The complement system in neurodegenerative diseases.

Complement is an important component of innate immune defence against pathogens and crucial for efficient immune complex disposal. These core protective activities are dependent in large part on properly regulated complement-mediated inflammation. Dysregulated complement activation, often driven by persistence of activating triggers, is a cause of pathological inflammation in numerous diseases, including neurological diseases. Increasingly, this has become apparent not only in well-recognized neuroinflammatory diseases like multiple sclerosis but also in neurodegenerative and neuropsychiatric diseases where inflammation was previously either ignored or dismissed as a secondary event. There is now a large and rapidly growing body of evidence implicating complement in neurological diseases that cannot be comprehensively addressed in a brief review. Here, we will focus on neurodegenerative diseases, including not only the 'classical' neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, but also two other neurological diseases where neurodegeneration is a neglected feature and complement is implicated, namely, schizophrenia, a neurodevelopmental disorder with many mechanistic features of neurodegeneration, and multiple sclerosis, a demyelinating disorder where neurodegeneration is a major cause of progressive decline. We will discuss the evidence implicating complement as a driver of pathology in these diverse diseases and address briefly the potential and pitfalls of anti-complement drug therapy for neurodegenerative diseases.

Complement dysregulation is a prevalent and therapeutically amenable feature of long COVID.

BACKGROUND: Long COVID encompasses a heterogeneous set of ongoing symptoms that affect many individuals after recovery from infection with SARS-CoV-2. The underlying biological mechanisms nonetheless remain obscure, precluding accurate diagnosis and effective intervention. Complement dysregulation is a hallmark of acute COVID-19 but has not been investigated as a potential determinant of long COVID. METHODS: We quantified a series of complement proteins, including markers of activation and regulation, in plasma samples from healthy convalescent individuals with a confirmed history of infection with SARS-CoV-2 and age/ethnicity/sex/infection/vaccine-matched patients with long COVID. FINDINGS: Markers of classical (C1s-C1INH complex), alternative (Ba, iC3b), and terminal pathway (C5a, TCC) activation were significantly elevated in patients with long COVID. These markers in combination had a receiver operating characteristic predictive power of 0.794. Other complement proteins and regulators were also quantitatively different between healthy convalescent individuals and patients with long COVID. Generalized linear modeling further revealed that a clinically tractable combination of just four of these markers, namely the activation fragments iC3b, TCC, Ba, and C5a, had a predictive power of 0.785. CONCLUSIONS: These findings suggest that complement biomarkers could facilitate the diagnosis of long COVID and further suggest that currently available inhibitors of complement activation could be used to treat long COVID. FUNDING: This work was funded by the National Institute for Health Research (COV-LT2-0041), the PolyBio Research Foundation, and the UK Dementia Research Institute.

Complement-membrane regulatory proteins are absent from the nodes of Ranvier in the peripheral nervous system.

BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59 leading to loss of function have been described and, overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the localization and possible role of membrane-bound complement regulators, including CD59, in the peripheral nervous systems (PNS) of mice and humans. METHODS: We examined the localization of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy. RESULTS: We demonstrate that CD59a-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express myelin protein zero (P0), ankyrin G (AnkG), Caspr, dystroglycan, and neurofascin. Immunolabeling of WT nerves using antibodies to CD59 and myelin basic protein (MBP), P0, and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. CD59 was also detected in blood vessels within the nerve. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and CR1-related gene-y (Crry), rendering this area highly exposed to complement attack. CONCLUSION: The Nodes of Ranvier lack CD59 and are hence not protected from complement terminal attack. The myelin unit in human PNS is protected by CD59 and CD55, but not by CD46 or CD35. This renders the nodes and myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barré syndrome, as seen in CD59 deficiency.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Complement terminal pathway inhibition reduces peritoneal injuries in a rat peritonitis model.

Peritonitis and the resulting peritoneal injuries are common problems that prevent long-term peritoneal dialysis (PD) therapy in patients with end-stage kidney diseases. Previously, we have analyzed the relationship between the complement system and progression of peritoneal injuries associated with PD, particularly focusing on the early activation pathways and effects of the anaphylatoxins. We here utilized a novel mAb 2H2 that blocks assembly of the membrane attack complex (MAC) to investigate roles of the complement terminal pathway in PD-associated peritoneal injury. We intraperitoneally injected mAb 2H2 anti-C5b-7 (2.5 or 5 mg/rat) once or twice over the five-day course of the experiment to investigate the effects of inhibiting formation of MAC in a fungal rat peritonitis model caused by repeated intraperitoneal administration of zymosan after methylglyoxal pretreatment (Zy/MGO model). Rats were sacrificed on day 5 and macroscopic changes in both parietal and visceral peritoneum evaluated. Peritoneal thickness, the abundance of fibrinogen and complement C3 and MAC deposition in tissue and accumulation of inflammatory cells were pathologically assessed. The results showed that mAb 2H2, but not isotype control mAb, reduced peritoneal thickness and accumulation of inflammatory cells in a dose and frequency-dependent manner in the Zy/MGO model. These effects were accompanied by decreased C3, MAC, and fibrinogen deposition in peritoneum. In conclusion, in the rat Zy/MGO model, complement terminal pathway activation and MAC formation substantially contributed to development of peritoneal injuries, suggesting that MAC-targeted therapies might be effective in preventing development of peritoneal injuries in humans.

Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis.

Background: The extent of cortical pathology is an important determinant of multiple sclerosis (MS) severity. Cortical demyelination and neurodegeneration are related to inflammation of the overlying leptomeninges, a more inflammatory CSF milieu and with parenchymal microglia and astroglia activation. These are all components of the compartmentalised inflammatory response. Compartmentalised inflammation is a feature of progressive MS, which is not targeted by disease modifying therapies. Complement is differentially expressed in the MS CSF and complement, and complement receptors, are associated with demyelination and neurodegeneration. Methods: To better understand if complement activation in the leptomeninges is associated with underlying cortical demyelination, inflammation, and microglial activation, we performed a neuropathological study of progressive MS (n = 22, 14 females), neuroinflammatory (n = 8), and non-neurological disease controls (n = 10). We then quantified the relative extent of demyelination, connective tissue inflammation, complement, and complement receptor positive microglia/macrophages. Results: Complement was elevated at the leptomeninges, subpial, and within and around vessels of the cortical grey matter. The extent of complement C1q immunoreactivity correlated with connective tissue infiltrates, whilst activation products C4d, Bb, and C3b associated with grey matter demyelination, and C3a receptor 1+ and C5a receptor 1+ microglia/macrophages closely apposed C3b labelled cells. The density of C3a receptor 1+ and C5a receptor 1+ cells was increased at the expanding edge of subpial and leukocortical lesions. C5a receptor 1+ cells expressed TNFα, iNOS and contained puncta immunoreactive for proteolipid protein, neurofilament and synaptophysin, suggesting their involvement in grey matter lesion expansion. Interpretation: The presence of products of complement activation at the brain surfaces, their association with the extent of underlying pathology and increased complement anaphylatoxin receptor positive microglia/macrophages at expanding cortical grey matter lesions, could represent a target to modify compartmentalised inflammation and cortical demyelination.

Complement receptor 1 is expressed on brain cells and in the human brain.

Genome wide association studies (GWAS) have highlighted the importance of the complement cascade in pathogenesis of Alzheimer's disease (AD). Complement receptor 1 (CR1; CD35) is among the top GWAS hits. The long variant of CR1 is associated with increased risk for AD; however, roles of CR1 in brain health and disease are poorly understood. A critical confounder is that brain expression of CR1 is controversial; failure to demonstrate brain expression has provoked the suggestion that peripherally expressed CR1 influences AD risk. We took a multi-pronged approach to establish whether CR1 is expressed in brain. Expression of CR1 at the protein and mRNA level was assessed in human microglial lines, induced pluripotent stem cell (iPSC)-derived microglia from two sources and brain tissue from AD and control donors. CR1 protein was detected in microglial lines and iPSC-derived microglia expressing different CR1 variants when immunostained with a validated panel of CR1-specific antibodies; cell extracts were positive for CR1 protein and mRNA. CR1 protein was detected in control and AD brains, co-localizing with astrocytes and microglia, and expression was significantly increased in AD compared to controls. CR1 mRNA expression was detected in all AD and control brain samples tested; expression was significantly increased in AD. The data unequivocally demonstrate that the CR1 transcript and protein are expressed in human microglia ex vivo and on microglia and astrocytes in situ in the human brain; the findings support the hypothesis that CR1 variants affect AD risk by directly impacting glial functions.

Mechanism and intervention of murine transfusion-related acute lung injury caused by anti-CD36 antibodies.

Anti-CD36 Abs have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36 Ab-mediated TRALI, and potential therapies have not yet been identified. Here, we developed a murine model of anti-CD36 Ab-mediated TRALI to address these questions. Administration of mouse mAb against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab')2 fragments, induced severe TRALI in Cd36+/+ male mice. Predepletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 Abs increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab')2, antioxidant (N-acetyl cysteine, NAC), or C5 blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab')2 after TRALI induction, significant improvement was achieved when mice were treated postinduction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.

