scholarly journals Deposition of C3b and iC3b onto particulate activators of the human complement system. Quantitation with monoclonal antibodies to human C3.

1985 ◽  
Vol 161 (6) ◽  
pp. 1414-1431 ◽  
Author(s):  
S L Newman ◽  
L K Mikus
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Concentration ◽  
Human Serum ◽  
Molecular Form ◽  
Alternative Pathway ◽  
Classical Pathway ◽  
Sds Page ◽  
Predominant Form ◽  
Hemophilus Influenzae Type B ◽  
Type 3

Monoclonal antibodies were used to determine the number and molecular form of C3 bound to particulate activators of the complement (C) system by human serum. Sheep erythrocytes (E) coated with IgM (EIgM) and IgG (EIgG) were used to study activation of the classical pathway (CP). Yeast (Y), rabbit erythrocytes (ER), and five species of bacteria (Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae type 3, Streptococcus pyogenes, and Hemophilus influenzae type b) were used to study activation of the alternative pathway (AP). The deposition of C3b onto EIgM and EIgG incubated in C7-deficient human serum was dependent on the serum concentration. At all serum concentrations tested, there was complete conversion of C3b to iC3b. Kinetic analysis of C3b deposition and conversion to iC3b indicated that these events occurred almost simultaneously; the reaction was completed by 15 min. The deposition of C3 onto the AP activators ER and Y was also dependent on serum concentration, and ER, but not Y, required the presence of Mg-EGTA and thus the activation of only the AP. C3b deposition and conversion to iC3b on Y was complete in 15 min, with 82% of bound C3 converted to iC3b. For ER, maximum C3 deposition required 30 min in both the presence and absence of Mg-EGTA. However, after 1 h of incubation, 74% of bound C2 was iC3b in the absence of Mg-EGTA, compared with only 52% in the presence of Mg-EGTA. Thus, even on AP activators, a large portion of C3b may be converted to iC3b, and this conversion is probably controlled by elements on the particle's surface. Studies with the five species of bacteria yielded similar results. Approximately 3-5 X 10(4) molecules of C3 were bound per microorganism, with opsonization being completed in 30 min. Remarkably, only 16-28% of bound C3 was in the form of iC3b, even after 2 h of incubation. The presence or absence of Mg-EGTA, or the addition of purified CR1 to the reaction mixture, did not significantly effect the ratio of C3b to iC3b. Finally, SDS-PAGE and autoradiography of particle-bound 125I-C3 fragments confirmed that there was no conversion of iC3b to C3d,g or C3d. The data obtained about the opsonization of bacteria suggest that the predominant form of C3 that is encountered by inflammatory phagocytes may be C3b.

scholarly journals A role for complement receptor-like molecules in iron acquisition by Candida albicans.

1992 ◽  
Vol 175 (6) ◽  
pp. 1643-1651 ◽  
Author(s):  
M A Moors ◽  
T L Stull ◽  
K J Blank ◽  
H R Buckley ◽  
D M Mosser
Keyword(s):  
Candida Albicans ◽  
Human Serum ◽  
Iron Acquisition ◽  
Alternative Pathway ◽  
Protoporphyrin Ix ◽  
Surface Proteins ◽  
Pathogenic Potential ◽  
Opportunistic Fungal Pathogen ◽  
Iron Binding Proteins ◽  
Type 3

Candida albicans, an opportunistic fungal pathogen of humans, is dependent upon iron for growth. Consequently, human serum inhibits C. albicans growth due to the presence of high affinity iron-binding proteins that sequester serum iron, making it unavailable for use by the organism. We report that in the inhibitory environment of human serum, the growth of C. albicans can be restored by the addition of exogenous hemoglobin or heme, but not by protoporphyrin IX, the heme precursor that does not contain iron. We further report that C. albicans can utilize cell surface proteins that are homologues of the mammalian complement receptors (CR) to rosette complement-coated red blood cells (RBC) and obtain RBC-derived iron for growth. The ability of Candida to acquire RBC-derived iron under these conditions is dependent upon Candida-RBC rosetting mediated by CR-like molecules. Unopsonized RBC do not support Candida growth in serum, and restoration of Candida growth in serum by complement-opsonized RBC is inhibited by monoclonal antibodies to the human CR type 3 (CR3). In addition, activation of the human alternative pathway of complement by Candida leads to "bystander" deposition of C3 fragments on the surface of autologous, unopsonized RBC, generating the ligands necessary for Candida-RBC rosetting. These results suggest that C. albicans has evolved a unique strategy for acquiring iron from the host, which exploits the host complement system, and which may contribute to the pathogenic potential of the organism.

