Anticomplementary Properties Research Articles

In vivo effect of quantified flavonoids-enriched extract of Scutellaria baicalensis root on acute lung injury induced by influenza A virus

BackgroundScutellaria baicalensis root is traditionally used for the treatment of common cold, fever and influenza. Flavonoids are the major chemical components of S. baicalensis root. PurposeTo evaluate the therapeutic effects and action mechanism of flavonoids-enriched extract from S. baicalensis root (FESR) on acute lung injury (ALI) induced by influenza A virus (IAV) in mice. MethodsThe anti-influenza, anti-inflammatory and anti-complementary properties of FESR and the main flavonoids were evaluated in vitro. Mice were challenged intranasally with influenza virus H1N1 (A/FM/1/47) 2 h before treatment. FESR (50, 100 and 200 mg/kg) was administrated intragastrically. Baicalin (BG), the most abundant compound in FESR was given as reference control. Survival rates, life spans and lung indexes of IAV-infected mice were measured. Histopathological changes, virus levels, inflammatory markers and complement deposition in lungs were analyzed. ResultCompared with the main compound BG, FESR and lower content aglycones (baicalein, oroxylin A, wogonin and chrysin) in FESR significantly inhibited H1N1 activity in virus-infected Madin-Darby canine kidney (MDCK) cells and markedly decreased nitric oxide (NO) production from lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In vitro assays showed that FESR and BG had no anti-complementary activity whereas baicalein, oroxylin A, wogonin and chrysin exhibited obvious anti-complementary activity.Oral administration of FESR effectively protected the IAV-infected mice, increased the survival rate (FESR: 67%; BG: 33%), decreased the lung index (FESR: 0.90; BG: 1.00) and improved the lung morphology in comparing with BG group. FESR efficiently decreased lung virus titers, reduced haemagglutinin (HA) titers and inhibited neuraminidase (NA) activities in lungs of IAV-infected mice. FESR modulated the inflammatory responses by decreasing the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1), and increasing the levels of interferon-γ (IFN-γ) and interleukin-10 (IL-10) in lung tissues. Although showing no anti-complementary activity in vitro, FESR obviously reduced complement deposition and decreased complement activation product level in the lung . ConclusionFESR has a great potential for the treatment of ALI induced by IAV and the underlying action mechanism might be closely associated with antiviral, anti-inflammatory and anti-complementary properties. Furthermore, FESR resulted in more potent therapeutic effect than BG in the treatment of IAV-induced ALI.

Certification of Human Immunoglobulin Standard Samples for Determination of Anticomplementary Activity

An evaluation and stability study of the original Russian human immunoglobulin standard intended for determining the anticomplementary activity and assessing the stability and acceptability of test results are reported. The certified values of the experimental anticomplementary activity of the negative control standard fell in the range 37.9 – 45.1%; of the positive control, 73.6 – 82.8% (for P = 0.95). Ashelf life of 1.5 years was justified based on the stability data of the standard sample. Introduction of the certified reference materials into the analysis of human immunoglobulin pharmaceutical preparation for intravenous administration during evaluation and quality control under government projects and confirmation of compliance with regulatory requirements will ensure that the determined anticomplementary properties are accurate.

Аттестация стандартного образца иммуноглобулина человека для определения антикомплементарной активности

Представлены материалы по аттестации и изучению стабильности впервые разработанного отечественного стандартного образца иммуноглобулина человека для определения антикомплементарной активности, применяемого для оценки стабильности и приемлемости результатов испытания. По результатам проведенных исследований аттестованные значения антикомплементарной активности отрицательного контроля стандартного образца находятся в интервале от 37,9 до 45,1 %, положительного контроля — в интервале от 73,6 до 82,8 % (с доверительной вероятностью 0,95). На основании материалов по изучению стабильности обоснован срок годности 1,5 года. Внедрение аттестованного стандартного образца в фармацевтический анализ лекарственных препаратов иммуноглобулинов человека для внутривенного введения при проведении экспертизы и контроля качества в рамках государственных заданий, а также с целью подтверждения соответствия требованиям нормативной документации позволит обеспечить достоверность определения антикомплементарных свойств.

Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides

Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosorbent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELISA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of suramin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC50 between Bupleurum smithii var. parvifolium polysaccharides (1.055mg/mL) and Bupleurum chinense polysaccharides (0.98mg/mL) showed that, similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti-complementary properties against the lectin pathway. The results demonstrate that the described ELISA assay can compensate for the shortcomings of the hemolytic assay in lectin pathway.

Antithrombotic and anticomplementary properties of a very low molecular mass dermatan sulfate

Introduction Dermatan sulfate, a sulfated glycosaminoglycan, acts as an anticoagulant by accelerating the inhibition of thrombin by heparin cofactor II. Materials and methods A low molecular mass dermatan sulfate was obtained by peroxy-radical depolymerization from a parent dermatan sulfate. Chemical characterization of this low molecular mass dermatan sulfate shows a material of ∼ 5 kDa that conserves sulfated sequences (2- O-sulfation of the iduronic acid units and/or 4 or 6 positions of galactosamina N acetyl) essential for dermatan sulfate–heparin cofactor II interaction with more sulphated proportion (27.7 ± 1.9 μg% vs 11.5 ± 0.8 μg%, P < 0.05 n = 6, low molecular mass dermatan sulfate vs dermatan sulfate). Results After a single intravenous administration of low molecular dermatan sulfate in rats, fibrinolytic activity increased simultaneously with thrombin clotting time prolongation. Low molecular dermatan sulfate showed an inhibitory effect on classical complement activation pathway reaching a maximum during the first hour. Furthermore, low molecular dermatan sulfate was as effective as dermatan sulfate to prevent thrombus formation and to diminish thrombus weight in a rat venous thrombosis model. Conclusions The results indicate that peroxy-radical depolymerization of dermatan sulfate produced a low molecular dermatan sulfate with profibrinolytic, thrombolytic, antithrombotic and anticomplement properties. We conclude that low molecular dermatan sulfate may be an effective adjunct in the management of thrombotic events.

Characterization of Selected Strains of Pneumococcal Surface Protein A

Several proteins, in addition to the polysaccharide capsule, have recently been implicated in the full virulence of the Streptococcus pneumoniae bacterial pathogen. One of these novel virulence factors of S. pneumoniae is pneumococcal surface protein A (PspA). The N-terminal, cell surface exposed, and functional part of PspA is essential for full pneumococcal virulence, as evidenced by the fact that antibodies raised against this part of the protein are protective against pneumococcal infections. PspA has recently been implicated in anti-complementary function as it reduces complement-mediated clearance and phagocytosis of pneumococci. Several recombinant N-terminal fragments of PspA from different strains of pneumococci, Rx1, BG9739, BG6380, EF3296, and EF5668, were analyzed using circular dichroism, analytical ultracentrifugation sedimentation velocity and equilibrium methods, and sequence homology. Uniformly, all strains of PspA molecules studied have a high alpha-helical secondary structure content and they adopt predominantly a coiled-coil structure with an elongated, likely rod-like shape. No beta-sheet structures were detected for any of the PspA molecules analyzed. All PspAs were found to be monomeric in solution with the exception of the BG9739 strain which had the propensity to partially aggregate but only into a tetrameric form. These structural properties were correlated with the functional, anti-complementary properties of PspA molecules based on the polar distribution of highly charged termini of its coiled-coil domain. The recombinant Rx1 PspA is currently under consideration for pneumococcal vaccine development.

Effects of triterpenoids from Pueraria lobata on immunohemolysis: beta-D-glucuronic acid plays an active role in anticomplementary activity in vitro.

The anticomplementary properties of kaikasaponin III (4) and soyasaponin I (8) from Pueraria lobata and their hydrolytic analogs were investigated in vitro. Diglycosidic saponins [kaikasaponin I (3), soyasaponin III (7)] showed most potent anticomplementary activities, followed by monoglycosidic saponins [soyasapogenol B monoglucuronide (6), sophoradiol monoglucuronide (2)] and triglycosidic saponins [soyasaponin I (8), kaikasaponin III (4)], whereas sophoradiol (1) and soyasapogenol B (5) showed enhancement of hemolysis under the presence of serum on the classical pathway of complement system. But all of them showed very weak or no anticomplementary activities on the alternative pathway of complement system. The anticomplementary activity of the saponins was influenced by the nature of glucuronic acid, where the free acid forms (-COOH) showed much more potent activity than the sodium salt forms (-COO-Na+) or methyl ester forms (-COOCH3), and the reduced forms (-CH2OH) decreased the activity significantly.

