Complement Receptor Type Research Articles

TT30, a Novel Human Complement Inhibitor in Development for Paroxysmal Nocturnal Hemoglobinuria and Other Hemolytic Disorders, Demonstrates Red Blood Cell Surface Targeting and Retention In a Model of Complement Alternative Pathway-Mediated Hemolysis

AbstractAbstract 638Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases, especially genetically linked hemolytic disorders such as paroxysmal nocturnal hemoglobinuria (PNH) and thrombotic microangiopathy (TMA) disorders such as atypical hemolytic uremic syndrome (aHUS) and thrombotic thrombocytopenic purpura. The complement system can be activated through three unique pathways (classical, lectin/mannose and alternative). In PNH, the lack of CD55 on RBC allows CAP-initiated complement C3 activation by C3 convertases, while the lack of CD59 allows C5 activation by C5 convertase to proceed to formation of the membrane attack complex (MAC; C5b-9), resulting in intravascular hemolysis (IVH). Treatment of patients with the anti-C5 monoclonal antibody (mAb) eculizumab abrogates IVH; however, because eculizumab does not inhibit CAP activity prior to C5, covalently bound C3 fragments (C3frag) and both C3 and C5 convertases continue to accumulate on PNH red blood cells (RBCs). Clearance of PNH RBCs that are C3frag-coated by complement receptors within the reticuloendothelial system (RES) is the putative cause of continued extravascular hemolysis (EVH) in patients who receive eculizumab. Continued anemia and transfusion requirements are found in a substantial proportion of eculizumab-treated patients, and correlate with PNH RBC-bound C3frag. High levels of C5 convertases on the same cells may also contribute to intermittent escape from eculizumab control of IVH due to pharmacodynamic breakthrough. To selectively modulate CAP activity on PNH RBC and replace the CD55-mediated control of CAP activation, we developed TT30, a novel therapeutic fusion protein linking the C3frag-binding domain of human complement receptor type 2 (CR2/CD21) with the CAP inhibitory domain of human factor H (fH). TT30 delivers cell surface-targeted (via CR2) inhibition of CAP activity (via fH) and blocks the ex vivo hemolysis of PNH RBCs, while at the same time retaining the normal ability of the complement system to efficiently activate C3 through the classical and lectin pathways. We studied the mechanism of TT30 prevention of hemolysis by control of CAP activity in human serum using: 1) an in vitro model of CAP-mediated hemolysis in which rabbit RBCs are exposed to normal human serum under conditions promoting CAP activation (Mg++/EGTA) and the extent of hemolysis is quantified by measuring hemoglobin release; 2) flow cytometric phenotyping of C3frag accumulation on rabbit RBCs exposed to normal or C5-deficient human serum using mAbs specific for human iC3b (A710, Quidel) or C3d (A702, Quidel); 3) flow cytometric demonstration of TT30 binding to C3frag+ rabbit RBCs with a noncompeting mAbs against CR2 (HB5, Taligen) or fH (A255, Quidel); and 4) an in vitro model of CAP-mediated MAC formation in which human serum is exposed to an LPS-coated surface in the presence of Mg++/EGTA and CAP activation through to the MAC is quantified by detection of a neoantigen in poly-C9 by ELISA. The results demonstrate that TT30 efficiently inhibits CAP-mediated MAC formation (IC50 of 3.2 ug/ml) and hemolysis (IC50 of 50.1 ug/ml) and that both of these activities are dependent upon targeting to C3frag+ surfaces by CR2, as evidenced by complete reversal of TT30 inhibitory activity in the presence of a 2-fold molar excess of a competing anti-CR2 mAb (1048, Taligen). Rabbit RBCs were shown to become coated with C3frag in the presence of normal and C5-deficient serum and to undergo lysis with normal serum. TT30 was readily demonstrated to be bound to C3frag+ RBCs during prevention of hemolysis and to remain detectable on RBCs for at least 24 hours. The amount of bound TT30 was proportional to the accumulation of C3frags. Collectively, these results demonstrate that TT30 displays targeted control of cell surface CAP activation, with both effective and prolonged blockade of MAC formation, and dose-dependent inhibition of hemolysis. Therefore, the CAP-specific novel therapeutic TT30 has potential utility for the treatment of human complement-mediated diseases, such as PNH and aHUS, in which modulation of CAP activation is predicted to be clinically beneficial. Disclosures:Fridkis-Hareli:Taligen Therapeutics: Employment. Storek:Taligen Therapeutics: Employment. Risitano:Taligen Therapeutics: Consultancy, Research Funding. Lundberg:Taligen Therapeutics: Employment, Membership on an entity's Board of Directors or advisory committees. Horvath:Taligen Therapeutics: Employment. Holers:Taligen Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Delineation of the Complement Receptor Type 2–C3d Complex by Site-Directed Mutagenesis and Molecular Docking