Alternative pathway dysregulation in tissues drives sustained complement activation and predicts outcome across the disease course in COVID-19.

Complement, a critical defence against pathogens, has been implicated as a driver of pathology in COVID-19. Complement activation products are detected in plasma and tissues and complement blockade is considered for therapy. To delineate roles of complement in immunopathogenesis, we undertook the largest comprehensive study of complement in COVID-19 to date, comprehensive profiling of 16 complement biomarkers, including key components, regulators and activation products, in 966 plasma samples from 682 hospitalized COVID-19 patients collected across the hospitalization period as part of the UK ISARIC4C (International Acute Respiratory and Emerging Infection Consortium) study. Unsupervised clustering of complement biomarkers mapped to disease severity and supervised machine learning identified marker sets in early samples that predicted peak severity. Compared to healthy controls, complement proteins and activation products (Ba, iC3b, terminal complement complex) were significantly altered in COVID-19 admission samples in all severity groups. Elevated alternative pathway activation markers (Ba and iC3b) and decreased alternative pathway regulator (properdin) in admission samples were associated with more severe disease and risk of death. Levels of most complement biomarkers were reduced in severe disease, consistent with consumption and tissue deposition. Latent class mixed modelling and cumulative incidence analysis identified the trajectory of increase of Ba to be a strong predictor of peak COVID-19 disease severity and death. The data demonstrate that early-onset, uncontrolled activation of complement, driven by sustained and progressive amplification through the alternative pathway amplification loop is a ubiquitous feature of COVID-19, further exacerbated in severe disease. These findings provide novel insights into COVID-19 immunopathogenesis and inform strategies for therapeutic intervention.

Complement dysregulation and Alzheimer's disease in Down syndrome.

INTRODUCTION: Down syndrome (DS) is associated with immune dysregulation and a high risk of early onset Alzheimer's disease (AD). Complement is a key part of innate immunity and driver of pathological inflammation, including neuroinflammation in AD. Complement dysregulation has been reported in DS; however, the pattern of dysregulation and its relationship to AD risk is unclear. METHODS: Plasma levels of 14 complement biomarkers were measured in 71 adults with DS and 46 controls to identify DS-associated dysregulation; impact of apolipoprotein E (APOE) ε4 genotype, single nucleotide polymorphisms (SNPs) in CLU and CR1, and dementia on complement biomarkers was assessed. RESULTS: Plasma levels of complement activation products (TCC, iC3b), proteins (C1q, C3, C9), and regulators (C1 inhibitor, factor H, FHR4, clusterin) were significantly elevated in DS versus controls while FI and sCR1 were significantly lower. In DS with AD (n = 13), C3 and FI were significantly decreased compared to non-AD DS (n = 58). Neither APOE genotype nor CLU SNPs impacted complement levels, while rs6656401 in CR1 significantly impacted plasma sCR1 levels. CONCLUSIONS: Complement is dysregulated in DS, likely reflecting the generalized immune dysregulation state; measurement may help identify inflammatory events in individuals with DS. Complement biomarkers differed in DS with and without AD and may aid diagnosis and/or prediction. HIGHLIGHTS: Complement is significantly dysregulated in plasma of people with DS who show changes in levels of multiple complement proteins compared to controls. People with DS and dementia show evidence of additional complement dysregulation with significantly lower levels of C3 and factor I compared to those without dementia. rs6656401 in CR1 was associated with significantly elevated sCR1 plasma levels in DS.

Therapeutic Intervention with Anti-Complement Component 5 Antibody Does Not Reduce NASH but Does Attenuate Atherosclerosis and MIF Concentrations in Ldlr-/-.Leiden Mice.

BACKGROUND: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. METHODS: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. RESULTS: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). CONCLUSION: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression.

Discovery of functionally distinct anti-C7 monoclonal antibodies and stratification of anti-nicotinic AChR positive Myasthenia Gravis patients.

