Terminal Membrane Attack Complex Research Articles

Advances and challenges in the management of complement-mediated thrombotic microangiopathies.

Complement activation plays a major role in several renal pathophysiological conditions. The three pathways of complement lead to C3 activation, followed by the formation of the anaphylatoxin C5a and the terminal membrane attack complex (MAC) in blood and at complement activating surfaces, lead to a cascade of events responsible for inflammation and for the induction of cell lysis. In case of ongoing uncontrolled complement activation, endothelial cells activation takes place, leading to events in which at the end thrombotic microangiopathy can occur. Atypical haemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy characterized by excessive complement activation on the surface of the microcirculation. It is a severe, rare disease which leads to end-stage renal failure (ESRF) and/or to death in more than 50% of patients without treatment. In the first decade of the second millennium, huge progress in understanding the aetiology of this disease was made, which paved the way to better treatment. First, protocols of plasma therapy for treatment, prevention of relapses and for renal transplantation in those patients were set up. Secondly, in some severe cases, combined kidney and liver transplantation was reported. Finally, at the end of this decade, the era of complement inhibitors, as anti-C5 monoclonal antibody (anti-C5 mAb) began. The past five years have seen growing evidence of the favourable effect of anti-C5 mAb in aHUS which has made this drug the first-line treatment in this disease. The possible complication of meningococcal infection needs appropriate vaccination before its use. Unfortunately, the worldwide use of anti-C5 mAb is limited by its very high price. In the future, extension of indications for anti-C5 mAb use, the elaboration of generics and of mAbs directed towards other complement factors of the terminal pathway of the complement system might succeed in reducing the cost of this new valuable therapeutic approach and render it available worldwide for patients from all social classes.

Complement-Targeted Therapies in Lupus

Systemic lupus erythematosus is a complex disease affecting most organs in the body. Deficiency of early components of the classical complement pathway is a key predisposing genetic factor for lupus in both mice and humans. Activation of the complement cascade downstream of C3 is a key factor in tissue inflammation and damage in lupus. Thus, lupus provides an interesting puzzle for how and when to block complement activation as a therapy. Interventions blocking C5, the alternative pathway, the lectin pathway, and the terminal membrane attack complex are all effective in treating murine models of lupus. Over 20 complement-directed therapies are in clinical trials for various diseases. Despite the promising results regarding efficacy of complement inhibition in murine lupus, the overall lack of effective therapies in lupus, and the availability of a number of complement inhibitors for human trials, there have been and currently are no trials of complement inhibitors for treating lupus. The complexity of lupus and the failure of many recent clinical trials in lupus have perhaps discouraged industry sponsors from testing their complement therapies in lupus. There may also be concern that blocking complement alone may not be effective in treating lupus as other inflammatory mediators are also involved in disease pathogenesis. Despite these possible confounders, it is clear that complement is a key mediator of tissue inflammation and damage in lupus. Complement inhibitors may not be effective at long-term chronic management of lupus, but would appear to be very attractive as an acute intervention during disease flares, especially in lupus nephritis. Ongoing efforts by the NIH and by the lupus organizations to bring new lupus drugs to patients will hopefully lead to clinical trials of complement inhibitors in lupus in the near future.

Dissecting the complement pathway in hepatic injury and regeneration with a novel protective strategy.

Liver resection is commonly performed under ischemic conditions, resulting in two types of insult to the remnant liver: ischemia reperfusion injury (IRI) and loss of liver mass. Complement inhibition is recognized as a potential therapeutic modality for IRI, but early complement activation products are also essential for liver regeneration. We describe a novel site-targeted murine complement inhibitor, CR2-CD59, which specifically inhibits the terminal membrane attack complex (MAC), and we use this protein to investigate the complement-dependent balance between liver injury and regeneration in a clinical setting of pharmacological inhibition. CR2-CD59 did not impact in vivo generation of C3 and C5 activation products but was as effective as the C3 activation inhibitor CR2-Crry at ameliorating hepatic IRI, indicating that the MAC is the principle mediator of hepatic IRI. Furthermore, unlike C3 or C5 inhibition, CR2-CD59 was not only protective but significantly enhanced hepatocyte proliferation after partial hepatectomy, including when combined with ischemia and reperfusion. Remarkably, CR2-CD59 also enhanced regeneration after 90% hepatectomy and improved long-term survival from 0 to 70%. CR2-CD59 functioned by increasing hepatic TNF and IL-6 levels with associated STAT3 and Akt activation, and by preventing mitochondrial depolarization and allowing recovery of ATP stores.

