Academic literature on the topic 'C3 complement deposition'

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2·06 μg/ml (±0·18, SEM), 0·57% of the mean value found in serum (360 μg/ml). This proportion was comparable to that of serum albumin (0·63% of serum value) but less than the proportion of C3 in milk (2·71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 °C for 3 min or 50 °C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 °C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

Background. This study aimed to investigate the clinical significance of complement 3 (C3) deposition in renal tubules of children with primary nephrotic syndrome (PNS). Methods. The clinical and pathological characteristics of PNS were retrospectively reviewed in 99 PNS pediatric patients, who were divided into the C3 deposition and the non-C3 deposition groups. Results. A total of 39 patients (39.39%) had renal tubule C3 deposition. In the C3 deposition group, the ratios of urine N-acetylglucosaminidase/creatinine (UNAG/Cr), urine β2 microglobulin/creatinine (Uβ2MG/Cr), and urine transferrin/creatinine (UTRF/Cr) were significantly higher than those of the non-C3 deposition group. The patients of the C3 deposition group had lower serum total protein and albumin, higher cholesterol and D-dimer (DD), lower proportion of CD3+CD8+ cells, and higher proportion of CD19+CD23+ cells. The number of the patients with interstitial fibrosis, renal cell vacuolar degeneration, renal tubular immunoglobulin deposition, and severe tubulointerstitial injury in the C3 deposition group was higher than that of the non-C3 deposition group. The C3 deposition intensity was positively correlated with the number of recurrences. Conclusion. PNS pediatric patients with C3 deposition in renal tubules have more severe disease condition, tubulointerstitial injury, and recurrence suggesting a worse long-term prognosis.

ABSTRACT Our previous studies showed that Blastomyces dermatitidis yeast activates the human complement system, leading to deposition of opsonic complement fragments onto the yeast surface. This report examines the influence of altered surface expression of glucan or BAD1 protein (formerly WI-1) on the yeast's ability to activate and bind C3. Compared to the wild type, a glucan-deficient mutant yeast delayed initiation of C3 deposition and reduced C3-binding capacity by 50%. Linkage of baker's-yeast β-glucan to the glucan-deficient yeast restored initial C3 deposition kinetics to the wild-type level and partially restored C3-binding capacity, suggesting that β-glucan is an initiator of complement activation and a C3 acceptor. The role of BAD1 in B. dermatitidis yeast-complement interaction was also assessed.BAD1 knockout yeast initiated faster C3 deposition and increased C3-binding capacity compared to the wild-type yeast or aBAD1-reconstituted yeast, suggesting either a lack of an intrinsic ability in BAD1 or an inhibitory role of BAD1 in complement activation and binding. However, both complement activation and the capacity for C3 binding by the wild-type yeast were enhanced in normal human serum supplemented with an anti-BAD1 monoclonal antibody (MAb) or in immune sera from blastomycosis patients. Microscopic analysis revealed that more initial C3-binding sites were formed on yeast in the presence of both naturally occurring complement initiators and exogenous anti-BAD1 MAb, suggesting that anti-BAD1 antibody enhanced the ability of B. dermatitidis yeast to interact with the host complement system. Thus, glucan and BAD1 have distinctly different regulatory effects on complement activation by B. dermatitidis.

ABSTRACT Streptococcus pneumoniae infection is a frequent cause of pneumonia, otitis media, meningitis, and septicemia. Pneumococcal surface protein A (PspA) is an important virulence factor on the pathogen surface, and it is known to interfere with complement activation. In this study, flow cytometry was used to study the effects of PspA and antibodies to PspA on the deposition of complement C3 on the surface of a capsular type 3 strain, WU2, and its PspA− mutant, JY1119. Using naive mouse serum as a complement source, measurable deposition of C3 was observed within 4 min on PspA− pneumococci, and the amount of surface-bound C3 accumulated rapidly as the amount of serum was increased. In contrast, very little C3 was deposited on the PspA+ strain. In nonimmune mouse serum, the classical pathway was the dominant activation pathway triggered by PspA− pneumococci. Accordingly, EGTA blocked almost all of the complement activation. Moreover, a significant amount of C3 was still deposited on the PspA− strain when serum from factor B-deficient mice was used. This deposition was not observed on the PspA+ pneumococci, indicating that PspA may inhibit complement deposition via the classical pathway. Furthermore, under the conditions we tested, PspA also inhibited C3 deposition when the classical pathway was initiated by antibodies to capsular polysaccharide. Antibodies to PspA could overcome the anticomplementary effect of PspA, allowing for increased complement activation and C3 deposition onto PspA+ bacteria.

