheromatous plaque was demonstrable by radioimmunoelectrophoresis. In contrast to the diseased artery, the normal artery did not synthesize IgG and did not contain vascular bound IgG or complement. However, the normal artery was capable of fixing IgG and B1C eluted from the diseased artery. The present studies suggested that the IgG contained and synthesized by the plaque might represent an immune response to an endogenous or exogenous antigen closely associated with plaque collagen. IgG and B1C either alone or in the form of an immune complex also may play an important role in phagocytosis in the plaque and thereby influence the course of atherosclerosis. The proteolytic inhibitors, alpha 1-antitrypsin and alpha 2-macroglobulin, found in relatively high concentrations in the plaque, could enhance fibrosis of the lesion because of thier known inhibitory effects on collagenase and elastase.

Aorta↗
PubMed1989

Kinetics of granulocyte phagocytosis: rate limited by cytoplasmic viscosity and constrained by cell size.

Micromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37 degrees C; calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37 degrees C to above several min/particle below 15 degrees C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10(-8) cm2 at 37 degrees C to greater than 8 sec/10(-8) cm2 at 15 degrees C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the "apparent" viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion↗
PubMed1978

The application of complement fixation to antibody weight estimates I. To soluble antigens.

A method is described for estimating early mouse IgG antibodies to the dinitrophenyl (DNP) determinant in absolute weight units. The antibodies to be measured are adsorbed quantitatively onto an appropriate immunoadsorbent such as DNP polyacrylamide. The particulate immune complexes are then incubated with a standard dilution of a potent rabbit antiserum to mouse IgG. Removal of the rabbit anti-mouse IgG (RaM) immunoglobulins by the complexes decreases the C fixing ability of the RaM supernatants reacted with C and mouse IgG. The decrements in C fixation are linearly related to the logarithm of the weight of mouse IgG. The decrements in C fixation are linearly related to the logarithm of the weight of mouse IgG anti DNP added to the immunoadsorbent. A calibration curve is constructed with known amounts of mouse IgG )anti-NDP as measured by quantitative precipitation. Concurrent assays with the early mouse anti-DNP sera permit conversion of the losses in C fixing potencies to weight units of mouse IgG anti-DNP by interpolation on the calibration curve.

Animals↗
PubMed1988

Clinical application of new technique that measures C4d for assessment of activation of classical complement pathway.

A new laser nephelometric technique that measures C4d for the assessment of the activation of the classical complement pathway was developed. C4d was isolated from other larger C4 related molecules at a final concentration of polyethylene glycol of 12% and then quantitated by laser nephelometry using a commercially available antiserum, which reacts with C4d determinants. C4d standard (100%) was produced by exhaustive activation of the classical pathway in pooled normal human serum using heat aggregated human immunoglobulin. Serial dilutions of the standard provided a reference curve against which clinical samples were read. Patients with rheumatoid arthritis showed significantly higher C4d values (mean 53.8%) than controls (21.7%; p less than 0.001). The technique proved accurate, rapid, and suitable for the routine laboratory evaluation of complement activation through the classical pathway, and it may be useful in the management of those conditions in which complement activation has a pathogenic role.

Adolescent↗
PubMed1984

Pathogenic synergy: mixed intra-abdominal infections.

In this article we review our researches into the pathogenesis of mixed infections. These may conveniently be divided into in vitro and in vivo studies. In vitro we confirmed that interference with the killing of aerobes by polymorphonuclear leucocytes (PMN's) is a property of the Bacteroides strains tested and appears to depend on competition for opsonins i.e. complement factors. Further studies are in progress to define which complement factors and which bacterial structures are involved. The influence of B. fragilis on chemotaxis has also been studied. Our preliminary data suggest that B. fragilis is itself poorly chemotactic and reduces the chemoattractivity of Proteus mirabilis. This observation is surprising when we consider that abscess formation is the hall-mark of B. fragilis infections and needs clarification. In vivo we have developed a skin infection model in mice which is economical and gives reproducible and quantitative results. In this model we have demonstrated pathogenic synergy between Escherichia coli and B. fragilis. Further studies are planned to assess the role of complement and bacterial factors in this in vivo synergy.

Animals↗
PubMed1996

Changes in neutrophil actin and shape during sequestration induced by complement fragments in rabbits.

