Control Complement Activation Research Articles

Eculizumab Therapy Leads to Rapid Resolution of Thrombocytopenia in Atypical Hemolytic Uremic Syndrome

Eculizumab is highly effective in controlling complement activation in patients with the atypical hemolytic uremic syndrome (aHUS). However, the course of responses to the treatment is not well understood. We reviewed the responses to eculizumab therapy for aHUS. The results show that, in patients with aHUS, eculizumab therapy, when not accompanied with concurrent plasma exchange therapy, led to steady increase in the platelet count and improvement in extra-renal complications within 3 days. By day 7, the platelet count was normal in 15 of 17 cases. The resolution of hemolytic anemia and improvement in renal function were less predictable and were not apparent for weeks to months in two patients. The swift response in the platelet counts was only observed in one of five cases who received concurrent plasma exchange therapy and was not observed in a case of TMA due to gemcitabine/carboplatin. In summary, eculizumab leads to rapid increase in the platelet counts and resolution of extrarenal symptoms in patients with aHUS. Concurrent plasma exchange greatly impedes the response of aHUS to eculizumab therapy. Eculizumab is ineffective for gemcitabine/carboplatin associated TMA.

The yin and yang of the terminal non-lytic complement pathway

Event Abstract Back to Event The yin and yang of the terminal non-lytic complement pathway Kheir Eddine KERBOUA1*, Zineb Hadjidj2, Warda MEZIANE2, Amina BOUMEDIENE1 and Mourad Aribi2 1 Unite of Immunology, Military University Hospital of Oran, Algeria 2 Laboratory of Applied Molecular Biology andn Immunology, University of Tlemcen, Algeria The anaphylatoxins C5a and C3a are bioactive products generated for the terminal non-lytic complement pathway, known for their ability to recruit inflammatory cells, induce oxidative stress, trigger signaling of survival and anti apoptotic responses, control innate and adaptive immunity, and take part in the body’s growth and maintenance. Firstly, we highlight their benefic role in the brain by activating MAPK and AKT signaling in leukocytes and protect neurons against cell death and their help for spur microglia to eliminate the damaged connections; then we see their implication in skeleton, illustrated by the 3MC syndrome; and finally in spurs damaged organs like liver injured which repair itself by C3a and C5a in via the upregulatation of the hepatocyte growth factor-c-Met axis. Secondly, we highlight the dark side of these anaphylatoxins via their implication in various illnesses, including immune related conditions such as sepsis, rheumatoid arthritis, and organ transplant rejection; and non immune related conditions such as osteoarthritis and age-related macular degeneration. Compelling evidence based on the cancer immunoediting theory has been reported emerging data on the relationship between complement and cancer progression where tumor cells under the immunological pressure develop a variety of strategies to avoid complement-mediated damage and the expansion of new tumor populations that are able to control complement activation. Here we will review the contribution of The C5a/C5aR axis to lung cancer progression, emphasizing the upregulatation of phospholipase C2, phospholipase D, cyclins E and D1 and the transactivation of epidermal growth factor receptor. At the end, we illustrate the importance of these biomolecules by the armory of therapeutic inhibitors that has been proposed like eculizumab, cinryze, compstatin, TT30, and the C5a/C5aR axis inhibitor PMX-53 Keywords: C5a, C3a, benefic role, dark side, therapeutic inhibitors Conference: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014), Tlemcen, Algeria, 17 Oct - 20 Oct, 2014. Presentation Type: Poster Presentation Topic: Immunodeficiencies Citation: KERBOUA K, Hadjidj Z, MEZIANE W, BOUMEDIENE A and Aribi M (2014). The yin and yang of the terminal non-lytic complement pathway. Front. Immunol. Conference Abstract: The First International Congress of Immunology and Molecular Immunopathology (CIMIP2014). doi: 10.3389/conf.fimmu.2014.04.00032 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 01 Dec 2014; Published Online: 01 Dec 2014. * Correspondence: Mr. Kheir Eddine KERBOUA, Unite of Immunology, Military University Hospital of Oran, Orna, Algeria, K.K.Eddine@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kheir Eddine KERBOUA Zineb Hadjidj Warda MEZIANE Amina BOUMEDIENE Mourad Aribi Google Kheir Eddine KERBOUA Zineb Hadjidj Warda MEZIANE Amina BOUMEDIENE Mourad Aribi Google Scholar Kheir Eddine KERBOUA Zineb Hadjidj Warda MEZIANE Amina BOUMEDIENE Mourad Aribi PubMed Kheir Eddine KERBOUA Zineb Hadjidj Warda MEZIANE Amina BOUMEDIENE Mourad Aribi Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

