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PubMed2005

Baculovirus GP64-pseudotyped HIV-based lentivirus vectors are stabilized against complement inactivation by codisplay of decay accelerating factor (DAF) or of a GP64-DAF fusion protein.

The eventual development of efficient gene transfer vectors for in vivo gene delivery will require the development of a number of important new technologies such as stabilization of vectors against protective mechanisms that destroy or otherwise inactivate foreign infectious agents like gene transfer vectors. It is known that the baculovirus envelope protein GP64 of Autographa californica nucleopolyhedrovirus can efficiently pseudotype lentivirus vectors and that modified forms of the baculovirus envelope protein GP64 can also assemble efficiently into baculovirus particles to display functional foreign proteins on the baculovirus surface. In the present study we have combined these techniques to prepare HIV-based lentivirus vectors pseudotyped with GP64 envelope protein and coexpressing a fusion protein of GP64 with the complement-regulatory, decay accelerating factor (DAF, CD55). In addition, we have also prepared GP64-pseudotyped vectors in the presence of a DAF expression plasmid to allow the incorporation of DAF protein into viral particles. Our results demonstrate both the efficient expression and the high-titer production of GP64/GP64-DAF and GP64/DAF-pseudotyped particles and their stability against inactivation by human and nonhuman primate serum.

CD55 Antigens↗
PubMed1982

Complement activation in chronic liver disease.

Patients with HBsAg positive chronic active liver disease (CALD) and primary biliary cirrhosis (PBC) exhibit increased C3d concentrations and changes in the serum concentrations of the complement components consistent with activation of the classical and alternative pathways. In these patients the concentrations of the regulatory proteins, C3b inactivator (C3bINA) and beta IH globulin, are normal. Patients with HBsAg negative CALD and alcohol induced liver disease (ALD) exhibit no evidence of an increased level of complement system activation. In these patients diminished serum concentrations of complement components appear to be related to diminished hepatic synthetic function. C4 synthesis may be specifically reduced in autoimmune chronic active liver disease.

Complement Activation↗
PubMed1990

Inhibition of the alternative C3 convertase and classical C5 convertase of complement by group A streptococcal M protein.

When Streptococcus pyogenes group A type 3 strain C203 (M+) and its M-protein-lacking derivative, strain C203S (M-), were treated with normal human serum in the presence of magnesium-EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], virulent M+ bacteria bound only 10 to 30% as much C3 and factors B and P as did avirulent M- bacteria. After treatment of M+ bacteria with trypsin, which inactivates M protein, their binding of these substances was similar to that of M- bacteria. Pretreatment of M+ bacteria with the Fab fragment of rabbit immunoglobulin G anti-M antibody also increased their binding of C3 in the absence of Ca2+. Therefore, M protein inhibits the alternative C3 convertase. In contrast, in the presence of Ca2+ and Mg2+, M+ bacteria bound 75% as much C3 as M- bacteria. This binding was mostly mediated by classical pathway activation, because M+ bacteria bound much smaller amounts of factors B and P than did M- bacteria but consumed amounts of C4 and C2 comparable to those consumed by M- bacteria. On the other hand, the amount of C5 bound to M+ bacteria was much less than that bound to M- bacteria, and the consumption of C5 and C8 by M+ bacteria was also much less than that by M- bacteria. Therefore, M protein does not inhibit the classical C3 convertase but does inhibit the classical C5 convertase. When M+ and M- streptococci were incubated with normal human serum containing radiolabeled C3 in the presence of Ca2+ and Mg2+, more than 85% of the C3 bound to either type of streptococcus was extractable by sodium dodecyl sulfate and alkali treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the C3 extracted from both strains showed that it was mostly C3b and iC3b. The proportions of C3b and iC3b, respectively, were 7.5 and 71.9% on M+ bacteria and 18.9 and 58.4% on M- bacteria. These results support and extend previous findings that the antiphagocytic activity of streptococcal M protein may be due to complement inhibition mediated by the binding of factor H.

Animals↗
PubMed1989

FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.

We have previously found low levels of C1 and C4 INH in the sera of chronic lymphocytic leukaemia (CLL) patients. Hypocomplementaemia was supposed to be the consequence of a permanent activation of the classical pathway. We have compared the levels of C1 INH-C1rC1s and C1q-FN complexes in the sera of 95 CLL patients and 100 healthy controls, because these complexes are known to be formed in the early stage of classical pathway activation. A significant increase in the level of both types of complexes was found in sera of CLL patients as compared to the controls. These findings support the assumption that the classical complement pathway is activated in the patients with CLL.