Longitudinally extensive NMO spinal cord pathology produced by passive transfer of NMO-IgG in mice lacking complement inhibitor CD59

Spinal cord pathology with inflammatory, demyelinating lesions spanning three or more vertebral segments is a characteristic feature of neuromyelitis optica (NMO). NMO pathogenesis is thought to involve binding of immunoglobulin G anti-aquaporin-4 autoantibodies (NMO-IgG) to astrocytes, causing complement-dependent cytotoxicity (CDC) and secondary inflammation, demyelination and neuron loss. We investigated the involvement of CD59, a glycophosphoinositol (GPI)-anchored membrane protein on astrocytes that inhibits formation of the terminal C5b-9 membrane attack complex. CD59 inhibition by a neutralizing monoclonal antibody greatly increased NMO-IgG-dependent CDC in murine astrocyte cultures and exvivo spinal cord slice cultures. Greatly increased NMO pathology was also found in spinal cord slice cultures from CD59 knockout mice, and invivo following intracerebral injection of NMO-IgG and human complement. Intrathecal injection (at L5-L6) of small amounts of NMO-IgG and human complement in CD59-deficient mice produced robust, longitudinally extensive white matter lesions in lumbar spinal cord. Pathology was most severe at day 2 after injection, showing loss of AQP4 and GFAP, C5b-9 deposition, microglial activation, granulocyte infiltration, and demyelination. Hind limb motor function was remarkably impaired as well. There was partial remyelination and recovery of motor function by day 5. Our results implicate CD59 as an important modulator of the immune response in NMO, and provide a novel animal model of NMO that closely recapitulates human NMO pathology. Up-regulation of CD59 on astrocytes may have therapeutic benefit in NMO.

Targeted mouse complement inhibitor CR2-Crry protects against the development of atherosclerosis in mice

Atherosclerosis is a chronic inflammatory and immune vascular disease, and clinical and experimental evidence has indicated an important role of complement activation products, including the terminal membrane attack complex (MAC), in atherogenesis. Here, we investigated whether complement inhibition represents a potential therapeutic strategy to treat/prevent atherogenesis using CR2-Crry, a recently described complement inhibitor that specifically targets to sites of C3 activation. Previous studies demonstrated that loss of CD59 (a membrane inhibitor of MAC formation) accelerated atherogenesis in Apoe deficient (Apoe(-/-)) mice. Here, both CD59 sufficient and CD59 deficient mice in an Apoe deficient background (namely, mCd59 ab(+/+)/Apoe(-/-) and mCd59 ab(-/-)/Apoe(-/-)) were treated with CR2-Crry for 4 and 2 months respectively, while maintained on a high fat diet. Compared to control treatment, CR2-Crry treatment resulted in significantly fewer atherosclerotic lesions in the aorta and aortic root, and inhibited the accelerated atherogenesis seen in mCd59 ab(+/+)/Apoe(-/-) and mCd59 ab(-/-)/Apoe(-/-) mice. CR2-Crry treatment also resulted in significantly reduced C3 and MAC deposition in the vasculature of both mice, as well as a significant reduction in the number of infiltrating macrophages and T cells. The data demonstrate the therapeutic potential of targeted complement inhibition.

A Novel C5a-neutralizing Mirror-image (l-)Aptamer Prevents Organ Failure and Improves Survival in Experimental Sepsis

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.

