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Biomedical subjects

M R Daha

Publications and source records attributed to M R Daha.

At least 433 records · Page 24Linked to original sources

Regulation of immune complex-mediated complement activation by autoantibodies (F-42) isolated from sera of patients with systemic lupus erythematosus.

Sera of some patients with systematic lupus erythematosus (SLE) contain IgG autoantibodies (F-42) which have been shown to stabilize the cell bound classical pathway C3 convertase of complement, C42. C42 is susceptible to inactivation by the plasma protein C4bp while stabilized C42 is relatively resistant to C4bp. The present study demonstrates that F-42 by itself does not induce activation of the classical pathway in vitro but that it is able to modulate the immune complex-induced consumption of C2 and C3 in whole serum. Incubation of incremental concentrations of F-42 with normal human serum (NHS) for 30 min at 30 degrees C did not result in detectable consumption of C1q, C4, C2 and C3. However, when soluble immune aggregates or immune complexes were incubated in NHS together with 100 u/ml of F-42, a significant increase in consumption of C3 was seen as compared to the reaction mixture containing immune complexes or F-42 alone. In addition the presence of F-42 during the immune complex mediated consumption of complement was associated with relative protection of C2 consumption. (Fab)'2 and Fab' fragments of F-42 behaved as intact F-42, except that their activities on a molar basis were less than that of intact F-42. The results presented in this paper suggest that F-42 may play a regulatory role in the immune complex-mediated consumption at C2 and C3 in vivo.

Antigen-Antibody Complex↗

Relative importance of C4 binding protein in the modulation of the classical pathway C3 convertase in patients with systemic lupus erythematosus.

Serum concentrations of C1q, C4, C4 binding protein (C4bp), C3 and C2 haemolytic activity have been measured in 110 samples from 20 patients with systemic lupus erythematosus (SLE). Significant reductions in comparison to normal levels were found in the mean serum concentrations of C4, C3 and C4bp as well as C2 haemolytic activities. For patients serum concentrations of C4 correlated with C2 haemolytic activities (r = 0.91) and C4bp (r = 0.79); the C2 haemolytic levels correlated with the concentration of C4b (r = 0.72). It is concluded that serum concentrations of the complement components C4 and C2, which are the constituents of the classical pathway C3 convertase, are regulated by C4bp in vivo. Further metabolic studies are required to determine the causes of decreased serum concentrations of C4bp in patients with SLE.

Carrier Proteins↗

Activation of C1 by soluble IgG aggregates as detected by a novel one-step hemolytic assay that specifically measures the proenzyme form of C1s.

A new hemolytic assay is described that specifically measures the precursor form of the C1s subcomponent of the complement system. The assay employs a C1s-depleted reagent obtained by immunoadsorption of fresh human plasma on immobilized goat anti-human C1s antibodies. Linear Z plots are obtained with nanogram levels of precursor C1s, whereas C1s completely fails to induce hemolysis in the assay. Because low concentrations of C1s do not interfere with the activity of precursor C1s, the assay can be used for the stoichiometric measurement of C1 activation. The precursor C1s assay was applied to the study of C1 binding and activation by soluble aggregates of human IgG (AIgG). Incubation of purified human C1 with AIgG caused a temperature-independent consumption of whole C1 hemolytic activity, indicating binding of C1, but almost no consumption of the total (precursor + activated) C1s activity. On the other hand, activation of C1, measured as the time- and temperature-dependent consumption of precursor C1s, could greatly exceed the binding of C1. These findings can be explained by using recent findings concerning the association-dissociation equilibrium between C1q and the tetrameric complex of C1r and C1s.

Animals↗

C1- inactivator: its efficiency as a regulator of classical complement pathway activation by soluble IgG aggregates.

The role of C1- inactivator (C1(-)-In) during activation of the classical complement pathway by soluble immune complexes was studied using purified human complement components C1, C4 and C1(-)-In, and stabilized soluble aggregates of normal human IgG as a model for soluble immune complexes. The C4-consuming ability that could be generated by incubation of precursor C1 with IgG aggregates was abolished completely by the presence of a large excess of C1(-)-In during the C1 activation step. Kinetic studies confirmed that this inhibition was due to a second-order reaction between C1- and C1(-)-In resulting in the irreversible inactivation of C1-. When aggregates of various sizes were enabled to induce C4 conversion in mixtures of C1, C4 and a variable concentration of C1(-)-In, the presence of C1(-)-In had two effects. Firstly, the efficiency of the aggregates in causing C4 consumption was reduced remarkably. At a C1(-)-In:C1 ratio of 8, which can be found in normal human serum, approximately eight to ten times as many aggregates were required for a given level of C4 consumption as when no C1(-)-In was present. Secondly, C1(-)-In diminished the maximum C4 consumption that could be achieved, especially with smaller aggregates. Thus, a complete or partial C1(-)-In deficiency probably facilitates complement activation by soluble immune complexes in two ways: it may enhance the efficiency of classical pathway activation by all C1-activating complexes, and it may enable small complexes, which normally cannot overcome the C1(-)-In barrier, to activate the classical pathway to the C4 level.