The interactions between the complement receptor type 2 (CR2) and the C3 complement fragments C3d, C3dg, and iC3b are essential for the initiation of a normal immune response. A crystal-derived structure of the two N-terminal short consensus repeat (SCR1-2) domains of CR2 in complex with C3d has previously been elucidated. However, a number of biochemical and biophysical studies targeting both CR2 and C3d appear to be in conflict with these structural data. Previous mutagenesis and heteronuclear NMR spectroscopy studies directed toward the C3d-binding site on CR2 have indicated that the CR2–C3d cocrystal structure may represent an encounter/intermediate or nonphysiological complex. With regard to the CR2-binding site on C3d, mutagenesis studies by Isenman and coworkers [Isenman, D. E., Leung, E., Mackay, J. D., Bagby, S. & van den Elsen, J. M. H. (2010). Mutational analyses reveal that the staphylococcal immune evasion molecule Sbi and complement receptor 2 (CR2) share overlapping contact residues on C3d: Implications for the controversy regarding the CR2/C3d cocrystal structure. J. Immunol. 184, 1946–1955] have implicated an electronegative “concave” surface on C3d in the binding process. This surface is discrete from the CR2–C3d interface identified in the crystal structure. We generated a total of 18 mutations targeting the two (X-ray crystallographic- and mutagenesis-based) proposed CR2 SCR1-2 binding sites on C3d. Using ELISA analyses, we were able to assess binding of mutant forms of C3d to CR2. Mutations directed toward the concave surface of C3d result in substantially compromised CR2 binding. By contrast, targeting the CR2–C3d interface identified in the cocrystal structure and the surrounding area results in significantly lower levels of disruption in binding. Molecular modeling approaches used to investigate disparities between the biochemical data and the X-ray structure of the CR2–C3d cocrystal result in highest-scoring solutions in which CR2 SCR1-2 is docked within the concave surface of C3d.

Biophysical Investigations of Complement Receptor 2 (CD21 and CR2)-Ligand Interactions Reveal Amino Acid Contacts Unique to Each Receptor-Ligand Pair

Human complement receptor type 2 (CR2 and CD21) is a cell membrane receptor, with 15 or 16 extracellular short consensus repeats (SCRs), that promotes B lymphocyte responses and bridges innate and acquired immunity. The most distally located SCRs, SCR1-2, mediate the interaction of CR2 with its four known ligands (C3d, EBV gp350, IFNalpha, and CD23). To ascertain specific interacting residues on CR2, we utilized NMR studies wherein gp350 and IFNalpha were titrated into (15)N-labeled SCR1-2, and chemical shift changes indicative of specific inter-molecular interactions were identified. With backbone assignments made, the chemical shift changes were mapped onto the crystal structure of SCR1-2. With regard to gp350, the binding region of CR2 is primarily focused on SCR1 and the inter-SCR linker, specifically residues Asn(11), Arg(13), Ala(22), Arg(28), Ser(32), Arg(36), Lys(41), Lys(57), Tyr(64), Lys(67), Tyr(68), Arg(83), Gly(84), and Arg(89). With regard to IFNalpha, the binding is similar to the CR2-C3d interaction with specific residues being Arg(13), Tyr(16), Arg(28), Ser(42), Lys(48), Lys(50), Tyr(68), Arg(83), Gly(84), and Arg(89). We also report thermodynamic properties of each ligand-receptor pair determined using isothermal titration calorimetry. The CR2-C3d interaction was characterized as a two-mode binding interaction with K(d) values of 0.13 and 160 microm, whereas the CR2-gp350 and CR2-IFNalpha interactions were characterized as single site binding events with affinities of 0.014 and 0.035 microm, respectively. The compilation of chemical binding maps suggests specific residues on CR2 that are uniquely important in each of these three binding interactions.