The Alternative Complement Pathway Propagates Inflammation and Injury in Murine Ischemic Stroke

There is mounting evidence indicating an important role for complement in the pathogenesis of cerebral ischemia-reperfusion injury, or ischemic stroke. The role of the alternative complement pathway in ischemic stroke has not been investigated, and there is conflicting data on the role of the terminal pathway. In this study, we show that compared with wild-type mice, mice deficient in the alternative pathway protein factor B or mice treated with the alternative pathway inhibitor CR2-fH have improved outcomes after 60-min middle cerebral artery occlusion and 24-h reperfusion. Factor B-deficient or CR2-fH-treated mice were protected in terms of improved neurologic function and reduced cerebral infarct, demyelination, P-selectin expression, neutrophil infiltration, and microthrombi formation. Mice deficient in both the classical and lectin pathways (C1q/MBL deficient) were also protected from cerebral ischemia-reperfusion injury, and there was no detectable C3d deposition in the ipsilateral brain of these mice. These data demonstrate that the alternative pathway is not alone sufficient to initiate complement activation and indicate that the alternative pathway propagates cerebral injury via amplification of the cascade. Deficiency of C6, a component of the terminal cytolytic membrane attack complex, had no effect on outcome after ischemic stroke, indicating that the membrane attack complex is not involved in mediating injury in this model. We additionally show that the protective effect of factor B deficiency and CR2-fH treatment is sustained in the subacute stage of infarct development, adding to the clinical relevance of these findings.

Use of Anti-C5 Monoclonal Antibody Eculizumab in the Treatment of a Patient with Refractory Idiopathic Thrombotic Thrombocytopenic Purpura (TTP)

Abstract Abstract 4666 Background: Case reports and two prospective, controlled trials document the efficacy of anti-C5 monoclonal antibody eculizumab in the treatment of refractory atypical hemolytic uremic syndrome (aHUS) as well as in aHUS patients requiring chronic plasma exchange. It has shown efficacy in the presence and absence of defects in complement (C) and C regulatory proteins. However, eculizumab has not been previously used in TTP associated with low ADAMTS13 activity, despite the many shared clinical and pathologic features of TTP with aHUS. Case report: A 27 y.o. white male with a history of inflammatory bowel disease presented with fever, thrombocytopenia, and microangiopathic hemolytic anemia, and rapidly developed renal failure, seizures, and coma. ADAMTS13 activity was <5% of normal (IgG anti-ADAMTS13 inhibitor titer of 160), and LDH, 476 on presentation, steadily rose to 1321 by day 8 (Figure). Daily plasma exchange, initiated on hospital day 1, produced no improvement in platelet count, LDH, creatinine, schistocyte levels on peripheral blood smear, mental status, or seizure activity through day 8. Admission stress dose steroids were then increased to 60mg/day prednisone, and vincristine, N-acetylcysteine, and weekly rituximab infusions were initiated, with no change in clinical or laboratory status (Figure). Skin biopsies on day 21 revealed intense granular deposits of C3d, C4d, and the terminal membrane attack complex C5b-9 within and around vessels, including endothelium and pericytes, suggesting a role for local vascular C activation. Hence, eculizumab (900mg IV once a week for 4 weeks, with two supplemental weekly doses of 600mg to adjust for some loss of antibody with plasma exchange, and a final 1200mg dose) was added to the treatment regimen. Platelets increased to 71 and LDH declined to 293 by day 31, and ADAMTS13 activity normalized by day 54 (85%). Plasma exchange and eculizumab were discontinued, and a steroid taper initiated on day 56 when the platelet count was 213K, LDH 145, and creatinine 1.07. The beginning of a rapid decent in platelet count was noted on day 85, accompanied by a decrease in ADAMTS13 activity to 9%. Eculizumab alone (900mg) was restarted. Platelet count, Hb, and creatinine returned to normal within 9 days. Relevant clinical data: Figure and C1q and complement regulatory proteins: factors H, I, and B: normal. Outcome: In remission four months after initial presentation, receiving biweekly eculizumab infusions as an outpatient. Conclusion: Eculizumab was associated with remission induction in a patient with classic idiopathic TTP refractory to plasma exchange. Although multiple, overlapping initial therapies obfuscate assignment of efficacy to a single agent during initial treatment, remission obtained with eculizumab alone on relapse suggests its efficacy. Eculizumab should be considered in patients with refractory TTP. Eculizumab treatment is ongoing in this patient, but the duration of the treatment period remains to be elucidated. Our study also emphasizes that vascular C activation, possibly triggered by endothelial cell injury, may be a critical factor in at least some cases of TTP. C5b-9 induces a secretory response in endothelium to elaborate ultra high molecular weight von Willebrand factor multimers which would be ineffectually cleaved in patients with antibodies to ADAMTS13. This could lead to a dysregulated thrombotic microangiopathy. Blocking C5 formation may thus address a critical upstream pathway relevant to TTP as well as aHUS. Disclosures: Off Label Use:Eculizumab treatment of TTP is an off-label use.

