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PubMed1975

Effect of corticosteroids on the human monocyte IgG and complement receptors.

A quantitative in vitro assay was employed to directly assess the effect of corticosteroids on the IgG and complement receptor function of human mononuclear phagocytic cells. In this system corticosteroids were solubilized with cholesterol-phospholipid sonicated dispersions before exposure to mononuclear cells. Solubilized corticosteroids at concentrations between 10(-4) and 10(-3) M inhibited both IgG and complement receptor activity in a dose-response fashion. Inhibition was dependent upon the time of interaction of the mononuclear cells with corticosteroids and was half-maximal by 15 min. The inhibitory effect at all concentrations of hydrocortisone was partially overcome by increasing the number of IgG molecules per erythrocyte. Hydrocortisone also inhibited the binding of erythrocytes coated with both IgG and C3, despite the fact that when both were on the erythrocyte surface a synergistic effect on binding to mononuclear cells was observed. At the steroid concentrations employed, the capacity of mononuclear cells to exclude trypan blue and to take up latex particles and neutral red was unaffected. Mineralocorticoids also inhibited receptor activity, but the sex hormones were less effective. These studies demonstrate an effect of steroid hormones on cell membrane receptor function, and they suggest that an inhibition of the recognition system for IgG and C3 in vivo may explain, in part, the effect of corticosteroids in man.

Adrenal Cortex Hormones↗

PubMed1987

Quantitative evaluation of the terminal C5b-9 complement complex by ELISA in human atherosclerotic arteries.

A quantitative ELISA using monoclonal and polyclonal antibodies against neoantigens of the terminal C5b-9 complement complex was used to evaluate the presence of terminal complexes in 68 human arterial samples with or without atherosclerotic involvement. Plasma levels of SC5b-9 were directly compared with the corresponding levels eluted from the femoral arteries in six patients undergoing surgical procedures. The plasma concentration of SC5b-9 in these donors was in the range of 30-90 arbitrary units (AU)/ml, equivalent to 100-300 ng/ml SC5b-9 or 45-130 AU/100 mg plasma protein. All the arterial samples contained detectable amounts of C5b-9. The aortic normal and fatty streaks intimae presented a minimum mean value of 65 +/- 12 AU/100 mg total protein, in the range of normal plasma SC5b-9 levels. The corresponding media contained significantly higher amounts of terminal complexes (115 +/- 30 AU/100 mg protein). Markedly increased levels of C5b-9 were eluted from aortic intimal thickenings (350 +/- 100 AU/100 mg protein) and the corresponding media (300 +/- 53 AU/100 mg protein). Similar concentrations were found in aortic fibrous plaques (340 +/- 80 AU/100 mg protein). The observed correlation between C5b-9 levels and atherosclerotic alterations in arterial walls is suggestive of chronic complement activation with involvement of the terminal complement sequence at these sites. These processes may contribute to progression of the arteriosclerotic lesions.

Adult↗

PubMed1984

Fixation of complement by anti-F(ab')2 antibodies or ssDNA when added to systemic lupus erythematosus sera.

A solid phase total hemolytic complement assay was used to quantitatively determine the % complement fixation by anti-F(ab')2 antibodies or ssDNA when added to serum from patients with systemic lupus erythematosus (SLE). Both autologous and homologous anti-F(ab')2 antibodies with demonstrated anti-anti-ssDNA activity fixed complement when added to SLE sera and normal human complement. Furthermore, comparable amounts of anti-F(ab')2 antibodies with minimal anti-anti-ssDNA activity fixed zero or minimal amounts of complement when added to the same lupus sera. When measured by the same assay with the same sera, ssDNA fixed greater amounts of complement than did comparable amounts of any of the anti-F(ab')2 antibodies.

Antibodies, Anti-Idiotypic↗

PubMedDate not supplied

Quantitative determinations of immunoglobulins and complement components in human aortic atherosclerotic wall.

Saline and acid eluates of intima with only fatty streaks, fibrous plaque and intima surrounding the fibrous plaque were obtained from 42 human aortae. IgG, IgA, IgM, C1q, C3c, C4, C9, C3A, C-reactive protein, alpha-1-antitrypsin, alpha-2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined in these eluates using the radial immunodiffusion technique. Saline extracted IgG and IgA were significantly higher in the fibrous plaque and adjacent tissue than in the fatty streak intima, where IgM was the highest. IgG and IgA were only present in acid eluates. The complement components were present in all saline eluates, but only traces of C3c and C1q in some acid eluates. Statistically significant differences were found for C1q and C9. C-reactive protein was present in 23 samples. The results could suggest an involvement of immune mechanisms in the progression of the atherosclerotic lesion.