Myasthenia Gravis (MG) is mediated by autoantibodies against acetylcholine receptors that cause loss of the receptors in the neuromuscular junction. Eculizumab, a C5-inhibitor, is the only approved treatment for MG that mechanistically addresses complement-mediated loss of nicotinic acetylcholine receptors. It is an expensive drug and was approved despite missing the primary efficacy endpoint in the Phase 3 REGAIN study. There are two observations to highlight. Firstly, further C5 inhibitors are in clinical development, but other terminal pathway proteins, such as C7, have been relatively understudied as therapeutic targets, despite the potential for lower and less frequent dosing. Secondly, given the known heterogenous mechanisms of action of autoantibodies in MG, effective patient stratification in the REGAIN trial may have provided more favorable efficacy readouts. We investigated C7 as a target and assessed the in vitro function, binding epitopes and mechanism of action of three mAbs against C7. We found the mAbs were human, cynomolgus monkey and/or rat cross-reactive and each had a distinct, novel mechanism of C7 inhibition. TPP1820 was effective in preventing experimental MG in rats in both prophylactic and therapeutic dosing regimens. To enable identification of MG patients that are likely to respond to C7 inhibition, we developed a patient stratification assay and showed in a small cohort of MG patients (n=19) that 63% had significant complement activation and C7-dependent loss of AChRs in this in vitro set up. This study provides validation of C7 as a target for treatment of MG and provides a means of identifying patients likely to respond to anti-C7 therapy based on complement-activating properties of patient autoantibodies.

Terminal complement pathway activation drives synaptic loss in Alzheimer's disease models.

Complement is involved in developmental synaptic pruning and pathological synapse loss in Alzheimer's disease. It is posited that C1 binding initiates complement activation on synapses; C3 fragments then tag them for microglial phagocytosis. However, the precise mechanisms of complement-mediated synaptic loss remain unclear, and the role of the lytic membrane attack complex (MAC) is unexplored. We here address several knowledge gaps: (i) is complement activated through to MAC at the synapse? (ii) does MAC contribute to synaptic loss? (iii) can MAC inhibition prevent synaptic loss? Novel methods were developed and optimised to quantify C1q, C3 fragments and MAC in total and regional brain homogenates and synaptoneurosomes from WT and AppNL-G-F Alzheimer's disease model mouse brains at 3, 6, 9 and 12 months of age. The impact on synapse loss of systemic treatment with a MAC blocking antibody and gene knockout of a MAC component was assessed in Alzheimer's disease model mice. A significant increase in C1q, C3 fragments and MAC was observed in AppNL-G-F mice compared to controls, increasing with age and severity. Administration of anti-C7 antibody to AppNL-G-F mice modulated synapse loss, reflected by the density of dendritic spines in the vicinity of plaques. Constitutive knockout of C6 significantly reduced synapse loss in 3xTg-AD mice. We demonstrate that complement dysregulation occurs in Alzheimer's disease mice involving the activation (C1q; C3b/iC3b) and terminal (MAC) pathways in brain areas associated with pathology. Inhibition or ablation of MAC formation reduced synapse loss in two Alzheimer's disease mouse models, demonstrating that MAC formation is a driver of synapse loss. We suggest that MAC directly damages synapses, analogous to neuromuscular junction destruction in myasthenia gravis.

Development, Characterization, and in vivo Validation of a Humanized C6 Monoclonal Antibody that Inhibits the Membrane Attack Complex.

Damage and disease of nerves activates the complement system. We demonstrated that activation of the terminal pathway of the complement system leads to the formation of the membrane attack complex (MAC) and delays regeneration in the peripheral nervous system. Animals deficient in the complement component C6 showed improved recovery after neuronal trauma. Thus, inhibitors of the MAC might be of therapeutic use in neurological disease. Here, we describe the development, structure, mode of action, and properties of a novel therapeutic monoclonal antibody, CP010, against C6 that prevents formation of the MAC in vivo. The monoclonal antibody is humanized and specific for C6 and binds to an epitope in the FIM1-2 domain of human and primate C6 with sub-nanomolar affinity. Using biophysical and structural studies, we show that the anti-C6 antibody prevents the interaction between C6 and C5/C5b by blocking the C6 FIM1-2:C5 C345c axis. Systemic administration of the anti-C6 mAb caused complete depletion of free C6 in circulation in transgenic rats expressing human C6 and thereby inhibited MAC formation. The antibody prevented disease in experimental autoimmune myasthenia gravis and ameliorated relapse in chronic relapsing experimental autoimmune encephalomyelitis in human C6 transgenic rats. CP010 is a promising complement C6 inhibitor that prevents MAC formation. Systemic administration of this C6 monoclonal antibody has therapeutic potential in the treatment of neuronal disease.