Variable Sensitivity of Oral Isolates of Eikenella Corrodens to Serum Bactericidal Activity: Role of Antibody

1988 ◽  
Vol 2 (2) ◽  
pp. 346-353 ◽  
Author(s):  
C. Chen ◽  
M.E. Wilson
Keyword(s):  
Human Serum ◽  
Host Defense ◽  
Potential Role ◽  
Reference Strain ◽  
Alternative Pathway ◽  
Immune Defense ◽  
Classical Pathway ◽  
Eikenella Corrodens ◽  
Gram Negative

Eikenella corrodens is a facultatively anaerobic Gram-negative bacterium which is among the predominant cultivable microflora of periodontal lesions characterized by loss of attachment level. In the present study, we examined the potential role of complement-mediated killing in host defense against this periodontopathic organism. Seven clinical isolates obtained from human subgingival plaque and one reference strain of E. corrodens were characterized with respect to (a) susceptibility to the bactericidal properties of pooled human serum and (b) the role of the classical and/or alternative pathway(s) of complement in effecting killing of sensitive strains. Six strains, including the reference strain, were found to be variably serum-sensitive, exhibiting 1-12.5% survival after two hr of incubation in the presence of 20% pooled human serum. The remaining two isolates were serum-resistant. Both serum-resistant and serum-sensitive strains consumed complement via the classical pathway in normal but not in hypogammaglobulinemic serum, thus ruling out an antibody-independent mechanism of classical pathway activation. Four of six serum-sensitive strains exhibited little or no loss of viability following incubation with serum depleted of the classical pathway component Clq. One strain which was resistant to killing by normal human serum was, nevertheless, highly susceptible to complement-mediated killing in the presence of rabbit immune serum. Two additional serum-sensitive strains were killed, albeit to a lesser extent, in Clq-depleted serum, indicative of a role of the alternative pathway in killing of some serum-sensitive strains. These results indicate a potential role for complement-mediated killing in host defense against Gram-negative periodontal bacteria such as E. corrodens. However, the ultimate contribution of this immune defense mechanism may be defined, at least in part, by the presence of a humoral response to key bacterial membrane constituents.

scholarly journals In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1120-1124 ◽  
Author(s):  
DE Stepan ◽  
RM Bartholomew ◽  
TW LeBien
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Classical Pathway ◽  
Normal Human ◽  
Human Leukemic Cells ◽  
Murine Monoclonal Antibodies

Abstract We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.

scholarly journals Impaired Opsonization with C3b and Phagocytosis of Streptococcus pneumoniae in Sera from Subjects with Defects in the Classical Complement Pathway

2008 ◽  
Vol 76 (8) ◽  
pp. 3761-3770 ◽  
Author(s):  
Jose Yuste ◽  
Ashwin Sen ◽  
Lennart Truedsson ◽  
Göran Jönsson ◽  
Liang-Seah Tay ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Human Serum ◽  
Alternative Pathway ◽  
Antibody Activity ◽  
Classical Pathway ◽  
Complement Factor ◽  
Complement Pathway ◽  
Classical Complement Pathway ◽  
C2 Deficiency ◽  
Classical Complement

ABSTRACT Results from studies using mice deficient in specific complement factors and clinical data on patients with an inherited deficiency of the classical complement pathway component C2 suggest that the classical pathway is vital for immunity to Streptococcus pneumoniae. However, the consequences of defects in classical pathway activity for opsonization with C3b and the phagocytosis of different S. pneumoniae serotypes in human serum are not known, and there has not been a systematic analysis of the abilities of sera from subjects with a C2 deficiency to opsonize S. pneumoniae. Hence, to investigate the role of the classical pathway in immunity to S. pneumoniae in more detail, flow cytometry assays of opsonization with C3b and the phagocytosis of three capsular serotypes of S. pneumoniae were performed using human sera depleted of the complement factor C1q or B or sera obtained from C2-deficient subjects. The results demonstrate that, in human serum, the classical pathway is vital for C3b-iC3b deposition onto cells of all three serotypes of S. pneumoniae and seems to be more important than the alternative pathway for phagocytosis. Compared to the results for sera from normal subjects, C3b-iC3b deposition and total anti-S. pneumoniae antibody activity levels in sera obtained from C2−/− subjects were reduced and the efficiency of phagocytosis of all three S. pneumoniae strains was impaired. Anticapsular antibody levels did not correlate with phagocytosis or C3b-iC3b deposition. These data confirm that the classical pathway is vital for complement-mediated phagocytosis of S. pneumoniae and demonstrate why subjects with a C2 deficiency have a marked increase in susceptibility to S. pneumoniae infections.