Mapping the Intermedilysin-Human CD59 Receptor Interface Reveals a Deep Correspondence with the Binding Site on CD59 for Complement Binding Proteins C8α and C9

CD59 is a glycosylphosphatidylinositol-anchored protein that inhibits the assembly of the terminal complement membrane attack complex (MAC) pore, whereas Streptococcus intermedius intermedilysin (ILY), a pore forming cholesterol-dependent cytolysin (CDC), specifically binds to human CD59 (hCD59) to initiate the formation of its pore. The identification of the residues of ILY and hCD59 that form their binding interface revealed a remarkably deep correspondence between the hCD59 binding site for ILY and that for the MAC proteins C8α and C9. ILY disengages from hCD59 during the prepore to pore transition, suggesting that loss of this interaction is necessary to accommodate specific structural changes associated with this transition. Consistent with this scenario, mutants of hCD59 or ILY that increased the affinity of this interaction decreased the cytolytic activity by slowing the transition of the prepore to pore but not the assembly of the prepore oligomer. A signature motif was also identified in the hCD59 binding CDCs that revealed a new hCD59-binding member of the CDC family. Although the binding site on hCD59 for ILY, C8α, and C9 exhibits significant homology, no similarity exists in their binding sites for hCD59. Hence, ILY and the MAC proteins interact with common amino acids of hCD59 but lack detectable conservation in their binding sites for hCD59.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

Structural and Functional Analysis of a C3b-specific Antibody That Selectively Inhibits the Alternative Pathway of Complement

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

Direct Antiglobulin Test, Ferritin and GPI-Negative Cell Populations in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) during Eculizumab Therapy: Extravascular Hemolysis Unmasked by Efficient Eculizumab-Mediated Inhibition of Intravascular Hemolysis.

