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PubMed1991

Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans.

The capsule of Cryptococcus neoformans is a powerful activator of the alternative complement pathway. This study examined the manner in which the cryptococcal capsule influences initiation of and early events in complement activation by C. neoformans. These studies examined the effects of the classical and alternative pathways on the kinetics and early sites for deposition of C3 fragments on encapsulated cryptococci, nonencapsulated cryptococci, and zymosan. The results showed that nonencapsulated cryptococci and zymosan are qualitatively and quantitatively similar in the manner in which they initiate complement activation. Both utilize the classical and alternative pathways. Initiation via the classical pathway occurs suddenly and simultaneously at sites distributed over the entire cell surface. Initiation of the alternative pathway by zymosan and nonencapsulated cryptococci is characterized by a lag of 6 to 8 min before appreciable amounts of C3 accumulate on the cells. Alternative pathway initiation by zymosan and nonencapsulated cryptococci occurs at a limited number of focal initiation sites that expand with alternative pathway amplification to cover the cell surface. Presence of the cryptococcal capsule blocks classical pathway initiation, which would normally occur at the cryptococcal cell wall, and produces an initiation that is dependent solely on the alternative pathway. Initiation of the alternative pathway by the cryptococcal capsule is characterized by a lag in C3 accumulation and the appearance of a limited number of focal initiation sites which resemble those observed when the alternative pathway is activated by zymosan and nonencapsulated cryptococci.

Complement Activation↗
PubMed1975

The effect of antibody and complement on perfused hearts: a model for the study of xenograft rejection phenomenon.

An in vitro heart perfusion system was developed to study the effect of antibody and complement on hearts in vitro, as a model to quantitatively evaluate factors involved in xenograft rejection. Both guinea pig and rabbit hearts were tested with various reagents. Perfusion of hearts with normal autologous or "homologous" sera did not induce rejection, while "heterologous" sera, normal or immune, arrested cardiac function. Either the inactivation of certain complement components or the absence of antibodies against heart tissue prevented rejection as determined by lack of change in electrocardiogram and alterations in ultrastructure. Antibody alone had no effect. A lethal effect of C6-deficient rabbit serum on guinea pig hearts was prevented by 2 antihistamines, Chlortripolon and Benadryl.

Animals↗
PubMed1989

Factor B activation products in patients with systemic lupus erythematosus. A marker of severe disease activity.

Complement activation patterns were determined in a group of 51 patients with systemic lupus erythematosus (SLE), and the clinical outcomes of these patients at 2 years were correlated with the complement activation patterns. Activation of the classical pathway was monitored by analysis of C4a desArg levels and total C4 levels, and activation of the alternative pathway was monitored by isoelectric focusing/immunofixation and quantitative analyses of Factor B. Activation of C3 (a general measure of complement activation) was determined by an enzyme-linked immunosorbent assay for C3d and by quantitative analysis of C3. Patients were stratified into 3 groups: those with C4 but not C3 activation; those with C4 and C3 but not Factor B activation; and those with C4, C3, and Factor B activation. At the end of 2 years, there was a statistically significant difference in the morbidity and mortality rates of the third group of SLE patients compared with those in the other 2 groups. There was also a statistically significant association between the presence of Ba and cutaneous vasculitis. Unlike the patterns seen with in vitro-activated serum or with membrane-activated plasma, the Bb activation fragment was not present in the activated plasma samples from the SLE patients. These data suggest that activation of the alternative complement pathway may be a marker for severe SLE and that the Bb fragment may be playing a role in the development of this more serious pathologic condition.

Biomarkers↗
PubMed1982

Activation of the alternative pathway of bovine complement by Trypanosoma congolense.

Homogenates of Trypanosoma congolense and particulate and supernatant fractions thereof were, by two different techniques, shown to activate bovine complement via the alternative pathway. (1) All three preparations converted complement factor Bbov to its split product B gamma 2 as demonstrated (a) qualitatively by immunoelectrophoresis and (b) quantitatively by radial immunodiffusion. (2) Lysis of guinea-pig erythrocytes by bovine complement via the alternative pathway was inhibited by whole homogenates and particulate fractions of T. congolense organisms.

Animals↗
PubMed1991

In vitro activation of human complement by nitrogen bubbles.

