Antibody-sensitized Sheep Erythrocytes Research Articles

Preclinical Characterization of BCX9930, a Potent Oral Complement Factor D Inhibitor, Targeting Alternative Pathway-Mediated Diseases Including Paroxysmal Nocturnal Hemoglobinuria (PNH)

INTRODUCTION: Complement factor D is a serine protease that specifically catalyzes the cleavage of factor B [bound to C3(H2O) or C3b], its only natural substrate, to Ba and Bb. Factor D has the lowest concentration in plasma among all the complement proteins and is the rate-limiting enzyme of the alternative pathway (AP) of complement. Factor D plays a key role in both AP-initiated C3 convertase formation and the amplification loop of the complement cascade. Therefore, targeting factor D is a promising therapeutic strategy to inhibit AP activation for treatment of AP-mediated diseases such as PNH, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Inhibition of terminal complement C5 by eculizumab or ravulizumab results in sustained inhibition of intravascular hemolysis in patients with PNH by blocking formation of the membrane attack complex (C5b-9). However, many PNH patients treated with eculizumab continue to have symptomatic anemia, with up to half of the patients requiring blood transfusions due to extravascular hemolysis as a result of ongoing AP activation and C3 opsonization on erythrocytes. BioCryst Pharmaceuticals has developed a small-molecule, orally bioavailable human factor D inhibitor that is currently under evaluation as oral monotherapy for the treatment of patients with PNH in a Phase 1 study (BCX9930-101; NCT04330534). Here we demonstrate that BCX9930 is a potent and highly selective factor D inhibitor, and completely blocked AP activation on PNH erythrocytes in vitro. METHODS: Factor D enzymatic activity was analyzed in vitro in an esterolytic assay with a synthetic substrate and in a proteolytic assay using its natural substrate factor B bound to C3b. The selectivity was assessed in substrate-specific assays using other human serine proteases. The inhibition of AP-mediated hemolysis was assessed in vitro with rabbit erythrocytes incubated with 10% normal human serum (NHS). The Ham test was used to evaluate the inhibition by BCX9930 of AP-mediated hemolysis of erythrocytes from PNH patients with 20% acidified NHS. Inhibition of AP-mediated C3 fragment deposition was evaluated by flow cytometry using PNH erythrocytes incubated with acidified C5-depleted NHS. Activity of the classical pathway (CP) of complement l was evaluated by measuring 50% Hemolytic Complement (CH50) activity of serum and by assay of CP-mediated hemolysis of antibody-sensitized sheep erythrocytes. Pharmacodynamic effects in rhesus monkeys dosed orally with BCX9930 (100 and 200 mg twice-a-day) were assessed ex vivo with the AP Wieslab assay for the determination of C5b-9 generation after AP activation. RESULTS: BCX9930 potently inhibited esterolytic activity of purified human factor D with a mean 50% inhibitory concentration (IC50) of 14.3 nM. BCX9930 also inhibited its proteolytic activity against factor B bound to C3b with a mean IC50 of 28.1 nM. BCX9930 inhibited AP-mediated hemolysis of rabbit erythrocytes with a mean IC50 of 29.5 nM. Using the Ham test with erythrocytes from three PNH patients, BCX9930 completely suppressed AP-mediated hemolysis with a mean IC50 of 35.4 nM. Furthermore, BCX9930 suppressed C3 fragment deposition on PNH erythrocytes with a mean IC50 value of 39.3nM. BCX9930 did not inhibit enzyme activity of the human serine proteases thrombin, activated protein C, tissue plasminogen activator and trypsin up to 28 μM, and activated factor X and activated factor XII up to 50 μM. The mean IC50 values of BCX9930 inhibition of C1s and plasmin were 936 and 2850 nM, respectively. BCX9930 at a concentration up to 3 μM had no effect on CP activity in in vitro assays. Oral dosing of BCX9930 in monkeys completely suppressed AP activity of serum measured by AP-dependent generation of C5b-9. CONCLUSIONS: BCX9930 inhibitory activity on Factor D in vitro was potent and highly specific. BCX9930 completely blocked hemolysis of PNH cells in vitro and suppressed the accumulation of C3 fragments on PNH erythrocytes, indicating that BCX9930 has the potential to inhibit both intravascular and extravascular hemolysis. After oral dosing of BCX9930 in rhesus monkeys, AP activity was completely suppressed in ex vivo assays. These data demonstrate that BCX9930, an orally administered investigational drug, is a potent and selective factor D inhibitor, and that targeting factor D with BCX9930 has potential for the treatment of patients with PNH and other alternative pathway mediated diseases. Disclosures Chen: BioCryst Pharmaceuticals, Inc.: Current Employment. Cheng:BioCryst Pharmaceuticals, Inc.: Current Employment. Liu:BioCryst Pharmaceuticals, Inc.: Current Employment. Muppa:BioCryst Pharmaceuticals, Inc.: Current Employment. Parker:BioCryst Pharmaceuticals, Inc.: Current Employment. Zhu:BioCryst Pharmaceuticals, Inc.: Current Employment. Raman:BioCryst Pharmaceuticals, Inc.: Current Employment. Kotian:BioCryst Pharmaceuticals, Inc.: Current Employment. Babu:BioCryst Pharmaceuticals, Inc.: Current Employment.