Anti-complement activity of tiliroside from the flower buds of Magnolia fargesii.

As part of the search for anticomplementary active components from natural products, the anticomplementary properties of methanolic extracts from the flower buds of Magnoliafargesii have been investigated. Bioassay-guided chromatographic separation of the active constituents led to the isolation of compound 1, whose structure was identified by spectroscopic methods to be kaempferol 3-O-beta-D-(6"-O-coumaroyl)glucopyranoside (tiliroside). Tiliroside showed very potent anti-complement activity (IC50=5.4 x 10(-5) M) on the classical pathway of the complement system, even higher than rosmarinic acid, which is a well-known inhibitor against the complement system. On the other hand, the hydrolysates of tiliroside, kaempferol, astragalin and p-coumaric acid showed very weak activity on this system.

Sulfonated dextran inhibits complement activation and complement-dependent cytotoxicity in an in vitro model of hyperacute xenograft rejection

In the present study, we demonstrate that a substituted soluble dextran derivative bearing 73% carboxylic groups and 15% benzylamide sulfonate groups, termed CMDBS25, inhibits complement activation and complement-mediated damage in an in vitro model of xenogeneic rejection. Incubation of porcine aortic endothelial cells with normal human serum resulted in time-dependent complement consumption as assessed by C3a generation in the fluid phase and deposition of activated complement fragments C3, C5 and of C5b-9 on target cells. The presence of C5b-9 membrane attack complex was associated with 51Cr release from prelabelled endothelial cells. The addition of 5–25 mg of CMDBS25/ml under the experimental conditions used, inhibited complement activation and C3a generation in a dose-dependent fashion. CMDBS25 (25 mg/ml) totally suppressed iC3b, C5 and C5b-9 cytolytic complex deposition on cells and inhibits by 42% lysis of target endothelial cells. Native dextran had no effect. Our observations document the anti-complementary properties of sulfonated dextran derivatives and their potential as therapeutic agents for the prevention of complement-dependent hyperacute xenograft rejection.

Effect of substituted dextran derivative on complement activation in vivo

A soluble dextran derivatized with carboxylic groups (73%) and benzylamide sulphonate groups (15%), termed CMDBS 25, exhibited significant anticomplementary activity in the absence of anticoagulant activity. The polysaccharide inhibited both classical and alternative pathway-dependent complement activation in human and rat serum in vitro. Simultaneous administration of CMDBS 25 (100 mg) and crushed Sephadex G25 (20 mg) into normal Lewis rats suppressed systematic complement consumption that was induced by Sephadex in the animals by 98% for 1 h. Two consecutive injections of 100 mg of CMDBS at 1 h interval resulted in total suppression of systemic complement activation for 2 h and in 50% suppression for an additional 2 h. Infusion of CMDBS alone was well tolerated and had no effect on CH50 in serum in vivo. Our results demonstrate that CMDBS 25 exhibits anticomplementary properties In vivo and suggest that the polymer represents a potential therapeutic agent for pathological conditions associated with complement activation.

Anticomplementary Properties of Plasmin-Treated Human G Immunoglobulin and Its Components

Plasmin-treated human gamma-globulin, isolated plasmin residual portion (PRG), Fab and Fc fragments were tested for anticomplementary properties. The plasmin digest had very low anticomplementary activity while some of its components were anticomplementary, the latter probably due to alterations resulting from the isolation procedures employed. The highest activity was found in the isolated Fc fragment, no activity being detected in the isolated Fab fragment. Various mixtures of the isolated components were also tested, the results indicating that the increase in anticomplementary activity of the PRG and the Fc fragment was markedly lower when the Fab fragment was present.