Antigen-Antibody Complex↗
PubMed2006

Structure-function mapping of BbCRASP-1, the key complement factor H and FHL-1 binding protein of Borrelia burgdorferi.

Borrelia burgdorferi, a spirochaete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease, the most frequent vector-borne disease in Eurasia and North America. Sporadically Lyme disease develops into a chronic, multisystemic disorder. Serum-resistant B. burgdorferi strains bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochaete surface. This binding is dependent on the expression of proteins termed complement-regulator acquiring surface proteins (CRASPs). The atomic structure of BbCRASP-1, the key FHL-1/FH-binding protein of B. burgdorferi, has recently been determined. Our analysis indicates that its protein topology apparently evolved to provide a high affinity interaction site for FH/FHL-1 and leads to an atomic-level hypothesis for the functioning of BbCRASP-1. This work demonstrates that pathogens interact with complement regulators in ways that are distinct from the mechanisms used by the host and are thus obvious targets for drug design.

Amino Acid Sequence↗
PubMed1996

Inflammatory reaction and capillary leak syndrome related to cardiopulmonary bypass in neonates undergoing cardiac operations.

We studied the inflammatory reaction related to cardiopulmonary bypass in 24 neonates (median age 6 days) undergoing the arterial switch operation for simple transposition of the great arteries, with respect to the development of postoperative capillary leak syndrome. Complement proteins, leukocyte count, tumor necrosis factor-alpha, and histamine levels were determined before, during, and after cardiopulmonary bypass. Additionally, protein movement from the intravascular into the extravascular space during cardiopulmonary bypass was assessed by the measurement of plasma concentrations of proteins with molecular weights ranging from 21,200 to 718,000. Capillary leak syndrome developed in 13 of the 24 neonates. Patients with capillary leak syndrome, as compared with those without, had preoperatively higher C5a levels (C5a, 3.0 +/- 0.6 microgram/L vs 0.9 +/- 0.2 microgram/L) (mean +/- standard error of the mean) (p < 0.05) and higher leukocyte counts (leukocytes, 17.9 +/- 2.1 X 10(3) cells/ml versus 11.7 +/- 0.8 X 10(3) cells/ml) (p < 0.05), suggesting in these neonates a preoperative inflammatory state. Preoperative clinical and operative data were identical in both patient groups. Before cardiopulmonary bypass, serum protein concentrations were similar in all patients. Ten minutes after institution of cardiopulmonary bypass, protein concentrations fell to significantly lower values in patients with capillary leak syndrome than in those without: albumin (19% +/- 1.5% vs 30% +/- 6% of the prebypass value, p < 0.05), immunoglobulin G (17% +/- 1.5% vs 29% +/- 5.5%, p < 0.001), and alpha 2-macroglobulin (15% +/- 1.2% vs 25% +/- 4%, p < 0.02). During cardiopulmonary bypass, albumin concentrations remained significantly lower in patients with capillary leak syndrome than in those without, whereas hematocrit values were similar in both groups. During cardiopulmonary bypass, patients with capillary leak syndrome also had lower concentrations of complement proteins C3 and C4 but not C1 inhibitor. C3d/C3 ratio and C5a levels were similar in both patient groups. In contrast, histamine liberation during cardiopulmonary bypass was significantly more pronounced in patients with capillary leak syndrome than in those without (725.2 +/- 396.7 pg/ml vs -54.1 +/- 58.4 pg/ml, p < 0.05). Tumor necrosis factor-alpha levels after protamine administration were also significantly higher in patients with capillary leak syndrome (38.1 +/- 10.0 pg/ml vs 15.3 +/- 3.4 pg/ml, p < 0.05). Leukocyte count during and after cardiopulmonary bypass was similar in both patient groups. This study demonstrates increased protein leakage as early as 10 minutes after initiation of.

Acute-Phase Reaction↗
PubMed2000

Different regulation of factor H and FHL-1/reconectin by inflammatory mediators and expression of the two proteins in rheumatoid arthritis (RA).