Скрининг-тест для определения функциональной активности классического пути системы комплемента

Цель настоящей работы - разработка доступного теста для скрининга функциональной активности системы комплемента по классическому пути. Материалы и методы. В работе исследовали сыворотки крови 90 относительно здоровых лиц с избыточной массой тела (ИМТ >25) без метаболического синдрома. Иммуноферментный анализ функциональной активности системы комплемента по классическому пути проводили с использованием коммерческого набора. Реакцию лизиса эритроцитов барана, сенсибилизированных антителами (ЕА), оценивали турбидиметрически по снижению оптической плотности суспензии при длине волны 620 нм, степень лизиса определяли по калибровочному графику. Результаты. Разработан простой скрининг-тест для определения функциональной активности классического пути системы комплемента. Корреляционный анализ показал высокую степень сходимости результатов (r = 0,496), полученных разными методами. Заключение. Скрининг-тест для определения функциональной активности классического пути системы комплемента является быстрым, информативным и доступным для рутинных исследований. The aim of this work was to develop an accessible test for screening the functional activity of the complement system classic pathway. Materials and methods. Blood serum from 90 relatively healthy, overweight (BMI>25) individuals without the metabolic syndrome was studied. Enzyme immunoassay of the complement system functional activity following the classic pathway was performed using a commercial kit. The lysis of antibody-sensitized sheep erythrocytes was evaluated turbidimetrically by a decrease in suspension optical density at a wavelength of 620 nm; the lysis intensity was determined by the calibration curve. Results. A simple screening test was developed to determine the functional activity of the complement classic pathway. A high degree of correlation (r = 0.496) was observed between results obtained by different methods. This screening test for evaluating the functional activity of classic complement system pathway is fast, informative, and available for routine research.

Modulation of Human Complement System by Antimicrobial Peptide Arenicin-1 from Arenicola marina.

Antimicrobial peptides from marine invertebrates are known not only to act like cytotoxic agents, but they also can display some additional activities in mammalian organisms. In particular, these peptides can modulate the complement system as was described for tachyplesin, a peptide from the horseshoe crab. In this work, we investigated the influence on complement activation of the antimicrobial peptide arenicin-1 from the marine polychaete Arenicola marina. To study effects of arenicin on complement activation in human blood serum, we used hemolytic assays of two types, with antibody sensitized sheep erythrocytes and rabbit erythrocytes. Complement activation was also assessed, by the level of C3a production that was measured by ELISA. We found that the effect of arenicin depends on its concentration. At relatively low concentrations the peptide stimulates complement activation and lysis of target erythrocytes, whereas at higher concentrations arenicin acts as a complement inhibitor. A hypothetical mechanism of peptide action is proposed, suggesting its interaction with two complement proteins, C1q and C3. The results lead to the possibility of the development of new approaches for therapy of diseases connected with complement dysregulation, using peptide regulators derived from natural antimicrobial peptides of invertebrates.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte–macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte–macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.

