Alternative Complement Pathway Inhibition Abrogates Pneumococcal Opsonophagocytosis in Vaccine-Naïve, but Not in Vaccinated Individuals.

To assess the relative contribution of opsonisation by antibodies, classical and alternative complement pathways to pneumococcal phagocytosis, we analyzed killing of pneumococci by human blood leukocytes collected from vaccine-naïve and PCV13-vaccinated subjects. With serotype 4 pneumococci as model, two different physiologic opsonophagocytosis assays based on either hirudin-anticoagulated whole blood or on washed cells from EDTA-anticoagulated blood reconstituted with active serum, were compared. Pneumococcal killing was measured in the presence of inhibitors targeting the complement components C3, C5, MASP-2, factor B or factor D. The two assay formats yielded highly consistent and comparable results. They highlighted the importance of alternative complement pathway activation for efficient opsonophagocytic killing in blood of vaccine-naïve subjects. In contrast, alternative complement pathway inhibition did not affect pneumococcal killing in PCV13-vaccinated individuals. Independent of amplification by the alternative pathway, even low capsule-specific antibody concentrations were sufficient to efficiently trigger classical pathway mediated opsonophagocytosis. In heat-inactivated or C3-inhibited serum, high concentrations of capsule-specific antibodies were required to trigger complement-independent opsonophagocytosis. Our findings suggest that treatment with alternative complement pathway inhibitors will increase susceptibility for invasive pneumococcal infection in non-immune subjects, but it will not impede pneumococcal clearance in vaccinated individuals.

Complement in neurological disorders and emerging complement-targeted therapeutics.

The complement system consists of a network of plasma and membrane proteins that modulate tissue homeostasis and contribute to immune surveillance by interacting with the innate and adaptive immune systems. Dysregulation, impairment or inadvertent activation of complement components contribute to the pathogenesis of some autoimmune neurological disorders and could even contribute to neurodegenerative diseases. In this Review, we summarize current knowledge about the main functions of the complement pathways and the involvement of complement in neurological disorders. We describe the complex network of complement proteins that target muscle, the neuromuscular junction, peripheral nerves, the spinal cord or the brain and discuss the autoimmune mechanisms of complement-mediated myopathies, myasthenia, peripheral neuropathies, neuromyelitis and other CNS disorders. We also consider the emerging role of complement in some neurodegenerative diseases, such as Alzheimer disease, amyotrophic lateral sclerosis and even schizophrenia. Finally, we provide an overview of the latest complement-targeted immunotherapies including monoclonal antibodies, fusion proteins and peptidomimetics that have been approved, that are undergoing phase I-III clinical trials or that show promise for the treatment of neurological conditions that respond poorly to existing immunotherapies.

Clinicopathological features of C3 glomerulopathy in children: a single-center experience.

BACKGROUND: C3 glomerulopathy (C3G) is defined by dominant glomerular deposition of C3 and minimal or no immunoglobulin, with two subtypes-dense deposit disease (DDD) and C3 glomerulonephritis (C3GN)-distinguished by features on electron microscopy (EM). Given that this rare disease has generally unfavorable yet highly variable outcomes, we sought out to review the histopathology, complement/genetic studies, and renal outcomes of pediatric patients with C3G at our institution. METHODS: All native kidney biopsies performed in a single pediatric hospital over a 10-year period were reviewed for features of C3G. Of 589 biopsy reports, we identified 9 patients fulfilling the diagnostic criteria for C3G and retrospectively reviewed their clinical chart and renal biopsy findings. RESULTS: We identified 4 patients with DDD, 4 with C3GN, and 1 indeterminate case, with features of both C3GN and DDD. Five patients were positive for one or more nephritic factors (C3NeF, C4NeF, C5NeF) with 1 patient additionally positive for complement factor H (CFH) autoantibody. Genetic testing done in 5 of the 9 patients failed to identify any causative mutations. Three patients showed progressive renal dysfunction over a mean follow-up period of 33 months. CONCLUSIONS: Complement and genetic studies are now routinely recommended for patients with a histopathological diagnosis of C3G. Careful interpretation of these studies and their prognostic and therapeutic implications in conjunction with biopsy findings is needed to further understand the pathophysiology of this rare disease in children.

Complement C5b-9 and Cancer: Mechanisms of Cell Damage, Cancer Counteractions, and Approaches for Intervention.