Abstract Background Complement activation has been highlighted in immunoglobulin (Ig) A nephropathy pathogenesis. However, whether the complement system can affect the downstream phenotype of IgA nephropathy remains unknown. Herein, we investigated the association of mesangial C3 deposition with the Oxford classification and their joint effects on worsening kidney function. Methods We investigated 453 patients with biopsy-proven IgA nephropathy. C3 deposition was defined as an immunofluorescence intensity of C3 ≥2+ within the mesangium. The subjects were classified according to the combination of C3 deposition and Oxford classification lesions. The primary endpoint was a composite of ≥30% decline in the estimated glomerular filtration rate or an increase in proteinuria ≥3.5 g/g during follow-up. Results Among the Oxford classification lesions, mesangial hypercellularity (M1), segmental glomerulosclerosis (S1) and tubulointerstitial fibrosis (T1–2) and crescentic lesion significantly correlated with C3 deposition. During a median follow-up of 33.0 months, the primary endpoint occurred more in patients with M1, S1, T1–2 and mesangial C3 deposition than in those without. In individual multivariable-adjusted Cox analyses, the presence of M1, S1, T1–2 and C3 deposition was significantly associated with higher risk of reaching primary endpoint. In the combined analyses of C3 deposition and the Oxford classification lesions, the hazard ratios for the composite outcome were significantly higher in the presence of C3/M1, C3/S1 and C3/crescent than in the presence of each lesion alone. Conclusions Complement deposition can strengthen the significance of the Oxford classification, and the presence of both components portends a poorer prognosis in IgA nephropathy.

AbstractAtypical hemolytic uremic syndrome (aHUS) may be associated with mutations in the C-terminal of factor H (FH). FH binds to platelets via the C-terminal as previously shown using a construct consisting of short consensus repeats (SCRs) 15 to 20. A total of 4 FH mutations, in SCR15 (C870R) and SCR20 (V1168E, E1198K, and E1198Stop) in patients with aHUS, were studied regarding their ability to allow complement activation on platelet surfaces. Purified FH-E1198Stop mutant exhibited reduced binding to normal washed platelets compared with normal FH, detected by flow cytometry. Washed platelets taken from the 4 patients with aHUS during remission exhibited C3 and C9 deposition, as well as CD40-ligand (CD40L) expression indicating platelet activation. Combining patient serum/plasma with normal washed platelets led to C3 and C9 deposition, CD40L and CD62P expression, aggregate formation, and generation of tissue factor-expressing microparticles. Complement deposition and platelet activation were reduced when normal FH was preincubated with platelets and were minimal when using normal serum. The purified FH-E1198Stop mutant added to FH-deficient plasma (complemented with C3) allowed considerable C3 deposition on washed platelets, in comparison to normal FH. In summary, mutated FH enables complement activation on the surface of platelets and their activation, which may contribute to the development of thrombocytopenia in aHUS.

Abstract Tumor cells may inhibit the induction of a complement-mediated inflammatory response through overexpression of membrane-bound regulators of complement activation. Therefore, it is of interest to determine the most efficient approach to block these membrane-bound complement regulators on tumor cells. In the present study, we first generated a bispecific mAb directed against both CD55, using the functional blocking mAb MBC1, and the highly expressed HLA class I molecule as a model for a tumor-associated Ag, using the mAb W6/32. Tumor cells opsonized with bispecific mAb W6/32*MBC1, then exposed to complement and subsequently stained for C3 deposition, were assessed by flow cytometric analysis. We found that opsonization with W6/32*MBC1 resulted in a 92% enhancement of C3 deposition on renal tumor cells as compared with opsonization with W6/32 alone and a 17% enhancement of the C3 deposition as compared with incubation with a mixture of both parental mAb. Based on these results, we developed a bispecific mAb recognizing both CD55 and the relatively low expressed renal tumor-associated Ag G250. Increasing concentrations of the bispecific mAb G250*MBC1 resulted in a 25 to 400% increase in C3 deposition on renal tumor cells as compared with C3 deposition in the presence of the parental mAb G250 alone. G250*MBC1 enhanced C3 deposition by 21% in comparison with a mixture of both parentals. Furthermore, opsonization of tumor cells with G250*MBC1 rendered these cells more sensitive to complement-mediated lysis. In conclusion, the bispecific mAb G250*MBC1 induces deposition of C3 and tumor cell lysis more efficiently than G250 alone.