Progress on solving high-resolution 3D structure of site 1 of complement receptor type 1

Progress on solving high-resolution 3D structure of site 1 of complement receptor type 1

The human complement receptor type 2/factor H fusion protein TT30, an alternative pathway specific complement inhibitor, prevents hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria by membrane-targeting of factor H activity

The human complement receptor type 2/factor H fusion protein TT30, an alternative pathway specific complement inhibitor, prevents hemolysis of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria by membrane-targeting of factor H activity

Differential expression and function of complement receptor type 1 (CD35) and 2 (CD21) on human B lymphocytes

Differential expression and function of complement receptor type 1 (CD35) and 2 (CD21) on human B lymphocytes

Characterizing the complement receptor Type 2–C3d complex by site-directed mutagenesis and molecular modeling

Characterizing the complement receptor Type 2–C3d complex by site-directed mutagenesis and molecular modeling

Analysis of complement receptor Type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms

The KN blood group system, which consists of nine antigen specificities, is located on complement receptor Type 1 (CR1/CD35). CR1, a complement regulatory protein, acts as a vehicle for immune complex clearance. CR1 exhibits a red blood cell (RBC) density polymorphism. CR1 sites on RBCs in normal individuals range from 150 to 1200 molecules per cell. CR1 density polymorphism is regulated by HindIII restriction fragment length polymorphism and Q981H and P1786R polymorphisms in Caucasians. Yet, the role of the different polymorphisms in determining the CR1 density on RBCs remains unknown. The "null" serologic KN phenotype, known as Helgeson phenotype, was reported to be related with a very low CR1 density, less than 150 molecules per cell. The aim of this work was to investigate whether the KN-negative phenotype displayed by 60 individuals was related to the CR1 density by performing the phenotypic and genetic analysis of CR1 and to investigate the molecular background associated with the KN system. We showed that the Helgeson-like phenotype had a prevalence of 12% in this population. The overall genotype/phenotype concordance was 90%. Among individuals with a KN-negative phenotype, the prevalences of Kn(a-), McC(a-), Sl1-negative, Sl3-negative, and KCAM-negative deduced phenotype were 37, 12, 29, 7, and 24%, respectively. From our data, we suggest that the definition of the Helgeson phenotype must be revised, since the latter may be due not only to a very low CR1 density on RBCs, but also to the absence of expression of a high-prevalence KN antigen.

A novel trifunctional IgG-like bispecific antibody to inhibit HIV-1 infection and enhance lysis of HIV by targeting activation of complement.