The interactions of cancer cells with components of the complement system are highly complex, leading to an outcome that is either favorable or detrimental to cancer cells. Currently, we perceive only the "tip of the iceberg" of these interactions. In this review, we focus on the complement terminal C5b-9 complex, known also as the complement membrane attack complex (MAC) and discuss the complexity of its interaction with cancer cells, starting with a discussion of its proposed mode of action in mediating cell death, and continuing with a portrayal of the strategies of evasion exhibited by cancer cells, and closing with a proposal of treatment approaches targeted at evasion strategies. Upon intense complement activation and membrane insertion of sufficient C5b-9 complexes, the afflicted cells undergo regulated necrotic cell death with characteristic damage to intracellular organelles, including mitochondria, and perforation of the plasma membrane. Several pro-lytic factors have been proposed, including elevated intracellular calcium ion concentrations and activated JNK, Bid, RIPK1, RIPK3, and MLKL; however, further research is required to fully characterize the effective cell death signals activated by the C5b-9 complexes. Cancer cells over-express a multitude of protective measures which either block complement activation, thus reducing the number of membrane-inserted C5b-9 complexes, or facilitate the elimination of C5b-9 from the cell surface. Concomitantly, cancer cells activate several protective pathways that counteract the death signals. Blockage of complement activation is mediated by the complement membrane regulatory proteins CD46, CD55, and CD59 and by soluble complement regulators, by proteases that cleave complement proteins and by protein kinases, like CK2, which phosphorylate complement proteins. C5b-9 elimination and inhibition of cell death signals are mediated by caveolin and dynamin, by Hsp70 and Hsp90, by the mitochondrial stress protein mortalin, and by the protein kinases PKC and ERK. It is conceivable that various cancers and cancers at different stages of development will utilize distinct patterns of these and other MAC resistance strategies. In order to enhance the impact of antibody-based therapy on cancer, novel precise reagents that block the most effective protective strategies will have to be designed and applied as adjuvants to the therapeutic antibodies.

Paramyxovirus activation and inhibition of innate immune responses.

Paramyxoviruses represent a remarkably diverse family of enveloped nonsegmented negative-strand RNA viruses, some of which are the most ubiquitous disease-causing viruses of humans and animals. This review focuses on paramyxovirus activation of innate immune pathways, the mechanisms by which these RNA viruses counteract these pathways, and the innate response to paramyxovirus infection of dendritic cells (DC). Paramyxoviruses are potent activators of extracellular complement pathways, a first line of defense that viruses must face during natural infections. We discuss mechanisms by which these viruses activate and combat complement to delay neutralization. Once cells are infected, virus replication drives type I interferon (IFN) synthesis that has the potential to induce a large number of antiviral genes. Here we describe four approaches by which paramyxoviruses limit IFN induction: by limiting synthesis of IFN-inducing aberrant viral RNAs, through targeted inhibition of RNA sensors, by providing viral decoy substrates for cellular kinase complexes, and through direct blocking of the IFN promoter. In addition, paramyxoviruses have evolved diverse mechanisms to disrupt IFN signaling pathways. We describe three general mechanisms, including targeted proteolysis of signaling factors, sequestering cellular factors, and upregulation of cellular inhibitors. DC are exceptional cells with the capacity to generate adaptive immunity through the coupling of innate immune signals and T cell activation. We discuss the importance of innate responses in DC following paramyxovirus infection and their consequences for the ability to mount and maintain antiviral T cells.

Specific acquisition of functional CD59 but not CD46 or CD55 by hepatitis C virus.

Viruses of different families encode for regulators of the complement system (RCAs) or acquire such RCAs from the host to get protection against complement-mediated lysis (CML). As hepatitis C virus (HCV) shares no genetic similarity to any known RCA and is detectable at high titers in sera of infected individuals, we investigated whether HCV has adapted host-derived RCAs to resist CML. Here we report that HCV selectively incorporates CD59 while neither CD55, nor CD46 are associated with the virus. The presence of CD59 was shown by capture assays using patient- and cell culture-derived HCV isolates. Association of CD59 with HCV was further confirmed by Western blot analysis using purified viral supernatants from infected Huh 7.5 cells. HCV captured by antibodies specific for CD59 remained infectious for Huh 7.5 cells. In addition, blocking of CD59 in the presence of active complement reduced the titer of HCV most likely due to CML. HCV produced in CD59 knock-down cells were more significantly susceptible to CML compared to wild type virus, but neither replication, assembly nor infectivity of the virus seemed to be impaired in the absence of CD59. In summary our data indicate that HCV incorporates selectively CD59 in its envelope to gain resistance to CML in serum of infected individuals.

