Complement activation

Mycobacterium tuberculosis-specific antibodies possess immunomodulatory effects during tuberculosis infection. Prior sensitization to environmental mycobacteria is known to suppress immune responses against BCG and M. tuberculosis. Mycobacteria-induced antibodies can influence events such as complement activation and phagocytosis during infectious process. In the present study role of anti-M. tuberculosis IgG (anti-M. tb IgG) antibody during interaction between M. tuberculosis and human macrophages mediated through complement has been examined in vitro. Anti-M. tb IgG antibody significantly enhanced complement activation by M. tuberculosis. Phagocytosis of M. tuberculosis by macrophages increased significantly in the presence of complement and/or antibody. Moreover, antibody enhanced phagocytosis in the presence of complement. Addition of antibody alone or in combination with complement also augmented intracellular viability of bacilli within macrophages. Results of this study showe...

and Transplantation (SIUT) from April 2007 to March 2008. The study was approved by the ethical review committee of SIUT. Two hundred consecutive patients of NS, both children and adults, were included in the study. Patients' demographic and laboratory data including age, sex, renal function and 24 hour urinary protein were collected.

The four human IgG isotypes are highly conserved in amino acid sequence, but show differential ability to activate complement (C'): IgG3 and IgGl are very active. IgG2 is active under certain conditions, and IgG4 is inactive. Although the second constant domain (C,2) is critical for C' activation, the individual amino acids that confer isotype-specific activity have not been identified. We have generated a series of mutants between IgG2 and IgG3, resulting in the exchange of the four N-terminal and six C-terminal polymorphic residues within C"2. Mutants containing the N-terminus of the C,2 of IgG3 were as effective as wildtype IgG3 in Clq binding, Cl activation and terminal complex (MAC) formation, but had reduced ability to effect C/-mediated lysis. IgG2 and mutants containing the N-terminal portion of the CH2 of IgG2 were reduced compared to IgG3 in activating Cl, binding Clq and inducing assembly of the MAC, and were inactive in mediating lysis of target cells. Thus, the amino acid sequence differences in the N-terminus of C,,2 play a critical role in determining the relative abilities of IgG2 and IgG3 to bind Clq and activate the C' cascade although additional residues of C,2 must be involved in mediating optimal target cells lysis. The sequence of the N-terminus of C,2 was less critical in determining C4 and C3 binding. Characterization of domain exchange mutants suggests that intermediate steps may be partly dependent on domains other than C,2. IgGs that do not direct target cell lysis nevertheless activate intermediate steps in the pathway, which may contribute to immune complex-associated disorders. pi;, 1997 Elsevier Science Ltd. All rights reserved.

Monoclonal antibodies (mAbs) are being increasingly used in cancer therapy owing to their ability to recognize specifically cancer cells and to activate complement-and cell-mediated cytotoxicity and/or to induce growth arrest or apoptosis. The therapeutic potential of anticancer antibodies is significantly limited due to the ability of cancer cells to block killing by complement. Of the multiple resistance strategies exploited by cancer cells, the expression of membrane complement regulatory proteins (mCRPs), such as CD46 (membrane cofactor protein (MCP)), CD55 (decay-accelerating factor (DAF)), CD35 (complement receptor type-1 (CR1)) and CD59, has received most attention. CD46, CD55 and CD35 block the complement cascade at the C3 activation stage and CD59 prevents assembly of the membrane attack complex of complement (MAC). These proteins protect normal tissues from accidental injury by activated complement, but also confer resistance on cancer cells, thereby limiting the effect of complement-fixing monoclonal antibodies. Expression of mCRPs on malignant cells is highly variable, yet there is clear indication that certain tumors express higher mCRP levels than the normal tissue from which they have evolved. mCRP level of expression and cellular location may also vary during malignant transformation and between differentiated and undifferentiated tumors. Neutralizing anti-mCRP mAbs have been used in vitro to elucidate the significance of mCRP expression to the tumor complement resistance phenotype. In general, CD59 appears to be the most effective mCRP protecting tumor cells from complement-mediated lysis. Nevertheless, it acts additively, and in certain tumors even synergistically, with CD55 and CD46. It is envisaged that treatment of cancer patients with mCRP blocking antibodies targeted specifically to cancer cells in combination with anticancer complement-fixing antibodies will improve the therapeutic efficacy.