BackgroundThe complement system is not only a key component of innate immunity but also provides a first line of defense against invading pathogens, especially for viral pathogens. Human immunodeficiency virus (HIV), however, possesses several mechanisms to evade complement-mediated lysis (CoML) and exploit the complement system to enhance viral infectivity. Responsible for this intrinsic resistance against complement-mediated virolysis are complement regulatory membrane proteins derived from the host cell that inherently downregulates complement activation at several stages of the cascade. In addition, HIV is protected from complement-mediated lysis by binding soluble factor H (fH) through the viral envelope proteins, gp120 and gp41. Whereas inhibition of complement activity is the desired outcome in the vast majority of therapeutic approaches, there is a broader potential for complement-mediated inhibition of HIV by complement local stimulation.Presentation of the hypothesisOur previous studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that another new activator of complement, consisting of two dsFv (against gp120 and against C3d respectively) linked to a complement-activating human IgG1 Fc domain ((anti-gp120 × anti-C3d)-Fc), can not only target and amplify complement activation on HIV virions for enhancing the efficiency of HIV lysis, but also reduce the infectivity of HIV through blocking the gp120 and C3d on the surface of HIV.Testing the hypothesisOur hypothesis was tested using cell-free HIV-1 virions cultivated in vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified (anti-gp120 × anti-C3d)-Fc proteins. As a control group, viruses were incubated with normal human serum under the same conditions. Virus neutralization assays were used to estimate the degree of (anti-gp120 × anti-C3d)-Fc lysis of HIV compared to untreated virus.Implications of the hypothesisThe targeted complement activator, (anti-gp120 × anti-C3d)-Fc, can be used as a novel approach to HIV therapy by abrogating the complement-enhanced HIV infection of cells.

Overexpression of complement receptor type I (CR1, CD35) on erythrocytes in patients with hemoplasma infection

In the present study, 12 patients with fever of undetermined origin, anemia and icterus were diagnosed with hemoplasma infection by light microscopy, transmission electron microscopy examination and PCR assay after being excluded from other usual febrile diseases. Complement receptor type I (CR1, CD35) expression on the surface of erythrocytes was assessed by flow cytometry using mouse anti-human CD35 antibody. Compared with healthy volunteers, the level of CD35 was significantly elevated in patients with severe hemoplasma infection at diagnosis, and decreased sharply after treatment. However, in latent infection cases without clinical manifestations, CD35 expression showed an ascending trend but had no statistical difference compared to the healthy controls. The present study demonstrated that hemoplasma infection can induce high levels of expression of CR1 on the membrane of red blood cells, which may be a reaction to the immunity challenge.

Renal Inflammation: Targeted Iron Oxide Nanoparticles for Molecular MR Imaging in Mice

To determine the feasibility of T2-weighted magnetic resonance (MR) imaging in the noninvasive quantification of renal inflammation by using superparamagnetic iron oxide (SPIO) nanoparticles targeted to tissue-bound C3 activation fragments in a mouse model of lupus nephritis. All animal procedures were approved by the University of Colorado-Denver animal care and use committee. SPIO nanoparticles were encapsulated by using amine-functionalized phospholipids. A recombinant protein containing the C3d-binding region of complement receptor type 2 (CR2) was then conjugated to the surface of the SPIO nanoparticle. Five MRL/lpr mice (a model of lupus nephritis) and six C57BL/6 wild-type mice were assessed with T2-weighted MR imaging at baseline and after SPIO injection. The same five MRL/lpr mice and three C57BL/6 mice also underwent MR imaging after injection of CR2-targeted SPIO. A series of T2-weighted pulses with 16 echo times was used to enable precise T2 mapping and calculation of T2 relaxation times in the cortex and outer and inner medulla of the kidneys, as well as in the spleen, muscle, and fat. The effects of treatment and animal genotype on T2 relaxation times were analyzed with repeated-measures analysis of variance. At baseline, the T2-weighted signal intensity in the kidneys of MRL/lpr mice was higher than that in the kidneys of wild-type mice. Injection of untargeted SPIO did not alter the T2-weighted signal in the kidneys in either strain of mice. Injection of CR2-targeted SPIO in MRL/lpr mice, however, caused a significant accumulation of targeted iron oxide with a subsequent decrease in T2 relaxation times in the cortex and outer and inner medulla of the kidneys. No changes in T2 relaxation time were observed in the wild-type mice after injection of targeted SPIO. Injection of CR2-conjugated SPIO caused a significant reduction in T2-weighted MR imaging signal and T2 relaxation time in nephritic kidneys.