ABSTRACT The polysaccharide capsule of Cryptococcus neoformans is a powerful activator of the complement system. The goal of the present study was to assess serum and cellular variables that influence the sites for C3 binding within the capsular matrix. Confocal microscopy using fluorophore-labeled polyclonal anti-C3 and anticapsular monoclonal antibodies and rosetting of fluorescent microspheres coated with anti-C3 were used to identify sites of C3 binding relative to the capsular edge. The results showed that the source of serum was a major variable influencing localization of C3. C3 bound at or very near the capsular edge in the case of human serum. C3 deposition was further from the capsule edge with guinea pig and rat sera; in the case of mouse serum, there was no binding of C3 in the outer region of the capsule. Addition of human C3 to mouse serum led to deposition of the C3 at the capsular edge, indicating that distinct properties of mouse and human C3 account for the differential localization of C3. Finally, the density of the capsular matrix was an important variable in determining sites for C3 deposition. Yeast cells with a high concentration of polysaccharide near the capsule edge supported deposition of mouse C3 at or near the capsular edge, whereas cells with a low matrix density showed deposition well beneath the edge. Taken together, these results indicate that the spatial deposition of C3 within the capsular matrix is a complex process that is influenced by the serum source and the density of the capsular matrix.

In this study, direct visual evidence for local opsonization of L. donovani by macrophage (M phi)-derived complement components was obtained using immunoelectron microscopy. C3 deposition was detected on the surface of both promastigotes and amastigotes after 20 min serum-free incubation with murine resident peritoneal M phi (RPM), followed by fixation and incubation first with specific antibody directed against C3 and then with gold-labelled protein A. Gold deposition was not observed around either form of the parasite if the anti-C3 antibody was omitted. For promastigotes, the degree of C3 deposition under serum-free conditions was comparable with that observed in the presence of an exogenous (serum) source of C3, but did not result in the same severe damage to the parasite as did the latter. Addition of sodium salicyl hydroxamate, which prevents covalent binding of C3 to activator surfaces, abrogated promastigote binding. Hence, although the anti-C3 antibody did not distinguish between native C3 and its breakdown product iC3b, these data support our earlier conclusion that promastigote binding to the CR3 of murine RPM is complement dependent. For amastigotes, gold deposition and binding to murine RPM were not eliminated by sodium salicyl hydroxamate. The presence of normal mouse serum resulted in increased gold deposition, but did not mediate either enhanced binding to M phi or damage to the amastigote. These data suggest that a proportion of C3 binding to the amastigote surface may be via noncovalent linkages, and that the C3 bound may not be in the correct form to mediate binding to CR3.

C3 glomerulopathy is a recently defined entity that encompasses a group of kidney diseases caused by abnormal control of complement activation with deposition of complement component C3 in glomeruli leading to variable glomerular inflammation. Before the recognition of the unique pathogenesis of these cases, they were variably classified according to their morphological features. C3 glomerulopathy accounts for roughly 1% of all renal biopsies. Clear definition of this entity has allowed a better understanding of its pathogenesis and clinical course and is likely to lead to the design of rational therapies over the next few years.