The activation of complement via the mannan-binding lectin (MBL) pathway is initiated by the MBL complex consisting of the carbohydrate binding molecule, MBL, two associated serine proteases, MASP-1 and MASP-2, and a third protein, MAp19. In the present report we used an assay of complement activation specifically reflecting the physiological activity of the MBL complex to identify biological and synthetic inhibitors. Inhibitor activity towards the MBL complex was compared to the inhibition of the classical pathway C1 complex and to a complex of MBL and recombinant MASP-2. A number of synthetic inhibitors were found to differ in their activities towards complement activation via the MBL pathway and the classical pathway. C1 inhibitor inhibited both pathways whereas a 2 -macroglobulin (a 2 M) inhibited neither. C1 inhibitor and a 2 M were found to be associated with the MBL complex. Upon incubation at 37°C in physiological buffer, the associated inhibitors as well as MASP-1, MASP-2, and MAp19 dissociated from MBL, whereas only little dissociation of the complex occurred in buffer with high ionic strength (1 M NaCl). The difference in sensitivity to various inhibitors and the influence of high ionic strength on the complexes indicate that the activation and control of the MBL pathway differ from that of the classical pathway. MBL deficiency is linked to various clinical manifestations such as recurrent infections, severe diarrhoea, and recurrent miscarriage. On the other hand, impaired control of complement activation may lead to severe and often chronically disabling diseases. The results in the present report suggests the possibility of specifically inhibiting of the MBL pathway of complement activation.

Background: Cancer patients treated with the anticancer drug, paclitaxel (Taxol) often experience mild to severe hypersensitivity reactions. It is not known how these reactions are induced and whether the inducer is paclitaxel or its vehicle (i.e., Cremophor EL in 50% ethanol). Molecules present in Cremophor EL are similar in structure to certain nonionic block copolymers that activate complement proteins (i.e., proteins involved in various immune processes). To explore the role of complement in the observed hypersensitivity reactions, we studied the effects of paclitaxel and Cremophor EL plus ethanol on human complement in vitro. Methods: Serum specimens from healthy individuals and cancer patients were incubated with paclitaxel or with relevant control compounds (Cremophor EL with ethanol, ethanol only, docetaxel, and cyclosporine), and markers of complement activation (SC5b-9 and Bb) were measured by enzyme-linked immunosorbent assay. Similar incubations were performed in the presence of inhibitors of complement activation (i.e., EGTA/Mg 2+ and soluble complement receptor type 1 [sCR1]). Results: Paclitaxel in Cremophor EL plus ethanol caused increased formation of SC5b-9 in serum specimens from 10 of 10 healthy control subjects and from five of 10 cancer patients. Experiments with one or more individual sera indicated the above effect was due to Cremophor EL plus ethanol, that increased formation of Bb also occurred, that the drug-induced rise in SC5b-9 was inhibited by sCR1, and that EGTA/Mg 2+ partially inhibited SC5b-9 formation and stimulated Bb formation. Implication: The role of complement activation in hypersensitivity reactions associated with administration of paclitaxel in Cremophor EL plus ethanol should be studied in vivo. [J Natl Cancer Inst 1998;90:300-6] Paclitaxel (100 g/mL) was incubated with sera from two healthy donors (subjects 1 and 2) and two cancer patients (subjects 3 and 4) in the presence or absence of 10 mM EGTA plus 2.5 mM Mg 2+ . Serum levels of SC5b-9 and Bb were determined by enzyme-linked immunosorbent assay, as described in the ''Materials and Methods'' section. Entries are mean values with 95% confidence interval (CI) for three to four wells, followed by exact P values obtained by Student's two-sample t test (two-tailed) for differences between phosphate-buffered saline and paclitaxel and between paclitaxel and paclitaxel + EGTA/Mg 2+ . †Serum from subject 1 was incubated with 8.8 mg/mL Cremophor EL with 0.8% ethanol instead of paclitaxel.

20. A mean visuomotor processing time of 171 ms was estimated for both 1g and -1g trials, on the basis of methods described in . 21. Both the 1g model and the t model use on-line visual information about target position and velocity to trigger an interceptive movement when TTC(t) [see ] reaches a time threshold equal to the visuomotor processing time . However, the 1g model computes T TC(t) under the assumption that the target is accelerated by gravity [Eq. 1 in ], whereas the t model assumes that the target moves at constant speed [TTC(t) 0 ĥ w (t)=v w (t), see (4, 23)]. Therefore, if the brain uses the 1g model to predict target motion, the responses to 1g trials will be correctly timed. By contrast, the t model overestimates the TTC(t) of 1g trials and predicts that the corresponding responses should be systematically late. As a quantitative test, the time of response was computed separately for the five flight durations and was averaged across all trials of all subjects for a given flight duration. For 1g trials, the response times predicted by the 1g model were highly correlated with the experimental values in all subjects (mean r 0 0.998), whereas those predicted by the t model systematically failed to fit the experimental values (mean r 0 -0.5). The root mean square error (RMSE) of the fit to 1g trials was worse by a factor of 1.7 for the t model than for the 1g model. For -1g trials, the response times predicted by the t model were highly correlated with the experimental values in all subjects (mean r 0 0.973), and adequacy of the fit was demonstrated by the random distribution of the residuals around the mean over the data range. Instead, the 1g model fitted the responses to -1g trials less adequately, as shown by a systematic bias of the residuals around the mean (thus r value was not computed for this fit). Accordingly, the RMSE of the fit was 3.6 times worse for the 1g model than for the t model.

