C3 Conversion Research Articles

Hemolytic, cytotoxic and complement inactivating properties of extracts of different species of Aspergillus.

Some of the biological properties of saline extracts of the mycelia of several species of the Aspergillus genus, namely, A. fumigatus, A. flavus, A. niger, A. nidulans, A. parasitucus and A. glaucus, were studied. Only the extract prepared from A. fumigatus was found to be hemolytic for sheep red blood cells. In contrast, all the extracts with the only exception of that of A. glaucus, had cytotoxic effects on Vero cells. Both, the hemolytic and cytotoxic constituents of the extracts were removable by adsorption with activated carbon. Heating of the extracts at 100 degrees C for 30 minutes also resulted in detoxification. In vivo studies, performed only with detoxified extracts of A. fumigatus, showed these were capable of depleting complement levels in guinea pigs. Complement inactivation was also found to occur in vitro and was cuased by all the extracts tested. Also triggered by the extracts was the conversion of serum C3 but not of purified C3, indicating that other serum factors are essential in the process. Despite the similarity in this respect with cobra venom factor, differences in activity after heating-negative in cobra venom factor-indicate that the complement inactivating substance/s present in the Aspergillus extracts differ from those of the snake product.

Requirement for Complement Components and Fibrinogen in the Zymosan-Induced Release Reaction of Human Blood Platelets

Abstract As shown earlier, zymosan particles incubated with human citrated plasma develop the ability to induce a release reaction in platelets. Thus when incubated zymosan is shaken for 4 to 15 min with human citrated platelet-rich plasma, ADP, ATP, and 14C-serotonin are released and the ADP causes platelet aggregation. Plasma depleted in vitro of C3 or GBG failed to generate activity on zymosan unless at least 65 µg of C3 or 100 µg of glycine-rich β-glycoprotein (GBG) were added/ml plasma. Plasmas from individuals with congenital deficiency of C3 or C3 inactivator (and hence GBG) were also inactive unless either purified C3 or hydrazine-treated plasma was added. Afibrinogenemic plasma was inactive unless at least 80 µg of fibrinogen was added/milliliter, a requirement previously reported for serum. Zymosan particles washed after incubation with normal plasma bore more fibrinogen (determined immunologically) than particles incubated with afibrinogenemic or ethylenediaminetetraacetic acid plasma and washed. The amount of zymosan-associated fibrinogen was not necessarily less on particles incubated with C3- or GBG-deficient plasma than on those incubated with normal plasma but was greater when purified C3 or GBG was added to the appropriate deficient plasma before incubation. Properdin-depleted plasma could not generate release-promoting activity on zymosan; activity was not restored by purified active properdin. When plasma from two patients with C̄1 inhibitor deficiency was depleted of C4 by incubation, one sample generated activity on zymosan normally. Serum from a guinea pig with C4 deficiency generated activity on zymosan when fibrinogen was added. Inulin and endotoxin did not develop release-promoting activity when incubated with normal plasma although they caused as much conversion of C3 and GBG as zymosan. Plasma incubated with inulin and endotoxin generated activity when subsequently incubated with zymosan, whereas plasma incubated with zymosan lost its ability to generate activity when incubated with fresh zymosan. We conclude that the alternate pathway of complement activation and fibrinogen are involved in generating release-promoting, platelet-aggregating activity on zymosan, and that activity may develop only during activation of properdin.

Activation of complement in anaphylactoid reactions in connection with infusion of dextran.

Complement components were investigated in 12 patients who had developed anaphylactoid shock symptoms on preoperative infusion of dextran for prevention of postoperative thromboembolism. An activation of C3 proactivator to C3 activator and a conversion of C3 were found in 5 of the 12 patients. Quantitation of C3, C3 proactivator, and C3 did not discriminate between these patients and the controls No precipitating antibodies were detected, and no quantitative changes of the earlier reacting complement components. O1q and C4, were found.