Novel Small-Molecule Inhibitors Targeting Complement Factor D for Therapy of Paroxysmal Nocturnal Hemoglobinuria

INTRODUCTIONThe complement system is a pivotal player in multiple hematological conditions that include paroxysmal nocturnal hemoglobinuria (PNH). The current standard of care for PNH is intravenous infusion of eculizumab, a humanized monoclonal antibody that targets the terminal complement protein C5 and thereby efficiently impairs intravascular hemolysis. However, a significant fraction of PNH patients in clinical practice respond incompletely to eculizumab due to extravascular hemolysis by immune cells as eculizumab does not prevent erythrocyte opsonization. Additionally, a non-responsive sub-population has been identified with a rare genetic polymorphism in C5 that renders the variant incapable of binding eculizumab. Therefore, an unmet medical need remains for regimens that improve efficacy and that can be administered orally. To achieve this goal, we initiated a discovery program for small molecule inhibitors of complement factor D, a serine protease that is the rate limiting enzyme of the alternative complement pathway. We have obtained high resolution (1.5 Å) crystal structure of inhibitor-bound factor D. Through structure-guided optimization, we have discovered novel inhibitors of factor D that possess high potency and specificity as well as pharmacokinetic properties suitable for oral administration. Herein, we present a biochemical characterization of lead compounds and studies of their activities in various complement-mediated processes. METHODSThe binding affinity to factor D was determined by surface plasmon resonance (Biacore) analysis. The inhibitory effect on factor D protease was evaluated biochemically using both a nonspecific synthetic substrate and a natural substrate consisting of C3b and the complement factor B. The alternative pathway (AP) hemolytic assay was conducted with unsensitized rabbit erythrocytes, 8% human or non-human primate serum, and 10 mM MgEGTA. The classical pathway (CP) hemolytic assay was conducted with antibody-sensitized sheep erythrocytes and 0.5% human serum. To assess inhibition of CP activation through the AP-dependent amplification loop, inhibition of terminal complement complex (TCC) formation by 50% human serum was quantitated following activation by the CP-specific activator HAIGG. Inhibition of complement opsonization and lysis of PNH-like erythrocytes exposed to acidified serum (Ham test) was assessed ex vivo. Finally, selected lead compounds were administered to non-human primates to evaluate the efficacy of factor D inhibitors for blockade of alternative pathway-mediated events. RESULTSLead compounds obtained through optimization bound to factor D with Kd values below 1 nM. Tight binding to factor D of the compounds resulted in potent inhibition of factor D proteolytic activity assessed using a synthetic nonspecific substrate as well as a natural substrate. When evaluated in an AP hemolytic assay with human serum, the most potent compounds inhibited cell lysis with EC50 values below 10 nM. Similar inhibitory activity was observed when compounds were assessed in AP hemolytic assays with non-human primate serum, but not with rodent serum. The compounds showed no activity in a CP hemolytic assay using highly diluted human serum and antibody-coated erythrocytes, indicating specificity for factor D. When evaluated in the presence of 50% human serum and with TCC formation as an endpoint, however, the compounds blocked approximately 75% of TCC formation upon CP activation, a consequence of blockage of the AP-dependent amplification loop. Furthermore, the compounds prevented both opsonisation and lysis of PNH-like cells in the Ham test. Finally, when selected compounds were administered to non-human primates, potent inhibition of TCC formation following ex vivo AP activation was observed with EC50 values below 50 nM. CONCLUSIONSHighly potent and specific factor D inhibitors that block both the terminal pathway and opsonization were discovered using structure-guided design. These orally deliverable inhibitors hold promise as a novel therapeutic approach for treatment of complement-mediated hematological conditions including PNH. DisclosuresMorgan:Swedish Orphan Biovitrum: Consultancy, Research Funding; Glaxo Smith Kline: Consultancy; Achillion Pharmaceuticals: Consultancy, Research Funding. Thanassi:Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Wiles:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

A novel method for direct measurement of complement convertases activity in human serum.

Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function leads directly or indirectly to pathological conditions, including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischaemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and, thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. The use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of haemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong the half-life of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.

Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators

Adjuvant research is being oriented to TLR-agonists, but complement activation has been relatively unexplored. In previous studies it was demonstrated that poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (PVMA NPs) used as adjuvant differentially activate dendritic cells through toll like receptors (TLR) stimulation, however, a high dose of these NPs was used. Now, we demonstrated a dose–response effect, with a concentration as low as 20μg/mL able to stimulate TLR2 and TLR4 transfected dendritic cells. In addition, we investigated whether PVMA NPs are able to exploit also the immunomodulatory benefits of complement activation. Results indicated that the hydroxylated surface of these NPs highly activated the complement cascade, as measured by adsorption studies and a complement fixation bioassay. Stable binding of C3b to NPs was confirmed as indicated by lability to SDS treatment after washing resistance. Complement consumption was confirmed as the lytic capacity of complement exposed to NPs was abolished against antibody-sensitized sheep erythrocytes, with a minimal inhibitory concentration of 50μg NPs, equivalent to a surface of 1cm2. On the contrary, nanoparticles prepared with poly(lactic-co-glycolic acid) (PLGA), used as a reference, did not consume complement at a concentration ≥3mg NPs (≥40cm2). Complement consumption was inhibited when PVMA NPs were cross-linked with diamino groups (1,3-diaminopropane), indicating the role of hydroxyl groups as responsible of the phenomenon. These results favour a model whereby PVMA NPs adjuvant activate complement on site to attract immature antigen presenting cells that are activated through TLR2 and TLR4.

Low-molecular-weight heparin inhibition in classical complement activaton pathway during pregnancy

Introduction Low-molecular-weight heparin is used clinically for the prevention of pregnancy complications associated with prothrombotic disorders, particularly anti-phospholipid syndrome. Nevertheless, recent studies have suggested that heparin may exert direct effects on the placental trophoblast, independently of its anticoagulant activity. In addition, heparin prevents complement activation in vivo and protects mice from pregnancy complications. Materials and Methods The inhibition of the classical complement activation pathway by heparin was analyzed by means of in vitro assays and in pregnant women receiving prophylaxis with therapeutic doses (40 mg/day) of subcutaneous low molecular weight heparin by haemolysis of antibody-sensitized sheep erythrocytes (CH 50 assay). Results The specific interaction between low-molecular-weight heparin and the C1q subunit of the C1 complex of the complement cascade allowed the isolation of a small subpopulation of heparin ( 8.03 ± 1.20 μg %), with an anti-activated factor X activity more than four times greater than the starting material. This subpopulation could be responsible for the in vitro inhibition of the classical complement activation pathway evaluated by the total haemolysis of antibody-sensitized sheep erythrocytes. About 60 µg/ml of low molecular weight heparin was needed to achieve 50% of haemolysis. The detection of the classical complement pathway inhibition in pregnant women treated with heparin required a first activation with aggregated human IgG. Conclusions We concluded that the interaction between low-molecular-weight heparin and C1q could be relevant not only in the complement-dependent, but also in the complement-independent inflammation mechanisms responsible for the prevention of pregnancy loss.

Live Cell Imaging of Outward and Inward Vesiculation Induced by the Complement C5b-9 Complex