An improved antigen for the paratuberculosis complement fixation test

Methanol extraction of acetone-dried Mycobacterium paratuberculosis gave an antigen preparation which fixed complement, was highly active, stable and lacked anticomplementary properties. Serological trials using cattle, sheep, rabbit and guinea-pig antisera showed a high degree of correlation between the Maltaner-Wadsworth antigen and the methanol antigen.

Media exchange method for demonstrating killing of L-phase variants in solid media by normal human serum.

Normal human serum has been shown to kill L-phase variants in a fluid system by the action of antibody plus complement. Previous studies, however, failed to demonstrate such killing in solid media. This failure probably resulted from inhibition of complement by medium components such as agar and NaCl. A method was developed which circumvents the problems of the anticomplementary properties of agar media and the requirement of some L-phase variants for concentrations of salt that inhibit complement. Using this method, we have demonstrated in solid media the killing by normal human serum of the L-phase variants of Staphylococcus aureus, Proteus mirabilis, Streptococcus pyogenes, S. faecalis, Pseudomonas aeruginosa, and Salmonella typhi. This method provides a relatively simple and graphic means for studying host humoral factors lethal for L-phase variants.

Immunochemical Properties of Fc Fragment of Rabbit γG-Globulin

Summary Various fractions comprising Fc fragment appear to be similar in their reactivity to horse and goat anti-rabbit γG-globulin serum, as determined by immunodiffusion and quantitative immunoprecipitation techniques. These fractions show marked differences in their anti-complementary properties. These results indicate that the anti-complementary site of Fc fragment may be different from its major antigenic determinant.

A new method for isolation and fractionation of complement fixing antigens from Plasmodium knowlesi.

SummaryA new method is presented for nonchemical isolation and fractionation of the malaria parasite Plasmodium knowlesi. Since the mechanical fragility of erythrocytes is considerably greater than that of the Plasmodia, it was possible to preferentially fragment the red cell membrane without apparent alteration of the parasites. This was achieved by utilizing the precisely controlled conditions obtained with a French pressure cell operated at 1500-2000 psi. Parasites isolated by this method were subsequently fragmented at higher pressures (20,000 psi) and the solubil-ized substances fractionated by gel filtration. Fractions showing the major complement fixing activity reacted strongly with P. knowlesi antisera, were colorless, exhibited no anticomplementary properties and showed no serologic evidence of red cell contaminants. The findings indicate that the method may provide essential material for further studies on plasmodial composition and be of particular value for isolation of other intra-erythro-cy...

New complement fixing antigen for serodiagnosis of gonorrhoea.

A new method of preparing gonococcal antigen for use in the complement fixation test is presented. Briefly, the procedure consists of extracting Neisseria gonorrhoeae cells with pyridine and then exposing the washed sediment to ultrasonic treatment.The new method is superior to that of Price by yielding highly potent, genus-specific and stable antigens, which are free of anticomplementary properties and possess a broader antigenic valence and a longer range of working antigenic power. It is simpler and more practical than Torrey's modification of the Price method since the lengthy and laborious procedure of strain selection is eliminated.In routine use for the past several years, the new antigen has proved to be specific and reliable in the complement fixation test. The test itself provides a useful supplementary aid to the laboratory diagnosis of gonorrhoea.

Studies of Aggregated γ-Globulin

Summary Molecular aggregation occurs when solutions of γ-globulin are heated to 56°C. Aggregated γ-globulin, when concentrated by salt fractionation, demonstrated marked anticomplementary properties. Preliminary C′ studies suggested that absorption of guinea pig serum with solid γ-globulin resulted in C′ component destruction that resembled C′ inactivation by immune systems.

Serologic Analysis of Ehrlich Tumor-Cell Induced Ascites Fluid.

SummaryAscites fluid induced in mice by intraperitoneal injection of Ehrlich tumor cells contained specific antibody to S. typhimurium when mice were immunized against this organism. The serum and ascites were identical in their anticomplementary properties and neither contained detectable complement. The serum had normal properdin levels. but the ascites did not contain detectable properdin under the assay conditions employed.

Removal of Anticomplementary Properties of Serum with Kaolin

Removal of Anticomplementary Properties of Serum with Kaolin

A practical method for removal of anticomplementary properties from human serum.

A practical method for removal of anticomplementary properties from human serum.