Complement activation may be responsible for some of the symptoms of decompression sickness. In the present study, complement activation was studied by exposing human sera with or without red blood cells to nitrogen bubbles. Nitrogen bubbles activated human complement as measured by generation of the fluid-phase, complement-split product C5a des Arg. In addition, we found that complement activation continued after exposure to bubbles was stopped. This continued complement activation may explain the failure of recompression treatment in some patients. Complement activation induced by nitrogen bubbles in human sera was enhanced when red cells were present. Red cells may be able to provide a stable membrane surface on which complement activation by bubbles can occur more efficiently. Complement activation by nitrogen bubbles in serum also led to the binding of activated C3 to red cells. Quantitation of bystander red-cell-bound C3d may allow the assessment of complement activation occurring by nitrogen bubbles in individuals undergoing decompression.

Complement Activation↗
PubMed1996

A soluble deletion mutant of the human complement receptor type 1, which lacks the C4b binding site, is a selective inhibitor of the alternative complement pathway.

The human complement receptor type 1 (CR1, CD35), is a single-chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. The SCR themselves, considered in groups of seven, form long homologous repeats (LHR) which have been designated LHR-A, -B, -C, and -D for the most common human allotype of CR1. A soluble deletion mutant of CR1 which lacks the first seven N-terminal SCR (LHR-A) as well as the transmembrane and cytoplasmic domains was produced and characterized. The resulting protein, designated sCR1[desLHR-A], lacks the C4b binding site found in LHR-A, but retains the two C3b binding sites found in LHR-B and -C, respectively. The functional activities of sCR1[desLHR-A] were quantitatively compared in vitro to those of soluble complement receptor type 1 (sCR1) which has been shown to retain all known functions of the native cell surface receptor. sCR1[desLHR-A] and sCR1 competed equally for the binding of dimeric C3b to erythrocyte CR1. sCR1[desLHR-A] and sCR1 were similar in their capacity to serve as a cofactor in the factor I-mediated degradation of the C3b and C4b alpha chains. sCR1[desLHR-A] and sCR1 were comparable in their capacity to inhibit erythrocyte lysis and anaphylatoxin production mediated by the alternative complement pathway. sCR1[desLHR-A], however, was significantly less effective an inhibitor of erythrocyte lysis and anaphylatoxin production than sCR1 under conditions which allow classical pathway activation. These results demonstrate sCR1[desLHR-A] to be a selective inhibitor of the alternative complement pathway in vitro.

Binding, Competitive↗
PubMed2026

Pathogenic properties of myasthenia gravis AChR autoantibodies associate with clinical response to efgartigimod.

BACKGROUND: Efgartigimod, a neonatal Fc receptor (FcRn) blocker, effectively reduces total IgG, including pathogenic acetylcholine receptor (AChR) autoantibodies in myasthenia gravis (MG); however, clinical responses vary. To investigate this variability, we studied how efgartigimod impacts AChR-specific autoantibody profiles and associated pathogenic mechanisms, including complement activation, AChR internalisation and ACh-binding site blockade. METHODS: Serum samples (N=150) were sourced from 50 AChR autoantibody-positive generalised MG patients participating in the phase 3 ADAPT study, randomised to receive efgartigimod (N=40) or placebo (N=10) in cycles of 4-weekly infusions. Samples were collected at baseline, day 29 and day 57 during the first cycle. Live cell-based assays quantified AChR-specific IgG subclasses and isotypes and assessed their capacity to mediate pathomechanisms. RESULTS: Efgartigimod decreased all detectable AChR-specific IgG subclasses. At baseline, AChR autoantibody-mediated C3b deposition, AChR internalisation and ACh-binding site blockade were detected in 42 (84%), 41 (82%) and 10 (20%) patients, respectively. After 4-weekly infusions of efgartigimod, the magnitude of all three pathomechanisms was significantly decreased. However, the extent of this reduction varied across individuals. Double responders on both MG-activities of daily living and quantitative MG scores demonstrated a greater reduction in complement activity and AChR internalisation compared with patients who responded on only one score or were double non-responders. In addition, efgartigimod reduced IgG-dependent IgM binding to AChR. CONCLUSIONS: These findings suggest that clinical efficacy may be more closely associated with functional modulation of the AChR-specific autoantibodies than with their absolute quantity alone. These results support the evaluation of mechanistic pathway monitoring as a potential strategy to predict or guide efgartigimod treatment response.