An ELISA assay with two monoclonal antibodies allows the estimation of free factor H and identifies patients with acquired deficiency of this complement regulator

Complement factor H (FH) serum levels can be affected by the presence of immune complexes of FH with autoantibodies like in autoimmune forms of atypical haemolytic uraemic syndrome (aHUS) or with C3b in homozygous factor I (FI) deficiency. These complexes reduce the amount of free functional circulating FH. In this study we aimed to determine whether FH levels measurement is disturbed in some pathological conditions and to establish a method for quantifying free and total FH in serum. For that purpose, FH levels were measured in serum samples from aHUS patients having anti-FH autoantibodies or mutations in FH gene, in patients with homozygous FI deficiency, and in healthy controls. Two anti-FH monoclonal antibodies, OX24 and A229, recognizing different functional regions in FH, were used as capture antibodies in an ELISA assay. In the control group and in the group of patients with FH mutations, the FH levels obtained with the two monoclonal antibodies were similar. In patients with anti-FH autoantibodies or with homozygous FI deficiency, however, FH levels measured with both antibodies were significantly different. As these patients had complexes of FH with autoantibodies or C3b, we interpreted that OX24 was detecting total FH and A229 was recognising free FH. Therefore, quantification of FH in plasma using these two monoclonal antibodies provides not only total FH level but also gives an estimation of how much FH circulates free and is thus available to properly control complement activation.

SP0012 Complement in the Rheumatic Disease: Complementopathies

Complement is an integral component of immunity. Inherited deficiencies in the ability to activate complement increases susceptibility to bacterial infection and autoimmunity. Examples include: meningitis and terminal component deficiency; systemic...

Eculizumab in the treatment of atypical haemolytic uraemic syndrome and other complement-mediated renal diseases

This review considers the use of eculizumab in the treatment of atypical haemolytic uraemic syndrome (aHUS) as well as the other complement-mediated renal diseases, including dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). In addition, a brief discussion of the effectiveness of eculizumab for the prevention of antibody-mediated rejection (AMR) in the setting of renal transplant and the treatment of shiga toxin associated haemolytic uraemic syndrome (STEC HUS) is also provided. No randomized controlled trials exist to support the use of eculizumab in renal disease. The results of two unpublished, prospective adult and adolescent trials support its utility in aHUS, whereas retrospective data support the effectiveness in paediatric aHUS. These two data sets form the basis of the sole renal indication for eculizumab. One small, single-centre trial and a growing number of case reports support the use of eculizumab in C3 glomerulopathy (C3G). There are limited trial data in AMR and renal transplant. Finally, there are conflicting data for the use of eculizumab in STEC HUS. The cumulative published data establish the effectiveness of eculizumab in a select group of renal diseases that have at the centre of their disease either abnormal complement control or maladaptive complement activation.

Complement inhibitory proteins expression in placentas of thrombophilic women

Factors controlling complement activation appear to exert a protective effect on pregnancy. This is particularly important in women with thrombophilia. The aim of this study was to determine the transcript and protein levels of complement decay-accelerating factor (DAF) and membrane cofactor protein (MCP) in the placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women.Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia.The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western blot. We observed a higher transcript (p < 0.05) and protein (p < 0.001) levels of DAF and MCP in the placentas of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of proteins that control activation of complement control proteins is only seemingly contradictory to the changes observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.

Complement inhibitory proteins expression in placentas of thrombophilic women

Factors controlling complement activation appear to exert a protective effect on pregnancy. This is particularly important in women with thrombophilia. The aim of this study was to determine the transcript and protein levels of complement decay-accelerating factor (DAF) and membrane cofactor protein (MCP) in the placentas of women with acquired and inherited thrombophilia. Also, we assessed immunohistochemistry staining of inhibitors of the complement cascade, DAF and MCP proteins, in the placentas of thrombophilic women. Placentas were collected from eight women with inherited thrombophilia and ten with acquired thrombophilia. The levels of DAF and MCP transcripts were evaluated by qPCR, the protein level was evaluated by Western blot. We observed a higher transcript (p &lt; 0.05) and protein (p &lt; 0.001) levels of DAF and MCP in the placentas of thrombophilic women than in the control group. DAF and MCP were localized on villous syncytiotrophoblast membranes, but the assessment of staining in all groups did not differ. The observed higher expression level of proteins that control activation of complement control proteins is only seemingly contradictory to the changes observed for example in the antiphospholipid syndrome. However, given the hitherto known biochemical changes associated with thrombophilia, a mechanism in which increased expression of DAF and MCP in the placentas is an effect of proinflammatory cytokines, which accompanies thrombophilia, is probable.