Factor H and the FHL-1/reconectin protein are two human plasma proteins that act as important regulators of the alternative complement pathway. Each protein is encoded by a unique transcript, but both mRNAs are derived from the factor H gene by means of alternative processing. In order to address potential functional differences between the two proteins we analysed their expression in hepatic and non-hepatic cells and studied their regulation by inflammatory mediators. We demonstrate that factor H and FHL-1/reconectin transcripts which are regulated by the same gene promoter and are initiated at the same transcription start site are differently expressed. Expression of the molecules is induced and regulated by the inflammatory mediators interferon-gamma (IFN-gamma) and the anti-inflammatory glucocorticoid dexamethasone. Both factor H and FHL-1/reconectin are expressed and secreted by synovial fibroblasts and are present in synovial fluid derived from patients suffering from rheumatoid or reactive arthritis. The local synthesis in synovial fibroblasts and their induction by IFN-gamma and dexamethasone, but not by tumour necrosis factor-alpha, suggests for each of the two complement regulators a protective role in RA.

Alternative Splicing↗
PubMed1982

Double immunofluorescence studies of immunoglobulins, complement components and their control proteins in patients with IgA nephropathy.

A study on double immunofluorescent staining of immunoglobulins, complement components, and their control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy. These biopsy specimens were stained with anti sera to human C1q, C4-binding protein, and beta 1H globulin by indirect immunofluorescent staining. These samples were then stained with rhodamine-conjugated anti human IgG, IgM, and IgA. Although C1q and C4-binding protein do not combine with IgA, they ubiquitously combine with IgG and/or IgM. beta 1H globulin also combine with IgG, IgM and/or IgA. It was demonstrated that the complement system activated in IgA nephropathy was via both alternative and classical pathways. The presence of C4-binding protein in glomeruli appeared to be a more sensitive indicator of classical pathway activation than the presence of C4.

Carrier Proteins↗
PubMed1988

Inhibition of C1q functions by RHP, a protein elevated in sera from patients with rheumatoid arthritis.

We have previously shown that serum levels of C1q, unbound to C1r X C1s, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the C1q-anti C1q precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of C1q, disaggregates C1, and inhibits the ability of C1q bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

Adsorption↗
PubMed1989

Interaction of Neisseria gonorrhoeae with classical complement components, C1-inhibitor, and a monoclonal antibody directed against the Neisserial H.8 antigen.

Strains of Neisseria gonorrhoeae were used to evaluate bactericidal and opsonic properties of McAb 10 directed against the Neisserial outer membrane antigen, H.8. Gonococci were either serum resistant in the absence but serum sensitive in the presence, of McAb 10, or serum sensitive or serum resistant regardless of the presence of McAb 10. Strain JS3, which fell in the former category, was used in subsequent studies. C1 zymogen formed by reassociation of isolated C1 subunits was not directly activated by JS3 in the presence or absence of C1-inhibitor. JS3 thus was unable to directly activate the classical pathway independently of antibody. When purified classical pathway components were used to deposit C3 on JS3 in the absence of serum regulatory proteins or antibodies, added C1-inhibitor reduced C3 binding to background levels. When McAb 10 was present, C3 binding was unaffected by C1-inhibitor. Covalently bound, large molecular weight C3 alpha-chain-gonococcal complexes were disbanded by methylamine release of ester linkages. Released 125I-C3 migrated as C3b without degradation by gonococcal proteases. Purified classical components alone or McAb 10 alone facilitated JS3 killing by neutrophils; when combined, the two provided maximal killing. Levels of McAb 10 that only slightly increase C3 deposition on JS3 are bactericidal in serum and maximally opsonic in combination with purified classical pathway components.

Antibodies, Monoclonal↗
PubMed1987

Concentrations of serum proteins and erythrocyte sedimentation rate in patients with different histological types of gastric carcinoma.

ESR and serum concentrations of IgG, IgA, IgM, C3, C4, C1-INH and CEA were quantified preoperatively in 195 patients with gastric carcinoma. The values were grouped according to the extent of disease (T1-3N0M0, T2-3N + M0, T4AnyNM0, AnyTAnyNM1) and according to the histological type of tumor (intestinal-type, diffuse and unclassifiable). The data were analysed using a two-way analysis of variance with unequal cell sizes. ESR, C4, C1-INH, IgG and CEA varied with the extent of disease. When the data were adjusted for this variation, we found that the values of ESR, C4 and CEA were different between the various histological types. The values were highest in patients with the intestinal-type tumor and lowest in those with diffuse tumor. The concentrations of IgG and C1-INH were not different between the histological types. Our results are relevant when ESR, C4 and CEA are used in the evaluation of patients with gastric carcinoma.