Adolescent↗

PubMedDate not supplied

[The activation of the complement system in various connective tissue diseases].

A knowledge of the complement system in patients with collagen diseases is of interest as it may serve to indicate the existence of activity or non-activity in these diseases. We have studied the existence or non-existence of the activity of the complement system through evaluation of the levels of C3, as well as the implicated mechanism of the classical or alternate pathways through the measure of C'4 and C'3 proactivator components. Quantitation of complement components was obtained by electroimmunodiffusion, employing specific antiserum. From the results obtained, we observed that the complement activity in patients with SLE, the classic pathway was affected more (7 patients of the 24 studied), than the alternate pathway (3 patients). However, in those patients affected by sclerodermia, there seems to be an equal incidence of the activation of complement through both pathways (2 patients with activation through each way). Also of interest is the existence of a patient with SLE and another affected by sclerodermia in which a selective reduction of C'3 was observed. This observation suggests the possible existence of a selective destruction, perhaps mediated through a factor type such as the nephritic factor. However, there may exist other patients of whom we have already commented on, with a parallel reduction in the three components which suggests a change in the synthesis or in the simultaneous activation in both of the pathways. When realizing the comparative studies of the state in which the immunoglobulins (IgG, IgA and IgM) and complement components are to be found, the conclusion is that there is no significative correlation between the two. This is particularly interesting as it assumes the necessity of knowing the state of the complement and not only of the immunoglobulin, in order to know the state of activity in which the process is.

Complement C3↗

PubMed1979

Fluctuation of serum complement levels in children with neuroblastoma.

Serum samples from eleven children with neuroblastoma were drawn at diagnosis and again every 15 days or a month after the beginning of treatment over the course of 17 months observation. Twenty healthy children of the same age with no appreciable clinical manifestation were also studied as controls. Investigations included quantitative serum complement levels (total complement hemolytic activity, C3 and C4), anticomplementary serum activity and urine catecholamine levels (VMA and HVA). Complement levels of tumor patients were significantly higher than those of healthy subjects, but fluctuations were seen at different stages of the disease; patients at admission with active tumors or tumor recurrence had higher complement levels than those of patients in remission. Temporary recurrences of the disease were usually accompanied by fluctuations of urinary catecholamines, serum complement levels (mainly C3) and anticomplementary serum activity. At the terminal phase of the disease a drop in complement levels was usually seen, while urinary catecholamines were progressively increasing.

Bacterial Infections↗

PubMed1992

Time for new concepts about measurement of complement activation by cardiopulmonary bypass?

Fifty-one patients admitted for routine coronary bypass operations were randomized to cardiopulmonary bypass with a membrane oxygenator (Capiox) or a bubbler (Polystan or William Harvey). Complement activation was measured using enzyme immunoassays for concentrations of C3 activation products and the terminal complement complex. From 5.8 to 8.1 arbitrary units (AU)/mL (medians), the plasma concentrations of C3 activation products increased by 119.9 AU/mL (Capiox), 124.6 AU/mL (Polystan), and 79.5 AU/mL (William Harvey) to a peak at closure of the sternum (not significant when related to baseline concentrations). The increase in C3 activation products and baseline C3 activation were linearly correlated (R2 = 0.30; p less than 0.0001). From 5.5 to 6.1 AU/mL, the plasma terminal complement complex concentrations increased by 45.2 AU/mL (Capiox), 15.4 AU/mL (Polystan), and 17.4 AU/mL (William Harvey) to a peak before termination of cardiopulmonary bypass. Maximal terminal (C5-C9) activation was significantly higher in the membrane oxygenator group (p less than 0.0001) and showed no relationship to C3 activation. Measurement of C3 activation only gives no information about C5-C9 activation. At present, terminal complement complex quantitation is probably the best index of C5-C9 activation during cardiopulmonary bypass.

Cardiopulmonary Bypass↗

PubMed1985

Quantitation of activation of the human terminal complement pathway by ELISA.