The pathogenic bacterium Staphylococcus aureus counteracts the host immune defense by excretion of the 85 residue staphylococcal complement inhibitor (SCIN). SCIN inhibits the central complement convertases; thereby, it reduces phagocytosis following opsonization and efficiently blocks all downstream effector functions. In this study, we present the crystal structure of SCIN at 1.8 Å resolution and the identification of its active site. Functional characterization of structure based chimeric proteins, consisting of SCIN and the structurally but nonfunctional homologue open reading frame-D, indicate an 18-residue segment (Leu-31-Gly-48) crucial for SCIN activity. In all complement activation pathways, chimeras lacking these SCIN residues completely fail to inhibit production of the potent mediator of inflammation C5a. Inhibition of alternative pathway-mediated opsonization (C3b deposition) and formation of the lytic membrane attack complex (C5b-9 deposition) are strongly reduced for these chimeras as well. For inhibition of the classical/lectin pathway-mediated C3b and C5b-9 deposition, the same residues are critical although additional sites are involved. These chimeras also display reduced capacity to stabilize the C3 convertases of both the alternative and the classical/lectin pathway indicating the stabilizing effect is pivotal for the complement inhibitory activity of SCIN. Because SCIN specifically and efficiently inhibits complement, it has a high potential in anti-inflammatory therapy. Our data are a first step toward the development of a second generation molecule suitable for such therapeutic complement intervention.

The outbreak of severe acute respiratory syndrome COVID-19 caused by SARS-CoV-2 in China represents a significant threat to global health. Unfortunately, effective therapeutic drugs and vaccines to cure SARS-CoV-2 are still lacking. Convalescent Plasma therapy which induces passive immunization seems a successful treatment for COVID-19 patients and also proves to be of huge value in terms of saving the severely ill patient. Plasma therapy acts by Blocking the Virus (Neutralization), Enhancing Phagocytosis (Opsonization), Immune System Activation (Complement Activation), and finally killing Virus (Antibody-Dependent Cell-mediated Cytotoxicity). Food and Drug Administration (FDA) of the United States was also given the stamp of approval for use of plasma therapy on 23 August 2020. In this review, we introduced the effective level of antibodies, potential mechanisms of CPT, WHO criteria for donating the plasma as well as future uncertainties of convalescent plasma therapy in the treatment of COVID-19 patients.

Background: Lutzomyia longipalpis is the vector of Leishmania infantum in the New World, and its saliva inhibits classical and alternative human complement system pathways. This inhibition is important in protecting the insect´s midgut from damage by the complement. L. longipalpis is a promiscuous blood feeder and must be protected against its host's complement. The objective of this study was to investigate the action of salivary complement inhibitors on the sera of different host species, such as dogs, guinea pigs, rats and chickens, at a pH of 7.4 (normal blood pH) and 8.15 (the midgut pH immediately after a blood meal). We also investigated the role of the chicken complement system in Leishmania clearance in the presence and absence of vector saliva. Results: The saliva was capable of inhibiting classical pathways in dogs, guinea pigs and rats at both pHs. The alternative pathway was not inhibited except in dogs at a pH of 8.15. The chicken classical pathway was inhibited only by high concentrations of saliva and it was better inhibited by the midgut contents of sand flies. Neither the saliva nor the midgut contents had any effect on the avian alternative pathway. Fowl sera killed L. infantum promastigotes, even at a low concentration (2%), and the addition of L. longipalpis saliva did not protect the parasites. The high body temperature of chickens (40°C) had no effect on Leishmania viability during our assays. Conclusion: Salivary inhibitors act in a species-specific manner. It is important to determine their effects in the natural hosts of Leishmania infantum because they act on canid and rodent complements but not on chickens (which do not harbour the parasite). Moreover, we concluded that the avian complement system is the probable mechanism through which chickens eliminate Leishmania and that their high body temperature does not influence this parasite.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5Gl. 1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5Gl. 1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5Gl. 1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5Gl. 1 scFv also effectively blocked C5a generation. Finally. an intact humanized h5Gl.l antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5Gl. 1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G 1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases. (0 1997 Elsevier Science Ltd. All rights reserved.

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not an CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.

Spirochetes were first observed in the oral cavity in humans by van Leeuwenhoek (34, 101, 102, 182). Spirochetes are gram-negative microorganisms with long spiral-shaped cells which achieve motility by means of periplasmic flagella (Fig. 1). Members of the phylum "Spirochaetes" include the etiologic agents of syphilis, Lyme disease and other borrelial relapsing fevers. The phylum consists of Spirochaetaceae, Serpulinaceae and Leptospiraceae (3, 39). The first group consists of Spirochaeta,