Invasive disease caused by capsular group B Neisseria meningitidis (MenB) is life threating disease causing hundred thousands of deaths every year, still remaining an unmet medical need in many countries. Although disease can be observed at all age groups, infants and adolescents are the most at risk populations showing the highest incidence in case numbers. Since the MenB capsule was not-immunogenic the development of a MenB vaccine which makes the use of other antigens becomes necessary. 4CMenB is a multicomponent vaccine against serogroup B N. meningitidis composed by three major protein antigens, factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA), combined with outer membrane vesicles (OMVs) from the New-Zealand epidemic strain (NZ98/254). Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein expressed by all N. meningitidis strains analyzed so far and is composed of two major domains, a highly variable amino-terminal (N-term) domain which anchors the protein on the bacterial outer membrane through the lipobox motif, and a highly conserved carboxyl-terminal (C-term) domain. These domains are separated by a short and quite conserved Arginine-rich (Arg-rich) motif which has been reported to be involved in different mechanisms that mediate meningococci adhesion, infection and survival within the host’s blood stream. NHBA is susceptible to cleavage by NalP, a bacterial protease which has its cleavage site upstream of the arginine region. Moreover human proteases such as human lactoferrin (hLf) and kallikrein are able to process NHBA downstream the the Arg-rich region. Both bacterial and human proteases-mediated cleavage releases the C-term of NHBA in the supernatant, while the N-term of the protein remains anchored on the bacterial surface. NalP cleavage did not impact SBA titers elicited by anti-NHBA antibodies but little is known about the impact that host’s proteases have on bactericidal titers. Based on sequence analysis it has been reported that NHBA has two major alleles, the so called “short” and “long” variants, which differentiate by the presence or absence of a 190 bp long fragment. Despite its sequence variability, NHBA is able to induce a robust and broad immune response against meningococcal strains expressing vaccine homologous and heterologous variants. Although anti-NHBA antibodies are able to induce bacterial killing when tested in serum bactericidal activity assay (SBA), the regions involved in eliciting cross protective immune response remain still unknown. Aims of this study were to use monoclonal antibodies (mAbs) raised against the NHBA vaccine variant peptide 2 (NHBAp2) to (i) map the NHBA regions involved in eliciting the functional response, (ii) test their ability to induce cross protection against strains expressing epidemiologically relevant homologous and heterologous NHBA variants, and (iii) investigate the molecular mechanism of NHBA-mediated bactericidal activity. To this end we used a panel of anti-NHBA mAbs selected to recognize different regions of the protein. Our results showed that only anti-N-term mAbs were able to induce killing of bacterial strains expressing the homologous NHBAp2 and closely related heterologous NHBA variants. Synergy between monoclonal antibodies targeting the N-term and the C-term of NHBA resulted in a significant increase of bactericidal titers but cross protection remained restricted to closely phylogenetic NHBA variants. Anti C-term mAbs were not able to induce SBA activity when tested individually, but surprisingly they became bactericidal when tested in combination. Moreover they were able to induce full cross protection against a panel of strains expressing phylogenetically distant heterologous NHBA variants. Our results suggest that the partial release of the NHBA C-terminal portion upon NalP and serum proteases could explain why anti-C-term mAbs are not able to induce complement mediated bactericidal killing when tested individually. However, the simultaneous binding of C-term mapping mAbs on the same NHBA molecule can induce the formation of a very stable ternary complex that probably allows a more efficient C1q engagement and C3 deposition, thus leading to the observed co-operative bactericidal activity. These results suggest that synergy between anti-NHBA antibodies is at the basis of the mechanism of NHBA-induced bactericidal activity, which could explain the robust and cross-protective immune response elicited by anti-NHBA polyclonal antibodies following immunization. Collectively, the body of experimental data suggests that both domains of NHBA are required to elicit complement mediated bactericidal activity against strains expressing the vaccine homologous and heterologous NHBA variants.

Membranoproliferative glomerulonephritis (MPGN) is synonymous with mesangiocapillary glomerulonephritis and refers to light microscopic appearances of a kidney biopsy in which there are increased mesangial cells and matrix with thickening of the glomerular capillary walls, often with a double contour appearance. MPGN represents morphological appearances caused by a wide range of diseases, most of which are systemic and involve activation of the immune system. It commonly presents as nephrotic syndrome, alternatively with proteinuria, haematuria, and varying degrees of hypertension and renal dysfunction. MPGN was historically characterized into types 1–3 according to the location of immune deposits, but a more useful classification is by whether the underlying disorder results in prominent glomerular immunoglobulin deposition (with secondary complement deposited) or if there is glomerular complement, but scanty or no immunoglobulin deposited. The immunoglobulin group includes MPGN caused by infections, autoimmunity, cryoglobulinaemia, and paraprotein production. The complement group (C3 glomerulopathy) includes dense deposit disease and other complement disorders. Similar light microscopic appearances without deposition of immunoglobulin or complement are sometimes seen in patients with chronic thrombotic microangiopathies. Management and prognosis depend on aetiology.

Antiglomerular basement membrane disease (anti-GBM disease, also known as Goodpasture’s disease) is a rare autoimmune disease caused by pathogenic autoantibodies directed against the noncollagenous, C-terminal domain of the α‎-3 chain of type IV collagen (α‎3(IV)NC1). Immunohistology is characteristic, with linear deposition of IgG (sometimes with IgA or IgM) and complement C3 along the GBMs....