Functional recruitment of the human complement inhibitor C4BP to Yersinia pseudotuberculosis outer membrane protein Ail.

Ail is a 17-kDa chromosomally encoded outer membrane protein that mediates serum resistance (complement resistance) in the pathogenic Yersiniae (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis). In this article, we demonstrate that Y. pseudotuberculosis Ail from strains PB1, 2812/79, and YPIII/pIB1 (serotypes O:1a, O:1b, and O:3, respectively) can bind the inhibitor of the classical and lectin pathways of complement, C4b-binding protein (C4BP). Binding was observed irrespective of serotype tested and independently of YadA, which is the primary C4BP receptor of Y. enterocolitica. Disruption of the ail gene in Y. pseudotuberculosis resulted in loss of C4BP binding. Cofactor assays revealed that bound C4BP is functional, because bound C4BP in the presence of factor I cleaved C4b. In the absence of YadA, Ail conferred serum resistance to strains PB1 and YPIII, whereas serum resistance was observed in strain 2812/79 in the absence of both YadA and Ail, suggesting additional serum resistance factors. Ail from strain YPIII/pIB1 alone can mediate serum resistance and C4BP binding, because its expression in a serum-sensitive laboratory strain of Escherichia coli conferred both of these phenotypes. Using a panel of C4BP mutants, each deficient in a single complement control protein domain, we observed that complement control protein domains 6-8 are important for binding to Ail. Binding of C4BP was unaffected by increasing heparin or salt concentrations, suggesting primarily nonionic interactions. These results indicate that Y. pseudotuberculosis Ail recruits C4BP in a functional manner, facilitating resistance to attack from complement.

Staphylococcus aureus virulence is enhanced by secreted factors that block innate immune defenses.

Staphylococcus aureus is a leading human pathogen that causes a large variety of diseases. In vitro studies have shown that S. aureus secretes several small proteins that block specific elements of the host innate immune system, but their role in bacterial pathogenicity is unknown. For instance, the extracellular complement-binding protein (Ecb) impairs complement activation by binding to the C3d domain of C3. Its homolog, the extracellular fibrinogen-binding protein (Efb), is known to block both complement activation and neutrophil adhesion to fibrinogen. Here, we show that targeted inactivation of the genes encoding Ecb and Efb strongly attenuates S. aureus virulence in a murine infection model: mice experienced significantly higher mortality rates upon intravenous infection with wild-type bacteria (79%) than with an isogenic ΔEcbΔEfb mutant (21%). In addition, Ecb and Efb are both required for staphylococcal persistence in host tissues and abscess formation in the kidneys (27% for wild-type vs. 7% for the ΔEcbΔEfb mutant). During staphylococcal pneumonia, Ecb and Efb together promote bacterial survival in the lungs (p = 0.03) and block neutrophil influx into the lungs. Thus, Ecb and Efb are essential to S. aureus virulence in vivo and could be attractive targets in future vaccine development efforts.

Advances in understanding the structure, function, and mechanism of the SCIN and Efb families of Staphylococcal immune evasion proteins.

Our understanding of both the nature and diversity of Staphylococcal immune evasion proteins has increased tremendously throughout the last several years. Among this group of molecules, members of the SCIN and Efb families of complement inhibitors have been the subject of particularly intense study. This work has demonstrated that both types of proteins exert their primary function by inhibiting C3 convertases, which lie at the heart of the complement-mediated immune response. Despite this similarity, however, significant differences in structure/function relationships and mechanisms of action exist between these bacterial proteins. Furthermore, divergent secondary effects on host immune responses have also been described for these two protein families. This chapter summarizes recent advances toward understanding the structure, function, and mechanism of the SCIN and Efb families, and suggests potential directions for the field over the coming years.

Functional recruitment of human complement inhibitor C4B-binding protein to outer membrane protein Rck of Salmonella.