Interaction of C-Reactive Protein Complexes with the Complement System

Abstract The reaction of C-reactive protein (CRP)-positive sera and crystalline CRP with pneumococcal C-polysaccharide at equivalence induced consumption of human complement and C3 conversion in the absence of detectable antibody. Sera with negative or trace reaction for CRP most often gave no or minimal consumption of complement on addition of C-polysaccharide. Analysis of complement components consumed in the reaction of CRP-positive sera with C-polysaccharide, in the absence of detectable antibody, demonstrated consumption of 80% or more of total hemolytic complement, C1, C4, and C2, and 42 to 66% consumption of C3-9. Quantitative estimates of the minimal amount of CRP in acute phase sera which permitted fixation of complement closely approximated the minimal value obtained with crystalline CRP. All normal human sera tested permitted consumption of complement by CRP complexes, including sera from infants 5 to 7 months of age with physiologic hypogammaglobulinemia and sera previously absorbed with pneumococcal cell walls to remove any presumptive trace of antibody to C-polysaccharide. Both the CRP-C-polysaccharide precipitation and complement consumption reactions were 100% inhibited by phosphoryl choline but were not at all inhibited by N-acetyl galactosamine, the major determinant of antibody specificity. CRP reacted with phosphoryl choline of the choline phosphatides, lecithin and sphingomyelin, by complement fixation and by flocculation. Immunodiffusion analysis of the washed complexes obtained by reaction of CRP-positive sera with C-polysaccharide and with lecithin demonstrated strong lines of precipitation for CRP, C1q, and C3, whereas immunoglobulins were absent or detectable only in faint traces. These combined data have permitted the conclusion that complement consumption was attributable to CRP complexes and that it proceeded via the classical pathway independent of the presence of specific antibody or immunoglobulin. The specificity of CRP for choline phosphatides and its capacity to activate the complement system are considered in relation to the possible functions of CRP in the inflammatory process.

INFUSION OF HEPATITIS-B ANTIBODY IN ANTIGEN-POSITIVE ACTIVE CHRONIC HEPATITIS

The effects of infusion of an immunoglobulin preparation with a high titre of antibody to hepatitis-B antigen (HBAg) have been investigated in six patients with active chronic hepatitis, in five of whom HBAg was persistently detected in the serum. The intravenous infusions were well tolerated and two patients only showed minor and transient evidence suggesting an immunecomplex reaction. Complexes containing HBAg were demonstrable after the infusion by electron microscopy in four cases, but in three of these complexes were also present beforehand. Complement levels and C3 conversion products were consistent with the presence of immune complexes. Clearance of antigen from the circulation for up to 9 days was achieved in the two cases with low initial titres of HBAg.

The association of respiratory infection, recurrent hematuria, and focal glomerulonephritis with activation of the complement system in the cold.

The study of the activation of C3, C5, and C7 associated with the conversion of C3 in the serum of a 9-yr old girl after incubation at 0 degrees C for 6-8 h without utilization of C1, C4, and C2 is described. The patient has upper respiratory infections associated with recurrent gross hematuria, focal glomerulonephritis, and transient renal insufficiency. Histological lesions demonstrated the presence of B1c globulin IgA and properdin in the glomeruli. The activation of complement (C) in the cold requires the patient's IgA. Removal of IgA from the serum by immunoadsorption prevents activation and conversion of C3. Bactericidal and phagocytic activity is also impaired after incubation. C3 proactivator (C3PA) level is reduced before and after incubation. Properdin level drops after incubation. These findings suggest that the activation of C3 which is demonstrable in vitro may be a continuous process in vivo.

Polyacrylamide Gel Electrophoresis (PAGE) of Reduced and Dissociated C3 and C5: Studies of the Polypeptide Chain (PPC) Subunits and Their Modifications by Trypsin (TRY) and C4̄2̄-4̄2̄3̄.

Abstract Our previous PAGE studies (according to Weber and Osborn) revealed an α and a β PPC of C5 of the approximate m.w. of 120,000 and 80,000, respectively. Our present studies show a similar structure of human C3, with an α and a β PPC of approximately 110,000 and 70,000 m.w. respectively. Furthermore, the effects of TRY and of - on C3 and C5 were explored by immunoelectrophoresis (IEP) and by PAGE. C3b (TRY) and C3b () were both faster than C3 and indistinguishable by IEP. C5b (TRY) was distinctly faster than C5b () which was, however, only slightly faster than C5 by IEP. PAGE analysis indicated that the modifications observed by IEP were all related to degradations of the α PPC:s of C3 and C5: conversion of C3 to C3b (TRY) or C3b () coincided with a reduction of the m.w. of the α PPC of C3 by approximately 7000 to 8000; conversion of C5 to C5b (TRY) coincided with the reduction of the α PPC of C5 by approximately 18,000, whereas conversion of C5 to C5b () coincided with a reduction by less than 18,000.