A complement-IL-4 regulatory circuit controls liver regeneration.

The involvement of IL-4 in liver regeneration has not yet been recognized. In this article, we show that IL-4, produced by NKT cells that accumulate in regenerating livers after partial hepatectomy, contributes to this process by regulating the activation of complement after liver resection in mice. The mechanism of this regulation was associated with the maintenance of an appropriate level of IgM in mouse blood, because IgM deposited in liver parenchyma most likely initiated complement activation during liver regeneration. By controlling complement activation, IL-4 regulated the induction of IL-6, thereby influencing a key pathway involved in regenerating liver cell proliferation and survival. Furthermore, the secretion of IL-4 was controlled by complement through the recruitment of NKT cells to regenerating livers. Our study thus reveals the existence of a regulatory feedback mechanism involving complement and IL-4 that controls liver regeneration.

Specific collaboration between rat membrane complement regulators Crry and CD59 protects peritoneum from damage by autologous complement activation

The peritoneal cavity is isolated from the outside and is usually a sterile environment. Patients on peritoneal dialysis (PD) have PD fluid (PDF) infused into the peritoneal cavity. We previously showed that unregulated complement activation could contribute to the development of peritoneal inflammation in yeast peritonitis in PD therapy. In that situation, suppression of local complement activation is essential to protect the host from further injury. The membrane complement regulators (CRegs), Crry, CD55 and CD59, are expressed in the rat peritoneum, especially along the mesothelial cell layer. We investigated CRegs' functional roles in the peritoneal cavity using blocking mAb against each CReg and complement activation in different PDFs. Blockade of any single CReg did not cause spontaneous peritoneal injury in rat. Combined blockade of Crry and CD59 induced focal peritoneal tissue injury and heavy accumulation of inflammatory cells with peritoneal edema at 24 h. Deposits of C3 and C5b-9 were found on the peritoneal surface after combined blocking of Crry and CD59. Systemic complement depletion by cobra venom factor abrogated these inflammatory changes. When combined blockade of Crry and CD59 was performed with PDF of different pH and glucose concentration in rats, the peritoneal injuries were enhanced with lower pH and higher glucose concentration. These results were confirmed by in vitro experiments using primary rat mesothelial cell culture. Rat CRegs, Crry and CD59, specifically collaborate to control complement activation in rat peritoneum. During PD, impairment of CReg might contribute to the development of severe peritoneal inflammation.

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD.MethodologySerum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. Principal FindingsFourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients.ConclusionsThe comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage.

Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5.

The opportunistic human pathogenic fungus Aspergillus fumigatus is a major cause of fungal infections in immunocompromised patients. Innate immunity plays an important role in the defense against infections. The complement system represents an essential part of the innate immune system. This cascade system is activated on the surface of A. fumigatus conidia and hyphae and enhances phagocytosis of conidia. A. fumigatus conidia but not hyphae bind to their surface host complement regulators factor H, FHL-1, and CFHR1, which control complement activation. Here, we show that A. fumigatus hyphae possess an additional endogenous activity to control complement activation. A. fumigatus culture supernatant efficiently cleaved complement components C3, C4, C5, and C1q as well as immunoglobulin G. Secretome analysis and protease inhibitor studies identified the secreted alkaline protease Alp1, which is present in large amounts in the culture supernatant, as the central molecule responsible for this cleavage. An alp1 deletion strain was generated, and the culture supernatant possessed minimal complement-degrading activity. Moreover, protein extract derived from an Escherichia coli strain overproducing Alp1 cleaved C3b, C4b, and C5. Thus, the protease Alp1 is responsible for the observed cleavage and degrades a broad range of different substrates. In summary, we identified a novel mechanism in A. fumigatus that contributes to evasion from the host complement attack.