Resistance to complement mediated killing, or serum resistance, is a common trait of pathogenic bacteria. Rck is a 17 kDa outer membrane protein encoded on the virulence plasmid of Salmonella enterica serovars Typhimurium and Enteritidis. When expressed in either E. coli or S. enterica Typhimurium, Rck confers LPS-independent serum resistance as well as the ability to bind to and invade mammalian cells. Having recently shown that Rck binds the inhibitor of the alternative pathway of complement, factor H (fH), we hypothesized that Rck can also bind the inhibitor of the classical and lectin pathways, C4b-binding protein (C4BP). Using flow cytometry and direct binding assays, we demonstrate that E. coli expressing Rck binds C4BP from heat-inactivated serum and by using the purified protein. No binding was detected in the absence of Rck expression. C4BP bound to Rck is functional, as we observed factor I-mediated cleavage of C4b in cofactor assays. In competition assays, binding of radiolabeled C4BP to Rck was reduced by increasing concentrations of unlabeled protein. No effect was observed by increasing heparin or salt concentrations, suggesting mainly non-ionic interactions. Reduced binding of C4BP mutants lacking complement control protein domains (CCPs) 7 or 8 was observed compared to wt C4BP, suggesting that these CCPs are involved in Rck binding. While these findings are restricted to Rck expression in E. coli, these data suggest that C4BP binding may be an additional mechanism of Rck-mediated complement resistance.

Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

How to avoid complement attack in baculovirus-mediated gene delivery.

Serum inactivation of baculovirus vectors is a significant barrier to the development of these highly efficient vectors for therapeutic gene delivery. In this review we will describe the efforts taken to avoid complement attack by passive or active measures. Evidently good targets for baculovirus-mediated gene delivery include immunoprivileged tissues, such as eye, brain and testis. Similarly baculovirus vectors have also proven their efficacy in an ex vivo setting for tissue engineering. Active measures to inhibit complement include the use of pharmacological inhibitors of complement as well as surface engineering of the baculoviral vectors through the use of synthetic polymers, pseudotyping or display of complement inhibitors. Lessons learned from these studies will significantly increase the possibility of using baculovirus vectors for therapeutic applications.

C1q aggregate binding for the determination of anti-complementary activity of immunoglobulin products.

Aggregates in human immunoglobulin (Ig) products can develop due to employed manufacturing, formulation and storage conditions and can cause adverse reactions in patients. The test for anti-complementary activity (ACA) recommended by the European Pharmacopoeia (EP) is insensitive, variable and time consuming. We have optimised a commercial assay for the detection and quantitation of C1q binding aggregates in intravenous and intramuscular IgG preparations. The generation of C4d, iC3b and SC5b-9 induced by aggregates in vitro was measured by enzyme-linked immunosorbent assays (ELISA). In establishing the sensitivity of the C1q aggregate binding assay to detect IgG aggregates in comparison to turbidity and ACA, pure IgG at neutral and acidic pH was heated for various lengths of time to generate varying amounts of aggregates. The level of C1q binding aggregates was 7 fold greater in intramuscular samples. These aggregates were capable of activating complement in vitro and correlated with an increase in ACA. C1q aggregate binding was apparent before any quantifiable turbidity and ACA in the heat-treated samples. Furthermore, the C1q binding assay could discriminate between different levels of aggregates where ACA had reached a plateau. C1q aggregate binding is a sensitive, convenient, specific and robust means of detecting aggregates with a propensity for complement activation.

Lack of complement inhibitors in the outer intracranial artery aneurysm wall associates with complement terminal pathway activation.

Inflammation and activation of the complement system predispose to intracranial artery aneurysm (IA) rupture. Because disturbances in complement regulation may lead to increased susceptibility to complement activation and inflammation, we looked for evidence for dysregulation of the complement system in 26 unruptured and 26 ruptured IAs resected intraoperatively. Immunohistochemical and immunofluorescence results of parallel IA sections showed that deposition of the complement activation end-product C5b-9 was lacking from the luminal part of the IA wall that contained complement inhibitors factor H, C4b binding protein, and protectin as well as glycosaminoglycans. In contrast, the outer, less cellular part of the IA wall lacked protectin and had enabled full complement activation and C5b-9 formation. Decay accelerating factor and membrane cofactor protein had less evident roles in complement regulation. The Factor H Y402H variant, studied in 97 IA patients, was seen as often in aneurysm patients with or without aneurysm rupture as in the control population. The regulatory capacity of the complement system thus appears disturbed in the outer part of the IA wall, allowing full proinflammatory complement activation to occur before aneurysm rupture. Insufficient complement control might be due to matrix remodeling and cell loss by mechanical hemodynamics and/or inflammatory stress. Apparently, disturbed complement regulation leads to an increased susceptibility to complement activation, inflammation, and tissue damage in the IA wall.