Cerebrospinal Fluid C

Abstract Determination of C3 concentration in unconcentrated cerebrospinal fluid by electroimmunodiffusion was possible only when an immunofixation layer was added by using antibody to the original gel antibody against C3. Recently we have been able to measure C4 in this manner, but in this situation the original running time is critical and must not be excessive since then the precipitate dissolves. This technique was applied to the study of paired sera and cerebrospinal fluids. Native C3 and C3 conversion products may be visualized by antigen-antibody crossed electrophoresis after concentration of cerebrospinal fluid 40-fold by ultrafiltration, providing the immunofixation procedure is used. Ten normal specimens of cerebrospinal fluid had a mean (± 1 S.D.) of 378 ± 159 µg/100 ml for C3, with a mean C3/albumin of 1.49 ± 0.18%. Eight normal spinal fluids had a mean for C4 of 274, with a mean C4/albumin of 1.23%.

Automated Two-Dimensional Immunoelectrophoresis and its Application to the Analysis of C3 and C4 in Rheumatoid Arthritis and Systemic Lupus Erythematosus (S.L.E.)

A semi-automated two-dimensional immunoelectrophoretic method has been developed which is quicker and cheaper to use than the original system, making it more suitable for routine use. It is accurate and reproducible and is suitable for complement conversion studies where C3 and C4 conversion can be measured with 5 and 10% reproducibility respectively. Conversion of C3 and C4, observed in both S.L.E. and rheumatoid arthritis, is an early and sensitive index of disease activity. Total C3 levels, although fluctuating widely in the course of the disease, were not shown to be as sensitive an index.

Further Investigations on the Inherited Polymorphism of the Third Component of Complement (β<sub>1</sub>C/β<sub>1</sub>A-Globulin)

C3 polymorphism has been studi ed in a sample of 1,204 Germans, 961 Spaniards, 216 families having 420 children, and 310 mother/child combinations. Further studies regarding C3 conversion and possibleidentity with the posttransferrin (β₁A ?) polymorphism are reported.

Immunoconglutinin and complement studies in congenital nephrotic syndrome and nephritis of Henoch-Schönlein purpura in children.

Evidence of complement activation in 6 children with congenital nephrotic syndrome and 9 children with nephritis associated with Henoch-Schönlein purpura was sought by estimating the concentration and by immunoelectrophoretic analysis of the complement component C3, and by immunoconglutinin titration (IK: an autoantibody to reacted complement components). IK and quantitative C3 levels did not differ significantly from control values in either disease; <i>in vivo</i> C3 conversion was not detected in plasma from congenital nephrotic patients, but was present in 2 of 9 patients with Henoch-Schönlein nephritis. The data do not provide consistent evidence of complement involvement in either disease.