Abstract Introduction: Eculizumab is a humanized monoclonal antibody inhibiting the cleavage of complement factor C5 and preventing terminal complement activation. Therapy with eculizumab leads to a highly significant reduction of complement-mediated intravascular hemolysis and related morbidities including thromboembolic events in Paroxysmal Nocturnal Hemoglobinuria (PNH). It is a new targeted, disease-modifying treatment of PNH. In a placebo-controlled, double-blind randomized study of eculizumab more than 50% of patients (pts) achieved transfusion independence with the remaining pts showing a 44% reduction in the number of red blood cell (RBC) units transfused. In this study we analyzed the proportion of cells with deficiency of GPI-anchored proteins, i.e. PNH cell populations, during eculizumab therapy. Furthermore, we examined a potential mechanism to explain residual hemolysis in some pts despite complete blockade of the terminal complement cascade. Methods: We performed flow cytometric analysis of GPI-anchored proteins, measured levels of hemolysis (LDH), direct bilirubin, hemoglobin and ferritin, and carried out a direct antiglobulin test in samples from 15 (7 female, 8 male) PNH pts during eculizumab therapy. To identify PNH cells, flow cytometric analyses of CD58/CD59 on reticulocytes and erythrocytes, CD66b/CD24/CD16 on granulocytes as well as CD14/CD48 on monocytes were performed. For comparison, the direct antiglobulin test was also performed in 22 (11 female, 11 male) PNH pts without eculizumab therapy. Results: Flow cytometric results of the 15 PNH pts before eculizumab (baseline) and at the time of latest follow-up during eculizumab were compared: GPI-deficient erythrocyte population doubled (29.3% +/−19.3% vs. 56.9% +/−24.0%). The proportion of GPI-deficient reticulocytes (79.1% +/−6.8% vs. 84.6% +/−8.7%), granulocytes (87.5% +/−9.7% vs. 85.5% +/−12.4%) and monocytes (79.0% +/−20.6% versus 73.6%+/−16.2%) did not change significantly. We observed a mean decrease in LDH from 2802 U/l +/− 1015 U/l to 240 U/l +/− 62 U/l; while total bilirubin was stable (47.2 +/− 33.3 μmol/l versus 45.0 +/− 27.8 μmol/l), direct bilirubin showed a threefold increase (3.2 +/− 3.2 μmol/l versus 9.6 +/− 2.4 μmol/l). An increase of ferritin levels was also observed (270 +/− 609 ng/ml versus 651 +/− 652 ng/ml). We performed a direct antiglobulin test in the control group of 22 pts not receiving eculizumab therapy and in 12 pts that received eculizumab. 10 of the 12 pts were tested before and during eculizumab therapy. Only three of all 34 pt who had not received eculizumab at time of antiglobulin test showed a positive antiglobulin test with polyspecific antiglobulin serum, two of them with monospecific anti-IgG and one of them was positive with monospecific anti-C3d. In contrast, all but one of the 12 pts during eculizumab therapy reacted positive with polyspecific antiglobulin serum and monospecific anti-IgG and anti-C3d; in addition, one pt was positive with anti-C3c. The only patient with a negative antiglobulin test during eculizumab therapy was treated for PNH-related thromboembolic events without having transfusion dependent hemolytic anemia. Two of the 12 pts had anti-erythrocytic allo-antibodies (anti-D, -C, -E, -Lua, and -Kpa) before initiation of eculizumab therapy and one of them had additional warm auto-antibodies. Conclusions: 1) A positive direct antiglobulin test (due to C3d loading of PNH RBC) seems to be a consistent finding during eculizumab therapy, which has therefore to be taken into account as a new differential diagnosis of a positive direct antiglobulin test. 2) Eculizumab does not block the early stages of complement activation and therefore C3 cleavage products may occur and bind to CD55/CD59-deficient RBC. The inhibition of terminal membrane attack complex formation by eculizumab allows the survival of these cells which otherwise would undergo a rapid intravascular hemolysis due to C5b-9 formation. 3) The endogenous population of GPI–anchored protein deficient RBC increases during eculizumab therapy. 4) Our data are preliminary and need to be confirmed in larger patient groups: For this purpose, we recommend the monitoring of signs of extravascular hemolysis and ferritin during eculizumab therapy.

Functions of the complement components C3 and C5 during sepsis

Activation of the complement system is a key event in the pathogenesis of sepsis. Nevertheless, the exact mechanisms remain inadequately understood. In the current study, we examined the role of complement C3 and C5 in sepsis in wild-type and C3- or C5-deficient mice induced by cecal ligation and puncture. When compared to wild-type mice, C5(-/-) showed identical survival, and C3(-/-) presented significantly reduced survival. Interestingly, this was associated with significant decreases in plasma levels of proinflammatory mediators. Moreover, although septic C3(-/-) animals displayed a 10-fold increase of blood-borne bacteria, C5(-/-) animals exhibited a 400-fold increase in bacteremia when compared to wild-type mice. These effects were linked to the inability of C5(-/-) mice to assemble the terminal membrane attack complex (MAC), as determined by complement hemolytic activity (CH-50). Surprisingly, although negative control C3(-/-) mice failed to generate the MAC, significant increases of MAC formation was found in septic C3(-/-) mice. In conclusion, our data corroborate that hemolytic complement activity is essential for control of bacteremia in septic mice. Thus, during sepsis, blockade of C5a or its receptors (rather than C5) seems a more promising strategy, because C5a-blockade still allows for MAC formation while the adverse effects of C5a are prevented.