Antigen-Antibody Complex↗

Degradation of soluble immunoglobulin aggregates in vitro by monocytes from normal subjects and from patients with systemic lupus erythematosus.

The capacity of human peripheral monocytes to degrade soluble immunoglobulin (IgG) aggregates (AIgG) was studied in vitro. Under serum-free conditions peripheral monocytes from normal donors were able to degrade soluble AIgG in a linear and time-dependent fashion. Addition of fresh human or fresh guinea-pig serum to the incubation mixtures caused a marked increase in degradation of the amount of soluble AIgG available. The stimulatory effect of fresh serum was complement-mediated, because it was abolished by heat treatment of the serum and was not seen when C4- or C3-deficient sera were tested. Functional inactivation of C3 receptors on the phagocytes by trypsin also abolished the complement-mediated stimulation, suggesting cooperation between Fc and C3 receptor in degradation of soluble AIgG. No significant differences were found between monocytes from normal donors and those from patients with systemic lupus erythematosus, as far as degradation is concerned in the presence of complement.

Animals↗

Regulation of the C3 nephritic factor stabilized C3/C5 convertase of complement by purified human erythrocyte C3b receptor.

Activation of complement may result in the generation of the amplification convertase C3bBb. This convertase can be stabilized by properdin (P) or C3NeF. C3bBbP is susceptible to inactivation by beta 1H, while C3bBbNeF is relatively resistant. Since it has been shown that the human erythrocyte C3b receptor (CR1) is able to inactivate C3bBbP, the inactivating action of CR1 on C3bBbNeF was investigated CR1 is at least five times more efficient than beta 1H in inactivating C3bBbNeF. Kinetic studies revealed that CR1 induces an enhanced biphasic kinetics of decay of C3bBbNeF; further purification of this C3NeF preparation by cation exchange chromatography showed that this phenomenon is dependent on the population of C3NeF. Finally CR1 is also able to inactivate fluid phase C3bBbNeF.

Complement Activating Enzymes↗

Antibody, immune complexes, and complement activity fluctuations in kittens with experimentally induced feline infectious peritonitis.

Humoral changes were studied in 6 specific-pathogen-free kittens during experimental infection with feline infectious peritonitis virus. Although the incubation period and the duration of the disease differed widely, a similar pattern of rectal temperatures, serum complement values, circulating immune complexes, and antibody titers was found for all kittens during the last 15 to 20 days of life. Antibody formation started 8 to 13 days before death and was accompanied by the appearance of circulating immune complexes with subsequent increased concentrations of complement followed by complement depletion. The data are discussed as evidence for an immune complex pathogenesis of feline infectious peritonitis.

Animals↗

Increased IgM and IgM immune complex-like material in the circulation of renal transplant recipients with primary cytomegalovirus infections.

Twelve of 60 consecutive adult recipients of cadaver kidney transplants had increased polyethyleneglycol (PEG) precipitable IgM immune complex-like material in their circulation in the first 4 months after transplantation. All 10 recipients with primary CMV and two of four with secondary CMV infections had significant elevations in PEG precipitable IgM that coincided with rises in their CMV antibody titres. Ultracentrifuge analysis demonstrated two peaks of PEG precipitable material with sedimentation rates of about 20S and 40S. Total IgM immunoglobulin levels also were increased in transplant recipients with CMV infections, but this was less specific and occurred in patients without CMV infections. The Clq binding assay, which is more sensitive for IgG than IgM containing complexes, was positive in only three of 10 patients with primary CMV and none of four with secondary CMV. Granular deposits of IgM, but not IgG, were detected in the glomeruli of six of seven transplants biopsied during CMV infection. The PEG-IgM assay was not influenced by rejection or prednisone therapy. Thus, transplant patients, who develop primary CMV infections, produce elevated levels of circulating IgM and IgM immune complex-like material. These findings may help to differentiate CMV infection from transplant rejection as well as to increase our understanding of the special pathogenic properties of CMV in transplant recipients.

