Antibacterial Fusion Proteins Enhance Moraxella catarrhalis Killing.

Moraxella catarrhalis is a human-specific commensal of the respiratory tract and an opportunistic pathogen. It is one of the leading cause of otitis media in children and of acute exacerbations in patients with chronic obstructive pulmonary disease, resulting in significant morbidity and economic burden. Vaccines and new immunotherapeutic strategies to treat this emerging pathogen are needed. Complement is a key component of innate immunity that mediates the detection, response, and subsequent elimination of invading pathogens. Many pathogens including M. catarrhalis have evolved complement evasion mechanisms, which include the binding of human complement inhibitors such as C4b-binding protein (C4BP) and Factor H (FH). Inhibiting C4BP and FH acquisition by M. catarrhalis may provide a novel therapeutic avenue to treat infections. To achieve this, we created two chimeric proteins that combined the Moraxella-binding domains of C4BP and FH fused to human immunoglobulin Fcs: C4BP domains 1 and 2 and FH domains 6 and 7 fused to IgM and IgG Fc, respectively. As expected, FH6-7/IgG displaced FH from the bacterial surface while simultaneously activating complement via Fc-C1q interactions, together increasing pathogen elimination. C4BP1-2/IgM also increased serum killing of the bacteria through enhanced complement deposition, but did not displace C4BP from the surface of M. catarrhalis. These Fc fusion proteins could act as anti-infective immunotherapies. Many microbes bind the complement inhibitors C4BP and FH through the same domains as M. catarrhalis, therefore these Fc fusion proteins may be promising candidates as adjunctive therapy against many different drug-resistant pathogens.

Complement-Independent Modulation of Influenza A Virus Infection by Factor H.

The complement system is an ancient innate immune defense mechanism that can recognize molecular patterns on the invading pathogens. Factor H, as an inhibitor of the alternative pathway, down-regulates complement activation on the host cell surface. Locally synthesized factor H at the site of infection/injury, including lungs, can act as a pattern recognition molecule without involving complement activation. Here, we report that factor H, a sialic acid binder, interacts with influenza A virus (IAV) and modulates IAV entry, as evident from down-regulation of matrix protein 1 (M1) in H1N1 subtype-infected cells and up-regulation of M1 expression in H3N2-infected A549 cells. Far-western blot revealed that factor H binds hemagglutinin (HA, ~70 kDa), neuraminidase (NA, ~60 kDa), and M1 (~25 kDa). IAV-induced transcriptional levels of IFN-α, TNF-α, IL-12, IL-6, IFN-α, and RANTES were reduced following factor H treatment for the H1N1 subtype at 6 h post-infection. However, for the H3N2 subtype, mRNA levels of these pro-inflammatory cytokines were enhanced. A recombinant form of vaccinia virus complement control protein (VCP), which like factor H, contains CCP modules and has complement-regulatory activity, mirrored the results obtained with factor H. Both factor H (25%), and VCP (45%) were found to reduce luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles. Factor H (50%) and VCP (30%) enhanced the luciferase reporter activity for H3N2, suggesting an entry inhibitory role of factor H and VCP against H1N1, but not H3N2. Thus, factor H can modulate IAV infection and inflammatory responses, independent of its complement-related functions.

CFH exerts anti-oxidant effects on retinal pigment epithelial cells independently from protecting against membrane attack complex.

Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions' structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.

Elevated surface-bound complement FH alters the function of platelets and monocytes in FHR1/3 null healthy individuals.

