Absence of Distinct Immunohistochemical Distribution of Annexin A5, C3b, C4d, and C5b-9 in Placentas From Patients With Antiphospholipid Antibodies, Preeclampsia, and Systemic Lupus Erythematosus.

INTRODUCTION: In pregnancy, the presence of preeclampsia (PEC), systemic lupus erythematosus (SLE), and/or antiphospholipid antibody syndrome (APLS) is characterized by poor obstetric outcomes, with potential adverse effects for both mother and fetus. Although the histopathologic changes observed in these entities have been well established, the pathogenic mediators associated with tissue injury are poorly understood. METHODS: Forty placentas were evaluated, including 10 patients with preeclampsia, 9 with SLE, 11 with APLS, and 10 disease-free controls. Each case was subjected to a panel of immunohistochemical markers including C3b, C4d, Annexin A5, and C5b-9. Staining was graded on intensity and distribution. RESULTS: C4d staining was distinctly different among disease groups and controls. Moreover, 6/10 PEC cases, 3/9 SLE cases, and 4/11 APLS cases showed at least focal staining for C4d. All controls were negative. Annexin A5 (AnxA5) staining showed intrinsic variability in all disease groups, while 10/10 controls showed diffuse, strong staining (2+ or 3+). C3b staining was heterogeneous among groups. DISCUSSION: Previously, antiphospholipid antibody (aPLA)-associated pregnancy complications have been thought to be a consequence of a unique aPLA-mediated pathogenic mechanism. However, the immunohistochemical similarity (increased complement and decreased AnxA5 staining) observed in placentas from patients with APLS, PEC, and SLE suggests that aPLA-associated pregnancy complications may reflect a more general autoimmune mechanism.

The MFHR1 Fusion Protein Is a Novel Synthetic Multitarget Complement Inhibitor with Therapeutic Potential.

The complement system is essential for host defense, but uncontrolled complement system activation leads to severe, mostly renal pathologies, such as atypical hemolytic uremic syndrome or C3 glomerulopathy. Here, we investigated a novel combinational approach to modulate complement activation by targeting C3 and the terminal pathway simultaneously. The synthetic fusion protein MFHR1 links the regulatory domains of complement factor H (FH) with the C5 convertase/C5b-9 inhibitory fragment of the FH-related protein 1. In vitro, MFHR1 showed cofactor and decay acceleration activity and inhibited C5 convertase activation and C5b-9 assembly, which prevented C3b deposition and reduced C3a/C5a and C5b-9 generation. Furthermore, this fusion protein showed the ability to escape deregulation by FH-related proteins and form multimeric complexes with increased inhibitory activity. In addition to substantially inhibiting alternative and classic pathway activation, MFHR1 blocked hemolysis mediated by serum from a patient with aHUS expressing truncated FH. In FH-/- mice, MFHR1 administration augmented serum C3 levels, reduced abnormal glomerular C3 deposition, and ameliorated C3 glomerulopathy. Taking the unique design of MFHR1 into account, we suggest that the combination of proximal and terminal cascade inhibition together with the ability to form multimeric complexes explain the strong inhibitory capacity of MFHR1, which offers a novel basis for complement therapeutics.

Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

AAV-mediated expression of human PRELP inhibits complement activation, choroidal neovascularization and deposition of membrane attack complex in mice.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Approximately 50% of AMD patients have a polymorphism in the negative regulator of complement known as Factor H. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroid and retinal pigment epithelium relative to individuals homozygous for the wild-type allele. An inability to form MAC due to a polymorphism in C9 is protective against the formation of choroidal neovascularization (CNV) in AMD patients. Hence, blocking MAC in AMD patients may be protective against CNV. Here we investigate the potential of human proline/arginine-rich end leucine-rich repeat protein (PRELP) as an inhibitor of complement-mediated damage when delivered via the subretinal route using an AAV2/8 vector. In a fluorescence-activated cell sorting (FACS) lysis assay, PRELP inhibited normal human serum-mediated lysis of Hepa-1c1c7 cells by 18.7%. Unexpectedly, PRELP enhanced the formation of tubes by human umbilical vein endothelial cells (HUVECs) by approximately 240%, but, when delivered via an AAV vector to the retina of mice, PRELP inhibited laser-induced CNV by 60%. PRELP reduced deposition of MAC in vivo by 25.5%. Our results have implications for the development of complement inhibitors as a therapy for AMD.

Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway.

Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.

Acute inflammation with induction of anaphylatoxin C5a and terminal complement complex C5b-9 associated with multiple intra-articular injections of hylan G-F 20: a case report.

OBJECTIVE: The purpose of this case report was to investigate local immune mechanisms present during an acute inflammatory flare initiated by viscosupplementation with hylan G-F 20 in a patient with osteoarthritis (OA) and past meniscectomy. EXPERIMENTAL DESIGN: A patient with a history of bilateral OA and partial left knee meniscectomy, who had received three injections of hylan G-F 20, was diagnosed with an acute flare reaction in the left knee. Her chart was evaluated for clinical, radiological, and laboratory findings and for clinical follow-up. Histopathological synovial examination and real-time polymerase chain reaction (RT-PCR) for genes with major roles in local inflammation and enzyme-linked immunosorbent assays (ELISAs) for markers of complement activation and cytokines were performed. To study the impact of the inflammatory and immune features we compared the case patient with groups of three representative OA and three rheumatoid arthritis (RA) patients. RESULTS: The patient exhibited evidence of highly increased acute phase reactant C-reactive protein (CRP) in the blood. The pathological examination of the synovial membrane identified abundant fibrinous exudate with numerous particles of hyaluronan surrounded by a dense infiltrate of neutrophils and eosinophils. The synovium had moderate hypertrophy and sclerosis as well as an inflammatory infiltrate predominantly composed of T lymphocytes and macrophages with scattered perivascular eosinophils and neutrophils. Immunoperoxidase staining identified numerous deposits of C5b-9 in the fibrinous exudates and the synovial membrane of the patient. Similar findings were observed in the RA patients, whereas deposits were rare in OA synovial samples. In addition, both anaphylatoxin C5a and the terminal complement complex C5b-9 were present at high levels, comparable to those in RA patients. The levels of mRNA for interleukin-1 beta (IL-1ß), IL-6, and the neutrophil marker myeloperoxidase (MPO) were markedly increased compared to those in the RA and OA patients. CONCLUSIONS: This present study is indicative of a pseudo-septic acute inflammatory reaction in response to local accumulation of hylan G-F 20 with the activation of complement and local invasion of pro-inflammatory cells.

C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.

Complement factor B expression profile in a spontaneous uveitis model.

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Salicylic acid and a chitin elicitor both control expression of the CAD1 gene involved in the plant immunity of Arabidopsis.

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

Dynamic expression of the membrane attack complex (MAC) of the complement system in failing human myocardium.

Inflammatory cytokine-mediated pathways are activated in heart failure and participate in the pathogenesis and progression of the disease. Another major response to inflammation is mediated through the complement system with the production of the membrane attack complex (MAC), a protein known to cause cell lysis and mediate apoptosis. It was postulated that the complement system is activated in patients with heart failure, and this study investigated whether hemodynamic conditions regulate this pathway. The expression of the MAC was assessed in myocardial biopsy samples of normal and failing hearts by immunohistochemistry and Western blot analysis. Myocardial samples from failing hearts were obtained before and after left ventricular assist device implantation. Immunohistochemical staining and Western blot analysis identified increased MAC expression in failing but not normal myocardium. After hemodynamic unloading with left ventricular assist device support, MAC expression returned to levels found in normal controls. In failing hearts, MAC expression did not differ between ischemic and nonischemic causes of heart failure. In conclusion, increased MAC expression in failing human hearts indicates that the complement system is activated in the heart failure milieu. Its removal after hemodynamic normalization is evidence of dynamic regulation, suggesting a pathogenic role for the MAC. These findings identify the complement system as part of a novel pathophysiologic path in heart failure that can potentially be targeted by future therapy.

Coagulation cascade activation causes CC chemokine receptor-2 gene expression and mononuclear cell activation in hemodialysis patients.

