Formation Of Membrane Attack Complex Research Articles

Complement inhibition abrogates nerve terminal injury in Miller Fisher syndrome

A large body of clinical and experimental data indicate that complement activation is an important mechanism for neuronal and glial injury in Guillain-Barré syndromes. Inhibition of complement activation therefore might be expected to limit the progression of the disease. Using in vitro and in vivo models of the Guillain-Barré syndrome variant, Miller Fisher syndrome, we have shown previously that anti-GQ1b ganglioside antibodies target the presynaptic motor nerve terminal axon and surrounding perisynaptic Schwann cells, thereby mediating destructive injury through deposition of membrane attack complex. Here, we have used this model to investigate the effects of a novel therapeutic inhibitor of complement activation, APT070 (Mirococept), both in vitro and in vivo. In these models, APT070 completely prevents membrane attack complex formation, and thereby has a major neuroprotective effect at the nerve terminal, as assessed by immunohistology of perisynaptic Schwann cell and axonal integrity. These data provide a rationale for considering clinical trials of APT070 in Guillain-Barré syndrome, its variant forms, and other complement dependent neuromuscular disorders.

Selection of GM2, fucosyl GM1, globo H and polysialic acid as targets on small cell lung cancers for antibody mediated immunotherapy.

Glycolipids GM2, GD2, GD3, fucosyl GM1, sialyl Lewis a (sLe(a)) and globo H, and polysialic acid on embryonal NCAM, are cell-surface antigens expressed on small cell lung cancer (SCLC) biopsy specimens. They are all candidates for inclusion in a polyvalent, antibody-inducing vaccine or for adoptive therapy with monoclonal antibodies (mAbs) against SCLC. To identify the minimum optimal combination of target antigens on SCLC and to confirm that antibodies against this combination might be able to mediate complement activation and lysis in the majority of cases, we tested ten SCLC cell lines with fluorescence activated cell sorter (FACS) and complement dependent cytotoxicity (CDC) assays using mAbs against these seven target antigens individually or pooled in different combinations. We find that (1) none of these mAbs demonstrated strong FACS reactivity with more than 6 of the 10 cell lines, (2) no mAb had strong CDC reactivity with more than 4 of the cell lines, (3) when the mAbs were pooled, nine cell lines were strongly positive by FACS and nine cell lines were strongly positive by CDC, and (4) mAbs against GM2, FucGM1, globo H and polysialic acid was the minimum optimal combination for inducing FACS reactivity. The addition of mAbs against sLe(a), GD2 and GD3 had no additional impact by FACS and only minimal additional impact in CDC assays. H345, the only cell line that had less than 30% CDC with the four mAb pool was strongly positive by FACS. To understand the lack of correlation between FACS and CDC in the case of H345, the ten cell lines were screened for expression of complement resistance factors CD55 and CD59. Three cell lines were strongly positive for CD55 and eight were strongly positive for CD59. Overall, no correlation was seen between expression of either of these factors on the ten cell lines and sensitivity to CDC. In the case of H345 however, complement resistance of H345 is demonstrated to be mediated primarily by CD59, and in the presence of mAb against CD59, the four mAb MEM-43 pool induced strong (94%) CDC. CD59 inhibits membrane attack complex formation but not activation of earlier complement components. Consequently, all ten cell lines are good targets for complement activation by the four antibody pool and for elimination by effector mechanisms including complement mediated inflammation and opsonization. These findings support our plan to develop a tetravalent vaccine against SCLC targeting GM2, fucosyl GM1, globo H and polysialic acid.