Humans↗
PubMed1989

In vitro fixation of C3d and C5b-9 on platelets by human platelet reactive antibodies.

Most platelet-reactive autoantibodies and alloantibodies are not able to fix complement in vitro. However, exceptions have been found. These antibodies are usually characterized by the conventional platelet complement fixation test. A recently developed competitive enzyme immunoassay for quantitation of platelet-associated immunoglobulins and a modification thereof allowed the quantitative study of fixation of C3d and the membrane attack complex (C5b-9) on platelets by HLA antibodies, human platelet autoantibodies, and drug-dependent antibodies (ddab). The highest amounts of both complement products were fixed through ddabs, whereas autoantibodies only showed moderate complement fixation. This enzyme immunoassay is a valuable tool for the characterization of the complement-fixing properties of platelet-reactive antibodies.

Antibodies↗
PubMed1984

Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity.

We have used cell fusion and mutant reversion analysis to study a collection of Chinese hamster ovary (CHO) cell mutants that are unable to bind and internalize low density lipoprotein (LDL). Pairwise cell fusions show that these LDL receptor-deficient mutants fall into three recessive complementation groups, ldlA, ldlB, and ldlC. Complementation was detected by observing the uptake of fluorescent LDL and was quantitated by measuring the degradation of 125I-labeled LDL by isolated hybrid cells. Previous studies had defined a fourth recessive complementation group, ldlD. Complementation tests between CHO cells and human fibroblasts suggested that the defects in mutants of the ldlA complementation group are analogous to those in a patient with homozygous familial hypercholesterolemia. A revertant of an ldlA mutant was isolated and appeared to be heterozygous at the ldlA locus. The phenotype of this revertant was similar to that of cells from patients with the heterozygous form of familial hypercholesterolemia. Together with recent DNA transfection studies, these results suggest that the ldlA locus is the structural gene for the LDL receptor in CHO cells. Mutants in the ldlB, ldlC, and ldlD complementation groups must have defects in genes that are required for either the regulation, synthesis, transport, recycling, or turnover of LDL receptors.

Animals↗
PubMed1993

Cloning of a recA-like gene from the diazotroph Herbaspirillum seropedicae strain Z78.

A recombinant plasmid, pBMR5, carrying a recA-like gene of Herbaspirillum seropedicae, was isolated from a H. seropedicae genomic library by intergeneric complementation of Escherichia coli recA mutant strain HB101. Quantitative survival experiments showed that pBMR5 restored the ultraviolet radiation and methyl methanesulfonate resistances and recombinational proficiency of this strain. Hybridization studies showed that there is DNA sequence homology between the recA gene of E. coli K12 and that of H. seropedicae. Restriction sites for EcoRI, HindIII, BamHI, and Bg/II were found in the DNA insert derived from H. seropedicae in pBMR5. A Tn5 insertional mutant of pBMR5, called pBMR26.2, failed to restore recombination proficiency and methyl methanesulfonate and ultraviolet resistance to recA mutants of E. coli.

Cloning, Molecular↗
PubMed1980

[Studies into specificity for dye test (DT), complement fixation (CF), and intradermal test (ST) in diagnosis of toxoplasmosis (author's transl)].

Reference is made to an earlier study into the usefulness of the intradermal test. Now, an account is given of the specificities of tests used at present for the detection of toxoplasmosis, with reference being made to 4,341 intradermal tests, 1,332 dye tests, and 880 complement fixations. -- The three methods are qualitatively and quantitatively compared in great detail, with the close correlations between them explained. Reference is also made to the different origins of the antibody types detectable by the methods concerned. -- It seems to be quite obvious that the production of those antibody types differs in time and site, just as their lives are different in length. These findings actually are used as a point of departure for more detailed methodical proposals that should be followed in the search for an acute infection with Toxoplasma gondii. -- Various effects which might lead to unspecific tests are described in some length. -- All the above studies and findings have been used to compile a diagnosis diagram which may be a good basis for clinical decision-making on therapy.