Genetic control of the alternative pathway of complement in humans and age-related macular degeneration

Activation of the alternative pathway of complement is implicated in common neurodegenerative diseases including age-related macular degeneration (AMD). We explored the impact of common variation in genes encoding proteins of the alternative pathway on complement activation in human blood and in AMD. Genetic variation across the genes encoding complement factor H (CFH), factor B (CFB) and component 3 (C3) was determined. The influence of common haplotypes defining transcriptional and translational units on complement activation in blood was determined in a quantitative genomic association study. Individual haplotypes in CFH and CFB were associated with distinct and novel effects on plasma levels of precursors, regulators and activation products of the alternative pathway of complement in human blood. Further, genetic variation in CFH thought to influence cell surface regulation of complement did not alter plasma complement levels in human blood. Plasma markers of chronic activation (split-products Ba and C3d) and an activating enzyme (factor D) were elevated in AMD subjects. Most of the elevation in AMD was accounted for by the genetic variation controlling complement activation in human blood. Activation of the alternative pathway of complement in blood is under genetic control and increases with age. The genetic variation associated with increased activation of complement in human blood also increased the risk of AMD. Our data are consistent with a disease model in which genetic variation in the complement system increases the risk of AMD by a combination of systemic complement activation and abnormal regulation of complement activation in local tissues.

Factor H–related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation

AbstractHomozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides

The aim of this study was to evaluate a new approach to inhibit complement activation triggered by biomaterial surfaces in contact with blood. In order to inhibit complement activation initiated by the classical pathway (CP), we used streptococcal M protein-derived peptides that specifically bind human C4BP, an inhibitor of the CP. The peptides were used to coat polystyrene microtiter wells which served as a model biomaterial. The ability of coated peptides to bind C4BP and to attenuate complement activation via the CP (monitored as generation of fluid-phase C3a and binding of fragments of C3 and C4 to the surface) was investigated using diluted normal human serum, where complement activation by the AP is minimal, as well as serum from a patient lacking alternative pathway activation. Complement activation (all parameters) was significantly decreased in serum incubated in well surfaces coated with peptides. Total inhibition of complement activation was obtained at peptide coating concentrations as low as 1–5 μg/mL. Successful use of Streptococcus-derived peptides shows that it is feasible to control complement activation at a model biomaterial surface by capturing autologous complement regulatory molecules from plasma.

Efficacy of Eculizumab in a Plasmatherapy-Dependent Patient with Atypical Hemolytic Uremic Syndrome with C3 Mutation Following Plasmatherapy Withdrawal

Atypical hemolytic uremic syndrome (aHUS) is a rare microangiopathic hemolytic anemia characterized by chronic intravascular hemolysis, consumptive thrombocytopenia, microvascular glomerular thrombosis and acute renal failure. Atypical HUS develops as the result of unregulated complement activation either through genetic abnormalities in one or more complement proteins or more rarely the development of autoantibodies to complement factor H. Complement dysregulation has been shown to cause cause subendothelium exposure and activation of platelets resulting in a chronic proinflammatory and prothrombotic state. The prognosis for aHUS is poor as 25% of patients die during acute phases of the disease and 50% progress to end-stage-renal disease. In addition, the majority of renal transplants result in loss of the graft. Plasmatherapy (PT), either plasmapheresis, plasma infusion, or both, is currently used in an attempt to control complement activation and thereby reduce the thrombotic microangiopathy (TMA) and declining renal function, but this therapy is cumbersome and not effective in all patients. Eculizumab, an antibody targeting complement C5, blocks activation of terminal complement and generation of the proinflammatory and prothrombotic molecules C5a and C5b-9. In previous studies eculizumab significantly blocked complement-mediated hemolysis in patients with paroxysmal nocturnal hemoglobinuria, subsequently reducing thrombotic events and improving renal function. In this study, we report the first case of eculizumab treatment in a patient with recurrent aHUS after renal transplantation who refused further PT. The patient is a 42-year-old female diagnosed with a familial form of aHUS with a C3 mutation leading to a binding defect between C3b and the complement control molecules factor H and membrane cofactor protein. The patient showed reduced serum levels of C3c (670 mg/L) suggesting C3 consumption. The patient had received 2 previous renal transplants, the last of which was performed in 2004; aHUS recurred after each transplant and required PT. In March 2007 the patient experienced an acute episode of aHUS and received 2 intensive PT sessions (60 treatments over 9 mos) to resolve the recurrence. In April 2008, the patient presented with septicemia and acute renal failure and was hospitalized for 10 days. In May 2008 her platelet count dropped to 170 ×109/L, haptoglobin became undetectable (< 0.15 g/L), and schistocytes increased to 3.7% suggesting an acute TMA exacerbation, confirmed by renal biopsy. Plasmatherapy was initiated with a course of high dose steroids and IV immunoglobulins. The administration of frequent PT treatments (16 treatments over 5 weeks) resulted in an improvement in the ongoing TMA. However, despite intensive PT, the patient continued to suffer from severe fatigue and daily episodes of diarrhea and chose to discontinue this therapy. As a result, disease deterioration was observed (see 10 Days of No PT in Table). The clinical deterioration established the need for an alternative treatment to reduce TMA and stabilize renal function. PT (3 treatments) was performed as a bridging treatment to eculizumab. Treatment with eculizumab was initiated 4 days following the last PT. The patient received a meningococcal vaccine 4 days prior to treatment with eculizumab and then prophylactic antibiotics (ciprofloxacin) after the vaccination. The patient received 4 doses of eculizumab, 900 mg IV approximately every 7 days, and then 1200 mg 7 days later, and is scheduled to receive chronic dosing at 1200 mg every 14 days. Platelet count, hemolysis and renal function were monitored. After one month of eculizumab treatment, and without concomitant PT, platelet count increased (range from 227 to 284 ×109/L), schistocytes decreased to 0.8% and haptoglobin increased to within normal limits (1.5 g/L; see “Ecu Dose 5”). Levels of C3c fluctuated between 420 and 690 mg/L, creatinine levels were stable and no further episodes of diarrhea were reported. In summary, the data suggest that chronic blockade of complement C5 with eculizumab maintained renal function and reduced platelet consumption and hemolysis without PT in a patient with aHUS previously dependent on frequent PT. Based on these results clinical trials are warranted to confirm the activity of eculizumab for the treatment of patients with recurrent aHUS that are dependent on PT. [Display omitted]