We have devised an enzyme-linked immunosorbent assay (ELISA) to quantitate fluid phase terminal complement pathway activation. Upon activation to form C5b-9, terminal complement components express neoantigens not present in the unassembled individual components. Expression of one of these neoantigens occurs at the step of C9 activation. C9 neoantigen is present in fluid phase SC5b-9 complexes, membrane-bound MC5b-9 complexes, and in in vitro polymerized C9. Under physiologic conditions, the presence of C9 neoantigen indicates that the terminal complement pathway is activated through the terminal component C9. In our assay for C9 neoantigen, we used rabbit antiserum to polymerized C9 rendered specific for C9 neoantigenic determinants by serial absorption with human serum, human C9, and other terminal complement components bound to Sepharose. Using the IgG from this antiserum, we devised a sandwich ELISA to bind SC5b-9 from solution onto polystyrene plates. The ELISA plates were developed with the use of goat antiserum to native C9 epitopes followed by a swine anti-goat IgG-alkaline phosphatase conjugate. Quantitation of SC5b-9 in solution was performed by comparing sample OD to a standard curve generated with human SC5b-9 that was purified from zymosan-activated serum. The assay was sensitive to as little as 100 ng of SC5b-9/ml and should be useful for screening plasma, serum, cerebrospinal fluid, or other biological fluids for the presence of terminal complement pathway activation.

Antibody Specificity↗

PubMed1991

Quantitative and antigenic differences in complement component C4 between American blacks and whites.

A population of 98 healthy Black Americans was studied in order to determine normal ranges for total C4, C4A and C4B. Mean total C4 in Blacks measured by an enzymelinked immunoassay (EIA) was 44 +/- 12.8 mg/dl which was significantly different (p less than 0.001) from Caucasian levels of 31.7 +/- 11.5 mg/dl. The difference in total C4 was due to increased levels of C4B in Blacks (means = 24.4 mg/dl) as compared to Whites (means = 15.7 mg/dl; p less than 0.001). These results remained significantly different even when 8 samples having the C4A 91 phenotype were excluded. Since EIAs using monoclonal antibodies with Ch 1 specificity may yield false results, C4 allotyping is recommended when quantitating C4A and C4B levels in Blacks.

Alleles↗

PubMedDate not supplied

[Guerreiro Machado reaction. Chromatographic analysis of Trypanosoma cruzi antigens].

The aqueous extract of Trypanosoma cruzi, previously treated by benzene, was filtered through a column of Sephadex G-200. The eluted fractions were tested by complement-fixation with the chagasic reference serum. It was found that the CF antigens were eluted in the first fractions and the contaminants in the last ones. The aqueous antigen, when dried and extracted with methanol, showed a very high titer in the complement-fixation quantitative test. This antigen is free of contaminants and can be recommended for use in the serological test for the diagnosis of Chagas' disease.

Animals↗

PubMed1983

Complement activation during cardiopulmonary bypass: quantitative study of effects of methylprednisolone and pulsatile flow.

Forty four patients undergoing open heart surgery were divided into three groups. Group 1 (17 patients) underwent routine anaesthesia and surgery; group 2 (17 patients) received two doses of methylprednisolone (30 mg/kg), one during induction of anaesthesia and the other immediately before induction of cardiopulmonary bypass; and group 3 (10 patients) received pulsatile flow while undergoing pulsatile perfusion by the heart-lung machine. A modification of the previously described technique was used to detect and measure complement activation in plasma before and during the bypass period using crossed immunoelectrophoresis. About 45% of all patients showed measurable complement activation (greater than 4.5%) during cardiopulmonary bypass and the mean activation in this group was 6.4%. There was no significant difference between the three groups in complement activation. In group 2, however, women showed significantly more complement activation than men (p less than 0.05). It is suggested that neither corticosteroids nor pulsatile flow affect complement activation, but caution should be exercised in women receiving methylprednisolone.

Animals↗

PubMed1987

Simplified methods for the purification, quantitation, and functional estimation of human complement C-1-inhibitor (C-1-INH) with a monoclonal anti-C-1-INH antibody.

New methods have been developed for the isolation, quantitative detection, and functional measurement of human complement C-1-inhibitor (C-1-INH). The two-step purification procedure for C-1-INH from human plasma or serum employs affinity chromatography with a monoclonal anti-C-1-INH antibody coupled to CNBr-activated Sepharose 4B followed by fractionation on a FPLC Mono Q HR 5/5 column. It yields functionally active, homogeneous C-1-INH with about 40% recovery. For quantitative estimation of C-1-INH an ELISA was performed. ELISA plates were coated with a polyclonal anti-C-1-INH antibody, serum or plasma was added and bound C-1-INH was detected with the monoclonal anti-C-1-INH antibody. The method has a sensitivity of 0.4 ng C-1-INH per assay corresponding to 20 ng/ml. For the detection of functionally active C-1-INH an ELISA was developed using C1-s-coated microtiter plates. After incubation with serum or plasma, C1-s-bound C-1-INH was monitored with the monoclonal anti-C-1-INH antibody. With this method it is possible to measure as little as 0.3 ng of functionally active C-1-INH in 20 microliter of a biological sample. All methods described in the present paper are easy to perform, rapid, sensitive, and highly reproducible.