Recombinant C345C and Factor I Modules of Complement Components C5 and C7 Inhibit C7 Incorporation into the Complement Membrane Attack Complex

Complement component C5 binds to components C6 and C7 in reversible reactions that are distinct from the essentially nonreversible associations that form during assembly of the complement membrane attack complex (MAC). We previously reported that the approximately 150-aa residue C345C domain (also known as NTR) of C5 mediates these reversible reactions, and that the corresponding recombinant module (rC5-C345C) binds directly to the tandem pair of approximately 75-residue factor I modules from C7 (C7-FIMs). We suggested from these and other observations that binding of the C345C module of C5 to the FIMs of C7, but not C6, is also essential for MAC assembly itself. The present report describes a novel method for assembling a complex that appears to closely resemble the MAC on the sensor chip of a surface plasmon resonance instrument using the complement-reactive lysis mechanism. This method provides the ability to monitor individually the incorporation of C7, C8, and C9 into the complex. Using this method, we found that C7 binds to surface-bound C5b,6 with a K(d) of approximately 3 pM, and that micromolar concentrations of either rC5-C345C or rC7-FIMs inhibit this early step in MAC formation. We also found that similar concentrations of either module inhibited complement-mediated erythrocyte lysis by both the reactive lysis and classical pathway mechanisms. These results demonstrate that the interaction between the C345C domain of C5 and the FIMs of C7, which mediates reversible binding of C5 to C7 in solution, also plays an essential role in MAC formation and complement lytic activity.

Role of complement and complement membrane attack complex in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV) is the hallmark of exudative age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been experimentally explored. A reliable way to produce CNV in animals is to rupture Bruch’s membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(−/−) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser-induced choroidal angiogenesis in mice. —Hans E. Grossniklaus

Complement Inhibitors Targeted to the Proximal Tubule Prevent Injury in Experimental Nephrotic Syndrome and Demonstrate a Key Role for C5b-9

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Porcine complement regulators protect aortic smooth muscle cells poorly against human complement‐induced lysis and proliferation: consequences for xenotransplantation

Accelerated atherosclerosis after transplantation has been observed and is characterized by smooth muscle cell proliferation in the graft. Porcine cells are frequently used in models of atherosclerosis and porcine organs are considered for use in transplantation. Complement (C) activation is known to play a major role in rejection of xenografts and is also considered to play a role in the development of atherosclerosis. The aim of this study was to investigate the expression and function of membrane bound regulators of complement (CReg) on porcine aortic smooth muscle cells (PASMC). The PASMC were assessed for expression of CReg and susceptibility to lysis by human C by flow-cytometry. The effect of various cytokines on CReg expression and C-susceptibility was investigated. The ability of human C to induce cell proliferation was assessed using the Alamar blue assay. The PASMC only express the CReg membrane cofactor protein (MCP) and CD59 on their cell surface. MCP expression was increased by interleukin (IL)-4. In contrast to porcine aortic endothelial cells (PAEC), PASMC were found to be surprisingly sensitive to C-mediated lysis, mainly due to a low level of expression of CD59. Human C-induced proliferation of PASMC, which was dependent on complete membrane attack complex (MAC) formation. Endogenously expressed CReg on PASMC poorly protect these cells to human C. Human C can induce proliferation of PASMC. In order to prevent accelerated atherosclerosis in porcine xenografts, increased levels of CReg not only have to be obtained on the endothelial cells but also on the smooth muscle cells.

Humoral Theory of Transplantation: Mechanism, Prevention, and Treatment

We discuss the potential mechanisms of antibody-induced primary endothelium injury, which includes complement-dependent pathway (membrane attack complex formation, recruitment of inflammatory cells, and complement-complement receptor-mediated phagocytosis) and complement independent pathway antibody-dependent cell cytotoxicity. Secondary to endothelium injury, the following pathological reactions are found to be responsible for progressive tissue injury and final graft function loss: platelet activation and thrombosis, pathological smooth muscle and endothelial cell proliferation, and humoral and/or cellular infiltrate-mediated parenchyma damage after endothelium injury. We also introduce three categories of therapeutic strategy in the prevention and treatment of antibody-mediated rejection: (1) inhibition and depletion of antibody producing cells (immunosuppressants, antilymphocyte antibodies, splenectomy); (2) removal or blockage of preexisting or newly developed antibodies (immunoadsorption, plasmapheresis/plasma exchange, intravenous immunoglobulin); and (3) impediment or postponement of antibody-mediated primary and secondary tissue injury (anticoagulation, glucosteroids). In conclusion, because alloantibodies have destructive effect on allografts, alloantibody monitoring becomes extremely important. It will help clinicians to determine a patient’s humoral responses against allograft and will therefore direct clinicians to optimize and/or minimize immunosuppressive drug therapy.