Analysis of Complement Gene Expression, Clinical Associations, and Biodistribution of Complement Proteins in the Synovium of Early Rheumatoid Arthritis Patients Reveals Unique Pathophysiologic Features.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial hyperplasia and inflammation. The finding of autoantibodies in seropositive RA suggests that complement system activation might play a pathophysiologic role due to the local presence of immune complexes in the joints. Our first objective was to explore the Pathobiology of Early Arthritis Cohort (PEAC) mRNA sequencing data for correlations between clinical disease severity as measured by DAS28-ESR (disease activity score in 28 joints for erythrocyte sedimentation rate) and complement system gene expression, both in the synovium and in blood. Our second objective was to determine the biodistribution using multiplex immunohistochemical staining of specific complement activation proteins and inhibitors from subjects in the Accelerating Medicines Partnership (AMP) RA/SLE study. In the PEAC study, there were significant positive correlations between specific complement gene mRNA expression levels in the synovium and DAS28-ESR for the following complement genes: C2, FCN1, FCN3, CFB, CFP, C3AR1, C5AR1, and CR1 Additionally, there were significant negative correlations between DAS28-ESR and Colec12, C5, C6, MASP-1, CFH, and MCP In the synovium there were also significant positive correlations between DAS28-ESR and FcγR1A, FcγR1B, FcγR2A, and FcγR3A Notably, CFHR4 synovial expression was positively correlated following treatment with the DAS28-ESR at 6 mo, suggesting a role in worse therapeutic responses. The inverse correlation of C5 RNA expression in the synovium may underlie the failure of significant benefit from C5/C5aR inhibitors in clinical trials performed in patients with RA. Multiplex immunohistochemical analyses of early RA synovium reveal significant evidence of regional alterations of activation and inhibitory factors that likely promote local complement activation.

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

Targeting complement in neurodegeneration: challenges, risks, and strategies.

Evidence implicating complement in neuroinflammatory and neurodegenerative diseases (NDDs) has accumulated over the past decade, revealing complement as a driver of pathology across these diverse diseases. Over the same period, there has been an explosion of interest in the development of complement-modulating drugs, first for a few rare complement dysregulation diseases but recently also for more common diseases where complement contributes to the disease process. To date, there has been little attention paid to the potential role of anticomplement drugs in neurodegeneration and the current landscape does not feature drugs that can enter the central nervous system (CNS), a prerequisite in most NDDs. Here we summarise the evidence implicating complement in neurodegeneration, build the case for testing anticomplement drugs, and discuss how drugs may be modified or designed de novo to inhibit complement in neurodegeneration.

The association between neurodegeneration and local complement activation in the thalamus to progressive multiple sclerosis outcome.

The extent of grey matter demyelination and neurodegeneration in the progressive multiple sclerosis (PMS) brains at post-mortem associates with more severe disease. Regional tissue atrophy, especially affecting the cortical and deep grey matter, including the thalamus, is prognostic for poor outcomes. Microglial and complement activation are important in the pathogenesis and contribute to damaging processes that underlie tissue atrophy in PMS. We investigated the extent of pathology and innate immune activation in the thalamus in comparison to cortical grey and white matter in blocks from 21 cases of PMS and 10 matched controls. Using a digital pathology workflow, we show that the thalamus is invariably affected by demyelination and had a far higher proportion of active inflammatory lesions than forebrain cortical tissue blocks from the same cases. Lesions were larger and more frequent in the medial nuclei near the ventricular margin, whilst neuronal loss was greatest in the lateral thalamic nuclei. The extent of thalamic neuron loss was not associated with thalamic demyelination but correlated with the burden of white matter pathology in other forebrain areas (Spearman r = 0.79, p < 0.0001). Only thalamic neuronal loss, and not that seen in other forebrain cortical areas, correlated with disease duration (Spearman r = -0.58, p = 0.009) and age of death (Spearman r = -0.47, p = 0.045). Immunoreactivity for the complement pattern recognition molecule C1q, and products of complement activation (C4d, Bb and C3b) were elevated in thalamic lesions with an active inflammatory pathology. Complement regulatory protein, C1 inhibitor, was unchanged in expression. We conclude that active inflammatory demyelination, neuronal loss and local complement synthesis and activation in the thalamus, are important to the pathological and clinical disease outcomes of PMS.

Efficacy of an Experimental Gonococcal Lipooligosaccharide Mimitope Vaccine Requires Terminal Complement.

A safe and effective vaccine against multidrug-resistant gonorrhea is urgently needed. An experimental peptide vaccine called TMCP2 that mimics an oligosaccharide epitope in gonococcal lipooligosaccharide, when adjuvanted with glucopyranosyl lipid adjuvant-stable emulsion, elicits bactericidal immunoglobulin G and hastens clearance of gonococci in the mouse vaginal colonization model. In this study, we show that efficacy of TMCP2 requires an intact terminal complement pathway, evidenced by loss of activity in C9-/- mice or when C7 function was blocked. In conclusion, TMCP2 vaccine efficacy in the mouse vagina requires membrane attack complex. Serum bactericidal activity may serve as a correlate of protection for TMCP2.