Adolescent↗

Isolation, characterization, and mechanism of action of rat beta 1H.

beta 1Hrat was purified to homogeneity from fresh rat plasma by precipitation with 28.6% ammonium sulfate followed by sequential chromatography on DEAE-Sephacel, Biorex-70, and gel filtration on Sephacryl-S300. The final material was homogeneous on SDS-PAGE analysis and had an apparent m.w. of 150,000. Reduction with dithiothreitol did not affect its m.w., suggesting that the molecule is composed of one polypeptide chain. The recovery of beta 1H was approximately 10%. A monospecific antibody against beta 1Hrat was obtained from immunized rabbits, which recognized beta 1Hrat as a protein with beta-electrophoretic mobility upon immunoelectrophoresis of fresh rat plasma. The concentration of beta 1H in plasma of normal 4-mo-old Wistar rats was 243.5 +/- 36.3 micrograms/ml (mean +/- S.D.). beta 1Hrat in this study was detected by its capacity to inhibit formation of the P-stabilized cell-bound amplification C3/C5 convertase composed of cell-bound C3bhu and Bbhu. Purified beta 1Hrat produced a dose-related, first-order loss of convertase function and release of 126I-Bbhu from the P-stabilized C3bhuBbhu convertase, indicating a mechanism of action by decay-dissociation of Bbhu from the complex C3bhuBbhuP. beta 1Hrat was at least four times less effective than beta 1Hhu in release of 125I-Bbhu from the homologous convertase composed of C3bhu and Bbhu. On the other hand beta 1H was twice as effective in releasing 125I-Bbrat from the convertase composed of C3bratBbratP when compared to beta 1Hhu. These differences are presumably dependent upon the species-specific affinity of beta 1H from humans or rats for C3bhu or C3brat, respectively.

Animals↗

Combined hereditary deficiency of the sixth component of complement and factor VIII coagulant activity in a Dutch family.

Prompted by previous observations of defective blood clotting in rabbits deficient in the sixth component of complement (C6), and the discovery of a patient with both C6 and factor VIII deficiency, an evaluation was made of the haemostatic functions in this individual and his family members. The family contained three members homozygous for C6 deficiency (C6D); two of them were deficient also in factor VIII. In addition, one other member of the family was only deficient in factor VIII. The only C6D member without haemophilia A had a normal recalcification time without clinical symptoms of a bleeding disorder. Reconstitution of factor VIII and C6 deficient plasma from the various members of the family in this study with purified human C6 did not result in a change in the recalcification time. The results obtained from this study also indicate that there is no linkage between the inheritance of C6 and factor VIII.

Adult↗

Stimulation of the intracellular killing of Staphylococcus aureus by monocytes: regulation by immunoglobulin G and complement components C3/C3b and B/Bb.

The intracellular killing of Staphylococcus aureus by human monocytes requires continuous stimulation by extracellular serum factors interacting with the cells via membrane binding sites. At least 75% of the intracellular killing in the presence of fresh serum was accounted for by the combined stimulatory activities of IgG, C3/C3b, and B/Bb. C3b is a more potent stimulator than C3, and Bb stimulates the killing to the same degree as B. The stimulation of intracellular killing by C3 and C3b occurs by interaction of the stable binding site of this molecule with the C3b receptor in the monocyte membrane. The stimulation of intracellular killing by B and Bb is most probably mediated via a binding site on these proteins interacting with a receptor site in the monocyte membrane. Both complement receptors, i.e., for C3b and B, are pronase sensitive. However, only the C3b receptor can be inhibited by (Fab')2 fragments of anti-C3 receptor antibodies, indicating that the binding sites for C3/C3b and B/bb are different.

Blood Bactericidal Activity↗

Influence of aggregate size on the binding and activation of the first component of human complement by soluble IgG aggregates.