Neonatal Porcine Sertoli Cells Inhibit Human Natural Antibody-Mediated Lysis1

Sertoli cells protect cotransplanted cells from allogeneic and xenogeneic rejection. Additionally, neonatal porcine Sertoli cells (NPSCs) survive long-term as xenografts in nonimmunosuppressed rodents. This has led to the hypothesis that NPSCs could be used to prevent cellular rejection in clinical transplantation, thereby eliminating the need for chronic immunosuppression. Prior to transplantation of NPSCs in humans it is necessary to determine whether they are also protected from humoral-mediated xenograft rejection. The presence of Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (alphaGal epitope) as well as binding of human immunoglobulin G (IgG) and IgM to NPSCs was examined by immunocytochemical and fluorescence-activated cell sorter analysis. alphaGal was detected on 88.5% +/- 3.0% of NPSCs. Consistent with this, 71.7% +/- 1.0% and 65.4% +/- 5.2% of NPSCs were bound by IgG and IgM, respectively. When cultured NPSCs underwent an in vitro cytotoxicity assay by incubation with human AB serum plus complement, no increase in cellular lysis was observed, while controls--porcine aorta endothelial cells--were shown to contain > 60% dead cells. Finally, activation of the complement cascade was examined by immunohistochemistry. C3 and C4 were deposited on the surface of the NPSC membrane, indicating activation of complement. Although the complement cascade was activated, the membrane attack complex (MAC) was not formed. These data demonstrate that despite expression of alphaGal, binding of xenoreactive antibodies, and the activation of complement, NPSCs survive human antibody and complement-mediated lysis by preventing MAC formation. This suggests that NPSCs may be able to survive humoral-mediated rejection in a clinical situation.

215 COMPARISON OF STRONG ENDOTHELIAL CELL-SPECIFIC PROMOTERS FOR EXPRESSION OF HUMAN COMPLEMENT REGULATORY PROTEINS IN PORCINE XENOGRAFT ENDOTHELIAL CELLS

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue cells. Activation of complement pathways results in the formation of the membrane attack complex (MAC). Several cell surface regulators known as membrane complement regulatory proteins (mCRPs) prevent the formation of MAC. For blocking the formation of MAC in the xenotransplanted cells, we tried to overexpress mCRPs in the endothelial cells to prevent the hyperacute rejection (HAR), because one of the major obstacles to xenotransplantation of vascularized organs was reported to be HAR. For this purpose, we isolated human MCP (membrane cofactor protein, CD46) (1.5 kb) and DAF (decay-accelerating factor, CD55) (1.1 kb) promoter regions from human genomic DNA isolated from the human embryonic kidney cell line HEK293, and cloned into pGL3 plasmid for luciferase reporter assay. We also inserted other known endothelial cell-specific promoters, such as Flk-1 (fetal liver kinase-1) and ICAM-2 (intercellular adhesion molecule-2), thrombomodulin promoters, into pGL3 vector to compare their promoter activities. We transfected the plasmids into several endothelial cell lines such as bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC), and mouse pancreatic microvascular endothelial cells (MS1). We also used two epithelial cell lines, human embryonic kidney epithelial cells (HEK293) and human epidermal keratinocytes (HaCaT). The endothelial specific expression of the promoters were compared with those of SV40, CMV, and EF-2a (elongation factor) promoters which are generally used in mammalian gene expression. Luciferase assays showed that among the endothelial cell-specific promoters, the 1.1 kb DAF promoter was the strongest, followed by the Flk-1 promoter. The promoter activity of 1.1 kb DAF was about 2-fold that of the Flk-1 promoter. Moreover, the 1.1 kb DAF and Flk-1 promoters were chosen as the best endothelial specific promoters, as both showed about 5 times more luciferase activity in the endothelial cells compared to that in the epithelial cell lines. We also found that the 5′-flanking region between −1126 and −968 of the 1.1 kb DAF promoter was very important for the endothelial-specific strong gene expression. On the 0.2 kb region of DAF promoter, we could detect several conserved nucleotide sequences interacting with the specific transcription factors, such as GATA-1, USF, CdxA, SRY, Sox-5, and Sp1. Interestingly, deletion of the GATA-1 motif between −1113 and −1104 reduced the promoter activity of the DAF promoter by about 25%. We conclude that the 1.1 kb DAF promoter is a suitable candidate promoter for strong endothelial cell-specific expression of mCRP genes.