Cells resist death induced by the complement membrane attack complex (MAC, C5b-9) by removal of the MAC from their surface by an outward and/or inward vesiculation. To gain an insight into the route of MAC removal, human C9 was tagged with Alexa Fluor 488 and traced within live cells. Tagged C9-AF488 was active in lysis of erythrocytes and K562 cells. Upon treatment of K562 cells with antibody and human serum containing C9-AF488, C9-AF488 containing MAC bound to the cells. Within 5-10 min, the cells started shedding C5b-9-loaded vesicles (0.05-1 mum) by outward vesiculation. Concomitantly, C9-AF488 entered the cells and accumulated in a perinuclear, late recycling compartment, co-localized with endocytosed transferrin-Texas Red. Similar results were obtained with fixed cells in which the MAC was labeled with antibodies directed to a C5b-9 neoepitope. Inhibition of protein kinase C reduced endocytosis of C5b-9. Kinetic analysis demonstrated that peripheral, trypsin-sensitive C5b-9 was cleared from cells at a slower rate relative to fully inserted, trypsin-resistant C5b-9. MAC formation is controlled by CD59, a ubiquitously expressed membrane complement regulator. Analysis at a cell population level showed that the amount of C5b-9-AF488 bound to K562 cells after complement activation was highly heterogeneous and inversely correlated with the CD59 level of expression. Efficient C9-AF488 vesiculation was observed in cells expressing low CD59 levels, suggesting that the protective impact of MAC elimination by vesiculation increases as the level of expression of CD59 decreases.

Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni

Schistosomes are believed to evade complement-mediated damage by expression of complement inhibitory proteins. Our previous results [Deng, J., Gold, D., LoVerde, P.T., Fishelson, Z., 2003. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect. Immun. 71, 6402–6410.] have demonstrated that paramyosin (Pmy) of the blood fluke S. mansoni binds to the human complement proteins C8 and C9, inhibits complement activation at the terminal stage and protects the parasite from complement-mediated damage. In order to locate the Pmy binding site to C8 and C9, various fragments of Pmy cDNA were PCR-cloned into a pET28a bacterial expression vector. Recombinant His-tagged Pmy fragments were expressed in BL21 Escherichia coli and purified over a nickel–nitrilotriacetic acid column. Binding assays by Western blotting with monoclonal anti-His antibody demonstrated that PmyCC (Pmy amino acids 744Asp– 866Met) was the only Pmy fragment that bound to human C8 and C9. Functional analyses demonstrated that PmyCC inhibited hemolysis of rabbit erythrocytes and of antibody-sensitized sheep erythrocytes by human complement. Importantly, PmyCC inhibited in vitro killing of trypsin-sensitized schistosomula of S. mansoni by human complement. In the presence of PmyCC, Zn 2+-induced C9 polymerization was inhibited. Most of the immunodominant B-cell antigenic epitopes of Pmy are present in the PmyCC region, as antibodies collected from mice immunized with recombinant Pmy bound primarily to PmyCC. Taken together, this study has mapped the complement regulatory domain in Pmy, capable of binding to C8 and C9 and preventing polyC9 formation, to its C-terminal region.

Loxosceles Sphingomyelinase Induces Complement-Dependent Dermonecrosis, Neutrophil Infiltration, and Endogenous Gelatinase Expression

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Müllerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Mullerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.

The cleavage site of C5 from man and animals as a common target for neutralizing human monoclonal antibodies: in vitro and in vivo studies.

The isolation of an anti-C5 single-chain fragment variable (scFv) antibody, TS-A12/22, from a human phage display library, is described. This antibody inhibits the activation of C5 and the assembly of the terminal complement complex implicated in cell and tissue damage. Using antibody-sensitized sheep erythrocytes and rabbit red cells as target cells in hemolytic assays, we found that TS-A12/22 inhibited the activation of C5 by the convertases of both classical and alternative pathways. Western blot analysis and competition experiments with synthetic peptides showed that TS-A12/22 reacted with the alpha chain of C5 and recognized the cleavage site of this complement component by the C5 convertase. As a result, the antibody prevented splitting of C5 and inhibited the generation of C5a and of the terminal complement complex. The identification of the TS-A12/22 recognition site as a conserved sequence in man, mouse, rat and rabbit enabled the demonstration of in vitro inhibition of complement activity in these species. The scFv TS-A12/22 was tested in a rat model of antigen-induced arthritis and proved to be effective in preventing influx of polymorphonuclear cells into the knee joint and C9 deposition on synovial tissue.