Complement Fixation Tests↗
PubMed2006

Global and region-specific analyses of apparent diffusion coefficient in dentatorubral-pallidoluysian atrophy.

BACKGROUND AND PURPOSE: Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant spinocerebellar ataxia. Techniques for the quantitative assessment of neurodegenerative lesions remain to be established in this disease. We attempted to quantify global and region-specific neurodegeneration in DRPLA using analysis of apparent diffusion coefficient (ADC) maps. METHODS: Diffusion-weighted images (b = 1000 s/mm(2)) by echo-planar sequences were obtained with the use of a 1.5T clinical scanner. Whole-brain histogram and region of interest (ROI) analyses of ADC values as well as conventional MR imaging studies were performed in 6 patients with genetically confirmed DRPLA. RESULTS: Histograms demonstrated significantly higher mean ADC values in the patients than in age- and sex-matched control subjects (P < .01). ROI analysis revealed that the patients had significantly higher ADC values in the cerebellum and globus pallidus, preferentially affected regions (P < .05), but not in the thalamus, the region relatively spared in this disease. ADC values in the white matter were higher only in patients with adult-onset disease. Histogram analyses could more sensitively identify abnormalities than ROI analyses, because the former avoided errors associated with setting ROIs and thus had smaller P values on statistical analysis than the latter. CONCLUSIONS: Histogram ADC analyses were more sensitive for the detection of neurodegeneration in DRPLA than ROI analyses, whereas ROI analyses revealed regional alterations reflecting the distribution of pathologic changes. Thus, histogram and ROI analyses complement each other and may permit the sensitive, quantitative evaluation of neurodegeneration in DRPLA, especially that involving the globus pallidus showing normal T2 signals.

Adult↗
PubMed1983

Competitive enzyme immunoassay for detection of complement-fixing antibodies in diagnostic virology.

A competitive enzyme immunoassay was developed to detect serum complement-fixing antibodies in virus diseases. This assay utilized conglutinin-covered plastic beads as the solid phase to detect specific antibody-antigen complexes that competed for complement with a probe complex comprised of Escherichia coli beta-galactosidase and its specific antibody. Binding to the solid phase is C3bi mediated, and when specific antibody-antigen complexes are not present the probe is bound and an enzymatic reaction ensues. This type of competitive assay was introduced in the field of immunopathology for investigating circulating immune complexes by Manca et al. (Clin. Immunol. Immunopathol. 16:131-141, 1980). The assay gave satisfactory results in terms of specificity, reproducibility, and handling, enabling laboratories to obtain results in 5 h. The sensitivity of the method coincided with that of the complement fixation test. However, this technique offers several advantages over conventional complement fixation because it requires less time, is easier to perform, and gives more reliable quantitative results. The data obtained indicated that this competition assay offers a feasible alternative to conventional complement fixation tests and should be useful in routine diagnostic applications and in seroepidemiological surveys.

Antibodies, Viral↗
PubMed1982

Alternatives to the standardized laboratory branch complement fixation test for detection of antibodies to Coccidioides immitis.

Antibody titers to Coccidioides immitis, using coccidioidin antigen, were determined by three methods: the standardized Laboratory Branch complement fixation method (LBCF), a modified version of the Viral and Rickettsial Disease Laboratory complement fixation test (VRDL-CF), and a quantitative immunodiffusion test (QID). Of the 133 samples evaluated, 72 were negative by each method and 57 (42 serum samples, 15 cerebrospinal fluid samples) were positive by all three methods. Four additional specimens (1 serum sample, 3 cerebrospinal fluid samples) were positive by QID alone. All positive patients were diagnosed clinically as having pulmonary or extrapulmonary coccidioidomycosis or both. When titers from two methods were compared, the agreement within +/- 1 dilution was VRDL-CF/QID, 88.5%; VRDL-CF/LBCF, 85.2%; and LBCF/QID, 82.0%. The agreement of these methods within +/- 2 dilutions was VRDL-CF/QID, 98.4%; VRDL-CF/LBCF, 96.7%; and LBCF/QID, 93.4%. The VRDL-CF and QID methods are simpler to perform; however, they are yet unrecognized as suitable alternatives to the more cumbersome LBCF. Our data show that they should be considered as options for C. immitis serology.