Co-expression of human complement regulatory proteins DAF and MCP with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects

C3 convertase regulatory proteins, decay accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46), have complementary function and transfected into non-human cells might confer protection against human complement. This may be an effective strategy to alleviate C-mediated cell damage by combining the two activities. In this study, we constructed a dicistronic mammalian expression vector pcDNA3-MCPIRESDAF using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV), and stable cell lines were obtained by G418 screening. Integration of extraneous genes was identified by PCR. RT-PCR and Western blotting analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hMCP and hDAF in NIH3T3 cells stably transfected with pcDNA3-MCPIRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and MCP proteins could provide more significant protection against complement-mediated cytolysis than either hMCP or hDAF alone. These results suggest that DAF and MCP synergize the actions of each other, and the IRES-mediated polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery. The pcDNA3-MCPIRESDAF vector has potential therapeutic value for effectively controlling complement activation, thereby increasing the possibility of inter-species transplantation.

Gpm1p Is a Factor H-, FHL-1-, and Plasminogen-binding Surface Protein of Candida albicans

The human pathogenic yeast Candida albicans utilizes host complement regulators for immune evasion. Here we identify the first fungal protein that binds Factor H and FHL-1. By screening a protein array of 4088 proteins of Saccharomyces cerevisiae, phosphoglycerate mutase (ScGpm1p) was identified as a Factor H- and FHL-1-binding protein. The homologous C. albicans Gpm1p (CaGpm1p) was cloned and recombinantly expressed as a 36-kDa His-tagged protein. Purified CaGpm1p binds the host complement regulators Factor H and FHL-1, but not C4BP. The CaGpm1p binding regions in the host proteins were localized; FHL-1 binds via short consensus repeats (SCRs) 6 and 7, and Factor H utilizes two contact regions that are located in SCRs 6 and 7 and in SCRs 19 and 20. In addition, recombinant CaGpm1p binds plasminogen via lysine residues. CaGpm1p is a surface protein as demonstrated by immunostaining and flow cytometry. A C. albicans gpm1(-/-) mutant strain was generated that did not grow on glucose-supplemented but on ethanol- and glycerol-supplemented medium. Reduced binding of Factor H and plasminogen to the null mutant strain is in agreement with the presence of additional binding proteins. Attached to CaGpm1p, each of the three host plasma proteins is functionally active. Factor H and FHL-1 show cofactor activity for cleavage of C3b, and bound plasminogen is converted by urokinase-type plasminogen activator to proteolytically active plasmin. Thus, the surface-expressed CaGpm1p is a virulence factor that utilizes the host Factor H, FHL-1, and plasminogen for immune evasion and degradation of extracellular matrices.