Blood Proteins↗
PubMed2002

Immune evasion of Borrelia burgdorferi: insufficient killing of the pathogens by complement and antibody.

The innate immune system and, in particular, the complement system play a key role in the elimination of micro-organisms after entrance in the human host. Like other pathogens, borreliae must develop strategies to inactivate host defence mechanisms. By investigating serum (NHS)-susceptibility of borreliae, we found that mainly B. afzelii isolates are serum-resistant, whereas the majority of B. burgdorferi s. s. isolates display an intermediate serum-sensitive phenotype. In contrast, B. garinii isolates are killed effectively by complement and therefore are classified as serum-sensitive. Up to now, we have identified two distinct proteins of 27.5 kDa and 20.7 kDa expressed on the outer surface of borreliae, which interact directly with FHL-1/reconectin and factor H, the two major regulators of the alternative complement pathway. These borrelial proteins are termed CRASPs (complement regulator-acquiring surface proteins). CRASPs are detectable only on serum-resistant borreliae and, accordingly, binding of FHL-1/reconectin and factor H only occur with serum-resistant borrelial isolates. We conclude from these results that the control of complement activation on the borrelial surface is due to the interaction of borrelial CRASPs with host complement regulatory proteins. Thus, CRASPs represent an important mechanism of immune evasion on the part of borrelial isolates belonging mostly to the genospecies B. afzelii. By analysing the humoral adaptive immune response of patients, we detected sera that killed NHS-resistant borreliae. Borreliacidal activity is observed most frequently with sera of patients at stage III of the disease. The killing of NHS-resistant isolates by these immune sera always requires the combination of antibodies and complement. Bactericidal activity, however, is not detected in all immune sera at the different disease stages, although specific anti-Borrelia antibodies are present according to serological test results. This observation suggests that not all borrelial antigens are able to induce a borreliacidal immune response. In an extensive analysis of 24 immune sera, we identified up to 12 borrelial antigens, including OspC, which possess the greatest potential for the induction of borreliacidal antibody. The borreliacidal potential of anti-OspC antibodies was tested directly on an OspC-expressing borrelial wild-type isolate and a corresponding variant lacking OspC. In these studies, only the wild-type isolate expressing OspC on its surface proved positive for the lytic complement complex, thereby indicating the great importance of this antigen for the control of the infection. Additional studies are required to identify further "protective" antigens among these 12 proteins, all of which are candidates for infection control according to our studies involving patient immune sera. These antigens may include the recently detected CRASPs.

Antibodies, Bacterial↗
PubMed1979

Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane.

An activity that is inhibitory to the properdin-stabilized amplification C3 convertase (C3b,Bb,P) was solubilized from human erythrocyte (E(hu)) membranes by Nonidet P-40 and purified to homogeneity. The inhibitory membrane glycoprotein had an apparent M(r) of 1-1.2x10(6) on gel filtration in the presence of Nonidet P-40. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis it presented a single stained band with an apparent M(r) of 205,000, with or without prior reduction of disulfides. The inhibitory protein of the E(hu) membrane produced a dose-related, first-order decay of C3b,Bb,P function on sheep erythrocytes (E(s)) and released (125)I-labeled Bb from these sites, indicating a mechanism of inhibition by decay-dissociation of the amplification C3 convertase. The 50% inhibitory dose of the E(hu) membrane protein was not altered by removal of sialic acid from the E(s) bearing C3b,Bb,P sites. E(hu) membrane protein also serves as a cofactor for C3b inactivator-induced cleavage of the alpha polypeptide chain of C3b. Thus, the inhibitory membrane protein can abrogate the activity of amplification convertase sites that have formed and also can prevent generation of such sites by augmenting irreversible inactivation of C3b.Discrimination between cells by the alternative complement pathway occurs after initial deposition of C3b and is related to the modulation by surface constituents of the capacity of bound C3b to function as a subunit of the amplification C3 convertase. The existence in the E(hu) membrane of a protein that can impair the functions of membrane-bound C3b and C3b,Bb,P could represent a molecular basis for preventing inappropriate self-recognition.