Role of Complement and Complement Membrane Attack Complex in Laser-Induced Choroidal Neovascularization

Choroidal neovascularization (CNV), or choroidal angiogenesis, is the hallmark of age-related macular degeneration and a leading cause of visual loss after age 55. The pathogenesis of new choroidal vessel formation is poorly understood. Although inflammation has been implicated in the development of CNV, the role of complement in CNV has not been explored experimentally. A reliable way to produce CNV in animals is to rupture Bruch's membrane with laser photocoagulation. A murine model of laser-induced CNV in C57BL/6 mice revealed the deposition of C3 and membrane attack complex (MAC) in the neovascular complex. CNV was inhibited by complement depletion using cobra venom factor and did not develop in C3(-/-) mice. Anti-murine C6 Abs in C57BL/6 mice inhibited MAC formation and also resulted in the inhibition of CNV. Vascular endothelial growth factor, TGF-beta2, and beta-fibroblast growth factor were elevated in C57BL/6 mice after laser-induced CNV; complement depletion resulted in a marked reduction in the level of these angiogenic factors. Thus, activation of complement, specifically the formation of MAC, is essential for the development of laser- induced choroidal angiogenesis in mice. It is possible that a similar mechanism may be involved in the pathophysiology of other angiogenesis essential diseases.

Inhibition of C5 or absence of C6 protects from sepsis mortality

Inhibiting complement anaphlytoxin C5a during sepsis may prevent sepsis mortality. Although human anti-C5 antibodies exist, their therapeutic use in microbial sepsis has been avoided because of the hypothesis that inhibiting C5b will prevent formation of the bactericidal membrane attack complex (MAC) and worsen clinical outcome. We wished to test the hypothesis that inhibition of C5 would improve outcomes in sepsis. Sepsis was induced in rats by laparotomy and cecal ligation and puncture (CLP) by an IACUC-approved protocol. Sham animals underwent laparotomy without CLP. Following CLP rats were randomized to receive a single IV dose of purified IgG ant-C5 antibody (Ab) or control IgG Ab. Anti-C5 Ab treated rats ( n = 20 ) had significantly lower mortality vs. controls ( n = 21 ) , 20% vs. 52% ( P = 0.019 , log-rank). Analysis of bacterial load by culture of spleen and liver homogenates showed a reduction in colony forming units in anti-C5 Ab treated rats vs. control IgG ( P = 0.003 and 0.009, respectively). Anti-C5 treatment reduced lung injury as measured by total MPO content of lung tissue ( P = 0.024 ) . Finally, rats genetically deficient in C6 production, unable to form MAC but capable of producing C5a and C5b, were protected from CLP-induced sepsis mortality. Our results show that an anti-C5 antibody therapy prevents CLP sepsis-induced mortality and improves lung injury. Inhibition of the complement MAC does not increase bacterial load or mortality, therefore, the use of anti-C5 therapy may be beneficial rather than detrimental in sepsis.

Glycation inactivation of the complement regulatory protein CD59: a possible role in the pathogenesis of the vascular complications of human diabetes.