scholarly journals Characterization of Anticapsular Monoclonal Antibodies That Regulate Activation of the Complement System by theCryptococcus neoformans Capsule

1998 ◽  
Vol 66 (4) ◽  
pp. 1538-1546 ◽  
Author(s):  
Thomas R. Kozel ◽  
Bouke C. H. deJong ◽  
Matthew M. Grinsell ◽  
Randall S. MacGill ◽  
Kevin K. Wall
Keyword(s):  
Monoclonal Antibodies ◽  
Capsular Polysaccharide ◽  
Alternative Pathway ◽  
Variable Region ◽  
Group Iv ◽  
Classical Pathway ◽  
Antibody Isotype ◽  
Group Iii ◽  
Alternative Pathways ◽  
Group Ii

ABSTRACT Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.

A Novel Approach to Treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH): Both Hemolysis and C3 Deposition Are Blocked by a Monoclonal Antibody (mAb) Specific for the Alternative Pathway of Complement (APC) C3/C5 Convertase.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 157-157
Author(s):  
Ronald P Taylor ◽  
Margaret A Lindorfer ◽  
Andrew W Pawluczkowycz ◽  
Charles J Parker
Keyword(s):  
Flow Cytometry ◽  
Human Serum ◽  
Conflicts Of Interest ◽  
Degradation Products ◽  
Alternative Pathway ◽  
Complement C3 ◽  
Classical Pathway ◽  
Immune Functions ◽  
Intravascular Hemolysis

Abstract Abstract 157 In PNH, red blood cells (RBCs) lack key complement control proteins, CD55 and CD59 and are therefore sensitive to complement activation and intravascular hemolysis. These regulatory proteins function at two different steps in the complement cascade; CD55 (decay accelerating factor, DAF) controls the formation and stability of the APC C3 and C5 convertases, while CD59 (membrane inhibitor of reactive lysis, MIRL) blocks formation of the cytolytic membrane attack complex (MAC). The intravascular hemolysis of PNH can be inhibited in vivo by eculizumab, a humanized mAb that binds complement C5, thereby preventing formation of the MAC. However, PNH patients treated with eculizumab continue to manifest evidence of ongoing hemolysis as they remain anemic with an elevated reticulocyte count and low serum haptoglobin concentration, and approximately 50% of eculizumab-treated patients require transfusion. This observation is consistent with the hypothesis that, in patients treated with eculizumab, PNH RBCs undergo extravascular hemolysis as a consequence of C3 opsonization because eculizumab does not compensate for deficiency of DAF. Recent studies (Risitano et al., Blood, 2009) support this hypothesis as patients undergoing treatment with eculizumab were found to have a positive Coombs test for C3 but not IgG, and flow cytometry demonstrated C3 activation and degradation products bound to the PNH RBCs. This process appeared clinically relevant as transfusion requirement correlated with the percentage of C3 opsonized PNH RBCs. These observations suggest that blocking the APC C3/C5 convertase would be a better way to treat the hemolysis of PNH because this approach has the advantage of blocking both extravascular hemolysis by inhibiting C3 opsonization and preventing intravascular hemolysis by inhibiting MAC generation. We have developed a mAb 3E7 and its deimmunized chimeric humanized derivative H17 that specifically block the APC C3/C5 convertase by binding to a neoepitope expressed when complement C3 is activated. In vitro, 3E7/H17 prevents APC-mediated lysis of rabbit RBCs in human serum and blocks deposition of human C3 activation fragments on APC activator substrates such as zymosan (Mol Immunol, 2006; J Immunol, 2007). We now report that mAb H17/3E7 blocks lysis in acidified normal human serum (aNHS) (a process mediated by the APC) of RBCs from patients with PNH (n=5). Representative results for patients 1 and 2 are as follows: 60% and 40% of RBCs were lysed after a one hour incubation at 37°C; lysis was reduced to 10% and 6%, respectively, at 80 ug/ml of mAb H17, and to 1% lysis (both patients) at 170 ug/ml of mAb H17. We also showed that mAb H17/3E7 blocks deposition of C3 activation fragments on PNH RBCs. After lysis in aNHS, blood samples from PNH patients were probed with Al488 mAb 1H8, specific for C3b/iC3b/C3dg. Flow cytometry experiments revealed C3 fragment deposition on lysed cells corresponding to 30,500 molecules of equivalent soluble fluorochrome (MESF) compared to a background signal of 225 MESF on unlysed RBCs in the same sample. Addition of mAb H17 blocked C3 fragment deposition not only on the unlysed cells but also on the small number of recovered ghosts . Importantly, mAb H17/3E7 inhibits the APC specifically. PNH RBCs, opsonized with IgM in serum from a patient with chronic cold agglutinin disease, were lysed in NHS by the classical complement pathway, and this lysis was not inhibited by mAbH17/3E7. Together, these experiments demonstrate that both hemolysis and C3 opsonization of PNH RBCs can be inhibited by a novel mAb that specifically blocks the APC C3/C5 convertase while leaving intact the classical pathway of complement. These findings suggest an approach to therapy of PNH in which both intravascular and extravascular hemolysis can be inhibited while preserving the important immune functions of the classical pathway of complement. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Lysis of RNA tumor viruses by human serum: direct antibody-independent triggering of the classical complement pathway.