The term immunoglobulin A nephropathy (IgA nephropathy or IgA N) refers to a primary glomerular disease characterized by the dominant or co-dominant, diffuse, and generalized mesangial deposition of IgA, often accompanied by deposition of IgG and the C3 component of complement in a similar distribution (Donadio and Grande, 2004; Barratt and Feehally, 2005; Tomino, 2007; Glassock, 2008; Lai, 2008). In the past, it has also been referred to as Berger's disease, to signify the senior author of the original publication describing the disorder that first appeared more than 4 decades ago in September of 1968 (Berger and Hinglais, 1968). IgA N is most likely the commonest primary glomerular disease in the developed world (D’Amico, 1987). The disease is characterized principally by episodic glomerular hematuria often with persistent proteinuria of a variable degree. It usually runs an indolent course, but may lead to end-stage renal disease (ESRD) in about 30–50% of cases after 25 years or more of follow-up.

Platelets are routinely stored for transfusion at room temperature in autologous, citrated plasma. We have demonstrated previously that these conditions do not completely block activation of plasma enzyme systems, as indicated by generation of thrombin activity (Vox Sanguinis, JL1:192,1986). Here, we demonstrate the conversion of large amounts of complement factor C3 during storage of citrated PC by using radioimmunoassay quantitation of the activation peptide C3a des-Arg (Upjohn Diagnostics). Supernatant samples from stored PC and from citrated platelet-poor plasma (PPP) stored under the same conditions showed a rapid linear increase in C3a levels over time with no significant difference (paired t-test, p<0.5) between PC and PPP (see table). The values at Day 10 represent conversion of approximately 11% of the native C3. Possible effects on stored platelets of C3 conversion in the surrounding plasma include:activation of platelets by C3a des-Arg (M.Polley and R. Nachman; J.Exp.Med. 158:603, 1983) and deposition of C3b on the cell surface as "innocent bystanders" (A. Salama and C. Mueller-Eckhardt; Transfusion 25:528,1985).In contrast, <10 ng/mL C5a was found in all samples tested, representing less than 0.2%conversion of C5a.Nephelometricassay of native C5 levels in PC samples showed a slight but significant difference by a paired t-test (p=0.04) between fresh PC (mean=117 ug/mL±12.0, n=6)and P stored for 10 days(nean=108 ug/mL±9.7). Nochange in C5 levels was observed in stored PPP (106 ug/mL to 107 ug/mL). Radiolabelled monoclonal antibodies to C3 fragments showed less than 600 molecules bound per platelet. This study demonstrates for the first time the extent of complement activation in stored platelet concentrates.

Paroxysmal nocturnal hemoglobinuria (PNH)-is frequently complicated by thrombosis. It has been suggested that the abnormal interactions of PNH platelets with complement contribute to thrombosis. Using purified complement proteins, we have previously demonstrated that the platelets from some patients with PNH do not demonstrate elevated activity of C3bBb, the alternative pathway C3 amplification enzyme complex, even though they lack the C3bBb regulatory protein, decay accelerating factor (DAF). As measured by fluorescence flow cytometry, washed platelets from both normal donors and PNH patients released the fluid phase C3bBb regulatory protein, factor H, in response to the deposition of purified complement proteins. Platelet factor H was localized to the alpha granules by immunocytochemical techniques. A quantitative radioimmunoassay demonstrated that normal platelets released 54 ± 6 ng factor H/108 platelets in response to thrombin stimulation. PNH platelets contained less factor H (22 ±7 ng/108 platelets) than normal platelets. Thrombin stimulated platelets from patients with elevated C3bBb activity released less than half of the factor H measured in detergent extracts. However, thrombin stimulated platelets from PNH patients exhibiting normal C3bBb activity released nearly all their factor H. The release of factor H from normal platelets was blocked by treating the platelets with metabolic inhibitors. In the absence of factor H release, the activity of the C3bBb complex increased three-fold. In addition, the number of molecules of 1251-factor B bound per C3b increased from 0.40 to 0.92 when factor H release was blocked. The inhibition of DAF by anti-DAF had no effect on the activity of C3bBb if factor H could be released from the platelets. However, when factor H release was blocked by treatment with metabolic inhibitors, the inhibition of DAF by anti-DAF increased the activity of C3bBb by 40%. Therefore, in the absence of DAF, platelets can regulate complement activation by the alternative pathway via the release of platelet factor H. Since factor H is an alpha granule protein, platelet release in the presence of activated complement may contribute to the occurrence of thrombosis.