The interaction between small aggregates of human IgG and the first component of human complement was studied. Stabilized soluble IgG aggregates of restricted size were prepared by heat aggregation of human IgG, followed by sucrose-density ultracentrifugation. Human C1 was isolated in its precursor form by euglobulin precipitation, followed by gel filtration and immunoadsorption. A C1 preparation was obtained of which more than 90% was still in its unactivated form. Soluble aggregates containing 20, 10 or 5 molecules IgG, and monomeric IgG were tested for their ability to bind and to activate C1. The binding of C1 was determined by C1 consumption, whereas the activation of C1 was measured as the increased ability of the C1 preparation to consume purified human C4 after the incubation with the aggregates. The three aggregates tested and monomeric IgG were all able to bind and to activate C1, but the efficiency of both processes markedly increased with increasing aggregate-size. Furthermore, it was found that all four preparations activated an appreciable amount of C1 at concentrations that did not result in any detectable C1 fixation. These results confirm earlier suggestion that C1 can be activated during a short, transient binding to small aggregates or immune complexes that have a low avidity for C1, after which the activated form, C1, is released into the medium.

Binding Sites↗

The role of cellular Fc and C3 receptors on the complement-dependent degradation of stable soluble immunoglobulin aggregates by normal and trypsin-treated peritoneal macrophages.

The experiments described in this paper were designed to determine the degradation of soluble IgG aggregates (AIgG) bearing C3b by guinea-pig peritoneal macrophages with different numbers of functional C3b receptors. The number of functional Fc receptors for AIgG containing 40 molecules of IgG per aggregate (AIgG40) were kept constant during these experiments. By increasing the concentration of trypsin for the treatment of normal peritoneal macrophages a dose-dependent inactivation of C3b receptors was achieved as determined by the binding of tetrameric [125I]-C3b([125I]-AC3b). Normal peritoneal macrophages bound 148,500 AIgG40 and 60,300 AC3b per cell. Treatment of the macrophages for 15 min at 37 degrees with incremental amounts of trypsin from 60 to 1000 micrograms/ml did not affect the binding of AIgG40 to macrophages but caused a dose-related loss of up to 95% of AC3b binding, indicating functional inactivation of C3b receptors. Degradation of trichloroacetic acid non-precipitable protein of [125I]-AIgG40 by 10(6) macrophages was 38 +/- 6% in medium alone and 60 +/- 8% in the presence of 10% fresh guinea-pig serum (NGPS) after 60 min at 37 degrees. The inactivation of 25%, 50%, 72% and 94% of C3b receptors by trypsin did not affect the degradation of AIgG40 in medium alone, but decreased degradation from 69% to 58%, 47%, 32% and 13% of AIgG40 respectively, in the presence of 10% GPS. Macrophages (10(6)) in medium alone degraded 40%, 47%, 65% and 68% of AIgG40 bearing 0, 5, 10, 16 and 20 C3b molecules per aggregate, indicating a dose-dependent enhancement of degradation by bound C3b; conversely, inactivation of 95% of C3 receptor on the macrophages resulted in 39%, 30%, 12%, 5% and 6% degradation of these AIgG40-C3b, indicating a dose-related inhibition by aggregate-bound C3b in the absence of cellular C3 receptor. These experiments stress the importance of Fc and C3b receptor co-operation and immune complex bound C3b.

Animals↗

The clinical implications and the pathogenetic significance of circulating immune complexes in infective endocarditis.

Circulating immune complexes were determined with the 125I-Clq binding assay and the conglutinin binding assay in a prospective, longitudinal study of 40 patients with infective endocarditis, 34 patients with endocardial defects and nonseptic fever and 25 patients with septicemia without endocarditis. Fourteen patients with uncomplicated valvular lesions constituted a control group. Upon admission, 63 percent of the patients with infective endocarditis had a positive 125I-Clq binding assay versus 9, 12 and 7 percent, respectively, of the other three groups (p less than 0.001). The incidence of positive conglutinin binding assays became significantly higher during the course of infective endocarditis (53 percent) than during the course of nonseptic fever (21 percent), but, upon admission, this difference was not significant. The high incidence of Clq-binding immune complexes among patients with infective endocarditis could be attributed mainly to those patients with the characteristic features of subacute endocarditis. The incidence of circulating immune complexes in acute endocarditis was low and did not contribute to making the clinically important distinction from septicemia without endocarditis. A rise in the 125I-Clq binding assay levels during the course of infective endocarditis correlated significantly (p less than 0.01) with failure of antibiotic treatment. With the 125I-Clq binding assay, significantly higher levels were found in patients with signs of renal involvement of cutaneous vasculitis than in patients without these extracardiac manifestations of endocarditis. These results show that the determination of circulating immune complexes has clinical implications for both the diagnosis and the management of infective endocarditis and that circulating immune complexes are probably involved in the development of glomerulonephritis and vasculitis.

Adolescent↗