Complement factor H (FH) and FH-related proteins (FHRs), structurally similar proteins are involved in the regulation of complement activation. Homozygous deletion of FHR 1 and 3 proteins (FHR1/3-/-) is known as a risk factor for disorders such as aHUS and SLE, characterised by thrombo-inflammatory complications. Interestingly, FHR1/3-/- genotype also exists as polymorphism in healthy population of various ethnicities around the world including 8-10% Indians. In an effort to understand the functional role of this polymorphism, we describe in this study an elevated surface-bound FH on platelets and monocytes, but not other blood cells in FHR1/3 -/- healthy individuals. The FHR1/3-/- platelets displayed diminish ability to form aggregates in response to agonists in vitro. The FHR1/3-/- monocytes displayed elevated secretion of TNFα, IL1ß, IL6 and IL10 in response to TLR ligands. However, exogenous FH limits platelet aggregates formation as well as cytokine secretion in monocytes. Therefore, observations together suggest a differential regulation of platelets and monocytes by FH-FHR1/3 axis in healthy individuals. While these findings will need more detailed investigation, it is clear that the connection between FH-FHR axis and thrombo-inflammatory complications is likely to be complex in diseases including aHUS and SLE, and provide interesting new directions for future study.

Kallikrein Cleaves C3 and Activates Complement.

The human plasma contact system is an immune surveillance system activated by the negatively charged surfaces of bacteria and fungi and includes the kallikrein-kinin, the coagulation, and the fibrinolytic systems. Previous work shows that the contact system also activates complement, and that plasma enzymes like kallikrein, plasmin, thrombin, and FXII are involved in the activation process. Here, we show for the first time that kallikrein cleaves the central complement component C3 directly to yield active components C3b and C3a. The cleavage site within C3 is identical to that recognized by the C3 convertase. Also, kallikrein-generated C3b forms C3 convertases, which trigger the C3 amplification loop. Since kallikrein also cleaves factor B to yield Bb and Ba, kallikrein alone can trigger complement activation. Kallikrein-generated C3 convertases are inhibited by factor H; thus, the kallikrein activation pathway merges with the amplification loop of the alternative pathway. Taken together, these data suggest that activation of the contact system locally enhances complement activation on cell surfaces. The human pathogenic microbe Candida albicans activates the contact system in normal human serum. However, C. albicans immediately recruits factor H to the surface, thereby evading the alternative and likely kallikrein-mediated complement pathways.

Factor H specifically capture novel Factor H-binding proteins of Streptococcus suis and contribute to the virulence of the bacteria.

Factor H (FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins (FHBPs) of Streptococcus suis serotype 2 (SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 pathogenesis. Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identified proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 pre-incubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with FH. Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results first indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoting SS2 to resist killing and leading to enhance virulence.

Painting factor H onto mesenchymal stem cells protects the cells from complement- and neutrophil-mediated damage.

Mesenchymal stem cells (MSCs) are undergoing intensive testing in clinical trials as a promising new therapy for many inflammatory diseases and for regenerative medicine, but further optimization of current MSC-based therapies is required. In this study, we found that in addition to direct complement-mediated attack through the assembly of membrane attack complexes (MACs) that we and others have recently reported, of the released complement activation products, C5a, but not C3a, activates neutrophils in the blood to further damage MSCs through oxidative burst. In addition, we have developed a simple method for painting factor H, a native complement inhibitor, onto MSCs to locally inhibit complement activation on MSCs. MSCs painted with factor H are protected from both MAC- and neutrophil-mediated attack and are significantly more effective in inhibiting antigen-specific T cell responses than the mock-painted MSCs both in vitro and in vivo.