Structure of an integrin with an αI domain, complement receptor type 4

We report the structure of an integrin with an alphaI domain, alpha(X)beta(2), the complement receptor type 4. It was earlier expected that a fixed orientation between the alphaI domain and the beta-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the alphaI domain is highly flexible, enabling two betaI domain conformational states to couple to three alphaI domain states, and greater accessibility for ligand recognition. Although alpha(X)beta(2) is bent similarly to integrins that lack alphaI domains, the terminal domains of the alpha- and beta-legs, calf-2 and beta-tail, are oriented differently than in alphaI-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the beta(2)-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

Modulation of immune response by combined targeting of complement receptors and low-affinity Fcγ receptors

Immune complexes (ICs) induce effective pathogen-specific innate and humoral immune response via immunecomplex-binding receptors, such as murine complement receptor type 1 and 2 (mCR1/2) and murine low-affinity Fc receptors for IgG (mFcγRII and III). The exact function of mCR1/2 in cooperation with mFcγRII/III in modulation of humoral immunity has not yet been adequately clarified. The aim of this study was to target these receptors by specific single-chain fragments of antibody (scFv), either individually or in combination, thus modelling the action of IC. For targeting, we used scFv derived from the well-characterized 7g6 and 2.4g2 monoclonal antibodies recognizing mCR1/2 and mFcγRII/III, respectively. These scFvs were monobiotinylated and conjugated to streptavidin or streptavidin-coated microspheres. Such complexes were investigated with respect to target receptor recognition and in vivo localization. Antibody response against the constructs was measured by ELISA and ELISPOT. Results show that targeting streptavidin complexes to mFcγRII/III induces stronger IgG1 response than targeting to mCR1/2 yet both strategies enhance the antibody response compared to the control group immunized with non-targeted peptide–streptavidin complexes. Moreover, the immunogenicity of coupled antigens increased using microspheres as carrier, instead of using soluble streptavidin. In summery, our in vivo experiments reveal that mFcγRII/III is more potent a target than CR1/2 and show that combined targeting of CR1/2 and FcγRII/III receptors does not result in cumulative enhancement of the antigen specific immune response. In addition, microparticle-mediated enhancement of immunization can be further improved by FcγRII/III targeting.

TT30, a Novel Human Protein Therapeutic, Selectively Modulates the Complement Alternative Pathway by Targeted Supplementation of Local Factor H Activity.