Pneumolysin, a member of the thiol-activated cytolysin family of toxins, is a virulence factor from the Gram-positive bacterium Streptococcus pneumoniae. The toxin forms large oligomeric pores in cholesterol-containing membranes of eukaryotic cells. A plethora of biochemical and mutagenesis data have been published on pneumolysin, since its initial characterization in the 1930s. Here we present an homology model of the monomeric and oligomeric forms of pneumolysin based on the recently determined crystal structure of perfringolysin O and electron microscopy data. A feature of the model is a striking electronegative surface on parts of pneumolysin that may re¯ect its cytosolic location in the bacterial cell. The models provide a molecular basis for understanding the effects of published mutagenesis and biochemical modi®cations on the toxic activity of pneumolysin. In addition, spectroscopic data are presented that shed new light on pneumolysin activity and have guided us to hypothesise a detailed model of membrane insertion. These data show that the environment of some tryptophan residues changes on insertion and/or pore formation. In particular, spectroscopic analysis of a tryptophan mutant, W433F, suggests it is the residue mainly responsible for the observed effects. Furthermore, there is no change in the secondary structure content when the toxin inserts into membranes. Finally, the basis of the very low activity shown by a pneumolysin molecule from another strain of S. pneumoniae may be due to the movements of a key domaindomain interface. The molecular basis of pneumolysin-induced complement activation may be related to the structural similarity of one of the domains of pneumolysin to Fc, rather than the presumed homology of the toxin to C-reactive protein as previously suggested.

During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the classical complement cascade, is expressed by postnatal neurons in response to immature astrocytes and is localized to synapses throughout the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retinogeniculate connections and the retention of excess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregulated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and suggest that complement-mediated synapse elimination may become aberrantly reactivated in neurodegenerative disease.

The aim of the research was to evaluate the effects of immunoglobulin G (IgG) colostrum concentration on goat kid immune status when the total amount of IgG fed was constant. Majorera goat kids (n = 56) were randomly assigned to 1 of 4 groups, and kids received 4 g of IgG per kg of body weight of atomized colostrum at 4 different IgG concentrations: 20 (AC-20), 40 (AC-40), 60 (AC-60), and 80 (AC-80) mg/mL. Blood samples were obtained on d 0, 1, 2, 3, 4, and 5 postpartum. Immunoglobulin G, IgA, and IgM plasma concentrations, apparent efficiency of absorption of IgG, plasma chitotriosidase activity, plasma complement activity, and plasma proteinogram were measured. Plasma IgG and IgM concentrations were highest on d 1 in AC-80 animals, and IgA plasma concentration was lower in AC-20 than in AC-80. The apparent efficiency of absorption was higher in AC-80 (24.4%) than in the other treatment groups (by an average of 13.8%). Chitotriosidase plasma activity on d 5 (1,488 nmol/mL per hour) was higher than on d 0 and 1 (average of 1,183 nmol/mL per hour). There were no effects of colostrum IgG concentration on complement activity and plasma protein distribution, but γ-globulin and α-globulin were lower on d 0 than on d 1, 2, 3, 4, and 5. Increasing the immunoglobulin concentration in colostrum using atomized colostrum improves the immunoglobulin absorption at the same amount of immunoglobulin fed.

Complement receptor (CR) 1 on human erythrocytes (Es) and complement factor (CF) H on rodent platelets perform immune adherence, a function which allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1-Tg/CFH −/− mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing antigen, IgG and C3 bound to Es in CR1-Tg/CFH −/− mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and 0022-1759/$ -see front matter D 2004 Published by Elsevier B.V. (M.R. Daha). 1 This publication represents the results of an EU sponsored collaboration between European academic partners and an industrial partner. Journal of Immunological Methods 296 (2005) 187 -198 www.elsevier.com/locate/jim defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form. D 2004 Published by Elsevier B.V.

Immunosafety analysis of pharmaceutical surfactants is an important step in understanding the complex mechanisms by which they induce side effects in susceptible patients. This paper provides experimental evidences that polyethoxylated surfactants, Cremophor-EL and Tween-80, also known as Polysorbate-80, activate the complement system in vitro, in normal human serum and plasma. They appeared to be more efficient reactogens than their structural homolog, Tween-20. Cremophor-EL and Tween-80 promoted the generation of biologically active complement products, C3a, C5a and C5b-9. Consistently, Paclitaxel and Taxotere (Docetaxel), pharmaceuticals formulated in Cremophor-EL and Tween-80, activated the complement system in similar extent. Moreover, comparison of serum reactivity against the drug-loaded and drug-free formulations exhibited a significant linear correlation. Taken together, these results are consistent with the hypothesis that therapeutic side effects, such as acute hypersensitivity and systemic immunostimulation, caused by intravenous nanomedicines containing polyethoxylated detergents such as Cremophor-EL and Tween-80, can be attributed to complement activation-derived inflammatory mediators.