ADAM10-mediated Release of Complement Membrane Cofactor Protein during Apoptosis of Epithelial Cells

Membrane cofactor protein CD46 controls complement activation on cells, is a receptor for several pathogens, and modulates immune responses by affecting CD8(+) T cells. Cells can release CD46 in an intact form on membrane vesicles and in a truncated form by a metalloproteolytic cleavage. The mechanism of shedding and its relationship to cell physiology has remained unclear. We have found using RNA interference analysis that a disintegrin and metalloproteinase (ADAM) 10 is responsible for the regulated shedding of the ectodomain of CD46 in apoptotic cells. The shedding of CD46 was initiated with staurosporine and UVB. Exposure of cell cultures to either UVB or staurosporine resulted in changes of cell morphology and detachment of cells from their matrices within 8-24 h. During this process CD46 was released both in apoptotic vesicles (vCD46) and proteolytically (sCD46) into the medium. Both the metalloproteinase inhibitor GM6001 and RNA interference of ADAM10 completely prevented the release of sCD46 and increased the expression of vCD46 on HaCaT cell vesicles, suggesting that ADAM10 releases sCD46 from the apoptotic vesicles. To explore whether the release of sCD46 is associated with apoptosis we analyzed the effects of caspase inhibitors. As expected, the inhibition of caspase activity attenuated the characteristic features of apoptosis and also decreased the release of sCD46. Our results reveal ADAM10 as an important regulator of CD46 expression during apoptosis. The ADAM10-mediated release of CD46 from apoptotic vesicles may represent a form of strategy to allow restricted complement activation to deal with modified self.

A Review of Current Knowledge of the Complement System and the Therapeutic Opportunities in Inflammatory Arthritis

The complement activation system, a key component of the innate immune system, protects the host from microorganisms such as bacteria, and other foreign threats including abnormal cells. However, it is also double-edged in that it can have negative effects in the host; excessive complement activation damages the host and can even kill in anaphylactic shock and septic shock. Regulation of the complement system is a useful strategy to control inflammatory diseases, including inflammatory arthritis. Rheumatoid arthritis is a common inflammatory disease worldwide. Many medicines are developed to control inflammation, including recently developed biological response modifiers such as anti-TNF and IL-6 agents. Nevertheless, in some patients disease remains difficult to control because of complications, side effects and tolerance of medicines. In inflammatory arthritis, including rheumatoid arthritis, there is abundant evidence implicating complement activation in humans and animal models. Therefore, anti-complement agents might be beneficial as part of clinical treatment. However, at present, there are still no applicable agents for therapeutic regulation of excessive complement activation in chronic disease. Novel agents in development might be useful as a strategy to control complement activation. Here I describe recent knowledge of the complement system in inflammatory arthritis, the recent developments in anti-complement agents and their considerable potential for the future.

De novo gene conversion in the RCA gene cluster (1q32) causes mutations in complement factor H associated with atypical hemolytic uremic syndrome

Many of the complement regulatory genes within the RCA cluster (1q32) have arisen through genomic duplication and the resulting high degree of sequence identity is likely to predispose to gene conversion events. The highest degree of identity is between the genes for factor H (CFH) and five factor H-related proteins--CFHL1, CFHL2, CFHL3, CFHL4, and CFHL5. CFH mutations are associated with atypical hemolytic uremic syndrome (aHUS). In the Newcastle cohort of 157 aHUS patients we have identified CFH mutations in 25 families or individuals. Eleven of these 25 independent mutations are either c.3226C>G,Q1076E; c.3572C>T,S1191L; c.3590T>C,V1197A or combined c.3572C>T,S1191L/c.3590T>C,V1197A. Sequence analysis shows that all four of these changes could have arisen as a result of gene conversion between CFH and CFHL1. Analysis of parental samples in two patients with S1191L/V1197A has shown that the changes are de novo thus providing conclusive evidence that gene conversion is the mutational mechanism in these two cases. To confirm that S1191L and V1197A are disease predisposing we examined their functional significance in three ways - analysis of the C3b/C3d binding characteristics of recombinant mutant S1191L/V1197A protein, heparin affinity chromatography and haemolytic assays of serum samples from aHUS patients carrying these changes. The results showed that these changes resulted in impaired C3b binding and a defective capacity to control complement activation on cellular surfaces. We, therefore, provide conclusive evidence that gene conversion is responsible for functionally significant CFH mutations in aHUS.