THE ANTIGENIC AND MOLECULAR ALTERATIONS OF C3 IN THE FLUID PHASE DURING AN IMMUNE REACTION IN NORMAL HUMAN SERUM

During the reaction of an immune precipitate with fresh human serum, C3 undergoes a number of molecular alterations with the formation of conversion products differing from those obtained when purified components react. Those products which remain in the fluid phase, the subject of the present paper, have been identified by their reaction with monospecific antisera to the three antigenic determinants of C3, A, B, and D, after electrophoresis in agar or polyacrylamide gel. When purified C3 reacts with EAC1,4,2, C3i is found in the fluid phase. C3i, a loose complex of C3a and C3b, is in a conformational state whereby only the A and D antigens, present on its C3b portion, will consume antibody. The B antigen, present on the C3a portion of C3i, is unavailable for combination with antibody until C3i dissociates. In the fluid phase of the reaction of an immune precipitate with whole serum, C3i, C3a, and C3b, formed when purified components react, cannot be found. Instead the end products of the reaction appear to be C3c, which contains the A antigen, and C3d, which contains the D antigen. C3c and C3d are similar to the beta1A and alpha2D produced by the aging of serum but differ in their mobilities in acrylamide gel and in agar. The C3c and C3d generated by an immune precipitate also differ slightly from the C3c and C3d produced by the reaction of trypsin with C3 in whole human serum. As human serum reacts with an immune complex, native C3 appears to undergo a primary alteration before conversion. This alteration results in a molecular species of C3 which is labile at 56 degrees C for 30 min, fails to expose additional A and D antigenic sites upon aging, and which forms beta1A and C3d rather than beta1A and alpha2D during aging. In addition to this altered form of native C3, a new conversion product, C3x, is formed as whole serum reacts with an immune complex. C3x is not found in systems utilizing pure complement components. C3x is like C3 in that it bears all three antigenic determinants but differs in that it has a slightly faster mobility in polyacrylamide gel than does native C3. C3x is not only found in the fluid phase but is also bound to the immune precipitate. Finally, the fluid-phase kinetics of each of the antigens of C3 have been determined as normal human serum reacts with an immune precipitate. These illustrate that nearly the entire population of native C3 molecules undergoes conversion rapidly as manifested by the disappearance of the B antigen from the fluid phase. Moreover, the kinetics of the fluid-phase A and D antigens reflect that the conversion of C3 in serum is quantitatively not the same as when purified C3 reacts with C4,2.

Generation of Chemotactic Activity from Human Serum and Purified Components of Complement by Serratia Proteinase

Abstract The ability of culture filtrates of Serratia marcescens to effect immunoelectrophoretic (IEP) conversion of C3 in human serum in the presence of EDTA or of purified C3 correlated with elaboration of Serratia proteinase. This was confirmed with purified proteinase (supplied by Dr. C. Decedue). Higher concentrations of purified proteinase were required for partial IEP conversion of C3 in serum (500 µg proteinase/ml) than purified C3 (1 to 2 µg/ml), indicating the presence of a serum inhibiter which was localized in a 36% to 57% saturated ammonium sulfate fraction of pseudoglobulin. The extent of IEP conversion of purified C3 was a function of proteinase concentration and incubation time and was associated with loss of hemolytic activity and cleavage of C3 with appearance of two light fragments (estimated molecular weights 34,000 and 5,600). Chemotaxis for rabbit neutrophils was found in the slowly sedimenting region of sucrose gradients.

Isolation of the Precursor and the Active Form of the C3 Activator from Human Serum

Abstract It had previously been shown that a protein isolated from the venom of Naja naja, the cobra factor, is capable of forming a complex with a β globulin present in human serum. This complex is able to cleave C3 in free solution and apparently activates the terminal complement components beginning with C3. The C3 proactivator (C3PA) which participates in these reactions by forming the enzymatically active complex with cobra factor was isolated from human serum. It was found to be homogeneous on disc- and immunoelectrophoresis. Cleavage of the C3PA into at least two fragments was observed by treatment of serum with a variety of naturally occurring lipopolysaccharides and polysaccharides such as endotoxin, zymosan, inulin or agar. This reaction was accompanied in all instances by the conversion of C3 to its electrophoretically faster form. The larger of the two C3PA fragments (m.w. approximately 60,000) has γ globulin mobility and is antigenically deficient.

Protein A from Staphylococcus aureus complement fixation by aggregates between protein A and IgG1 or IgG2 from guinea pig

Protein A from Staphylococcus aureus is known to react with the Fc fragment (**) of IgGl andIgG2 from guinea pig [ I]. It has also been shown that protein A is able to evoke anaphylactic reactions in normal guinea pigs [2] as well as the Arthus reaction in rabbits [3]. Since the Arthus reaction is known to depend on the activation of complement, we have investigated the interaction between protein A and complement (C). It was found that the protein A-IgG complex will fix guinea pig [4], human and rabbit [5] complement. Furthermore it was shown that protein A added to human serum resulted in conversion of complement factor C3 [6]. It was concluded that protein A-human IgG complexes act on human complement in the same way that ordinary antigen-antibody complexes act. Different biological activities have been ascribed to the two subclasses of guinea pig IgG; IgGl and IgG2 [7-91. These differences are thought to depend on structural differences in the Fc fragments [IO] . Recently it has been claimed that some biological activities of IgGl and IgG2 can not be strictly differentiated [ 11, 121 because preformed aggregates between antigen and IgG2 as well as between antigen and lgG1 are able to fix complement. The present communication confirms that protein A precipitates both IgGl and lgG2 [ 13, 141. Both IgGl

