Terminal Membrane Attack Complex Research Articles

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Increased Serum Terminal Complements Complex Levels in Attention Deficit Hyperactivity Disorder Children.

Attention deficit hyperactivity disorder (ADHD) is a widespread neuropsychiatric disorder in both children and adolescents, which is associated with social isolation and poor academic performance. Complement proteins are regarded as a major player in inflammation and disease development for several neuropsychiatric diseases such as schizophrenia and bipolar diseases. As clarified by previous data, increased levels of complement molecules and other immunological markers as cytokines were demonstrated in these disorders. Limited studies have investigated complement proteins particularly terminal complement complex or membrane attack complex (C5b-9) among ADHD patients. The present research aims to elucidate the association between C5b-9 complex protein and ADHD. This is a cross-sectional study. Sera were collected from Al-Hussain Teaching Medical City in Holy Karbala, Iraq, during 2019-2020. Sera were tested for C5-b9 using commercial kits by enzyme-linked immunosorbent assay (ELISA). In 90 participants included in the study, a significant increment in C5b-9 levels among ADHD patients (P=0.019) was observed. Patients with positive C5b-9 levels had a 2.76 times higher risk of developing ADHD than control subjects. The diagnostic utility for C5b-9 was statistically significant with 71.11% sensitivity, 55.6% specificity, and a high negative predictive value (97.3%). The study concluded elevation of the C5b-9 terminal complements complex levels in ADHD patients, which could point to the association of complement proteins as inflammatory markers with the ADHD disease process.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Six-week-old wildtype (WT+ONA) and βB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

C9 regulates the complement-mediated cell lysis in association with CD59 to resist bacterial infection in a primary animal

Complement component 9 (C9), as an essential component of terminal membrane attack complex of complement system, plays an important role in innate immune defense. However, the function and regulatory mechanism of C9 in the antimicrobial immune response of teleost fish remain unclear. In this study, the open reading frame of Nile tilapia (Oreochromis niloticus) C9 (OnC9) gene was amplified. The mRNA and protein expression of OnC9 were significantly changed upon infection with Streptococcus agalactiae and Aeromonas hydrophila in vivo and in vitro. Upon bacterial challenge, the OnC9 knockdown could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by re-injection of OnC9, which restored the healthy status of the knockdown tilapia. Further, the OnC9 was an essential component in complement-mediated cell lysis and associated with OnCD59 to regulate the efficiency of lysis. Overall, this study indicates that OnC9 is involved in host defense against bacterial infection, and provides a valuable reference for further exploration of the molecular regulatory mechanism of C9 in innate immune defense in a primary animal.

Ultra-rare complement factor 8 coding variants in families with age-related macular degeneration.

Genome-wide association studies have uncovered 52 independent common and rare variants across 34 genetic loci, which influence susceptibility to age related macular degeneration (AMD). Of the 5 AMD-associated complement genes, complement factor H (CFH) and CFI exhibit a significant rare variant burden implicating a major contribution of the complement pathway to disease pathology. However, the efforts for developing AMD therapy have been challenging as of yet. Here, we report the identification of ultra-rare variants in complement factors 8A and 8B, two components of the terminal complement membrane attack complex (MAC), by whole exome sequencing of a cohort of AMD families. The identified C8 variants impact local interactions among proteins of C8 triplex invitro, indicating their effect on MAC stability. Our results suggest that MAC, and not the early steps of the complement pathway, might be a more effective target for designing treatments for AMD.

Aberrant activation of the complement system in renal grafts is mediated by cold storage.

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation.NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.

Is primary membranous nephropathy a complement mediated disease?

Membranous nephropathy (MN) is an immune complex mediated disease. Although limited to the kidney, in up to 20% of patients MN is associated with other autoimmune, infectious or malignant diseases. The initial pathogenetic event in what is still considered “primary” MN is the binding of circulating autoantibodies to proteins (autoantigens) expressed in glomerular podocytes. This antibody binding leads to the formation of immune complexes in the glomerular basement membrane. There is clinical and experimental evidence that these immune deposits lead to the activation of the complement system. Experimental studies in the MN model of Heymann’s nephritis show that the terminal membrane attack complex (MAC) of the complement system induces a disturbance of the glomerular filtration barrier and leads to proteinuria, the clinical hallmark of MN. After the discovery of the phospholipase A2 receptor 1 and thrombospondin type 1 domain containing protein 7A as endogenous antigens, it is assumed that IgG4 antibodies directed against these proteins induce MN in over 85% of patients with primary MN. As a result, the role of complement in the pathogenesis of MN needs to be defined in light of these developments. In this review we describe the current knowledge on the function of the complement system in primary MN and discuss the open questions, which have to be solved for a better understanding of the potential role of complement in the pathophysiology of primary MN.