A Targeted Disruption of the Murine Complement Factor B Gene Resulting in Loss of Expression of Three Genes in Close Proximity, Factor B, C2, and D17H6S45

Factor B is a serine protease, essential for the function of the alternative pathway of complement activation. To study further the importance of the alternative pathway of complement activation in vivo and to help elucidate any additional functions of factor B or its activation fragments we developed, by homologous recombination in embryonic stem cells, mice with a disrupted factor B gene. Factor B-deficient mice produced no detectable factor B mRNA or protein and had no detectable factor B enzymatic activity or alternative pathway function in their serum. Further studies revealed that the two adjacent genes, complement component C2 and D17H6S45, had been down regulated as a result of the disruption. The down-regulation of C2 gene expression was sufficient to cause a complete loss of classical pathway function as determined by the failure of sera from the deficient mice to opsonize antibody-sensitized sheep erythrocytes and by impairment of immune complex processing in vivo. The resulting mouse is deficient in both factor B and C2, and hence the alternative and classical pathways of complement activation, and adds to the repertoire of models for studying the in vivo role of complement in the immune system.

Stability of C3 convertase in the rat classical complement pathway.

It has been reported that rat serum complement causes efficient hemolysis of antibody-sensitized sheep erythrocytes (EA) at 20 C but not at 37 C. In connection with this, we demonstrated that C3 convertase of rat complement was significantly unstable at 37 C using purified components of rat complement.

Purification and characterization of the eighth and ninth components of carp complement

Complement components corresponding to mammalian C8 and C9 were isolated from carp ( Cyprinus carpio) serum. Carp C8 ( M r 146 000) proved to be a γ-globulin composed of three polypeptide chains (α-chain, M r 62 000; β-chain, M r 62 000); γ-chain, M r 22 000). The α-chain was disulfide-linked to the γ-chain and the β-chain was non-covalently associated with the α-γ chain, in fair agreement with mammalian C8. However, the N-terminal amino acid sequences of the three subunits showed no homology with those of human C8. Carp C9 was an α-globulin composed of a single polypeptide ( M r 91 000) and the N-terminus was blocked. Carp serum depleted of C8 did not hemolyse either carp antibody-sensitized sheep erythrocytes or non-sensitized rabbit erythrocytes, while C9-depleted carp serum did not hemolyse the former, but did hemolyse the latter target cells, as in the case of C9-depleted human serum.

Regulation of complement activity by immunoglobulin. I. Effect of immunoglobulin isotype on C4 uptake on antibody-sensitized sheep erythrocytes and solid phase immune complexes.

Intravenous Ig, composed principally of IgG, prevents complement attack by inhibiting C3 and C4 uptake onto target cells and tissues. Using two different models, Ab-sensitized SRBC and BSA-anti BSA solid phase immune complexes, we have examined the complement inhibitory capacity of three Ig classes (IgG, IgM, IgA) focussing on inhibition of C4 uptake. It was found that on both a weight and molar basis, monomeric serum IgA and IgM were far more active than IgG (weight efficiency ratios were 1.0, 20.8, and 236.3, and molar efficiency ratios 1.0, 24.0, and 1382.9 for polyclonal IgG, IgA1, and IgM, respectively). Monoclonal IgM were less active than polyclonal IgM (50% inhibition was achieved in SRBC model by 0.022, 0.30, 1.6, and 1.6 mg/ml of polyclonal IgM and monoclonal IgM from patients Lew, Will, and Pri). Secretory IgA was less active than serum IgA1 and similar in inhibitory activity to IgG (weight and molar efficiency ratios 1.5 and 0.6 compared with IgG). All tested preparations were less active in the solid phase immune complex model than in the sensitized cell model. A mixture of Igs of different isotypes was somewhat more active than any isotype alone. These results suggest that polyclonal serum IgA and IgM can also be considered for active therapy in diseases accompanied by the activation of classical complement pathway.

Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies.

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.

Functional Properties of Heterogeneous Human Asialo-C4 and Its Isotypes C4A and C4B

The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37°C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of T max of human C2, using asialo-C4 or buffer-treated C4 on {ieEAC1gp} and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active {ieC42} complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to {ieEAC1gp} (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQO; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B 1.