Protein A from Staphylococcus aureus conversion of complement factor C3 by aggregates between IgG and protein A

Protein A from Staphylococcus aureus conversion of complement factor C3 by aggregates between IgG and protein A

Studies in vivo and in vitro on an abnormality in the metabolism of C3 in a patient with increased susceptibility to infection.

In a patient with increased susceptibility to infection, lowered serum C3 concentration, and continuously circulating C3b, it was shown that purified (125)I-labeled C3 was converted to labeled C3b shortly after intravenous administration. The fractional catabolic rate of C3 was approximately five times normal at 10% of the plasma pool per hr. The synthesis rate and pool distribution of C3 were normal. Despite this evidence of C3 instability in vivo, no accelerated inactivation of C3 was found in vitro. Similarly, no free proteolytic activity could be detected in the patient's serum, and serum concentrations of known protease inhibitors were normal.Complement-mediated functions, which were markedly deficient in the patient's serum, could be restored partially or completely by the addition of a 5-6S heat-labile beta pseudoglobulin from normal serum. The C3 proinactivator, which has these physicochemical characteristics, was also shown to be either absent or nonfunctional in the patient's serum. An unidentified 6S beta pseudoglobulin to which a monospecific antiserum was available was not detectable in the patient's serum. This last protein appeared not to be a complement component, nor was it the C3 inactivator or proinactivator. Finally, the substance or substances necessary for the conversion of C3b to C3c were missing from the patient's serum. The administration of 500 ml of normal plasma to the patient corrected all of his abnormalities partially or completely for as long as 17 days. The changes in C3 were dramatic; serum concentration rose from 8 to 70 mg/100 ml, and C3b could no longer be detected. A second metabolic study during this normalization period showed a decrease in fractional catabolic rate toward normal. The patient's histamine excretion was constantly elevated but increased further after a warm shower and after receiving normal plasma; at both times he had urticaria. These observations were consistent with the endogenous production of C3a and the resulting histamine release from mast cells. The inactivating mechanism for C3a was apparently intact in the patient's serum. The difference in the electrophoretic mobilities of C3b and C3c was shown as well as the electrophoretic heterogeneity of C3c. Suggestive evidence was also presented that the form of C3 with an activated combining site for red cells, previously postulated by others, is a transient C3 conversion product with an electrophoretic mobility slower than that of C3 on agarose electrophoresis.

Metabolic control in Acinetobacter sp. I. Effect of C4 versus C2 and C3 substrates on isocitrate lyase synthesis.

The comparative effects of various substrates serving as sole carbon and energy source or as a supplemental nutrient on the synthesis of isocitrate lyase by a species of Acinetobacter have been investigated. Previous work has shown that succinate, as carbon source, allows some late, limited induction of enzyme synthesis. No increase in synthesis is seen above the basal level, however, in cultures growing in a medium containing L-malate as a sole carbon source. The addition of acetate to cultures growing in media containing either of the C4 intermediates results in rapid enzyme induction. Further, Acinetobacter grows very well in pyruvate medium and isocitrate lyase is synthesized to a significant extent, indicating that the glyoxylate cycle is acting anaplerotically under these conditions. Phosphoenolpyruvate synthetase activity has been demonstrated in this organism; levels comparable to those observed in Escherichia coli have been detected; the levels of NAD- and NADP-linked "malic enzyme" and phosphoenolpyruvate carboxykinase, enzymes functioning in C4 to C3 conversion, do not fluctuate with the various carbon sources tested; i.e. no correlation between the in vitro specific activity of these enzymes and the levels of isocitrate lyase activity may be made. All of the data are consistent with the hypothesis that, in this aerobic organism, as opposed to the facultative E. coli, the C4 intermediates of the tricarboxylic acid cycle may be more direct "coarse" control metabolites regulating the rate of the glyoxylate cycle.