Abstract 3021Poster Board II-997The complement system is activated through three pathways: classical, lectin/mannose and alternative. Polymorphisms and mutations that promote Complement Alternative Pathway (CAP) activity are associated with human diseases including atypical hemolytic uremic syndrome (aHUS) and age-related macular degeneration (AMD). The complement system is also centrally involved in many hemolytic disorders, including paroxysmal nocturnal hemoglobinuria (PNH) where the CAP initiates complement activation resulting in intravascular hemolysis (IVH) after engagement of C5 and formation of the membrane attack complex (MAC). Systemic neutralization of C5 with the anti-C5 monoclonal antibody, eculizumab, abrogates IVH when plasma concentrations are maintained above the minimal efficacious concentration (Cmin = 35 μg/mL). However, because eculizumab does not inhibit CAP activity prior to C5, C3 fragments (C3frag) continue to covalently bind to and accumulate on PNH red blood cells (RBCs). Clearance by the reticuloendothelial system of PNH RBCs that are C3frag-coated is a putative cause of extravascular hemolysis (EVH) in eculizumab-treated patients.In order to selectively modulate CAP activity, we developed TT30, a novel therapeutic 65kD fusion protein linking the first four short consensus repeat (SCR) domains of human complement receptor type 2 (CR2/CD21) with the first five SCR of human factor H (fH). CR2 SCR1-4 encompasses the antigen-fixed C3frag (iC3b, C3dg and C3d) binding domain. Factor H is the primary soluble phase, negative regulator of CAP activity functioning via the SCR1-5 domains. The unique mechanism of TT30 utilizes CR2 SCR1-4 to recognize and bind to C3frag on cells in which complement activation is occurring, thus delivering cell surface-targeted inhibition of CAP activity via fH SCR 1-5. TT30 both prevents CAP-dependent hemolysis of rabbit RBCs in human serum and blocks accumulation of C3frag on the RBC surface.By design, TT30 should also be a potent inhibitor of the CAP, but with minimal inhibition of the complement classical (CCP) and mannose (lectin; CMP) pathways. To test this hypothesis, we utilized sensitive pharmacodynamic assays that allow in vitro or ex vivo assessment in an ELISA format of individual complement pathway activity present in human serum. In this format, TT30 is a potent and selective inhibitor of CAP activity in normal human complement-preserved serum, with EC50 and EC100 values of ∼0.1 and 1 μg/mL serum. As predicted by the use of fH in its construction, TT30 is a much less potent inhibitor of the CCP and CMP, with EC100 values of ∼65 μg/mL. By contrast, in these assays a monoclonal and polyclonal anti-C5 antibody each demonstrate non-selective inhibition of CAP and CCP activity at all effective concentrations.TT30 activity is dependent upon CR2 binding to C3frag, as an anti-CR2 monoclonal antibody reverses the surface inhibition of CAP activity. This surface-targeting approach to delivering fH SCR1-5 results in a molecule with a 10-fold potency gain in CAP inhibition relative to added purified fH and an ∼30-fold potency gain relative to the total fH present in the serum used in the assay.TT30 administered as a single IV injection at 20 mg/kg to rats, rabbits and monkeys results in Cmax values of ∼400, 500 and 300 μg/mL and concentration-dependent inhibition of CAP activity. At serum concentrations of TT30 that induced maximal (100%) inhibition of systemic CAP activity for up to 12 hours, CCP activity is modestly (∼35-60%) inhibited for only 2 hours. CAP activity returns to baseline levels in a predictable fashion. Pharmacokinetic analysis indicates no gender-related differences and the expected scaling of parameters across species. TT30 is pharmacologically active in monkeys, rabbits and mice. TT30 administered as a single subcutaneous injection at 20 mg/kg to monkeys results in Cmax values of ∼25 μg/mL, and EC100 values identical to those observed with IV administration, but with a 3-fold prolongation of the maximal pharmacodynamic effect.The novel therapeutic TT30 has been shown in vitro and ex vivo to deliver cell surface-targeted control of CAP activation with minimal CCP and CMP inhibition and effective blockade of C3frag accumulation and MAC formation. As a result, TT30 has potential utility for the treatment of complement-mediated diseases such as PNH, AMD and aHUS, in which cell surface-targeted control of CAP activation may be clinically beneficial. DisclosuresHolers:Taligen Therapeutics: Employment, Equity Ownership, Patents & Royalties, Research Funding. Mazsaroff:Taligen Therapeutics: Employment. Akana:Taligen Therapeutics: Employment. Smith:Taligen Therapeutics: Employment. Emlen:Taligen Therapeutics: Employment, Equity Ownership. Marians:Taligen Therapeutics: Employment. Horvath:Taligen Therapeutics: Employment.

The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35)

The complement system is comprised of soluble and cell surface associated proteins that recognize exogenous, altered, or potentially harmful endogenous ligands. In recent years, the complement system—particularly component C3 and its receptors—have been demonstrated to be a key link between innate and adaptive immunity. Complement receptor type 1 (CR1), the receptor for C3b/C4bcomplement peptides, has emerged as a molecule of immense interest in gaining insight to the susceptibility, pathophysiology, diagnosis, prognosis and therapy of such diseases. In this review, we wish to briefly bring forth the structure, genetic polymorphisms, expression and biological functions of CR1.