Priming of the coagulation cascade during hemodialysis (HD) leads to the release of activated factor X (FXa). The binding of FXa to its specific receptors, effector protease receptor-1 (EPR-1) and protease-activated receptor-2 (PAR-2), may induce the activation of peripheral blood mononuclear cells (PBMC) and promote a chronic inflammatory state that is responsible for several HD-related morbidities. In the attempt to elucidate the mechanisms underlying the coagulation-associated inflammation in HD, 10 HD patients were randomized to be treated subsequently with a cellulose acetate membrane (CA) and Ethylen-vinyl-alcohol (EVAL), a synthetic membrane that has been shown to reduce FXa generation. At the end of each experimental period, surface FXa and thrombin receptors (EPR-1 and PAR-1, -2, and -4) and CCR2 (monocyte chemoattractant protein-1 receptor) gene expression in isolated PBMC were examined. the ability of dialytic membranes to activate protein-tyrosine kinases and the stress-activated kinase JNK and to modulate the generation of terminal complement complex (TCC) was also investigated. EPR-1 and PAR-2 and -4 mRNA expression, barely detectable in normal PBMC, were significantly upregulated in HD patients, particularly in those who were treated with CA. A striking increase of tyrosine-phosphorylated proteins and JNK activation was observed at the end of HD only in CA-treated patients. Simultaneously, an increased gene expression for both splicing isoforms of CCR2, A and B, only in PBMC from CA-treated patients was demonstrated. The increased CCR-2 mRNA abundance was followed by a significant increase in its protein synthesis. The high expression of CCR2 was associated with an increased generation of plasma TCC and a significant drop in leukocyte and monocyte count. By contrast, EVAL treatment slightly lowered TCC generation and normalized leukocyte count. In vitro FXa induced CCR2 A and B expression and JNK activation in freshly isolated PBMC. FXa-induced CCR2 mRNA expression was completely abolished by JNK and tyrosine kinase inhibition. In conclusion, these data suggest that subclinical clotting activation may cause an increased CCR2 gene and protein expression on uremic PBMC, contributing to HD-related chronic microinflammation. The use of the less coagulation-activating membrane, EVAL, may reduce PBMC activation through the modulation of the stress-activated kinase JNK.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis.

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.

Soluble human complement receptor 1 limits ischemic damage in cardiac surgery patients at high risk requiring cardiopulmonary bypass.

BACKGROUND: This study was undertaken to determine whether soluble human complement receptor type 1 (TP10), a potent inhibitor of complement activation, would reduce morbidity and mortality in high-risk patients undergoing cardiac surgery on cardiopulmonary bypass (CPB). METHODS: This was a randomized multicenter, prospective, placebo-controlled, double-blind study in which 564 high-risk patients undergoing cardiac surgery on CPB received an intravenous bolus of TP10 (1, 3, 5, 10 mg/kg) or placebo immediately before CPB. The primary endpoint was the composite events of death, myocardial infarction (MI), prolonged (> or =24 hours) intra-aortic balloon pump support (IABP), and prolonged intubation. RESULTS: TP10 significantly inhibited complement activity after 10 to 15 minutes of CPB and this inhibition persisted for 3 days postoperatively. However, there was no difference in the primary endpoint between the 2 groups (33.7% placebo versus 31.4% TP10; P=0.31). The primary composite endpoint was, however, reduced in all male TP10 patients by 30% (P=0.025). TP10 reduced the incidence of death or MI in males by 36% (P=0.026), the incidence of death or MI in CABG males by 43% (P=0.043) and the need for prolonged IABP support in male CABG and valve patients by 100% (P=0.019). There was, however, no improvement seen in female TP10 patients. There were no significant differences in adverse events between the groups. CONCLUSIONS: TP10 effectively inhibits complement activation during CPB; however, this was not associated with an improvement in the primary endpoint of the study. Nevertheless, TP10 did significantly decrease the incidence of mortality and MI in male patients.

The role of complement activation in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease in which dyslipidemia, inflammation, and the immune system play an important pathogenetic role. A role in atherogenesis was demonstrated for monocyte/macrophages, complement system, and T-lymphocytes. Complement activation and C5b-9 deposition occurs both in human and experimental atherosclerosis. Complement C6 deficiency has a protective effect on diet-induced atherosclerosis, indicating that C5b-9 assembly is required for the progression of atherosclerotic lesions. The maturation of atherosclerotic lesions beyond the foam cell stage was shown to be strongly dependent on an intact complement system. C5b-9 may be responsible for cell lysis, and sublytic assembly of C5b-9 induces smooth muscle cell (SMC) and endothelial cell (EC) activation and proliferation. All these data suggest that activation of the complement system plays an important role in atherogenesis.