Complement Deficiencies Result in Surrogate Pathways of Complement Activation in Novel Polygenic Lupus-like Models of Kidney Injury.

Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.

Atypical Hemolytic Uremic Syndrome Successfully Treated with Eculizumab

Atypical hemolytic uremic syndrome (aHUS) is a rare syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal injury, which results from uncontrolled complement activation. Delayed diagnosis and treatment of aHUS may result in end-stage renal disease (ESRD) and an associated dependence on dialysis. In extreme cases, it may cause death due to multi-organ failure. Eculizumab, a humanized monoclonal antibody against C5, inhibits the formation of the terminal membrane attack complex and is used to treat aHUS. Here, we report a 46-year-old male patient who suffered from aHUS relapse, despite prior treatment with repeated plasma exchange and hemodialysis. Eculizumab therapy improved his hematologic findings without use of hemodialysis.

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

SUMMARYRetinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

Upregulation and release of soluble fms-like tyrosine kinase receptor 1 mediated by complement activation in human syncytiotrophoblast cells.

Antiangiogenic molecule soluble fms-like tyrosine kinase receptor 1 (sFLT1) released from trophoblast cells is associated with pregnancy-specific hypertensive disorder pre-eclampsia. Cause of elevated sFLT1 in pre-eclampsia patients is not well understood. Despite evidence of excess systemic and placental complement activation in pre-eclampsia patients, its role in pathophysiology is not clear. If the complement activation plays a role in upregulation and secretion of sFLT1 is not known. Human trophoblast cells were isolated from term placentas and allowed to syncytialize. Complement was activated in vitro at sublethal levels on syncytiotrophoblast cells. Effect of complement activation on expression and release of sFLT1 was assessed by comparing its levels in these cells with and without complement activation. Sublethal level of complement activation on syncytialized human trophoblast cells induced upregulation of sFLT1 mRNA and protein. Complement also induced secretion of sFLT1 in a manner depending on degree of activation. Anaphylatoxins C3a induced upregulation but not the release of sFLT1. Release of terminal membrane attack complex (MAC) was associated with sFLT1 secretion. Complement activation plays a major role in both the expression and secretion of sFLT1 from syncytial trophoblast cells. The terminal MAC complex is involved in its secretion. Increased levels of sFLT1 in pre-eclampsia patients may be due to complement-induced upregulation and secretion.

Abstract PD6-02: Immunological differences between primary and metastatic breast cancer