Micro- and macrovascular diseases are major causes of morbidity and mortality in the diabetic population, but the cellular and molecular mechanisms that link hyperglycemia to these complications remain incompletely understood. We proposed that in human diabetes, inhibition by glycation of the complement regulatory protein CD59 increases deposition of the membrane attack complex (MAC) of complement, contributing to the higher vascular risk. We report here 1) the generation and characterization of an anti-glycated human CD59 (hCD59) specific antibody, 2) the detection with this antibody of glycated hCD59 colocalized with MAC in kidneys and nerves from diabetic but not from nondiabetic subjects, and 3) a significantly reduced activity of hCD59 in erythrocytes from diabetic subjects, a finding consistent with glycation inactivation of hCD59 in vivo. Because hCD59 acts as a specific inhibitor of MAC formation, these findings provide a molecular explanation for the increased MAC deposition reportedly found in the target organs of diabetic complications. We conclude that glycation inactivation of hCD59 that leads to increased MAC deposition may contribute to the extensive vascular pathology that complicates human diabetes.

CD59a Deficiency Exacerbates Ischemia-Reperfusion Injury in Mice

The terminal complement components C5a and the membrane attack complex are involved in the pathogenesis of ischemia-reperfusion injury in many organs. CD59 is the major regulator of membrane attack complex formation. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in renal ischemia-reperfusion injury. Unilateral ischemia-reperfusion injury was induced by clamping the left renal pedicle for 30 minutes under general anesthetic. Mice were studied at 72 hours and 2 weeks after ischemia-reperfusion injury. mCd59a-/- mice developed significantly greater tubular injury (P = 0.01), tubulointerstitial apoptosis (P = 0.02), and neutrophil influx (P = 0.04) than controls at 72 hours after ischemia-reperfusion. Two weeks after ischemia-reperfusion, mCd59a-/- mice exhibited more severe tubular damage predominantly in a corticomedullary distribution than controls (P = 0.02). Quantification of interstitial leukocytes revealed significantly greater numbers of infiltrating lymphocytes (but not macrophages) in mCd59a-/- mice than controls (P = 0.04) at 2 weeks. At both time points, significantly more C9 (as a marker of membrane attack complex) deposition occurred in a peritubular distribution in mCd59a-/- mice than controls. In conclusion, these results demonstrate that the lack of CD59a, by allowing unregulated membrane attack complex deposition, exacerbates both the tubular injury and the interstitial leukocyte infiltrate after ischemia-reperfusion injury in mice.

Anti-disialoside antibodies kill perisynaptic Schwann cells and damage motor nerve terminals via membrane attack complex in a murine model of neuropathy.

Anti-disialoside antibodies (Abs) that bind NeuAc(alpha2-8) NeuAc epitopes on GQ1b and related gangliosides are found in human autoimmune neuropathy sera and are considered to be pathogenic. In a model system in mice, one mechanism by which anti-disialoside Abs have been demonstrated to induce paralysis is through a complement dependent blocking effect on transmitter release at the neuromuscular junction, similar to the effects of alpha-latrotoxin. Although direct targeting of presynaptic neuronal membranes occurs in this model, concomitant injury to perisynaptic Schwann cells (pSC) could indirectly contribute to this paralytic effect by influencing nerve terminal function and survival. To examine this possibility and the specific complement components that might mediate these effects, we exposed neuromuscular junctions in vivo and in vitro to an anti-disialoside Ab in conjunction with intact and selectively deficient complement sources. Using immuno-electron microscopy, we observed Ab deposits equally distributed on both neuronal and pSC membranes, and ultrastructural evidence of injury at both sites. Presynaptic neuronal injury was demonstrated functionally with microelectrode recordings and histologically as neurofilament loss. As hypothesized, concomitant pSC injury occurred, as indicated by abnormal uptake of ethidium dimer into pSC nuclei. The pSC and nerve terminal damage indicators correlated well with deposition of the pore-forming terminal complement component, membrane attack complex (MAC) in pSC and nerve terminal membranes. Furthermore, both neuronal and pSC injury were exacerbated in tissues from mice lacking the inhibitory complement regulator, CD59, where MAC formation is increased. These data demonstrate that both presynaptic neuronal membranes and pSCs are targets for anti-disialoside Abs, and that the injury to both sites is mediated by MAC and further regulated by CD59. This is the first demonstration that complement mediated pSC injury occurs in a model of autoimmune neuropathy and provides a rationale for investigating the possibility of pSC injury in equivalent conditions in man.