Complement fragment-induced sequestration of neutrophils within the lungs may be mediated by stimulus-induced decreases in the deformability of neutrophils, prolonging their lung capillary transit times. As changes in deformability often occur through changes in cytoskeletal proteins, this study determined whether the distribution of actin within intracapillary neutrophils was altered by intravascular complement fragments and whether sequestered neutrophils were less deformed. Ultrathin cryosections of lung tissue from rabbits given an infusion of complement fragments or saline were immunolabeled with anti-actin antibodies. The number of gold particles/microvillus and the density of gold particles/microgram 2 cytoplasm in the submembrane and the central region of intracapillary neutrophils was quantitated. Neutrophil shape was evaluated using laser confocal microscopy. In control rabbits, the ratio of submembrane/central gold was always greater than one and most neutrophils were elongated, 97% having shape factors > 1.10. The ratio of submembrane/central gold was greater in complement-treated rabbits (5.1 +/- 0.9) than controls (2.6 +/- 0.4; P < 0.026). The number of gold particles/microvillus was also increased in complement-treated rabbits (3.9 +/- 0.5) compared with controls (2.3 +/- 0.5; P < 0.045). Neutrophils were more often spherical when rabbits received complement fragments for 1.5 minutes than in control lungs or after 15-minute infusions. These data suggest that complement fragments induce a rapid redistribution of actin from the central to the submembrane region and the microvilli and result in more round neutrophils. This redistribution may decrease the deformability of neutrophils by altering the stiffness of the submembrane region and/or by preventing the microvilli from flattening.

Actins↗
PubMed1974

Characterization of the precipitin bands detected in the immunodiffusion test for paracoccidioidomycosis.

In order to characterize the precipitin bands detected in the immunodiffusion test for paracoccidioidomycosis, a study was undertaken in 54 patients with the disease. On the basis of the pattern of known control sera, the three commonly observed lines of precipitate were designated as 1, 2, and 3 according to their location in the immunodiffusion plate. At time of diagnosis, 28 of the patients exhibited all three bands, 16 gave two bands, and 10 showed only one precipitin line. Over 50 of the sera with three bands had high complement fixation titers (above 1:512), whereas those with one band exhibited lower titers. A similar picture was obtained with the quantitative agar-gel techniques, where titers of 1:64 and above were more commonly observed in sera with three precipitin lines. Follow-up studies carried out in 18 patients revealed that band 3 disappeared first, followed by band 2, and, finally, by band 1. At the end of 2 to 3 years, 85.7% of the patients had lost band 3, 75% band 2, and only 27.7% band 1. Cross-reactions with histoplasmin were found in eight patients who gave the M precipitin line with this antigen. It was found that the latter band and our paracoccidioidin band 3 fused, producing lines of identity. Bands 1 and 2 were specific. The implications of these findings are discussed.

Blastomycosis↗
PubMed1985

Grip strength test and infrared thermometry as non-invasive methods to complement acute toxicity data in mice.

Acute toxicity testing in-vivo seeks a new approach minimising the number of animals used and increasing the number of quantitative and qualitative observations. The measurement of grip strength of mice (forelimbs), a classical tool for the evaluation of muscular force and the emitted heat measurement, as a tool for evaluation of basic metabolism, have been used. The aim of acute toxicity was, up to now, to determine LD50 level or eventually a minimal lethal dose. These non-invasive tests give an idea of general health condition and allow the determination of the minimal toxic dose while in the search for the minimal lethal dose. A computer-assisted grip strength test and an infrared thermometry test have been developed and calibrated with Na+ pentobarbital, theophylline and diazepam given once orally or intraperitoneally to 10 male NMRI (MAI) mice/dose. The tests were performed within one hour after administration and daily until the values of the surviving animals went back to normal. Dose-response curves were obtained and plotted against control animals (n = 10). Oral and intraperitoneal administration in ascending doses of Na+-pentobarbital, theophylline and diazepam (i.p. only) up to the lethal level have a parallel influence on grip strength and heat emission; the correlation with mortality is clear for Na+-pentobarbital and diazepam but not for theophylline.

Animals↗
PubMed1971

Quantitative immunology of immune hemolytic anemia. I. The fixation of C1 by autoimmune antibody and heterologous anti-IgG antibody.

The fixation of the first component of complement (C[unk]1) by rabbit and goat anti-IgG antibodies reacting with auto- or isoimmune antibodies attached to red cells has been investigated. Two molecules of the rabbit IgG anti-IgG were required to fix a single molecule of C[unk]1, whereas only one molecule of goat IgM anti-IgG was required. The relationship between the number of auto- or isoimmune antibody molecules attached to the red cells and the amount of C[unk]1 fixed by anti-IgG was determined by the concentration of anti-IgG. A concentration of anti-IgG was found such that the number of molecules of C[unk]1 fixed was directly proportional to the concentration of auto- or isoimmune antibody. By this method a sensitive, reproducible minimum estimate of the amount of cell-bound and serum antibody could be made.

Anemia, Hemolytic, Autoimmune↗