Complement analysis in the clinic is usually associated with the quantification of C3 and C4, measurement of C1-inhibitor and screening for complement activity. These analyses have been available in routine diagnostic laboratories for decades. In recent years, however, the field of complement analysis has expanded considerably, with the introduction of novel assays to detect complement activation products, and spreading still further towards genetic analysis to reveal the basis of complement deficiencies and identify mutations and polymorphisms associated with defined diseases such as atypical haemolytic uraemic syndrome and age related macular degeneration. Here we review the current status of complement analysis, including assays for the quantification of complement activity and complement activation products, together with genetic methods for the detection of deficiencies, mutations and polymorphisms. This is an area where significant developments have been made recently, paralleling the research advances into the role of complement in human disease. It is clear, however, that there is a need for consensus and standardisation of analytical methods. This will be a major challenge for the complement society in the future.

Membrane based medical therapies have increased considerably in recent years. At the same time, awareness of the side-effects caused by blood material incompatibility has grown. The necessity of mass production at low cost has also contributed to the formation of demands placed on any membrane used in medical applications today. These demands are application specific transport characteristics, high clearance, blood compatibility and a design enabling cost efficient ways of production. A theoretical approach relating membrane structure to transport properties suggests that the deciding factors are pore radius, porosity, tortuosity, diffusion coefficients, pore shape and protein adsorption potential. A hollow fiber structure, which fits these demands for hemodialysis, hemodialfiltration and hemofiltration, is a three-layer structure consisting of an inner blood facing skin layer, followed by a sponge structure and a macroporous finger structure. The influence of the membrane surface on protein adsorption, leading to a change in permeability, has also been investigated. Two concepts for limiting protein and cell interaction with artificial surfaces are introduced. Copolymerization and blending of hydrophobic and hydrophilic polymers have been successful approaches. To limit blood-membrane interactions, an optimized microdomain surface structure formed by hydrophilic patches in a hydrophobic matrix has evolved. In vitro test methods for measuring activation levels of coagulation, kallikrein-kinin pathways, cell stimulation and complement activation were necessary for the development of highly optimized artificial membranes made for hemodialysis, hemodiafiltration and hemofiltration. The Polyflux S membranes, consisting of the hydrophobic polymers polyamide and polyarylethersulfone as well as hydrophilic poylvinylpyrrolidone, with their integral three-layer microdomain structures performed well in all of the mentioned in vitro tests.

Reducing the complement-activating capacity of a polymer surface is important in improving its blood compatibility. Polystyrene surfaces bearing hydroxymethyl (CH2OH) groups activate the alternative pathway of complement. This activation depends strongly on the density of the groups. Polystyrene surfaces bearing sulphonate (SO3-) groups adsorb proteins, resulting in an apparent activation. Polystyrene surfaces bearing both types of groups in close proportions are not activators in human serum, due to the adsorption of a protein of the alternative pathway, which has a protecting effect, not found when a polymer surface bearing hydroxyl groups is mixed in serum with another polymer surface bearing SO3- groups. In the presence of purified proteins of alternative pathway, C3 convertase activity can be created on each of these surfaces by deposition of C3b, but their susceptibility to inactivation by regulatory proteins H and I depends on the types of chemical groups present on the surfa...

Fish have many non-specific and specific, humoral and cellular mechanisms to resist bacterial diseases. Non-specific humoral factors include growth inhibiting substances, e.g. transferrin and antiproteases; lysins, e.g. lysozyme, C-reactive protein (CRP), bactericidal peptides and most importantly, complement which has lytic, proinflammatory, chemotactic and opsonic activities thus making a link with non-specific phagocyte responses. These are primarily executed by neutrophils and macrophages. The phagocytes contain many hydrolytic enzymes and when stimulated by bacteria produce reactive oxygen species (ROS), especially the hydroxyl radical, via generation of superoxide anions and nitric oxide. ROS are highly bactericidal. In addition, the neutrophils possess large quantities of myeloperoxidase (MPO) which generates bactericidal hypohalite ions from halide ions and H 2 O 2 . Macrophages can receive MPO from neutrophils and this may enhance their bactericidal activity. Antibody constitutes a specific humoral defence by inhibiting bacterial adherence or invasion of non-phagocytic host cells and neutralising bacterial toxins. Antibody can act as an opsonin and by activating complement by the classical pathway may lyse the bacterial cell, activate inflammation, phagocyte influx and further enhance phagocytosis. Macrophage bactericidal activity can be markedly increased by cytokines released by T lymphocytes on contact with their specific antigen. Pathogenic bacteria have evolved many ways of subverting these defence mechanisms of fish. These include the in vivo production of high a$nity iron uptake systems (e.g. siderophores and their surface receptors, in Aeromonas salmonicida, Vibrio anguillarum). Some bacteria e.g. V. anguillarum strains of serotype 01, while being resistant to the alternative pathway of complement activation in normal serum, are killed in immune serum. In other bacteria, surface layers, capsules and elongated lipopolysaccharide (LPS) 0-antigens protect against classical complement-mediated killing (e.g. A. salmonicida, Photobacterium piscicida, V. anguillarum serotype 02 respectively). While some important pathogens (e.g. P. piscicida) are readily killed by macrophages, others (A. salmonicida) possess specialised superoxide dismutase and catalase to resist ROS and are toxic themselves to macrophages. Renibacterium salmoninarum uses antibody, complement and other serum components to activate the respiratory burst of macrophages on immediate contact thus exhausting this macrophage bactericidal mechanism before the bacterium is phagocytosed. Subsequently, R, salmoninarum is taken up by the macrophage where it multiplies as an intracellular pathogen. However, these two highly adapted pathogens, A. salmonicida and R. salmoninarum are both killed by activated macrophages indicating that the whole concerted e#orts of specific and non-specific, humoral and cellmediated immune responses are required for immunity against these two important pathogens. 1999 Academic Press 291