A Novel Factor H-Fc Chimeric Immunotherapeutic Molecule against Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, has developed resistance to almost every conventional antibiotic. There is an urgent need to develop novel therapies against gonorrhea. Many pathogens, including N. gonorrhoeae, bind the complement inhibitor factor H (FH) to evade complement-dependent killing. Sialylation of gonococcal lipooligosaccharide, as occurs in vivo, augments binding of human FH through its domains 18-20 (FH18-20). We explored the use of fusing FH18-20 with IgG Fc (FH18-20/Fc) to create a novel anti-infective immunotherapeutic. FH18-20 also binds to select host glycosaminoglycans to limit unwanted complement activation on host cells. To identify mutation(s) in FH18-20 that eliminated complement activation on host cells, yet maintained binding to N. gonorrhoeae, we created four mutations in domains 19 or 20 described in atypical hemolytic uremic syndrome that prevented binding of mutated fH to human erythrocytes. One of the mutant proteins (D to G at position 1119 in domain 19; FHD1119G/Fc) facilitated complement-dependent killing of gonococci similar to unmodified FH18-20/Fc but, unlike FH18-20/Fc, did not lyse human erythrocytes. FHD1119G/Fc bound to all (100%) of 15 sialylated clinical N. gonorrhoeae isolates tested (including three contemporary ceftriaxone-resistant strains), mediated complement-dependent killing of 10 of 15 (67%) strains, and enhanced C3 deposition (≥10-fold above baseline levels) on each of the five isolates not directly killed by complement. FHD1119G/Fc facilitated opsonophagocytic killing of a serum-resistant strain by human polymorphonuclear neutrophils. FHD1119G/Fc administered intravaginally significantly reduced the duration and burden of gonococcal infection in the mouse vaginal colonization model. FHD1119G/Fc represents a novel immunotherapeutic against multidrug-resistant N. gonorrhoeae.

Complement factor H in its alternative identity as adrenomedullin-binding protein 1.

Complement factor H has been extensively studied since its discovery 50 years ago, and its role in the complement system is quite well established. It has another role, however, as a binding protein for the regulatory peptide adrenomedullin. Part of this role appears to be protection of adrenomedullin from proteolytic degradation. The binding interaction is unusual and merits further investigation. Adrenomedullin has potential therapeutic uses in diseases affecting the vasculature, and factor H has been administered with adrenomedullin in some animal models of disease.

Combination of adrenomedullin with its binding protein accelerates cutaneous wound healing.

Cutaneous wound continues to cause significant morbidity and mortality in the setting of diseases such as diabetes and cardiovascular diseases. Despite advances in wound care management, there is still an unmet medical need exists for efficient therapy for cutaneous wound. Combined treatment of adrenomedullin (AM) and its binding protein-1 (AMBP-1) is protective in various disease conditions. To examine the effect of the combination treatment of AM and AMBP-1 on cutaneous wound healing, full-thickness 2.0-cm diameter circular excision wounds were surgically created on the dorsum of rats, saline (vehicle) or AM/AMBP-1 (96/320 µg kg BW) was topically applied to the wound daily and wound size measured. At days 3, 7, and 14, skin samples were collected from the wound sites. AM/AMBP-1 treated group had significantly smaller wound surface area than the vehicle group over the 14-day time course. At day 3, AM/AMBP-1 promoted neutrophil infiltration (MPO), increased cytokine levels (IL-6 and TNF-α), angiogenesis (CD31, VEGF and TGFß-1) and cell proliferation (Ki67). By day 7 and 14, AM/AMBP-1 treatment decreased MPO, followed by a rapid resolution of inflammation characterized by a decrease in cytokines. At the matured stage, AM/AMBP-1 treatment increased the alpha smooth muscle actin expression (mature blood vessels) and Masson-Trichrome staining (collagen deposition) along the granulation area, and increased MMP-9 and decreased MMP-2 mRNA expressions. TGFß-1 mRNA levels in AM/AMBP-1 group were 5.3 times lower than those in the vehicle group. AM/AMBP-1 accelerated wound healing by promoting angiogenesis, collagen deposition and remodeling. Treatment also shortened the days to reach plateau for wound closure. Thus, AM/AMBP-1 may be further developed as a therapeutic for cutaneous wound healing.

Fusion protein comprising factor H domains 6 and 7 and human IgG1 Fc as an antibacterial immunotherapeutic.