Autoimmunizing Mechanisms in Thymoma and Thymus*

Autoimmunizing mechanisms are very hard to study in humans, so we have focused on vital clues in thymomas and hyperplastic thymuses in myasthenia gravis (MG). According to our multi-step hypothesis: thymic epithelial cells (TEC) present epitopes from the isolated acetylcholine receptor (AChR) subunits they express, and autoimmunize helper T cells; subsequently, these evoke "early antibodies" that then attack rare thymic myoid cells expressing intact AChR; in the resulting germinal centers, autoantibodies diversify to recognize native AChR. We have studied: 1) thymomas, to identify autoimmunizing cell types, focusing on IFN-alpha, against which many patients have high titer autoantibodies, as in another highly informative autoimmune syndrome. Although IFN-alpha is much easier to label than the sparse and delicate AChR subunits, we have not yet located obviously autoimmunizing micro-environments; 2) hyperplastic MG thymuses, where we find (a) upregulation of complement receptors and regulators on hyperplastic TEC and deposition of activated C3b complement component on them, (b) absence of complement regulators from almost all myoid cells, indicating vulnerability to attack, and (c) deposition of C3b, and even of the terminal membrane attack complex, especially on the myoid cells close to the infiltrating germinal centers. The changes are very similar in over 50% of the so-called seronegative patients with generalized MG (SNMG) but without detectable autoantibodies against AChR or MuSK, consistently with other evidence that they belong to the spectrum of AChR-seropositive MG. Together, moreover, our findings implicate both myoid cells and TEC in autoimmunization, and thus strongly support our hypothesis.

Decay-accelerating factor attenuates remote ischemia–reperfusion-initiated organ damage

Complement activation contributes to the expression of local and remote organ injury in animal models of ischemia-reperfusion (IR). We demonstrate here that a soluble form of decay-accelerating factor (DAF) protects normal C57Bl/6 and autoimmunity-prone B6.MRL/lpr mice subjected to hindlimb IR from remote intestinal and lung injury without affecting the degree of local skeletal muscle injury. In addition, DAF treatment attenuates remote organ injury in mice subjected to mesenteric IR. Soluble DAF allowed the deposition of complement 3 in local and remote injury sites while it limited the presence of terminal membrane attack complex and did not increase animal susceptibility to sepsis. These data provide evidence that soluble DAF might offer clinical benefit to patients suffering remote intestinal or lung damage in response to muscle or other organ injury.

Pexelizumab Does Not “Complement” Percutaneous Coronary Intervention in Patients With ST-Elevation Myocardial Infarction

TIMELY RESTORATION OF CORONARY ARTERY BLOOD flow using thrombolytic therapy, percutaneous coronary intervention (PCI), or coronary artery bypass graft (CABG) surgery salvages threatened myocardium and decreases cardiac morbidity and mortality. Reperfusion of ischemic tissues can, however, be associated with life-threatening reperfusion injury that can cause arrhythmias, myocardial stunning, microvascular dysfunction, and cell death. Accordingly, therapies that modulate reperfusion injury would be expected to enhance the effectiveness of thrombolysis and primary percutaneous coronary intervention for preserving myocardium and reducing mortality in patients with ST-elevation myocardial infarction (STEMI). Complement activation plays a key role in the acute inflammatory response associated with ischemia and reperfusion injury. The anaphylotoxins, C3a and C5a, and the C5b-9 membrane attack complex that are formed during complement activation promote tissue injury by increasing vascular permeability, activating endothelial and inflammatory cells, activating hemostasis, inducing apoptosis, and causing cell lysis. Targeting the complement pathway to reduce inflammation and subsequent tissue injury in patients undergoing reperfusion therapy is thus an attractive therapeutic strategy. Pexelizumab is a recombinant humanized singleantibody chain fragment that targets and binds to the human C5 complement component with high affinity, thereby blocking generation of the C5a anaphylotoxin and the formation of the C5b-9 terminal membrane attack complex. Impressive results of early studies in patients with STEMI undergoing reperfusion therapy, particularly those undergoing percutaneous coronary intervention, and in patients undergoing coronary artery bypass graft surgery prompted the evaluation of pexelizumab in 2 large phase 3 studies: The Assessment of Pexelizumab in Acute Myocardial Infarction (APEX AMI) trial, reported in this issue of JAMA, and the Pexelizumab for Reduction in Myocardial Infarction and Mortality in Coronary Artery Bypass Graft Surgery (PRIMO CABG-II) study, which has been presented but is, as yet, unpublished. The APEX AMI trial investigators planned a large, simple trial to investigate the efficacy of pexelizumab for preventing death in 8500 patients with STEMI presenting within 6 hours of symptom onset and undergoing primary percutaneous coronary intervention (PCI). The trial was designed to have 80% power to detect a 24% reduction in all-cause mortality at 90 days. However, recruitment was subsequently curtailed to 5745 patients, resulting in a markedly underpowered trial that did not demonstrate a significant effect of pexelizumab on mortality or any other efficacy outcome. Two factors were central to the decision to stop the APEX AMI trial before originally planned. First, initial samplesize calculations were based on a predicted 6.5% mortality rate in the placebo group at 90 days. At the time of the first interim analysis (September 2005), the overall mortality rate at 90 days was much lower than expected and it was estimated that the sample size would have to be increased to more than 11 000 patients to maintain study power. Second, it was reported in November 2005 that the parallel phase 3 trial, the PRIMO-CABG II trial of Pexelizumab in CABG surgery, did not achieve its primary objective. The latter results together with the lower-than-expected event rates observed in the APEX AMI trial reportedly led the steering committee, in conjunction with the study sponsors, to formulate a revised study plan to discontinue the study after half of the anticipated number of events was accumulated. The rationale for targeting one half of the number of anticipated events is unclear, but the revised study plan provided only about 50% power to detect a 24% reduction in mortality at 30 days. The study authors attributed the lower-than-expected event rates observed in the APEX AMI trial to a high standard of care delivery with widespread use of effective therapies. Indeed, a very high proportion of patients did receive evidence-based treatments, both in hospital and at discharge, and most patients underwent timely reperfusion. However, the lower-than-expected mortality also may be explained by the tendency to enroll low-risk patients in clini-