Binding Sites↗
PubMed1993

Activation of the complement system in baboons challenged with live Escherichia coli: correlation with mortality and evidence for a biphasic activation pattern.

Activation of the complement system was studied in baboons that were challenged with live Escherichia coli. In the group challenged with a lethal dose (n = 4), the complement activation parameters C3b/c, C4b/c, and C5b-9 increased 13, 5, and 12 times the baseline value, respectively, during the first 6 h after the E. coli infusion, whereas in the group challenged with a sublethal dose (n = 10), they increased only moderately, by 2 to 3 times the baseline value. However, in this latter group, a more pronounced activation occurred at 24 h. Subsequent experiments showed that this second phase in complement activation started at 6 h after the challenge, at which time infused microorganisms had been cleared from the circulation. The simultaneous increase in C-reactive protein with this second phase suggested an endogenous activation mechanism involving this acute-phase protein. Levels of inactivated (modified) C1 inhibitor also increased in both groups, with peak levels of 2.5 times the baseline value at 24 h in the sublethal group and of 4 times at 6 h after the challenge in the lethal group. Thus, activation of complement in this animal model for sepsis occurs in a biphasic pattern, the initial phase mediated by the bacteria and the later phase mediated by an endogenous mechanism possibly involving C-reactive protein. The differences in complement activation between animals with lethal or sublethal sepsis support the hypothesis that complement activation contributes to the lethal complications of sepsis.

Animals↗
PubMed1992

In vitro biosynthesis of complement factor I by human endothelial cells.

We have studied the secretion of the complement regulatory protein factor I by human umbilical vein endothelial cells (HUVEC). Northern and Western blot analysis and biosynthetic labeling experiments indicate that HUVEC secrete factor I at very low levels in basal conditions and that this secretion is significantly enhanced by interferon-gamma. Analysis of the proteolytic inactivation of C3b by HUVEC supernatants show that factor I is secreted in a functional form and can promote the specific proteolytic inactivation of C3b to iC3b. Together with previous studies establishing the secretion of complement factor H by HUVEC, this work demonstrates that the endothelial cell is able to secrete in its environment two complement regulatory proteins, factor I and factor H, which can mediate the degradation of C3b to iC3b. The secretion of factor I by HUVEC provides a useful in vitro model to analyze the modulation of this secretion and may be relevant to the local deposition of iC3b at the surface of the endothelium during the inflammatory reaction.

Cells, Cultured↗
PubMed2001

Immune evasion of Borrelia burgdorferi by acquisition of human complement regulators FHL-1/reconectin and Factor H.

To understand immune evasion mechanisms of Borrelia burgdorferi we compared serum-resistant B. afzelii and serum-sensitive B. garinii isolates for their capacity toacquire human complement regulators. Here we demonstrate that the two borrelial genospecies show different binding of the two important human complement regulators, FHL-1/reconectin and Factor H. All serum-resistant B. afzelii isolates bound FHL-1/reconectin and also Factor H, and all analyzed serum-sensitive B. garinii isolates showed no or a significantly lower binding activity. Using recombinant deletion mutants, the binding domains were localized to the C terminus of FHL-1/reconectin to short consensus repeats 5-7. The borrelial binding proteins were located in the surface of the bacteria as demonstrated by immunofluorescence staining of intact, serum-exposed bacteria and by enrichment of outer membrane proteins. The surface-attached complement regulators maintained complement regulatory activity as demonstrated in a cofactor assay. By ligand blotting two different borrelial binding proteins were identified that were responsible for the surface attachment of FHL-1/reconectin and Factor H. These borrelial complement regulators acquiring surface proteins (CRASP) were further characterized as either CRASP-1, a 27.5-kDa molecule which preferentially binds FHL-1/reconectin and which was present in all serum-resistant borreliae, or CRASP-2, a 20/21-kDa protein which interacts preferentially with Factor H and the expression of which was more restricted, being detected in four of the six isolates analyzed. In summary, we describe a new immune evasion mechanism of B. burgdorferi, as these bacteria acquire human complement regulators to control complement activation on their surface and to prevent formation of toxic activation products.

Adsorption↗
PubMed2001

Further characterization of complement regulator-acquiring surface proteins of Borrelia burgdorferi.

The three genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B. afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674-1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived from B. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37 degrees C compared with those cultured at 20 degrees C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.

Bacterial Proteins↗