Animals↗

PubMed1988

Lymphocyte and complement abnormalities in splenectomized patients with hematologic disorders.

Twenty-two patients, splenectomized 1 to 26 years earlier for hematologic disorders, were studied to determine possible defects in immunologic function or complement levels. Quantitation of B cells and T-cell subsets revealed slight decreases in the proportions of CD3 and CD4 cells but normal or increased absolute numbers of all cell populations. IgM synthesis in vitro by peripheral blood mononuclear cells was markedly diminished, but IgG synthesis was normal. Fractionation studies, in which various B-cell- and T-cell-enriched populations from controls and patients were combined, demonstrated diminished B-cell function in the patients. Sickle cell patients, who were functionally asplenic, showed similar deficits. Complement levels in splenectomized and sickle cell patients in both the classical and alternative pathways were generally normal. A modest decrease in component H in the alternative pathway in splenectomized and sickle cell patients probably was not clinically significant. In summary, splenectomized patients have a diminished capacity for IgM synthesis that can be attributed primarily to defective B-cell function. This may be partially responsible for their increased susceptibility to infection by encapsulated organisms.

Adolescent↗

PubMedDate not supplied

Quantitation of red cell-bound immunoglobulin and complement using enzyme-linked antiglobulin consumption assay.

Various techniques have been described for quantitating IgG or complement (C3) on red cells (RBCs). The techniques either are cumbersome, as the complement consumption test, or use radioactivity. This paper describes an antiglobulin consumption assay using an enzyme-linked immunosorbent method that can be used to quantitate IgG, IgM, and C3. With this technique RBCs from normal, healthy donors gave a mean value of 106 +/- 60 molecules of IgG per RBC, 4.5 +/- 3 molecules of IgM per RBC, and 37 +/- 28 molecules of C3 per RBC, respectively. The RBCs of hospital patients, particularly of those with infections or inflammatory conditions, contain increased amounts of nonspecifically bound immunoproteins. The availability of a common method to quantitate RBC-associated IgG, IgM, and C3 allows easy monitoring or study of the immune mechanism of autoimmune hemolytic anemia.

Adult↗

PubMed1978

Eosinophilic gastroenteritis with obstruction. Immunological studies of seven patients.

We studied 7 patients with eosinophilic gastroenteritis presenting with obstruction of the gastrointestinal tract. Five had required surgical treatment for relief of obstructive symptoms or because of diagnostic uncertainty. Immunological studies, including serum immunoglobulins, serum complement, lymphocyte quantitation, and lymphocyte response to nonspecific mitogens revealed no striking abnormalities. None was clinically allergic or food sensitive, and serum IgE was normal in all. A review of the literature since 1970 suggests that the obstructive form of eosinophilic gastroenteritis accounts for about half of the reported cases. Allergy, especially hypersensitivity to food, is not required for the diagnosis.

Adolescent↗

PubMed1975

Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.

Immunofluorescence absorption methods are described which permit quantitative estimation and differentiation of Epstein-Barr virus (EBV)-associated antigens (virus capsid antigen, VCA, early antigen, EA and EBV-determined nuclear antigen, EBNA) in cell extracts. EBNA was present in all cell lines (producer and non-producer) which carried the EBV-genome, while VCA and EA were present in producer lines only. All the antigens were absent from a lymphoid cell line (MOLT-4) which lacked the EBV-genome, as well as from leukemia cells from peripheral blood. The techniques demonstrated antigenic identity of the various antigens when prepared from different cell lines.

Absorption↗

PubMed1995

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.

Amino Acid Sequence↗

PubMed1979

Complement proteins and macrophages. 1. Quantitative estimation of factor B produced by mouse peritoneal macrophages.

Cobra venom factor was used for the detection of factor B synthesized by mouse peritoneal macrophages. This method was shown to be specific for factor B assay by neutralization by antimouse factor B antibody. The amount of factor B in the culture supernatant, assessed by this method, was found to be dependent on the medium used for cultivation of macrophages. The addition of 25% L cell-conditioned medium to minimal essential medium (LCM-MEM) enhanced the production of factor B and also of lysozyme. Kinetic analysis in LCM-MEM showed that factor B produced by 6 x 10(4) cells/cm2 increased up to 72 hr and reached a plateau at 96 hr. The amounts of factor B and lysozyme produced in LCM-MEM depended upon the number of macrophages. Production of factor B was completely inhibited by 1 microgram of cycloheximide per ml and was restored by its removal.

Animals↗