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue-engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin. Natural biopolymers such as collagen and fibronectin have been investigated as potential sources of biomaterial to which cells can attach. The first generation of degradable polymers used in tissue engineering were adapted from other surgical uses and have drawbacks in terms of mechanical and degradation properties. This has led to the development of synthetic degradable gels primarily as a way to deliver cells and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is usually accompanied by fibrotic reactions that result in the production of a scar. Certain mammalian tissues, however, have a capacity for complete regeneration without scarring; good examples include embryonic or foetal skin and the ear of the MRL/MpJ mouse. Investigations of these model systems reveal that in order to achieve such complete regeneration, the inflammatory response is altered such that the extent of fibrosis and scarring is diminished. From studies on the limited examples of mammalian regeneration, it may also be possible to exploit such models to further clarify the regenerative process. The challenge is to identify the factors and cytokines expressed during regeneration and incorporate them to create a smart matrix for use in a skin equivalent. Recent advances in the use of DNA microarray and proteomic technology are likely to aid the identification of such molecules. This, coupled with recent advances in non-viral gene delivery and stem cell technologies, may also contribute to novel approaches that would generate a skin replacement whose materials technology was based not only upon intelligent design, but also upon the molecules involved in the process of regeneration.

Our failure to produce truly non-thrombogenic materials may reflect a failure to fully understand the mechanisms of biomaterialassociated thrombosis. The community has focused on minimizing coagulation or minimizing platelet adhesion and activation. We have infrequently considered the interactions between the two although we are generally familiar with these interactions. However, we have rarely considered in the context of biomaterial-associated thrombosis the other major players in blood: complement and leukocytes. Biomaterials are known agonists of complement and leukocyte activation, but this is frequently studied only in the context of inflammation. For us, thrombosis is a special case of inflammation. Here we summarize current perspectives on all four of these components in thrombosis and with biomaterials and cardiovascular devices. We also briefly highlight a few features of biomaterial-associated thrombosis that are not often considered in the biomaterials literature: * The importance of tissue factor and the extrinsic coagulation system. * Complement activation as a prelude to platelet activation and its role in thrombosis. * The role of leukocytes in thrombin formation. * The differing time scales of these contributions.

Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) play essential role in DNA synthesis, repair and cell division by catalyzing two subsequent reactions in thymidylate biosynthesis cycle. The lack of either enzyme leads to thymineless death of the cell, therefore inhibition of the enzyme activity is a common and successful tool in cancer chemotherapy and treatment of other diseases. However, the detailed mechanism of thymidylate synthesis cycle, especially the interactions between cycle enzymes and its role remain unknown.

The presence of albuminuria or proteinuria constitutes a sign of kidney damage and, together with the estimation of glomerular filtration rate, is based on the evaluation of chronic kidney disease. Proteinuria is a strong marker for progression of chronic kidney disease, and it is also a marker of increased cardiovascular morbimortality.

Activation of the complement system contributes significantly to the pathogenesis of numerous acute and chronic diseases. Recently, a monoclonal antibody (5Gl. 1) that recognizes the human complement protein C5, has been shown to effectively block C5 cleavage, thereby preventing the generation of the pro-inflammatory complement components C5a and C5b-9. Humanized 5Gl. 1 antibody, Fab and scFv molecules have been produced by grafting the complementarity determining regions of 5Gl. 1 on to human framework regions. Competitive ELISA analysis indicated that no framework changes were required in the humanized variable regions for retention of high affinity binding to C5, even at framework positions predicted by computer modeling to influence CDR canonical structure. The humanized Fab and scFv molecules blocked complement-mediated lysis of chicken erythrocytes and porcine aortic endothelial cells in a dose-dependent fashion, with complete complement inhibition occurring at a three-fold molar excess, relative to the human C5 concentration. In contrast to a previously characterized anti-C5 scFv molecule, the humanized h5Gl. 1 scFv also effectively blocked C5a generation. Finally. an intact humanized h5Gl.l antibody blocked human complement lytic activity at concentrations identical to the original murine monoclonal antibody. These results demonstrate that humanized h5Gl. 1 and its recombinant derivatives retain both the affinity and blocking functions of the murine 5G 1.1 antibody, and suggest that these molecules may serve as potent inhibitors of complement-mediated pathology in human inflammatory diseases. (0 1997 Elsevier Science Ltd. All rights reserved.