Mechanisms of self-inactivation in anergic T cells

Las células T autorreactivas que escapan al proceso de selección negativa en el timo han de ser inactivadas o eliminadas en la periferia. En respuesta a una estimulación parcial o subóptima, las células T se vuelvenanérgicas e incapaces de proliferar y producir citocinas en respuesta aencuentros posteriores con el antígeno. Las señales mediadas por calcio tienen un papel importante en la inducción de anergia, por medio de la activación de un programa de auto-inactivación intrínseco a la célula dependiente de calcio/calcineurina/NFAT. Esta revisión se centra en la descripción de nuestros conocimientos actuales acerca de los mecanismos reguladores de la expresión de un programa de expresión génica específico dela anergia de las células T, y cómo las proteínas codificadas por esos genesimponen un estado funcional falta de respuesta a nuevos estimulos. Estose lleva a cabo mediante la localización y la modulación de la actividadde sucesos cruciales para la activación de las células T, incluyendo fenó-menos como la atenuación de las señales del receptor de linfocitos T (TCR)y la inhibición de la transcripción de citocinas (AU)

Self-reactive T cells that escape negative selection in the thymus mustbe inactivated or eliminated in the periphery. In response to a partial orsuboptimal stimulation, T cells become anergic and unable to proliferate and express cytokines in response subsequent re-encounters with antigen. Calcium signaling plays a central role in the induction of anergy, causing the activation of a calcium/calcineurin/NFAT-dependent cell-intrinsic program of self-inactivation. This review will focus on our currentknowledge on the mechanisms that regulate the expression of an anergyspecific program of gene expression in T cells, and how the proteins encoded by those genes impose a state of functional unresponsiveness by targeting and modulating the activity of crucial events required for the activation of T cells, which include downregulation of TCR signaling andinhibition of cytokine transcription (AU)

Induction of antibodies by Staphylococcus aureus nasal colonization in young children.

In order to develop novel antistaphylococcal strategies, understanding the determinants of carriage and how humans respond to Staphylococcus aureus exposure is essential. Here, the primary S. aureus-specific humoral immune response and its association with nasal colonization was studied in young children. Sera from 57 colonized or non-colonized children, serially collected at birth and at 6, 14 and 24 months, were analysed for IgG, IgA and IgM binding to 19 staphylococcal proteins, using flow cytometry-based technology. The antibody responses showed extensive inter-individual variability. On average, the levels of antistaphylococcal IgA and IgM increased from birth until the age of 2 years (p <0.05), whereas the levels of IgG decreased (p <0.001). Placentally transferred maternal IgG did not protect against colonization. In colonized children, IgG and IgA levels for a number of proteins were higher than in non-colonized children. At both 14 and 24 months, the levels of IgG against chemotaxis inhibitory protein of S. aureus (at 24 months; median fluorescence intensity, 4928 vs. 24, p <0.05), extracellular fibrinogen-binding protein (987 vs. 604, p <0.05), and iron-responsive surface determinant H (62 vs. 5, p <0.05) were significantly higher in colonized children. The levels of IgA against CHIPS, IsdH and IsdA were higher (p <0.05). Therefore, CHIPS, Efb, IsdA and IsdH seem to play a role in nasal colonization of young children.

Recent developments in low molecular weight complement inhibitors.

As a key part of the innate immune system, complement plays an important role not only in defending against invading pathogens but also in many other biological processes. Inappropriate or excessive activation of complement has been linked to many autoimmune, inflammatory, and neurodegenerative diseases, as well as ischemia-reperfusion injury and cancer. A wide array of low molecular weight complement inhibitors has been developed to target various components of the complement cascade. Their efficacy has been demonstrated in numerous in vitro and in vivo experiments. Though none of these inhibitors has reached the market so far, some of them have entered clinical trials and displayed promising results. This review provides a brief overview of the currently developed low molecular weight complement inhibitors, including short peptides and synthetic small molecules, with an emphasis on those targeting components C1 and C3, and the anaphylatoxin receptors.

Complement regulators and inhibitory proteins.

The complement system is important for cellular integrity and tissue homeostasis. Complement activation mediates the removal of microorganisms and the clearance of modified self cells, such as apoptotic cells. Complement regulators control the spontaneously activated complement cascade and any disturbances in this delicate balance can result in damage to tissues and in autoimmune disease. Therefore, insights into the mechanisms of complement regulation are crucial for understanding disease pathology and for enabling the development of diagnostic tools and therapies for complement-associated diseases.

Structural and functional implications of the alternative complement pathway C3 convertase stabilized by a staphylococcal inhibitor.

Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.