Complement synthesis and activation in the brain of SIV-infected monkeys

Complement is one of the most critical defence tools against cerebral infections, but uncontrolled complement biosynthesis and activation can induce profound brain tissue damage. To clarify the role of complement in the pathogenesis of AIDS-associated neurological disorders, we analysed the synthesis of complement in the brains of SIV-infected rhesus macaques. Using immunohistochemical staining we could show that the cerebral synthesis of complement factors C1q and C3 was strongly upregulated in SIV-infected monkeys compared to the spontaneous synthesis in uninfected control monkeys. Astrocytes, neurons, microglia, infiltrating macrophages and multinuclear giant cells all contribute to the high amounts of C1q and C3 in the brain. Secreted C1q and C3 are also deposited on the membrane of neurons, a prerequisite for formation of the membrane-driven lytic membrane attack complex. The membrane deposition thus might suggest complement-induced lysis of bystander neurons as a potential mechanism for cell damage during viral infection of the brain.

The Possibilities and Pitfalls for Anti-Complement Therapies in Inflammatory Diseases

The complement system is a key component of innate immunity, acting to protect the host from micro-organisms such as bacteria and other "foreign" threats, including tumor cells. However, excessive activation of complement can injure the host and can even be life threatening. These toxic effects are caused primarily by the excessive production of the anaphylatoxins C3a and C5a during complement activation and excessive formation of membrane attack complex on the host cell membrane. Many inflammatory diseases, including rheumatoid arthritis and glomerulonephritis, are thought to involve excessive activation of complement, both for their development and perpetuation. Uncontrolled complement activation is also implicated in post-ischemic inflammation and tissue damage and in sepsis. Therefore, it is important to regulate the complement system to treat disease. There are still no broadly applicable agents for the therapeutic regulation of excessive complement activation. However, there are now some agents in the development that might provide useful anti-complement therapies in the near future. Current strategies include the use of neutralizing antibodies, small synthetic antagonists, soluble recombinant forms of the natural complement regulators, and gene therapies to control excessive complement activation. Here we describe these new agents, their strengths and weaknesses and progress in testing the agents in relevant animal models.

The classical and alternative pathways of complement activation play distinct roles in spontaneous C3 fragment deposition and membrane attack complex (MAC) formation on human B lymphocytes.

The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous serum in the absence and presence of selective inhibitors of the AP and CP, respectively. While the total amount of C3 fragments deposited was relatively unaffected by blocking either pathway individually, deposition was virtually abrogated by their combined blockade. A marked difference was observed, however, in the nature of the fragments deposited as a result of CP and AP activation: C3b fragments deposited via the CP were extensively ( approximately 90%) converted to the terminal degradation product, C3dg, whereas about 50% of those deposited by the AP persisted as C3b/iC3b fragments. The extent of MAC formation was also found to be highly pathway dependent, with the AP being about 15-fold more efficient at initiating this process than the CP. A model accounting for the effectiveness of the AP in both preserving C3 fragment integrity and initiating MAC is presented.