Acute motor axonal neuropathy (AMAN) is a pure motor axonal subtype of Guillain-Barré syndrome (GBS) that was identifi ed in the late 1990s. In Asia and Central and South America, it is the major subtype of GBS, seen in 30-65% of patients. AMAN progresses more rapidly and has an earlier peak than demyelinating GBS; tendon refl exes are relatively preserved or even exaggerated, and autonomic dysfunction is rare. One of the main causes is molecular mimicry of human gangliosides by Campylobacter jejuni lipo-oligosaccharides. In addition to axonal degeneration, electrophysiology shows rapidly reversible nerve conduction blockade or slowing, presumably due to pathological changes at the nodes or paranodes. Autoantibodies that bind to GM1 or GD1a gangliosides at the nodes of Ranvier activate complement and disrupt sodium-channel clusters and axoglial junctions, which leads to nerve conduction failure and muscle weakness. Improved understanding of the disease mechanism and pathophysiology might lead to new treatment options and improve the outlook for patients with AMAN.

Coexistence of viruses and their hosts imposes an evolutionary pressure on both the virus and the host immune system. On the one hand, the host has developed an immune system able to attack viruses and virally infected cells, whereas on the other hand, viruses have developed an array of immune evasion mechanisms to escape killing by the host's immune system. Generally, the larger the viral genome, the more diverse mechanisms are utilized to extend the time-window for viral replication and spreading of virus particles. In addition, herpesviruses have the capacity to hide from the immune system by their ability to establish latency. The strategies of immune evasion are directed towards three divisions of the immune system, i.e., the humoral immune response, the cellular immune response and immune effector functions. Members of the herpesvirus family are capable of interfering with the host's immune system at almost every level of immune clearance. Antibody recognition of viral epitopes, presentation of viral peptides by major histocompatibility complex (MHC) class I and class II molecules, the recruitment of immune effector cells, complement activation, and apoptosis can all be impaired by herpesviruses. This review aims at summarizing the current knowledge of viral evasion mechanisms.

Entada africana is a tree used in traditional medicine in Mali. The root is, for example, used for wound-healing. Since polysaccharides from other plants are thought to play a role in the wound-healing process, we wanted to study the polysaccharides present in the root of this species. The polysaccharides were extracted with water at 50 and 100°C and were further separated by anion exchange chromatography. The acidic fractions were finally purified by affinity chromatography on a Con A column. The fraction denoted Ea100 acidic I had the highest activity in the complement fixation test system, while the other acidic fractions had minor activities and the neutral fractions were almost negative. Ea100 acidic I has a structure resembling the arabinogalactan-protein type II polymer, which also was demonstrated by the abilities to precipitate with the Yariv reagent. The biological activity was reduced considerably after removal of arabinofuranoside residues by weak acid hydrolysis. The main core of the other polysaccharides extracted with 100°C were pectins resembling the rhamnogalacturonan type I. These fractions also contain arabinogalactan type II structures, shown by the formation of precipitates with the Yariv reagent.

The human antibody immunoglobulin G1 (IgG1) b12 neutralizes a broad range of human immunodeficiency virus-type 1 (HIV-1) isolates in vitro and is able to protect against viral challenge in animal models. Neutralization of free virus, which is an antiviral activity of antibody that generally does not require the antibody Fc fragment, likely plays an important role in the protection observed. The role of Fc-mediated effector functions, which may reduce infection by inducing phagocytosis and lysis of virions and infected cells, however, is less clear. To investigate this role, we constructed a panel of IgG1 b12 mutants with point mutations in the second domain of the antibody heavy chain constant region (CH2). These mutations, as expected, did not affect gp120 binding or HIV-1 neutralization. IgG1 b12 mediated strong antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of HIV-1-infected cells, but these activities were reduced or abrogated for the antibody mutants. Two mutants were of particular interest. K322A showed a twofold reduction in Fc␥R binding affinity and ADCC, while C1q binding and CDC were abolished. A double mutant (L234A, L235A) did not bind either Fc␥R or C1q, and both ADCC and CDC functions were abolished. In this study, we confirmed that K322 forms part of the C1q binding site in human IgG1 and plays an important role in the molecular interactions leading to complement activation. Less expectedly, we demonstrate that the lower hinge region in human IgG1 has a strong modulating effect on C1q binding and CDC. The b12 mutants K322A and L234A, L235A are useful tools for dissecting the in vivo roles of ADCC and CDC in the anti-HIV-1 activity of neutralizing antibodies.