The emergence of antimicrobial resistance among several medically important pathogens represents a serious threat to human health globally and necessitates the development of novel therapeutics. Complement forms a key arm of innate immune defenses against invading pathogens. A mechanism of complement evasion employed by many pathogens is binding of complement inhibitors, including factor H (FH), a key downregulator of the alternative pathway. Most FH-binding bacteria engage FH through regions in FH spanned by domains 6 and 7 and/or 18 through 20. We created a chimeric protein that comprised human FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/HuFc) and tested its activity as an immunotherapeutic against Neisseria meningitidis, which binds FH through domains 6 and 7. FH6,7/HuFc bound to meningococci and effectively blocked FH binding to bacteria. FH6,7/HuFc enhanced human C3 and C4 deposition and facilitated complement-mediated killing in a dose-responsive manner; complement activation and killing were classical pathway dependent. To investigate in vivo efficacy, infant Wistar rats were treated intraperitoneally (IP) with different doses of FH6,7/HuFc and challenged 2 h later with serogroup C strain 4243 given IP. At 8 to 9 h after the challenge, the FH6,7/HuFc-treated rats had >100-fold fewer CFU per ml of blood than control animals pretreated with phosphate-buffered saline (PBS) or FH18-20/HuFc, which does not bind to meningococci (P < 0.0001). These data provide proof of concept of the utility of FH/Fc fusion proteins as anti-infective immunotherapeutics. Because many microbes share a common binding region(s) in FH, FH/Fc chimeric proteins may be a promising candidate for adjunctive therapy against drug-resistant pathogens.

A potential anti-coagulant role of complement factor H.

Anti-phospholipid syndrome (APS) is a complex autoimmune disease, associated with recurrent venous and arterial thrombosis in various tissues. APS is associated with specific antibodies against plasma beta-2 glycoprotein 1 (ß2-GP1), and these antibodies react with ß2-GP1 bound to negatively charged phospholipids (e.g. cardiolipin) on cell membranes. Some APS patients also have autoantibodies to complement factor H (FH), a homologue of ß2-GP1, which also binds to anionic phospholipids. ß2-GP1 has earlier been shown to inhibit the intrinsic (contact) activated blood coagulation pathway, promoted by anionic phospholipids. Here we examine whether FH could have similar anti-thrombotic properties. In vitro experiments with surface-bound phospholipids and human plasma, in the presence of FH, confirm this hitherto unreported property of FH.

Generation of multiple fluid-phase C3b:plasma protein complexes during complement activation: possible implications in C3 glomerulopathies.

The complement system is tightly regulated to safeguard against tissue damage that results from unwanted activation. The key step of C3 cleavage to C3b is regulated by multiple mechanisms that control the initiation and extent of activation. This study demonstrated that C3b:plasma protein complexes form in the fluid-phase during complement activation. Several different plasma proteins displayed a discrete high molecular SDS-resistant band when any of the three complement activating pathways were triggered in normal human serum or plasma. Serum depleted of individual complement proteins revealed that C3 and factors B and D were essential for complex formation. Inactivation of the thioester bond in C3 also prevented complex formation. In vitro, complexes could be generated using four purified proteins-C3, factor B, factor D, and target protein-and Mg(2+) to allow C3 convertase formation. These studies showed that the complexes consisted of a plasma protein covalently bound to C3b in a 1:1 molar ratio; the C3b portion was rapidly degraded by factors H and I. Analysis of plasma samples from patients with dense deposit disease and C3 glomerulonephritis demonstrated that C3b:protein complexes form spontaneously in the blood of patients with dense deposit disease and, to a lesser extent, in C3 glomerulonephritis patients, but not in healthy controls. This finding supports the underlying hypothesis that these C3 glomerulopathies are diseases of fluid-phase complement dysregulation. These complexes could normally function as a passive mechanism to intercept C3b from depositing on host cells. However, excessive generation and/or defective clearance of fluid-phase C3b:protein complexes may have pathological consequences.

A method of purifying intact complement factor H from human plasma.