The Terminal Complement Membrane Attack Complex Is Not Required for Survival from CLP Sepsis: An Anti-C5 Antibody Protects from Mortality in Polymicrobial Sepsis

The Terminal Complement Membrane Attack Complex Is Not Required for Survival from CLP Sepsis: An Anti-C5 Antibody Protects from Mortality in Polymicrobial Sepsis

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.

Complement in Ischemia Reperfusion Injury

In the current issue of The American Journal of Pathology Stahl et al describe an important role for the alternative complement pathway in ischemia reperfusion (I/R) of the intestine, using factor D-deficient mice. 1 The requirement for the complement system in I/R-induced tissue injury has been established, but the contribution of the various complement pathways (classical, alternative, and lectin), is largely unknown because, until recently, the tools for such an investigation were not available. The complement split product, C5a, and the terminal membrane attack complex, C5b-9 (MAC), are believed to be responsible for complement-mediated I/R-induced tissue injury, and, therefore, may be targets for therapeutic interventions in clinical settings. More precise understanding of the relevant complement pathway(s) involved in the final generation of C5a and MAC will define the options for therapeutic intervention.

Complement and Dilated Cardiomyopathy: A Role of Sublytic Terminal Complement Complex-Induced Tumor Necrosis Factor-α Synthesis in Cardiac Myocytes

Dilated cardiomyopathy is a syndrome characterized by cardiac enlargement and impaired systolic function of the heart. Tumor necrosis factor (TNF)-alpha, a pleiotropic cytokine, seems to play a central role in the progression of dilated cardiomyopathy. Recent data suggest that ongoing inflammation in the myocardium may, in many cases, contribute to the development of disease. Chronic generation of autoantibodies to myocardial antigens or, in some cases, viral infection are pathobiologically involved. Although both antibodies and some viruses activate the complement system, the role of innate immunity in dilated cardiomyopathy has as yet not been investigated systematically. In this study we demonstrate by analysis of myocardial biopsies from 28 patients that C5b-9, the terminal membrane attack complex of complement, accumulates in human myocardium in dilated cardiomyopathy. C5b-9 significantly correlates with immunoglobulin deposition and myocardial expression of TNF-alpha. In vitro, C5b-9 attack on cardiac myocytes induces nuclear factor (NF)-kappaB activation as well as transcription, synthesis, and secretion of TNF-alpha. We conclude that chronic immunoglobulin-mediated complement activation in the myocardium may contribute in part to the progression of dilated cardiomyopathy via C5b-9-induced TNF-alpha expression in cardiac myocytes.