Abstract Background: Little is known about how the immune microenvironment of breast cancer evolves during disease progression. Immunological differences between primary and metastatic lesions may explain discordant results of clinical trials that showed low tumor response rates with immune checkpoint therapy in metastatic breast cancer but high rates of pathologic complete response in early stage disease. The goal of this project was to examine TIL counts, PD-L1 protein expression and immune gene mRNA expression in primary tumors (P) and metastatic lesions (M). Methods: FFPE blocks of primary breast cancers and core needle biopsies of matching distant metastases from 54 patients (n=104 samples). TIL count was assessed on H&E slides for 39 paired tissues and is reported as % of TIL in the stroma. PD-L1 protein expression was detected with immunohistochemistry (IHC, E1L3N antibody) in 36 pairs, samples with > 1% cells showing staining were considered PD-L1 positive which was determined separately for tumor and stroma. The expression of 770 immune-related genes was measured using the Nanostring PanCancer Immune Gene Panel in 31 P and 17 M, including 10 paired cases. Genes were organized into 14 immune cell type (total T, Th1, Treg, Total CD8, exhausted CD8, Cytotoxic T, B, NK, NK-CD56, Mast cell, CD45, Dendritic cell, macrophage, neutrophil) and 22 immune function metagenes. Differences in mean expression in P and M were assessed using Fisher exact and Mann-Whitney tests without adjustment for multiple comparisons due to overlap in metagene membership. Results: Mean TIL counts (14% vs 20%, p=0.026) and stromal PD-L1 positivity by IHC (14% vs 54%, p=0.004) were significantly lower in M. PD-L1 positivity in tumor cells was similar (25% in M vs 42%, in P p=0.14). The total TIL gene expression score (2.48 vs 2.8 p=0.018) and all immune cell type metagenes, except neutrophils, had lower absolute expression levels in M. The relative abundance of neutrophils (0.035 vs -0.38, p=0.0001) and macrophages (0.62 vs 0.38 p=0.0013) increased in M. Among the 22 immune function metagenes, T, B and NK cell functions, cytotoxicity, chemokine and TNF superfamily expression, regulation and pathogen defense were significantly lower in M and none showed significantly increased expression in M. Pro-inflammatory/immune-activating cytokines of IL-6, CCL-5,-12,-19,-22 and CXCL-5,-9,-10,-11 were all significantly lower in M. The greatest drop was seen for CXCL-9 (2243 vs 422, p<0.0001) and CCL-19 (2537 vs 309, p<0.0001). No cytokine showed increased expression in M. Only 6 immune genes (C7, GPI, MAPK1, TAB1, TLR5, PVR) showed significantly higher (p<0.05) expression in M, the greatest increase was for Complement C7 (member of the terminal complement pathway membrane attack complex; 484 vs 3499, p=0.03) and GPI (glucose 6-phosphate isomerase that induces immunoglobulin secretion; 1967 vs 3011, p=0.01). Conclusions: Breast cancer metastases exist in an attenuated immune microenvironment. Most immune cell subtypes, immune functions, and immune-associated gene expression are lower in M compared to P, consistent with immune escape. Metastatic lesions have higher relative abundance of macrophages and neutrophils, which suggest new therapeutic opportunities. Citation Format: Szekely B, Bossuyt V, Li X, Baine M, Silber A, Sanft T, Hofstatter E, Mougalian S, Baghwagar S, Neumeister V, Pelekanou V, Hatzis C, Pusztai L. Immunological differences between primary and metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD6-02.

Identifying the Role of Complement in Triggering Neuroinflammation after Traumatic Brain Injury.

The complement system is implicated in promoting acute secondary injury after traumatic brain injury (TBI), but its role in chronic post-traumatic neuropathology remains unclear. Using various injury-site targeted complement inhibitors that block different complement pathways and activation products, we investigated how complement is involved in neurodegeneration and chronic neuroinflammation after TBI in a clinically relevant setting of complement inhibition. The current paradigm is that complement propagates post-TBI neuropathology predominantly through the terminal membrane attack complex (MAC), but the focus has been on acute outcomes. Following controlled cortical impact in adult male mice, we demonstrate that although inhibition of the MAC (with CR2-CD59) reduces acute deficits, inhibition of C3 activation is required to prevent chronic inflammation and ongoing neuronal loss. Activation of C3 triggered a sustained degenerative mechanism of microglial and astrocyte activation, reduced dendritic and synaptic density, and inhibited neuroblast migration several weeks after TBI. Moreover, inhibiting all complement pathways (with CR2-Crry), or only the alternative complement pathway (with CR2-fH), provided similar and significant improvements in chronic histological, cognitive, and functional recovery, indicating a key role for the alternative pathway in propagating chronic post-TBI pathology. Although we confirm a role for the MAC in acute neuronal loss after TBI, this study shows that upstream products of complement activation generated predominantly via the alternative pathway propagate chronic neuroinflammation, thus challenging the current concept that the MAC represents a therapeutic target for treating TBI. A humanized version of CR2fH has been shown to be safe and non-immunogenic in clinical trials.SIGNIFICANCE STATEMENT Complement, and specifically the terminal membrane attack complex, has been implicated in secondary injury and neuronal loss after TBI. However, we demonstrate here that upstream complement activation products, generated predominantly via the alternative pathway, are responsible for propagating chronic inflammation and injury following CCI. Chronic inflammatory microgliosis is triggered by sustained complement activation after CCI, and is associated with chronic loss of neurons, dendrites and synapses, a process that continues to occur even 30 d after initial impact. Acute and injury-site targeted inhibition of the alternative pathway significantly improves chronic outcomes, and together these findings modify the conceptual paradigm for targeting the complement system to treat TBI.