The aim of this study was to establish a method of purifying intact complement factor H (CFH) from human plasma. CFH was isolated from human plasma by polyethylene glycol (PEG) precipitation, following three sequential chromatographic columns, which consisted of l-lysine Sepharose column, Resource Q column and Sephacryl S-300 High Resolution HiPrep 16/60 column. All the above steps were performed at 4°C by Fast Protein Liquid Chromatography (FPLC) AKTA Purifier 10 with Frac-900. Identification of the purified CFH was confirmed by SDS-PAGE and Western blot. The following functions of the purified CFH were further analyzed compared with the commercial CFH in vitro: (1) binding ability with C3b; (2) binding ability with mCRP; (3) the protecting function of the hemolysis of sheep red blood cells; (4) the cofactor role for complement factor I-mediated proteolytic inactivation of C3b. Homogeneous CFH was purified from the plasma fraction through the above four steps. The purity and the functions of the purified CFH were comparable to the commercial CFH. The yield of CFH was 26±3% in our study. Compared with previous methods, our method was high yield with high purity. We established a stable and feasible system for purifying intact CFH, which could be used in the lab and clinical investigations.

Complement factor H-derived short consensus repeat 18-20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells.

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18-20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18-20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18-20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18-20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18-20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies.

Rational engineering of a minimized immune inhibitor with unique triple-targeting properties.

Inadequate control of the complement system is the underlying or aggravating factor in many human diseases. Whereas treatment options that specifically target the alternative pathway (AP) of complement activation are considered highly desirable, no such option is available in the clinic. In this study, we present a successful example of protein engineering, guided by structural insight on the complement regulator factor H (FH), yielding a novel complement-targeted therapeutic (mini-FH) with clinical potential. Despite a 70% reduction in size, mini-FH retained and in some respects exceeded the regulatory activity and cell surface-recognition properties of its parent protein FH, including the recently described recognition of sites of oxidative stress. Importantly, the chosen design extended the functional spectrum of the inhibitor, as mini-FH showed increased binding to the surface-bound opsonins iC3b and C3dg when compared with FH. Thus, mini-FH is equipped with a unique and clinically valuable triple-targeting profile toward diseased host cells, through its binding to sites of ongoing complement activation, markers of oxidative damage, and host surface-specific polyanions. When assessed in a clinically relevant AP-mediated disease model of paroxysmal nocturnal hemoglobinuria, mini-FH largely outperformed FH and indicated advantages over clinically evaluated AP inhibitors. Thus, the rational engineering of a streamlined FH construct not only provided insight into the function of a key complement regulator, but also yielded a novel inhibitor that combines a triple-targeting approach with high AP-specific inhibitory activity (IC50 ~ 40 nM), which may pave the way toward new options for the treatment of complement-mediated diseases.

The complement receptor 2/factor H fusion protein TT30 protects paroxysmal nocturnal hemoglobinuria erythrocytes from complement-mediated hemolysis and C3 fragment.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement-mediated intravascular hemolysis because of the lack from erythrocyte surface of the complement regulators CD55 and CD59, with subsequent uncontrolled continuous spontaneous activation of the complement alternative pathway (CAP), and at times of the complement classic pathway. Here we investigate in an in vitro model the effect on PNH erythrocytes of a novel therapeutic strategy for membrane-targeted delivery of a CAP inhibitor. TT30 is a 65 kDa recombinant human fusion protein consisting of the iC3b/C3d-binding region of complement receptor 2 (CR2) and the inhibitory domain of the CAP regulator factor H (fH). TT30 completely inhibits in a dose-dependent manner hemolysis of PNH erythrocytes in a modified extended acidified serum assay, and also prevents C3 fragment deposition on surviving PNH erythrocytes. The efficacy of TT30 derives from its direct binding to PNH erythrocytes; if binding to the erythrocytes is disrupted, only partial inhibition of hemolysis is mediated by TT30 in solution, which is similar to that produced by the fH moiety of TT30 alone, or by intact human fH. TT30 is a membrane-targeted selective CAP inhibitor that may prevent both intravascular and C3-mediated extravascular hemolysis of PNH erythrocytes and warrants consideration for the treatment of PNH patients.