1976 ◽  
Vol 144 (4) ◽  
pp. 970-984 ◽  
Author(s):  
N R Cooper ◽  
F C Jensen ◽  
R M Welsh ◽  
M B Oldstone
Keyword(s):  
Human Serum ◽  
Defense Mechanism ◽  
Alternative Pathway ◽  
Feedback Mechanism ◽  
Classical Pathway ◽  
Classical Complement Pathway ◽  
Natural Defense ◽  
Absolute Requirement ◽  
Recognition Function

In earlier studies we found that human serum, but not serum from multiple other species, inactivated and lysed oncornaviruses from a number of diverse sources in the apparent absence of antibody. A detailed analysis of the role of the human complement (C) system in mediating this lytic process indicates that human C1q interacts directly, in the absence of immunoglobulin, with oncornaviruses. Binding of C1 via C1q in this manner leads to activation of C1r, C1s, and thus of the classical C pathway. Integrity of the classical pathway is an absolute requirement for lysis although activation of the alternative pathway considerably amplifies the amount of lysis obtained, possibly through involvement of the C3b-dependent feedback mechanism. Activation of C is accompanied by deposition of C components on the viral surface and lysis on completion of the C reaction sequence. Thus in this system, the C1q subunit of C1 subserves a specific recognition function normally associated with antibody. This ability of human serum to inactivate oncornaviruses may represent a natural defense mechanism operative in vivo which deters expression of intact oncornaviruses in human malignancies.

scholarly journals Inactivation of classical and alternative pathway-activated bactericidal activity of human serum by sodium polyanetholsulfonate

1977 ◽  
Vol 5 (3) ◽  
pp. 278-284
Author(s):  
W H Traub ◽  
I Kleber
Keyword(s):  
Human Serum ◽  
Bactericidal Activity ◽  
Alternative Pathway ◽  
Inhibitory Effect ◽  
Final Concentration ◽  
Classical Pathway ◽  
Control Strain ◽  
E Coli ◽  
Alternative Complement ◽  
And Control

Sodium polyanetholsulfonate (SPS) at a final concentration of at least 250 microng/ml (0.025%) was required for inhibition of the bactericidal activity of 80% (vol/vol) of fresh human serum against "promptly serum-sensitive" strains of Serratia marcescens and control strain Escherichia coli C, i.e., for inhibition of the classical pathway of complement activation. In contrast, SPS at 125 microng/ml (0.0125%) was sufficient for neutralization of the bactericidal activity of 80% (vol/vol) fresh human serum against "delayed serum-sensitive" strains of S. marcescens known to activate the alternative pathway of human complement. Addition of up to 500 microng of SPS per ml to 80% (vol/vol) fresh human serum failed to neutralize transferrin-mediated, "late" bacteriostasis against control strain E. coli C, an effect that was demonstrable only after prolonged, i.e., overnight, incubation of the test strain. However, this late inhibitory effect against E. coli C was not observed in SPS-treated 20% (vol/vol) fresh human serum or in 10 or 20% (vol/vol) conventionally heat-inactivated human serum. Immunoelectrophoretic examination disclosed that SPS did not precipitate transferrin from either fresh or heat-inactivated human serum. Thus, SPS, at 250 microng/ml, was demonstrated to be sufficient for the inhibition of both classical and alternative complement pathway-activated bactericidal activity of 80% (vol/vol) human serum. However, SPS at a concentration of 500 microng/ml failed to antagonize one antimicrobial system of 80% (vol/vol) human serum, namely transferrin-mediated bacteriostasis.

scholarly journals In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement

Blood ◽  
1984 ◽  
Vol 63 (5) ◽  
pp. 1120-1124
Author(s):  
DE Stepan ◽  
RM Bartholomew ◽  
TW LeBien
Keyword(s):  
Monoclonal Antibodies ◽  
Human Serum ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Target Cells ◽  
Classical Pathway ◽  
Normal Human ◽  
Human Leukemic Cells ◽  
Murine Monoclonal Antibodies

We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.

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