Absence of CD59 in Guinea Pigs: Analysis of the Cavia porcellus Genome Suggests the Evolution of a CD59 Pseudogene.

CD59 is a membrane-bound regulatory protein that inhibits the assembly of the terminal membrane attack complex (C5b-9) of complement. From its original discovery in humans almost 30 years ago, CD59 has been characterized in a variety of species, from primates to early vertebrates, such as teleost fish. CD59 is ubiquitous in mammals; however, we have described circumstantial evidence suggesting that guinea pigs (Cavia porcellus) lack CD59, at least on erythrocytes. In this study, we have used a combination of phylogenetic analyses with syntenic alignment of mammalian CD59 genes to identify the only span of genomic DNA in C. porcellus that is homologous to a portion of mammalian CD59 and show that this segment of DNA is not transcribed. We describe a pseudogene sharing homology to exons 2 through 5 of human CD59 present in the C. porcellus genome. This pseudogene was flanked by C. porcellus homologs of two genes, FBXO3 and ORF91, a relationship and orientation that were consistent with other known mammalian CD59 genes. Analysis using RNA sequencing confirmed that this segment of chromosomal DNA was not transcribed. We conclude that guinea pigs lack an intact gene encoding CD59; to our knowledge, this is the first report of a mammalian species that does not express a functional CD59. The pseudogene we describe is likely the product of a genomic deletion event during its evolutionary divergence from other members of the rodent order.

Vascular changes and perifascicular muscle fiber damage in dermatomyositis: another question of the chicken or the egg that is on our mind.

Dermatomyositis (DM) is a multisystem disease with symptoms spanning diverse body functions including the skin, the joints, the heart and the lungs. The skin manifestations involve a sun-sensitive rash on the face, neck and upper trunk, and erythema of knees, elbows and knuckles. In classical cases, where these typical skin manifestations combine with muscle weakness, diagnosis can be set on clinical grounds. In other cases, diagnosis is confirmed by assessing the level of serum muscle enzymes, electromyographic findings, and a muscle biopsy (1). In the skeletal muscle tissue of DM, the primary target of the immune response is the vascular endothelium of perimysial and perifascicular blood vessels, and inflammatory infiltrates often occur at perivascular sites. Complement deposition of the terminal C5b-9 membrane attack complex (MAC) leads to blood vessel necrosis (2). The trigger that initiates complement activation remains however unclear.

Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction.

A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption.

Critical appraisal of eculizumab for atypical hemolytic uremic syndrome.

The biology of atypical hemolytic uremic syndrome has been shown to involve inability to limit activation of the alternative complement pathway, with subsequent damage to systemic endothelial beds and the vasculature, resulting in the prototypic findings of a thrombotic microangiopathy. Central to this process is the formation of the terminal membrane attack complex C5b-9. Recently, application of a monoclonal antibody that specifically binds to C5, eculizumab, became available to treat patients with atypical hemolytic uremic syndrome, replacing plasma exchange or infusion as primary therapy. This review focuses on the evidence, based on published clinical trials, case series, and case reports, on the efficacy and safety of this approach.

C5 Polymorphism in a Dutch Patient with Paroxysmal Nocturnal Hemoglobinuria (PNH) and No Asian Ancestry, Resistant to Eculizumab, but in Vitro Sensitive to Coversin