Antibodies, Fungal↗
PubMed1982

The complement fixing ability of putative circulating immune complexes in rheumatoid arthritis and its relationship to extra-articular disease.

The hypothesis that the pathogenicity of putative circulating immune complexes (CIC) in rheumatoid arthritis (RA) is related to their ability to fix complement was investigated. Three assays for CIC were employed; (a) the 125I C1q binding assay (C1q BA), (b) the C1q solid phase assay (C1q SP) and (c) the Raji cell assay (RCA). Evidence for hypercatabolism of complement was obtained by using a highly sensitive quantitative assay for C3d (a breakdown product of C3) by rocket immunoelectrophoresis. One hundred and fifty-two patients with classical or definite RA were studied; 54 had clinical evidence of extra-articular disease including vasculitis, nodules, scleritis, neuropathy and lung disease; 98 patients had clinical evidence of joint disease alone. Plasma levels of C3d were significantly elevated in the RA group as a whole 16.7 +/- 4.4 mg/l (mean +/- 1 s.d.) compared with 13.1 +/- 3.25 mg/l in a group of 55 normal controls (P less than 0.01). Elevated levels of C3d were found in 26% of all patients but occurred significantly more often in the extra-articular disease group (P less than 0.05). Fifty-four percent of patients had at least one positive assay for CIC although no individual assay was positive in more than 36% of the group as a whole. The prevalence of positive CIC was significantly greater in those patients with extra-articular disease than in those with joint disease alone (P less than 0.005). Of the total of 82 patients with putative CIC, 30 (37%) had a raised C3d level. The coincident finding of positive tests for CIC and an elevated C3d level was very significantly correlated with the presence of extra-articular disease (chi 2 = 12.7 P = 10(-3)). Whilst putative CIC are frequent in RA (54%) these findings in contrast to previous work, suggest that the majority are not associated with abnormal complement activation and may account for the relative infrequency of clinically detectable active extra-articular disease.

Adolescent↗
PubMed1975

Complement activation in seropositive and seronegative rheumatoid arthritis. 125I-C1q binding capacity and complement breakdown products in serum and synovial fluid.

1. The detection and quantitation of immune complex-like material in synovial fluid and in serum from patients with joint diseases was done through the measurement of the capacity to bind radiolabeled C1q. It was found that 65% of synovial fluid samples from seropositive or seronegative RA patients had a high C1q binding capacity as compared to other joint diseases. Immune complex-like material was also detected in 63% of serum samples from seropositive RA patients. 2. The existence of C3 or C3PA breakdown products in synovial fluid from most of the synovial fluids from RA patients probably reflects an activation of the complement system occurring in both forms of the disease. C3PA breakdown products were never found in degenerative or post-traumatic joint diseases and only occasionally in other inflammatory arthritis. Apart from their pathogenic significance, these results may have some interest for the clinical investigation of patients with joint diseases.

Arthritis, Rheumatoid↗
PubMed1972

Effect of rheumatoid factor on complement-mediated phagocytosis.

The frequency and amount of IgM rheumatoid factor (RF) in the blood of patients with rheumatoid arthritis (RA) correlate with the severity of the disease and the number of complications. Though previous studies of RF in subacute bacterial endocarditis have shown that RF inhibits phagocytosis of microorganisms by granulocytes, the presence of low levels of complement (C) in blood and synovial fluid of patients with the highest titers of RF suggests that an interaction between RF and C may contribute to the inflammatory process in RA. We thus employed a quantitative methodology to examine the effect of RF on complement-dependent phagocytosis of sheep erythrocytes by rabbit granulocytes. Addition of 2500 molecules of IgM RF to sheep cells heavily coated with IgG antibody (195,000 molecules per cell) resulted in virtually complete inhibition of uptake of C3 (beta(1c)) and prevention of phagocytosis, an effect resulting from inhibition of uptake of C1 by the cells. When erythrocytes coated with only 34,000 molecules of IgG antibody were employed, phagocytosis was similarly inhibited. However the effect of RF on such cells was shown to be primarily mediated through inhibition of C4 rather than C1 uptake. Although the results do not exclude the participation of an IgM RF of higher avidity, present only in the tissues in rheumatoid inflammation, circulating IgM RF probably does not play a potentiating role in rheumatoid inflammation.

Animals↗