The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35)

The structure, genetic polymorphisms, expression and biological functions of complement receptor type 1 (CR1/CD35)

Soluble complement receptor type 1 inhibits complement system activation and improves motor function in acute spinal cord injury

Spinal cord injured rat model, treated with soluble complement receptor type 1 (sCR1). Experimental Animal Department of China Medical University, Shenyang, China. Soluble CR1 is a powerful inhibitor of complement activation. In this study, we investigate the effectiveness of sCR1 on spinal cord injury (SCI) in rats. Spinal cord injury was induced in Sprague-Dawley rats. Three experimental groups were examined; the sCR1 group was administered sCR1 at 1 h after the SCI, whereas the control group was administered saline at 1 h after SCI and the sham group underwent a sham operation without SCI or administration. The expressions of C9 and CD59 in the injured spinal cords were evaluated by immunohistochemistry, and numbers of positive cells counted. Furthermore, myeloperoxidase (MPO) activity and motor function were evaluated in each group. At all postoperative time points, the numbers of C9- and CD59-positive cells in the sCR1 group were reduced compared with the control group and MPO activity was significantly decreased compared with both other groups. Moreover, the Basso, Beattie and Bresnahan score for the sCR1 group was significantly improved as compared with that of the control group after 7 days postoperatively. Soluble CR1 decreases inflammation reactions by inhibiting activation of the complement system and improves motor function after acute SCI.

A novel approach to inhibit HIV-1 infection and enhance lysis of HIV by a targeted activator of complement

BackgroundThe complement system is one of the most potent weapons of innate immunity. It is not only a mechanism for direct protection against invading pathogens but it also interacts with the adaptive immunity to optimize the pathogen-specific humoral and cellular defense cascades in the body. Complement-mediated lysis of HIV is inefficient but the presence of HIV particles results in complement activation by the generation of many C3-fragments, such as C3dg and C3d. It has been demonstrated that activation of complement can enhance HIV infection through the binding of special complement receptor type 2 expression on the surface of mature B cells and follicular dendritic cells.Presentation of the hypothesisPrevious studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that a new activator of complement, consisting of a target domain (C3-binding region of complement receptor type 2) linked to a complement-activating human IgG1 Fc domain (CR2-Fc), can target and amplify complement deposition on HIV virions and enhance the efficiency of HIV lysis.Testing the hypothesisOur hypothesis was tested using cell-free HIV-1 virions cultivated in vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified CR2-Fc proteins. As a control group, viruses were incubated with normal human serum under the same conditions. Virus neutralization assays were used to estimate the degree of CR2-Fc-enhanced lysis of HIV compared to untreated virus.Implications of the hypothesisThe targeted complement activator, CR2-Fc, can be used as a novel approach to HIV therapy by abrogating the complement-enhanced HIV infection of cells.

Structural comparison between functional sites 1 and 2 of complement receptor type 1

Structural comparison between functional sites 1 and 2 of complement receptor type 1

Spontaneous complement activation on human B cells results in localized membrane depolarization and the clustering of complement receptor type 2 and C3 fragments

While our previous studies have demonstrated that complement activation induced by complement receptors type 2 (CR2/CD21) and 1 (CR1/CD35) results in C3-fragment deposition and membrane attack complex (MAC) formation in human B cells, the consequences of these events for B-cell functions remain unknown. In the present study, we show that CR2-induced complement activation results in membrane depolarization, as indicated by annexin V binding, with kinetics similar to those of C3-fragment deposition and different from those of MAC formation. On the other hand, like MAC formation, depolarization requires activation of complement via the alternative pathway, as indicated by total inhibition upon neutralization of factor D, and is abrogated by combined blockade of CR1 and CR2, but not of either receptor alone. The membrane depolarization is not associated with the apoptosis of B cells, as examined by co-staining with APO-2.7 or by the TdT-mediated biotin-dUTP nick-end labelling (TUNEL) assay. Confocal microscopy revealed that depolarization and C3 deposition, unlike MAC deposition, are limited to restricted areas on the B-cell surface. Double staining revealed a close association between the C3-fragment patches and membrane depolarization, as well as redistribution of lipid rafts to these areas. We propose that these events may play a role in the regulation of B-cell signalling and cross-talk with T cells.