Eculizumab (Soliris®) is a humanized monoclonal antibody that targets complement factor C5and inhibits the production of C5a and formation of the terminal complement membrane attack complex. It is registered for the treatment of PNH and aHUS. Eculizumab results in significant reduction of hemolysis in PNH, improves symptoms and reduces the incidence of PNH related thrombosis . A poor response, defined as sustained high levels of LDH during treatment with eculizumab irrespective of improvement of clinical symptoms, has been reported in a subgroup of PNH patients (Nishimura et al, NEJM 2014;370:632-639). These patients had a genetic variant of C5 which occurs in approximately 3.5% of the Japanese and 1% of the Chinese Han populations and interferes with binding of eculizumab to C5. We describe the first patient with no known Asian ancestry with a poor response to eculizumab, who subsequently had a good in vitro response to protein rEV576 (syntheticOrnithodoros moubata complement C5 binding) or coversin. Coversin is a recombinant small protein derived from a tick salivary molecule which binds to C5 and, through steric hindrance, interferes with the access of C5 convertase to the active site and thus prevents cleavage to C5a and C5b in a similar fashion to eculizumab.Our 30-year old male patient commenced treatment with Eculizumab because of PNH (granulocyte clone size: 90%), severe haemolysis (LDH 3-6x ULN, and peak value of 17xULN), transient renal failure, extreme fatigue and erectile dysfunction. He had no history of thrombosis and no underlying bone marrow disease. During eculizumab treatment (dosed 600 mg iv every 7 days, weeks 1-4 and 900 mg biweekly starting in week 5) he felt better, seemed less fatigued and experienced less erectile dysfunction. However, laboratory examination showed sustained elevated markers of hemolysis. Other causes of hemolysis were excluded. Underdosing of eculizumab was ruled out by demonstrating sustained high LDH levels at different time points in between subsequent eculizumab infusions and by measuring trough levels of eculizumab (>100ug/ml). In vitro terminal complement complex blockage by eculizumab through antibody-coated chicken red blood cell lysis was indicative of ongoing active hemolysis in our patient's serum. The presence of Human Anti-Drug Antibodies was excluded using an illuminescent MSD®assay. Treatment was discontinued when the patient experienced increased hemolysis (LDH 9x ULN) and macroscopic hemoglobinuria one day after receiving a dose of 900 mg eculizumab. As expected, discontinuation did not result in further increase of hemolysis parameters or clinical change.To investigate whether a mutation of complement C5 might explain the eculizumab resistance in our patient, DNA analysis of the coding region of C5 was performed. This showed a single C5 heterozygous missense mutation, c.2653C>A, which predicts p.Arg885Ser. This new mutation was very similar to variants previously found in Japanese patients (c.2654G>A, which predicts p.Arg885His) and an Argentinian patient with Asian ancestry (c.2653C>T, which predicts p.Arg885Cys), indicating the importance of this amino acid in C5 recognition by eculizumab. The same mutation was demonstrated in the DNA of our patient's healthy father.Since coversin binds to an epitope on C5 remote from the eculizumab binding site, we hypothesised that it might block C5 cleavage in our patient. Serum samples from our patient and 6 healthy controls were spiked with ascending doses of either eculizumab or coversin and complement activity was measured using a commercially available CH50 Equivalent ELISA (Quidel Corporation ®). In agreement with our in vivo observation eculizumab was incapable of inhibiting CH50 activity in the patient's serum beyond approximately 75%, even at concentrations of 100ug/ml. In contrast coversin, even in concentrations of 10 ug/ml inhibited complement activity completely, both in serum of our patient and serum of healthy controls. We conclude that coversin may prove a useful alternative to eculizumab for patients with resistance due to C5 polymorphisms. [Display omitted] DisclosuresNishimura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Weston-Davies:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Nunn:Volution Immuno Pharmaceuticals: Employment, Equity Ownership. Kanakura:Alexion Pharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mackie:Volution Immuno Pharmaceuticals (Uk) Ltd: Research Funding. Muus:Alexion Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Targeted complement inhibition and microvasculature in transplants: a therapeutic perspective.

Active complement mediators play a key role in graft-versus-host diseases, but little attention has been given to the angiogenic balance and complement modulation during allograft acceptance. The complement cascade releases the powerful proinflammatory mediators C3a and C5a anaphylatoxins, C3b, C5b opsonins and terminal membrane attack complex into tissues, which are deleterious if unchecked. Blocking complement mediators has been considered to be a promising approach in the modern drug discovery plan, and a significant number of therapeutic alternatives have been developed to dampen complement activation and protect host cells. Numerous immune cells, especially macrophages, develop both anaphylatoxin and opsonin receptors on their cell surface and their binding affects the macrophage phenotype and their angiogenic properties. This review discusses the mechanism that complement contributes to angiogenic injury, and the development of future therapeutic targets by antagonizing activated complement mediators to preserve microvasculature in rejecting the transplanted organ.