ONO-6818, a novel, potent neutrophil elastase inhibitor, reduces inflammatory mediators during simulated extracorporeal circulation

Background Among the serine proteases, neutrophil elastase is a powerful cytotoxic enzyme and plays a pivotal role in the inflammatory response associated with cardiopulmonary bypass. This study assesses the effects of the specific inhibition of neutrophil elastase by a novel, potent, low-molecular-weight neutrophil elastase inhibitor, ONO-6818. We hypothesized that ONO-6818 reduces inflammatory mediators and modulates adhesion molecules and the deformability of neutrophils during simulated extracorporeal circulation. Methods Simulated extracorporeal circulation was established by recirculating fresh heparinized (3.75 U/mL) human blood for 120 minutes in a membrane oxygenator and a roller pump with and without 1.0 μmol/L of ONO-6818 (n = 9 for control group, n = 7 for ONO-6818 group). The neutrophil adhesion molecules, CD11b and L-selectin, and the cytoplasmic F-actin of neutrophils were measured by flow cytometry. Neutrophil deformability was evaluated using simulated silicon microcapillaries. Neutrophil elastase, interleukin 8, and C5b-9 were measured using enzyme immunoassay. Results Neutrophil elastase levels were significantly lower in the ONO-6818 group. ONO-6818 significantly reduced interleukin 8 and C5b-9 production. ONO-6818 did not modulate changes of CD11b and L-selectin during recirculation. Cytoplasmic F-actin content and changes of neutrophil deformability did not significantly differ between the groups. Conclusions Inhibition of neutrophil elastase activity with ONO-6818 reduces further interleukin 8 production and the formation of the complement membrane attack complex, and this results in a reduction of neutrophil elastase levels during simulated extracorporeal circulation. This study suggests that specific neutrophil elastase inhibition with ONO-6818 is a feasible therapeutic option to attenuate the exaggerated inflammatory response associated with cardiopulmonary bypass.

Chronic progression of tubulointerstitial damage in proteinuric renal disease is mediated by complement activation: a therapeutic role for complement inhibitors?

The mechanisms by which increased urinary protein concentrations lead to nephrotoxic injury are certain to be multifactorial and involve complex interactions between numerous pathways of cellular damage mediated by both cellular and humoral pathways. These may include a major role for the podocyte in glomerular diseases leading to chronic renal failure, the loss of microvascular endothelium, the albumin-induced upregulation of renal cytokines and growth factors that promote tubulointerstitial injury by inflammation and fibrogenesis, and the role of complement-mediated tubulointerstitial injury due to proteinuria. This review will focus on the last mechanism, and emphasize recent studies implicating a primary role for activation of complement in proteinuric urine as the principal mediator of tubulointerstitial damage and progressive renal disease in various experimental animal models of nephrosis. It will be our contention that intraluminal activation of the terminal complement cascade leading to the formation of the C5b-9 membrane attack complex is the principal mediator of chronic progressive interstitial damage and progressive renal failure irrespective of the type of primary glomerular injury. This paradigm has important implications for the potential therapeutic role of complement inhibitors that are currently being developed.

Loss of CD59 expression in breast tumours correlates with poor survival.

CD59 (protectin), a phosphatidylinositol-anchored glycoprotein, is a member of the cell membrane-bound complement regulatory proteins that inhibits the formation of the terminal membrane attack complex (MAC) of complement. In this study, the expression of CD59 was evaluated in 520 breast carcinomas from patients with a mean follow-up of 87 months. This expression was correlated with clinicopathological features and patient survival. Marked variation in the intensity of CD59 expression, which correlated with histological grade and Nottingham prognostic index (NPI), was found, with higher expression of CD59 found more often in well and moderately differentiated tumours and those of good prognosis (NPI < or = 3.4). In contrast, high grade and poor prognosis (NPI > 5.4) carcinomas significantly demonstrated lack of CD59 expression (p < 0.001). Moreover, it was found that the percentage of CD59-positive cells correlated significantly with patient survival, ie patients with a high percentage of positive cells (>50%) had a better overall survival (p = 0.006). A correlation was also found between the percentage of CD59-positive cells and tumour type and also the development of distant metastases. No association was found between either the intensity or the percentage of cells expressing CD59 and vascular invasion, lymph node stage, tumour size, patient age or menopausal status. In multivariate analysis, CD59 percentage positivity was of independent prognostic significance with grade and lymph node stage. These findings indicate that loss of CD59 may offer a selective advantage for breast cancers, resulting in more aggressive tumours and conferring a poor prognosis for patients.