The complement system is a component of the innate immune system. Its main function was initially believed to be limited to the recognition and elimination of pathogens through direct killing or stimulation of phagocytosis. However, in recent years, the immunoregulatory functions of the complement system were demonstrated and it was determined that the complement proteins play an important role in modulating adaptive immunity and in bridging innate and adaptive responses. When the delicate mechanisms that regulate this sophisticated enzymatic system are unbalanced, the complement system may cause damage, mediating tissue inflammation. Dysregulation of the complement system has been involved in the pathogenesis and clinical manifestations of several autoimmune diseases, such as systemic lupus erythematosus, vasculitides, Sjögren's syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis, and rheumatoid arthritis. Complement deficiencies have been associated with an increased risk to develop autoimmune disorders. Because of its functions, the complement system is an attractive therapeutic target for a wide range of diseases. Up to date, several compounds interfering with the complement cascade have been studied in experimental models for autoimmune diseases. The main therapeutic strategies are inhibition of complement activation components, inhibition of complement receptors, and inhibition of membrane attack complex. At present, none of the available agents was proven to be both safe and effective for treatment of autoimmune diseases in humans. Nonetheless, data from preclinical studies and initial clinical trials suggest that the modulation of the complement system could constitute a viable strategy for the treatment of autoimmune conditions in the decades to come.

The rapid proliferation of many different engineered nanomaterials (defined as materials designed and produced to have structural features with at least one dimension of 100 nanometers or less) presents a dilemma to regulators regarding hazard identification. The International Life Sciences Institute Research Foundation/Risk Science Institute convened an expert working group to develop a screening strategy for the hazard identification of engineered nanomaterials. The working group report presents the elements of a screening strategy rather than a detailed testing protocol. Based on an evaluation of the limited data currently available, the report presents a broad data gathering strategy applicable to this early stage in the development of a risk assessment process for Published: 06 October 2005 Particle and Fibre Toxicology 2005, 2:8 (page number not for citation purposes)

Background: Snake bite is one of the most neglected public health issues in poor rural communities worldwide. In addition to the clinical effects of envenoming, treatment with antivenom frequently causes serious adverse reactions, including hypersensitivity reactions (including anaphylaxis) and pyrogenic reactions. We aimed to investigate the immune responses to Sri Lankan snake envenoming (predominantly by Russell's viper) and antivenom treatment.

Factor I deficiency causes a permanent, uncontrolled activation of the alternative pathway resulting in an increased turnover of C3 and consumption of factor B, factor H and properdin. Factor I deficiency is clinically associated with recurrent bacterial infections already in early infancy, mainly affecting the upper and lower respiratory tract, or presenting as meningitis or septicemia. We here report on a Brazilian family (n = 9) with known consanguinity, where in 3/7 children, suffering from chronic otitis, meningitis, and respiratory infections, a complete factor I deficiency was recognized. One of the patients died after fulminant sepsis. Hemolytic activity of the alternative pathway was not detectable in the patients' sera due to decreased plasma concentrations of C3, factor B and properdin. As a consequence of factor I deficiency, C3b could not be metabolized with the result that no C3-derived split products (C3dg/C3d) were detectable in the patients' sera. In vitro reconstitution with purified factor I restored the regulatory function in the patients' sera with the subsequent cleavage of C3b to C3c and C3dg. Factor H levels were decreased in all patients' sera and found to be tightly complexed with C3b resulting in a modified electrophoretic mobility. Upon factor I reconstitution, factor H was released from C3b regaining its /3~ electrophoretic mobility. Complement-mediated biological functions like opsonization of bacteria, chemotactic activity and phagocytosis in these patients were impaired. The parents (cousins, 2nd degree) and 3/4 siblings had significantly reduced factor I plasma levels without further alteration in their complement profile. 3 of these obviously heterozygously deficient family members suffered from recurrent bacterial infections of different frequency and severity.

TL1A, a TNF-like ligand, mediates signaling via its cognate receptor DR3, a death receptor whose activation was known to induce both death and survival factors. TL1A, like TNF, is also presumed to circulate as a homotrimeric soluble form. To identify soluble TL1A in the immune system, we have developed a quantitative ELISA by pairing a monoclonal antibody (MAb) with a biotinylated anti-TL1A polyclonal antibody (PAb) as capture and detection antibodies, respectively. The assay had a detection limit of 32 pg/ml. Overnight culture of human umbilical vein endothelial cells (HUVEC) expressed up to 160 pg/ml of TL1A, and that proinflammatory cytokines, IL-1 and TNF, significantly increased TL1A mRNA and soluble protein up to several folds in contrast to IL-6 and IL-11, which induced neither protein nor mRNA in the cells. IL-1 appeared to be a better stimulator of TL1A than TNF-a, and the induction of soluble TL1A in HUVEC in response to IL-1 was dose and timedependent. We have also purified soluble TL1A from a large volume of IL-1 stimulated HUVEC conditioned medium using an anti-TL1A PAb-coupled affinity column. The protein eluted from the column was further reacted with anti-TL1A MAbs in Western blot: 30-kDa and 32-kDa under non-reducing and reducing conditions, respectively. Taken together, our results indicated that ELISA might be useful in studying soluble TL1A regulation in certain inflammatory conditions. D Signal transduction by DR3, a death domain-containing receptor related to TNFR-1 and CD95. Science 274, 990.