The critical role of adrenomedullin and its binding protein, AMBP-1, in neuroprotection.

Chronic neurodegenerative disorders and acute injuries of the central nervous system exert a prohibitive economic burden, which is aggravated by an unmet medical need for the development of effective neurotherapeutics. The evolutionarily conserved neuropeptide, adrenomedullin (AM), and its binding protein, AMBP-1, also known as complement factor H, play important roles in brain physiology, and their expression is altered in brain pathology. In this review, we discuss the molecular regulation of AM and AMBP-1 and the pivotal roles they play in neuroprotection following brain injury. We assess the reciprocal synergistic effects of AM and AMBP-1 and make suggestions for the design of a novel combination neurotherapy devoid of the potential hypotensive effects of AM while optimizing its neuroprotective property.

Peripheral administration of human adrenomedullin and its binding protein attenuates stroke-induced apoptosis and brain injury in rats.

Stroke is a leading cause of death and the primary medical cause of acquired adult disability worldwide. The progressive brain injury after acute stroke is partly mediated by ischemia-elicited inflammatory responses. The vasoactive hormone adrenomedullin (AM), upregulated under various inflammatory conditions, counterbalances inflammatory responses. However, regulation of AM activity in ischemic stroke remains largely unknown. Recent studies have demonstrated the presence of a specific AM binding protein (that is, AMBP-1) in mammalian blood. AMBP-1 potentiates AM biological activities. Using a rat model of focal cerebral ischemia induced by permanent middle cerebral artery occlusion (MCAO), we found that plasma levels of AM increased significantly, whereas plasma levels of AMBP-1 decreased significantly after stroke. When given peripherally early after MCAO, exogenous human AM in combination with human AMBP-1 reduced brain infarct volume 24 and 72 h after MCAO, an effect not observed after the treatment by human AM or human AMBP-1 alone. Furthermore, treatment of human AM/AMBP-1 reduced neuron apoptosis and morphological damage, inhibited neutrophil infiltration in the brain and decreased serum levels of S100B and lactate. Thus, human AM/AMBP-1 has the ability to reduce stroke-induced brain injury in rats. AM/AMBP-1 can be developed as a novel therapeutic agent for patients with ischemic stroke.

Molecular characterization of the interaction between sialylated Neisseria gonorrhoeae and factor H.

Human factor H (HufH), a key inhibitor of the alternative pathway of complement, binds to Neisseria gonorrhoeae and constitutes an important mechanism of human-specific complement evasion. The C-terminal domain 20 of HufH contains the binding site for sialylated gonococci. We exploited differences in amino acid sequences between human and non-binding chimpanzee fH domain 20 to create cross-species mutations to define amino acids important for binding to sialylated gonococci. We used fH/Fc fusion constructs that contained contiguous fH domains 18-20 fused to Fc fragments of murine IgG2a. The Fc region was used both as a tag for detection of each fusion molecule on the bacterial surface and as an indicator for complement-dependent killing. Arg-1203 was critical for binding to both porin (Por) B.1A and PorB.1B strains. Modeling of the R1203N human-to-chimpanzee mutation using the crystal structure of HufH19-20 as a template showed a loss of positive charge that protrudes at the C terminus of domain 20. We tested the functional importance of Arg-1203 by incubating sialylated gonococci with normal human serum, in the presence of wild-type HufH18-20/Fc or its R1203A mutant. Gonococci bound and were killed by wild-type HufH18-20/Fc but not by the R1203A mutant. A recombinant fH/Fc molecule that contained chimpanzee domain 20, humanized only at amino acid 1203 (N1203R) also bound to sialylated gonococci and restored killing. These findings provide further insights into the species specificity of gonococcal infections and proof-of-concept of a novel therapeutic approach against gonorrhea, a disease rapidly becoming resistant to conventional antibiotics.