Role of complement activation in atherosclerosis.

PURPOSE OF REVIEW: Atherosclerosis is characterized by a strong inflammatory component. One factor contributing to inflammation in the arterial intima is the complement system. Here we summarize recent progress in the field of complement research on atherogenesis. RECENT FINDINGS: The complement system is activated in human atherosclerotic lesions and is actively regulated by the local synthesis of complement components and of complement regulatory proteins. Potential triggers of complement activation in the arterial intima include immunocomplexes, C-reactive protein, modified lipoproteins, apoptotic cells, and cholesterol crystals. Complement activation releases anaphylatoxins, and anaphylatoxin receptors have been identified in human atherosclerotic lesions. However, experiments on genetically engineered mice with severe hyperlipidemia have been unable to show a major role for complement in experimental atherogenesis. SUMMARY: In humans there is extensive circumstantial evidence for a role of complement in atherosclerosis, which is somewhat contradictory to recent modest or negative findings in atherosclerosis-prone genetically engineered hyperlipidemic mice.

Formation of complement membrane attack complex in mammalian cerebral cortex evokes seizures and neurodegeneration.

The complement system consists of >30 proteins that interact in a carefully regulated manner to destroy invading bacteria and prevent the deposition of immune complexes in normal tissue. This complex system can be activated by diverse mechanisms proceeding through distinct pathways, yet all converge on a final common pathway in which five proteins assemble into a multimolecular complex, the membrane attack complex (MAC). The MAC inserts into cell membranes to form a functional pore, resulting in ion flux and ultimately osmotic lysis. Immunohistochemical evidence of the MAC decorating neurons in cortical gray matter has been identified in multiple CNS diseases, yet the deleterious consequences, if any, of MAC deposition in the cortex of mammalian brain in vivo are unknown. Here we demonstrate that the sequential infusion of individual proteins of the membrane attack pathway (C5b6, C7, C8, and C9) into the hippocampus of awake, freely moving rats induced both behavioral and electrographic seizures as well as cytotoxicity. The onset of seizures occurred during or shortly after the infusion of C8/C9. Neither seizures nor cytotoxicity resulted from the simultaneous infusion of all five proteins premixed in vitro. The requirement for the sequential infusion of all five proteins together with the temporal relationship of seizure onset to infusions of C8/C9 implies that the MAC was formed in vivo and triggered both seizures and cytotoxicity. Deposition of the complement MAC in cortical gray matter may contribute to epileptic seizures and cell death in diverse diseases of the human brain.

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease.

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.

Narrowing in on the causative defect of an intriguing X-linked myopathy with excessive autophagy.

BACKGROUND: X-Linked myopathy with excessive autophagy (XMEA) is a childhood-onset slowly progressive disease of skeletal muscle with no cardiac, nervous system, or other organ involvement. Pathology is distinctive: membrane-bound autophagic vacuoles, multifold reduplication of the basement membrane, and intense deposition of membrane attack complex and calcium at the myofiber surface. XMEA has been linked to the most telomeric 10.5 cM of Xq28. The authors now report identification of new families, refinement of the locus, mapping of genes to the region, and screening of candidate genes for mutations. METHODS AND RESULTS: Seven new families were ascertained, including an American family with XMEA. Using 11 new microsatellite genetic markers, the authors fine-mapped a recombination in this family and a common ancestral haplotype in two French families, which localized the gene in a 4.37-Mb region. Sequence data were assembled from public and private databases and a near-continuous sequence derived for the entire region. With this sequence, a gene map of 82 genes and 28 expressed sequence tag clusters was constructed; to date, 12 candidate genes have been screened for mutations. CONCLUSIONS: This study doubles the number of reported families with XMEA and more firmly establishes its distinctive clinicopathologic features. It also advances the search for the XMEA causative defect by reducing the disease locus to approximately half its previous size, assembling an almost complete sequence of the refined region, identifying all known genes in this sequence, and excluding the presence of mutations in 10% of these genes.